Schistosomiasis

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TROPICAL MEDICINE

Science and Practice

Edited by

Adel AF Mahmoud

1

V'i

r *^k

Series editors Geoffrey Pasvol and Stephen L Hoffman Imperial College Press

\

Schistosomiasis

Tropical Medicine: Science and Practice Series Editors: Geoffrey Pasvol Dept. of Infection & Tropical Medicine Imperial College School of Medicine, UK Stephen L Hoffman Naval Medical Research Institute Rockville, Maryland, USA Published Vol. 1 Lymphatic Filariasis Edited by Thomas B Nutman Vol. 2

Amebiasis Edited by Jonathan I Ravdin

TROPICAL MEDICINE Science and Practice Volume 3

Schistosomiasis

edited by

Adel AF Mahmoud President, Merck Vaccines, Merck & Co, Inc

Series editors:

Geoffrey Pasvol and Stephen L Hoffman

Imperial College Press

Published by Imperial College Press 57 Shelton Street Covent Garden London WC2H 9HE Distributed by World Scientific Publishing Co. Pte. Ltd. P O Box 128, Farrer Road, Singapore 912805 USA office: Suite IB, 1060 Main Street, River Edge, NJ 07661 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

SCHISTOSOMIASIS Copyright © 2001 by Imperial College Press All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN

1-86094-146-X

Printed in Singapore by U t o P r i n t

V

About the Series Editors Geoffrey Pasvol is Professor of Infection and Tropical Medicine at Imperial College School of Medicine, London. He graduated from the University of Cape Town and went to Oxford in 1974 as a Rhodes Scholar to study the relationship between malaria and the hemoglobinopathies at the Nuffield Department of Clinical Medicine. From 1974 to 1979 he worked at the Medical Research Council Laboratories at Fajara in the Gambia on the cellular interactions between the malaria parasite and the red cell, and from 1989 to 1991 on clinical aspects of malaria at the KEMRI Wellcome Unit at Kilifi on the Kenyan coast. He was a Medical Research Council visiting fellow at Harvard Medical School in 1984. He has a special interest in the cellular and molecular interactions in the pathogenesis of malaria and has more recently been involved in the immunopathology of tuberculosis. Professor Pasvol is currently Director of the Wellcome Trust Centre for Clinical Tropical Medicine at Imperial College. Stephen L Hoffman is Director of the Malaria Program at the Naval Medical Research Center in Silver Spring, Maryland. After studies at the University of Pennsylvania, Cornell University Medical College, and the London School of Hygiene and Tropical Medicine, and residency training at the University of California, San Diego (UCSD), he cofounded the Tropical Medicine and Traveler's Clinic at the UCSD in 1979. In 1980 he joined the US Navy and moved to Indonesia, where he was Director of the Department of Clinical Investigation and Epidemiology at the Naval Medical Research Unit-2 from 1980 to 1984, and conducted studies on typhoid fever, tropical splenomaly syndrome, severe malaria, cholera, and lymphatic filariasis. In 1985 he joined the malaria vaccine development team at the Naval Medical Research Institute (NMRI) and the Walter Reed Army Institute of Research, and has been Director of the NMRI program since 1987, during which time he has studied the epidemiology, diagnosis, treatment, and prevention of

vi

About the Series Editors

malaria, and the mechanisms of immunological protection against malaria in Asia, Africa, and South America. However, his major recent scientific efforts have been in the development of multiantigen, multistage DNA-based malaria vaccines, the sequencing of the genomes of malaria parasites, and the development of the technological capabilities to exploit genomic sequence data for vaccine, drug, and diagnostic development. Dr Hoffman is an adjunct professor at the Uniformed Services University of the Health Sciences in Bethesda, Maryland, a visiting professor at the Faculty of Tropical Medicine, Mahidol University, Bangkok, and a member of numerous national and international advisory and steering committees. He is currently President of the American Society of Tropical Medicine and Hygiene.

Vll

List of Contributors Robert Bergquist UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) CH-1211 Geneva 27, Switzerland E-mail: [email protected] Ronald Blanton Division of Geographic Medicine Case Western Reserve University School of Medicine Cleveland, OH 44106, USA E-mail: [email protected] Daniel G Colley Division of Parasitic Diseases National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, GA, USA David Dunne Department of Pathology, University of Cambridge Tennis Court Road Cambridge CB2 1QP, UK E-mail: [email protected] Taha El-Khoby Ministry of Health and Population Office of the Minister Cairo, Egypt E-mail: [email protected]

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Schistosomiasis

Alan Fenwick Schistosomiasis Vaccine Development Project (SVDP) Egyptian Organisation for Vaccine Production 51 Wezarat El Zaeraa St Agouza, Cairo, Egypt E-mail: [email protected] Gloria R Franco Departamento de Bioquimica e Imunologia, ICB, UFMG Av. Antonio Carlos 6627, Pampulha Belo Horizonte, MG, Brazil E-mail: [email protected] Stephanie L James Parasitology and International Programs Branch Division of Microbiology and Infectious Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA E-mail: [email protected] Charles H King Associate Professor of Medicine and International Health Division of Geographic Medicine Department of Medicine Case Western Reserve University School of Medicine and University Hospitals Cleveland, OH 44106, USA E-mail: [email protected] Christopher L King Division of Geographic Medicine Case Western Reserve University School of Medicine Cleveland, OH 44106, USA E-mail: [email protected]

List of Contributors

Adel AF Mahmoud President, Merck Vaccines, Merck & Co, Inc One Merck Drive, PO Box 100 Whitehouse Station, NJ 08889-0100, USA Formerly: John H Hord Professor Chairman and Physician-in-Chief Case Western Reserve University and University Hospitals of Cleveland, USA E-mail: [email protected] Adrian Mountford Department of Biology, University of York PO Box 373 Heslington, York Y010 5YW, UK Remigio M Olveda Associate Professor of Medicine University of the Philippines, College of Medicine Director, Research Institute for Tropical Medicine Department of Health, Philippines E-mail: [email protected] John Ouma Division of Vector Borne Diseases PO Box 20750, Nairobi, Kenya E-mail: [email protected] Aluizio Prata Fac. de Med. do Triangulo Min. Med. Trop., Caixa Postal 118 Uberaba Minas Gerais 38001-970, Brazil E-mail: [email protected] Alan Sher Immunobiology Section, Laboratory of Parasitic Diseases National Institutes of Health Building 4, Room 126, Bethesda, MD 20892, USA E-mail: [email protected]

ix

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Schistosomiasis

Andrew JG Simpson Ludwig Institute for Cancer Research Rua Prof. Antonio Prudente 109, 4th floor, 01509-010, Sao Paulo, SP, Brazil E-mail: [email protected] Robert F Sturrock Department of Infectious and Tropical Diseases London School of Hygiene and Tropical Medicine Keppel Street, London WC1E 7HT, UK E-mail: [email protected]

Preface Progress in fundamental knowledge of schistosomes and their associated disease syndromes has accelerated during the past two decades. Remarkable discoveries have been made at the genetic, subcellular and molecular levels. These achievements resulted from the influx of a new generation of "young turks," many of whom have contributed to this volume. They and others brought modern scientific tools to investigations of schistosomiasis and expanded the opportunities to explore new control strategies. The new millennium, however, raises major concerns about the level and intensity of efforts in the field of schistosomiasis. The global focus is shifting to significant efforts regarding HIV as well as tuberculosis and malaria. While undeniably important, scientific progress, funding and recruitment of new talents for fields such as schistosomiasis have slowed down. This is serious, in view of the magnitude of the tasks ahead. There is a very narrow spectrum of chemotherapeutic agents used for treatment of schistosomiasis and the threat of antmelminth resistance is ever-increasing. Control efforts in the major endemic areas are achieving mixed and definitely inconsistent results. Furthermore, two factors are working against all good intentions: rapid population growth in endemic regions a n d the necessity to develop agricultural and irrigation schemes. To put it bluntly, there is no single example of a sustainable, effective and affordable country-wide control program that may be pointed at as a model. The success of schistosomiasis control in Japan may be an unaffordable exception. At the turn of the century, hopes for vaccines are there but the effort is fragmented and its future accomplishments are uncertain. The assessment is therefore simple — we have entered the new millennium without a single scientifically based and comprehensive control strategy. This pessimistic view is intended as a sign of concern. It is a view not shared by "official" announcements from governments or multinational organizations. The debate, which we hope this volume will contribute to,

xii Preface

is intended to serve as a warning, and not to undermine all the good efforts of the numerous individuals and organizations. We have assembled a group of contributors who are at the cutting edge of their respective fields and who have firsthand knowledge of schistosomiasis at the bench, bedside or field. The goal is a statement of where the field of schistosomiasis stands at the dawn of a new century and a bird's eye view of future challenges. The book provides students, researchers, clinicians and public health workers as well as policymakers with easy access to the relevant information. It may be used as the first step toward a global dialog: what is needed to energize the field of investigations, basic and applied, that will ultimately impact on the prevalence of infection and the burden of illness due to schistosomiasis. Finally, I would like to dedicate this volume to the memory of the pioneers, particularly Kenneth S Warren.

Adel AF Mahmoud

Xlll

Contents About the Series Editors List of Contributors Preface

v vii xi

Chapter 1 Schistosomiasis: Setting the Stage Adel AF Mahmoud

1

Chapter 2 The Schistosomes and Their Intermediate Hosts Robert F. Sturrock

7

Chapter 3 The Structure and Expression of the Schistosome Genome Gloria R Franco and Andrew JG Simpson Chapter 4

85

Epidemiology of Schistosomiasis: Determinants of Transmission of Infection Charles H King

115

Resistance to Infection in Humans and Animal Models David Dunne and Adrian Mountford

133

Chapter 6 Initiation and Regulation of Disease in Schistosomiasis Christopher L King

213

Chapter 5

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Schistosomiasis

Chapter 7

Disease in Schistosomiasis Haematobia Charles H King

265

Chapter 8

Disease in Schistosomiasis Mansoni in Brazil Aluizio Prata

297

Chapter 9

Disease in Schistosomiasis Mansoni in Africa John Ouma, Taha El-Khoby, Alan Fenwick and Ronald Blanton

333

Chapter 10 Disease in Schistosomiasis Japonica Remigio M Olveda

361

Chapter 11 Disease Due to Schistosoma mekongi, S. intercalatum and Other Schistosome Species Charles H King

391

Chapter 12 Strategies for Control of Infection and Disease: Current Practice and Future Potential Robert Bergquist

413

Chapter 13 Progress in Vaccine Development Stephanie L James and Daniel G Colley Chapter 14 Immunology of Schistosomiasis: Towards a Consensus Viewpoint Alan Sher and Adel AF Mahmoud

469

497

1

Chapter 1 Schistosomiasis: Setting the Stage Adel AF M a h m o u d

Human infections with the parasitic helminths of the family Schistosomatidae represent a significant segment of the global burden of illness. Members of this family are estimated to infect 200-300 million people and to be endemic in more than 70 countries. The schistosomes share the major biological and epidemiological features of macroparasites (1-4). The central theme of this concept is the recognition that macroparasites, in general, do not multiply within the definitive host. In the case of schistosomes, increase in the population of the pathogen occurs only in the snail intermediate host while adult worms in humans or experimental animals are unable to expand their numbers without acquisition of new infections from the environment. The second fundamental concept concerns the host-parasite relationship in schistosomiasis, as well as other helminthic infections: it relates to the dependence of the outcome of infection and disease on numbers of parasites in the host. This correlation however is not exact, as there has been recognition in recent years of the multiplicity of other factors such as the genetics of the parasite and/or host, the role of other infections and perhaps host nutrition in shaping or modifying the outcome of host-parasite interaction. Our current appreciation indicates that infection and disease in schistosomiasis is a multifactorial process, although the intensity of infection seems to be the dominant factor. Over the past several decades, the study of the epidemiology of infection and disease in schistosomiasis has been introduced into modern methodology by several investigators (5-11). The new fundamental focus relates to evaluation of schistosome infection and disease in populations rather than infected individuals or hospitalized patients. The monumental studies of

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Schistosomiasis

Forsythe in Zanzibar (5), Ongom and Bradley in the West Nile district of Uganda (6), Warren and his colleagues in Kenya (7), Egypt (8), the Philippines (9) and China (10), and of Kloetzel in Brazil (11) established the basis for appreciating the population dynamics of the schistosomes in human as well as snail populations. Adult schistosomes are not distributed randomly in their human host; in any endemic area where infection was examined in populations, the distribution of helminths was found to be aggregated. Most infected populations harbor low worm burdens while heavy infection is limited to a small segment of infected individuals. While the etiology of this phenomenon is not completely understood, it is important to appreciate its epidemiological and clinical consequences, which are essential in approaching control strategies. This volume presents an up-to-date and detailed examination of infection and disease due to schistosomiasis. This helminth is one of the better-studied human worm infections, as evidenced by the knowledge of their biology, biochemistry and molecular organization (Chapters 2-4). Similarly, the mechanisms underlying pathogenesis and immunity in infected humans and experimental animals have been extensively dissected (Chapters 5, 6). This fundamental knowledge of the host-parasite interaction has resulted in better understanding of the disease syndromes caused by the different species of schistosomes capable of infecting humans (Chapters 7-11). Basic to our modern appreciation of the mechanisms of disease in schistosomiasis is the description of egg granulomas both in the livers of infected animals and in the isolated egg pulmonary model. This pioneering discovery by von Lichtenberg (12) and Warren (13) paved the way for our current understanding of the initiation, regulation and sequelae of the host responses to schistosome eggs impacted in its tissues. Warren led the way in immunologic investigations as he and his colleagues in the late 1960's and early 1970's established that granuloma formation around S. mansoni eggs injected into the pulmonary parenchyma of mice is an immunologic response of the delayed hyper-sensitivity type. They then proceeded to define the role of cell-mediated and humoral factors as well as the role of complement and other immunologic manipulations. Warren and Boros had the foresight to examine the mediators of the host immune responses by exploring the egg antigens involved in the initiation and regulation of the granulomatous response (14). During the past two decades many more investigators have applied modern immunologic techniques to achieve a detailed understanding

Schistosomiasis: Setting the Stage

3

of the mechanisms and regulation of immunopathology in schistosomiasis (15-18). Most significant among these is the dissection of T lymphocyte subsets, the cytokine profile and mediators of fibrosis (Chapter 6). Studies on acquired resistance to schistosomiasis have progressed during the past three decades to the exciting stage of contemplating trials of defined antigens as possible vaccines (Chapters 5, 13, 14). Originally the excitement was due to Smithers' studies on concomitant immunity and investigations of the protective components of the host immune response (19). The early 1970's witnessed two major developments: Butterworth et al. developed an in vitro system to examine effector mechanisms of destruction of schistosomula of S. mansoni (20); simultaneously, Mahmoud et al. developed the first monospecific antieosinophil sera (21). The two systems were combined to demonstrate that eosinophils play a major part in the killing of schistosomula in vitro, and more excitement was associated with the report that antieosinophil sera in vivo are capable of ablating the acquired immunity to schistosomiasis, trichinella and several other helminthic infections (2224). At last, the long-established association of eosinophilia and tissue helminthiases has acquired a mechanistic significance. The 1980's and 1990's were spent examining the mediators of this antibody-dependent eosinophilmediated cytotoxicity as well as the role of other cells, particularly host macrophages (26). The current understanding of the process of parasite killing in immune hosts indicates a multifactorial system that is responsible for the release of oxidative, nonoxidative and NO-dependent mediators which result in parasite killing (22-29). In parallel with these investigations, a major effort was devoted to defining and characterizing parasite antigens that elicit protective responses. Several have been well established, paving the way for serious efforts in the vaccine field (Chapter 13). Schistosomiasis is the second-best-studied human parasitic infection, next to malaria. This effort has resulted in significant advances which impacted positively on control efforts of infection and disease in endemic areas. The results, however, are not uniform and successes are hard to find in situations where population growth and efforts to expand water utilization for agrarian use result in increased prevalence of infection and disease (30). A unifying approach to control is urgently needed, using today's technology and watching for the new and exciting. Unless this is achieved, we will leave a legacy very similar to the schistosome eggs that were found in the ancient Egyptian mummies of 1250 BC.

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Schistosomiasis

References 1. Anderson RM and May RM (1985). Adv. Parasitol. 24: 1. 2. Anderson RM and May RM (1991). Infectious Diseases of Humans: Dynamics and Control (Oxford University Press, New York), 538 pp. 3. Mahmoud AAF (1989). Science 246: 1015. 4. Fulford AJC, Butterworth AE, Ouma JH and Sturrock RF (1995). Parasitology 10: 307. 5. Forsyth DM (1969). Bull. WHO 40: 771. 6. Ongom VL and Bradley DJ (1972). Trans. R. Soc. Trop. Med. Hyg. 66(6): 835. 7. Siongok TKA, Mahmoud AAF, Ouma JH, Warren KS, Muller AS, Handa AK and Houser HB (1976). Am. } . Trop. Med. Hyg. 25: 273. 8. Abdel Salam E, Abdel Khalik A, Abdel Meguid A, Barakat N and Mahmoud AAF (1986). Tissue Antigens 23: 142. 9. Domingo EO, Tin E, Peters PA, Warren KS, Mahmoud AAF and Houser HB (1980). Am. J. Trop. Med. Hyg. 29: 858. 10. Weist PM, Wu G, Zhang S, et al. (1992). Trans. R. Soc. Trop. Med. Hyg. 86: 47. 11. Kloetzel K (1963). Am. J. Trop. Med. Hyg. 12: 334. 12. Lichtenburg FV (1962). Am. J. Pathol. 41: 711. 13. Warren KS, Domingo ES and Cowan RBT (1967). Am. J. Pathol. 51: 735. 14. Boros DL and Warren KS (1970). /. Exp. Med. 132(3): 488. 15. Wynn TA, Marawetz R, Schaston-Kursten T, et al. (1997). /. Immunol. 159: 5014. 16. James SL and Sher A (1990). Curr. Topics Microbiol. Immunol. 155: 21. 17. Farah IQ, Nyindo M, Suleman MA, et al. (1997). Exp. Parasitol. 86: 93. 18. Kaplan MG, Whitefield JR, Boros DL and Grusby MJ (1998). /. Immunol. 160: 1850. 19. Butterworth AE (1994). Parasitol. Today 10: 378. 20. Butterworth AE, Sturrock RF, Homba V, et al. (1975). Nature 256: 727. 21. Mahmoud AAF, Warren KS and Peters PA (1975). /. Exp. Med. 142: 805. 22. Grove DI, Mahmoud AAF and Warren KS (1977). /. Exp. Med. 145: 755. 23. Mahmoud AAF (1981). /. Infect. Dis. 145: 613. 24. Capron M and Eapson A (1994). Science 264: 1876. 25. Peck CA, Carpenter MD and Mahmoud AAF (1983). /. Clin. Invest. 71: 66.

Schistosomiasis: Setting the Stage

5

26. James SL (1995). Microbiol. Rev. 59(4): 533. 27. Kazura JW, Fanning M, Blumer JT and Mahmoud AAF (1981). /. Clin. Invest. 67: 93. 28. Mahmoud AAF (1993). Immunology and Molecular Biology of Parasitic Infections, 3rd edition, ed. Warren KS (Blackwell), pp. 23-24. 29. James SL, Cheever AW, Casper P and Wynn TA (1998). Infection and Immunity 66: 3510. 30. World Health Organization (1993). WHO Technical Report Series 830: 1.

7

Chapter 2 The Schistosomes and Their Intermediate Hosts Robert F Sturrock

Introduction and Historical Background General accounts of various aspects of schistosomes and their intermediate hosts are given by Ansari (3), Brown (24), Doumenge et al. (56), Jordan (99), Jordan and Webbe (100), Jordan, Webbe and Sturrock (101), Mandahl-Barth (127), Mahmoud (125), Mott (140), Sobhon and Upatham (193), Rollinson and Simpson (179) and Warren (234). They should be consulted for fuller coverage than space allows in this chapter. Schistosomiasis, also known as bilharzia(sis), is a helminthic disease of human beings and both domestic and wild animals in many tropical and subtropical countries. The infection is sometimes called snail fever, because certain aquatic, freshwater snails serve as intermediate hosts in the parasites' life cycle. The adult flukes, depending on their species, inhabit blood vessels, mostly those of the hepatic portal system or the vesical plexus of the definitive, mammalian hosts. Related trematodes utilize birds as their definitive hosts. Schistosomes have infected man for over four millennia. Papyrus of Kuhn (-2000 BP — before the present) hieroglyphics were believed to refer to hematuria, a clinical sign of urinary schistosomiasis, and the Ebers Papyrus was said to give remedies for its cure (54), but this interpretation is questionable. In fact, they may have been penis sheaths or an early form of condom (149). Whatever the truth, schistosome eggs have certainly been found in Egyptian (183) and Chinese (252) mummies (3000+ BP and 2100 BP respectively). Immunological and molecular methods are now being used to investigate the paleoepidemiology of Egyptian schistosomiasis as far back as 5000 BP (42).

8

Schistosomiasis

The modern study of schistosomiasis began with two independent events in the middle of the 19th century: the descriptions of the Katayama syndrome during the early stages of infection in Japan (65) and of the adult worms found during postmortem investigations in Egypt (18). Sixty years of controversial and often acrimonious debate passed before the involvement of aquatic snails in the life cycle was finally proved in Japan (136) and in Africa (118). The latter study also confirmed unequivocally that two distinct species occurred in Egypt, Schistosoma mansoni and S. haematobium, compared with a single species in Japan, S. japonicum. Once the life cycle was known, it was possible to develop rational control programs. From the start, their outcome was limited by the available control methods, especially their effectiveness, safety, cost and acceptability to the communities involved. Some programs were successful initially but few have achieved permanent control. Differing national perceptions of the importance of bilharzia affect the political will to maintain control efforts. When the problem is no longer perceived to be of public health importance, relaxation of control is usually followed by a resurgence of transmission. In addition, aquatic snail habitats continue to increase in number with the construction of large and small scale water developments for hydroelectric power, agricultural irrigation and provision of much-needed drinking water for domestic stock. These developments invariably attract large numbers of immigrants searching for agricultural land or work, especially in those areas where the human population explosion is greatest. Wars, civil disturbances and natural disasters such as famine due to floods and droughts have exacerbated the situation (123). Provision of proper sanitation and safe water supplies simply cannot keep pace with rising and shifting populations. Increasing h u m a n contamination of and contact with snail habitats is usually accompanied by the enhancement or introduction of schistosome transmission. Estimates of the number of people currently exposed to or infected by schistosomiasis are inevitably imprecise, because the necessary data are rarely collected in a suitably standardized manner. Stoll (200) calculated that some 114 million people were infected with bilharzia worldwide. Wright (250) estimated that approximately 40 million of 160 million at risk in Asia and the Americas were infected, but refrained from calculating equivalent figures for Africa. In the late 1970s, Iarotski and Davis (86) concluded from responses

The Schistosomes and Their Intermediate Hosts 9 -Hepatic portal i veins ADULT FLUKES S. haematobium in ve«is of bladder

MAN IS THE DEFINITIVE HOST

S. mmsoni & S.japooicum n mesenteric veins of bowel

EGGS a. Retained in tissue

-^fc*

Liver T Arteries

Piired ^ - < ^ \mature (kikes

T Heart

— • " - DISEASE

or b. Excreted

i\ •

— * - TRANSMISSION ^

COMMON EXTERNAL ENVIRONMENT IN WATER

)Lungs cercariae penetrate skin

SuNnussp £ naamstobtun fiiomphafaria sp S. ranson/ Oicomebnia sp S.japonicvm

©— o —

ovum

rriracidium

snail THE INTERMEDIATE HOST

cercana

Figure 1. The basic life cycle of the principal schistosomes infecting man. (Drawn by Dr G. Barnish; courtesy CAB International.)

to a questionnaire from 59 endemic countries, excluding China, that nearly 200 million of the 600 million people at risk were infected, figures echoed by Doumenge et al. (56) and Savioli et al. (185), who also considered that 20 million suffer from severe complications. Explosive population growth over the last half century in most endemic countries and failures in maintaining long term control programs make it probable that these figures are optimistically low.

Basic Life Cycle Schistosomes are digenetic trematodes of the family Schistosomatidae. Unlike other trematodes, the adults are dioecious with separate sexes. A dorsoventrally flattened male clasps a cylindrical female in a ventral groove, the gynaecophoric canal (also called the schist, from which the name

10

Schistosomiasis

"schistosome" is derived) formed by infolding his lateral margins, so that the pair assume a nematoid, worm like form suited to life within the vascular system. Both sexes have oral and ventral suckers, but only the male ventral sucker is sufficiently muscular and well developed to allow him to hold firmly onto the blood vessel walls. Despite differences in detail, all schistosomes of medical importance follow essentially the same life cycle (101, 179). Juvenile flukes develop rapidly in the nutrient-rich medium within the hepatic sinusoids of the liver. Pairing occurs as they approach maturity and the paired worms then leave the liver with the male carrying the female against the blood flow of the hepatic portal vein to the final site of predilection. Depending on the species, this is either in the proximal branches of the hepatic portal vein around the intestine or to veins around the bladder. Eggs laid within these veins must traverse their walls, enter the lumen of the intestine or bladder and be voided in the feces or urine, respectively. They must then reach fresh water to hatch and release the free-living miracidium. Miracidia continue development only if they penetrate a compatible freshwater snail to begin a process of asexual multiplication within the intermediate host. During penetration the miracidium changes into a primary sporocyst within which a number of secondary sporocysts develop over a period of one to two weeks. When released, secondary sporocysts migrate to the nutrient-rich milieu of the digestive glands. A second phase of asexual multiplication follows when each secondary sporocyst eventually produces large numbers of cercariae. (NB. The offspring of each egg all have the same sex: therefore the use of "primary" and "secondary" rather than "mother" and "daughter" is to avoid the logical absurdity of male mothers and daughters.) Cercariae break out of the snail into the water but die unless they penetrate the unbroken skin of a definitive host. Successful penetrants transform within the skin into schistosomulae and enter the vascular system directly (or, for a small proportion, indirectly via the lymphatics) to migrate through the left heart to the lungs, the right heart and the systemic circulation to the splanchnic vasculature of the hepatic portal system, eventually reaching the sinuses of the liver to complete the cycle. Stable temperatures of ~37°C in the warm-blooded (homeothermic) definitive host produce fairly stable parasite development times within the normal limits of biological variability of each species, although there are some

The Schistosomes and Their Intermediate Hosts

11

differences between species. However, ambient temperature is a major determinant of the survival of the free-living, aquatic stages and the development of intramolluscan stages within cold-blooded (poikilothermic) snails. In general, parasite metabolism and development times are inversely related to temperature within the range 15-35°C, although mortality tends to rise for both the parasites and snails at 30+°C. The optimum range is generally around 25 + 2°C, substantially lower than within the definitive host. Clearly, the parasite has to adjust several times during its life cycle to substantially different environmental conditions. Elimination of water acquired by osmosis presents little difficulty for parasitic stages living in isotonic conditions within the hosts but becomes a major problem for the free-living stages in hypotonic conditions. These stages cannot feed and use energy from glycogen reserves by oxidative metabolism. Parasitic stages probably use anaerobic respiration for energy production, obtaining presynthesized elements from the host for biochemically deficient pathways. Few of the offspring of one pair of worms ever reach another definitive host. The high fecundity of the female schistosomes and the asexual amplification phase within the snail allow for substantial losses. In low density, rural human populations, the balance between the numbers of people, snails, snail habitats and parasites allows enough parasites to complete the cycle to maintain their population without causing disease, even allowing for the clumped worm distribution within the definitive hosts to enhance the chance of transmission (249). If the human and snail populations, and the levels of human contamination of and contact with snail sites increase, so, too, will the levels of transmission: human prevalence and intensity will rise with a greater probability of severe disease developing. The objective of control programs is to reduce transmission to a level where the risk of developing disease is eliminated. Defining this level is a task that has exercized mathematical modelers for nearly half a century. The taxonomy of schistosomes is fairly well established, with some 19 species in the genus Schistosoma recognized by conventional methods and supported by more recent molecular studies (10, 20,121,178). Rollinson and Southgate (180) listed four general groups: the haematobium group, with terminal spined eggs transmitted by pulmonate snails of the genus Bulinus; the mansoni group, with lateral spined eggs transmitted by pulmonate snails of the genus Biomphalaria; the indicum group, with terminal or subterminal spined eggs transmitted by pulmonate snails, mainly of the genus

12

Schistosomiasis

Indoplanorbis; and the japonicum group, with eggs bearing a reduced lateral spine and transmitted by prosobranch snails, mainly of the genus Oncomelania. Not all species fit easily into these groups and molecular techniques are being used to clarify some of the anomalies (52). Although these groups remain valid, partial 28S ribosomal gene sequences show clear differences between the haematobium, mansoni and japonicum groups but suggest that the indicum group is most closely related to the haematobium group (121). Members of the japonicum group show intraspecific variations in repetitive sequences within the first internal transcriber spacer (ITS1) of ribosomal DNA (225). This intraspecific variation is absent in the mansoni and haematobium groups (103). Five Schistosoma spp. complete normal development in humans and some of the other species may undergo partial development.

Schistosomes Affecting Humans and Their Distribution All five human species may, potentially, give rise to heavy infections, pathology and serious disease. The three with the most extensive distributions (S. japonicum, S. mansoni and S. haematobium) are usually considered more important than the two with more restricted distributions (S. intercalatum and S. mekongi). Their worldwide and local distribution is determined by the availability of suitable habitats for the freshwater snails that act as their intermediate hosts. Such habitats often have a patchy distribution reflected by the snails and the schistosomes, even within endemic countries. Discontinuity is a major feature in the epidemiology of schistosomiasis. Nonhuman schistosomes can complicate epidemiological studies in several different ways. Cercariae of mammal and bird schistosomes try to penetrate humans. Many die in the skin but others may progress to various levels of development, sometimes even reaching maturity, especially if hybrid pairing occurs in mixed infections (171, 181). Such zoonotic infections do not, generally, cause severe disease although they may cause minor pathology. However, they can confuse the results of immunodiagnostic tests, especially for antibodies, by triggering immune responses causing cross-reactions (108). It has also been suggested that they might provoke heterologous immunity ameliorating the severity of normal human schistosome infections (146). Eating animals infected with nonhuman schistosomes, especially their livers, may result in exotic schistosome eggs being found by parasitological stool examinations, giving rise to spurious reports of human infections with these


13

species (109). Snails transmitting human schistosomes also serve as intermediate hosts of nonhuman schistosomes: this complicates transmission studies because the different cercariae are hard to distinguish under field conditions.

Species of Widespread Importance S.

japonicum

S. japonicum is the major cause of human schistosomiasis in Southeast Asia and some islands of the Western Pacific. A major difference from the other human schistosomes is the wide range of domestic and wild mammals which the parasite can use as a definitive host. These zoonotic infections considerably complicate control activities. S. japonicum was formerly widespread in mainland China, on Japan, on a number of islands in the Philippines, and in an isolated focus on Sulawesi. A completely zoonotic strain on the island of Taiwan seems incapable of maturing in humans (250). Successful control programs have eliminated S. japonicum from Japan (206) and much, though not all, of China (182). Elsewhere, transmission persists in the Philippines and in the Sulawesi focus, despite extensive, prolonged research and control activities. Strain differences within S. japonicum and its intermediate hosts have been reported in China (80, 93, 168). Strains of S. japonicum from the Philippines appear to have diverged considerably from those in China (81, 131, 248). There seems to be very little difference in randomly amplified polymorphic DNA (RAPD), or ribosomal ITS2 and mitochondrial COl DNA sequences (22). Triosephosphate isomerase cDNA and paramyosin sequences showed much greater differences (82, 83) and there were intraspecific variations in nuclear ribosomal internal transcribed spacer 1 (ITSl), though less than intraspecific differences from other Asian schistosomes (225). ITSl variations have not been found in African schistosomes (103), although variations in ribosomal DNA occur in Brazilian S. mansoni strains (232). The adults worms live around the gut in the mesenteric branches of the hepatic portal vein. Their reported preference for the superior mesenteric veins may be an artifact due to studies using very high infections in experimental animals: late pathological lesions of the human colon suggest a wider distribution (33). Typical, oval eggs with a lateral spine reduced

14

Schistosomiasis

Table 1. Comparison of principal features of Schistosoma spp. infecting man (after Sturrock (102)*). S. japonicum S. mekong?

S. mansoni

S. haematobium

S. intercalatum

Mesenteric veins

Mesenteric veins

Mesenteric veins

Vesical plexus

Mesenteric veins

Medium

Medium

Very long

Short

Short

Male: Length (mm) Width (mm) No. of testes

10-20 0.55 6-7

-15 0.41 6-7

10-15 0.90 4-5

Tubercles

Absent

Absent?

6-13 1.10 4-13 (6-9) b Coarse

Fine

11-14 0.3-0.4 2-7 (4-5) b Fine

20-30 0.30

-12 0.23

10-20 0.16

16-26 0.25

10-14 0.15-0.18

Middle half

Rear third

Front third

Rear half

Rear half

Front half Short 50-200 3500

Front half Short 10+

Front half Very short 1-2 100-300

Front two thirds Long 10-50 20-300

Front two thirds Long 5-60 150-400

Feces Round 60 x 100 Lateral (reduced) +ve

Feces Round 57x66 Lateral (reduced) ?

Feces Ovoid 61 x 140 Lateral (prominent) +ve

Urine Ovoid 62 x 150 Terminal (prominent) -ve

Feces (and urine) Ovoid 61 x 176 Terminal (prominent) +ve

Oncomelania

Neotricula

Biomphalaria

Bulinus

Bulinus

Item Adult worms: Location of adult in host Length of post gut cecum

Female: Length (mm) Width (mm) Ovary — Position in body Uterus — Position in body Length No. of eggs Eggs/female/day Mature egg: Normally passed in: Shape Size (fi) Spine Ziechl-Neelson c Intermediate host snail: a

?

From experimental animal infections Hjsual range c Reaction of egg shell to stain in histological sections *Published with permission from CABI


15

or absent are voided in the feces, and the molluscan phase of the life cycle takes place in (semi)amphibious prosobranch snails of the genus Oncomelania. Human disease, as with all human schistosomes, is primarily associated with those eggs that fail to escape from the definitive host and the various mechanisms used in their entrapment, isolation and destruction. These mechanisms involve the formation and resolution of granulomata around such eggs, particularly those swept to the liver and lungs. In S. japonicum infections, granulomata typically contain several eggs adhering to each other due to a sticky exudate they produce. However, because they are smaller than those of most other schistosomes, individual eggs can pass the lung vasculature to enter the general circulation before being trapped in distal capillary beds elsewhere in the body. Neurological complications due to granulomata formation in the brain are believed to be more frequent in S. japonicum than in other schistosome infections. There is evidence that acute disease (Katayama syndrome) early in S. japonicum infections is more severe than for other schistosomes and that it can recur after superinfection later in life among people with chronic infections (167).

S. mansoni S. mansoni causes intestinal schistosomiasis in most African countries south of the Sahara, including Madagascar offshore in the Indian Ocean, and extends northwards down the Nile Valley to the Mediterranean coast and northeast across the Arabian peninsula as far north as Saudi Arabia in southwest Asia. Its has spread recently in the coastal regions of Egypt (124), Mozambique (220), Ghana (237), Mauritania and Senegal (160). It also occurs in the New World, possibly introduced from Africa by the slave trade, in parts of Brazil, Surinam and Venezuela on the South American mainland and discontinuously among the Caribbean islands. Although S. mansoni infections can easily be maintained in a wide range of mammals, such mammals rarely sustain field transmission in the absence of infected human populations, except, perhaps, for some South American rodents. Thus, they are anthropozoonoses where the infection spills over from man to the other mammals. Zoonotic infections are far less important than for S. japonicum. Control programs have greatly reduced the importance of S. mansoni in the Caribbean foci but neither it, nor its intermediate hosts, have been totally eliminated (68, 166, 221). Numerous control programs on the African and South American mainlands

16

Schistosomiasis

have enjoyed variable levels of temporary success but there are few instances of sustained, long term control. Like S. japonicum, S. mansoni adult worms inhabit the mesenteric branches of the hepatic portal vein around the gut. Typical, lateral-spined eggs are voided in the feces and the molluscan phase of the life cycle takes place in aquatic pulmonate snails of the genus Biomphalaria. Although most eggs are passed in the feces, a small proportion of patients also pass eggs in the urine. It has been speculated that juvenile males of terminal-spined schistosomes mate with S. mansoni females during development in the liver and transport them to atypical sites (171). However, up to 10% of patients on the West Indian island of St Lucia, where terminal-spined schistosomes are absent, passed S. mansoni eggs in their urines (43). Serious h u m a n pathology a n d disease resembles that caused by S. japonicum and is similarly caused by reactions to eggs trapped in the human body. Early, acute signs and symptoms are unusual in indigenous inhabitants of endemic areas, nor do they recur with subsequent exposures. Because the larger eggs are filtered out more efficiently in the liver and lungs, neurological complications due to egg granulomata alone are unusual and are normally due to ectopic worm pairs (165). There is, however, a possible association of brain granulomata and a neurotrophic factor that causes neuropathological dysfunction (254).

S.

haematobium

S. haematobium causes u r i n a r y schistosomiasis in m o s t African countries north and south of the Sahara and in adjacent islands in the Indian and Atlantic Oceans. [Bilharzia capense, described from South Africa (78), is now considered a synonym of S. haematobium.] Its distribution, like S. mansoni, extends northeastwards into South West Asia as far as Iran. The widespread presence of the snail B. truncatus in this area renders those countries currently

free of the parasite vulnerable to its introduction by infected immigrants, as recently reported in Jordan (184). In the past, it was also transmitted in Portugal on the European mainland. There are controversial reports from the Indian subcontinent regarding the identity of an isolated transmission focus at Gimvi near Bombay and of eggs reported in fecal examinations elsewhere (17). It is possible that zoonotic infections are involved as the snail genus Bulinus does not occur on the Indian subcontinent (25). Genuine


17

S. haematobium infections may be found among immigrant workers returning from working in endemic Arabian countries. There are no authenticated reports of endemic S. haematobium in the New World, even though there was probably a greater likelihood that slaves originating from West Africa were infected with S. haematobium — the absence of Bulinus spp. prevented its establishment in the Americas. Complete eradication by control programs appears to have been achieved in Israel and Portugal, and, possibly, in some isolated African oases on the northern borders of the Sahara and in some Arabian countries. Elsewhere, as for S. japonicum and S. mansoni, local control activities have enjoyed only temporary success. Adult S. haematobium worms live in the veins of the vesical plexus around the bladder, especially the upper trigone, and along the ureters. The characteristic terminal-spined eggs traverse the walls of these veins and the bladder or ureter to pass out of the body in the urine. As with S. mansoni, some patients may pass S. haematobium eggs in their feces. The molluscan intermediate hosts throughout most of the range of S. haematobium belong to aquatic pulmonate snails of the genus Bulinus. Possible exceptions are two other pulmonates, the Portuguese helisomatid Planorbarius metidjensis and the Indian ancylid (freshwater limpet) Ferrissea tenuis (but see intermediate hosts below). The elimination of eggs is accompanied by some blood loss, which, early in an infection, is sufficient to stain the urine red, and hematuria is a characteristic sign of S. haematobium infection, especially among children. The condition is usually recognized by inhabitants of endemic areas and given a specific name in the local language. (Transient, bloody diarrhea may also occur early in heavy infections of both S. japonicum and S. mansoni, but there are so many other potential causes that specific local names are rarely, if ever, given.) Human pathology and disease is again caused by those eggs trapped within the body. The urogenital system, predominantly the bladder, ureters and kidneys, is involved because of the location of the adult worms. Although, in theory, eggs could be swept along the inferior vena cava and through the right heart to the lung capillaries, pulmonary complications are not commonly reported. Transverse myelitis is a more frequent complication, due to pressure from reactions around eggs lodging alongside the spinal cord. Katayama-like conditions sometimes occur in naive immigrants on initial exposure to a heavy infection in an endemic area but are unusual among the local population.

18

Schistosomiasis

Species of Local Importance S. mekongi A parasitic infection resembling that caused by S. japonicum was first described over 40 years ago in a patient from Khong island in the Mekong Delta on the Laos-Kampuchea (Cambodia) border (230). Pomatiopsid snails of the genus Oncomelania are not found in Indochina but members of the related aquatic trichulinid family, now placed in the genus Neotrichula, were eventually incriminated. Subsequent human surveys suggested that the parasite was quite widely distributed in Kampuchea and, especially, among Laotian boat people living on the Mekong river (87), and that dogs served as an important reservoir host. Human prevalences were generally low and for a while S. mekongi was considered mainly a local curiosity of limited public health importance. More recently, though, very high prevalences and intensities accompanied by severe disease have been discovered among Laotian communities living in the Mekong river (198). Although S. mekongi will probably remain a local problem, the spread of the infection within Indochina is feared if dams across the river extend potential Neotrichula habitats (6, 213).

S. intercalatum Fisher (62) named intestinal schistosome infections of terminal-spined eggs passed in human feces in the Kisangani District of the Belgian Congo (now Zaire) in Central Africa as S. intercalatum. The parasite has since been been found to be endemic in restricted areas of northeast Zaire, Gabon and Cameroon, with additional cases reported from other parts of Central and West Africa, including the Central African Republic, Chad, Nigeria and Upper Volta, mainly from tropical rain forest areas where both S. mansoni and S. haematobium were absent, although a recent report from Western Uganda is abnormal in this respect (150), and it is present on the island of Sao Tome (177). Bulinid snails were incriminated as the intermediate hosts; B. africanus s.l. along the Lualuba river in northeastern Zaire and B.forskalii in Cameroon and Gabon (19). Hybrids of the two strains will infect either snail species. Natural zoonotic infections were found in the rat, Hypobomys univitatus (189), but the high susceptibility of the Nile rat, Arvicanthus niloticus, and the mulitimammate mouse, Mastomys huberti, suggests they may be implicated: both are widespread in the wild (89).


19

S. intercalatum pairs with S. haematobium in experimental infections (26, 210). Eggs of hybrid offspring were passed in either urine or feces, depending on their parentage. Adult females produced eggs parthenogenetically (210). In the field, natural hybrids with S. haematobium have been found using biochemical, molecular and morphological methods (212). Earlier fears that they might eventually displace the parent species (194) do not appear justified. Most hybrids are of low viability (11) and the predilection of males for mates of their own species minimizes miscegenation and allows the parental species to maintain their integrity under field conditions (211).

Other Schistosome Infections Schistosomes

of Domestic

Mammals

Many domestic and farm animals have their own schistosomes, some of local economic importance in their own right. Because of the close and sometimes intimate association of humans and their stock, these schistosomes may also confuse human epidemiological studies. Their geographical distribution differs somewhat from that of human schistosomes, but the two have considerable areas of overlap. In Africa, cattle, sheep and goats are infected by three schistosome species, S. mattheei, S. bovis and S. curassoni, which, although terminal-spined like S. haematobium, normally inhabit the mesenteric veins around the gut so that their eggs are passed in the feces. Asian domestic animals are exposed to three different schistosome species, S. indicum, S. spindale and S. nasale, in the Indian subcontinent and southeast Asia (Indochina and Indonesia but not, apparently, China). The eggs of these species have terminal or subterminal spines but, as in Africa, are passed in the feces. Adults of S. indicum and S. spindale live in the mesenteric vessels around the gut but those of S. nasale dwell in the veins of the nasal mucosa. Occasional reports of h u m a n schistosome infections from the Indian subcontinent probably represent spurious infections with eggs ingested in infected meat. Schistosomes specific for domestic animals are apparently absent from the Americas.

Africa S. mattheei is transmitted by snails of the genus Bulinus in southern Africa. Its economic effect is not usually considered significant but serious

20

Schistosomiasis

epidemics have been reported in sheep in South Africa (116) and studies in Zambia indicate adverse effects on the growth rate of cattle (48). Human infections diagnosed from eggs in feces may have been from hybrids with S. haematobium, as experimental infections in humans failed to produce eggs (115, 244). S. mattheei extends northwards towards the equator, where it overlaps the southern limits of another schistosome of cattle, sheep and goats, S. bovis. Hybrid eggs of S. mattheei and S. bovis have been described from Tanzania and Zambia (51). S. bovis occurs in most African countries north of the equator and extends northwards to the Mediterranean island of Sardinia and Spain on mainland Europe and northeast into the Middle East as far as Iraq. It is transmitted by snails of the genus Bulinus except in Spain, where the intermediate host was identified as Planorbarius metidjensis. S. bovis is reported to be of considerable economic significance in domestic animals in some areas, but it does not normally infect humans — reported infections are probably spurious (109), although abnormal eggs from human Kenyan biopsy material (Dr P. Rees, personal communication) suggest hybridization with S. haematobium a n d / o r S. mansoni. S. curassoni, a schistosome of domestic ruminants with a limited distribution in West Africa (Senegal, Mali, Mauritania and Northern Nigeria), is transmitted by Bulinus spp. Its distribution overlaps that of S. bovis and the two may be present in the same animal, sometimes as heterologous pairs. It does not seem to cause serious pathology in sheep (229). Initial reports, based on egg morphology and intermediate host preference, that S. curassoni can coinfect people with S. haematobium have since been questioned in the light of further molecular studies (178).

Asia S. indicum occurs in the Indian subcontinent. The adults infect horses and buffaloes as well as sheep, goats and camels, probably causing limited economic losses, especially among sheep and horses. Transmission is by pulmonate snails of the genus Indoplanorbis. The terminally spined eggs, though somewhat smaller on average, superficially resemble those of S. haematobium. Periodic reports of human S. haematobium infections in India could conceivably be due to spurious infections with S. indicum.


21

S. spindale is present throughout the Indian subcontinent, including Sri Lanka, extending eastwards into Malaysia, Thailand, Vietnam and Indonesia. Adults infect most domestic ruminants, occasionally causing severe pathology or death, and the spindle-shaped, terminal-spined eggs are quite distinctive. Buffaloes are believed to play a major role in transmission because of their close association with rice paddies and irrigation channels harboring Indoplanorbis spp. S. spindale infects undomesticated mammals in Peninsular Malaysia (90) and is probably an important cause of human schistosomal dermatitis in these areas. S. nasale is widespread in the Indian subcontinent and extends into Myanmar (Burma). It affects cattle, sheep, goats and, especially, buffaloes, but not man. The thin, sinuous, terminal-spined eggs have an almost nematoid appearance. It causes "snoring disease" by cauliflower-like growths that impede breathing around eggs trapped in the nasal mucosa. Natural intermediate hosts belong to the genus Tricula.

Schistosomes

of Wild

Mammals

The likelihood of exposure of humans to schistosomes of wild mammals, though in some respects less than that to those infecting domestic animals, should not be ignored. All often share the same water source infested by potential intermediate hosts which, if infected, may allow exposure to exotic cercariae. Eating infected game meat can cause the diagnosis of spurious infections. At least eight schistosome species have been described; five from Africa and three from Asia.

Africa Two species, S. margreboxviei and S. leiperi, infect antelope in south and central Africa and have been studied recently in rodent models (51). S. margreboxviei is found from Bot wana in the south through Zambia and Zaire to Chad, and possibly Mali, in the north. The lechwe (Kobus leche kafuensis) and puku (K. vardoni), both swamp-loving antelope, are the principal definitive hosts, although infections have been recorded from many other antelope. S. leiperi occurs from Bot wana through Zambia, Tanzania and Uganda northwards to Sudan, in antelope, zebra and bush-buck inhabiting

22

Schistosomiasis

savanna and open plains as well as antelope dwelling in swamps. Many infections of domestic stock are documented. Both species are transmitted by snails of the genus Bulinus, but by snails from different groups (B.forskalii, B. scalaris and B. tropicus for S. margrebowiei; B. africanus and B. reticulatus for S. leiperi). The eggs of the two species are easily distinguished: those of S. margrebowiei resemble S. japonicum but the spine, when visible, is terminal, not lateral; S. leiperi eggs have a clear terminal spine and extended poles at each end. Mixed infections of the two species occur naturally in other mammals, occasionally accompanied by S. bovis and S. mattheei, but neither, apparently, is recorded as developing in humans. A possible exception is a report of S. japonicum-like eggs in the stools of fishermen in Zaire (233) and the finding of S. mansoni, S. haematobium and S. margrebowiei-hke eggs together in rectal biopsy samples from Mali (180). S. rodhaini is a rodent schistosome recorded in tropical Africa (Kenya, Uganda, Ruanda, Burundi and Nigeria). The eggs are variable in shape but have a distinctive subterminal spine. Its intermediate hosts belong to the genus Biomphalaria and its cercariae are shed nocturnally. Although it will hybridize with S. mansoni in experimental infections, its potential epidemiological importance lies, perhaps, in the complication of field studies of S. mansoni transmission by snails. Two schistosomes have been described from adult worms recovered from the African hippopotamus (Hippopotamus amphibius) in Lake Edward (Uganda): S. edwardiense from the hepatic vasculature; and S. hippopotami in the mesenteric vasculature. S. hippopotami eggs had a subterminal spine but were smaller than S. mansoni. There is no information on its intermediate host. It may be an aberrant form of S. mansoni in an unusual host and not a valid species according to Pitchford and Visser (163), who, however, considered S. edwardiense from South African hippopotamuses in the Kruger National Park to be a valid species. Except for the rudimentary lateral, not terminal, spine, its eggs resemble those of S. margrebowiei. B. salinarium was successfully infected but the cercariae, shed nocturnally, failed to infect two rodent species (Saccostomus campestris and Praomys natalensis).

Asia S. incognitum (including S. suis, which is considered a synonym) occurs on the Indian subcontinent, in Indochina and on some Indonesian islands.


23

Its was first described from a (probably spurious) human infection (30), but it appears to be a parasite of rodents though it does mature in domestic animals, especially pigs and dogs, but less well in ruminants although sheep and goat infections do occur. The egg is oval, with a sub-terminal spine. The intermediate host in India is the lymnaeid, Lymnaea luteola, but elsewhere is Radix auricularia rubiginosa. Natural infections have been found mixed with S. indicum and S. spindale in India and Thailand, and with S. japonicum on Sulawesi. S. sinensium occurs in Szechuan Province, China, and Northern Thailand. It appears to be primarily a parasite of the mesenteric vessels of rodents (rats). Its egg is reminiscent of S. mansoni, with a clear lateral spine (153), but the intermediate hosts are trichulinid prosobranchs (Trichula or Neotrichula spp.) (7, 114). S. malayensis eggs were first found in hepatic biopsy material from a group of West Malaysian aborigines (144). They were oval, with a reduced lateral spine, like S. japonicum or S. mekongi, but caused no significant pathology. The parasite was found naturally in rats and wild cervids (deer). Neotrichula spp. can be infected in the laboratory (72) but its natural intermediate host is the trichulinid operculate snail, Robertsiella kaporensis.

Other Genera Related to Schistosoma Several genera of parasites related to Schistosoma spp. exist. They are transmitted mostly by lymnaeid snails and can cause cercarial dermatitis in humans but do not mature. They may also develop in domestic animals as well as in their normal feral hosts. Examples include the rodent parasites Schistosomatium douthitti in temperate regions and Heterobilharzia americana in subtropical and tropical areas of North America; Bivetellobilharzia loxodontae and B. nairi in African and Asian elephants respectively; Orientobilharzia turkestanicum in both domestic and wild artiodactyls (including cattle, buffaloes, sheep and goats) in temperate and subtropical Asian countries from Turkey to Mongolia; and other species in tropical areas of India (O. dattai and O. bomfordi ) and Thailand (O. harinasutai). Several less closely related, avian "schistosomes" have an almost worldwide distribution in tropical and temperate areas and cause Swimmers Itch (e.g. Australobilharzia spp., Gigantobilharzia spp., Microbilharzia spp., Ornithobilharzia spp. and Trichobilharzia spp., and they are transmitted by

24

Schistosomiasis

TJ C 01 C

o OH

OH

to

X, bO C

'% o

X

J 3 60


25

various freshwater and marine gastropods of several genera, including Littoraria spp., Lymnaea spp., Physa spp., Planorbis spp., Polyplis spp. and Chilinia spp.).

Anatomy and Biology of the Schistosome Life Cycle Stages Eggs The eggs of most schistosome species are either ovoid or spherical, although in some species the poles may be extended along the longitudinal axis (S. intercalatum, S. matiheei, S. bovis, S. spindale and S. nasale) or at an angle to it (S. leiperi, S. rodhaini and S. incognitum). The external spine, if present, is usually terminal (S. haematobium and related species) or lateral (S. mansoni and related species), but may be reduced and difficult to see, especially in spherical eggs (S. japonicum, S. mekongi, S. malayensis, S. margrebowiei and S. edwardiense). In other species (S. rodhaini, S. hippopotami and S. incognitum), though, the spine may be subterminal, i.e. situated neither terminally nor clearly laterally. Similar deformities sometimes occur in immature tissue eggs or after treatment in normally clear-cut species. The eggs range in size from 60 to 400 \im in length and are immature when laid, either singly (S. mansoni) or in clusters (S. haematobium and S. japonicum), but mature rapidly during their 7-10 days' passage to the exterior, by which time they are fully embryonated. Eggs trapped within the definitive host die within 2 - 3 weeks. Although the products of autolysis are cleared within days or weeks, the shells, especially if they become calcified, may persist for months or years, as does fibrosis and, sometimes, calcification associated with the healing of the lesions formed by the granulomatous reactions. All schistosome eggs are nonoperculate. The shell, which is either clear or yellow in colour, is composed of sclerotin-tanned protein and is perforated by numerous micropores through which can pass enzymes produced by the enclosed miracidium. The spines, or sticky mucus secretions in the case of S. japonicum, may help adherence to the blood vessel wall at the start of this process, but other factors are involved (129). Infection induces endothelial cell adhesion molecules which may assist this process (54, 61, 147) but cytokines also play a part. Maturing worms induce an initial,

26 Schistosomiasis

Figure 3. Human schistosome eggs: (a) S. mansoni, (b) S. japonicum, (c) S. intercalahim and (d) S. haematobium. Parasites in human feces. {Courtesy H. Melhorn.)

protective Thl cytokine profile (elevated ry, IL-12 and TNFa). This is modulated at the onset of egg laying by the production of Th2 cytokines (IL-4 and IL-10) but elevated TNFa, even that stimulated by unisexual infections (32), appears to be a necessary trigger for a normal granulomatous response (119). Enzymes from the eggs lyse the intermediate tissues to help their passage from the lumen of the blood vessel to that of the gut or bladder. They also probably provoke immune mechanisms, particularly the initial stages of granuloma formation, to facilitate this process. This critical step in the life cycle is immune-dependent as egg excretion is reduced in hosts with impaired immune systems. Although most work was done in rodents, its relevance to humans is indicated by reduced egg excretion of HIV patients (105). However, granuloma formation also has a protective effect. In S. mansoni infections, a large proportion of eggs are flushed from the gut to downstream organs and trapped in capillary beds or the presinusoidal vessels of the liver. The granulomatous reaction destroys these eggs and protects the surrounding tissue from the lytic enzymes. Eggs of terminal-spined schistosomes do not have the same heptotoxic effect (54). If eggs are voided and reach fresh water, hatching is stimulated by reduced osmotic pressure, by light and by warmth in the temperature range 15-30"C. Mucilaginous material within the egg swells by osmosis until rising internal pressure splits the shell. Increased miracidial activity hastens the


27

process. S. haematobium eggs will hatch in undiluted urine exposed to light in tropical temperatures (> 30°C). Their hatching may be suppressed for a week or more if stored on ice in the dark in normal saline with added antibiotics to prevent bacterial attack. S. mansoni eggs in feces remain alive for a week or more if chilled in a refrigerator. Under field conditions, depending on the consistency of the feces, they remain viable in shaded sites with high humidities for 6-8 days, but are killed rapidly by high temperatures and especially desiccation of the feces (223). In countries where night soil is an essential fertilizer, composting kills S. japonicum eggs if sufficiently high temperatures are achieved. Alternatively, chemicals may be used (206).

Miracidia Miracidia, the first free-living stages in the life cycle, are approximately 160 |im by 65 |xm, the size varying both within and between species. Each miracidium has an anterior, apical sensory papilla or terebratorium covered with membranes shown by electron microscopy to be folded like either a "honeycomb" (S. haematobium and S. intercalatum) or a "rosette" (S. mansoni and S. japonicum). The body is covered by four rows of epidermal plates bearing cilia of equal length that propel it through the water. The subepithelium is lined by arched cells surrounding muscle layers and

Figure 4. Interference contrast photograph of a recently hatched Schistosoma mansoni miracidium. (Courtesy A. E. Butterzvorth.)

28

Schistosomiasis

parenchymatous cells within which lie the main organs. These include a neural mass, germinal cells and two pairs of flame cells of the excretory system, essential for removing water taken up by osmosis. The characteristic flickering movement of the fibrils within the flame cells can be seen in the miracidium before eggs hatch and provides evidence of their viability. The gut is not functional and the organism relies on its limited glycogen reserves for energy. The most prominent organs are a pair of Periodic Acid Shiff (PAS) positive penetration glands connected by ducts to anterolateral apertures at the base of the apical papilla. Lytic enzymes secreted by these glands diffuse through the egg shell to aid both egg passage to the exterior of the definitive host and, later, miracidial penetration of the snail. Because they cannot feed, miracidia are short-lived, but they swim actively at speeds of up to 2 mm per second for up to 8-12 hours in controlled laboratory conditions, although their infectivity starts to drop rapidly after 4 - 6 hours as they exhaust their food reserves. Factors hastening aging of miracidia include high temperatures raising the metabolic rate, ultraviolet irradiation, high turbidity, excessive water flows and turbulence, and the duration of exposure to chemical and other stimulants in the absence of actual snails. Depending on the degree of stimulation, food reserves will be used more or less rapidly so that the physiological and temporal ages of the miracidium will not necessarily correspond. Unless it penetrates a suitable snail, its movement eventually ceases and it drops to the bottom, swells and disintegrates. Most miracidia tend to be positively phototactic and negatively geotactic, but these taxes are sometimes reversed within and between different h o s t parasite combinations to increase the likelihood of miracidia contacting an appropriate aquatic snail living on floating or submerged plants or other substrates within a habitat. Newly hatched miracidia scan the habitat by swimming in straight lines but, later, turning motions increase around watersubstrate interfaces, trapping and causing them to accumulate close to aquatic snails. It is considered unlikely that miracidia actively follow chemical gradients towards snails but, rather, that they respond to changes in the microenvironment by more frequent, klinokinetic turning motions. A role in this process has been proposed for miraxones (molecules such as fatty acids, amino acids and, possibly, the neurogenic product seratonin from snails). Ecdysteroids may play this role (186) but altered ratios of simple inorganic ions such as Na, Ca and K may have a similar effect (34).


29

Once contact is made, there may be a prolonged exploration period before the miracidium begins active penetration of any exposed region of the soft parts of the snail: head, foot, mantle or within the mantle cavity. The process is initiated by boring movements of the apical papilla, the folds on which may aid attachment. Secretions of the penetration glands and, possibly, the primordial gut may assist attachment and penetration. The remaining miracidial food reserves and the age of the intermediate host affect the speed of penetration, which takes only a few minutes once started. The stimulus for penetration is probably nonspecific, because miracidia also penetrate inappropriate snails and other aquatic organisms: "biological sponges" have been suggested for transmission control (35). It has been suggested that miracidia of different species differ in their ability to locate their own intermediate hosts on the grounds that field infection rates tend, for example, to be greater among snails transmitting S. haematobium than those transmitting S. mansoni. Even within the same species, it is commonly observed in controlled laboratory infections that some exposed snails fail to become infected, even when exposed individually to numerous miracidia, and many of the miracidia that do penetrate fail to develop further. In order to make comparisons of the compatibility of different species, strains or subspecific isolates of both schistosomes and snails, a standardized index of the total cercarial production of 100 exposed snails was proposed by Frandsen (64).

Primary and Secondary Sporocysts A miracidium loses its epidermal plates during penetration but the ciliated cells are retained and overgrown with microvillae. A successful penetrant remains close to the site of entry in all species except, possibly, S. japonicum. During the following 48 hours the muscle layers are lost and the remaining cells reorganized into an elongated sac, the primary sporocyst, bounded by a syncitial tegument and with central vacuoles surrounded by germinal cells. The sac elongates into a globular mass comprising a nonmotile, convoluted and coiled tube. Germinal cells bud off and grow into opaque, cylindrical secondary sporocysts covered anteriorly with spines. These sporocysts mature in about 8-10 days. For S. mansoni, estimated secondary sporocysts production from primary sporocysts varies wildly from < 40 to > 600. After breaking through the wall of the primary sporocyst, the vermiform secondary

30 Schistosomiasis

Figure 5. Schistosoma spp. cercaria. (Courtesy MEDDIA, Royal Tropical Institute, Amsterdam, and Bruijning CFA.) sporocysts migrate either actively through the snail tissues or passively through the blood sinuses to the digestive glands. There, they develop into nonmotile, racemose sacs in which the 50-100 germinal cells develop either into cercariae, sometimes with subdivision of the initial morula in a form of polyembryony allowing regeneration of exhausted germinal layers to maintain a continuous if fluctuating cercarial production from the secondary sporocysts or, according to some authors, a proportion of the secondary sporocyst germinal cells develop into tertiary sporocysts from which later waves of cercariae will be produced (102). The cercariae may either break directly through special areas of the sporocyst walls (S. haematobium and S. bovis) or, possibly, leave through a terminal birth pore (S. mansoni and S. japonicum). A single miracidium successfully infecting a snail is capable of producing hundreds or even thousands of cercariae, all of the same sex, over a period of weeks or months. Bisexual snail infections indicate successful infection by two or more miracidia. The proportions of unisexual and bisexual schistosomes in field snails generally follow a Poisson distribution, but may be distorted if other factors differentially affect the development of each sex (215).


31

The length of the prepatent period between the penetration of a snail and the first release of cercariae is affected by the ambient temperature (162). It ranges from 17 to 18 days at 30-35°C, the highest temperatures tolerated by snails, for S. mansoni in B. glabrata (156,157), to several months at < 15°C in other snail-parasite combinations in temperatures in subtropical latitudes. Similar delays occur among infected snails estivating during periods of drought. At average ambient temperatures, however, the normal prepatent period for schistosomes is 20-35 days in planorbid snails, compared with 40+ days for those in prosobranchs.

Cercariae Cercariae are the second free-living stage in the life cycle. Like miracidia, they do not feed and have to survive in fresh water. They die if their energy reserves of glycogen become exhausted before they locate and penetrate a definitive host. Survival times are affected by many factors, especially temperature and the frequency of external stimuli provoking swimming activity. In controlled laboratory conditions, they may survive up to 72 hours, especially if glucose is added to the medium. The proportion penetrating definitive hosts and developing into adult worms begins to diminish by 8 12 hours: few penetrants from cercariae over 24 hours old develop to maturity.

Figure 6. Biomphalaria spp. snail shedding S. mansoni cercariae. {Courtesy MEDD1A, Royal Tropical Institute, Amsterdam; H. P. Streibel, Ciba Geigy.)

32

Schistosomiasis

Schistosome cercariae all lack eye-spots and a pharynx in the primordial gut. They are about 0.5 m m in length and so similar that, when alive, that they are indistinguishable with the naked eye or at low-to-medium-power magnification. The timing of the diurnal shedding pattern and the subsequent behavior of the cercariae may be suggestive in some cases (228). Specific staining allows differentiation of fixed specimens of some species (14). All cercariae have a head (the body of the future adult worm) and a brevifurcate locomotory organ or tail. The head has an anterior organ, often called the oral sucker, and a prominent, muscular ventral sucker or acetabulum. The body is bounded by a syncitial tegument with an external lipid bilayer which, in electron micrographs, appears trilaminate and covered with an amorphous, external "glycocalyx." Hairs and spines on the tegument are especially dense along the posterior margin of the body, on the acetabulum and anteriorly. Argentophyllic papillae on the surface, revealed by silver nitrate staining, are of some taxonomic value as their distribution differs between species. The mouth penetrates the anterior organ subterminally and leads to a nonfunctional Y-shaped gut that lacks a pharynx. Internally, a basal lamina beneath the tegument covers a thick layer of interstitial material and both circular and longitudinal muscles fibers. The excretory system comprises three pairs of flame cells in the head and one pair at the base of the tail, from which tubules lead to a posterior excretory bladder whence the main excretory duct runs through the stem of the tail to two branches that open at the tips of the furcae. The most prominent internal organs of the head are the penetration glands and their ducts to openings on the edges of the oral "sucker." Two pairs of anterior, preacetabular glands contain alkaline alizarin-staining material thought to be primarily enzymatic for penetration of host tissues. Four pairs of posterior, postacetabular glands contain granular PAS-positive material which swells on contact with water into an adhesive, waterproof mucus. Stirewalt (199) suggested two functions for this mucus. First, its gradual secretion during the free-living stage provides the surface "glycocalyx" minimizing water uptake by osmosis. Secondly, it helps adhesion and forms a gasket to focus enzymes from the preacetabular gland on the target area of the skin during penetration of the definitive host. It has also been suggested that the postacetabular glands may also produce enzymes (77). Finally, there is an apical or head gland associated with the oral "sucker" which persists into the schistosomulum stage when its secretions may aid in the penetration of blood vessel walls at the start of vascular migration.


33

Cercarial shedding from snails exhibits a pronounced diurnal periodicity, usually with a single emission every 24 hours. For human schistosomes, increasing light intensity within the normal temperature range (15-30°C) stimulates the release of cercariae with peak emission some hours later, causing a buildup of cercariae in the water to coincide with periods of high human contact. The process is rather more protracted for S. haematobium (shedding starting after about 2 hours and peaking by 4 - 6 hours) than for S. mansoni and S. japonicum (starting in 1-2 hours and peaking by 2-4 hours). However, this pattern may vary locally and the chronobiology of the start, peak and duration of emission may be suggestive if several schistosomes are transmitted by the same snail species, e.g. S. bovis, S. curassoni, S. intercalatum and S. haematobium (141,142,152, 227, 228). A rat strain of S. mansoni is reported to shed cercariae at night when peak cercarial densities will coincide with the highest rodent contact (214). A similar nocturnal pattern is shown by the rodent schistosome, S. rodhaini, some isolates of S. japonicum from the Philippines (107) and Taiwan (92), and S. malayensis (224). However, other Chinese and Philippine isolates of S. japonicum cercariae exhibited normal day-time peaks in laboratory studies (138). Another strain of S. japonicum has two (morning and evening) peaks of emission within the same day (148), as does S. margrebowiei (173, 253) and, seasonally, S. mattheei (164). Such variations in cercarial shedding patterns may be evolutionary adaptations by schistosomes to alternative definitive hosts and may be under genetic control. In Guadaloupe, crossing "late" and "early" shedding strains of S. mansoni gave an Fl generation with an intermediate emission rhythm, whereas "early" Guadaloupe and "late" Brazilian strains produced offspring with twin emission peaks (215). The daily and lifetime schistosome cercarial production in laboratory conditions varies considerably and is affected by many different biotic and abiotic factors. Cercarial production is not necessarily related to the number of miracidia to which the snails were exposed: snails exposed to single miracidia often produce more than those exposed to greater numbers. Undoubtedly the age, size and nutritional status of the snail all play a part, as does the compatibility of the schistosome and snail strains involved. Generally, S. mansoni and S. haematobium cercariae are produced in greater n u m b e r s than those of S. japonicum. Some of the highest productivities recorded are for S. mansoni in B. glabrata (< 18,000 in a single day and > 68,000 over 18 weeks). However, daily production of 1500 and

34

Schistosomiasis

2000 a day is more usual for S. mansoni and S. haematobium, respectively, and considerably less may be shed by naturally infected field snails. Daily production of S. japonicum and S. mekongi infections averages between 15 and 30 cercariae a day. Cercariae move in several ways. The most common is intermittent bursts of swimming: rapid tail movements pull the head after it but, when the tail movement ceases, the cercaria sinks head first through the water column using the tail furcae as a "parachute." Cercariae tend to hang perpendicularly and thus rise and fall vertically when swimming. Some lateral movement occurs if the tail is not held completely vertically or when a cercaria reaches the surface meniscus at the air-water interface. However, significant lateral movement is probably passive, due to water currents caused by gravity, wind or thermal effects. In some species, cercariae sink to the bottom and remain motionless for prolonged periods: others cling with their suckers to either solid substrate surfaces or the meniscus. Both behaviors conserve energy until a potential host enters the water and renewed swimming is stimulated by sudden changes in light intensity (phototaxis) or by vibrations (thigmotaxis). They can also crawl over solid surfaces, using their suckers like an "inch-worm," by alternate muscular contraction and extension of their bodies. S. japonicum cercariae, in particular, increase their dispersal range by adhering to the surface film of flowing waters. S. japonicum cercariae may also be released into de drops on emergent plants by their amphibious hosts, exposing definitive hosts that brush such vegetation without actually entering the water. The distribution in the water column of cercariae of various schistosomes shed by snails in glass tubes can give clues to their identity. Cercariae of S. haematobium form an inverted pyramid near the base of the tube, whereas those of S. mansoni and, especially, S. japonicum, tend to accumulate near the surface. So, too, do cercariae of S. intercalatum, which aggregate in clumps because of excessive sticky secretions. The detection and distribution of cercariae in natural habitats have been studied in two ways: by exposing rodents such as mice and hamsters and recovering the maturing adult worms, and by cercariometry, in which cercariae are recovered directly from water of natural habitats. Both methods face sampling problems, especially in deciding when and where they should be used to maximize the chance of detecting cercariae. Animal exposure is slow, taking a month or more to obtain a definitive answer, and is logistically expensive, requiring adequate transport and animal


35

holding facilities to avoid high mortalities among the exposed rodents during the prepatent period, when accurate diagnosis of immature schistosomes is difficult. The precise relationship of the number of worms recovered to the number of cercariae actually present in the water is uncertain. However, recovery of adult worms proves that the cercariae were infective and allows relatively easy identification of species if bisexual infections producing eggs are obtained. An alternative to exposing laboratory animals is regular trapping and examination of suitable wild rodents if they occur in the study area (190). Cercariometry is more versatile and provides specific cercarial counts more rapidly than animal exposures. However, the most effective methods involve killing the larvae with formalin before filtration and staining them on filters unsuitable for the high power microscopy. Differential staining required to identify the cercarial species (31, 143) is almost impossible. The cercariae cannot be used to infect experimental animals to obtain adult worms and eggs. The best approach is to combine the two methods if possible. Penetration of the definitive host after initial contact is enhanced by stimulation with certain components of sebum (sweat), particularly certain fatty acids and their derivatives. The warmth of the homiothermic definitive host may also play a part. However, such stimulation is not essential, because cercariae often attempt to penetrate other natural and synthetic materials. The initial phase, involving firm adherence of the body to the skin with the oral sucker, precedes the discharge of the penetration glands. Violent tail movements, before it is shed, assist the head in its initial penetration of the skin. The whole process takes only a few minutes.

Schistosomula Experimental

Production

and Storage

Experimental procedures have been developed to mass-produce schistosomula for a range of experimental studies because they are widely considered to be the main target of the definitive host's protective immune mechanisms. To produce so-called natural "skin-transformed" schistosomula, a cercarial suspension is incubated at body temperature over a piece of excised rodent skin above an appropriate culture medium. Cercariae penetrate the skin, losing their tails in the process, and the heads sink within the culture medium, whence they may be harvested in large numbers after about 3-4 hours (37).

36

Schistosomiasis

Alternative, mechanical methods physically shear the tails from the heads by repeated passage through a fine-gauge hypodermic syringe needle or by vortex mixing. The cercariae are suspended in an appropriate culture medium before shearing and the detached heads are subsequently incubated at body temperature for about 3 hours (38, 41, 67). The transformation of the cercarial head into a schistosomulum involves far-reaching biochemical and physiological changes. Proof of transformation as opposed to mere detachment of the tail is important for interpreting experimental results using either skin- or mechanicallytransformed schistosomula. Fully transformed schistosomula placed in a hypotonic medium cannot control water uptake, swell rapidly and die: untransformed cercarial heads are unaffected when placed in water but, unlike schistosomula, become enveloped in immune complexes when incubated in serum containing anti-schistosome antibodies. Morphologically, true schistosomula lack a tail and the penetration glands are empty, though not the apical (cephalic or head) gland. Electron microscopy shows the loss of the cercarial glycocalyx and the replacement of the original, trilaminate tegumental membrane by a heptalaminate membrane, actually a double, bilipid layer linked by complex protein molecules. Although relatively large numbers of schistosomula may be produced for a specific experiment, interbatch variation in recovery rates and viability is a problem. As a possible solution to such problems, and to accumulate the large numbers required for vaccination experiments using irradiated schistosomula (and cercariae), cryopreservation methods were developed (94). While normal cryopreserved larvae successfully infect rodents and irradiated larvae give good protection, the use of cryopreserved vaccines in primates gave disappointing results (95). Moreover, live vaccines themselves produced pathology unacceptable in man (27), although they may be of value against S. bovis and S. japonicum in domestic stock (85, 209). In addition, studying responses to irradiated cercariae and schistosomula continues to throw light on the immune mechanisms involved in normal schistosome infections (175).

In Vivo Migration,

Development,

Morphology

and Biology

Complete transformation from cercariae to schistosomula takes 3-6 hours both in vitro and in vivo. In vivo, it occurs in the stratum corneum. Nanduri et al. (145) found different properties in the glycocalyx of the tail and the


37

cercarial head. It is possible that the glycocalyx shed from the cercarial head impedes the mammalian immune system by binding key elements, thereby reducing its effectiveness during a critical period while the outer, trilaminate membrane of the schistosomulum tegument transforms into a heptalaminate membrane. This membrane is necessary for survival in mammalian blood: schistosomula in which its development is biochemically prevented will not mature in vivo (240). There are numerous in vivo and in vitro studies of this transformation (e.g. 12, 130, 242). The outer lipid bilayer may be formed initially by secretions from the cercarial postacetabular glands but is derived later from corpuscles situated deep in the tegumental syncitium. When completed, the schistosomulum is surrounded by a relatively inert layer (241). The schistosomulum remains within the epidermis from 72 to 96 hours before entering the dermis to begin the migration phase. At this stage, the oral and ventral suckers and the Y-shaped gut are clearly visible. True growth involving cell division does not occur but rearrangement of cells accompanies a posterior elongation of the body before entry into the vascular system. The surface of the schistosomulum tegument has received considerable attention because it is in direct contact with the definitive host and is assumed to be the target of protective immune responses. Seventeen or more macromolecules, more than half glycosolated and ranging in size from 14 to > 150 kDa, have been found expressed on the surface of three-hour-old S. mansoni schistosomulum, but many disappear after long term (18+ hours) in vitro culture. They include oligosaccharides, glycolipids and glycoproteins, and some may act as enzymes. Many appear to be preformed during cercarial development and initially reach the surface in secretions of the penetration glands but later are passed up through the tegument as membraneous vesicles extruded from the cell bodies. Other molecules may be synthesized de novo or else acquired directly from the host, particularly lipoproteins characteristic of mammalian cells. Some act as epitopes to which host antibodies and complement will bind: their production and shedding may be a mechanism to divert immune mechanisms intended to damage the schistosomulum tegument. An analogous situation has recently been described in the bird schistosome, Trichobilharzia szidati (84). The route by which schistosomula leave the skin and migrate to the liver, a source of controversy for many years, is comprehensively described by Wilson (241). The problems were eventually resolved radiographically using 75 Se-labeled cercariae to infect experimental animals, which were then killed

38

Schistosomiasis

sequentially for whole animal or selected organ compression autoradiography to estimate the number and location of labeled schistosomula present during this phase of the life cycle. Labeled larvae recovered from various organs of one animal were transferred to a range of sites in others and their progress tracked. Despite technical limitations, it was possible to estimate the dynamics of schistosomulum migration through the key organs. It was concluded that significant schistosomulum mortality did not occur in the skin as had been believed, even in immunized hosts. Briefly, most schistosomula leave the skin by 90 hours, penetrating a vein or, in some cases, a lymphatic vessel. Lytic enzymes discharged by the apical gland probably aid penetration at this stage. Schistosomula entering the lymphatics may be delayed by, but eventually traverse, the draining lymph node to enter the venous system via the thoracic duct. Whether they enter the venous system directly or via the lymphatics, they are are carried passively through the right heart to the pulmonary capillary beds, where they are trapped temporarily. There, elongation continues, still without active cell division, until schistosomula can move actively through the capillaries into the pulmonary vein. Pulmonary capillaries have thin, unsupported walls that rupture easily so that some schistosomula enter the alveoli, where most become trapped and die, although a few may re-enter the pulmonary capillaries. Mortality may be higher in immune animals, because inflammatory reactions weaken the capillary walls and damage the schistosomula. Once in the p u l m o n a r y vein, schistosomula are carried passively by the blood through the left heart, into the systematic circulation and on to the next set of capillary beds. If these are the splanchnic vasculature (the mesenteric artery and the capillaries leading to the hepatic portal vein), schistosomula proceed to the liver, where further development takes place. If they traverse capillary beds elsewhere, they return to the heart to start another circuit. Because they cannot feed, few schistosomula can complete more than two circuits before exhausting their limited food reserves and dying. The proportion of systemic blood passed to the splanchnic vasculature varies between mammalian species: in small rodents it is < 50% but it is probably more in larger animals such as primates (243). In naive animals, the proportional recovery of adult worms from a given, infecting dose of cercariae correlates with the proportional splanchnic blood flow. Animals with abnormal hepatoportal vasculature (e.g. 129-strain mice) give a false appearance of innate resistance because most invading schistosomula


39

pass right through the liver (45). This situation is akin to that in animals with pre-existing infections. The pathological effects of egg-induced liver granulomata, especially a widening of the portovenal vasculature and the development of portocaval shunts, allows a greater proportion of challenge schistosomula to pass right through without any development. This "leaky liver" effect may give rise to a spurious appearance of partial resistance to reinfection. In some cases, such as Rattus rattus, even adult worms may escape from the splanchnic vasculature to the lungs by this route and reside there for prolonged periods, though they are probably incapable of significant egg production (175). On arrival in the liver, schistosomula lodge in a nutrient-rich environment in the smallest hepatic portal distributaries, beginning development involving cell division, and moving upstream to larger vessels as they grow. Changes over an initial 3-4-day adjustment period include a shortening of the body accompanied by a ridging of the tegument, a loss of motility, and the initiation of various metabolic activities. The subterminal area enlarges around the mouth, ingestion of erythrocytes begins as the oesophagus grows and the lateral ceca of the gut elongate, eventually uniting to form the posterior gut cecum. Spines reappear in small numbers in the tegument cytoplasm and new sensory nerve endings appear. By this stage, schistosomula have become immature "liver" worms and pairing takes place, 4-5 weeks postinfection for S. mansoni and S. japonicum, and a week or two later for S. haematobium. The male clasps the female in his gynecophoric canal and, bracing his body against the vessel walls and using his ventral sucker, moves against the blood flow to the oviposition site: the mesenteric or rectal veins around the intestine via the hepatic portal vessels for S. mansoni and S. japonicum; or via the inferior branch of the mesenteric vein, superior rectal vein and the rectal venous plexus to the vesical plexus around the bladder for S. haematobium.

Adult Worms The adult worms vary in size by species, and according to their age and the definitive host in which they mature. The dorsoventrally flattened males range from < 3 m m (S. edwardiense and S. incognitum) to - 2 0 mm (S. japonicum) in length, and 0.40 to 1.00 mm in width. Fully developed, cylindrical females are proportionately longer than the male, sometimes

40

Schistosomiasis

exceeding 30 mm (S. margrebowiei), but are slimmer at 0.25-0.50 m m in diameter. The lateral margins of the male S. mansoni fold around the female to form the gynecophoric canal and elongated, acuminate spines act like velcro to secure her firmly in place. Externally, both sexes have weakly developed oral suckers perforated by the mouth. The ventral sucker, situated anteroventrally, is more strongly developed in the male than the female. The gonopore opens ventrally, immediately behind the ventral sucker, and the excretory pore opens posterodorsally. The entire body is covered with a tegument which, like that of the schistosomulum, is a continuous syncitium bounded with an external heptalaminate membrane. Scanning and transmission ultramicroscopical examination of the tegument reveals spines and tuberculations of varying size on or in the tegument, some of which can be seen using light microscopy. The patterns vary among different schistosome species and can be of diagnostic value: the surfaces of S. japonicum, S. haematobium and S. mansoni appear smooth, finely tuberculate and coarsely tuberculate, respectively. The tegument is not just an inert, protective layer but a living tissue, capable of taking up simple nutrients such as amino acids, simple sugars, fatty acids, lipids and inorganic ions. Novel methods confirm this uptake

Figure 7. S. haematobium: muscular male clasping nematoid female worm. (Courtesy The Wellcome Trust.)


41

to be an active, energy-dependent process (175). The transport of such nutrients from male to female where their teguments are in intimate contact along the gynecophoric canal is essential for her to reach full maturity and, subsequently, to maintain high egg production (44, 57). Eicosanoidal lipids, triglycerides and cholesterol are not synthesized de novo by the male but acquired from the host serum. They were thought to be potential chemical messengers to stimulate the sexual maturation of the female. However, recent immunomolecular studies suggest that the process may be triggered by molecules involved in signal transduction pathways, such as specific GTPase proteins activated by the small G protein Ras which transmits signals to switch on mitogen-activated protein kinases. These molecules occur in males and in mature, paired, egg-laying females but disappear from females separated from males (187). Other molecules are found in both paired and separated males and females, including protein disulphide isomerase and cathepsin L, or preferentially in paired females, such as egg shell precursor proteins (106, 112), the egg-shell protein pl4, the iron storage protein Ferl and the mucin-like protein A l l . They may be regulated by physicochemical factors (236) but are not believed to be involved in the signaling process although they are needed for subsequent maturation of the reproductive system and egg production (74). The only female specific enzyme so far detected and not associated with the reproductive system is from the gastrodermis; an amidase probably involved in the digestion of haemoglobin (188). Besides its role in nutrition, the tegument is the first line of defence against the host immune system. Worms survive for years in the essentially hostile environment of the blood system. They neutralize the normal clotting mechanisms by inhibiting activated Hageman factor Xlla (222). Adults use a strategy of disguise: host molecules of the ABO and Duffy blood group systems adhere to or become inserted in the tegument and worms may themselves synthesize such molecules to avoid being recognized as foreign bodies (126). Recent studies suggest that worms produce antioxidants to neutralize toxic oxidants released during antibody-dependent, cell-mediated reactions (133, 196). The internal anatomy of adult schistosomes includes circular and longitudinal muscles below the tegument, as well as other specialized muscles, all coordinated by a network of neural fibers to permit contractions and other movements. Fine tubules link the protonephridial flame cells embedded in

42

Schistosomiasis

the mesenchymatous cells to the posterior excretory bladder, allowing excretion of excess water and soluble metabolic waste products. The digestive system lacks a pharynx: the short oesophagus leads directly from the mouth to the intestine which divides in front of the ventral sucker into two lateral gut ceca that reunite behind the gonads to form a single, blind posterior gut cecum. Blood ingested through the mouth is exposed to proteolytic enzymes secreted by cells lining the gut ceca. Tyrosine is released from serum globulins and the hemoglobin of red blood cells, leaving residual black hematin. This pigment is more obvious in the less muscular females, which also consume erythrocytes more voraciously than males (117). There is no anus and the contents of the gut are periodically regurgitated through the mouth. The vomitus contains both enzymes and the products of digestion. Phagocytic cells of the host liver and spleen eventually remove the black pigment from its bloodstream. Hematin from schistosome and malarial infections can be distinguished ultramicroscopically (139). The reproductive organs mostly lie between the lateral gut ceca except for numerous pairs of follicular vitallaria in the mesenchyme surrounding the posterior gut cecum of the female. A single, pyramidal ovary is situated just in front of the posterior junction of the gut ceca. The vitelline duct joins the oviduct just behind the ootype. There is a seminal receptacle at the base of the oviduct and Mehlis' gland lies immediately behind the ootype. In males, according to the species, up to 15 testes lie dorsally in one or more rows running back from the ventral sucker. Sperm is stored, probably only briefly, in a seminal vesical anterior to the testes before it is passed via the sperm duct to the gonopore. Ultramicroscopy is necessary clearly to reveal the highly infolded, spongelike cirrus which can be everted to engage the female genital pore. Paired worms are not in a state of perpetual copulation, but it probably occurs quite frequently because sperm does not usually accumulate in the seminal vesical. In S. mansoni, eggs formed in the ootype are passed one at a time along the simple, tubular uterus and emerge through the genital aperture. In other species, the uterus may contain many more eggs: < 10 in S. mekongi, < 50 or 60 in S. haematobium and S. intercalatum, and < 200 in S. japonicum. Daily egg production from a mature female varies with the species, from a few hundred to 3000 or more, representing nearly 10% of her total body weight in extreme cases. Female worms do not normally reach sexual maturity unless pairing occurs, despite reports of parthenogenetic egg production in experimental,


43

unisexual female infections (207). Pairing of different species is possible in mixed sex infections, especially as there is frequently an excess of males over females. Hybridization and parthenogenetic egg production have both been reported in such circumstances (210, 211). In the field, the distributions of closely related schistosome species often do not overlap. Examples include the absence of S. haematobium from Sardinia despite the presence of S. bovis, the predominance of S. haematobium over S. mansoni in much of the Nile Delta and on the coastal plains of east and west Africa; S. intercalatum occurring in the absence of S. haematobium in central Africa; and the general lack of overlap in the distribution of S. leiperi and S. margrebowiei in southern Africa. The distribution, ecology and behavior of both the intermediate and the definitive hosts, coupled with manmade water developments, probably suffice to explain these patterns, and heterologous immunity probably plays only a small part in nature. Laboratory studies show cross protection between species, but invariably less than homologous immunity due to earlier infections of the same species, and hybrid pairing between some species (209). Hybrid eggs may yield Fl adult worms capable of producing viable offspring (181), but F2 generation eggs may not be viable or only produce worms of reduced vigor (11). Although various chemical attractants, including ecdysteroids, cholesterol, other lipophilic substances and undefined pheromones have been suggested, the precise nature of the stimulus for pairing is not understood (57). It is probably common to most schistosome species, explaining the propensity for hybrid pairing. The female uterus, oviduct and ovary may develop completely before pairing occurs, but development of the highly metabolically active vitelline gland requires additional nutrient inputs from the male after pairing. Ca 2+ -dependent ATP-ase activity, already widespread in and around the tegument of single males, develops in the newly paired females and the role of Cathepsin L proteinases in egg production has recently been demonstrated (235). All schistosomes so far studied have a chromosome number of 2n = 16 and have similar chromosomal morphologies. Considerable progress has now been made in mapping the schistosome genome using a variety of methods, including DNA cloning, restriction enzyme analysis and various forms of nucleotide sequencing, in some cases inferring the function of specific genes.

44

Schistosomiasis

Schistosomes were originally believed to be essentially homolactate fermenters depending on anerobic pathways to obtain energy (15) but this view has been disputed (39, 191, 226). In vitro experiments with isolated mitochondria indicate that adults are capable of obtaining energy from carbohydrates by the normal, aerobic pathways of Kreb's cycle, but hexokinase levels may be limiting in vivo, where it is believed that the Embden-Meyerhoff pathway is used for the anerobic breakdown of glucose to lactic, acetic and propionic acids. (A functional citric acid cycle exists in unhatched miracidia but it is uncertain whether glycolysis or oxidative mechanisms are more important before hatching occurs: complete oxidation occurs after hatching in free-living miracidia and cercariae.) In vitro experiments with adult schistosomes indicate that they can synthesize pyrimadine de novo, but not purine, and must therefore rely on salvage pathways to produce nucleotides, assuming that results from isolated w o r m s or h o m o g e n a t e s can b e extrapolated to in vivo conditions (40). There is a considerable literature on isoenzymes found by starch electrophoresis and isoelectric focussing of extracts from adult worms (and other developmental stages), primarily to aid diagnosis of closely related species: over 30 enzyme-staining systems have been used. Isoelectric focussing of the enzymes glucose-6-phosphate dehydrogenase and phosphoglutamase reveals geographical differences in numerous isolates of S. haematobium, and the same isoenzymes allow differentiation of S. haematobium and S. mattheei. S. curassoni, S. haematobium and S. bovis were distinguished by different patterns of phosphoglutamase, phosphoglucose isomerase, hexakinase and acid phosphatase. Studies of these isoenzymes are of taxonomic value, but their roles in vivo and the functional significance of specific differences in the isoenzyme patterns are less certain, except to infer which biochemical processes are involved. The mechanisms by which schistosomicidal drugs affect worms are gradually being unraveled and studies on their mode of action throw some light on aspects of schistosomal biochemistry and physiology. Initial studies revealed that the original antimonial drugs inhibited phosphofructokinase activity; that both oxamniquine and hycanthone inhibit glycogen metabolism; and that hycanthone interchelates with DNA. Praziquantel disrupts membranes and causes an influx of Ca 2+ ions in muscle cells, making them contract violently (174). Dichlorvos, the metabolite of the organosphosphorus ester metrifonate, inhibits both choline and acetylcholine esterases. It is


45

effective against S. haematobium, but progressively less so against S. bovis, S. japonicum and S. mansoni. All species transport glucose across their teguments with the aid of acetylcholine esterases, surface receptors for which are especially abundant on S. haematobium (28, 29). The different biochemical p a t h w a y s and mechanisms revealed by these drugs are sometimes sex-specific. Hycanthone was less effective against females than males. Males may physically shield females from a drug, but this cannot be the only explanation: niridazole and antimonial d r u g s affect females m o r e than males, suggesting, not surprisingly, real differences in the metabolism of the two sexes. Stage specificity of some drugs suggests that these pathways are not functional throughout the entire life cycle. Praziquantel and oxamniquine kill adults but not immature worms developing within the liver. Presumably the target metabolic pathway is not functional unless rapid detoxicification of the drug by the liver prevents exposure of developing worms to lethal levels of the drug or its metabolites. Nontoxic drugs such as sodium phenobarbitone affect adult worms transiently so that they shift from blood vessels around the gut to the liver, but they later recover and return to the gut. Effective schistosomicides cause a similar liver shift but also damage worm, exposing receptors, normally hidden, to leucocytes and other elements of the host's immune system. Dying worms are often trapped and phagocytosed within the liver. The synergistic relationship between drugs and vaccines is of growing interest. Some candidate vaccines are enzymes and thus susceptible to drugs. A number of glutathione-S-transferase (GST) enzymes are currently being studied as vaccines. Glutathione is a tripeptide crucial for many biological processes, including detoxification of xenobiotics and endogenous toxic compounds. If it protects worms against immune responses, drugs affecting its function would remove this protection. The experimental antischistosomal drug, oltipraz, modified the state of glutathione in adult worms, decreasing levels of the reduced form but increasing levels of the oxidized form (137). Praziquantel forms complexes with GST (132), and also depletes glutathione levels in schistosomula in vitro, rendering them susceptible to immune attack (175). Immunological disruption of cystiene protease activity reduces both the survival of adult worms and their egg production (120) and could form another target for drug attack. Other enzymes of interest include proteases released during cercarial penetration, those expelled in the vomitus from the digestive system and

46

Schistosomiasis

others passed across the tegument. Among them are the metalloprotease leucine aminopeptidase (47), the proteases Legumain, and Cathepsin L-, D-, B- and C-like enzymes involved in catabolizing hemoglobin (23). These enzymes may differ in form between species, e.g. Cathepsin D expressed by S. japonicum and S. mansoni (245). Tolerance/resistance to hycanthone and oxamniquine has been observed in the field, and to praziquantel in the laboratory (59). RAPD (random amplification of polymorphic DNA), developed to solve taxonomic problems (52), is now being used to investigate its genetic basis. Resistance of S. mansoni to oxamniquine and hycanthone is associated with the absence of a gene found in normal worms (36, 158, 159). Praziquantel tolerance appears to be associated with overexpression of a gene coding for cytochrome oxidasec production, allowing greater reliance on oxidative metabolic pathways (155). Field reports of praziquantel tolerance for S. mansoni in Egypt (91) and Senegal (197) may be an artifact due to a combination of the limitations of the Kato egg-counting technique and heavy infections (50), supported by the ultimate efficacy of repeated praziquantel treatments in Senegal (160, 161). Not all potential vaccines are enzymes. Neuropeptides currently being studied may provide targets for drugs or vaccines (128). Paramyosin is a component of muscle, usually hidden within the worm, that may be expelled via the tegument unaltered or as its breakdown products (69,169). It is immunogenic and shows some promise as a vaccine. In fact, schistosomes produce a bewildering array of immunogenic molecules. However, none of those so far tested has given sufficient protection to justify further development (16). Most molecules being studied provoke strong, natural cellular and humoral responses, yet the adult worms survive within the host for many years. Doenhoff (55) cites Waksman's postulates to argue that greater success might be achieved using molecules that provoke weaker responses in vivo, such as larval proteases and high molecular weight egg and worm antigens.

The Intermediate Hosts General Taxonomy Schistosomes are transmitted by freshwater snails of the class Gastropoda in the phylum Mollusca. The intermediate hosts of all Oriental


47

human schistosomes belong to the family Pomatiopsidae in the subclass Prosobranchiata. They include one genus in the subfamily Pomatiopsinae, Oncomelania, which transmits S. japonicum, and two genera of the subfamily Triculinae, Neotrichitia, which transmits S. mekongi; and Robertsiella, which transmits S. malayensis. The intermediate hosts of the remaining human schistosomes all belong to the family Planorbidae in the subclass Pulmonata, including numerous species of the genera Biomphalaria and Bulimis. S. mansoni is transmitted by Biomphalaria spp. (synonyms Australorbis, Taphius and Tropicorbis in older American publications). Biomphalaria spp. also transmit S. mansoniS. intercalatum hybrids, S. rodhaini and S. edioardiense. Bulinus spp. transmit S. haematobium and also act as intermediate hosts for S. intercalatum and the nonhuman African schistosomes. Brown (25) is skeptical about transmission of S. haematobium by Planorbis metidjensis from an extinct Portuguese focus on the Iberian peninsula, where it transmits S. bovis, and by an ancylid (Ferissea tenuis) from a doubtful Indian focus. The remaining Asian schistosomes are transmitted by the planorbid Indoplanorbis exustus except for S. incognitum, transmitted by the lymnaeids Lymnaea luteola and Radix auricularia rubiginosa, and S. sinensium by the triculids Tricula humida, T. gregarina and T. bollingi.

Figure 8. Amphibious Oncomelania spp. snails clustered on a rock; are intermediate hosts of S. japonicum in China, Japan, the Philippines and Sulawesi. (Courtesy The Wellcome Trust.)

48

Schistosomiasis

This traditional classification of intermediate hosts is based on morphological characters of the shell and radula, supplemented by differences in their soft anatomy, especially of the reproductive system (127). Susceptibility to schistosome infection, though epidemiologically important, is not always a reliable taxonomic character, because compatibilities differ between various snail and parasite strains or isolates. Biochemical methods, such as paper chromatography of mucus, electrophoresis of egg proteins on cellulose acetate paper, isoelectric focussing of digestive gland extracts and Ochterlony diffusion of egg proteins against specific antisera, have all been used in snail taxonomy, with varying degrees of success. The latter approach was generally more successful for pulmonate than prosobranch snails. Isoenzyme analyses have also been used to unravel relationships within the genus Bulinus (201). Most planorbids have a chromosomal haploid number of n = 18. Biomphalaria spp. conform to this rule but some Bulinus spp. have n = 19-21 and others exhibit polyploidy with some populations having n = 36, 54, 72 or more. Numerical taxonomy using shell characters and measurements of relative shell dimensions has helped within the genus Bulinus. Cladograms based on mathematical descriptions of isoenzyme diversity show supraspecific relationships among oncomelanid species and indicate when specific separation occurred. Similar techniques are applicable to the accumulating mass of molecular biological data on all intermediate hosts. Molecular biological techniques have been used in Egypt to distinguish Biomphalaria spp. and to separate exotic from indigenous species (113, 251). Apparently refractory B. nasutus was separated from the susceptible B. globosus populations from Zanzibar, where S. haematobium is endemic, by restriction product analyses of ribosomal internal transcribed spacer regions, although amplification and analysis of 18S deoxyribonucleic acid showed no difference (201). In addition, specific cloned DNA probes to differentiate Schistosoma spp. can be used to identify prepatent infections within snails, within hours of infection if used in conjunction with the polymerase chain reaction (75). Brown (24) combines modern and classical methods for African snails. Sobhon and Upatham (193) discuss briefly their implications for Far Eastern host snails. The impact of similar studies (231) on the traditional classification of American Biomphalaria spp. (8) has yet to be assessed. It remains to be seen if this information will clarify or confuse taxonomical classifications of heterogeneous populations (219).


49

Taxonomy, Life Cycles and Biology of the Intermediate Hosts Snails Transmitting

Oriental

Schistosomes

The intermediate hosts of Oriental schistosomes are all prosobranch snails that obtain dissolved oxygen directly from the water by a gill. They are dioecious and must copulate to produce viable eggs. All have small, conical or subconical shells with 4 - 8 dextral whorls, and rarely exceed 10 mm in height. The shell surface is smooth, or may have fine axial growth lines or strong axial ribs. There may be a single paucispiral ridge or concentric rings on the corneous or calcareous operculum. All snails transmitting S. japonicum were originally classified as Oncomelania hupensis with six races or subspecies according to their geographical origin: O. h. hupensis (mainland China), O. h. nosophora (Japan), O. h. formosana and O. h. chui (Taiwan, originally Formosa), O. h. quadrasi (Philippines) and O. h. lindoensis (Sulawesi, Indonesia). With the possible exception of O. h. chiui, these subspecies should be raised to full species based on data accumulated from modern taxonomic methods (193): further subdivisions may be expected for different O. hupensis populations within the vast area of China (93, 168, 247). Oncomelania spp. exhibit various degrees of amphibious behavior, according to species, subspecies and race. Because of their dependence on water for oxygen, the most amphibious forms are restricted to areas of high rainfall and humidity where mud surfaces and emergent vegetation are covered by a persistent water film. Thus O. quadrasi is confined to the eastern side on those islands in the Philippines where the prevailing winds from the Pacific provide suitable conditions. O. hupensis on mainland China favors habitats flooded for 1-5 months during the rainy summer period, surviving the drier and cooler winter months in high humidities and residual water in cracks in the ground where cooler winter temperatures reduce both their metabolic rate and the saturation vapor deficit, protecting them from excessive desiccation. They are mainly present in shallow water with vegetative cover within a meter of the shoreline. They do not thrive in permanently flooded areas or in fast-flowing water, but flash floods can transport them to downstream habitats. Oncomelania spp. will copulate daily but isolated, inseminated females produce viable eggs for up to 12 weeks. Females lay eggs singly or in short chains, often coated in sand or organic debris, on solid substrates; either submerged m u d (O. nosophora and O. hupensis) or above the water level

50

Schistosomiasis

(O. quadrasi). Female O. quadrasi avoid sunlight by laying eggs mostly at night when temperatures are cooler and humidities higher. Within the tropics, egg production, though low (probably - 3 0 eggs per female during her lifetime), occurs throughout the year; in the subtropics, breeding is limited to the summer months. The hatchlings emerge after 10-30 days, depending on temperature, and remain in the water for several weeks before venturing onto land to feed. Initial growth rates of 0.25-0.50 mm (in height) per week decline as snails approach sexual maturity at 11-30 weeks, depending on the species. Life spans, possibly less for females than males, range from 6 to 9 months (O. quadrasi) to several years (O. nosophora) in the laboratory but are much less in the field (9 weeks for O. quadrasi; 16 weeks for O. nosophora). Outside the tropics, longevity may be prolonged by estivation when habitats dry out over winter. The intermediate host of S. mekongi, originally named Tricula aperta, was renamed Neotricula aperta, and it is proposed to split it into four species — N. aperta and three others, as yet unnamed. N. aperta (s.l.) achieves a height of 2-3 mm, with a large body whorl and a reduced spire. The snails are fully aquatic and infest protected microhabitats on stones within large rivers subject to prolonged, torrential flows during heavy rains (224). Populations survive under stones as juveniles and firmly attached eggs when seasonal

Figure 9. Neotricula aperta (a-strain) — a snail host of S. mekongi, though far less efficient than the P- and y-strains of N. aperta. (Courtesy S. W. Attwood.)


51

monsoon floods dislodge exposed adults. Hatching normally occurs after a month but may be delayed until the seasonal floods subside. Surviving juveniles and new hatchlings repopulate the sites and the young snails grow to sexual maturity in 4 - 5 months. Suitable habitats are determined by the geomorphology of a river: snails may be present in fast flows along a rocky bank but absent from sandy or silty substrates in slow flows on the opposite bank. Two snail species transmit S. malaysiensis: Robertsiella kaporensis and R. gismanni. Their shells are spired like Oncomelania spp. but much smaller, rarely exceeding 3 mm in height. Both species are fully aquatic and colonize restricted habitats associated with the tree Saraca thaipingensis, the roots of which project into forest streams. The adults tolerate a limited degree of desiccation. Eggs, laid singly on rocky surfaces or leaves, sometimes covered with a sand jacket, hatch within 3-4 weeks. The hatchlings grow slowly, achieving sexual maturity in about 4 months.

Figure 10. Top row: Bulinus forskalii and B. truncatiis. Bottom row: Biomphalaria pfeifferi and Bulinus globosus. Biomphalaria spp. transmit S. mansoni throughout its range. Bulinus spp. transmit S. haematobium and S. intercalatum (see text). (Courtesy V. Southgate.)

52

Schistosomiasis

Snails Transmitting

African and American

Schistosomes

Life cycles and biology of Biomphalaria and Bulinus All these schistosomes are transmitted by pulmonate snails that primarily obtain oxygen directly from the air, although dissolved oxygen can cross the epithelium of the exposed soft parts: some species have developed a pseudogill to extend this capability. An air bubble is trapped within the pulmonary chamber via the pneumostome, an extension of the mantle margin. Snails must return to the surface regularly to replenish it and it is large enough in some species for a snail simply to release its hold on a submerged surface and float to the surface. If a floating snail releases its bubble, it sinks and must regain the surface by climbing up plants or other submerged objects. Pulmonates are hermaphrodite although the male reproductive system may be incompletely developed — aphallic populations occur frequently in some species (127). When kept alone, most species can produce viable eggs by self-fertilization but, if possible, cross fertilization is usual. If snails of the same species but with different genetic characters (e.g. albino versus pigmented or susceptible versus resistant to schistosome infection) are isolated before sexual maturity, their offspring exhibit only one of the characters. After pairing, offspring are produced exhibiting both characters, generally in Mendelian proportions. Pulmonate snails are far more prolific than prosobranchs and can produce hundreds or thousands of eggs in their lifetime. Egg laying is unusual at much below 18°C but increases proportionally with temperature up to 30-35°C, above which snail (and egg mortality) increases. Breeding in the tropics is possible throughout the year but its intensity may still reflect seasonal changes. Pulmonate eggs are laid in clusters within a transparent, yellowish gelatinous mass; circular or oval in shape and attached to flat surfaces for Biomphalaria; more elongated and frequently wrapped around curved surfaces of aquatic plant stems for Bulinus spp. Egg mass size and the number of eggs present are proportional to the size of the parent snail: masses may exceed 1 cm in diameter or length and contain 30 or more eggs. Subsequent development is related to temperature. Hatching occurs within 5-10 days and the hatchlings are 0.5-1.0 mm in size. The subsequent growth curve is sigmoid with a logarithmic phase lasting 4-12 weeks until sexual maturity is reached when snails are about 5 mm in diameter or height. Thereafter, growth slows but never stops during the life span of the species. In the


53

laboratory, African Biomphalaria spp. rarely live much more than a year or reach a maximum diameter much more than 15-20 mm, whereas American Biomphalaria spp. can live for 2 years or more and achieve diameters in excess of 30 mm. Some field snails may reach or exceed these dimensions. The largest Bulinus spp. in the africanus group (see below) may exceed 20 m m in height under field conditions.

Taxonomy of Biomphalaria spp. Biomphalaria spp. occur throughout Africa, in the Middle East and in tropical and subtropical regions of the Americas. Most have been found naturally infected with one or more S. mansoni strains although there are variations in susceptibility between and within species. The few exceptions have mostly been infected experimentally. All have "ultradextral," discoid or lens-shaped, biconcave shells. Some species have an angular cross section due to a keel on the external perimeter of the shell. The height (thickness) of the shell varies from species to species, as does the number of whorls, usually between 3.5 and 7.0, according to the age and size of the specimen. The size (maximum diameter) and depth of the ventral depression (umbilicus) also vary between species, and may be especially pronounced in those where the external lip of the aperture is deflected ventrally. Shell color is highly variable, from a brownish, horn color to a deeper red, but may be obscured in field specimens by a layer of black or gry material according to the substrate, or bleached almost white in exposed sites. Shell sculpture includes curved, transversely arranged growth lines, sometimes crossed by faint, spiral lines. Populations of some species develop apertural lamellae, especially those that estivate during droughts: discontinuities in growth may be evident in such snails. Mandahl-Barth (127) recognized four African Biomphalaria groups on the relative proportions of the penis in fixed, contracted animals: prepuce much shorter than vergic sheath — alexandrina; just shorter — choanomphala; just longer — sudanica; much longer — pfeifferi. Brown (24) was unconvinced by these groupings. The pfeifferi group is widespread throughout Africa south of the Sahara and occurs on Madagascar in the Indian Ocean and in Aden, Yemen and southwest Saudi Arabia on the Arabian peninsula. The center of evolution of this group seems to be around Lakes Kivu and Tanganyika and, despite

54

Schistosomiasis

considerable variations in form among local populations, the African snails are classified as a single species, B. pfeifferi. A form from Zambia and southern Tanzania may justifiably be considered as a subspecies, B. pfeifferi rhodesiensis, but Brown (24) does recognize the B. ruppelli form in its northern range in Africa and Yemen as a distinct species. B. arabica represents the pfeifferi group in Saudi Arabia. Adults of the B. pfeifferi group have a maximum of 5 whorls, rarely exceed 15 mm and are invariably compatible with local strains of S. mansoni. The sudanica group is confined to the equatorial region of tropical Africa, with B. sudanica in the east and B. camerounensis in the west. MandahlBarth (127) subdivided each into two subspecies but Brown (24) rejects their validity. B. salinarium from Angola may be a member of the sudanica group although B. camerunensis also occurs there. Fully grown adults have over 6 closely coiled whorls and may exceed 20 m m in size. Originally considered poor hosts of S. mansoni, they are now known to be important locally, but often having limited cross compatibility with exotic parasite strains. The choanomphala group includes several species inhabiting the Central African great lakes, including B. choanomphala from Lakes Victoria and Albert, B. stanleyi from Lake Albert, and B. smithii from Lake Edward. Adults have < 5 whorls and rarely exceed 12 mm. They are susceptible to S. mansoni and are notable for their ability to live at depths of < 12+ meters. {Biomphalaria from around lakes in Rwanda, Cameroon and Chad are ecophenotypes of B. pfeifferi rather than members of the choanomphala group.) The alexandrina group contains two species: B. alexandrina, from the Nile Delta southwards to Lake Nasser and extending into Libya, and B. angulosa, which has a discontinuous distribution in mountain areas from southern Tanzania to South Africa. Brown (24) does not recognize B. a. wansoni from northern Zaire. Adults have 5+ whorls and grow to 15 mm. B. alexandrina is the principal intermediate host of S. mansoni throughout its range and naturally infected B. angulosa have been found in Malawi. At least 15 Biomphalaria spp. have been described in the Americas but, although molecular techniques may clarify the situation (231), there is as yet no subclassification equivalent to that of the African Biomphalaria spp. The most important species is B. glabrata, which occurs on the mainland in Brazil, Venezuela, French Guiana and Surinam, and the Caribbean islands (Dominica, Guadaloupe, Hispaniola, Martinique, Puerto Rico and St Lucia). It was deliberately eradicated from Martinique and Guadaloupe (166) and


55

seems to have disappeared spontaneously from Antigua, Aruba and St Kitts in response to natural and man-made ecological changes. B. straminea has a much wider distribution, from as far south as Argentina and Paraguay, eastwards to Chile and Peru and northwards through the Central American isthmus and across the Caribbean island chain. Another species, B. obstructa, extends into the southern United States. Following agriculturally induced ecological changes in Brazil, B. glabrata has been replaced by B. straminea in the northeast and by B. tenogophila further south. Of these species, B. glabrata is the most widespread and efficient intermediate host of S. mansoni, with high infection rates and heavy and prolonged cercarial production when exposed to compatible strains. B. straminea, originally believed to be refractory to S. mansoni, was considered as a potential biological control agent but susceptible strains have since been discovered in northeastern Brazil which, though less efficient than B. glabrata, are able to sustain transmission in the field (9). Similarly, Brazilian B. tenogophila maintains S. mansoni transmission in the field: populations in northern Argentina are at least partially susceptible to S. mansoni (21). B. obstructa, like other New World species such as B. amazonica, B. chilensis, B. helophila and B. sericea, is susceptible to laboratory infections with one or more S. mansoni strains, although natural infections of these species have yet to be reported. Because B. glabrata is so easily maintained, colonies have been established in research laboratories worldwide to maintain S. mansoni. This poses no problem in temperate countries, but there is now irrefutable evidence that it has escaped from laboratory colonies in Egypt and is spreading rapidly in field habitats of the Nile Delta where, though less susceptible than the native B. alexandrina to the local S. mansoni, it poses a serious problem for the local authorities because of its greater size and cercarial production (251). Other South American Biomphalaria spp. have been spread accidentally, probably on ornamental water plants: B. straminea is now firmly established in the field around Hong Kong in China (247).

Taxonomy of Bulinus spp. The genus Bulinus occurs in Africa, on nearby islands in the Indian and Atlantic Oceans, and in adjacent regions of the Mediterranean and the Middle East. Amongst its members are the principal hosts of the terminal-spined

56

Schistosomiasis

schistosomes, including S. haematobium. All bulinid shells are sinistral with 4 - 7 whorls. Size is measured as shell height. Shell color, like that of Biomphalaria spp., is often obscured by a gray or black coating and varies from white to dark brown. The shell surface may be smooth or sculptured, with transversely arranged growth lines, sometimes developed into pronounced ribs. A microsculpture of pits and nodules is often present on the (oldest) apical whorl, although it may be difficult to see on old specimens due to erosion. All bulinids are hermaphrodite but cross fertilization is normal except in aphallic populations. Bulinids inhabit a very wide range of habitats and some species show special adaptations to extreme conditions. They now include over 30 species, most of which were divided by Mandahl-Barth (127) into 4 species groups based on shell and radula characters, some features of the urogenital system, and, to a lesser extent, on their susceptibility to schistosome infection. As he predicted, new material and taxonomic methods have modified the exact composition of these groups, but they remain a convenient framework for considering this genus, as modified by Brown (24). All members of the africanus group have a ventral kidney ridge, a truncate columellar margin, sometimes associated with apertural lamella, and may have a spiral sculpture of small nodules or punctations on the upper whorls in addition to the delicate punctation on the embryonic whorls of all bulinid snails. The normal chromosome number is n = 18. The group is now not normally accorded the subgeneric status of Bulinus (Physopsis). Its members grow to 15-20 mm or more and are globose or ovate in spired forms. As a group, it provides the main intermediate hosts of S. haematobium south of the Sahara where B. globosus is present almost universally. B. africanus is widely distributed in east, central and southern Africa. Other species with more restricted ranges on the African mainland include B. abyssinicus from Ethiopia and Somalia; B. ugandae from Lake Victoria northwards to Ethiopia and Sudan; B. hightoni in northeastern Kenya; B. nasutus throughout east Africa; B. obtusus from Chad; and B. umbillicatus and B. jousseaumei from north of the equator in west Africa. The group is represented on Madagascar by B. obtusispira. Six of these species have been found naturally infected with S. haematobium and at least three are naturally or experimentally susceptible to S. bovis, S. mattheei, S. intercalatum or S. leiperi. Of the species not naturally infected with S. haematobium, B. hightoni has been infected experimentally and B. ugandae is experimentally susceptible to S. margrebowiei and S. bovis. The status of B. obtusus and B. umbillicatus remains unknown.


57

Members of the forskali(i) group lack a ventral kidney ridge and are normally small (less than 10 m m in height), with slender, high-spired shells. The chromosome number is n = 18. They inhabit a variety of habitats but are especially associated with small, shallow seasonal seepages and pools. The nominate species, B. forskali, occurs throughout most of tropical Africa but the remaining eight species have more restricted distributions. B. scalaris occurs focally in east and central Africa and is replaced by B. canascens in Angola and Zambia, B. crystallinus in Angola and B. senegalensis in west Africa. B. camerunensis is confined to lakes in west Cameroon. The group is represented by B. bavayi on Madagascar, by B. cernicus on Mauritius and by B. beccarii in the south of the Arabian peninsula. B. beccarii, B. cernicus and B. senegalensis transmit local strains of S. haematobium naturally and B. bavayi and B. crystallinus have been infected experimentally. The role of B. forskali is less certain: it is likely that infected B. senegalensis from the Gambia were misidentified as B. forskali. Its presence in endemic S. haematobium areas in the absence of other bulinid species suggests that it is an intermediate host despite numerous — with one exception (196) — unsuccessful attempts to infect it experimentally. However, B. forskali, like B. camerunensis, B. cernicus, B. crystallinus and B. scalaris, is susceptible to S. intercalatum and S. bovis. Apart from B. canascens, all members of the forskali group are experimentally susceptible to one or more nonhuman schistosomes and three species have been found naturally infected. The reticulatus group has only two members: B. reticulatus, from MandahlBarth's original forskali group and B. ivrighti, described some years later. Both are small, globose snails < 10 m m high, lacking a ventral kidney ridge and with a chromosome number of n = 18. They inhabit small, often ephemeral, habitats. B. reticulatus occurs focally in eastern Africa from Ethiopia to South Africa and is experimentally susceptible to S. haematobium and S. bovis. B. wrighti is a natural S. haematobium host in southern Arabia and is experimentally compatible with most terminal-spined schistosomes. Mandahl-Barth (127) recognized two other bulinid groups, both lacking a ventral kidney ridge. The tetraploid truncatus group (2n = 72) occurs throughout Africa and extends through Asia eastwards to Iran and included important hosts of S. haematobium north of the Sahara. The diploid tropicus group (2n = 36) did not transmit schistosomes and was confined to Africa south of the Sahara. Later studies showed some members of the truncatus group from south of the Sahara to be susceptible to S. haematobium, though

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not usually to local strains except in west Africa and, possibly, Madagascar. Likewise, some members of the tropicus group were experimentally susceptible to north African S. haematobium strains. Members of both groups, especially species in which polyploidy had occurred, were later found to be naturally and experimentally susceptible to other terminal-spined schistosomes. Interestingly, natural infections of one of the tropicus group occurred only in the presence of other trematodes (195). This blurring of the original distinction based on susceptibility to schistosomes, and other morphological and biochemical evidence of intermediate forms, led to the groups being combined as a truncatus/tropicus species complex in which polyploidy has evolved more than once. B. truncatus is the most important host of S. haematobium north of the Sahara and in Arabia but is less important south of the Sahara, where its range extends south to Malawi in the east and to Mauritania to the west. Other species naturally infected include the related B. rohlfsi, involved in the epidemic of urinary schistosomiasis following the completion of the Volta Dam in the late 1960s, in several west African countries, especially Ghana; the poorly studied B. guernei from the Gambia, Senegal and Mali; and, possibly, B. liratus on Madagascar. The remaining species include B. tropicus in east Africa from Ethiopia to South Africa; B. natalensis from eastern and southern Africa; B. coulboisi from in and around the African Rift Valley in east Africa; B. permembanaceus from Kenya; B. depressus from Central and Southern Africa; and B. angolensis from southwest Africa. Species from the great lakes include B. succinoides and some deep water species: B. nyassanus from Lake Nyassa; and B. transversalis and B. trigonis from Lake Victoria. Other species favor mountainous regions: B. hexaploidus from Ethiopia and B. octoploidus from Ethiopia and Aden. Despite being experimentally susceptible, these species are probably unimportant in field transmission of S. haematobium, but B. truncatus transmits S. bovis naturally over much of its range, B. tropicus transmits S. mattheei in Southern Africa and both, like S. octoploidus, are experimentally susceptible to S. margrebowiei.

Ecology and Control of Intermediate Hosts Introduction Water must be present continuously in sites for 1-2 months or more to establish breeding snail colonies and allow the completion of the entire


59

schistosome intramolluscan cycle, but infected immigrant snails can transform the most ephemeral habitat into a transmission site overnight. Water bodies within a single drainage basin are usually interconnected, although they may become isolated temporarily during droughts. Excessive rains dislodge snails from exposed sites but disperse them passively to downstream sites if they survive mechanical damage due to excessive turbulence over rocky substrates. Snail numbers typically fluctuate in time and space. Snail populations simply cling on during (often extended) unfavorable periods of the seasonal cycle until the return of more favorable conditions when they expand, sometimes explosively. Conditions will vary seasonally within a year and from year to year so that the necessary snail-parasite interactions for transmission are in a state of continuous flux. Consequently, field studies are needed for at least one seasonal cycle, preferably several, to obtain a complete picture of the snail population behavior and its role in schistosome transmission in an area.

Snail Population

Biology and Control

The behavior of snail populations is determined by the interaction between the birth and death rates. These two processes are summarized by r, the intrinsic rate of natural increase of a species, derived from the finite rate of population increase, Ro, which integrates age-specific natality and mortality under defined conditions. A second factor is k, the carrying capacity of a defined habitat, which determines the maximum number of snails it can support in terms of available food, space and egg-laying sites. The more stable a habitat, the lower the reproductive capacity needed by the snails to reach and maintain k. In unstable habitats, a greater reproductive capacity is required to reach k during brief periods of conditions optimal for snails. The Oriental prosobranchs, particularly Oncomelania spp., tend to be k strategists, long-lived and with relatively low reproductive rates, inhabiting stable habitats. Physiological changes and adaptive behavior patterns may modify this general principle. Thus, Neotricula spp. evade high river flows during the monsoon floods by seeking refuge beneath, and attaching their eggs to, rocks and stones. Hatching may be delayed until the floods subside. Pulmonates mostly occupy far less stable habitats and tend to be r strategists with very high reproductive outputs early in their lives. Again, adaptive behavioral and physiological changes help them to survive

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Schistosomiasis

extreme conditions, particularly estivation during droughts. Because they are hermaphrodites, a single survivor or immigrant is enough to repopulate a habitat. Their ability to recover rapidly from catastrophic, natural population declines due to climatic extremes, mainly floods or droughts, is characteristic of pulmonate snails. Their resilience to natural catastrophes also allows pulmonate snail populations to survive artificial attempts to reduce their populations. Rendering snail habitats unsuitable for snails might seem a sustainable control measure but presupposes a full knowledge of the snails' natural requirements. Among the earliest snail control measures attempted in Egypt was the temporary drainage of irrigation canals. Despite high snail kills, canals were quickly repopulated by snails when refilled, with a rapid resumption of transmission. Precautions can sometimes be built in during the construction phase of water development projects (97), but modification is unrealistically expensive for any but the smallest natural habitats. This resilience also affects chemical control measures: mollusciciding rarely eradicates pulmonates and repeated retreatments are needed for prolonged suppression of their numbers because of rapid repopulation by the survivors (often supplemented by immigration). Mollusciciding is best synchronized with natural adverse factors, such as rains, droughts or the onset of hot seasons, although this may not always be easy in practice. Adequate precontrol studies of the snail populations are essential for correct timing and successful transmission control (202-204).

Factors Affecting Snail

Populations

Abiotic Practically any body of water, flowing or still, large or small, natural or man-made, can support freshwater snails because they are "euryok," i.e. tolerant of a wide range of physicochemical conditions. In general, planorbid and bulinid snails thrive in impounded waters or habitats with gentle flows. Shallow habitats are usually preferred, with little turbidity but some organic matter, s u p p o r t i n g submerged, emergent or intrusive vegetation, though not so dense as to prevent the penetration by light necessary for microfloral growth. The amphibious prosobranchs favor shallow habitats on flood plains, in mountain seepages and around seasonal lakes.


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Various abiotic factors have been studied to try to explain the natural distribution of snails and whether their modification might render natural habitats unsuitable. Chemical analyses of water from snail-infested sites have not proved very helpful. Snails need calcium for shell production, and electrolytes for various biochemical and physiological processes, but they tolerate an extremely wide range of values in the field. Snail faunas tend to be poor in the soft waters of eroded granite shields in Africa with very low Ca and Mg values and ratios, abnormal C 0 3 and HCO3 ratios, low dissolved solids and conductivities < 20 mmho and < 150 mg.l - 1 . Conversely, although snails can survive high NaCl levels briefly (conductivities of 2000+ mmho equivalent to seawater), prolonged exposure to seawater is harmful. Biomphalaria spp. are rarely found in saline, estuarine conditions in west Africa. Low (acid) p H conditions are directly harmful to snail shells and coagulate mucus on exposed body surfaces. The paucity of snails in tropical forest regions of South America has been attributed to high tannic acid levels associated with decaying leaves. However, aquatic vegetation, diurnal fluctuations in CO2 content and the buffering effects of dissolved ions often provide suitable microhabitats within a larger habitat, hostile to snails, especially where human activities alter the natural conditions. The extensive use of calcium-rich coral sand for road foundations in Surninam neutralized high acidity locally, and permitted B. glabrata to colonize previously unsuitable areas. Because the underlying geology affects the chemistry of the surface water, snail fauna tended to be richer in areas where limestone predominates. Equally important, though, is the effect of the geomorphology of the landscape and the physical nature of water bodies. Hard, igneous rocks, resistant to erosion, are associated with fast-flowing streams down the sides of mountains with flows in excess of 30 cm.sec" 1 that dislodge most snails. Rocky sills across soft, easily eroded sedimentary rocks produce snail habitats within rivers by trapping pools of slow-flowing water (5). Substrates within sites range from fine silt or mud through sand, gravel and stones to bare rock, and affect the aquatic vegetation and nutrition available: a film of algae on rock or concrete is ideal for snails. The quality of the underlying rocks also determines the turbidity of the water, usually related to seasonal rainfall, sometimes hundreds of kilometers upstream in large river basins. Seasonal flooding may be beneficial for the productivity of a site by replenishing scarce nutrients (151). However,

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Schistosomiasis

excessively turbid water is harmful to snails. Silt deposits smother and kill snail eggs as well as submerged plants, limiting snail food available — an effect intensified by reduced light for plant growth. High colloidal turbidities do not kill young or adult snails that survive prolonged darkness if food is available, although reported negative phototaxis may be an artifact (see below). Snails transmitting schistosomiasis favor temperatures about 25±2°C. Growth and reproduction drop steadily with declining temperature and eventually cease, but mortality is low and populations remain stable above lethal temperatures of < 5°C. Snail growth accelerates above optimum temperatures, but so, too, does snail mortality as the thermal death point (35-40°C) is approached. Fecundity increases up to ~30°C, but it then declines and egg mortality increases. Laboratory studies often use constant temperatures but some have incorporated cyclic variation (156). These show that snails can survive brief exposures to very high temperatures but that repeated exposures have a cumulative effect, hastening the death of snails. Such experiments mimic conditions found in natural habitats where temperatures are rarely constant or uniform and the cumulative effects of high temperatures are important (4). Convection insures that deeper water is cooler than surface water exposed to direct sunlight, and shade from both aquatic and marginal vegetation limits insolation. Thus, snails can usually avoid extreme heat by moving to cooler microhabitats or deep water. Similarly, they can survive in sites where ice forms at night by moving to deeper water at 4°C. Prolonged exposure to high temperatures as shallow habitats dry out has the greatest adverse effect on snail populations. Bulinid snails generally tolerate higher temperatures rather better than planorbids; prosobranchs favor habitats protected from extremely high temperatures but can estivate during the colder winter months outside the tropics. Water temperatures are more stable than air temperatures, but diurnal variations of ~10°C occur at high altitudes. Seasonal changes occur, even within the tropics, related to the north-south passage of the sun but may be modified by cloud cover in highland areas. Breeding ceases in the subtropics during cooler, "winter" months and during extreme, hot temperatures within the tropics when the sun is overhead. Tropical temperatures are modified by altitude, dropping by 1°C for every 100 m on average: at the same latitude, temperature can be lethal for snails at sea level, ideal at higher altitudes but too cold in mountainous areas. Cloud cover, with or


63

without rainfall, also affects temperatures in snail habitats by reducing insolation. Within the tropics, rainfall is the probably the most important climatic factor affecting snail populations. Abnormal bursts of egg laying have been observed in field sites preceding or coinciding with the start of rainy seasons. It is uncertain whether this is due to snails detecting changes in temperature, nutrient availability or other physicochemical factors as rainfall dilutes salts within habitats. Any of these changes could trigger neurosecretions regulating the seasonality of snail reproduction. Regular rainfall replenishes water supplies within a catchment area but rarely falls evenly throughout the year: even if it does, irregular typhoons, hurricanes, cyclones or storms invariably occur from time to time and subject all water bodies to scouring floods. Seasonal tropical rainfall is due to the north-south movement of the InterTropical Disturbance Zone, but the amount varies from year to year. Again, in exceptionally heavy rainy seasons, floods dramatically alter snail sites, especially lentic (flowing) sites which may take months or years to return to their original state. Conversely, lotic (still water) sites shrink during droughts between rainy seasons and often dry out completely. If habitats stay unfilled in a succession of poor rainy seasons, as in the recent Sahel droughts, even' snails adapted to such conditions will eventually succumb.

Biotic factors The adverse effects of extreme abiotic factors are modified by various biotic factors, including food supplies, vegetation, behavioral and physiological adaptations to survive droughts, and the influence of pathogens, predators and competitors. Pulmonate and prosobranch snails are browsing animals, feeding continuously as they move, using their rasplike radula to loosen and ingest the microflora as well as organic detritus from decaying plants. Many unstable, temporary pools are devoid of higher plants but snails still thrive on the microflora of bacteria, diatoms and both blue and green algae. Correlation of specific algae with the presence of bulinid snails in such pools has proved difficult, but successful laboratory culture of prosobranch snails usually requires a suitable algal species such as the diatom Navicula luzonensis (138). Higher plants are not usually eaten directly: rather, their leaves and stems act as substrates, especially when they die and decay, for the development

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Schistosomiasis

of a microflora suitable for snails (216). Pulmonate snails also absorb nutrients directly through the epidermis, including simple, soluble sugars and amino acids released during the decay of higher vegetation. However, excessive amounts of decaying plant material cause eutrophication of habitats, rendering them unsuitable for snails. Substances toxic to snails may also be released (60). In laboratory culture, pulmonate snails feed on fresh lettuce leaves (possibly more avidly when they start to decompose) and survive well, but they may perform even better if they receive high protein supplements. The frequent association of snails with various higher plant species is usually for reasons other than nutrition. Higher plants are directly important because they provide suitable oviposition sites rich in dissolved oxygen needed by developing eggs. Plants also act indirectly by protecting snails from extreme levels of various abiotic factors (e.g. high temperatures, acid pH values, rapid water flows, desiccation). Today, various man-made substances, such as discarded plastics, serve the same purpose where high h u m a n populations occur around rural irrigation schemes and in periurban slums caused by urban drift. However, contamination with agricultural chemicals and industrial pollution may kill snails directly or indirectly by killing essential plants. Laboratory experiments show decreased survival and fecundity as snail density increases. This crowding effect has been attributed variously to increased competition for limited resources (e.g. essential ions, food, egg laying sites), modified behavior caused by increased "collisions" between snails (98), or substances emitted by snails themselves, including harmful excretory products, such as ammonia (217), or pheromones — substances to control population growth (37). All these factors interact to determine the carrying capacity of laboratory environments, analogous to k in natural habitats. The difference is that conditions rarely remain constant in natural habitats. Stable natural habitats are usually large but snails are distributed unevenly in microhabitats within them. If adequate mixing occurs, resources are effectively infinite and snail products will be continuously diluted: otherwise, substantial local fluctuations will occur. In smaller and, especially, temporary habitats, such fluctuations are normal: variable water volumes as well as biological successions cause the carrying capacity of a particular site to vary seasonally within years and from year to year. One extreme is when a site dries out. Amphibious prosobranchs can withstand brief periods out of water and low temperatures enhance the ability


65

of 0. hupensis to estivate during winter droughts in China. Paradoxically, some of the aquatic pulmonates have even greater powers of estivation. Various Biomphalaria spp. can estivate for several weeks and the the bulinids are even more proficient, especially B. forskali, B. productus, B. reticulatus, B. senegalensis and B. truncatus found in temporary sites that dry out for 2-9 months (228). Eggs are killed by a few hours out of water but snails can survive, especially young, sexually mature adults. They seek refuge from unfavorable conditions under vegetation or in muddy substrates if habitats dry out slowly over several days. They retract within their shells and seal their apertures with layers of mucus (pulmonates) or an operculum (prosobranchs) to minimize water loss and exclude predatory insects. The shell is protected from mechanical damage by apertural lamellae, common in populations exposed to repeated droughts (176). Periodic drying, especially rapid emptying of canals to strand snails out of water, may control snail populations on irrigation schemes unless they are capable of estivation, but the effects will be temporary. Most irrigation systems contain numerous inverted siphons, culverts and diversionary weirs where residual water provides refuges for snails that reinvade canals and drains as they refill (110). Despite a considerable literature on pathogens, predators and competitors of aquatic snails, mainly as potential biological control agents and predominantly from laboratory studies, relatively little is known about how they affect snail populations in the field. Natural, catastrophic snail population declines frequently occur, but it is rarely possible to attribute them to a specific pathogen. Facilities for isolating such pathogens are not usually available, and plausible, alternative explanations are available, usually climatic and associated with floods, droughts or high temperatures. An exception (134) was the microsporidian, Plistophera husseyi, which was so effective that it proved impossible to maintain in the laboratory. Subsequent attempts to isolate similar organisms have enjoyed little success, even though mathematical modelers claim that pathogens will prove to be the most effective biological control agents on theoretical grounds (239). Aquatic snails are parasitized by many nematodes and, especially, trematodes other than schistosomes. Infection undoubtedly affects individual snails adversely, but the fecundity of uninfected snails is such that a population is unlikely to be totally eliminated. Field studies frequently reveal snails shedding other trematode cercariae, sometimes mixed with schistosomes, but they are rarely associated with sudden declines in snail numbers. Since these trematodes

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need snails to maintain their life cycles, it is unlikely that they would cause sufficient mortality to eradicate them. Snails are essentially herbivores, relatively low in aquatic food webs. They form one food source for many different predators, including various crustaceans, insects, fish, amphibia, reptiles, aquatic and wading birds and mammals, especially rodents. However, most predators are omnivorous, eating snails when they are abundant but consuming alternative foods as they become scarce. Absolute dependence on a single prey species would result either in the extinction of the predator if the prey was eliminated, or oscillating predator populations controlled by the fluctuating abundance of the prey. At best, predators will suppress snail populations within habitats, especially artificial reservoirs including farm and fish ponds, but will never eradicate them. Most successful biological control agents so far discovered are other snails, such as Helisoma duryii, competing for the same, limited resources. Again, species effective in artificial laboratory conditions often prove ineffective in the field. The competitors either cannot cope with extreme conditions, particularly droughts and floods, or become segregated in different microhabitats from the target species so that competition is confined to areas suboptimal for both species: the target species persists, albeit in reduced numbers. There are a few exceptions to this situation. The natural displacement of B. glabrata by B. straminea and B. tenogophila in Brazil appeared to be examples of successful competition likely to control schistosomiasis until strains of both species were discovered to be susceptible to S. mansoni (9, 154). Marisa cornuarietis is a large, South American prosobranch that feeds voraciously on higher aquatic plants, accidentally ingesting pulmonate egg masses and juveniles. It acts as a facultative predator, but its primary effect is elimination of plants that normally provide the most favorable microhabitats for the target snail. It is easily dislodged by rapid flows and does not thrive in very high temperatures; nor does it colonize shallow, marginal seepages in which Biomphalaria spp. persist. Its successful use has depended on reseeding natural habitats at regular intervals from special "Marisa farms" (96). Another South American prosobranch, Pomacea glauca, is claimed to control Biomphalaria species in Brazil, but it coexisted with B. glabrata on the West Indian island of St Lucia. The southeast Asian prosobranch, Terebia (Thiara) granifera, was introduced to the Americas, probably accidentally on ornamental pond plants, and began


67

spreading southwards through the Caribbean islands in the 1950s. Despite extensive laboratory experiments, the precise mechanism by which is displaces Biomphalaria spp. remains a mystery although it has remarkable powers of reproduction and dispersion. It is now the most abundant aquatic snail species on many islands where it was introduced to control S. mansoni transmission (166). Pockets of B. glabrata still remain on some islands, but in remote sites unlikely to be involved in S. mansoni transmission (68, 221). It has now been introduced into S. mansoni-endemic areas of Venezuela and Brazil on the South American mainland (104). This development may be viewed with some apprehension, because T. granifera acts as the first intermediate host of the lung fluke, Paragonimus westermani, in southeast Asia. This infection is of no consequence in the Caribbean but is more significant in South America and Africa. The safety implications should be considered carefully before contemplating its introduction to Africa, where, moreover, the closely related African snail Melanoides tuberculata clearly does not control Bulinus or Biomphalaria spp.

Snail-Schistosome Interactions in the Field Susceptibility Schistosome infection rates approaching 100% can be obtained in the laboratory with many isolates of snails exposed to local parasite strains if sufficient miracidia and optimal conditions are used. With fewer miracidia and suboptimal laboratory conditions, the same combinations usually produce lower infection rates. Natural, field infection rates of these combinations may approach < 100% in individual sites, but are invariably < 5-10% when averaged over a number of sites. Several problems affect the precision of these field estimates. First, most studies report only patent infections, detected by forced cercarial shedding so that snails can be returned to sites in longitudinal studies. Prepatent infections are rarely estimated although they can be detected under low power microscopy as secondary sporocysts in field snails. Time-consuming, histological techniques are needed to detect primary sporocysts < 6-8 days old and are rarely attempted in field studies. New tools are being developed which may change this situation. Monoclonal antibodies specific to intramolluscan stages of schistosomes can detect

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infections more than 4 days old (75) and, more recently, PCR techniques to detect schistosome DNA in snails have identified infections less than 24 hours old (76). Secondly, there are problems in sampling field sites. The number of snails from individual sites is often small (< 20), with young snails invariably underrepresented. Therefore, accurate age-prevalence data are hard to obtain and published figures are sparse, although they are important epidemiologically and for testing the calculations of mathematical modelers. Thirdly, not all snail-schistosome combinations are equally compatible. Even using snails and parasites from the same area, some isolates are only partly compatible with their local schistosome (73) and selective breeding can separate resistant and susceptible lines (192). Such studies are difficult with hermaphrodite snails capable of self-fertilization. Understanding the mechanisms of resistance and susceptibility is clearly of interest. Molecular biological investigations may be revealing in this context.

Effect of Infection on Snails Susceptible snails are not visibly affected during miracidial penetration, apart from occasional trivial bleeding, or during the early prepatent period until the maturing primary sporocysts appear as swellings on tentacles or discolored spots on the foot. There may be increased mortality in newly hatched snails, but growth and survival do not otherwise differ from uninfected controls. Snails sexually immature at exposure may continue at a rapid growth rate; that ceases in uninfected control snails when egg laying begins, until cercarial shedding marks the onset of patency. Thereafter, growth slows appreciably. The slow growth of snails sexually mature at infection also declines once infections become patent. Thereafter, the mortality of infected snails usually exceeds that of uninfected controls where cercarial production persists until the death of the snail. Histological studies showed that the secondary sporocysts do not parasitize the ovotestis of infected snails but that this organ progressively degenerates. A simplistic explanation is that the developing parasite intercepts nutrients normally used for egg production. Biochemical observations support this idea: infected snails show signs of progressive starvation (218). However, the picture is undoubtedly more complex and snails must expend energy in

The Schistosomes and Their Intermediate Hosts 69

Figure 11. Communal bathing and laundry in a natural stream in Madagascar. These activities result in prolonged exposure, though soap, if used, may provide some protection. {Courtesy A. Degremont, Swiss Tropical Institute.)

combating infection. This is probably of less importance in "compatible" combinations, in which the parasite has presumably evolved mechanisms to avoid the host's defences, but may assume considerably greater importance in partially compatible and refractory host-parasite combinations (135). Partial compatibility is shown in a number of ways, including susceptibility diminishing with the age of the snails, abnormally long prepatent periods, low infection rates, reduced cercarial production and, in some cases, self-cure when cercarial production ceases and egg laying is resumed. In refractory combinations, miracidia may simply be unable to penetrate the snail although direct and histological observations show that some miracidia do penetrate snails but fail to complete their normal development. Internal defence mechanisms involving both innate and acquired immunity affect the development of the parasite and, eventually, kill it. The immune system of molluscs is gradually being unraveled. Cells such as hemocytes (amebocytes) play a role analogous to macrophages (1, 70). Humoral components include interleukin-like cytokines (71), fibrinogenrelated proteins (2), agglutinins (79) and other plasma factors that interact

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with the surface of the intramolluscan stages (46), possibly via glycan-binding lectins specific for schistosomes (128). Endocrine changes associated with sexual maturity (13) may affect ecdysteroids which occur in schistosome sporozoites (172,186). Tropomyosin epitopes shared by trematodes and snails indicate convergent evolution in the immune relationships of schistosomes and their intermediate hosts (238). The effects of stress on some immune responses have been studied (49).

Infection and Distribution

of Infected Snails in the Field

Natural snail infections require contamination of snail habitats with eggs from h u m a n excrement to provide miracidia to infect snails. In comparison with extensive water contact studies on human exposure (66, 111), surprisingly little is known about this aspect of transmission. Direct observational studies pose obvious difficulties. Indirect measurement of fecal coliform contamination has yielded little information of value. Miracidial behavior maximizes the chance of encountering snails. Sentinel snail exposures have provided qualitative, or at most semiquantitative, indices of the presence of miracidia (205), but the technique is time-consuming and not widely used. Directly sampling the natural snail populations is as informative and easier. Physicochemical factors affect the miracidium-snail interaction. Extreme values — high or low temperatures, acid or alkaline p H , high salinity, excessive turbidity and high water flows, alone or interacting with each other — diminish infection rates. However, conditions totally inimical to infection are unlikely to obtain throughout a natural field site; suitable microhabitats, probably those most likely to used by the target snails, will invariably be present for infection to occur. Droughts halt transmission by killing eggs in excrement and prevent the release of miracidia, although viable eggs may persist for a week or more in feces protected from intense heat and desiccation (223). Estivation causes higher mortalities among snails weakened by patent infections than among uninfected snails, but snails with prepatent infections have survived weeks or months to start shedding cercariae shortly after sites refilled. An equivalent pause in the maturation of infections has been reported during cool, winter conditions in South Africa (164).


Figure 12. Prolonged exposure of adults and children during fishing in Mali. (Courtesy P. Flint.)

Longitudinal field studies in which a number of sites are sampled repeatedly invariably show that, just like the snail population, patent infections detected by cercarial shedding are distributed unevenly in space and time. The critical factor at a particular field site is the infection pressure, which depends on human behavior. Very high human contact often correlates positively with a high likelihood of finding infected snails, but they are frequently rare or absent in many apparently suitable sites. In part, this situation is explicable by the failure to detect prepatent infections, especially in juvenile snails underrepresented in the collections, increased mortality in snails with patent infections and, possibly, the occurrence of self-cures. For more remote sites with below normal human exposure, intermittent contamination may be added to this list. Consequently, field snail prevalence data averaged over time and space is invariably lower than that seen in individual sites. This aggregated distribution, typical of many aspects of schistosomiasis and beneficial in maintaining the transmission cycle (249), makes it difficult to focus snail control measures on transmission sites without extensive precontrol studies.

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Am. J. Trop. Med. Hyg. 36: 345. 247. Woodruff DS, Mulvey M and Yipp MW (1985). Bull. WHO 63: 621. 248. Woodruff DS, Staub KC, Upatham ES, Viyanant V and Yuan HC (1988). Malacologia 29: 347. 249. Woolhouse ME, Hasibeder G and Chandiwana SK (1996). Trop. Med. Int. Health 1: 456. 250. Wright WH (1973). In Epidemiology and Control of Schistosomiasis (Bilharziasis), ed. Ansari N (S. Karger, Basel), pp. 32-249. 251. Yousif F, Ibrahim A, Kader AA and El-Bardicy S (1998). In The Final SRP International Conference on Schistosomiasis, March 1998. Ministry of Health, Cairo, p . 79. 252. Zhou D, Li YS and Yang X (1994). World Health Forum 15: 387. 253. Pitchford RJ and Du Toit JF (1976). Ann. Trop. Med. Parasitol. 70: 181. 254. Aloe L and Fiore M (1998). Neurosci. Lett. 228: 65.

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Chapter 3 The Structure and Expression of the Schistosome Genome Gloria R Franco a n d A n d r e w JG S i m p s o n

Introduction Schistosomes are dioecious digenetic trematodes the adult worms of which reproduce sexually in vertebrate hosts and present sexual dimorphism. The larval stages, on the other hand, reproduce asexually inside distinct molluscan hosts and are not sexually differentiated. The complexity of the life cycle of schistosomes is reflected in the structure of their genome. Thus, these organisms constitute good models for studies of differentiation and development. Nevertheless, little information concerning their genetic constitution is currently available and much of what is known comes from studies of candidate genes for vaccines, genes expressed differentially in females or genes involved in sexual maturation. The Gene Discovery Programs initiated this decade for Schistosoma mansoni and Schistosoma japonicum have significantly increased the information available concerning the expressed genes of these parasites and will aid understanding of fundamental aspects of schistosome physiology and development. Concomitantly, the cloning and sequencing of selected schistosome genes or genome regions, together with the use of powerful tools, such as random amplification of polymorphic DNA (RAPD), are promoting, along with classical approaches, advances in phylogenetic studies. These will contribute to characterization of molecular variations and understanding of the parasite evolution and distribution. Also of great importance are the test proteins expressed from cloned recombinant genes that are being analyzed for their potential to induce protective immunity. These studies are well advanced and represent a major hope for

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the production of a schistosome vaccine that could conceivably be available within ten years. Schistosoma, the only genus of the family Schistosomatidae that infects humans, comprises 19 species divided into three groups according to traditional criteria such as the morphology of the egg, the genus of the intermediate molluscan host and geographical distribution. The groups are the African species group S. mansoni (eggs with a lateral spine), the S. haematobium species group (eggs with a terminal spine) and the Asian species group S. japonicum (eggs with a minute spine or without a spine). A fourth group, the S. indicum species group, comprising species from India and Southeast Asia, can be considered distinct, since it does not contain any known species infecting man (reviewed by Johnston et al. (1) and McManus and Hope (2)). Most of the information concerning the schistosome genome has been obtained from studies with S. mansoni, since it is the species of greater global distribution and most easily maintained in the laboratory. This chapter will be mostly restricted to data obtained from this species.

Schistosome Genome Organization African and Asian schistosomes are diploid organisms and their DNA content is distributed into seven pairs of autosomes and one pair of heteromorphic sex chromosomes. The homogametic gender is the male (ZZ) and the female is heterogametic (ZW). The chromosomes are divided into three groups according to size: two large pairs, including the sex chromosomes (subtelocentric for African species and metacentric or submetacentric for Asian schistosomes), three middle-sized pairs (subtelocentric) and three small pairs (submetacentric or metacentric). The definition of karyotypes is possible using chromosome m o r p h o l o g y and C-banding patterns, which identifies heterochromatic regions in metaphasic chromosomes. The major differences between Asian and African schistosomes are located in the sex chromosomes: in Asian schistosomes, the W chromosome is much smaller than the Z chromosome and the heterochromatic block of the W chromosome in S. japonicum is also smaller than that of African schistosomes. Additionally, the C-banding pattern of the W chromosomes varies considerably among some African schistosomes (3-6). The size of the schistosome haploid genome is estimated to be 2.7 x 108 base pairs, about a tenth of the human genome. The genome consists of 60%

The Structure and Expression of the Schistosome Genome

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highly and moderately repetitive DNA and only 30% single copy sequences (7). Measurements of thermal desnaturation and buoyant density in cesium chloride gradients of S. mansoni, S. haematobium and S. japonicum DNA indicate that the average content of A + T is high (66%) (8). Based on the size of the schistosome genome and their evolutive position (9), it has been suggested that these organisms contain 15,000-20,000 expressed genes. However, very little of this information is currently available and the number of genes completely or partially sequenced (as opposed to ESTs) and deposited in GenBank amounts to just over 180 for S. mansoni and only 40 for S. japonicum (GenBank, release 106 of April 30, 1998). Meadows and Simpson (10) published the first paper reporting the analysis of codon usage in schistosomes. They used a total of 19 partial or complete gene sequences of S. mansoni, S. japonicum and S. haematobium. This first analysis was followed by one by Wada et al. (11), based on 21 genes of S. mansoni. More recently Milhon and Tracy (12) used a total of 93 unique S. mansoni sequences and 21 unique S. japonicum sequences to generate species-specific tables of codon usage. The authors confirmed the (A + T)rich nature of the open reading frames (ORFs) in schistosomes, which is 62.8% for S. mansoni and 58.4% for S. japonicum. In the latter study the authors also demonstrated that there was a bias for a purine in the first position of codons and a strong bias for A / T in the third codon position for both S. mansoni and S. japonicum. The existence of bias in the codon usage was also verified by Ellis and Morrison (13), when examining 20 genes differentially expressed in S. mansoni. Of these 20 genes, four were significantly biased, but the rest showed low levels of bias. The authors showed that the magnitude of the bias was dependent on the overall base composition of each gene and on the pattern of usage of synonymous codons, but was not related to the level of gene expression of the genes analyzed. Interestingly, all overrepresented codons contained A or T in the third codon position and most of the underrepresented codons contained G or C in this position. These findings support the proposal that the genome of S. mansoni, similar to other eukaryotic organisms, is compartmentalized. This means that the genome is a mosaic of long segments of DNA the composition of which is homogeneous — the so-called isocores. Thus, variations in the GC content of distinct isocores are reflected in the content of the GC content of the genes that comprise the isocores (14). Musto and his collaborators, in a series of papers, reached the same conclusion. When analyzing the CpG

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avoidance in translated regions of S. mansoni genes, they showed that the CpG frequency is correlated with the percentage of GC of the region containing the gene (15). Subsequently, they investigated several compositional correlations among GC levels of exons and of the third codon positions against flanking regions; correlations between dinucleotide frequencies of exons against flanking regions and GC levels of 5' against 3' regions. The correlations found strengthened the hypothesis that the genome of schistosomes is composed of isocores (16). Finally, and more recently, these authors extended their study to the analysis of 88 distinct protein coding genes from S. mansoni obtained from GenBank. The same compositional correlations were investigated and the authors proposed that the compartmentalized structure of the genome might be one of the most important factors shaping codon usage in schistosomes (17).

The Mitochondrial Genome The schistosome mitochondrial genome has not been characterized in detail as yet, but shows a high level of polymorphism, a characteristic that favors its use as a marker for phylogenetic studies (18-20). In strains of S. mansoni, there is significant length polymorphism with mtDNA sizes varying from 16,500 bp to 24,900 bp. This variation occurs in a single region of the molecule, which ranges from 1500 bp to nearly 10,000 bp and most probably consists of the control region containing the origin of mtDNA replication (20). A more recent article reports that the variable region of the mtDNA of S. mansoni has a complex minisatellite composed of two repeated sequences: a large invariable region of 558 b p and a small variable tandem repeat of 62 bp. Polymerase chain reaction (PCR) amplification of this region, using primers designed from the small repeats, generates a ladder that can distinguish different S. mansoni cercarial clones from the same strain (21). Amplification of this minisatellite under low stringency conditions (LSPCR) proved to be an efficient method of detecting S. mansoni infection in Biomphalaria glabrata snails (22). Interestingly, this minisatellite has partial homology with the nuclear gene SM750, in which the small 62-bp unit is repeated five times. This 62-bp unit has been termed the polymorphic repeated element (PRE). The PRE is apparently common in numerous transcripts of S. mansoni, as suggested by the hybridization pattern in Northern


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blotting analyses (23). These findings led Pena et al. (21) to suggest the existence of an unusual DNA transfer from the nucleus to the mitochondria. Half of the mtDNA molecule of S. mansoni has been cloned (fragments of 2800 bp and 5400 bp). The position of six genes (cytochrome b, NADH dehydrogenase 5 and 6, cytochrome oxidase I and the genes encoding the 12S and 16S ribosomal subunits), as localized on the mtDNA, by cross-hybridization with Xenopus laevis probes, suggests that the organization of the mitochondrial genome of schistosomes is peculiar and is different from those of the nematodes, echinoderms, arthropods and vertebrates (20). The first report on the sequencing of part of the mitochondrial DNA of schistosomes was published by Despres et al. (19). They used the data from the sequencing of a 290 nt region from the 3' terminus of the 16S rDNA of six species of schistosomes and the partial sequencing of the nuclear ribosomal gene (internal transcribed spacer 2-ITS2) for molecular phylogenetic studies. The phylogenetic trees obtained support the classical separation of schistosomes into three groups. From this analysis, it was proposed that a recent radiation of schistosomes is linked to hominid evolution and that the capture of the parasite by man took place in Africa between 1 and 10 million years ago. It was also proposed that the introduction of S. mansoni into America is very recent, perhaps as a consequence of the slave trade from different parts of Africa, which began 400 years ago (19). The assumption of the recent introduction of S. mansoni into America was also supported by phylogenetic analysis using mtDNA restriction fragment length polymorphism (RFLP) in six strains of the parasite (20). Other evolutionary studies have been undertaken through the sequencing of the mitochondrial gene encoding the enzyme cytochrome c oxidase I (COI) from differen species and strains of schistosome (2). In this study, the COI region of the mtDNA was amplified by PCR using degenerated primers designed from evolutionary conserved regions of Fasciola hepatica COI. It was demonstrated that there are substantial differences among S. mansoni, S. haematobium and S. japonicum and between S. japonicum and S. mekongi, but no differences in the COI sequences among strains of S. japonicum.

Repetitive DNA Sequences Early studies of kinetics of DNA reassociation showed that a great proportion of the schistosome genome is composed of highly repetitive

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(> 1000 copies) and middle repetitive DNA (-100 copies). One of the repeated sequences w a s cloned after restriction analysis of the g e n o m e and characterized as an arrangement of tandemly repeated sequences encoding part of the ribosomal RNA (rRNA) gene (7). The entire rRNA gene complex was further cloned and described in detail. It was shown that each repeated unit is 10 kb long and is present in approximately 100 copies per haploid genome. Each unit codes for the three conserved species of eukaryotic rRNA, the 18S (small subunit), 5.8S and 28S (large subunit). The coding region is alternated by spacer DNA (NTS, nontranscribed spacer; ETS external transcribed spacer; ITS internal transcribed spacer), which is less conserved than the coding regions (2, 24, 25). In S. mansoni, the rDNA repeat was localized to the secondary constriction of the short arm of chromosome 3 by in situ hybridization (26). The rDNA gene family is polymorphic, and about 10% of the repetitive units show size heterogeneity (24). This peculiarity, together with the abundance and degree of conservation of the rDNA gene complex, has led to the use of this region as a probe for hybridization studies and RFLP analysis in an attempt to discriminate among several species and strains (27-30), to detect inter- and intravariation in field isolates and laboratory strains (31) and to demonstrate genetic variation associated with drug resistance (32, 33). Sequencing of the ribosomal RNA gene complex has provided the most valuable information for phylogenetic analysis (extensively reviewed by Johnston et al. (1) and Rollinson et al. (34)). Since distinct regions of this gene complex show different rates of evolution (coding regions are more conserved than spacer regions, for example), sequences derived from the different parts of the gene can be used to examine relationships at different phylogenetic levels. The first report on the complete sequencing of the 18S rRNA gene of S. mansoni and prediction of the secondary structure of its product was by Ali et al. (9). Subsequent and more complete articles, using the S. mansoni sequence and sequences of 18S rRNA genes of the three other schistosome species groups, were able to show that S. spindale (a representative of the S. indicum species group) is a sister taxon of S. haematobium (i.e. it clusters with African schistosomes), and that the S. japonicum group is distantly related to the other three species groups and shows greater intravariability (35, 36). A partial sequence of the S. mansoni 28S rRNA had also been obtained earlier (37) and, more recently, Littlewood and Johnston (38) and Barker and Blair (39) have published partial sequences of 28S rRNA of other schistosomes

The Structure and Expression of the Schistosome Genome 91

and members of other families of blood flukes. The sequences derived from the 28S rRNA genes confirmed the grouping obtained when using 18S rRNA gene data. Also of interest is the report of a gap region in the schistosome 28S rRNA gene that, after posttranscriptional processing, divides the rRNA into a and p fragments (40). This gap region has the same length in S. mansoni and S. haematobium, but it is 10-13 nt longer in S. japonicum (30). The most divergent region of the rRNA gene complex is the internal transcribed spacer (ITS1,5.8S rRNA and ITS2). Several groups have reported the sequencing of ITS1 and ITS2 from various species of Schistosoma and used the generated data for phylogenetic analysis. Again, all studies support the traditional arrangement of schistosome taxa including the separation of Asian and African species. They also suggest that S. hippopotami is a sister taxon of other African schistosomes and that S. mekongi and S. malayensis (Asian species) are more related to each other than to S. japonicum (19, 4 1 46). The current status on the sequencing of different regions of the rRNA gene complex of schistosome species is shown in Fig. 1 (adapted from (34)).

ITS1 ITS2 NTS

1

18S 1 5 . 8 s i

28S

S. mansoni S. rodhaini S. hippopotami S. haematobium S. mattheei

f

S. margrebowiei S. bovis S. curassoni S. intercalatum S. japonicum S. malayensis

•

S. mekongi S. spindale

•^•—

^™"^™

Figure 1. Schistosoma rDNA sequences. Blocks indicate the existence of the sequence (full or partial) for the indicated region of the rRNA gene complex. Data source: (34) and GenBank (release 106 of April 30, 1998).

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Several other repetitive DNA sequences have been described in S. mansoni. Spotila et al. (47, 48) reported a family of short repetitive elements sharing some characteristics with the h u m a n Alu family of short interspersed reiterated sequences (SINES). These repeats can be classified as a class I transposable element, present in 7000-10,000 copies per haploid genome. This family, named smoc, comprises members dispersed on different chromosomes, ranging in size from 107 to 322 bp, and members forming tandem arrangements in the genome (331 and 335 bp), some of them localized on the euchromatic region within the heterochromatic block of the short arm of the W chromosome (26). Other members of the class I transposable elements are the retrotransposons, which are much longer middle-repetitive elements that contain coding regions for reverse transcriptase (RT) and virus-like proteins gag and pol (49). Recently, in S. mansoni, a member of the nonlong-terminal-repeat (non-LTR) retrotransposon family or long interspersed sequences (LINES) has been described. This retrotransposon, named SRI, is present in more than 200 copies and contains an open reading frame (ORF) coding for a reverse transcriptase homologous to the chicken repeat 1 (50). Other types of tandem repeats have been observed in S. mansoni. One of them constitutes at least 12% of the genome and consists of 121-bp repeats present in male and female worms. It has a species specificity which has been used to differentiate S. mansoni from S. haematobium and S. magrebowiei (51). Repetitive units of peculiar structure have been found as constituents of mRNA, present not only in untranslated but also in translated regions of transcripts (23, 52). One of them has previously been discussed (see the subsection "The Mitochondrial Genome," p. 88) and consists of the polymorphic repeated element (PRE), a 62-bp repetition found as a 5-copy tandem array at the 3' end of the SM750 gene transcript. The first PRE unit is part of the single short ORF present in this gene of unknown function (23). Hybridization of a nucleotide probe containing part of the PRE in Northern blots resulted in the recognition of a heterogeneous population of mRNAs. Even though the authors suggested that the PRE is present in many S. mansoni transcripts, no other class, except SM750, has been characterized as yet. However, G. R. Franco (data not published) has observed the existence of cDNAs of several sizes composed solely of different numbers of this repeat, after random selection of clones from an adult worm cDNA library (53). Although it is still not known if these findings have any biological significance,


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it could be that the presence of these transcripts accounted for the hybridization profile reported by Spotila et al. (23). Sex-specific repetitive sequences located on the W chromosome have also been identified and have proven to be very useful for determining the sex of larval stages. Walker et al. (29) isolated a DNA clone consisting of a 400-bp degenerative repeat present in approximately 75 copies in the genome of adult females and developed a dot blot assay for sexing cercariae without the need for DNA isolation. Webster et al. (54), using differential hybridization of female genomic DNA clones with total DNA of female or male worms, isolated a female-specific fragment of 476 b p (named W l ) that consisted of a degenerative repeat present in more than 500 copies in the female genome. The W l repeat is localized in a euchromatic gap region within the heterochromatic block in the chromosome W long arm (89). This W l probe was also used for determining the sex of cercariae by a dot blot assay (54) and further used in the design of primers for the development of a PCR method that would allow sexing single cercariae and miracidia (55). A test for distinguishing the sex of cercariae shed from a single larval infection of B. glabrata was also developed based on low stringency-PCR (LS-PCR) using primers for the W l repeat. With this approach, a series of nonspecific but reproducible bands could be amplified from both male and female genomes, in addition to the femalespecific band, which served as internal controls for the presence of DNA in the preparations (56). Although the W l repeat is considered a S. mansoni sex-specific sequence, Grevelding (57) has amplified it from the genome of both genders of a strain called Liberian. The male W l sequence is 9 1 96% homologous to the female sequence of the Liberian strain. Also, there are 2.3-fold less copies of the W l sequence in the genome of males than in females. The author suggests that the presence of this repeat in male worms could be a consequence of a nonhomologous translocation of parts of the heterochromatic region of the W chromosome to either the Z chromosome or an autosome. Recently a novel methodology of subtractive hybridization based on PCR, representational difference analysis (RDA), was used by Drew and Brindley (58) to isolate female-specific repeated sequences. They were able to isolate two new repetitive elements present as tandem arrays on the W chromosome. One of them showed 76% homology to the members of the sma family, and the other one, of 715 bp, represents a new element which was denominated

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W2. Up to now, it seems clear that all sequences used for sex determination are specific to S. mansoni. The development of sexing methods for other species of schistosomes based on female-specific sequences would be of great importance.

The Structure and Organization of Genes To date, there are not a great number of articles in the literature describing the structure of schistosome genes and gene families, the organization of transcriptional units in the genome or the regulation of gene expression. Most of the schistosome genes exhibit some characteristics found in genes of other eukaryotic organisms, such as the presence of introns and the type and arrangement of their regulatory elements. Since the majority of these studies have been conducted on S. mansoni, this and the next two subsections will describe the organization of genes and their expression in this species. The structure of some S. mansoni genes has been determined. They contain no or few introns, ranging in size from approximately 30 nt to several kilobases, as seen in the gene encoding the integral membrane protein SmIMP25, which possesses an intron larger than 9 kb (59). Promoter elements (TATA box, CAAT box, etc.), donor/acceptor splicing sites consensus and polyadenylation signals are also present in the genes studied (59-75). The existence of introns smaller than 50 bp in some genes (the minimum intron length believed to be required for the accommodation of the splicing machinery) is not novel, since they have previously been described in the nematodes Caenorhabditis elegans (76) and Onchocerca volvulus (77). The splicing mechanism for removing of the small introns remains to be elucidated. The promoter region of some S. mansoni genes has been characterized in detail. They contain czs-regulatory elements to which transcription factors bind. For example, the gene encoding GP22, a membrane glycoprotein, has two binding sites for the SPl factor (78). The 5' flanking region of the gene encoding the heat shock protein of 70 KDa (hsp 70) carries two copies of the heat shock element (HSE), shown to be responsible for activation of heat shock gene transcription in other organisms and three inverted CCAAT motifs (67). Analysis of the 5' flanking region of the gene coding for the detoxifying enzyme Glutathione S-transferase 28 KDa (SmGST28) revealed the presence of various consensus sequences that may act as the target of regulatory proteins, including three CCAAT motifs at positions -145, -126, e - 5 7 (79)


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and two consensus sites for the AP-1 factor at positions -231 e -105 (80). The API factor is known to function as a mediator in the expression of genes associated with growth, differentiation and cellular stress (81). The S. mansoni calcium-binding protein calreticulin is apparently involved in the control of cell proliferation as part of a cell-signaling pathway. The analysis of the 5' region of the calreticulin gene showed that it contained a TATA box, two CCAAT motifs (82-84) and a recognition site for the API factor (85). The gene F10 or pl4, which codes for a major eggshell protein (66, 86), contains at its 3' untranslated region a penta- and hexanucleotide sequence identical to the hormone-regulated element (HRE), which is present in steroid regulated genes (87). A small number of gene families have been described in S. mansoni. Bobek et al. (60) characterized a small gene family containing two intronless homologous genes coding for the eggshell protein precursors (pl4 and p48), which are expressed in vitelline cells of mature females. These genes are arranged in the same orientation in the genome and are 7.5 kb apart. The authors localized regulatory elements such as a multiple TATA box, a CAAAT sequence as well as several elements homologous to cis-regulatory elements found in the silkmoth chorion gene (essential for the expression of this gene in the insect) in the promoter region. The p l 4 and p48 genes were shown to be expressed at the same time, stage and tissue in the parasite (66, 88). Subsequently, the p l 4 gene was localized to the distal portion of the q arm of chromosome 2 and the p48 gene was localized to the proximal portion of the q arm of the same chromosome by in situ hybridization (89). Further gene families include those that code for a series of calcium binding proteins (present in muscles of adult worms), which are clustered in a region of 15 kb of the S. mansoni genome (90) and a family of genes encoding nearly identical major proteins present in eggs and miracidia (p40), which are differentially regulated during the life cycle of the parasite. These proteins show homology with a-crystallins and Drosophila small heat shock proteins (91, 92). There is also a family of small exons (27 bp), tandemly arranged in regions of 3 kb in length (93). Each set of three 27 bp-exons produces an 81 nt repetitive region within the 10-3 antigens that are differentially expressed in the various stages of the life cycle of the parasite. These transcripts are extremely similar to one another and contain variable numbers of the 81 nt repeats. Size differences between individual 10-3 antigens are due mainly to the insertion of new 27 bp-exons at the 5' end of the repeat, to the use

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of alternative transcription initiation sites and to differential splicing of the small exons during development.

Differential Expression of Genes Several genes that are either stage- or sex-specific have been identified in S. mansoni (62, 66, 86, 90, 94-106). Examples are the genes for the eggshell proteins p48 and p l 4 that were found only in mature vitelline cells of egglaying females, but not found in eggs deposited in the vertebrate host liver (66). Eggs are responsible for the major pathology of schistosomiasis, thus the characterization of genes that regulate the reproductive development of the female and subsequent egg production may be of direct clinical relevance. With the aim of shedding some light on the regulation of female development, a very elegant cloning strategy was used for the identification of femalespecific gene transcripts that involved the construction of libraries enriched for such molecules (107). The first female-specific gene that was not expressed in vitelline cells was recently cloned. It codes for an acidic protein, similar to murine, which is thought to protect the epithelium of the reproductive ducts and which might be involved in preventing the premature formation of the eggshell (108). Grevelding et al. (109), studying gene expression in adult females cultured in vitro, were able to show the influence of the male on the transcription of female-specific genes. They demonstrated that while female-specific genes were dependent on pairing for expression, this was not the case for genes expressed in both genders which were not affected by this process. The nature of the stimuli provided by the male schistosome is still unknown. However, Bostic and Strand (110) have cloned a gene encoding a protein present in the gynecophoral canal of male worms, which contains conserved repeated regions sharing some sequence similarity with the neural cell adhesion protein fasciclin I. The expression of this gene is drastically reduced in unpaired males, suggesting its importance in schistosome mating a n d / o r egg production. Almost 200 partially or totally sequenced S. mansoni genes have been deposited in public databases (GenBank release 106 of 30 April 1998). The proteins derived from these sequences have been defined either as antigens or in terms of their cellular function, such as maintenance of metabolism, structure, cell recognition, transport, storage, signaling, nuclear and


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cytoplasmic regulation, energy production. For some of these genes, expression studies in different phases of the life cycle have been undertaken in an attempt to understand the basis for regulation of gene expression and the differences observed in the metabolism of each developmental stage. The energy metabolism in S. mansoni changes within the parasite life cycle. Adult worms and schistosomula have basically a fermentative metabolism, and sporocists are facultative aerobes. On the other hand, the free-living forms, miracidia and cercariae, are aerobic and obtain energy from glycogen breakdown (111). The metabolic differences are related to the environment and are reflected at the level of gene expression in each stage of the life cycle. Adult worms exhibit high expression levels of the glycolytic enzymes such as hexokinase, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase (104). Other highly expressed proteins important for nutrition of the worm are hemoglobinase (112), ferritins, for storage of iron (102), glucose transporters (113) and the G-protein a-subunit, which appears to be involved in the stimulation of glucose metabolism after induction by serotonine (114). It has been shown that the expression of mitochondrial malate dehydrogenase (Krebs cycle) and cytochrome oxydase subunit 1 (electron transport) is higher in the tails of cercariae than in the body or in early transformed schistosomula (104). It has also been observed that the expression of various genes in cercariae is silenced due to a "developmental program," which is related to the constitutive expression of genes only in miracidia, sporocysts and adult worms. One example is the heat shock gene hsp70, which is not expressed in cercariae but is activated in newly transformed schistosomula reaching maximum levels six hours after transformation. It is proposed that the hsp70 gene has a role in adaptation of the parasite to physical stress (i.e. increase in temperature, changes in the salt concentration in the environment and tail loss) (67,115). Conversely there are other genes that are highly expressed only in cercariae, such as the gene encoding the calcium-binding protein (CaBP) which is expressed in cercaria but not in preceding life cycle stages (miracidia and sporocists) or in adult worms (62), and a serine protease (elastase) that facilitates larval penetration through the host skin (116).

Regulation of Gene Expression During its life cycle, the S. mansoni is exposed to different temperatures and environmental conditions. Modifications from one developmental phase

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to another include changes in the size, shape and physiology of the parasite. It is believed that such modifications are, for the most part, the direct reflection of the activation/inactivation of stage-specific genes. Studying stage-specific genes and other genes involved in development and sexual maturation represents a powerful approach to the understanding the biology of the parasite, as discussed above. Studies on gene regulation in other organisms have led to the identification of cz's-regulatory elements in the promoter regions and also to the characterization of DNA-binding proteins which control the levels of gene transcription (117,118). The analyses of regulatory mechanisms in S. mansoni are complicated by the lack of S. mansoni cell lineages and the short life of juvenile worm cells in culture (119). Consequently, little is known about the genetic structure and organization of transcriptional units in S. mansoni. However, as mentioned above, many of the S. mansoni genes characterized so far resemble other eukaryotic genes in terms of the type and arrangement of putative regulatory elements. Only a few genes coding for putative nuclear regulatory proteins of S. mansoni have been cloned. Among them is the SMNF-YA transcription factor that binds to the CCAAT motif present in the promoter region of SmGST 28 gene (120). SMNF-YA exhibits significant similarity to yeast HAP2 and HAP-3, which have been previously characterized as heteromeric factors that bind CCAAT (121). Other transcription factors that bind the motif CCAAT present in various eukaryotic gene promoters are represented by the Y-box binding protein (YBP) family (122). A new member of this family has been cloned from S. mansoni (74). The gene, named SMYB1, codes for a 217 aa protein containing a nucleic acid binding domain (the cold-shock domain) which is 64% identical to the corresponding domain in other YBPs. The protein, when heterologously expressed in bacteria, is able to bind to oligonucleotides consisting of parts of gene promoters of S. mansoni and other organisms that contain the CCAAT motif in different contexts (A. F. Valadao, unpublished). Most information on gene regulation in S. mansoni involves the binding of proteins present in worm extracts, or purified proteins, to regulatory elements in electrophoretic mobility shift assays (EMSA's), DNAse I footprinting assays or UV crosslinking (80, 83,123-127). These techniques have allowed, for example, the demonstration that some nuclear proteins (possibly steroid receptors) might interact with elements present in the F10 gene, suggesting its regulation by such molecules (126). In addition, such studies have resulted


in the demonstration of the binding of heat shock factors (HSFs) present in protein extracts to the HSEI and HSEII regions of the hsp70 gene (125). Recently, a gene encoding the S. mansoni HSF was cloned. It was found to be 12 kb long and to contain ten exons and nine introns (128). This factor has a DNA-binding domain that shares 39-58% similarity to other HSFs and two leucine zipper motifs possibly responsible for oligomerization of HSF. Interestingly, multiple HSFs mRNA species generated by alternative splicing seemed to be expressed in different developmental stages of the parasite. Khalife et al. (83) have demonstrated that the regulatory elements of the S. mansoni calreticulin gene are able to induce the expression of a reporter gene in a heterologous system (Jurkat human T-cells) after binding of human transcription factors. This study was the first to use a S. mansoni regulatory element in a reporter assay. Subsequently, the ability of the hsp70 promoter to direct transcription of a reporter gene in CHO cells after they have been heat-shocked for three hours at 42°C was demonstrated (129). More recently it has been shown that the promoter region of the SmGST28 gene can also promote transcription of a reporter gene in HeLa cells (130). Promoter deletion analyses of the SmGST28 gene showed that the region responsible for directing transcription of a reporter gene in a mammalian cell system lies between nucleotides -278 and +1 (79). As the site for the API is located at position -231, the transcriptional activity has been initially attributed to this site. It was suggested that the correct activity of the parasite gene promoters in the mammalian heterologous systems is due to the similarity in the sequences and in the promoter organization in these organisms. The use of alternative promoters to control the expression of a single copy gene has been reported in S. mansoni (78). The gene GP22, encoding a family of surface membrane glycoproteins, has two candidate promoters (PI and P2). It has been suggested that PI and P2 could be preferentially used at different stages of the parasite's life cycle. Each promoter has two transcription initiation sites that generate mRNAs with different translation start sites. The sizes of the gene products from the PI mRNAs are 182 and 175 aa, while translation from the two P2 mRNAs gives rise to proteins of 140 and 136 aa. All these proteins share a common 136-aa carboxy terminus which contains a membrane-spanning hydrophobic segment, an external domain and a putative leucine zipper motif. The differences in the various proteins are at their amino termini. The larger species have an extensive hydrophobic region linking the common carboxy terminus and the hydrophilic amino terminal end.

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The control of gene expression in eukaryotes is not limited to the transcriptional level. Several controlling mechanisms at the posttranscriptional level exist and include processing and maturation of the primary transcripts. Control can occur at the level of transport of the mature mRNA to the cytoplasm, regulation of the availability of the mRNA to the translational machinery or the mRNA half-life. In addition, posttranslation modifications act as regulators of expression since proper protein folding, maturation, compartmentalization and secretion are essential for perfect functioning. In S. mansoni very few of these mechanisms have been studied. At the posttranscriptional level alternative splicing has been reported. As an example, at least three variant transcripts of the schistosome homolog of the epidermal growth factor receptor (SER) gene are generated in addition to the complete SER transcript. The SER mRNA codes for a protein containing a signal peptide, a cysteine-rich extracellular domain, a hydrophobic transmembrane region and an intracellular tyrosine kinase domain. The variant mRNAs appear to encode either soluble, secreted proteins or a protein containing a hydrophobic C-terminus that may serve as a membrane anchor. The variant transcripts show different levels of expression in adult worms and cercariae (131). A frans-splicing mechanism has also been observed in S. mansoni. Transsplicing is a manner of processing mRNA in which the 5' end (Spliced Leader — SL) of a small nonadenylated RNA is donated to an RNA transcript (pre-mRNA), so that a common 5' exon appears in different mRNAs (132). This mechanism was initially described in Trypanosoma and is believed to be a mechanism of mRNA maturation in this parasite. In the nematode C. elegans, approximately 70% of the mRNAs are generated by frans-splicing (133). In contrast, in S. mansoni, only a relatively small proportion of mRNAs are frans-spliced. This class of S. mansoni transcripts undergo processing with the addition of a 36-nucleotide SL (derived from a nonadenylated 90 nt SL RNA) to their 5' end (134). Analyses of 30 different mRNAs bearing a 5' SL fail to reveal any common characteristic between the genes, mRNAs or the translated proteins. While the mRNA for the S. mansoni glycolytic enzyme enolase is frans-spliced, this mechanism was not shown to occur for any other glycolytic enzymes, indicating that frans-splicing does not prevail in this metabolic pathway. Also, frans-splicing does not seem to be related to specific cell types, tissues or parasite sex (70). In some genes the acceptor frans-splicing site may be not only in the first exon but also in internal exons. This was


observed for the gene coding for the enzyme 3-hydroxy-3-methylglutarylCoA reductase, the gene encoding a homologue of the sinaptobrevin protein and four other genes. The internal exons can be linked to the SL or to the immediately preceding exon by an alternate splicing mechanism (ris-splicing), giving rise to distinct transcripts (134). The reason for the choice of cis- or frans-splicing is not known. Additional studies are required in order to understand better the role of frans-splicing in gene regulation in S. mansoni. A very interesting discovery has recently been made by Davis and Hodgson (133), working with a polycistronic transcript derived from the non-trans-spliced gene encoding the ubiquinol binding protein (UbCRBP) and the trans-spliced gene coding for the glycolytic enzyme enolase. These two genes are very closely linked in the genome. The authors demonstrated for the first time that trans-splicing in S. mansoni may be associated with polycistronic transcripts, possibly acting as a mechanism to produce monocistronic mRNAs. This suggestion is supported by the existence of a similar mechanism in kinetoplastida and some nematode genes in which monocistronic, capped mRNAs are produced from a multicistronic transcript.

The Genetic Diversity of Schistosomes It has been known for a long time that there are variations among natural populations of schistosomes and that even a single naturally infected host can exhibit a variable n u m b e r of parasite genotypes (135). The heterogeneity of schistosome strains and populations can now be measured using a diverse variety of powerful molecular biology techniques, such as RAPD. This approach consists of the PCR amplification of anonymous regions of the genome using arbitrary primers under low stringency conditions (136, 137). Using this technique, Dias Neto et al. (138) and Barral et al. (139) revealed limited but easily detectable polymorphism among different strains of S. mansoni. While Kaukas et al. (140) demonstrated less intraspecific variation in S. mansoni than in S. intercalatum and S. haematobium, Bowles et al. (42) showed identical RADP profiles for two isolates of S. japonicum (Philippine and Chinese), although these were distinct from the S. mekongi profile. In contrast to these findings, analysis by RAPD of field isolates of S. mansoni has clearly demonstrated that this species has a higher genomic variability than expected. Barral et al. (141), verifying 212 individuals from 10 isolates of S. mansoni obtained from the natural host Rattus ratus, demonstrated that

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the hosts are able to harbor multiple parasite genotypes with a maximum of 28 genotypes per host and that this diversity is greater within hosts than it is between them. Also, clones of S. mansoni obtained from field isolates, when compared to strains that had been maintained in the laboratory for long periods, showed higher levels of polymorphism (142). Similar results were obtained through RFLP profiles using part of the rDNA gene complex as a probe, which demonstrated not only differences among the various isolates that could be grouped by geographical origin, but also variation among individuals from the same isolate (131). Genetic diversity among 14 strains of S. mansoni has also been characterized by Southern blotting analysis using the SM750 gene as a probe (143). This gene contains, as described before, five units of the polymorphic element PRE. The probe used allowed the identification of different strains based on the banding profile generated after hybridization. Intrastrain variability was generally low, with the exception of the NMRI strain, but significant levels of interstrain heterogeneity were observed. The authors also suggested a maternal inheritance of this polymorphic marker based on genetic crosses. It may be possible that a minisatellite homologous to SM750 on the mitochondrial genome (21), which is maternally inherited, is accounting for the pattern of inheritance observed. Subsequently, Minchella et al. (144), using the same approach, demonstrated that there is a great variety of parasite genotypes in naturally infected Biomphalaria glabrata snails and that a single snail can be infected by multiple miracidia. It has been suggested that the schistosome genome may suffer dynamic changes during the course of development and that the genome can incorporate host-related sequences. Nara et al. (145) demonstrated, by Southern blotting analysis, the deletion and / o r amplification of DNA sequences in S. mansoni adult worms and miracidia. Based on these results the existence of stage-specific DNA sequences in this parasite was proposed. On the other hand, the acquisition of host DNA sequences has been detected in different stages of S. mansoni and S. japonicum by the use of blot hybridization, PCR and in situ hybridization techniques. These sequences are related to mouse retroviruses and to the H-2 locus of the mouse class I major histocompatibility complex (MHC) (146-150). The existence of stage-specific or host-related DNA sequences in the genome of schistosomes is relevant to an increased plasticity of the schistosome genome as well as to the adaptation of the parasite to different environment conditions and evasion of immune


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attack. However, these observations have been the target of severe criticisms, since contamination with host DNA cannot be ruled out due to technical difficulties (151, 152).

The Schistosome Genome Project The Human Genome Project (HGP) was initiated in the late 80s and even today it is regarded as a very ambitious venture. The primary objective of the HGP is the identification and mapping of all human genes and subsequent sequencing of the three billion base pairs genome. The secondary objectives include the discovery of new diagnostic tools and treatments for genetic diseases as well as the transfer of knowledge to other areas that may ultimately help the development of modern biotechnology in fields of research such as agriculture or animal breeding. As part of the HGP the sequencing of other genomes, mainly of model organisms, was also started in parallel. Some of these model organisms have medical or evolutionary importance and their genomes are usually substantially smaller than that of man. Among the organisms that have been chosen for genome sequencing are the nematode Caernohabditis elegans, the fruit fly Drosophila melanogaster and the weed Arabdopsis thaliana. One of the approaches for gene discovery widely used in genome projects is the partial sequencing of randomly selected cDNAs from a library (153). This strategy has the advantage of selecting only expressed sequences. The sequenced portions obtained after single pass sequencing of cDNAs have been designated expressed sequence tags (ESTs). ESTs can be used for data base searches against either DNA or protein sequences in an attempt to identify the genes from which they are derived. More recently, genome projects of parasites have been initiated with the support of the World Health Organization. The S. mansoni Genome Project started in 1992 as a joint initiative between the laboratories of Dr. S. Pena at the Universidade Federal de Minas Gerais, Dr. A. Simpson then at the Centro de Pesquisas Rene Rachou-FIOCRUZ, in Belo Horizonte, Brazil, and Dr. C. Venter at The Institute for Genomic Research (TIGR) in Gaithersburg, MD, USA. Due to the large size of the S. mansoni genome (270 mb), it was decided that a large scale cDNA sequencing would be most appropriate for analyzing the expressed genome of the parasite. The initial approach chosen was the production of ESTs from an adult

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w o r m directional cDNA library. From this screening, 607 ESTs were obtained corresponding to 169 different genes of which 154 were novel (53). This pioneering work with S. mansoni was of the utmost importance, since it added to the databases a great number of newly identified genes of the parasite (less than 100 genes had been previously deposited in GenBank). In 1994, the S. mansoni Genome Project received additional support from the W H O / U N D P / W o r l d Bank Special Program for Research and Training in Tropical Diseases (TDR) and new laboratories joined this initiative (154). At present, the WHO genome network is co-ordinated by Dr. Phillip LoVerde (State University of New York at Buffalo, USA) and five laboratories are involved in the gene discovery program using the EST strategy: Universidade Federal de Minas Gerais, Brazil; Centro de Pesquisas Rene Rachou-FIOCRUZ, Brazil; Natural History Museum, UK; Theodor Bilharz Research Institute, Egypt; and State University of New York at Buffalo, USA. Since the beginning, the genome network has decided not to adopt a reference strain of S. mansoni as there is no evidence for chromosomal rearrangements among strains. It was also decided to use initially the existing cDNA libraries, available from the research community, instead of producing new ones what would in turn accelerate the generation of data. In the first year of the Schistosoma Genome Network (SGN) hundreds of ESTs were produced from adult worm cDNA libraries and deposited in the EST database — dbEST (http://www.ncbi.nlm.nih.gov/dbEST/index.html). Construction and sequencing of new cDNA libraries from different phases of the parasite life cycle allows comparisons of the gene expression patterns in the various stages. The construction of new libraries was mainly undertaken by the Egyptian group which was also in charge of distributing them to the other laboratories involved in the project. Recently an article describing a comparative study of the gene expression profile in different stages of the parasite life cycle (egg, cercariae, lung stage and adult) based on the EST approach was published (155). In this study the quality of each of seven cDNA libraries was evaluated with a view to subsequent large scale sequencing. It was demonstrated that most of them met the minimum criteria required for their use in programs of this nature. The libraries showed less than 20% of uninformative clones (chimeric clones, vector without inserts, rRNA, mtDNA or contaminant sequences), low rates of redundancy, and had more than 50% novel genes. A clustering analysis of 1123 sequences was performed using the program ICATOOLS (156). Clustering of ESTs is


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of great importance for determining the number of unique genes sequenced, identifying the most expressed genes and verifying the redundancy present in libraries. This analysis demonstrated that a total of 466 unique genes were obtained from the libraries. Of this total, 427 were novel S. mansoni genes, some of which were expressed in a stage-specific manner while others were considered housekeeping genes (155). In an attempt to overcome frequent problems generated by sequencing of cDNAs randomly selected from libraries, such as the frequent sequencing of uninformative clones and the high level of redundancy in the sequencing due to existing bias in the libraries, Dias Neto et al. (157) developed a technique based on the RNA arbitrarily primed PCR (RAP-PCR) to generate normalized cDNA minilibraries ready for use in EST generation. This approach increases the chances of sequencing cDNAs derived from rare transcripts and permits the use of trace amounts of mRNA for the construction of the libraries (1 ng of mRNA can generate approximately 100 ESTs). This is of particular interest, since there are limitations in obtaining sufficient quantities of biological material from some phases of the parasite life cycle, such as lung stage schistosomula or sporocyst. The WHO Schistosoma Genome Initiative also includes a gene discovery program for S. japonicum that has been conducted by collaborative groups in Australia, China and the Philippines (154). Adult worm cDNA libraries from Chinese and Philippine strains have been examined, as well as libraries from egg and miracidium stages (158). To date, 6249 ESTs from S. mansoni and 692 ESTs from S. japonicum are present in dbEST (release 081498 of August 14, 1998). Clustering analyses of 5223 S. mansoni sequences and 724 S. japonicum sequences were performed by D. Johnston (the SGN Secretary) using the Sequencher 3.0 software running on a MacOS system on 4/07/98, and the results can be obtained at the WHO Schistosoma Genome Network WWW site (http://www.nhm.ac.uk/hosted_sites/schisto/). The parameters for the analysis were set to group sequences that displayed more than 90% homology over > 60 bases. This analysis revealed 3216 and 538 unique genes of S. mansoni and S. japonicum, respectively. Taken together these numbers indicate that tags for almost 20% of the schistosome expressed gene content are already available. Some of the genes were obtained only once from the libraries (orphan sequences), but others were very frequent and were isolated several times from the same or from various libraries (sequences assembled in clusters). The overall rate of redundancy was low in the dataset of both

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organisms (38% for S. mansoni and 27% for S. japonicum), indicating that the potential of the libraries for a large scale sequencing program has not been exhausted and that generation of ESTs still remains a very efficient approach to gene discovery. Reviews of the progress of the schistosome genome project are available (34, 154, 159) and general information about the Network can be obtained from the SGN 1996/97/98 Meeting Reports and 98-99 Network Workplan (http://www.nhm.ac.uk/hosted_sites/schisto/). With the recent completion of sequencing of the Saccharomyces cerevisae genome, an international effort aimed at understanding the genome in functional terms has been initiated. So-called functional genomics involves mapping the proteome (the set of polypeptides encoded by the genome) (160) as well as structural and functional characterization of each protein. In the program of gene identification in S. mansoni various novel genes have been isolated, from which several are potential targets for chemotherapy or for the production of vaccines in addition to their importance for deepening our understanding of the biology of the parasite. Functional genomics seems to be the best choice for the characterization of some of these genes. Such work has started in some laboratories of the SGN. Physical mapping of the S. mansoni genome is also included in the genome project. This was initiated in 1992 by the group of Dr. Manami Tanaka in the University of Tsukuba, Tsukuba, Ibaraki, Japan. A YAC library made from cercarial DNA was constructed containing at least 3000 clones bearing inserts of 358 kb on average that covered 2.6 genomic equivalents (161). Markers such as repetitive elements, individual genes and ESTs have been used in the characterization of the library. Several YAC clones were ordered from their localization on metaphase chromosomes using fluorescent in situ hybridization (FISH), generating the first low resolution physical map of S. mansoni. The library is available for other users in the form of high density filters. The group of Dr. Tanaka also constructed a genomic cosmid library and proceeded with the assembly of contigs of chromosome-specific cosmids and YACs, for chromosomes 3, Z and W. The visualization of these contigs by FISH originated a second generation physical map. The significant progress in the genetic mapping of Schistosoma was possible only because of the development and refinement of a few techniques. Hirai and LoVerde (162) have published protocols for chromosome spreads and FISH for S. mansoni. Another technique, primed in situ (PRINS), which allows the localization of specific sequences through PCR, has also been adapted


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for S. mansoni chromosomes (H. Hirai, unpublished). Chromosomes can be isolated through microdissection of spreads and can then be utilized in the construction of chromosome-specific libraries (H. Hirai, unpublished). A modification of the FISH technique was achieved that allowed the use of plasmids, cosmids and YACs as probes; it was termed chromosomal in situ suppression hybridization — CISS (159). The use of CISS has contributed to the construction of higher resolution physical maps. The construction of good quality new libraries containing fragments of at least 100 kb in vectors such as bacterial artificial chromosomes (BAC) (163) is considered to be a high priority. Such libraries are very valuable not only as a source of inserts to be used in the assembly of contigs and in the construction of high resolution physical maps, but also for the generation of genomic sequences using the BAC end sequencing strategy. As in other WHO Genome Projects of Parasites, the construction of a database according to the AceDB model has begun — SchistoDB. This database serve as a repository of all information generated from sequencing of cDNAs, clustering analyses of ESTs and chromosomal mapping of cosmid and YAC clones by FISH as well as information concerning the groups and laboratories of the Schistosome Genome Network. More information about SchistoDB can be obtained at http://www.nhm.ac.uk/hosted_sites/schisto/ informatics/SchistoDB_info.html

Acknowledgments We thank Adlane Vilas Boas Ferreira and Analina Furtado Valadao for critical reading and help in some sections of this chapter.

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Chapter 4 Epidemiology of Schistosomiasis: Determinants of Transmission of Infection Charles H Bang

Introduction Within endemic populations, schistosomiasis is a chronic helminthic infection that is widely dispersed. Distinct from short-term infections caused by bacteria and viruses, this helminthic "macroparasite" establishes a longterm, multi-year association with its h u m a n host (1). Schistosomiasisassociated disease is then a function of both intensity and duration of infection as well as a variety of host responses (2, 3). The dynamics of transmission and host-parasite interaction are significantly different for helminths — it is the epidemiological features of parasite transmission that provide us with a key to understanding much of the natural history of human schistosomiasis. In any given region, one or a number of features of the schistosome life cycle serve to regulate its transmission potential. These features include: (1) Human excretory habits; that is, the likelihood that an egg expelled in feces or urine will reach fresh water to hatch a miracidium infectious for snails. (2) The availability of a suitable snail intermediate host in local bodies of fresh water. (3) The adequacy of rainfall to maintain water levels sufficient for cercarial release and snail reproduction. (4) The presence or absence of antischistosomal resistance in the snail host. (5) Human water use habits leading to cercarial exposure. (6) The presence or absence of antischistosomal immunity in the human host.

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(7) The availability of a suitable mate for the infecting schistosome within the human host. (8) The availability of sufficient ecological space for survival and reproduction within the human host. (9) The fecundity of the female schistosome. (10) The efficiency of viable egg translocation from the bloodstream to the lumen of the bowel or bladder. While it has been possible to study many of these factors, it is apparent that there can be significant local variation in transmissibility within a given endemic area, as well as significant variation in transmission over time (4). Several aspects of schistosome infection, such as human parasite burden, cannot be directly measured and must be estimated from indirect testing (5, 6). Although average rates of infection can be estimated, day-today variations in egg excretion, rainfall, snail populations and water use add unpredictable stochastic elements to the transmission of infection, such that an individual's likelihood of infection cannot be precisely predicted. This stochastic "noise," along with our inability to directly measure certain components of parasite transmission, has contributed to the continuing controversy regarding which factors have the greatest influence on schistosome transmission (6). There is evidence in favor of each of the factors enumerated above. Ultimately, the pressing operational question is: Which type of intervention (whether drug therapy, immunization, snail control, or provision of safe water and latrines (7)) will provide the greatest disruption of parasite transmission, resulting in the most effective disease prevention?

How Efficient Is Schistosome Transmission? Determination of Prevalence and Intensity of Schistosome Infection In endemic areas, prevalence of active infection is determined by parasitological examination for viable eggs in the urine (for S. haematobium) or stool (for S. mansoni, S. japonicum). Intensity of infection is quantified by counting the number of eggs in a fixed aliquot of urine (via filtration microscopy of 10 ml stirred urine (8)) or stool (Kato smear of 50 mg feces (9)). These egg detection techniques are not completely sensitive, as egg excretion may exhibit diurnal fluctuation (as with S. haematobium (10)) and

Epidemiology of Schistosomiasis: Determinants of Transmission of Infection 117

vary from day to day (11, 12). Estimated sensitivity of S. haematobium egg detection in urine is 55-85% for a single determination, and 90 and > 99% for repeated testing on two and six days, respectively (8). Sensitivity for S. mansoni egg detection in the stool is estimated to be 70-80% for a single exam and > 93% for two or more daily examinations (9). Sensitivity is typically lower for very light infections. Recent serologic surveys of infection prevalence utilizing detection of circulating parasite antigens indicate that standard parasitological testing may also be less sensitive for determining infection among older individuals (13). This may be due to the presence of fibrosis in infected tissues in long-standing schistosomiasis, with an associated impairment in the ability of parasite eggs to reach the visceral lumen (14). Alternatively there may be a decrease in fecundity of older worms, leading to decreased egg excretion per infecting worm pair (13). Seroprevalence studies typically indicate that almost all long-term residents in a schistosome-endemic area are exposed to infection and have developed antiparasite antibodies (6). Unfortunately, antibody levels cannot determine the presence or absence of active infection, nor do they predict the level of infection intensity. Studies of circulating parasite antigens — circulating cathodic antigen (CCA), circulating anodic antigen (CAA), and others — have shown a significant correlation between antigen levels in the blood and the presence and intensity of infection (15). However, the correlation is not sufficiently close (R2 values of 0.16-0.25) to accurately predict infection levels in a given subject.

Transmission of Schistosomiasis in Travelers and Other Nonimmune Populations Under the right circumstances, intense, brief (< 2 weeks), cercarial exposure can result in very high schistosomiasis transmission rates, especially among immunologically naive travelers and immigrants. An average infection rate of 39% was noted among U.S. and European travelers after only brief S. mansoni exposure during white water rafting trips on the Omo River in Ethiopia (16). Other S. mansoni outbreaks among travelers, dating back to 1975, have had attack rates of 55-100% (mean of 77%) when U.S. and European travelers have been exposed to S. mansoni for periods averaging two weeks (17). In all cases, risk of infection was significantly associated

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with fresh water contact (wading, bathing, swimming, washing or boating). Acute disease (Katayama fever) occurred in 76% of those who became infected (17). In a recent investigation of S. haematobium infection among expatriates and tourists who had visited Lake Malawi resorts (a new focus of infection), 33% were positive for antischistosomal antibodies, indicating probable infection (18). The development of seropositivity (up to 55% in some age groups) was associated with length of stay, participation in diving, wind surfing or swimming, and visits to a specific section of the lake (Cape Maclear National Park), although other Lake Malawi resort sites were also implicated (18). These findings indicate that, under optimal conditions, parasite transmission is highly efficient. This impression is supported by data on transmission of schistosomiasis among previously unexposed residents in areas where infection has been newly introduced. As an example, the Richard Toll region of Senegal experienced vary rapid spread of S. mansoni infection following modification of local water conditions by the Diama dam project (19). Within 2.5 years, the prevalence of infection had reached 60% among the residents there (20).

Infection and Reinfection in Endemic Populations Infection transmission and acquisition of new infection is more difficult to measure precisely in endemic populations. Given the imprecision of diagnosis based on egg detection (described in the subsection "Determination of Prevalence and Intensity of Schistosome Infection," p. 116), exact rates for the incidence of new infection and reinfection cannot be determined. Nevertheless, study of egg-negative to egg-positive conversion rates provides an estimate of transmission in the population at large, and allows comparison of seasonal and regional variation in transmission. Conversion rates in younger children likely reflect true incidence, as egg-negative older children (10-14 years old) are fewer in number (< 30% of those 10-14 years old) and are not as likely to be representative of the general population (6). Typical childhood infection/reinfection rates observed in field studies range from 7 to 26% per year in areas endemic for S. haematobium (21), from 15 to 30% per year in areas endemic for S. mansoni (6, 22) and from 4 to 23% per year for S. japonicum (23). Incidence rates vary according to age, with children 6-10 years old having the highest risk of infection

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(up to 60% annual conversion rate (6)). Although the children tested in these surveys are long-term residents of schistosomiasis-endemic areas, their transmission rates are sometimes lower than infection rates for travelers and nonimmune immigrants (described in the subsection "Transmission of Schistosomiasis in Travelers and Other Nonimmune Populations," p. 117). This difference in susceptibility supports the concept that risk for transmission is not uniformly high in an endemic area, and that seasonal variation in transmission, variations in water use, and resistance acquired early in life play a role in modulating schistosome transmission to endemic populations (24).

Estimation of Local Transmission Potential Snail Monitoring Transmission of schistosomiasis is dependent on the presence of an appropriate freshwater snail intermediate host. The different schistosome species require their own specific snail host species in order to complete their life cycles; S. haematobium is transmitted by Bulinus spp. snails, S. mansoni is transmitted by Biomphalaria snails, and S. japonicum is transmitted by Oncomelania snails (25). Snail colonization of local water habitats, whether rivers, streams, ponds, paddies or ditches, sets the stage for schistosome transmission to h u m a n s (6). Each snail genus has its own geographic distribution, and the prevalence of the corresponding schistosome species conforms to this vector distribution (25). As eggs from human excreta reach these water habitats, parasite miracidia hatch to infect the resident snails. Their subsequent transformation within the snail, then release as infectious free-swimming cercariae, establishes the condition necessary for human infection, i.e. larval penetration of the skin and entry to the human circulation. An area's transmission potential can therefore be estimated by monitoring water contamination (26), monitoring the numbers of infected local snails (snail monitoring) (6) or measuring cercarial numbers at local human water use sites (cercariometry) (6). Snail monitoring is effective if applied in a systematic and consistent manner by trained personnel. Snail sampling can be subject to inaccuracy if multiple sampling sites are not tested, if sampling sites do not reflect the actual human-water contact sites, or if sampling techniques do not accurately

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measure the number of infected snails (6). Sampling scoops may consistently miss the youngest, smallest snails, and underestimate the level of local schistosome transmission. Seasonal variation and patchiness of local ecology means that there will be significant variation in snail populations from one section of a water body to another (27). Careful speciation and individual testing for schistosome infection (by dissection or cercarial shedding in individual specimen tubes) is necessary to accurately measure snail infection levels (6). Even in areas of high transmission, the average snail infection rate may be only 0.5-1.5% (28). For this reason, large numbers of samples are required to establish sufficiently narrow confidence limits for the infection rate, so as to allow meaningful comparisons of transmission potential from year to year and also following the introduction of control measures (6). In some studies, transmission potential has been monitored by placing laboratory-bred sentinel snails into bodies of water where schistosome transmission occurs. Analysis of the speed and intensity of infections among the sentinel snails has also been used to estimate the impact of various control measures on local transmission (6, 27, 29).

Cercariometry Studies using water filtration or centrifugation to enumerate cercarial density have been performed in various control programs (6). Their results suggest that only a very small number of the cercariae shed each day will be successful in finding a suitable human host. Although cercarial density is typically greatest around midday (an average of 2.5 cercariae/liter of water, ranging up to 21/liter (30, 31)), infectivity studies for experimental mice indicate that the greatest per-cercaria infectivity is measured earlier in the morning, suggesting that newly shed cercariae are more infectious. Cercarial density is affected by water flow, which is in turn influenced by local rainfall. As flow diminishes during dry periods, both snail density and cercarial density tend to increase. Even with relatively low numbers of cercariae (< 1 per liter) in local water, transmission to humans may be quite efficient, in a manner independent of the extent of human body surface exposure (6). This efficiency of transmission likely reflects clustering of cercariae at defined water levels, and cercarial skin-seeking behavior (32).


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Environmental Factors Affecting Transmission The lessons of snail monitoring studies indicate that schistosome transmission in endemic areas is highly variable, fluctuating widely with seasonal changes in weather (6,28). Transmission is most affected by changes in rainfall, whether seasonal monsoon fluctuations or periodic drought/flood events. If water supplies dry up, transmission ends. However, schistosome sporocysts will persist in dormant snails as they "estivate" in dried watercourses and lakebeds, so that schistosomiasis transmission will resume when rainfall returns. Environmental temperature has less importance, except in determining the extent of the food habitat of the snail. Schistosomiasis is not strictly a "tropical" disease, in that Oncomelania vectors for S. japonicum may be found in lakes in China with water temperatures near 4°C.

The Epidemiological Profile of Chronic Schistosomiasis The net result of repeated cercarial exposure is chronic schistosomiasis, which can persist for decades. Initial infection typically occurs by the age of five, and increases significantly in intensity during the next decade. Populations endemic for schistosomiasis demonstrate highest infection prevalence and greatest intensity of infection in young adolescent age groups, i.e. those under 15 years of age (see Figs. 1 and 2). This phenomenon is observed for all three of the major schistosome species that are parasitic for humans (S. haematobium, S. mansoni and S. japonicum). As individuals reach adulthood, their infection intensity generally lessens, and prevalence declines (minimally to moderately) among older adults. Infection levels among adults may vary between the sexes according to differences in their water contact exposure to infection. In areas where exposure is primarily related to men's involvement in irrigation or fishing activities, adult males exhibit higher prevalence and greater intensity infection. In areas where women have greater occupational water contact, the adult female prevalence and intensity of infection are greater.

Age-Prevalence and Age-Intensity Curves Figure 1 demonstrates a characteristic age-prevalence curve and ageintensity distribution for S. haematobium in an endemic area of Coast Province, Kenya (33). The prevalence increases from under 10% in infancy to near 60%

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S. haematobium % Prevalence

0-4

5-9

Geometric mean intensity

10-14 15-19 2 0 - 2 4 25-29 3 0 - 3 9 4 0 - 4 9 5 0 - 5 9 6 0 - 6 9

Age Geom. mean intensity

• % Infected

Figure 1. S. haematobium infection prevalence and intensity (mean egg count) in an endemic population in the Msambweni area of Coast Province, Kenya.

S. mansoni % Prevalence

0-4

5-9

Geometric mean intensity

10-14

15-19

20-24

25-29

30-39

40-49

Age % Infected

I Geom. mean intensity

Figure 2. S. mansoni infection prevalence and intensity in the Machakos District of Kenya.


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between ages 10 and 14, then declines after the age of 15. The prevalence among middle-aged adults is less than 20%. Notably, the intensity of infection parallels this prevalence pattern, with the greatest intensity of infection measured in early adolescence, just at the time of the highest prevalence. Figure 2 demonstrates age-prevalence and age-intensity curves for S. mansoni in an untreated endemic region in central Kenya (34). Here the prevalence was over 80% for all age groups u p to the age of 40, when it fell slightly, to -60%. However, the age-intensity profile showed the same type of up-and-down variation seen with S. haematobium during the first two decades of life, i.e. rapid increase to high intensity infection in the 10-14-year age group, followed by significant reductions in intensity in older age groups. This convexity in age-prevalence and age-intensity curves has also been noted in S. japonicum (5, 23).

Contrast with Other Flukes The distribution of schistosome infection in an endemic population is undoubtedly a function of the efficiency of waterborne transmission, as well as host behavioral and immune factors. Figures 3 and 4 contrast the distribution of S. haematobium infection with that of another trematode parasite, the liver fluke Opisthorchis viverrini. Unlike schistosomiasis, O. viverrini infection is transmitted orally by ingestion of parasite metacercariae encysted in fish-containing foods (35). Note that the peak intensity for human opisthorchiasis is not reached until after the age of 40 (Fig. 3). Also note that while the distribution of infection intensity for Opisthorchis follows a normal curve, the intensity profile for schistosome infection is highly skewed (Fig. 4). The uneven distribution of schistosome infection is best modeled by a negative binomial distribution suggesting significant aggregation of worm numbers in a small subset of its human hosts (1). The vast majority (80-90%) of heavy infections are found in children. Theories put forward to explain the more intense levels of schistosome infection in younger age groups have varied, and have been vigorously debated (6). The causes likely include a combination of age-related changes in risk related to changes in water use, changes in skin composition, and experience-related development of parasite-specific immunity (see the section "Influence of Host Factors on Transmission," p. 125). Family studies have also indicated that

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Geom. mean sgg count

0-4

5-9

10-14

15-19 2 0 - 2 4 25-29 3 0 - 3 9 4 0 - 4 9 50-59

> 60

Age I Opisthorchis

I S. haematobium

Figure 3. Age-intensity profile of parasite infection in untreated communities endemic for Opisthorchis viverrini (Thailand (35)) and S. haematobium (Kenya(33)).

% of Population

BMs2S»L_ None

Light

Moderate

Heavy

Very Heavy

Intensity of Infection I H Opisthorchis

B W S. haematobium

Figure 4. Distribution of infection intensity categories for O. viverrini and S. haematobium.

Epidemiology of Schistosomiasis: Determinants of Transmission of Infection 125

there are immunity-related heritable human genetic influences that contribute to an individual's risk for heavy infection (36, 37).

Duration of Infection Adult Worm Survival in Human Populations Duration of infection has been primarily studied in troops and immigrants leaving an endemic area following a defined period of exposure (6, 38). Based on these and community studies of transmission dynamics, the estimated average life span of the adult schistosome is 3-10 years (39, 40).

Adult Fecundity Because, in humans, infection prevalence and intensity are indirectly measured by egg recovery in feces or stool, factors that influence egg recovery or fecundity will affect estimates of parasite density and survival (13). Catalytic models suggest that crowding may play a significant role in reducing both the life span and the fecundity of mature schistosomes (1). Recent studies utilizing parasite antigen detection suggest that the efficiency of egg output declines in older humans. This finding may be related to progressive fibrosis of the gut or urinary tract, preventing eggs from reaching the stool or urine for excretion (14, 41). An alternative explanation may be that older schistosomes have reduced fertility, resulting in lower egg outputs, or that acquired host immunity lowers female schistosome fecundity (13, 42).

Influence of Host Factors on Transmission Water Contact Behavior The nature and extent of an individual's water contact activities have a direct effect on his or her risk for schistosome infection. Careful observational monitoring studies have been performed in a number of areas around the world. These demonstrate that it is the duration of water contact exposure and the proportion of the body immersed that are primary correlates of

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the incidence of infection (6, 21, 43). In many areas, water use is influenced by local cultural factors, which determine who performs the various work activities involving water and when (e.g. fishing, farming, washing clothes, washing cooking materials, bathing, etc.). In specific areas, adult male or female prevalence will be higher depending on which sex has the greatest water contact. Invariably, childhood swimming and playing in freshwater sites carries the greatest risk for infection, and this activity is usually not gender-specific. Other activities involving minimal exposure, such as fording of streams or canals and performance of ritual ablutions, carry significantly lower risk of transmission (6, 21, 43).

Water Contamination Actual water contamination by human excreta has been less well studied. Analysis of egg burdens within a section of the population can predict a theoretical "transmission potential" for that subset of local residents (5). However, contamination of water bodies is not directly measured. Several studies have observed elimination activity and have attempted to quantify the numbers of eggs reaching fresh water (6, 26). These suggest that younger males (aged 15-19) are responsible for the highest rates of environmental contamination, and that heavier rainfall facilitates the flushing of schistosome eggs into snail-containing bodies of water.

Inherent and Acquired Host Resistance to Infection Some experts feel that changes in water contact behavior from childhood into adulthood are sufficient to explain the age-specific variation in the prevalence a n d / o r intensity of schistosomiasis, without invoking the effects of individual host susceptibility or acquired resistance (44, 45). However, the sharp convexity of the age-intensity curve in early adolescence implies to other workers that experience-related resistance to parasite infection develops after repeated exposure (1). Recent studies have indicated that the presence of specific forms of antischistosomal immunity is associated with reduced risk for infection (46-53). Early studies in Egypt and Japan and more recent studies in Brazil also indicate that there are heritable factors (located in chromosone 5) that alter risk for heavy schistosome infection (or disease).


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These factors play a role in determining levels of schistosomiasis in a manner that is independent of age-adjusted parasite exposure (36, 37, 54, 55).

Human Influence on the Ecology of Transmission The epidemiology of schistosomiasis can be significantly modified by human intervention. The effect may be intentional, i.e. part of an organized disease control program (7), or it may be unintentional, as has occurred with snail habitat modification in water development schemes(20,56,57). Although environmental modification can reduce schistosomiasis transmission, there are numerous examples of human environmental interventions where the result has been accelerated parasite transmission.

The Influence of Migration and Urbanization Population growth and migration is a significant factor in the spread of schistosomiasis to new areas. If local infrastructure and development are limited, or if rapid migration outstrips the ability of sanitation to cope with human sewage, then transmission will flourish so long as appropriate vector snails are present in the local water environment and humans continue to come into contact with cercaria-infested waters. Despite partial development, transmission can persist in urban and suburban environments, as has been documented in Egypt, South Africa, Tanzania, Zimbabwe, Brazil and Venezuela (58-63). Immigration and settlement of new areas by schistosome-infected populations has led to spread of the disease to regions that have not previously seen schistosome transmission. The presence of a suitable snail host is required, as is sufficient human population density to maintain a parasite reservoir and continue water contamination. In recent years, mining, construction, and large agricultural schemes based on irrigation have served to introduce schistosomiasis-infected peoples into many previously unpopulated or sparsely populated areas of the world (64, 65). This human influx initiates local transmission in areas where none had previously occurred — in previously settled urban and periurban areas, influx of rural immigrants or refugees often leads to construction of "unofficial housing" or shanty towns without adequate sanitation. Such an increase in

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human population pressure often leads to effective and extensive parasite transmission (60, 61). In other areas, noninfected populations have migrated from parasite-free areas to schistosomiasis-endemic regions, and have become infected for the first time(20, 57, 66). The epidemiological features of schistosomiasis transmission in newly infected populations are of great interest to researchers in the field of transmission dynamics and control (1). In the newly exposed population, unlike populations with long-term endemicity, one notes high levels of transmission to all age groups. The prevalence may reach as high as 100% (57), with infection intensity initially greatest among the oldest age groups (> 25 years old) (66). With time, the profile of age-specific intensity of infection changes, becoming greatest among 10-14-year-olds, as is more typical for communities with long-established transmission (64,66). This shift of the intensity pattern may take as little as 1-2 years (66). The prevalence of schistosome-related disease may be initially low, but would be expected to increase as infection becomes established in these communities over a period of decades (57).

The Impact of Water Source Modification H u m a n impact on snail habitats can be profound — it appears that relatively small changes in salinity, water flow, water quality, or seasonal fluctuations in water level can dramatically influence the species distribution and density of local snail populations. In some areas, these changes have served to reduce schistosome transmission, while in others, transmission has increased 2-5-fold (6). Deforestation and water diversion for drinking water systems can significantly reduce the flow of water to stream and river habitats for vector snails. This has been related to elimination of transmission in some areas of the Caribbean (7). Observations following the construction of the Aswan dams in Egypt indicate that the development of new irrigation areas enhanced the habitat of Biomphalaria snails responsible for transmitting S. mansoni. At the same time, loss of seasonal flooding of the Nile reduced the habitat of Bulinus spp. snails, with a consequent reduction in the transmission of S. haematobium (6). The creation of Lake Volta in Ghana resulted in significant increases in S. haematobium transmission in that area (56), whereas the creation of the Diama dam in Senegal has led to reduction in salinity,


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allowing Biomphalaria snails to flourish and the transmission of S. mansoni to increase rapidly (20, 57). Development of irrigation schemes and largescale farming often draws immigrant population from low-risk to high-risk snail habitat areas, and thus significantly fosters schistosomiasis transmission. The introduction of rice farming, in particular, has been associated with enhanced schistosome transmission in Africa due to the creation of water impoundments (for rice culture) which provide excellent habitat for vector snails (67, 68). In some areas, chemical or thermal pollution has served to destroy the snail habitat (69). In areas of marginal transmission, this has served to reduce or eliminate new infections. However, nitrogen pollution may also serve to improve local water vegetation, enhancing snail proliferation, and plastic flotsam can provide effective breeding sites for vector snails (69). For these reasons, pollution cannot be considered a desirable developmental solution to schistosomiasis transmission. Adequate water treatment of both sewage and industrial wastes remains the most appropriate means of suppressing parasite transmission.

Conclusions Schistosomiasis remains one of the most prevalent infections in the world, and one of the most significant causes of chronic morbidity. While the ecology of transmission has been well studied, and the contributing causes of human infection have been defined, eradication of schistosomiasis has been achieved in only a few endemic areas. Effective implementation of schistosomiasis control schemes requires a detailed understanding of the critical points of schistosome transmission, along with postintervention epidemiological monitoring to ensure utility of the outcomes.

Acknowl edgments I am indebted to Dr Peter (Pip) Jordan for his helpful discussions and many cogent writings on schistosome epidemiology. I am also indebted to Drs John Ouma and Eric Muchiri for sharing unpublished data from their schistosomiasis studies at the Division of Vector Borne Diseases, Ministry of Health, Kenya.

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References 1. Anderson RM and May RM (1991). In Infectious Diseases of Humans: Dynamics and Control (Oxford University Press, New York), pp. 467-496, 507-520. 2. King CH, et al. (1988). Am. f. Trop. Med. Hyg. 39: 361. 3. Subramanian AK, et al. (1999). Am. J. Trop. Med. Hyg. 61: 476. 4. Woolhouse MEJ, et al. (1997). Proc. Natl. Acad. Sci. USA 94: 338 5. Wilkins HA (1987). In The Biology of Schistosomes from Genes to Latrines, eds. Rollinson D and Simpson AJG (Academic, New York), pp. 379397. 6. Jordan P and Webbe G (1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford, UK), pp. 8 7 158. 7. Jordan P (1985). Schistosomiasis: The St. Lucia Project (Cambridge University Press, Cambridge). 8. Peters PAS, et al. (1976). Bull. WHO 54: 159. 9. Peters PAS and Kazura JW (1987). In Balliere's Clinical Tropical Medicine and Communicable Diseases, Schistosomiasis, ed. Mahmoud AAF (Bailliere Tindall, London), pp. 419-433. 10. Pugh RNH (1979). Ann. Trop. Med. Parasitol. 73: 89. 11. Warren KS, et al. (1978). /. Infect. Dis. 138: 849. 12. Savioli S, et al. (1990). Am. J. Trop. Med. Hyg. 43: 289. 13. Agnew A, et al. (1996). Am. J. Trop. Med. Hyg. 55: 338. 14. Smith JH and Christie JD (1986). Hum. Pathol. 17: 333. 15. Deelder AM, et al. (1994). Trop. Geog. Med. 46: 233. 16. Centers for Disease Control (1983). MMWR 32: 585. 17. Centers for Disease Control (1990). MMWR 39: 141. 18. Cetron MS, et al. (1996). Lancet 348: 1274. 19. Vercruysse J, et al. (1994). Trop. Geog. Med. 46: 220. 20. Talla I, Kongs A and Verle P (1992). Trans. R. Soc. Trop. Med. Hyg. 86:182. 21. Muchiri EM, Ouma JH and King CH (1996). Am.}. Trop. Med. Hyg. 55:127. 22. Olds GR, et al. (1999). /. Infect. Dis. 179: 996. 23. Olveda RM, et al. (1996). /. Infect. Dis. 174: 163. 24. King CL, et al. (1998). /. Immunol. 160: 3578. 25. Rollinson D and Southgate VR (1987). In The Biology of Schistosomes: From Genes to Latrines, eds. Rollinson D and Simpson AJG (Academic, New York), pp. 1-50.


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26. Ouma JH (1987). Transmission of Schistosoma mansoni in an endemic area of Kenya with special reference to the role of human defaecation behavior and sanitary practices, PhD thesis (University of Liverpool, Liverpool). 27. Woolhouse ME and Chandiwana SK (1990). Parasitology 100 Pt 2: 247. 28. Sturrock RF, et al. (1990). Trans. R. Soc. Trop. Med. Hyg. 84: 257. 29. Anderson RM and Crombie J (1984). Parasitology 89: 79. 30. Upatham ES (1976). Int. J. Parasitol. 6: 239. 31. Upatham ES (1974). Ann. Trop. Med. Parasitol. 68: 343. 32. Shift CJ, Cmelik SHW, Ley HE and Kriel RL (1972). /. Parasitol. 58: 476. 33. Hodder SL, et al. (submitted). 34. DeStigter KV, et al. (1989). Trans. Assoc. Am. Phys. 102: 209. 35. Upatham ES, et al. (1982). Am. J. Trop. Med. Hyg. 31: 1156. 36. Abel L, et al (1991). Am. } . Hum. Genet. 48: 959. 37. Marquet S, et al. (1996). Nature Genetics 14 October 1996, 181. 38. Warren KS, et al. (1974). Am. J. Trop. Med. Hyg. 23: 902. 39. Wilkins HA, Goll PH, Marshall TF and Moore PJ (1984). Trans. R. Soc. Trop. Med. Hyg. 78: 227. 40. Goddard MJ and Jordan P (1980). Trans. R. Soc. Trop. Med. Hyg. 74: 185. 41. Smith JH, et al. (1974). Am. }. Trop. Med. Hyg. 23: 1054. 42. Agnew AM, Murare HM and Doenhoff MJ (1993). Parasite Immunol. 15: 261. 43. Muchiri EM (1991). Association of water contact activities and risk of reinfection for S. haematobium after drug treatment in the Msambweni Area, Kenya, MS thesis (Case Western Reserve University, Cleveland), pp. 1-121. 44. Dalton PR and Pole D (1978). Bull. WHO 56: 417. 45. Kloos H (1995). Soc. Sci. Med. 40: 1497. 46. Crombie JA and Anderson RM (1985). Nature 315: 491. 47. Wilkins HA, Blumenthal UJ, Hayes RJ and Tulloch S (1987). Trans. R. Soc. Trop. Med. Hyg. 81: 29. 48. Hagan P, et al. (1991). Nature 349: 243. 49. Roberts M, et al. (1993). Infect. Immun. 61: 4984. 50. Dunne DW, et al. (1992). Eur. ]. Immunol. 22: 1483. 51. Dunne DW, et al. (1992). Mem. Inst. Oswaldo Cruz 87 (Suppl. 4): 99. 52. Dunne DW, et al. (1997). Parasite Immunol. 19: 79. 53. Kabatereine NB, et al. (1999). Parasitology 118: 101. 54. Kojima S, Yano A, Sasazuki T and Ohta N (1984). Trans. R. Soc. Trop. Med. Hyg. 78: 325.

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55. 56. 57. 58. 59. 60. 61. 62. 63.

Abdel-Salam E, et al. (1986). Tissue Antigens 27: 142. Scott D, Senker K and England EC (1982). Bull. WHO 60: 89. Gryseels B, et al. (1994). Trop. Geog. Med. 46: 209. King CH, et al. (1989). /. Infect. Dis. 160: 686. Kvalsvig JD and Schutte CH (1986). Ann. Trop. Med. Parasitol. 80: 13. Sarda RK, Simonsen PE and Mahikwano LF (1985). Acta Trop. 42: 71. Chandiwana SK (1983). Cent. Afr. ]. Med. 29: 23. Parraga IM, et al. (1996). Am. J. Trop. Med. Hyg. 55: 150. de Noya BA, Noya O, Balzan C and Cesari IM (1992). Mem. Inst. Oswaldo Cruz 87 (Suppl. 4): 227. Mungomba LM, Chandiwana SK, Sukwa TY and Marshall I (1998). Ann. Trop. Med. Parasitol. 92: 279. Pinheiro FP, et al. (1974). Bull. Pan Am. Health Organ 8: 111. Ouma JH, et al. (1998). Parasitology 117: 123. Brinkman UK, Korte R and Schmidt-Ehry B (1988). Trop. Med. Parasitol. 93: 182. Kazura JW, Neill M, Peters PA and Dennis E (1985). Am. }. Trop. Med. Hyg. 34: 107. Sturrock RF (1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford, UK), pp. 1-32.

64. 65. 66. 67. 68. 69.

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Chapter 5 Resistance to Infection in Humans and Animal Models David Dunne and Adrian Mountford

Introduction Schistosomes are typical macroparasites. They need to chronically infect their mammalian hosts to allow sufficient time for the maturation of male and female parasites, followed by mating and the excretion of large numbers of eggs over many years. In schistosomiasis endemic areas that have moderate to high transmission rates, children begin to accumulate their schistosome worm burdens as soon as they are old enough to come into contact with water that is contaminated with infected snails and, because of the chronic nature of the infection and continued susceptibility to reinfection, they can remain infected for most of their lives. However, worm burdens do not simply keep increasing with age. Typically, infection intensities peak in early teenage years and then decline without necessarily resulting in the complete elimination of the parasite. Schistosomes have coevolved with, and are highly adapted to live in, their definitive mammalian hosts. This is not only apparent in the array of immune evasion strategies that they seem to have at their disposal to allow them to survive in their host's blood (1, 2), but from the fact that the passage of the S. mansoni egg from the mesenteric blood vessels to the lumen of the gut, and thus the continued transmission of the parasite, is actually dependent on the host's immune response (3). Clearly, if an effective protective immune response developed in a host within a short time after initial schistosome infection, this would rapidly block further transmission of the parasite. Even if any such rapid responses induced only "concomitant immunity," characterized by Smithers and Terry (4) as

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immunity against incoming larvae with continued susceptibility to existing adult worms, it is difficult to see how the population of sexual mature worms needed for continued transmission could accumulate. In these circumstances, our expectation should be that any naturally occurring host protective mechanisms will either be slow to develop or be limited in their effect and, therefore, difficult to define or even identify in human populations exposed to infection in schistosomiasis endemic areas. No vaccine is available for use against human schistosomiasis. However, the development of such a vaccine is the long term goal of much of the current research on schistosomiasis and has been approached using two basic parallel strategies. One approach is to examine the factors that affect the distribution of schistosomiasis in human populations, and to identify the contribution from any form of acquired immunity that might be usefully manipulated for the future development of effective vaccines. The alternative strategy is to identify protective mechanisms that occur or can be induced in animal models of schistosomiasis, with the hope that such mechanisms can be well characterized and subsequently might be applicable to vaccine development in humans. Experimental approaches include studies on "natural" infection models and those where the host a n d / o r parasite is manipulated to circumvent mechanisms by which the parasite is protected from immune attack, thereby tilting the balance of the host-parasite relationship in favor of the host. Human studies and experimental animal studies should be thought of as complementary. Any attempt to transfer to man immunological mechanisms or putative vaccine antigens that have proved effective in experimental animal models must be able to call on detailed knowledge of human/schistosome interactions. Equally, any vaccine strategy suggested by human studies must undergo preclinical trials in animal models. It cannot be predicted whether it will be research based on human or experimental schistosomiasis that will eventually lead to the development of the first effective vaccine against human schistosomiasis. However, it is clear that whichever strategy germinates the initial idea, the integration of the two approaches will be required to bring it to fruition. Historically, research on human schistosomiasis and on experimental schistosomiasis have followed largely independent paths, often with only cosmetic attempts to cross-reference ideas and progress. This is not really surprising given the limitations that are inherent in the tortoise-like progress of h u m a n studies compared with the constantly advancing technical

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possibilities afforded by the "experimental hare." Thus, try as we might to combine these two aspects of schistosomiasis research within this chapter, we concluded that they had to be reviewed in separate sections. However, with recent moves towards the testing of potential vaccine molecules in man (5) the link between human and experimental studies will have to become much closer. Hopefully the juxtaposition of the accounts of resistance to infection in human schistosomiasis and animal models in this chapter, will allow the reader to relate one to the other and identify common themes and patterns. Combining resistance in human and animal models has resulted in the need to cover a large and diverse body of work. Although this is a long chapter, we have still had to be selective in our choice of material. In this, we have tended to favor more recent results, perhaps sometimes at the expense of some excellent older publications.

Patterns of Human Infection in Schistosomiasis Endemic Areas Studies of the patterns of the prevalence or intensity of schistosome infections in human populations are assessed by detecting or counting the number of parasite eggs being excreted in the feces or urine. In recent years this method has been augmented by the measurement of circulating parasite antigens that can be found in the blood or urine of infected individuals (6). The prevalence of infection in human populations in schistosomiasis endemic areas with low to medium transmission rates rises steadily with age, often peaking in the teenage years, and then declines with age. In high transmission areas, this decline in prevalence may not occur. In such situations, virtually 100% of the population can be affected by the start of the second decade of life, and this high prevalence of infection is maintained thereafter. However, if the intensities of infection are assessed on the population level, either in terms of the excretion of parasite eggs or by the levels of circulating parasite antigens, a convex age-intensity curve is found, with older individuals being much less heavily infected than young children (Fig. 1). This pattern has been described in areas endemic for all three of the most numerically important species of schistosome that infect man, i.e. S. mansoni, S. haematobium and S. japonicum (7-11). Interestingly, similar patterns of infection with age have been described in domestic animals naturally infected with S. bovis (12) and baboons infected with S. mansoni (13).

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Age (yr) Figure 1. S. mansoni egg excretion profiles with age from six communities in the schistosomiasis endemic Machakos District, Kenya, epg = eggs per gram of feces, plotted against mean age in years (yr). This data is reproduced from (26) (with the permission of Cambridge University Press).

Early debate centered on whether this age pattern of infection intensities was due to acquired immunity (14) or the age differences in exposure to infection (15), and this has remained a difficult issue to resolve. Both interpretations of the observed age-infection intensity profiles would concur that children accumulate an increasing worm burden during their first decade of life and, in due course, the worms acquired early in life start to die — the mean life span of S. mansoni worms in Kenyan populations has been estimated as 7 years (16) while that of S. haematobium in the Gambia is 3.4 years (8) — and are not replaced at a comparable rate in older individuals. However, this reduced rate of worm replacement could be because older individuals have less exposure to reinfection than children or because, with age, they develop an innate resistance or acquired immunity to reinfection (17-19).

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Evidence for Immunity to Infection in Untreated Human Populations If the convex age-infection intensity profiles found in endemic areas are due to development of a resistance that is dependent on a cumulative

experience of infection, probably encompassing acquired immunity, rather than some intrinsically age-dependent physiological process which may or may not have an immunologically component, then two predictions can be made. Firstly, that a shift in the peak of infection towards younger age groups should occur in the most heavily infected communities (20, 21) and, secondly, there should be a reduced heterogeneity of egg counts in older individuals (22). The concept of the "peak shift" comes from the recognition that patterns of infection intensity with host age are often characteristic in different host-parasite relationships. However, although this relationship may be characteristic of a certain parasite, it is often not invariant. In particular, if the age-infection patterns are compared between different host populations, it has been noted that the peak level of infection is higher and occurs at a younger age when the transmission rate is high, and lower at a later age when transmission is lower. This phenomenon has been reported for human schistosomiasis (23). Mathematical models have been used to demonstrate that such changes in peaks of infection could be explained by the development of protective immunity if it was gradually acquired as a result of an accumulated experience of infection in terms of a combination of infection duration and intensity (24). Amongst S. haematobium-miected populations in the Gambia, an infection prevalence peak shift from 15 to 10 years of age has been shown to occur when low and high transmission areas are compared (21), and a significant peak shift was found in a comparison between two endemically S. haematobium-miected populations in Zimbabwe (25). Similarly, for S. mansoni, Fulford and colleagues analyzed quantitative stool egg counts from 13,000 Kenyan children in 36 primary schools and found a negative correlation between the mean intensity of infection and the age of the peak intensity. A significant effect of age on the variance/ mean relationship was also found in this Kenyan study (26). It has been suggested that these relationships would be generated in models that did not require density-dependent effects such as acquired immunity (26, 27), and that age-related patterns of exposure to infection could contribute to observed peak shifts in schistosome-infected populations. However, peak

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shifts may occur in troops of baboons naturally infected with S. mansoni (13), and have been described for Wuchereria bancrofti (28), and hookworm infections (23) in comparisons between human populations suffering different levels of transmission. More comparative studies of schistosomiasis-infected human populations are required to build up a clear picture of whether or not the peak shift phenomenon is consistently observed in the field and, therefore, coincides with the predictions of mathematical models in relation to resistance to superinfection resulting from accumulated experience of infection.

Treatment-Reinfection Population Studies in Schistosomiasis Endemic Areas Generally, the study of human immunity through analysis of empirical age-intensity data as observed in an untreated population is an attempt to retrospectively account for the sum of exposure, infection and reinfection events that must have occurred over many years previous to the start of the study. It was pointed out by Butterworth and colleagues (29) that such studies suffer from three problems: (a) it is difficult to determine the amount of new infection that is occurring, and thus the degree of immunity to new infections in the continued presence of adult worms from existing infections that are dying spontaneously at unknown and possibly variable rates; (b) it is difficult to distinguish between immunity and lack of exposure as possible reasons for a lack of superinfection; (c) partial immunity is more difficult to detect than "sterile" immunity. In this respect it is worth noting that in all animal models of immunity to schistosomiasis, the degree of protection observed has been partial. If protective immunity were to occur in human populations, there was no reason to expect that the situation would be different. However, since the risk of clinical disease in human schistosomiasis is at least partly dependent on the intensity of infection, partial immunity might be important in greatly reducing the prevalence of schistosomiasis associated disease even in the absence of a major effect on reducing the prevalence of infection (29). In an attempt to overcome these problems, Butterworth and colleagues established an approach to studying S. mansoni in man that involved treating study cohorts to remove existing worm burdens and then recording the rates of subsequent reinfection among individuals


within the cohort, whilst carefully monitoring their degree of exposure to contaminated water (29). A similar approach has since been employed by others, for example Dessein and colleagues on S. mansoni in Brazil (30-32) and by Wilkins, Hagan and others on S. haematobium in the Gambia (33, 34). These studies used local observers at established sites of contact with contaminated water to record for each individual the details of their water contact, including the time of day, duration, nature, and extent of his or her contact; snail numbers, infection rates and cercarial numbers at the various sites were also observed. Preliminary studies of this type were carried out in the late 1970s in a S. mansoni e n d e m i c area of M a c h a k o s District, Kenya (35), a n d immunological studies were carried out on a cohort of infected schoolchildren who were treated and followed for reinfection over a two-year period (36). The striking finding from this work (29, 37), confirmed by others (30, 31, 33, 38), was the very strong age dependence of reinfection, with a sharp decrease in reinfection levels after the age of 12 years. The reinfection patterns from different studies, because they represent accumulated reinfection over relatively short periods of time, are much more similar to each other and more consistent than are the age-infection intensity profiles found in nontreated communities. Thus, the infection curves of the nontreated populations represent accumulated acquisition of infection over varying periods of time within which fluctuations in transmission will be reflected in the accumulated age patterns. In contrast, the consistency of the age pattern of reinfection after treatment is striking, particularly when results from different studies from geographically dissimilar environments (Fig. 2), even with different species of schistosome, are compared. Estimates of water contact, when they are carried out, are usually more variable than are the patterns of reinfection (19, 39). In Butterworth's original study (36), the total duration of water contact observed peaked later, between the ages of 16 and 24 years, than did reinfection intensities (29). Similar studies in different schistosomiasis endemic areas continue to follow this study design and to identify patterns of reinfection that are consistent with the occurrence of agedependent resistance to reinfection in human populations, for example against S. haematobium in Zimbabwe (42) and Mali (43) and S. japonicum in China (11). A somewhat different approach to detecting possible acquired immunity after chemotherapy was taken in longitudinal studies of communities living

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Age (yr) Figure 2. S. mansoni reinfection profiles reported in various treatment-reinfection studies from Brazil, Kenya and Uganda. The scale of mean epg (eggs per gram of feces) has been adjusted for each study, to allow age-reinfection intensity profiles to be compared, and plotted against age in years (yr). Brazil 2 is an exception as this was derived from reinfection incidence data. Brazil 1 is taken from (30); Brazil 2 is reinfection prevalence data taken from (32); Kitengei, lietune, Matithini, Study I and Study H are reinfection intensity data from published and unpublished Kenyan studies, including (29, 40, 41). Butiaba (12 mth) is unpublished data, showing mean reinfection intensities 12 months after chemotherapy, in a Ugandan fishing community studied by (39). We thank AJC Fulford for the use of this figure.

in the S. japonicum endemic island of Leyte in the Philippines. In an eightyear study of some 5000 individuals, failure time analysis was used to study the time-to-reinfection after parasitological cure (44). This showed that children became infected faster than adolescents who became infected faster than adults. The most interesting aspect of the analysis was found when children from this study p o p u l a t i o n were divided into two m a t c h e d


subgroups consisting of those who initially were, or were not, infected. Both groups were followed until reinfection/infection occurred. In children who were less than 14 years old, no difference in the time to infection was observed. However, in all groups above the age of 14 years, the previously infected individuals were more slowly reinfected than the previously uninfected group who became infected for the first time. Although water contact studies were not reported in relation to this study, there is no reason to suppose that the group that were initially infected and then cured, should have a lower exposure to infection than those who were initially uninfected. The authors concluded that this was evidence for the occurrence of an age-dependent acquired immunity that was dependent on previous infection (44).

The Influence of Exposure in Patterns of Infection Clearly, exposure to infection has to be an important factor influencing rates of infection and reinfection in schistosomiasis endemic areas, and age patterns of prevalence and intensities of infection have been shown to follow closely the age patterns of exposure in a number of studies (42,45). Although childhood water contact often appeared to carry the greatest risk of infection (46, 47), analysis of the influence of water contact is made more difficult by the fact that the type of water contact that children and adults engage in is different, and it is possible that different water contact behavior carries different risk factors (48). In almost all published treatment/reinfection studies on endemic populations that have attempted to observe simple water contact, or analyze the more complex question of exposure, it has been found that children have greater contact/exposure than adults and that contact time and contact behavior are both age-dependent. All such studies have used regression techniques (either logistic or least squares) to demonstrate that the differences in water contact would not fully account for the much greater differences in intensities of reinfection (11, 32, 33, 43, 47, 49). However, it is inevitable that these conclusions, to some extent at least, are weakened by statistical problems arising from the potential inaccuracy and imprecision in estimates of individuals' exposure combined with the tendency towards greater water contact amongst children, and age-dependent behavioral differences in the nature of adult and childhood water contacts (27, 29, 48, 50). Preliminary results of water contact and reinfection with S. mansoni after

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treatment amongst fishing communities on Lake Albert in Uganda hold out the promise of solving this problem once and for all (39). This study provided the opportunity to focus on communities in which the pattern of water contact was strongly related to occupation rather than domestic activities, as the sole economic activity was fishing with little or no agriculture. In this study, both male and female water contact activities were greater in adults than in children, and these patterns were little affected when allowance was made for behavioral differences which had complicated previous similar studies (48, 50). In contrast, the patterns of reinfection after treatment with praziquantel were very similar to those described in previous studies, peaking sharply around ten years of age and falling to much lower levels in adults (39). In this fishing community, water contact behavior alone could not explain the consistency of the reinfection pattern seen between this and previous studies in different geographical areas endemic for different schistosome species. This strongly suggests that a marked age-dependent human resistance to reinfection occurs in human populations in endemic areas, and this manifests itself in an abrupt reduction in susceptibility to reinfection in early teenage years. An interesting adjunct to this conclusion is contributed by observations made on subgroups of endemic populations who are hyperexposed to infection during the course of their work. A particularly good example of such studies is those carried out on Sudanese canal cleaners who are occupationally exposed to S. mansoni. Without treatment, these long-term, hyperexposed canal cleaners (with greater than five years' exposure) were found to have higher levels of infection compared with age-matched newly recruited workers (migrants with less than one year of exposure). Both groups were more intensely infected than normal exposed individuals within the same communities. However, despite apparently identical exposure, one year after treatment with praziquantel, the newly recruited group were found to have suffered more than double the prevalence of reinfection compared with their chronically hyperexposed counterparts (51). Thus, in this situation, a difference in susceptibility to reinfection was found between two groups of adults, who were matched for age but who differed in the length of time that they had been hyperexposed to infection. This suggests that despite the consistency in population reinfection age patterns, factors other then age per se may have some influence on human susceptibility and resistance to reinfection with schistosomes.


Acquired Versus Innate Resistance to Reinfection Comparing the results from different treatment-reinfection studies, it is very striking how similar the age-infection intensity profiles are and how sharply the reinfection rates decline in the early teenage years (Fig. 2). Although age-dependent exposure patterns may be a predominant influence on rates of reinfection in some of these endemic situations, this is not true in all cases, as discussed in the section "Treatment-Reinfection Population Studies in Schistosomiasis Endemic Areas" (p. 138). This sharp fall in susceptibility to reinfection coincides with puberty, and this has led to the suggestion that age-dependent host physiological changes may be responsible for this rapid change in susceptibility (17-19). Hormonal changes underlie most of the physiological changes around this age, and it has been pointed out that hormones could have three possible modes of action on schistosome infections: (1) a direct influence on parasite metabolism (52); (2) induction of host physiological or anatomical changes in the host that increase innate resistance to infection; (3) induction of changes to the immune system that increase the likelihood of acquiring a protective immune response, by upregulating protective mechanisms or downregulating "blocking" responses (19). Studies in animal models have shown that host hormones can influence the number of adult schistosomes that successfully develop from a cercarial challenge ((53) and the section "Innate Resistance to Schistosomes in Animal Models," p. 164), and that testosterone has an adverse effect on schistosome development (54). Changes in human susceptibility to infection at puberty could also be caused by increased innate resistance, for example increased skin thickness or subcutaneous fat deposition, or changes in the composition of skin fatty acids, which might interfere with cercarial skin penetration. If h o r m o n a l changes are involved in the development of human resistance around the age of puberty, then these changes should be common to the two sexes as the difference in susceptibility between children and adults is far greater than any observed differences b e t w e e n males and females (19). A d r e n a l a n d r o g e n s , in particular dehydroepiandrosterone (DHEA), increase with age in both sexes and have been shown to influence host immune responses. However, in the context of a recent S. mansoni treatment-reinfection study in a Ugandan fishing community, neither DHEA nor testosterone was found to explain a significant portion of the age dependence of resistance to reinfection observed (Fulford

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et ah, unpublished results). Nonetheless, the remarkable coincidence between adolescence and changes in susceptibility to reinfection with schistosomiasis in human populations, makes it important that all possible puberty-related physiological and immunological changes are investigated during the course of future human reinfection studies.

Acquired Immunity; Age per se or Cumulated Experience of Infection If acquired immunity is an important factor in the development of resistance to reinfection, then its development or expression may be slow, perhaps dependent on prolonged and cumulative exposure to the relevant antigens. In this case, the apparent age dependence of this response would merely reflect the time it takes to gain this experience. Alternatively, it is possible that the protective responses are intrinsically age-dependent, being modified by other age-related physiological processes, such as hormonal changes that occur around puberty. A study of immigrant watercress growers in Brazil was an early attempt to uncouple duration of exposure from age, and suggested that many years of exposure to infection rather then age per se was responsible for the development of resistance to S. mansoni (55). However, this study has been criticized for its small scale and its statistical methodology (18). Not until the epidemic outbreak of S. mansoni, caused by the damming of the Senegal River in 1986, was there another attempt to systematically study the acquisition of infection with age in a previously unexposed population. The expectation of this study was that if immunity required 10-15 years' duration of infection before it developed, then levels of newly acquired infection in a previously naive population would be high throughout the adult age range, rather than dramatically declining after childhood as they do in populations in endemic areas. Initial studies of the Senegal population commenced some three to four years after the start of the epidemic, and the pattern of infection was found to be remarkably similar to those found in endemic areas (56). Thus, in this community the difference in the rate at which children and adults acquired their infection did not depend on many previous years of exposure. A similar result was obtained from a study of an immigrant population in Kenya that moved en masse from a schistosome-free area into the schistosomiasis mansoni endemic Masongaleni area (57). This Kenyan study had the additional advantages


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that, unlike the Senegal study, the immigrant population were first surveyed very shortly after they were exposed to infection, before the endemic pattern of infection intensity became evident; and there was an existing, wellestablished resident community of the same ethnic origin already living in the Masongaleni area that acted as an endemic control population. Initially, the prevalence of infection amongst the immigrants was low, with fecal egg counts peaking atypically in adults around the age of 30 years. Over the next 12-18 months infection increased rapidly, especially among the children and young teenagers, producing a pattern of infection typical of endemic communities. Thus, this study considerably shortened the estimated time required to develop the typical "endemic area" age-intensity profile (57). Both these studies underlined the fact that slowly acquired immunity cannot be the only factor governing the decline in infection in adulthood, but neither can they preclude the possibility that it exists. Water contact was also strongly age-dependent in both the Senegal and Masongaleni studies, falling in the second decade of life (49,57). This, combined with a lack of knowledge about the relative risks of infection associated with the different forms of adult and childhood exposure, makes it very difficult to determine whether or not age-dependent exposure patterns in these two situations were not an overriding factor in influencing who became most intensely infected. However, in other endemic situations, where adults are more exposed than children, the presence of the same endemic pattern of age-intensity of infection indicates that exposure itself cannot always be the dominant influence on reinfection (39). Hence, the need for further analysis of water contact data in these two studies means that the issue of whether age per se or accumulated experience of infection has the most influence on the development of acquired immunity is not yet resolved.

Immunological Correlates of Resistance to Reinfection Age-infection intensity and treatment/reinfection studies conclude that factors other than exposure are required to explain the age-dependent difference in infection/reinfection seen in endemic areas, and that acquired immunity was likely to have some role in this. Most schistosome-infected individuals have a wide range of immunological responses to the parasite. It has been frequently observed that individuals who are susceptible to reinfection have vigorous responses to the parasite. This strongly suggests

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that many human antiparasite responses are at best irrelevant to protective immunity, or at worst might actually favor the parasite. The only realistic and ethically acceptable way of addressing the question of which immune mechanisms might be involved in protective immunity, is to attempt to correlate the extent of reinfection after treatment with the presence or absence of a variety of antiparasite immune responses. Butterworth and colleagues (36) were the first to do this in 1981 by treating a cohort of 129 Kenyan schoolchildren between the ages of 9 and 15 years and infected with S. mansoni, and then measuring the intensities of reinfection they subsequently suffered over a period of two years. The younger children suffered higher intensities of infection than the older children, and these rates of reinfection were compared with the children's pre- and post-treatment IgM and IgG subclass responses to schistosome egg and adult worm antigen preparations. This study showed that the susceptibility of younger children to reinfection did not involve a generalized failure to respond immunologically to the parasite. In fact, many of their immune responses were more vigorous than those of the older, more resistant children (58, 59). In this early study, no antibody response was found to negatively correlate with intensities of reinfection after treatment and, therefore, have the characteristics of a potentially protective response. This study did, however, identify responses that positively correlated with intensities of reinfection. Thus, high levels of IgM and IgG2 were found to be associated with subsequent susceptibility to reinfection (58). These IgG2 and IgM responses were found to be higher in young children than in adults and to be T-independent responses induced by egg polysaccharide antigens, and to be cross-reactive with glycoproteins on the surface of migrating schistosomula larvae (59-61). The association between these anticarbohydrate IgG2 and IgM responses, and subsequent susceptibility to high intensities of reinfection after chemotherapy, supported the hypothesis that the susceptibility of young children was due to the production of ineffective antibody isotypes that were unable to mediate protective immune effector mechanisms, and might even block other protective responses, either by competing for common epitopes or by steric hindrance (58, 59). Similar evidence was found for the presence of IgG2 and IgG4 blocking antibodies in S. mansoni-intected communities in Brazil (32). Experimental evidence which supported this hypothesis was provided that demonstrated that murine IgM monoclonal antibodies, directed against epitopes shared by glycoproteins on the surface of schistosomula and


T-independent polysaccharides from the parasite egg, were capable of blocking the in vitro killing of schistosomula by human eosinophils in the presence of serum from S. mansonz'-infected individuals (62, 63). While in vitro studies showed that human IgM, IgG2 and IgG4, purified from the serum of infected individuals, were also able to prevent eosinophilmediated killing of S. mansoni schistosomula of other IgG subclasses present in serum of human infection sera, IgG2 was itself able to mediate some larval killing in the presence of more highly activated human eosinophils (64,65). While the action of "blocking" antibodies can be convincingly demonstrated in vitro, and this is consistent with the hypothesis that some antibody responses are associated with human susceptibility to reinfection, human correlation studies cannot provide unambiguous evidence that these mechanisms are a dominant in vivo influence on the outcome of human exposure to infection (66). The blocking antibody hypothesis sought to explain the susceptibility of young children to reinfection in terms of the blocking of more effective antiparasite immune responses that were assumed to be present in older children and adults. However, no such potentially protective response had been identified until Hagan and colleagues demonstrated that parasite-specific IgE was negatively correlated with subsequent intensities of reinfection in human communities living in areas of the Gambia endemic for S. haematobium (38). In this study, a 40% sample was drawn from the full age spectrum of the study community, and the prevalence and intensity of reinfection were found to be higher in children than in adults. When analysis was carried out for exposure to reinfection, sex and age, children with high levels of exposure were found to be heavily reinfected, whereas adults who had equivalent levels of exposure were not. IgG subclass and IgE responses against adult worm and egg antigens were measured, and it was found that IgG4 responses were high amongst the children and lower in older individuals, whereas IgE responses were low in the susceptible children but high amongst the reinfection resistant adults. In a logistic regression analysis that allowed for the effects of exposure and age, it was found that those in the lowest quintile for IgE responses to worm antigens were 10.2 times more likely to be reinfected than those in the highest quintile. In contrast, reinfection was 10.7 times more likely in the highest quintile for IgG4 responders compared with those in the lowest quintile. These results, suggesting a protective role for parasite-specific IgE in protection against reinfection with S. haematobium, were followed closely by similar findings in separate reinfection studies of

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S. mansoni-miected communities in Brazil (67,68) and Kenya (41). The Kenyan study was carried out on a cohort of 140 infected individuals who were treated and then examined for reinfection over the next three years. Reinfection was strongly age-dependent, with little reinfection occurring in individuals aged above 16 years. Antibody isotype responses against the worm, egg and schistosomula stages of the parasite life cycle were assayed both before treatment and again six months after treatment. This second time point was before reinfection had started to occur, because of the seasonal nature of S. mansoni transmission in this region of Kenya. In this study, as in previous studies (58), IgM and IgG2 responses were associated with susceptibility to reinfection. In contrast, IgE responses against the adult worm, but not IgE against responses to other life cycle stages, gave a highly significant negative correlation with subsequent reinfection. The results of the similarly designed Brazilian studies not only confirmed the relationship between parasite-specific IgE responses and resistance to reinfection (67, 68), but extended the association between susceptibility to reinfection and IgG2 and IgG4 responses. When IgM, IgA, IgE and IgG subclass responses were tested by logistic regression in the presence of the explicative variables (water contact, age and sex), IgE (negatively correlated), IgG2 and IgG4 (both positively correlated) were found to be associated with intensities of reinfection after chemotherapy. As the opposite relationships of IgE and IgG4 could not be disassociated in the analysis, it was suggested that these antibody isotypes were probably antagonistic to each other in terms of human resistance to reinfection. The IgG2 effect was independent of the other two antibody isotype responses (32). This was considered to be consistent with the view that IgE and IgG4 were directed against peptide antigens, while IgG2 is an anticarbohydrate response (59, 61). Although relatively few population scale treatment/ reinfection studies have been reported for human populations living in S. japonicum endemic areas, a similar age pattern of reinfection after treatment has been observed (11) and IgE responses against S. japonicum egg antigens have been reported to be negatively correlated to levels of reinfection (69). Until recently, the practical constraints on large scale cellular studies have meant that immunological studies of reinfection with schistosomiasis after treatment in human populations have focused mainly on antibody responses in relation to infection. Only recently have attempts been made to increase the scale of cellular immunology studies to enable meaningful correlations with infection and reinfection to be made while allowing for factors such as


exposure and age. Interestingly, in a study cohort of 8-13-year-old Gambian children infected with S. haematobium treated with praziquantel, the first predictive immunological marker of resistance to reinfection to be reported was elevated peripheral blood eosinophil counts (70). In a high transmission area for S. mansoni, Roberts and colleagues (40) investigated the cell-mediated

responses of a well- defined group of 89 infected Kenyans, between 9 and 35 years of age, in relation to their intensities of reinfection in the 12 months following treatment. Peripheral blood mononuclear cell (PBMC) proliferation and production of IL-2, IL-4, IL-5, IFN-y, and TNF, in response to in vitro stimulation with either adult worm, schistosomula or egg antigens, were examined before and 3 and 12 months after treatment. Even after allowing for the effects of age, sex and exposure in multiple regression analysis, PBMC proliferative responses to adult worm and schistosomula antigens gave significant negative correlations with subsequent intensities of infection in older individuals (14-35 years) but not in younger children (9-13 years). Inverse relationships were found between IL-5 and IFN-y responses to all parasite antigens at all three time points and significantly higher IL-5 levels were found in the older (more resistant) age group. The inverse correlation between IL-5 and reinfection could not be separated from the effects of age and exposure (40). A recent study of a cohort of Egyptian boys exposed to S. haematobium revealed a significant association between worm antigen-specific IL-4 and IL-5 responses and resistance to reinfection. Thus, an initial survey showed a 51% prevalence of S. haematobium in the study cohort and an incidence of 44% 12-18 months after chemotherapeutic cure. Those who became reinfected and those who did not had similar water contact, but the resistant individuals had a 3.5- to 14-fold greater frequency of worm-specific IL-5 or IL-4 secreting lymphocytes and their peripheral blood lymphocytes produced more IL-5 or IL-4 compared with susceptible subjects. In contrast, worm antigen-induced IFN-y and IL-10 production and lymphocyte proliferation were similar between the two groups. Schistosome egg antigen and streptolysin O each stimulated similar cytokine production in susceptible and resistant boys (71). Other studies have attempted to identify potentially protective immunological responses by analyzing reinfection, but without necessarily taking into account the significant variables for age, sex or water contact in their analysis, or have attempted to relate immune responses to patterns of infection

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observed in untreated populations. IgA responses to a recombinant S. mansoni 28-kDa glutathione-S-transferase (Sm28GST) paralleled on acquisition of resistance to reinfection with age in a Kenyan study population, but the influence of age was not considered in this analysis (72). In a two-year study of 37 S. haematobium-infected individuals who had been treated, those who did not become reinfected had significantly higher levels of worm-specific IgE before treatment than those who became reinfected. The median age of those who became reinfected was also significantly younger than those who did not (73). Similarly, antibody isotype responses were measured in a group of Sudanese canal cleaners who were hyperexposed to S. mansoni. The specific antibody isotype responses of chronically infected canal cleaners, who were highly resistant to reinfection, and less resistant newly recruited canal cleaners (51) were compared, and this showed an association between IgE and IgGl responses to worm antigens and resistance to reinfection. In contrast, an association was observed between IgG2 and IgM responses to SEA and susceptibility to reinfection (73). Analysis of schistosome-specific helper T-cell clones from Brazilians considered to be either resistant or susceptible to reinfection with S. mansoni has also given results that are consistent with the conclusions drawn from the more systematic population treatment/reinfection studies. Thus, 28 CD3 + , CD4 + , CD8 + -parasite-specific T-cell clones from three infection-resistant individuals produced a higher ratio of IL-4 to IFN-y than did similar T-cell clones from a sensitized individual from a non-endemic area (74). Another approach to the identification of the influence of human immunity or resistance to infection led to the identification of so-called "endemic normal" individuals. This is analogous to the situation found in studies of filarial worm infections where it is assumed that the whole population is equally exposed to the insect vectors of infection, yet some individuals, who have distinctive immunological responses to the parasite, are nonetheless consistently found to be uninfected. These observations have led to such individuals being described as "putative immunes." Work in Brazil has led to the identification of uninfected individuals who have contact with S. mansoni transmission sites but are found, on repeated examination, not to be excreting parasite eggs (75). These schistosomiasis endemic normals appear to constitute a relatively small proportion of the endemic population, and are generally older than the mean age of infected members of the same communities. This makes it difficult to include age-matched controls when immunological


studies have been carried out on this interesting group of individuals (76, 77). Similar schistosomiasis endemic normals have not been identified by studies in other endemic areas. However, it is important to note that in early treatment/reinfection studies carried out Kenya, the study cohorts were selected to be positively infected before initial treatment (41, 59), thus inadvertently excluding any potential endemic normals from these studies. As the identified Brazilian endemic normals are usually older individuals, it is possible that they represent a relatively small number of individuals at the extreme end of the age-dependent development of resistance reported in other schistosomiasis areas (11,19, 29-31,38,39,43). When these Brazilian schistosomiasis endemic normal individuals were compared with infected individuals from the same area, both had detectable IgGl, IgG4 and IgM responses against schistosome egg, cercaria and, to a lesser extent, adult worm antigens. In general, the endemic normal individuals expressed higher levels of specific IgE as compared to IgG4, while the infected individuals had higher IgG4 as compared to IgE, and it appeared that the endemic normals had a distinctive pattern of antibody isotype responses against the parasite (77). Evaluation of the production of IFN-y in vitro by schistosome antigen-stimulated PBMC from endemic normals and infected individuals showed that the infected individuals produced little or no IFN-y, while the PBMC from the two groups produced comparable levels when exposed to phytohaemagglutinin. This implies that persons with patent infections had antigen-specific defects in their response to the parasite that was not shared by the putatively resistant endemic normals (76). Such suppressive effects of infection on immune responses to the parasite would make it difficult to interpret the results of immunological comparisons of infected individuals and endemic normals in relation to the identification of putative protective immune responses.

Schistosome Antigens Associated with Human Resistance to Reinfection Schistosome-infected individuals respond to a wide range of parasite antigens, and different antigens are recognized by different individuals (41, 61). Progress in identifying parasite antigens that may be acting as targets for protective human responses does not compare with the array of defined antigens that have been shown to protect against infection in various animal

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models (see the section "Resistance Induced by Native and Recombinant Schistosome Antigens," p. 182). Some antigens that are partially protective in animal models are recognized by infected h u m a n s ; for example, paramyosin was first identified as a potential vaccine antigen in a murine S. mansoni model, and is responded to by some S. mansoni- and S. japonicuminfected patients (75, 78). Human responses against several antigens that protect mice have patterns of recognition that are consistent with a possible role in human immunity. SmW68, a S. mansoni surface antigen that protects mice, was studied as a function of infection intensity in 85 chronically infected Egyptians. The antibody to SmW68 increased significantly with age, reaching a plateau at 15-19 years, 1-5 years after peak prevalence and infection intensity. Among infected individuals, responses showed a significant inverse correlation with infection intensity, with higher IgM but not IgG being associated with low parasite burden. However, it cannot be determined from the responses of infected populations whether parasite burdens suppress antibodies to a particular antigen or whether an enhanced response represents acquired immunity (79). Thus, in the absence of direct evidence from human vaccination studies, only a correlation between a particular response against a defined antigen and a subsequent lack of reinfection in the face of observed exposure, can serve as a useful indicator of a potentially protective antigen. Schistosome glutathione-S-transferase (GST), extensively studied as a potential vaccine antigen, was first identified from studies in a murine S. mansoni model, and GSTs from S. mansoni, S. japonicum and S. bovis have been tested in various experimental and domestic animal models (see the subsection "Glutathione S-Transferases," p. 183). Human antibody responses against recombinant S. mansoni GST, and synthetic peptides derived from GST, have been compared with intensities of reinfection after chemotherapy, and IgA responses against GST increase with age and are negatively correlated with reinfection intensities. This pattern of response, although not controlled for age, is compatible with a protective role for human IgA anti-GST (72). The recent WHO initiative to test for human immune responses against a selection of antigens that have been reported to afford partial protection to experimental animals (5) will be discussed in the context of the immunization of experimental animals (see the section "Resistance Induced by Native and Recombinant Schistosome Antigens," p. 182). There is general agreement about the nature of the human immune responses that are associated with resistance to reinfection, with T helper


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2 type responses, and parasite-specific IgE in particular, being identified in both treatment/reinfection studies and more anecdotal observations (38, 40, 41, 67, 70, 71, 80, 81). However, different studies have not agreed on which stages of the parasite life cycle are most significant in relation to these potentially protective Th2 responses. For example, in their original study of the relationships between antibody responses and reinfection with S. haematobium in the Gambia, Hagan and colleagues reported that IgE against both parasite egg and worm antigens was correlated with low reinfection intensities (38). For S. japonicum, correlations between IgE responses to egg antigen and human resistance have been reported (81). In contrast, Dunne and colleagues (41) found no association between any antibody isotype response against egg or schistosomulum antigens and reinfection with S. mansoni in Kenya, whereas IgE against a number of different antigens prepared from worm, all significantly correlated with resistance to reinfection after allowing for the effects of age (41). Reinfection studies in Brazil concentrated on antibody responses to S. mansoni schistosomula antigens and found significant correlations between reinfection intensities and IgE responses (67). This apparent diversity in life cycle stages recognized by responses associated with resistance could be due to a variety of causes. The absence of a significant correlation in a particular study may be due to assay inaccuracies or inadequate sample size, or differences in antigen preparations or assay methodologies. Equally, real variation between strains or species of parasite, or differences in human study populations may be involved. Nevertheless, in all treatment/reinfection studies that have reported immune correlates of resistance to reinfection have identified parasite-specific IgE responses and associated Th2-type responses. To identify potentially protective schistosome antigens, human responses correlated with resistance to reinfection have been used to screen for candidate antigens in different life cycle stages. Thus, sera from Brazilians, observed to be resistant, recognized a large number of antigens in immunoblots of schistosomula tegument (67). A comparison between resistant and susceptible individuals showed that IgE levels were on average six- to eight-fold higher in the sera of the most resistant adolescents, but that there was no difference in patterns of antigen recognition between study groups. In contrast to IgE, anti-larval IgG and IgM levels were either similar in both groups or higher in the least resistant subjects. IgG that competed with the binding of IgE was detected in most sera and its levels were highest in the least resistant

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group. As the differences were in levels of IgE rather than in antigens recognized, and as antigens recognized by IgE were also recognized by IgG, individuals were selected for their low or high susceptibility to reinfection, and their reactivity to individual larval surface antigens was determined. This showed that six antigens were the principal targets for IgG from resistant subjects, and immunoblotting and ELISA indicated that IgG reactivity against a 37 kDa antigen might be associated with resistance (30). This 37 kDa schistosomal antigen was subsequently cloned from a S. mansoni cDNA expression library. The amino acid sequence of the encoded polypeptide showed a 72.5% homology with the human glycolytic enzyme Glyceraldehyde-3Pdehydrogenase (82). A study of S. mansoni- and S. haematobium-infected patients in Egypt has also suggested that this enzyme could be a target of protective T- and B-cell-mediated immune responses (83), and it has been shown to have a protective role in murine infections [see the subsection "Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH)," p. 190]. IgE responses against various worm antigen preparations, including antigens that had been treated with sodium periodate to remove carbohydrate epitopes, and antigens derived from the outer tegument of S. mansoni adult worms, correlated with resistance to reinfection in a S. mansoni endemic area of Kenya (41). Therefore, worm antigens recognized by high antiworm IgE responders were examined in immunoblots. In this study, IgE was found to recognize a restricted range of antigens compared with IgG from the same sera. A predominant 22 kDa antigen band was recognized by IgE from most, but not all, of the high IgE responders, and it was found that individuals that recognized this antigen with IgE were significantly less likely to have become reinfected in a three-year period after treatment than those who had no detectable IgE against it. Moreover, if they did become reinfected, the levels of reinfection in the responders were significantly lower than those suffered by nonresponders (41). The purified native 22 kDa antigen was incorporated into ELISA and IgE responses, but no other isotype response was found to have a statistically significant correlation with subsequent low levels of post-treatment reinfection. This significant correlation was present after allowing for the confounding effects of age by multiple regression analysis. Rabbit anti-22kDa serum recognized the outer tegument, gut tegument, and the collecting ducts and flame cells of adult worms. The 22 kDa band antigen(s) was also present in "lung" and "postlung" schistosomula stages of S. mansoni, and in S. haematobium, S. bovis and S. japonicum adult


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worms. Metabolic labeling of schistosomula and worms demonstrated the in vitro synthesis and release of 22 kDa antigen (84). This tegument antigen was cloned (85) and found to be identical to a 22.6 kDa S. mansoni antigen that had been cloned previously (86, 87). Anti-rSm22.6 antibodies were also compared with intensities of reinfection following treatment, and a negative correlation between IgE responses to rSm22.6 and infection intensity was found. This relationship remained significant after allowing for age and other isotype responses to rSm22.6, in particular IgG4 (85). A homologous r22.6 kDa tegumental antigen from S. japonicwn has also been shown to be a major antigen recognized by IgE from a human population from a S. japonicwn endemic region of the Philippines. Interestingly, the IgE-binding epitopes of SJ22.6 were found to have sequence similarity to parvalbumin and (Jlactoglobin-related allergens (88). These associations between IgE and resistance suggest that schistosome antigens that induce this response may have a role in immunity, and a number of studies have attempted to identify schistosome allergens (89). An allergen of S. haematobium has been described as being a species-specific surface antigen that is a member of the serine protease inhibitor gene family. The purified recombinant S. haematobium serpin protein was responded to with marked IgG4 and IgE and a high degree of species specificity in sera from S. haematobium-mfected persons. There was no correlation between serpin-specific IgE and IgG4 levels in 41 infected persons and serpin-induced antibody production in vitro, indicating that IgE and IgG4 responses are not co-ordinately regulated (90). Couissineir-Paris and colleagues (74) identified and purified a T-cellstimulating antigen from S. mansoni schistosomula that was able to trigger a strong cutaneous immediate hypersensitivity reaction in most adults living in a S. mansoni endemic area of Brazil. Children reacted weakly to this antigen preparation both in blastogenesis and in skin tests, although they mounted a significant reaction to crude larval antigen preparations. This T-cell response developed gradually in children and adolescents, was apparently not restricted by the HLA haplotypes common in the study area, and allowed the production of parasite-specific IgE. Thus, this T-cell response had features in common with the responses associated with the protection of chronically exposed humans from reinfection (91). Molecular cloning and biochemical characterization of the active component identified a 10 kDa polypeptide that had 70% homology with the sequences of previously reported proteins of

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unknown function (92). Interestingly, this antigen appears to be identical to a tegumental molecule identified by Hoffmann and Strand (93) as being a 10.4 kDa cytoplasmic dynein light chain (DLC). S. mansoni 10.4 kDa DLC was found to be developmentally regulated and localized to the tegument in the schistosomula, lung stage worms and adult worms, but is not present in the cercariae or ciliated miracidia (93). A second S. mansoni tegumental 20.8 kDa antigen has also been identified as having sequence homology with DLC. Although the highest concentration of this molecule was found to be present in the cercarial stage, it was found to specifically interact with S. mansoni 10.4 kDa dynein light chain (94). DLC's are components of dynein, a eukaryote enzyme complex involved in various aspects of microtubulebased motility and as such are likely to be crucial to the maintenance and repair of the schistosome tegument. The 20.8 kDa schistosome DLC has sequence similarity to a range of previously described schistosome tegumental antigens, including Sm21.7 (95) and the Sm22.6 (94), IgE against which is associated with human resistance to reinfection (85). A tegumental antigen with 64% sequence identity to Sm21.7 has also been identified as a target for IgE responses in S. japonicum-iniected individuals (96). Thus, it seems likely that a family of tegumental antigens, probably associated with the maintenance and integrity of the tegument, induce IgE and cutaneous immediate hypersensitivity reactions in individuals that have been observed to be resistant to reinfection with schistosomes. It is important to note that despite the generally observed correlations between immune responses and resistance to reinfection, we do not know what mechanisms might be killing which stage of the parasite life cycle in man. In vitro and animal studies indicate that early schistosomula are more susceptible to immune damage than adult worms. However, direct anti-adult worm and anti-fecundity effects occur, particularly against terminal-spined schistosomes, in a variety of experimental and domestic animal models (see the subsections "Schistosoma haematobium in Rodent Models," p. 168, and "Schistosoma bovis in Rodents and Other Animals," p. 169). This observation is one of the few derived from animal models that has been specifically tested in human populations. Agnew and colleagues (97) showed that there is a difference in the relationship between circulating worm adult antigen (a marker for worm burden) and levels of excreted parasite eggs in S. mansoniand S. haematobium-infected human populations in Kenya. The relationship between egg excretion and circulating antigen was constant with age in


S. marcsom'-infected individuals, but S. haematobium-iniected adults have a lower ratio of eggs to circulating antigen than infected children. This suggests that, as in animal models (98), an anti-worm fecundity effect was occurring in S. haematobium-iniected adults, but that this is not occurring in S. mansoni infections (97).

Factors That Influence the Expression of Potentially Protective Immune Responses Individuals living in schistosomiasis endemic areas differ widely in their susceptibility to reinfection and in their immune responses to the parasite. However, when studied as populations, rather consistent age patterns of susceptibility to infection and immune responses are discernible. As in murine studies, human schistosome infections induce predominantly T helper 2 cytokine profiles, with suppression of IFN-y responsiveness (40, 99, 100), and egg antigens induce stronger Th2-like responses than do worm antigens (100). Younger individuals, and those more recently infected, tend to have higher cellular (101, 102) and antibody (58, 103, 104) responses to egg rather than worm antigens. Nonetheless, IgE against the adult worm is typically higher in adults than in children. Webster and colleagues (103,104) found similar age- and sex-dependent patterns of antibody isotype response to worm and egg antigens in human populations living in S. mansoni endemic areas of Kenya and Brazil and a S. japonicum endemic area in the Philippines. Isotype responses to worms broadly increased with age whereas isotype responses to eggs decreased, and a higher proportion of males than females had detectable IgE against worm. Most isotype responses to adult worm antigens and egg antigens correlated positively with the intensity of infection with S. mansoni. In all populations the predominant worm antigen recognized by IgE was the S. mansoni or S. japonicum tegumental 22.6 kDa antigen, but only those individuals with relatively high IgE titers specifically reactive with S. mansoni or S. japonicum whole worm preparations had detectable IgE against SJ22.6 or Sm22.6 (103, 104). The correlations between IgE responses against worm and resistance to reinfection in Kenya were identified in blood samples obtained from patients after treatment but before the occurrence of reinfection, transmission being seasonal in the study area. IgE responses against the same antigens

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before treatment did not significantly correlate with subsequent reinfection intensities (41). Chemotherapy may modulate immune responses against the parasite by alleviating the suppressive effects of infection, or the intravenous death of parasites may expose previously cryptic antigens. Both of these effects may occur with treatment of human schistosomiasis. PBMC from Egyptian patients, assayed before and for two years after treatment with praziquantel, were found to have increased post-treatment proliferation responses to egg, w o r m or cercaria antigens, b u t not to phytohaemagglutinin, or a nonschistosome antigen (105). Similar results have been reported from other schistosomiasis endemic areas (76, 106-108). IL-ip release by monocytes is suppressed by infection and restored by treatment (109) and there is evidence that IL-10 contributes to parasite-specific T-cell hyporesponsiveness in patients with chronic schistosomiasis haematobium and mansoni (110112). Treatment of S. mansoni infections with hycanthone or oxamniquine resulted in a 5-6-fold increase in circulating eosinophils three weeks later, which was accompanied by a rise in antiworm antibodies and a decrease with antiegg antibodies (113). Similarly, when S. mansoni-mtected Kenyan schoolchildren were examined before and from 5 weeks to 18 months after treatment, antiworm IgG antibodies and eosinophil levels peaked 5 weeks after treatment, but declined thereafter (37). A treatment effect was also seen when antibody responses of 148 S. haematobium-mtected schoolchildren were measured before and u p to 12 months after chemotherapy. One month after treatment, the levels of all antibody isotype responses against worm antigen were increased, but one year after treatment they returned to pretreatment levels (114). When the pre- and post-treatment antibody responses to S. mansoni worms were compared in a study population previously used to show that post-treatment IgE against worm correlates with resistance, it was found that IgG subclass responses to worm were lower after treatment whereas IgM and IgE were higher. The increased IgE response was observed with different worm antigen preparations, including worm tegument extract, and after either praziquantel or oxamniquine therapy. There was no significant difference between pre- and post-treatment responses to eggs following either therapy (115). Interstudy differences in the detail of the immunological changes induced by treatment may be due to variation in the exact times post-treatment that were examined. However, it is clear that antiworm immune responses, particularly IgE, are boosted by treatment. Interestingly, when specific


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responses were studied in 110 S. haematobium-iniected persons before and 5 weeks after chemotherapy, IgG4 and IgE to adult worm antigen and IgE to SEA increased after chemotherapy in children but remained unchanged in adults. One explanation of this could be that immunoregulatory mechanisms operative in S. haematobium-iniected adults differ from those in infected children (116). Another explanation could be that many of the worm antigens that induce IgE are only revealed to the host immune system by dying worms. It is possible that IgE responses in untreated populations increase with age because older individuals have been exposed to more spontaneously dying adult worms than children have. The proportionally greater increase in specific IgE provoked by treatment of children could be because the children are being exposed to dying adult worms for the first time. In this respect, treatment would accelerate children's experience of infection, making it more similar to that of adults. It may be pertinent that mice infected with S. mansoni for u p to two years do not respond to the tegumental Sm22.6 antigen, IgE against which is associated with human immunity (41, 85). However, treatment of murine infections with either oxamniquine or praziquantel results in the induction of antibody and cellular immune responses against this and other previously unrecognized tegumental antigens (Jones et al., unpublished results). Based on a study of 41 S. haematobiuminiected children, Mutapi and colleagues also suggested that treatment accelerates a switch in antibody responses, in this case from predominantly antiegg IgA to IgGl (117) and that age-related changes in antibody isotype responses generally occur more rapidly in communities suffering high rates of S. haematobium transmission (25). One effect of chemotherapy may be to make children's experience of infection, in terms of exposure to antigens released by dying adults worms, more like that of adults and, thus, accelerate the induction of more protective immune responses. It is possible that the effect of chemotherapy on the host's responses to the parasite is why the peak shift phenomenon reported in untreated populations (23) is not readily apparent in the age-reinfection profiles after chemotherapy (Fig. 2).

Genetic Analysis of Human Resistance to Reinfection Within the generally observed convexed age pattern of infection and reinfection, it has been reported that certain individuals appear to be predisposed to rapid and severe reinfection (30,118). Abel and colleagues (119)

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Schistosomiasis

used segregation analysis, from family data, to determine whether a major gene could be identified that determined human susceptibility to infection with S. mansoni. This study was performed in a hyperendemic focus of S. mansoni (30) on 20 Brazilian pedigrees (269 individuals) that had been randomly collected, using both the unified mixed model (using 44 nuclear families) and the regressive model of analysis. The raw phenotypic values were adjusted for the relevant factors known to influence the intensity of infection (water contact, age and sex) before being subjected to segregation analysis. The results were consistent with the hypothesis that there is a codominant major gene controlling human susceptibility/resistance to infection with S. mansoni. Parameter estimates indicated a frequency of 0.200.25 for the deleterious allele, indicating that about 5% of the population were predisposed to high infections, 60% were resistant, and 35% had an intermediate level of resistance (119). This gene was termed SMI and Dessein and colleagues went on to perform a genome-wide study on 142 Brazilian subjects belonging to 11 informative families, using 246 microsatellite markers, with an average marker interval of 15 centimorgans. Only two adjacent markers on chromosome 5 gave LOD scores greater than 1.9. When this area of chromosome 5 was analyzed in more detail, two closely linked markers were found to give maximum two-point LOD scores of +4.74 and +4.52. This was corroborated by multipoint analysis, and indicated that SMI is most likely to be located in close proximity to the colony-stimulating factor-1 receptor in the chromosome 5q31-q33 (120). The same study group were typed for HLA-A, B, C, DR and DQ antigens and no evidence of physical linkage of SMI to HLA was found (121). Recently a similar analysis has been carried out on 15 extended pedigrees (154 subjects) in a newly emerged, epidemic focus of S. mansoni in Northern Senegal. In this study the intensity of infection, as measured by the numbers of parasite eggs counted in fecal samples, was adjusted for sex, age and water contact using the same methods as Abel and colleagues (119). The distribution of the adjusted intensities of infection differed from those described in Brazil. Whereas in the Brazilian focus a minority of individuals with high intensities of infection formed a distinct subgroup, in Senegal the distribution of infection intensities was more evenly distributed, with no discernible subgroup (122). A dominant mode of inheritance was determined as the best fit model of the Senegal data from segregation analysis, but this assumption failed to give a significant LOD score when used for genome mapping. In contrast, methods that were


independent of a specified genotype-phenotype relationship confirmed the effect of SMI and its location in chromosome 5q31-33 (122). The failure of conventional LOD score analysis to provide a significant result may have been due to differences between the endemic Brazilian and more recently exposed Senegalese populations, including the different lengths of time they had been exposed to schistosomiasis, to a lower allele frequency of SMI in the Senegalese population, or to different environmental influences. Nonetheless, the Senegalese study represents an important independent confirmation of the presence of a "schistosomiasis resistance" gene on human chromosome 5q31-33 (122). Perhaps significantly, this region contains several candidate genes that contain immunological molecules that have been shown to play an important role in human resistance to infection with schistosomes, including the granulocyte-macrophage colony-stimulating factor, IL-3, IL4, IL-5 immune regulatory factor 1, and IL-13. In addition, the region 5q3133 has been linked with a locus regulating IgE level (123, 124), and a locus controlling bronchial hyperresponiveness in asthma (125) and childhood asthma/atopy (126).

What Is the Relationship Between Schistosomiasis Age-Infection Profiles and Acquired Immunity? There is a solid consensus about the type of immune responses that correlate with h u m a n resistance to reinfection in ethnically diverse populations, exposed to different species of schistosomes in widely differing environments. However, the influences of exposure to infection, innate resistance and acquired immunity, controlled by age or duration of exposure, remain difficult factors to disentangle in schistosomiasis endemic areas, and it is clear that the influence of these factors can vary in different epidemiological situations. Although accumulating data from systemic immunological and genetic studies all point to an important role of acquired immunity after accounting for the effects of age and exposure, it is not certain that this immunity is a major factor in the typical age-intensity profiles seen in endemic areas. This is because there are two types of variation in reinfection intensities in human reinfection study cohorts. This can be seen most clearly by comparing the mean reinfection values plotted in Fig. 2 from various treatment-reinfection studies, with the individual reinfection values from

162 Schistosomiasis

£ o

o

Age (yr) Figure 3. Individual reinfection intensities after chemotherapy plotted against age in years (yr) from a study in Machakos District, Kenya. Reinfection intensities are mean eggs per gram of feces (epg) from multiple egg counts. This data is taken from (41).

one such study plotted in Fig. 3. The mean reinfection values show the dramatic change in susceptibility to reinfection around the age of puberty and, while this is still clear in the individual plots (Fig. 3), variation in the intensities of reinfection amongst those of similar ages can also be seen in the individuals' data. The most systematic immunological and genetic studies have identified correlates of human resistance to reinfection that are still significant after controlling for the effects of age and exposure (29, 30, 38, 41, 43, 67, 85, 119, 120, 122). This is entirely necessary in order to identify meaningful relationships between immunological parameters and reinfection. However, it is possible that one of the effects of removing the effect of age is that these studies are actually focused more on the variance in levels of reinfection within age groups, rather than the large change in susceptibility seen in the early teenage years. It remains to be seen whether


163

these two "modes" of resistance to infection/reinfection are predominantly controlled by the same factors.

Why Use Experimental Animals? The major gaps in our knowledge of human resistance to schistosomiasis, the identity of effector mechanisms and target stage of the parasite life cycle, are questions that can be effectively addressed in animal models. Moreover, experimental animals have an important role in acting as the first preclinical host for the design and testing of candidate vaccines against human schistosomiasis. Many of the studies performed in experimental animals are not possible in the natural human host for practical and ethical reasons. Thus, local immune responses in tissues through which the parasite migrates (e.g. skin, lymph nodes and lungs), or where they mature (e.g. hepatic-portal tract), can be examined in experimental animals with ease but are not available for studies using human subjects. Manipulations of the host's immune response (e.g. by serum transfers, or disruptions of defined immune components using gene "knockout" hosts) are also practicable in experimental hosts and are valuable tools with which to dissect the mechanisms of potentially protective responses. In addition, experimental models provide the opportunity to chart the development of immunity immediately following infection of the naive host, which is rarely feasible in man, but has a direct consequence for the design of vaccines to prevent infection in previously uninfected individuals, particularly young children. Finally, although measurement of resistance is normally confined to egg output as an indicator of worm burden in man, direct enumeration of adult worm populations can also be determined in experimental hosts following perfusion of the portal system. This is a particular advantage when one is determining the effectiveness of candidate "antiworm" vaccines. Resistance to reinfection conventionally relates to the reduction in worm burden of challenge infections, and this has been the focus of most research using experimental models. As such, the immune responses must operate against larval or adult worms, thereby preventing the production of eggs which are the major cause of morbidity; however, it is unlikely that a vaccination protocol will be 100% effective. Nevertheless, any reduction in worm burden should result in a corresponding decrease in the number of eggs that are produced and consequently reduce the level of transmission.

164

Schistosomiasis

Two other approaches have been described which seek to alleviate morbidity. First, "antifecundity" vaccines aim to reduce the number of eggs that are produced per female worm (e.g. (127); also see the subsection "Glutathione S-Transferases," p. 183). Second, "antipathology" vaccines aim to ameliorate the adverse effects of egg granulomas by altering the characteristics of the immune response (e.g. (128)). A major caveat to the interpretation of results from experimental animals is the extent to which they reflect the response in the natural host, particularly man. Although less well studied, interesting work is also being carried out on schistosome parasites of domestic animals. These veterinary models are uniquely important, allowing as they do greater manipulation of the host and parasite than is ethnically permissible in human studies, but allowing study of natural host-parasite relationships including, if required, natural exposure of animals to infection in the field. Clearly, careful consideration is required for each model in order to justify its study. Nevertheless, many invaluable observations on the nature of resistance to schistosomes have been made using experimental animals. Below, we describe some of the more recent and pertinent findings to appear from studies using experimental animals exposed to schistosomes which infect man a n d / o r domestic animals.

Innate Resistance to Schistosomes in Animal Models As discussed in the section "Acquired Versus Innate Resistance to Reinfection" (p. 143), the relative roles of innate resistance and acquired immunity can be difficult to distinguish in human studies, particularly in relation to hormonal changes associated with puberty (19). Studies in experimental animals show that female mice are more susceptible to infection, with a greater number of worm pairs reaching maturity (129). The reason for the greater maturation in female mice is likely to be hormonal, particularly since oestrogen and progesterone have profound effects on the immune system by increasing macrophage activity and the development of Th2 cells, respectively (see (130)). Moreover, testosterone has been implicated in the lower levels of susceptibility in male mice, since castrated animals had higher worm burdens (54). It has also been found that female mice treated with dehydroepiandrosterone sulphate were partially protected from infection (131).


165

Further study of the mechanisms of innate resistance to schistosomes in experimental animals is warranted. Such mechanisms could also involve physiological changes (e.g. skin thickness) which may not be related to an immune response. However, it is intriguing that, although the extent of worm maturation in many strains of transgenic mice deficient for particular components of the immune system is not different compared to wild type mice (e.g. IL-12, (132); IL-4, (133); IFN-y receptor, (134)), the absence of CD28 on T-cells (135) or IgE (136) results in a significant increase in the primary worm burden. Moreover, in mice deficient for IL-7, the migration and development of schistosome worms was markedly disrupted (137). In these mice, fewer worms reached the liver, the fecundity of female worms was reduced, and worms were much smaller. These findings are significant, but it is unclear how IL-7, which is primarily responsible for lymphopoesis, is affecting schistosome worms. The innate immune response is mediated by a number of different leucocytes including macrophages, neutrophils, NK cells, 78 T cells and Bl lymphocytes. Their contribution towards resistance against schistosomes is poorly studied. However, an insight into the role of Bl (CD5+) cells has been given in a recent report which showed that Xid mice (deficient in Bl cells) have a higher susceptibility to S. mansoni as judged by increased egg tissue loads and elevated mortality (138). The range of possible innate immune responses, which could operate against the primary infection, are poorly understood and would greatly benefit from further research. Ultimately, innate resistance may explain differences in the permissiveness of different host species, or the levels of maturation in different genetic strains of the same host (53, 139).

Resistance to Reinfection Induced by Normal Infections Many studies have been performed in different experimental hosts to determine whether normal parasites can elicit a protective response. However, interpretation of the results has proved controversial, with investigators disagreeing on which is the most appropriate model for the development of resistance to natural infection in man. We have taken an inclusive approach, highlighting the advantages or disadvantages of various models in turn.

166

Schistosomiasis

Schistosoma

mansoni in Rodent Models

The ease with which the life cycle of S. mansoni can be maintained in the laboratory is the major reason why it is the most intensively studied schistosome species. It can infect a variety of rodent animal hosts (mice, rats, rabbits, hamsters and guinea pigs) and primates (e.g. baboons, chimpanzees, vervets and rhesus macaques), but the mouse is the most frequently studied since it is widely available and is fully permissive to infection. In addition, the mouse develops many pathological features which are characteristic of human schistosomiasis. Between 20 and 50% of invasive cercariae mature in the hepatic portal system of experimental mice, and viable eggs are produced from day 42 onwards; the majority of nonmaturing larvae are trapped and die in the lungs. Acquired immunity in this model was originally thought to operate via a mechanism termed "concomitant immunity" (140). In this situation, animals with a chronic infection retain their primary worm burden, but are completely immune to a challenge infection. However, this concept was questioned when it was observed that infections with cercariae of a single sex would not generate protective immunity (e.g. (141,142)) and that, although immunity was directly correlated with tissue egg burden, administration of large numbers of eggs failed to elicit protection (143). Moreover, immunity could not be passively transferred to naive animals, even by parabiotic union (144). It was later demonstrated that deposition of eggs in the mouse liver induces a sequence of pathological changes which breach the integrity of the portal system (reviewed by (145)). As a result, schistosomula, newly arrived in the portal vasculature, escape via porta-systemic anastomoses. This hypothesis was supported by investigations of 129 strain mice, of which a large proportion develop solid immunity to primary S. mansoni infection, demonstrating that natural porta-caval shunts prevented the maturation of larvae in the portal tract (146). Therefore, it was concluded that "concomitant immunity" as a cause of resistance to reinfection in the mouse is an artefact of pathology, and is not an acquired immune response (145). However, it is unclear whether this phenomenon occurs in infections with all schistosome species, or whether it occurs in all experimental and natural hosts. The problems of egg-induced pathology in mice obscure whether the immune responses induced by normal parasites can be host-protective. Prior to egg production, normal parasites elicit antigen-specific CD4 + Th cells in


167

the skin-draining lymph nodes (sdLN) (146), lungs (147) and spleen (148) capable of secreting both IFNg and IL-4/IL-5 suggestive of a mixed T h l / Th2, or ThO response. However, the T-cell responsiveness in the sdLN and lungs is transient and not sustained. Antibodies against both larval and adult worm antigens are also produced early after infection, and a faster increase in the levels of IgGl compared with IgG2a isotypes indicates that Th2 cytokines play an increasing influence in determining the antibody response prior to patency (149). The onset of egg deposition between 6 and 8 weeks triggers a marked shift towards the production of Th2-associated cytokines (148, 150). This is reflected in an increase in IgGl antibodies, particularly against soluble egg antigens (149). The formation of the granuloma around schistosome eggs is the major causative agent of pathology and is related to the intensity of infection. The development of the granuloma passes through an acute phase around weeks 8-10, when the reaction is at its most intense, before being down-modulated thereafter (reviewed by (151)). The immunological components of the granulomas which form in the liver (e.g. (152)), or in the lungs after intravenous egg delivery (e.g. (153)), are largely characteristic of Th2-type immune responses (154). In addition, IL-10 has a key role in the development and modulation of the granulomatous response (155) and is discussed in more detail elsewhere in this book. Unfortunately, there are few studies on the development of resistance to reinfection after drug treatment of schistosome infections in mice, where the problems of egg-induced pathology can be safely ignored, which may aid interpretation of similar studies in man. However, in general, it appears that drug-treated infections do not induce significant levels of protective immunity to reinfection (reviewed by (139, 156)). One exception is the treatment of larvae, shortly after infection, with the experimental drug R o l l 3128, leading to the development of high levels of CD4 + -mediated protection (157,158), but this drug did not proceed to clinical trials. Further investigation of the effect of drug treatment and resistance to reinfection on experimental animals may yield beneficial information. In contrast to the mouse, the laboratory rat (Rattus norvegicus) is considered semipermissive in that although larvae migrate to the portal tract, the majority of the worms are eliminated before maturation, in a self-cure response around day 28. The larvae are slightly faster in their migration compared to in the mouse but only 25-30% reach the liver. Since the

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Schistosomiasis

parasites fail to reach patency, egg-induced pathology does not complicate the assessment of acquired immunity which is readily induced. IgE has been directly implicated in this phenomenon (159), and schistosome-specific IgE is seen to increase over the self-cure period (160). Moreover, RT-PCR of cytokine mRNA has revealed significantly increased levels of Th2-associated IL-4 and IL-5, in the absence of elevated IFN-y, in the lungs and draining lymph nodes of infected rats up to day 21 (161). After a secondary infection, IL-4 and IL-5 secretion remained elevated, as did the production of Th2associated antibody classes (162). It has also been reported that the demise of the parasite is associated with pronounced hepatic mastocytosis (163). Degranulation of mast cells (bearing Fc epsilon receptors), evidenced by the release of rat mast cell protease II into the bloodstream, coincides with elimination of primary worms in the hepatic-portal distributaries. Moreover, the allergenic properties of two worm products (67 & 36-38 kDa) can be ascribed to the relative abundance of carbohydrate molecules, implicating glycans in triggering mast cell degranulation (160). Thus, immunity in the rat appears to be Th2-biased with the characteristics of an immediate hypersensitivity response. This might provide a model with which to design a protective vaccine mediated by IgE associated with resistance in humans (41, 67, 84). However, such a vaccine strategy will need to be carefully designed in order to avoid exacerbating other allergic reactions. Although the semipermissive laboratory rat (R. norvegicus) is conventionally used as an experimental model, the black rat (R. rattus) is a natural host for S. mansoni and is fully permissive. Responses to infection, including the development of porta-caval shunts, are therefore more similar to those observed in infected mice (164). Other immunological analyzes on this model have not yet been performed but may prove advantageous.

Schistosoma

haematobium

in Rodent Models

Laboratory mice have been used as experimental hosts for S. haematobium but it is not possible for the parasites to reach the bladder wall (the natural site of infection in man). Instead, eggs are deposited in the intestinal wall and passed in the feces. This, combined with the fact that the life cycle is more difficult to maintain in the laboratory, has led to few studies of this species in rodent hosts. In mice, the parasite has a slower migration to the lungs, a lower level of maturation (165) and a longer prepatency period than


169

S. mansoni (166-168). The number of eggs produced declines markedly after week 16 in normal compared with thymectomized animals, leading to the suggestion that some form of immunological process was operating late against adult worms (167). A similar phenomenon was observed in a murine model of the closely related S. bovis, but in neither case has the contribution of egg-induced pathology as a cause of the observed reduction in egg and worm numbers been discounted (167). Nevertheless, there may be a real difference in the immune mechanisms operating in S. haematobium versus S. mansoni infections in mice which reflects observations in natural hosts such as man (97). A recent study has been the first to examine cellular immune responses to S. haematobium in mice (168). Changes in responses were related to the long prepatent period, with a marked increase in Th-2-associated IgE and IgA after the start of egg laying from week 10. In contrast, levels of antiparasite IgG increased progressively from week 4. There was also an increase in the proliferation of splenocytes and IFN-y secretion to worm antigens u p to week 14, but both declined thereafter. This is indicative of immunological downregulation, possibly related to the production of IL-10.

Schistosoma

bovis in Rodents and Other Animals

Although S. bovis is a parasite primarily of cattle, it will infect mice, and recent studies have characterized the immune response to a normal infection. These show that the parasite elicits predominantly IgGl antibodies with a marked increase in IgE and IgA after the onset of egg deposition (169). In terms of the T-cell response, there is a similarity to S. mansoni, with Th-2 cytokines being produced by in ciiro-stimulated splenocytes after patency, although IL-10 was induced earlier in response to stimulation with adult worm antigens. From about 10 weeks after infection, there is a progressive loss of both adult worms which appear to be T-cell-dependent, suggestive of an immunological mechanism to adult worm attrition in this model (167). Early work on S. bovis infections in cattle was among the first to report an "antifecundity" effect caused by the presence of a prior schistosome infection. As such, infected cattle challenged with 70,000 cercariae developed less severe clinical symptoms than previously uninfected animals, and this was probably due to the suppression of egg production from challenge worms (170). The tissue egg densities were 78-100% lower in infected and

170

Schistosomiasis

challenged cattle compared with those receiving the challenge infection only (171). However, the challenged worms regained their ability to produce eggs if recovered and transferred to naive cattle (172). The immunological basis of the antifecundity effect is not completely clear although passive transfer of sera from infected animals has an effect on reducing the tissue egg load (172, 173). Recently, the goat has provided an alternative model for S. bovis infections. Egg excretion declines after an early peak, indicative of the progressive development an antifecundity immune response (174). Tissue egg counts did not increase after a challenge infection, further supporting this proposal (174). Moreover, with time after primary infection, egg-associated granulomatous reactions in the intestine increased and this may be related to a decrease in the number of eggs that are passed in the feces (175).

Schistosoma

japonicum

in Rodent Models

S. japonicum is unique amongst the major species that infect man, in that it is also a significant parasite of domestic animals, primarily cattle. As such, although rodents have been used as experimental animals, other species such as the pig have also been studied. In mice, the parasite undergoes a very rapid migration to the lungs (176) and discrete petechial hemorrhages are visible on the lungs during the first week after infection (177). Indeed, migration to the lung is faster than for many other schistosome species (178). Maturation is also relatively high (-45-75%) but this depends upon the geographic isolate studied (177). The granulomas which form around the S. japonicum eggs in the liver have similar characteristics to those that form around S. mansoni eggs. The granulomas reach peak size at week 6 but decrease gradually thereafter; eosinophils are a major cellular component of the granuloma recruited in response to a locally released chemotactic factor (179). Th-2 cytokine production by splenocytes also increases after egg deposition in the liver and is accompanied by the down-regulation of Th-1 cytokines (180). Moreover, exogenous recombinant (r)IL-12 (which inhibits Th-2 cytokine production) reduces the size of pulmonary egg granulomas (181), whilst anti-IL-4 treatment inhibits hepatic fibrosis and splenocyte release of IL-4 and IL-5 (182), confirming that the immune response is largely of the Th-2 type. Resistance to reinfection appears to be closely related to the number of eggs


and the extent of pathology (183, 184). Indeed, mice exposed to single sex infections which showed no overt pathology were not resistant to reinfection (185). Thus, any analysis of resistance in this model, by analogy with studies in S. mansoni, is complicated by egg-induced pathology. Recently, the pig has been examined as a laboratory model for S. japonicum, since it is a natural reservoir of this parasite and has several physiological and anatomic similarities to man (186). Examination of the effect of a primary infection appears to suggest that it prevents the establishment of challenge parasites (187), and results on the immunological status of this host are keenly awaited.

Schistosoma

mansoni and S. haematobium

in Primate Models

Primates provide good experimental models for S. mansoni which should be closer to the situation pertaining in man due to similarities in size and genetic constitution (188). Nevertheless, for obvious reasons there have only been a limited number of studies. It appears that schistosomula migrate faster in the olive baboon (Papio anubis) than rodents and there is a high level of maturation (-80%) (189, 190). Maturation is also high in the vervet monkey (Cercopithecus aethiops) (191). It is unclear whether normal cercariae elicit any form of immunity in this infection model and the immune response has not been fully characterized. Baboons provide a host where exposure to multiple doses of normal cercariae is feasible. It appears that resistance to reinfection which develops after multiple exposures is not the consequence of pathological changes which disrupt the integrity of the liver (192). In fact, protection of 59 and 80% was recorded for baboons which had been exposed respectively to either a single dose of 1000 cercariae, or to 10 doses of 100 cercariae/week, and then cured with Praziquantel prior to challenge with 1000 normal cercariae (193). Moreover, it was observed in this study that levels of antiworm IgE antibodies correlated with the development of resistance indicating that some form of acquired immunity had been induced by the normal infection. In another study, this group also showed that the development of hepatic granulomas and pathology was modulated more rapidly in baboons exposed to multiple, rather than a single, dose of infective larvae (194). The baboon has also been used as a model for S. haematobium (195,196) although, as for the mouse, there is an atypical site of parasitization

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Schistosomiasis

in the mesenteric veins. The best model of urinary pathogenesis is provided by the monkey Erythrocebus patas (197).

Resistance Induced by the Radiation-Attenuated (RA) Vaccine Exposure of infective cercariae to gamma (y), or UV, irradiation results in their attenuation such that, depending upon the radiation dose, parasites fail to mature and thus do not provoke egg-related pathology. An important feature of optimally attenuated larvae is that they can induce significant and consistently high levels of acquired immunity to challenge infection in a number of experimental hosts (mice, rats and primates) (reviewed by (198)). For many researchers, this experimental vaccine provides the best paradigm for the design of an effective vaccine against schistosomiasis in man (199). The RA vaccine cannot be used in man, since it is impossible to predict the dose of irradiation that would induce adequate protection but prevent a minority of attenuated larvae reaching sites such as the brain. However, the vaccine has been proven to be effective in controlling disease in domestic livestock.

RA Vaccine-Induced Resistance to Schistosoma Rodent Models Induction

of Protection

mansoni

in

by the RA Vaccine in Mice

In mice, up to 60-75% protection can be induced by a single exposure of 500 cercariae attenuated with - 2 0 krad radiation (132). The sole effect of the vaccine is a reduction in the number of challenge larvae that can mature, resulting in lower adult worm burdens and by implication the number of pathological reactions to eggs. There is no evidence, so far, that this vaccine in the mouse has an effect on worm fecundity, or on the size and composition of egg granulomas resulting from the few challenge worms that do mature (Mountford, unpublished observations). The level of protection depends upon the strain of mouse used, with C57BL/6 mice regarded as high responders (201, 202). The optimum dose of irradiation permits migration of attenuated larvae to the lungs but no


further (203). A higher dose of 80 krad prevents parasite migration to the lungs but the mice are not immune (204), and a dose lower than 20 krad permits some larvae to mature and produce eggs, thus resulting in complications from egg-induced pathology. It is thought that a key feature of the vaccine is that attenuated parasites and their antigens persist in sites such as the skin, sdLN and lungs, thus increasing the exposure of parasite-released material to the host's immune system in these tissues (205). In contrast, normal parasites migrate rapidly through these sites (203). Antigens must be processed by antigen-presenting cells (APCs) in the skin and sdLN, and then presented to naive T-cells to prime the necessary immune response. It is thought that antigens released by the parasite as part of its normal developmental process are the most likely candidates to stimulate the protective immune response since they are readily available (compared with somatic constituents) for endocytosis by APCs (206). Moreover, they correspond to the antigens which are most likely to be produced by live challenge parasites. However, it has been suggested that irradiation may induce abnormal, or normative, antigens that make these parasites more immunogenic (207). One limitation of this theory is that, to be effective, the memory T-cell population resulting from priming must be reactivated by antigens from normal parasites which will not have such altered epitopes. Optimally attenuated lung stage larvae are also highly immunogenic, since they can elicit high levels of protection if administered to naive mice via an intradermal route (208). This argues that antigens specific to the cercariae, or skin stage larvae, are not necessary for the induction of immunity (206). Moreover, antigens from lung stage larvae are clearly the most potent inducers of proliferation and cytokine production by cells from the sdLN and lungs of vaccinated mice (209). Attenuated larvae induce a dominant Th-1 cell population in the sdLN soon after vaccination, as judged by the secretion of abundant IFN-y, rather than IL-4, by in vitro cultured lymphocytes; the response also persists for much longer than after a normal infection (146, 205). In comparison, little is known about the events in the skin which may bias the phenotype of the immune response, but attenuated larvae in the dermis provoke the accumulation of MHC II + MAC-1 + cells, some of which are C D l l c + and may be dendritic cells (210). Cells with a Th-1 phenotype are recruited from the sdLN to the lungs by the arrival of attenuated larvae in this site (147, 205), and are thought to prime the lungs against the arrival of challenge parasites

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Schistosomiasis

(211). The Th-1 bias of the immune response in the sdLN and lungs is supported by analysis of the pattern of cytokine mRNA transcripts (212,213), which also indicate a potentially important role for IL-12. The role of IL-12 in the protective Th-1 response induced by attenuated larvae has been supported by data which show that administration of rlL12 to mice shortly after vaccination boosted the secretion of Th-1-associated IFN-y, and the production of IgG2a and IgG2b antibodies (132, 212, 213). Levels of protection were also elevated in these mice. In contrast, rIL-12 caused a decrease in IL-4, IL-5, IL-13, eosinophilia and serum IgE (132, 212, 214, 215). It has been shown that mice genetically deficient (KO) for the p40 subunit of functional IL-12 have markedly reduced levels of protection associated with an absence of IFN-y secretion (132). Administration of rIL-12 to these IL-12 KO mice shortly after vaccination was capable of completely restoring I F N - Y production, and the level of resistance was comparable with wild type mice. However, it was also noted that residual levels of resistance (-40%) in IL-12 KO mice occur where there are increased levels of Th-2-associated cytokines (IL-4, IL-5 & IL-13), serum IgE and pulmonary eosinophilia (132, 215).

Cell-Mediated

Immune Effector

Mechanisms

It is generally accepted that elimination of the majority of challenge parasites in vaccinated mice occurs in the lungs via a CD4 + -dependent cellmediated immune mechanism (198), and the peak cell population in the lungs coincides with the arrival of challenge parasites (147). Lymphocytes from the lungs of vaccinated and challenged normal mice secrete abundant IFN-y (147), and there is an increase in the mRNA transcripts for IFN-y, TNF-cx and IL-12p40 (134, 216). The challenge parasites elicit, and become surrounded by, dense cellular foci (217). The cellular influx is likely to be the primary cause of elimination for a significant proportion of challenge parasites in the lungs (198). There is no consensus regarding the mechanism of parasite killing. Macrophages are a possible agent, probably involving the production of nitric oxide (NO). Indeed, vaccinated mice have increased mRNA for inducible nitric oxide synthase (iNOS) in the lungs after percutaneous challenge, whilst administration of a specific iNOS inhibitor (N-monomethyl-L-arginine)


175

resulted in a modest decrease in the level of protection (216). However, two recent studies using mice genetically deficient for iNOS indicate that because protective immunity induced by the RA vaccine was only slightly lower (-30%) than in intact animals, cytotoxic killing by N O is not the major cause of parasite elimination (218, 219). In contrast, the role of IFN-y in the Th-1-biased effector response in the lungs and the generation of protective immunity, is largely undisputed. A series of in vitro and in vivo studies by James and colleagues were the first to highlight the importance of IFN-y in the immune effector response (reviewed by (220)). Furthermore, administration of monoclonal antibodies (mAb) against IFN-y to mice (after the Th-1 response has been primed by vaccination), over the period when challenge parasites are migrating through the lungs, eliminated immunity by more than 90% (221). Furthermore, resistance to reinfection in mice with a genetic disruption to the IFN-y receptor, or IFNy, was also markedly reduced (134,222). Recent experiments investigating the Th-1-associated inflammatory cytokine TNF-a show that it too is likely to play a crucial role in determining the effectiveness of the protective response. In this respect, it has proven impossible to vaccinate mice deficient for the TNF-a p55 receptor with the RA vaccine, although the precise mechanism underlying this failure remains to be determined (223). On the other hand, protective immunity induced by the RA vaccine remained intact in mice depleted of IL-5 (224), deficient for IL-4 (133), or deficient for IgE (136), suggesting that these components are not required for the protective immune response, and that Th-1 responses are sufficient to provide high levels of protection. However, in situations where Th-1 responses are prevented from being induced (i.e. in mice deficient for IL-12, or the IFNy receptor), polarized Th-2-type responses develop instead (215), and these may be responsible for the partial levels of resistance observed in these mice (132). It remains clear that the conventional response to the RA vaccine is Th-1-biased, with the formation of dense cellular aggregates in the lungs around the challenge worms. It has been suggested that these foci, rather than being cytotoxic, block onward migration of a significant proportion of challenge worms, since parasites recovered form the lungs of vaccinated and challenged mice can mature if transferred to the lungs of naive cohorts (225). In support of this hypothesis, mice with a disrupted IFN-y receptor gene develop large diffuse inflammatory foci which allow challenge parasites to

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negotiate and complete their migration (134). Thus, the precise mechanisms responsible for parasite elimination in the lungs remain to be fully elucidated. One potential explanation for the diffuse foci was the decreased expression of ICAM-1, which via LFA-1 mediates homotypic cell adhesion, in mice lacking a functional IFN-y receptor (226). However, in ICAM-1 gene disrupted mice, protective immunity to the RA vaccine was only reduced by a mean of 15% (226). The RA vaccine is a favored model for the development of a vaccine precisely because it induces a novel response not normally encountered by the challenge parasite, and against which it has not evolved an evasion escape mechanism (199, 227). The lung stage parasite therefore provides a rich source of antigens which should be capable of eliciting the desired Th1-mediated immune response (206). Indeed, as a first step in defining the production of a second generation vaccine which induces a Th-1 response against schistosomes, it was found that coimmunization of mice with soluble lung stage antigens plus rIL-12, induced abundant IFN-y in the sdLN and lungs, and resulted in the induction of significant levels of protective immunity (228).

Antibody-Mediated

Immune Effector

Mechanisms

Although it has been unequivocally demonstrated that immunity to the RA vaccine involves a Th-1 cell-mediated mechanism, antibodies are produced following vaccination. Indeed, there has been much debate as to their role in protection since cell-mediated immune responses are classically associated with weak antibody production. Antigen-specific IgG2a (Th1-associated) antibodies are produced in mice shortly after exposure to attenuated larvae but so are those of the IgGl isotype (149), which are generally thought to be dependent upon the production of IL-4 (229). Vaccination also boosts the levels of IgG2a, IgG2b (Th-1-associated) and IgGl antibodies by 5 weeks after challenge infection, compared to mice exposed to challenge parasites only, although levels of IgE are much reduced (132). However, in a recent study, the majority of (iMT mice (which lack B cells) exposed to a single dose of attenuated cercariae develop protective immunity equivalent to those in wild type (C57BL/6) mice (230). The nMT mice also develop the characteristic Thl-biassed immune response, so appearing to


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confirm that antibodies are not required for resistance induced by the RA vaccine. In a very similar study using |*MT mice exposed to a single dose of the RA vaccine, Jankovic et al. (222) found that the mean level of protection in all mice was reduced by about 40% compared to wild types. Moreover, they found that the residual level of protection in vaccinated nMT mice could be abrogated by administration of anti-IFN-y antibody, and that protection could be restored to vaccinated [xMT mice using sera from singly vaccinated wild type mice (222). These findings suggest that both antibody and cellmediated (IFN-y dependent) immune mechanisms operate in mice exposed to a single dose of the RA vaccine. However, the mechanisms by which antibodies might function are unclear since it has also been shown that mice deficient in the FcR y chain (fail to express FcyRI, FcyRIII and FceRI) develop high levels of protection to the RA vaccine (222). This would seem to rule out immune responses mediated by IgGl, IgG2a, IgG2b and IgE that signal through the common FcR y chain. On the other hand, in mice exposed to multiple doses of irradiated larvae, there is clear evidence that antibodies do play a significant role in protection. In this respect, although immunity to a single dose of the attenuated vaccine can be totally ablated by the use of anti-CD4 + antibodies, this was less effective in animals exposed to more than one dose (157). Multiple exposures of mice to the attenuated vaccine resulted in modest increases in the levels of immunity compared to that elicited by a single dose (214, 231, 232), but such an increase was not detected in multiply vaccinated mMt mice (222, 230). In fact, exposure of wild type mice to multiple doses of the vaccine promoted the production of Th-2-associated reactivity, measured by antigen-specific IL-4 and IL-5 production by sdLN cells, which is consistent with the increased production of IgGl antibodies (231). Multiple vaccination of mice genetically deficient for IFN-y (222), or a host of cytokines alone and in combination (i.e. IL-4, IL-12, IL-10, IL-10 & IL-4, IL-10 & IL-12) (232), increased the levels of protection compared to those obtained with a single vaccination. In most cases, the increases in protection could be attributed to an increase in the titre of one or more of the different antibody isotypes. The protective capacity of antibody in multiple- (but not singly) vaccinated mice can be confirmed because sera from such animals were capable of passively transferring protection to naive mice (e.g. (202, 214)). Interestingly, splenocytes from multiple-vaccinated mice can still secrete abundant IFN-y, indicating that the cell-mediated arm of the immune response had not been removed

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(231). In this respect, administration of rIL-12 to multiple-vaccinated mice simultaneously increased IFN-y production and serum levels of Th-1associated IgG2a and IgG2b but not Th-2-associated IgGl, or IgE antibodies (214). Serum from these mice was also highly effective in transferring resistance to naive mice. It remains to be established how antibodies from multiple-vaccinated mice operate to confer protective immunity, but it provides another route by which it may be possible to elicit protective immune response to a putative vaccine in man.

Antibody

and Cell-Mediated

Immune Effector

Mechanisms

The above studies have in the past two years brought about a reappraisal of the mechanism by which protective immunity to the RA vaccine operates in the mouse. It has been widely accepted that the Thl (cell-mediated) and the Th2 (humoral) responses to infectious agents are mutually antagonistic. In the context of schistosomes, it appears that both Thl and Th2 type responses are protective to different extents. In this respect, Wilson and Coulson (227) proposed a "Happy Valley Hypothesis" whereby schistosomes survive in the host where the immune response is relatively ineffective and comprises either a mixed T h l / T h 2 , or a ThO cell population. However, protective immunity against the parasite is achieved when the immune response evolves to a polar Thl (e.g. RA vaccine plus IL-12), or a Th2 (e.g. deficient in IFNy signaling) immune response. It appears that the cell-mediated immune response at the Thl pole is more effective and generates a higher level of protection, than one at the Th2 pole utilizing antibody. However, a ceiling to sterile protection appears to be present at both poles and may be associated with the relative abundance of a regulatory factor such as IL-10 or TGFp. The recent studies by Hoffman and Wynn, using mice deficient one or more cytokines (232), have led to a different conclusion as to the relative importance of Thl and Th2-type associated responses. Their study examined the protective effects of the RA vaccine in mice with extremely polarized Thl (i.e. deficient in both IL-10 & IL-4) and Th2 (i.e. deficient in both IL-10 & IL-12) cell populations. They concluded that although the actual effector mechanism may differ in mice polarized towards type 1, or type 2 responses, both mechanisms can confer protective immunity, particularly after multiple exposures to the RA vaccine. Moreover, they present data showing that mice


179

deficient only for IL-10 have elevated cell-mediated (IFN-7) as well as humoral responses compared to wild type mice, suggesting that protective Thl and Th2 responses can develop simultaneously in the same host (232). This raises the hypothesis that a vaccine which elicits both strong cellular and humoral responses might be more desirable than a strategy designed to elicit one or other polarized response. Nevertheless, it remains unclear how antibodies may contribute towards a protective effector response since signaling via the FcR y chain is not required for immunity to a single or multiple doses of the RA vaccine (222). On the other hand, the issue of how cell-mediated mechanisms operate in mice after a single dose of RA larvae also remains to be fully resolved (223). Resolution of these gaps in our knowledge on immune effector mechanisms would be beneficial in the design of eventual vaccination strategies with the appropriate antigen(s).

Induction

of Protection

by the RA Vaccine in Rats

Although not studied in recent years, a single dose of 20 krad irradiated cercariae is sufficient to induce 75% protection in Fischer strain rats (233). As for mice, it was shown that this vaccination regime led to the death of optimally attenuated larvae after they reached the lungs. Even greater levels of resistance were induced in rats given multiple doses of the vaccine, and resistance persisted for at least 25 weeks, unlike following a normal infection which declined to 21% by this time (233). In addition, serum from these immunized hosts can confer passive protection on naive animals if given at the time (days 5-7) challenge parasites migrate through the lungs (234). Although protection cannot be induced by irradiated cercariae in nude rats, suggesting that T-cells are required, serum from vaccinated normal rats passively conferred protection on these animals (235). This underlines the importance of antibodies in the effector response, whilst T-cells are necessary during the induction phase. In line with studies in mice (228), significant protection to challenge infection can be conferred in rats by the administration of a worm extract (S3) in conjunction with rIL-12 (236). This correlated with elevated IgG2b and the suppression of IgGl, but since protection could not be passively transferred using serum from S3 plus IL-12 immunized rats, it was concluded that the vaccination strategy might confer protection by cell-mediated mechanisms.

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RA-Vaccine-Induced Resistance to S. haematobium in Rodent and Other Animal Species

and S. bovis

Irradiation of S. haematobium cercariae can induce very high levels of resistance in mice to homologous challenge infection (165, 166) and against heterologous challenge with S. bovis (167), but not against S. mansoni (165, 237). However, vaccination with S. mansoni can protect against S. haematobium (165). It is, in addition, possible to elicit moderate levels of resistance in gerbils exposed to RA S. haematobium (238) but, as yet, no studies have been performed to determine the immunological basis of the protective response induced by irradiated S. haematobium in any rodent species. RA S. bovis larvae induce significant levels of immunity to homologous challenge in mice, and against S. haematobium, since these two species are phylogenically closely related (166). These studies followed earlier experiments in cattle where attenuated S. bovis larvae stimulated significant resistance to a laboratory challenge (239). The effectiveness of the RA vaccine was confirmed under field conditions by releasing immunized calves into an enzootic region (240). Ten months later, there was a lower mortality rate and faecal egg count in the test group, which after necropsy had a 60-70% reduction in their worm burdens and tissue egg counts. The application of recombinant vaccines against S. bovis, particularly for their ability to reduce worm fecundity, appears promising (see the subsection "Glutathione STransferases," p. 183). However, the immunological features of the protective immune response to the S. bovis RA vaccine in mice and cattle have not yet been analyzed.

RA-Vaccine-Induced Resistance to S. japonicum Other Animal Species

in Rodent and

UV irradiation is effective in attenuating larvae of S. japonicum, resulting in high levels of protection to percutaneous challenge in murine models (183, 241), although y-irradiation appears not to be so effective (242). Vaccination with attenuated S. japonicum does not, however, protect against S. mansoni, further demonstrating the species specificity of most of the RA vaccines (237). Attenuated S. japonicum larvae elicit marked changes in the B and CD4 + cell populations of the sdLN, which are comparable with those observed to S. mansoni larvae (243), but other studies on the cellular responses to the vaccine


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have not been performed. However, it appears that antibody-mediated responses are an important component of the protective response, since serum from vaccinated mice can confer passive protection on naive mice (184). Increasing the number of vaccinations elevates the protective capacity of the serum, particularly the IgG constituents, and the transfer of serum is most effective when administered around the lung phase of migration (244, 245). As for rodent models, UV light has been used to attenuate cercariae of S. japonicum to vaccinate buffaloes (246) and pigs (247), resulting in very high levels of protection to a laboratory challenge. Thus, although the RA vaccine cannot be used in man, it may be effective in reducing zoonotic transmission of S. japonicum.

RA-Vaccine-Induced Resistance to Schistosoma S. haematobium in Primate Models

mansoni and

Since primates are phylogenically closer to man, they have been used in a limited number of studies to test the efficacy of RA vaccines against S. mansoni and S. haematobium (189). It is often difficult to directly compare the results between studies due to differences in a number of experimental procedures (e.g. number of immunising cercariae and dose of irradiation). For instance, whilst three vaccinations with S. mansoni cercariae attenuated with 6 krad failed to elicit any protection in baboons (248), two vaccinations with 60 krad attenuated cercariae induced up to 70% protection (249). It is also apparent that, unlike the mouse, a single vaccination is not sufficient to induce significant protection, and consequently multiple exposures are the norm (190). In this context, four vaccinations with 30 krad irradiated cercariae were shown to induce more than 80% immunity in the baboon host (250). In this study, the degree of protection induced in individual animals closely correlated with the titer of antibody at the time of the challenge. This was also reported to be the case in a study where baboons, vaccinated three times with 30 or 60 krad attenuated cercariae, were protected to 50% (190). The titers of both IgG and IgM antibodies to larval and adult worm antigens were boosted following each successive vaccination, as was the proliferation of peripheral blood lymphocytes stimulated in vitro (190). However, after the challenge, the titers of both IgM and IgG antibodies were observed to decline. It has also been reported that intestinal pathology (smooth muscle hypertrophy and villous atrophy) in infected baboons was less marked

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in those animals which had previously been vaccinated with irradiated cercariae (251), probably as a result of the reduced tissue egg count, rather than an "antipathology" effect. A couple of studies have examined the induction of protection in the vervet monkey which is permissive to S. mansoni. Again, multiple exposure to attenuated cercariae is required for the induction of the highest levels (30-50%) of protection (191, 252). However, a reduction in the level of protection induced was observed when five doses, compared with three, were administered, and this was mirrored by a decrease in the proliferative responses of blood lymphocytes and the levels of IgG antibodies (191). High levels of protection to homologous challenge can also be conferred by three exposures to irradiated S. haematobium cercariae (253). Intramuscular injection of attenuated newly transformed schistosomula was also effective in inducing protection. However, although specific serum antibodies were induced by this vaccination approach, there was little correlation with the level of protection induced in individual animals (254). Finally, it was found that vaccination was more effective if the challenge parasites were administered as a trickle over several weeks rather than a single dose (196). This may be a valid approach, since it more closely resembles the situation which would face potentially vaccinated humans. There are strong arguments for performing further experiments using the RA vaccine in primates. However, standardization of experimental protocols would be desirable. Moreover, as immunological reagents become available, a detailed analysis of the immune responses to the vaccines to identify key features, particularly with regard to cellular reactivity, should become more feasible.

Resistance Induced by Native and Recombinant Schistosome Antigens One of the major goals of schistosomiasis research using experimental animals has been to identify candidate vaccine antigens. Indeed most of the antigens currently under investigation were originally identified from laboratory animals protectively infected, or immunized with live parasites, or their crude antigenic fractions. Subsequently, it has been shown that many of the isolated antigens are also recognized by human antibody a n d /


183

or peripheral blood lymphocyte responses, confirming their immunogenic potential (see the section "Schistosome Antigens Associated with Human Resistance to Reinfection," p. 151). The majority of these antigens have been tested in experimental animals for their "antiworm" potential as judged by decreased worm burdens in immunized compared to control mice, but others

may have "antifecundity" or "antipathology" effects. As yet, no schistosome antigen has been tested in man, although Phase 1 clinical trials are soon to be initiated (5). Moreover, some antigens have been used successfully to immunize domestic livestock against natural infections. In 1991-92, the World Health Organisation Special Programme for Research and Training in Tropical Diseases (WHO/TDR) selected six of the best-characterized schistosome antigens for testing in independent vaccine trials in experimental animals (255). The main criterion for inclusion in this trial was prior publication of experiments showing the induction of protective immunity; a brief summary of each of these protective antigens follows below. Unfortunately, "...the stated goal of consistent induction of 40% protection or better was not reached with any of these antigen formulations" (5). These findings have led to a reappraisal of the vaccine program, with revised goals for each of the six antigens concerned (5). One of the major conclusions of the trial was that attention should be paid to how candidate vaccine antigens are formulated in order to stimulate optimum immune responses correlating with protective immunity. Surprisingly few of the antigens have been rigorously tested in this respect and most have only been reported in the context of a single formulation. It might also be concluded that the most potent vaccine candidates have not yet been identified. As such, we also describe some of the more recently characterized vaccine candidates which were not included in the WHO/TDR trial but which have been reported to be protective.

Glutathione S-Transferases By far the best-characterized schistosome antigen is the 28 kDa glutathione S-transferase of S. mansoni (Sm28GST), although work has also been carried out on GST from other schistosome species and on the 26 kDa forms of this molecule. The family of GST enzymes are involved in detoxification and antioxidant pathways. Originally, GST was identified as a component of homogenized adult worms which in a purified form [plus Al(OH) 3 or

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Schistosomiasis

complete Freund's adjuvant, CFA] could elicit protective immunity in rats (50-70%) and mice (40-43%), as judged by reduced challenge worm burdens in immunized compared to control animals (256). A recombinant fusion protein of GST also conferred significant protection on rats, hamsters (257) and mice (258), and induced elevated IgE and IgA antibodies in rats which mediate in vitro ADCC killing of skin stage larvae by eosinophils (259). However, immunization of baboons with SmGST in conjunction with Al(OH) 3 induced highly variable levels (0-80%) of resistance which were unrelated to the level of anti-GST IgG antibodies (258). Synthetic peptides comprising amino acids 115-131 of GST, which incorporate both T- and B-cell epitopes, can also induce significant protection in rats associated with elevated antibody responses (260). It was thought that antibodies to the GST might operate to inhibit its enzyme activity, thus conferring a protective effect. However, when a mAb (S13) to the molecule was administered to mice, there was no effect on worm burden, but the tissue egg load was substantially reduced (127). This, combined with a marked decrease in the fecal egg output and female worm fecundity in baboons immunized with GST (258), provided the first indications that GST may operate as an "antifecundity" vaccine. The C-terminal (190-211) and to a lesser extent the N-terminal (10-43) regions of GST are associated with the enzymatic site of the protein and may be responsible for i n d u c i n g the antifecundity i m m u n e response (261). Furthermore, SmGST (in conjunction with Muramyl-di-peptide) was found to reduce egg excretion from Patas monkeys reinfected with S. haematobium although there was no reduction in the number of worms in immunized animals (197). Similar effects were observed in Patas monkeys immunized with Sh28GST, where the antigen in combination with CFA or BCG did not reduce worm burdens but caused a decrease in the number of tissue and excreted eggs, and the extent of bladder pathology (262). The antifecundity effect has also been reported in cattle exposed to S. bovis, where prior vaccination with the native molecule (SbGST) resulted in a highly significant reduction in fecal and tissue egg counts (263). On the other hand, immunization of goats, or sheep, with recombinant SbGST induced a reduction in challenge worm burdens but had no effect on fecundity (264, 265). SbGST also effected a reduction in the worm burdens (50%) and fecal egg output (89%) of cattle exposed to S. mattheei under field conditions, probably linked to the capacity of IgG and IgA antibodies to inhibit enzyme activity (266,


185

267) but the effect of Sb28GST immunization on the egg maturation and viability are much lower in studies performed in laboratory mice (268). Two monoclonal antibodies which bind amino acid residues 202-211 of Sb28GST were found to inhibit GST activity and when passively transferred into mice induced a significant reduction in egg hatching (269). Further evidence for the antifecundity effect of GST comes from studies on S. japonicum in which it was found that SJ26GST induced in mice a low reduction in worm burden but a pronounced decrease in egg numbers (270). Moreover, immunization of water buffaloes with SJ26GST lowered the egg tissue load (up to 50%) in challenged animals while the ability of excreted eggs to hatch into viable miracidia was also reduced (271). Although antibodies are likely mediators of the antifecundity effect, IFN-y produced by CD4 + (Thl) a n d / o r CD8 + cells has also been implicated (272). Nevertheless, it is thought that IgA antibodies may be the chief isotype responsible for affecting parasite fecundity, while IgE is the major component mediating protection against challenge worms (273). As such, this concept has provided the basis for a number of experimental vaccination strategies using GST which aim to stimulate these effector responses. In this context, vaccination via a mucosal route should stimulate secretory IgA. This has been achieved by delivering SmGST incorporated into liposomes to mice via an oral route (274). Elevated levels of antigen specific IgA in gut washes, and IgGl and IgG2b antibodies in the serum, after immunization were associated with parallel decreases in both worm and tissue egg burdens (274). An alternative approach has been to immunize mice with Bordetella pertussis expressing recombinant SmGST fused to filamentous hemagglutinin (FHA) specifically to stimulate IgA after intranasal priming (275). High levels of IgA in the fluid from the lungs of immunized mice, and IgG in the serum, were detected, particularly after an intranasal boost with SmGST, resulting in a decrease in both the worm and egg burdens (275, 276). More recently, a delivery system involving conjugation of SmGST to cholera toxin B subunit has been employed to target the mucosal immune response as well as stimulating T cell reactivity (277). Mice immunized intranasally with this construct had lower worm burdens and reduced numbers of eggs in the liver. Moreover hepatic granulomas were smaller, and DTH-type responses were reduced. Thus, Sun et al. suggest that this approach is capable of limiting infection and suppressing pathology. Other live bacterial expression vectors, such as Salmonella typhimurium (aroA strain) (278) and Mycobacterium bovis bacillus Calmette-Guerin (BCG)

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Schistosomiasis

(279), have been used successfully to express recombinant SmGST and induce anti-GST antibodies, but the effect on protection is not reported. BCG has also been used to express Sh28GST successfully, and after intranasal immunization of mice elicited strong antibody (IgGl, IgG2a, IgG2b and IgA) responses in the serum and IgA in bronchoalveolar lavage fluids (280). Such expression systems offer a distinct advantage over conventional adjuvants, in that expression of the recombinant antigen can occur over a prolonged period, continuously priming the immune system. The latter vehicle in particular is of interest, since BCG is a known inducer of Th-1 responses, it can be used to target antigens to the pulmonary tissues, and it is well tolerated in man as a vaccine against tuberculosis. As such, it may prove to be an appropriate vaccine expression vector to mimic the protective immune response stimulated by the RA schistosome vaccine in the lungs. The recent advent of "DNA vaccines" has provided another potential vaccination strategy to protect experimental hosts against schistosomiasis. This approach has been used successfully in the development of viral vaccines where neutralizing antibodies and CD8 immune responses are important. Nevertheless, an intradermal route of delivery for plasmid DNA encoding SmGST was observed to elicit IgG2a and IgG2b antibodies in rats which mediate ADCC killing of larvae in vitro, although resistance to reinfection was not reported (281). In contrast, plasmid vectors containing cDNA encoding the 28 or 26 kDa GSTs of S. japonicum failed to elicit specific antibodies and consequently did not have an effect on worm and egg burdens (282). A more promising approach may be to combine DNA vaccination with chemotherapy of an existing schistosome infection. In this respect Dupre et al. (283) found that the pathology in mice immunized with a Sm28GST DNA construct in combination with treatment of infected mice with Praziquantel was almost completely prevented and there was a large reduction in the number of eggs in the tissues. The mechanism these authors propose is that anti-GST antibodies induced by DNA vaccination bind to native GST on the surface of the schistosome worms after being unmasked by the Praziquantel treatment. This form of dual chemotherapy and vaccination would be applicable to treatment of both humans and animals in endemic regions where prior exposure to schistosomes is highly likely. In summary, the family of GSTs from different schistosome species represent the most intensively studied vaccine candidates. Emphasis has recently been placed on the formulation of this antigen to induce IgA activity


which is related to inhibition of the enzyme activity and antifecundity effect. Despite the reported association between the induction of IgE by GST and resistance to challenge infection, a vaccination strategy aimed specifically at eliciting this type of immune response has not so far been reported. Nevertheless, the data from vaccination studies in experimental animals and domestic livestock have prompted the decision to start Phase 1 trials for ShGST in man (284).

Triose Phosphate Isomerase (TPI) This candidate vaccine molecule was first identified using a mAb that conferred passive protection when administered to naive mice (285). The molecule is a 28 kDa enzyme involved in the glycolytic pathway and has been located in all stages of parasite development. The full length recombinant molecule was found to have enzymatic activity (286) and stimulated LN cells from infected mice to secrete both IL-2 and IFN-y, indicative of a Th-1 response (287). T- and B-cell epitopes from nonconserved regions of TPI were subsequently identified and engineered to form four-armed multiple antigenic peptides (MAPs). This construct, "MAP-4," is highly immunogenic when used to immunize naive mice, with the induction of significant T-cell proliferation and IFN-y production by LN cells when restimulated in vitro with the MAP, or the full length TPI molecule (287). TPI in its MAP formulation has been recommended for GMP production and Phase 1 clinical trials, although reformulation may be required (5). Recently a MAP containing peptides encompassing a T- and B-cell epitope of TPI, plus the 115-131 peptide from GST, has been constructed (288). Irrespective of the mouse strain immunized with this construct in CFA, the resulting T-cell response was directed against the TPI components, while the B-cell response was primarily against the peptide derived from GST (288). Although the protective efficacy of this latter construct is not known, it represents the first attempt to immunize a host against two different schistosome antigens targeting different arms of the effector response.

Sml4 The molecule Sml4 was initially identified from a mixture of antigens released by adult worms cultured in PBS which can induce u p to 75%

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Schistosomiasis

protection when administered to outbred mice in conjunction with CFA (289). Moreover, the recombinant form of Sml4 is reported to induce significant and high levels of protection in mice, even in the absence of adjuvant (290). The protein shares homology with fatty acid binding proteins and a similar molecule has been identified from Fasciola hepatica against which it can confer complete protection (290). This latter observation showed that Sml4 may have a particular role in controlling trematode infections of veterinary importance.

Sm23 The internal membrane protein Sm23 has been identified by a number of research groups from S. mansoni (291). The fact that it is present on the surface of lung stage schistosomula has provided special interest in Sm23 being an important vaccine candidate (285). Although the T- and B-cell epitopes of Sm23 have been mapped (292), there is no published information on its protective efficacy. The antigen was included in the WHO vaccine trials formulated in micelles, or as a MAP construct, but for production purposes may be suited to a DNA vaccine approach (5). However, immunization of mice with a DNA construct encoding Sj23 failed to induce protective immunity despite eliciting specific IgG antibodies (293).

Myosin Heavy Chain (IrV-5) As discussed earlier, exposure of mice to multiple doses of irradiated cercariae induces significant levels of protection, and sera from these mice can confer passive protection on naive mice (see the subsection "RA VaccineInduced Resistance to Schistosoma mansoni in Rodent Models," p. 172). Serum from twice-vaccinated mice recognized a particular schistosome antigen (294) of which a 62 kDa fragment was subsequently identified and cloned (i.e. rlrV5) (295). The molecule has amino acid homology with a myosin II heavy chain of 200 kDa and when combined with OMP (outer membrane protein of meningococcus) in the form of proteosome complexes, rIrV5 can induce protection up to 75% in mice (295), u p to 97.4% in rats (296) and between 0 and 54% in baboons (250). It is significant that in this last study, the level of protection achieved in individual animals was highly correlated with the


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titer of antiparasitic antibodies, suggesting that resistance induced by this antigen was highly dependent upon elicitation of a strong humoral component. Recently, a small fragment of schistosome myosin located upstream of the rIrV5 region has been reported to be a strong T-cell immunogen and capable of inducing abundant IFN-y secretion by cells from mice exposed to the RA vaccine (297). This small fragment of myosin was used to immunize mice in conjunction with IL-12 and IL-18 adsorbed on alum (298). However, despite inducing strong levels of IFN-y and Thl-associated antibodies, the vaccine was not protective. A full length S. japonicum homolog of the 62 kDa S. mansoni rIrV5 antigen has been cloned and expressed but neither this, nor a truncated 44 kDa fragment, was able to consistently induce protective immunity, regardless of the adjuvant used (299).

Paramyosin One special feature of this vaccine candidate is that it was initially identified using an immunization regime specifically designed to elicit cell-mediated immunity by coadministering freeze-thawed larvae with BCG as an adjuvant (300). Serum from these animals uniquely recognized a 97 kDa antigen which was identified as myofibrillar protein paramyosin. The protein is present in the muscle and tegument of the parasite but is not exposed on the surface of cercariae and adult worms (301, 302). A recent study has revealed that paramyosin may be expressed on the lung stage parasite, presenting a window of opportunity for the immune system to recognize this potent antigen (303). Immunization with either the native or recombinant protein induced significant T-cell-mediated protection to challenge infection (301, 304). Detailed mapping of the protective epitopes of paramyosin has not been performed. Nevertheless, this antigen has many characteristics which make it suitable for Phase 1 trials (5). Moreover, its formulation into a number of the newer vaccine delivery systems designed to elicit cell-mediated immune responses may prove particularly rewarding. Paramyosin from S. japonicum has also been studied for its protective effect, and vaccinations of mice without added adjuvant induce between 62 and 86% resistance to cercarial challenge (305). However, in another study, only low levels of protection could be achieved by immunizing mice subcutaneously with native Sj paramyosin in CFA (306). One approach to improving the immunogenicity of paramyosin has been to use plasmid DNA encoding the

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molecule (also from S. japonicum) to vaccinate mice. However, although IgG antibodies were elicited, the mice were not protected against cercarial challenge (282). In addition, it has been reported that paramyosin is the target of a protective IgE mAb (307), raising the possibility that IgE-mediated immune mechanisms might be protective to certain vaccine antigens.

Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH) One of the best-characterized vaccine candidates not included in the WHO-sponsored vaccine trial is a 37 kDa glycolytic enzyme G3DPH which is associated with resistance to reinfection in humans ((30); see the section "Schistosome Antigens Associated with Human Resistance to Reinfection," p. 151). Indeed, the failure to develop resistance to reinfection after drug treatment was linked to the relative absence of anti-37 kDa IgG antibodies. Five B-cell and two T-cell epitopes have been determined on the molecule (308). One B-cell determinant (Sm37-5) which is highly antigenic in human infections, coupled to ovalbumin and administered in CFA, induces significant protection in rats (23%) and mice (25%) (308). This epitope did not induce protective immunity when administered with Al(OH) 3 , but the incorporation of the cytokines GM-CSF, or IL-12, increased the titers of IgG antibodies and boosted the levels of protection up to 32-38% and 27% respectively (309). Recently, an antigen with homology to G3PDH has been identified on the basis of the immunological responsiveness of Egyptian patients to electrophoretically separated adult worm antigens. The 42 kDa molecules from S. haematobium and S. mansoni were cross-reactive and induced significant protection to challenge in mice and hamsters (83), further confirming G3PDH as a strong vaccine candidate.

Calpain The goal of identifying vaccine candidates based solely upon their reactivity with T-cells has proved a difficult task. Nearly all recombinant antigens are selected by screening cDNA libraries for expressed clones using antibodies. Consequently, antigens are limited to those that contain B-cell epitopes. Recently, Jankovic et al. (310) used a T-cell clone, derived from mice immunized with adult worm antigens plus BCG, to screen a panel of


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expressed cDNA clones. One clone which induced IFN-y production by the T-cell clone was identified as having homology with the large subunit of calpain (a Ca 2+ -activated neutral protease). The authors report that it is present in different stages of the worm and appears to be released. Moreover, adoptive transfer of the T-cell clone specific for calpain conferred 65% protection on an intraperitoneal challenge of schistosome larvae (310). As such, this is the first schistosome antigen identified solely on the basis of T-cell reactivity. In support of these findings, it has recently been reported that a calciumbinding protein fraction of an apical plasma membrane preparation, when administered with Syntex adjuvant formulation or CFA, can induce 56-57% protection in mice (311). In this context, one of the most immunogenic constituents of this fraction is calpain. Indeed, it has recently been reported that a fragment of calpain is an efficient stimulator of IFN-y production by sdLN cells from mice vaccinated with the RA vaccine (297). A baculovirus expression system was used to express recombinant Sm-p80, which encodes the large subunit of calpain, and in vaccination experiments it induced 2 9 39% protection (311). Recent studies have also shown that 3 gene gun inoculations of mice with a DNA vaccine encoding a mutant version of Smp80 was capable of stimulating significant protection (60%) to challenge infection and this was associated with an increase in IgGl antibodies (312).

Other Candidate Vaccine Antigens Over the past decade, a number of different schistosome molecules have been proposed as vaccine candidates yet remain poorly studied. Some of the more recent additions to the list are described below and warrant further investigation, particularly with regard to formulation: SmlO is a protein isolated from fractions of schistosomula and is a potent stimulator of T-cells from adults living in an endemic area ((91); see the section "Schistosome Antigens Associated with Human Resistance to Reinfection," p. 151). The recombinant molecule, a 10 kDa dynein light chain, induces significant protection (27%) in mice when delivered in conjunction with CFA (313). The reduced number of worms in the immunized mice were negatively correlated with the antibody titer in immunized animals. This molecule is identical to another molecule identified as being a cytoplasmic dynein light

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chain (DLC) and is present on the tegument of lung-stage and adult worms (93). It also has homology with the surface antigens Sm20.8 (94), Sm21.7 (95) and Sm22.6 (94), recognition of which by IgE which has been associated with resistance to reinfection in h u m a n populations ((85); see the section "Schistosome Antigens Associated with Human Resistance to Reinfection," p. 151). The recombinant form of the Sj22 antigen is able to elicit antibodies in mice and buffaloes (314), as was a DNA vaccine construct where Sj22 was fused to a murine immunoglobulin kappa chain secretory leader sequence (315). Such novel approaches in DNA vaccine design may be sufficient to allow the induction of high levels of antibody which hitherto have been difficult to obtain and might be important for the effective protective immune response based upon humoral activity. 9B is a 450 kDa antigen with two main subunits, and a 14-residue peptide of the 45 kDa subunit induced between 30 and 50% protection in mice when administered in CFA (316). However, a different peptide, differing only in the residue in position 7, was not protective. The antigen is present on the surface of schistosomula, including those at the lung stage, so a vaccine construct was devised to target the parasite at the lung stage of migration. The active peptide of 9B was expressed in the flagellin of a Salmonella vaccine strain, and the isolated recombinant flagella used to immunize mice intranasally (317). This resulted in a strong antibody response and significant protection of 42% against challenge. Moreover, when coupled to proteosomes derived from meningoccocal outer membrane, the 9B antigen induced u p to 60% protection (318). Sm480 is present at a number of different developmental stages of the schistosome and is glycosylated. Although poorly immunogenic in the mouse, antibodies raised in rabbits to the native molecule could passively transfer 23-40% protection to recipient animals (319).

Sm25 is a tegumental glycoprotein that has previously been characterized in several laboratories. In this most recent paper, the recombinant protein "rl40" was used to immunize rats and mice administered in combination with a range of different adjuvants. Although high levels of antibody were induced in these studies, there was no reduction in the number of worms or eggs (320).


Sm27 has been identified as a serine protease, produced by the cercariae as they penetrate the host, which induces a 47% reduction in the worm burden when used to immunize mice (321). It appears to be poorly immunogenic with regard to its ability to induce antibody responses but nevertheless may represent an ideal vaccine candidate (322). 44.7/56.8 kDa egg antigens have been isolated on the basis of reactivity with two human mAbs. Not only do the mAbs confer passive protection on challenge in mice, but the affinity purified proteins coadministered with CFA were able to induce 43% protection in mice (323). The results are intriguing, in that egg antigens appear to have an effect on the worm survival, and so further studies of the distribution and cross-reactivity of these antigens between different developmental stages would be useful. Sm74 is an antigen obtained from adult worms and is localized to the gut and tegument. An IgG2a monoclonal antibody recognizing this antigen can confer between 41-53% passive protection in Swiss mice (324). Furthermore, the native antigen administered to mice in conjunction with or without CFA elicited protection up to 50% in C57BL/6J mice, or up to 76% protection in Swiss mice (325).

Conclusions Much progress has been made in recent years in the study of human resistance to schistosomiasis. Age-dependent resistance to reinfection seems to be invariant in human populations in endemic areas, even though exposure to infection can be highly variable, so that, at its most extreme, when adults are more highly exposed than children, children still suffer much more severe reinfection. Meaningful studies of the genetics and cellular immunology of human resistance are now being added to parasitology- and serology-based epidemiology studies. In particular, RT-PCR for semiquantification of cytokine mRNA, FACScan methods for detecting intracellular cytokines, and whole blood culture assays for monitoring antigen-specific cellular responses, are now being applied to human population scale studies, either using frozen human cells or directly in field laboratories in endemic areas. There is already compelling evidence that acquired immunity, mediated by Th2-type immune

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mechanisms, has a role in human resistance to reinfection. However, there is much that is still unknown. We have no knowledge of which human immune effector mechanisms actually kill the parasite in resistant individuals, nor do we know which developmental stage is the target for human immune attack in vivo. Nevertheless, IgE or IgG subclass-eosinophil-mediated ADCC mechanisms are attractive candidates for this role due to the coincidence of high levels of IgE and eosinophils with human resistance in vivo, and the in vitro affect of these mechanisms on the parasite (see (159)). Th2-type responses are also hallmarks of patent S. mansoni infections in the mouse and the self-cure response in the brown rat, although the immunological correlates of resistance are much less well understood in experimental models of other schistosome species. Nevertheless, Thl type responses induced by the RA vaccine are highly effective mediators of acquired immunity in several experimental hosts. It is not clear whether it might be better to attempt to protect humans by inducing Thl-type responses that are effective in the mouse, or to try to enhance Th2 responses that are associated with resistance in man and the rat. We have no reason to believe that a human vaccine will achieve any more than the partial protection obtained in animal models. Therefore, there is a danger that Thl-dependent vaccine immunity may be down-regulated by Th2 cytokines induced by eggs from a few worms maturing in a partially protected human. On the other hand, Thl-type vaccines lend themselves to administration to children prior to natural schistosome exposure and it might be hoped that, with further research into appropriate formulations, the induction of sterile immunity may be achievable. Certainly, the idea of vaccines designed to induce IgE and associated Th2-type responses has yet to be studied seriously and would need to avoid exacerbation of atopic/allergic conditions. Therefore, new developments in vaccine design will be necessary to allow effective use of either Thl- or Th2-type responses to combat human schistosomiasis. A major conclusion of this review is that research on resistance in human schistosomiasis and in animal models should complement each other. Detailed studies involving the manipulation of host and parasite that are the essence of experimental animal models are not practically or ethically possible in man. In particular, the advent in recent years of transgenic and targeted gene knockout mice has produced a rapid acceleration in our knowledge of the cellular and cytokine-mediated control of immune mechanisms that


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can be induced to partially protect experimental animals, while DNA-based techniques allow the characterization of parasite antigens that can induce protective responses. Conversely, antigens that protect experimental animals cannot be tested in man in the absence of detailed knowledge of the human host-parasite relationship. Vaccine success in man will have to be evaluated in relation to other factors that influence human resistance, such as host genetics, age, sex, existing or previous schistosome infections; and how responses against schistosomes might be affected by coinfecting pathogens, malnutrition or other environmental factors. It is also important to know how naturally occurring acquired immunity or innate resistance might be affected if a particular immunoprophylactic strategy is adopted for the control of schistosomiasis in humans. We hope that this review has highlighted some of the excellent and substantial advances that have been made, particularly in the past decade, towards our understanding of resistance to reinfection in both h u m a n populations and experimental animals. It should be clear that many issues remain unresolved, but we predict that in the next few years there will be a substantial increase in the information on immunological aspects of the relationship between schistosomes and their human and animals hosts, especially in relation to cellular immune responses and the genetics of resistance in human populations. New discoveries are rapidly advancing our knowledge of basic immunological processes. Inevitably, such new discoveries will first be applied to schistosomiasis in the context of experimental models but, interesting as these new discoveries might be, established methodologies and a sound background knowledge of human schistosomiasis will be needed to allow any immunological breakthroughs to be evaluated in the field. The imminent start of Phase 1 clinical trials for ShGST (Bilhvax) in man (Capron, 1998) is an exciting new stage in our attempts to combat schistosomiasis but, if anything, this welcome development only serves to underline our urgent need for more information about the basic biology of resistance in both man and animals.

Acknowledgments APM is supported by a Wellcome Trust Fellowship. Thanks are due to AJC Fulford for providing the figures.

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Chapter 6 Initiation and Regulation of Disease in Schistosomiasis C h r i s t o p h e r L King

Overview Schistosomiasis is probably an ancient illness of humans. Its chronicity and transmission by water contact are well suited for maintaining infection in small and relatively isolated bands of h u m a n s that must have characterized most h u m a n evolution. As such, host and parasite coevolved to limit morbidity and prevent the death of its host. This requires a complex host-parasite interaction that maintains a balance to allow continued transmission of the parasite without deleterious effect on the host. During the course of schistosomiasis the host is exposed to migrating schistosomula, maturation of adult worms that subsequently release many excretory-secretory products and the continuous release of ova that also secrete many highly immunogenic antigens. Initial exposure to these all these antigens can produce acute illness, particularly among individuals exposed to schistosomiasis for the first time; however, the burden of illness occurs with chronic infection, primarily in response to ova trapped in tissues. Adult worms can live, on the average, for 5-10 years and some worms have been reported to live more than 30 years (1). Since the principal feature of disease in chronic schistosomiasis is the granulomatous responses surrounding ova trapped in tissues, the mechanisms associated with induction and modulation of this response will be the principal focus of this chapter.

214 Schistosomiasis

Infection vs. Disease in Human Schistosomiasis Like many other chronic diseases, many more individuals become infected than actually develop clinically overt disease. In highly endemic areas for schistosomiasis, 60-80% of individuals may be infected with schistosomiasis; however, only 5-10% are clinically ill. Because schistosomiasis is so widespread a disease, this means that approximately 800,000 people die per year of complications associated with schistosomiasis. Most infected individuals, however, remain relatively unimpaired by their schistosome infection. How the human host and the parasite maintain this long-term interaction is complex and it should be remembered that any attempts to prevent or alter infection and disease may impinge on this h o s t parasite relationship, with potentially adverse effects. One reason that little disease is observed in many individuals is the relatively low infection intensity in many subjects as determined by fecal or urine egg output (2-4). The risk of disease increases with the intensity of infection, though this is not a close relationship and many heavily infected individuals fail to develop clinical disease (Fig. 1) (5-7). Another risk factor

Intensity of Infection Figure 1. The relationship between infection intensity and disease among individuals infected with schistosomiasis. Although individuals with the heaviest infections are at the greatest risk for disease, most heavily infected individuals do not have clinically overt disease.

Initiation and Regulation of Disease in Schistosomiasis

215

for development of disease relates to -previous exposure and immunologic experience to the parasite. Individuals who move from nonendemic to endemic areas, such as migrants, travelers, or military personnel, often experience more severe acute and chronic schistosomiasis compared to those that grew up within endemic areas (3,8-10). The reason for this difference is unknown, but may related to perinatal exposure to host antigens that shape the host's subsequent immune response. Genetic differences to anti-schistosomal immune responsiveness, clinical responses to infection and to the intensity of infection also contribute to variation of infection and disease. The generalization concerning endemic and nonedemic populations may go beyond these individual contributing factors.

Perinatal Exposure to Schistosomiasis and Its Impact on the Immune Response Prenatal exposure to schistosomiasis can potentially have an important role in shaping the host's immune response to schistosome antigens. Fetal exposure to exogenous antigens can result in tolerance, by clonal deletion of Ag-reactive clones or preferential development of a Th2-type immune response with subsequent exposure to infection later in life (11). This altered immune response can have a profound effect in modulating the immunemediated illness associated with schistosomiasis. Schistosomiasis is a chronic blood-dwelling infection, like lymphatic filariasis and malaria, in which vast amounts of parasite antigens are released intravascularly with the potential to expose the fetus. Since, the peak prevalence and intensity of infection can occur during child-bearing years, many individuals that eventually become infected with schistosomiasis were born to mothers who harbored active schistosome infections during pregnancy. This is obviously not true of the vast majority of those from nonendemic areas who become infected. In utero immunological sensitization may occur by either transplacental passage of schistosome antigens or anti-idiotypic antibodies occurring both in humans and experimental animal models (12, 12a). Cord blood obtained from schistosome-infected mothers has been shown to contain schistosome Ag-specific T and B cells that, in some cases, have the same frequency of lymphocytes observed in adults (13,14). This in utero sensitization produces

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Schistosomiasis

immunologic memory that persists for at least one year of age and can alter the pattern of immune response to the BCG vaccine given at birth (15). Prenatal sensitization also occurs in offspring of schistosome-infected mice during pregnancy and affects the outcome of the host-parasite relationship. Newborn mice of infected mothers developed smaller granulomas with subsequent schistosome infections compared to offspring of mothers that had not been subsequently infected (16, 17). Prenatal exposure to filariasis or malaria in animal models has also been shown to alter their immune response and susceptibility to infection and disease upon subsequent infection (18, 19). No human studies, however, have directly shown that in utero exposure to schistosomiasis or to any other parasite infection affects the subsequent outcome of the disease. Although such studies are difficult, they represent a critical area for further research.

Genetic Effects on Susceptibility to Disease The genetic analysis of many diseases has exploded in the past decade. The search for genes responsible for susceptibility to infection and disease in schistosomiasis has just begun, but has produced some striking initial findings. The major approach in these studies is a systematic examination of the whole human genome using polymorphic microsatellite markers. These studies have recently identified a region in chromosome 5 that contains genes important for regulating the intensity of S. mansoni infection (20). This region of the genome contains genes that control a number of cytokines, such as IL-4, IL-5, IL-13 and CSF. This finding supports the hypothesis that these cytokines may contribute to differences in susceptibility to infection. Recently, segregation analysis suggests a gene or closely linked genes are risk factors for development of severe hepatic fibrosis. This major locus has been linked to gene encoding the receptor of the antifibrotic cytokine IFN-y (20a). Identification of the specific genes responsible for the increased susceptibility to infection and disease may still be a long way off, since mapping is still very insensitive. Because disease is primarily immune-mediated, both MHC class II and I genes have been examined with respect to disease. Certain class I and class II HLA alleles have been shown to be linked to various forms of schistosome-induced disease (21-23). Individuals with HLA B5 are at

Initiation and Regulation of Disease in Schistosomiasis 217

greater risk of developing periportal fibrosis a n d / o r hepatosplenomegally with S. mansoni infection (23-25). Overall, however, specific HLA class I alleles fail to consistently correlate with disease. Since the granulomatous response is mediated by CD4+ T cells that interact with antigens presented by products of the class II major histocompatibility complex (MHC), certain alleles of class II genes may better correlate with the severity of disease. Using serologic techniques, Assaad-Khalil determined that severe S. mansonz'-induced hepatosplenic disease was significantly associated with HLA-DR3 genotype and the presence of HLA-DQ appears to protect against disease (26). In another study, the allele DQbl*0201 positively correlated with severe disease (27). For the related parasite, S. japonicum, Ohta et al. (28) showed that the HLA DQw genotype was associated with immunosuppression of in vitro T-cell responses, though this finding was not correlated with the clinical status of infected donors. Susceptibility to schistosome-induced liver disease is likely to be regulated by multiple genes, and whether these genes account for a small or large portion of the variability in the expression of disease remains to be determined.

Disease Mediated b y N o n i m m u n e Mechanisms Although disease caused by schistosomiasis primarily results from the hosts' immune response, toxic substances released by the parasite itself or embolization of ova into vital organs can also cause disease. A catonic glycoprotein released by viable ova has been shown to be directly hepatotoxic in T-and/or B-cell-deficient mice (29, 30). These lymphocyte-deficient mice fail to develop granuloma that may serve to retard or prevent the release of antigen (31). Whether these egg-derived molecules are toxic to human hepatocytes is not known. Rarely, eggs can embolize to the spinal cord or brain to cause transient and occasionally permanent neurological damage. Such disease is most often observed with acute S. japonicum infection, but can also occur with other schistosome species. Chronically infected individuals with portal hypertension are also at increased risk for neurologic damage from schistosomiasis because of development of portovenous shunts that permit passage of ova into the systemic circulation that may ultimately embolize to the brain or spinal cord.

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Schistosomiasis

Immunopathology and Its Immunoregulation Acute Infection Acute schistosomiasis can be associated with heavy exposure to infective larvae (10,32). Internal exposures to large quantities of antigens begins upon maturation of adult male and female worms that continues throughout the course of infection. Acute, primary infection in endemic populations usually leads to little, if any, morbidity. In contrast, initial infections of visitors from nonendemic areas and urbanites in endemic countries who visit active transmission sites for the first time often result in a clinical syndrome of fatigue, fever, sweats, chills, headache, malaise, myalgias, lymphadenopathy and gastrointestinal discomfort. The intensities of infection that leads to clinically acute disease are highly variable. Three types of disease have been described with acute schistosomiasis: (1) an immediate hypersensitivity-type response (33, 34); (2) an allergy type pneumonitis characterized by cough and transient pulmonary infiltrates; (3) an acute febrile reaction beginning about 4 - 6 weeks after infection that can last for several days to weeks, which has been referred to as Katayama fever syndrome. This latter form of acute schistosomiasis has been most often seen with S. japonicum infection. Acute less often with S. mansoni infection and rarely with S. haematobium. Although rarely reported, similar acute episodes may occur with S. haematobium infection. Organomegaly has been described with acute infection and may result from florid granulomatous responses analogous to those observed in rodents and non-human primates (35). Needle biopsies of the liver at this time have shown pathological changes consistent with an acute granulomatous response. These studies, though suggestive, are very limited for obvious reasons and require further study. The schistosome dermatitis and pulmonary disease develops as a consequence of immediate hypersensitivity responses in which penetrating cercariae and lung stage schistosomules activate tissue mast cells and basophils (36-39). Mast cells and basophils possess high affinity IgE receptors bound to parasitespecific IgE (40). This IgE response, in part, is directed against serine proteases (41) released by penetrating cercariae and schistosomules that facilitate tissue migration. These proteases are potent allergens. Generally, 3-7 days after penetrating the skin, schistosomula travel passively by the intravenous circulation to the lungs, where they cross the pulmonary capillary beds


219

into the arterial circulation. This can produce an allergic pneumonitis and transient eosinophilia accompanied by a cough and pulmonary infiltrates. These symptoms may last 1-2 weeks and then spontaneously resolve as the schistosomula complete their migration from the lung. Dermatitis and allergic pneumonitis occur infrequently in endemic areas, though the full extent of these illnesses has not been adequately studied (42). More often, allergic pneumonitis and dermatitis occur in previously unexposed individuals without active infection. The failure to observe immediate hypersensitivity in many individuals from endemic areas may arise by several mechanisms to suppress allergic responses to migrating larvae. These include (a) production of counter-regulatory cytokines that suppress IL-4- and possibly IL-13-mediated IgE and mast degranulation (e.g. I F N - Y , IL-12) (43); (b) synthesis of nonspecific IgE (e.g. > 90% of the IgE rise associated with schistosomiasis is not antischistosomal) (44); (c) generation of blocking antibodies (e.g. IgG4 which recognize shared epitopes to that of IgE (45); a n d / o r (d) expression of molecules by the schistosomes themselves that may inhibit mast-cell degranulation and modulate mediators of allergic inflammation (46, 88). The acute toxemic phase, or Katayama fever syndrome, develops at the time adult worms begin to mature and with the onset of egg deposition (typically 1-3 months after exposure). Predominant symptoms include fever, cough along with headache, general muscular pain and gastrointestinal complaints. Again, pulmonary symptoms can worsen and produce a radiographic appearance on the chest radiograph of scattered pulmonary infiltrates. The mechanism of this illness is thought to be immune-complexmediated and is initiated at the time of egg deposition. The developing larvae and adults share many epitopes with ova, particularly oligosaccharides such as the LewisX trisaccharide (47). As larvae mature into adult worms, these cross-reactive epitopes stimulate antibody production. At this time, patients' antibody levels are rising in response to large amounts of newly deposited ova. This condition leads to a period of circulating Ag excess and forms immune complexes with some of these early antibody responses preferentially recognizing several carbohydrate antigens (48-50). Complement activation also occurs during the acute syndrome, and Clq-binding parallels both the severity (49) and intensity (48) of the disease. Individuals infected with S. japonicutn (in which adult females release more ova than the other species) are at greatest risk for the Katayama fever syndrome. The illness abates as

220

Schistosomiasis

the ratio of antigen to antibody changes to prevent formation of as many immune complexes. Alternatively the immune complexes themselves may be immunoregulatory (51). Cross-linking FcyR on APCs can suppress IL-12 production and down-regulate costimulatory molecules such as B7-1 and B7-2. Complement activated by immune complexes can in itself directly modulate B- and T-cell function (51a). It is possible that immune complexes may, in themselves, be critical for modulation of the granulomatous response (see below). Re-exposure in already infected individuals rarely develops acute schistosomiasis, probably because of these immunoregulatory mechanisms. The relative absence of Katayama fever manifestations in early infections in endemic populations is of particular interest. This suggests that individuals in endemic regions handle these initial infections differently from those residing in nonendemic areas. It is possible that children residing in endemic regions are not immunologically naive and have been sensitized in utero, as indicated above. This may generate the expression of less fulminant and more regulated response to schistosome antigens with acute as well as chronic infection.

The Role of Eosinophils

in Acute

Disease

Acute infection in both humans and experimental models of schistosomiasis typically produces high peripheral blood eosinophilia. Eosinophils dominate the cellular infiltrate in the acute granuloma and have been shown to participate in killing of adult worms due to highly reactive 0 2 radicals a n d / o r release of catonic or major basic proteins from the granules (5254). These same eosinophil-derived molecules may also cause direct damage to host tissues (55-57), such as endomycardial damage and pulmonary fibrosis. Although eosinophils play an important role in killing both adult worms and ova, compensatory pathways can develop in mice. Mice with a targeted deletion of IL-5 or treatment with anti-IL-5 have markedly reduced peripheral and tissue eosinophilia, but retain their ability to limit worm burdens and kill ova (58-60). This may occur because of increased neutrophil infiltrates in granulomas. These studies, however, do not detract from the importance of eosinophils in the regulation of schistosome infections and highlight the redundancy of the host immune response to the parasite.


221

Chronic Infection Chronic schistosomiasis, like other chronic infections, neoplasms, transplantation situations or autoimmune disease, results in continuous release of foreign antigens to the immune system. The amount of antigens produced can be staggering and the long-term interaction of host immune responses to parasite antigens can produce severe immunopathology and disease. However, in most instances, morbidity induced by chronic antigenic exposure is partially controlled by counterbalancing immunoregulatory responses that offset the most severe immunopathology. Indeed, chronic parasitic infection such a schistosomiasis may have been a primary evolutionary force in the development of regulatory capacities of the immune system (61). Chronic disease in schistosomiasis is primarily a consequence of the continuous release of antigens by adult worms and viable ova. Adult worm and ova death can release additional antigens and further boost immune response. Clearly the intensity of infection plays a critical role in how much antigen is released and likely influences the severity of chronic disease. The principal host inflammatory response to schistosomiasis results from egg deposition that produces a strong egg antigen-specific cell-mediated granulomatous response. In the case of S. mansoni and S. japonicum where most ova are released into the intestine, this produces areas of focal inflammation associated with granuloma that may be associated with symptoms of intermittent abdominal pain and occasionally bloody diarrhea (62-64). Release of ova by S. haematobium produces similar inflammation in the bladder wall to stimulate areas of local thickening, occasional bladder polyps and hematuria and proteinura (7, 65, 66). Ureteral obstruction can develop to produce hydronephrosis and kidney failure. The frequency of the intestinal schistosomiasis has been difficult to quantify because of problems in the measurement of intestinal pathology. Many other intestinal pathogens coexist that can produce similar signs and symptoms. The morbidity associated with S. haematobium can be high, with the majority of infected subjects having hematuria. This is not surprising, considering the smaller surface area of the bladder compared to the intestine. Hepatic

Disease

With chronic Schistososoma mansoni and japonica, ova are continuously released. Many ova embolize to the liver. This chronic granulomatous

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Schistosomiasis

response produces a presinusoidal inflammation and fibrosis called Symmers' clay pipe-stem fibrosis (Figs. 2 and 3). In the case of chronic Schistosoma mansoni and japonica, about 4 - 8 % of those infected develop this form of the disease. The risk of fibrosis is thought to arise from the unregulated hepatic granulomatous responses and periportal fibrosis (Fig. 3). The pathogenesis of severe schistosomiasis results from the slow, cumulative blockage of portal flow that results in portal hypertension, portal-systemic collateral circulation, esophageal varices and upper gastrointestinal bleeding. This is the most common morbid consequence of Schistosoma hepatic fibrosis. Normal liver function is generally preserved until very late in the disease. Experimental models of schistosomiasis suggest that the risk of periportal fibrosis is directly related with the cumulative effect of granulomas, fibrosis, coalescence of granulomas and periportal fibrosis that leads to the characteristic picture of fibrosis (67, 68). This relationship between infection intensity and its longevity is less clear in experimental infection of nonhuman primates and humans (69, 70) that suggests other factors that can affect development of pathology. Insights into the mechanisms of why some individuals develop severe hepatosplenic disease may be provided by inbred CBA/J mice chronically infected with S. mansoni that produced

Figure 2. Hepatic fibrosis in a patient that died from complications of S. mansoni infection. The left and middle panels show the nodular changes on the liver surface and the right panel shows advanced presinusoidal (Symmers' pipe-stem) fibrosis of the liver in cross-section.


223

Figure 3. Periportal inflammation (left panel) and periportal fibrosis (right panel) in a baboon chronically infected with S. mansoni. All sections had been strained with trichrome for collagen (blue). This animal had fibrosis in 42% of observed portal tracts similar to that shown in the right panel of the figure. Notable is the broadened portal spaces with increased fibroblast infiltration and enhanced connective tissue deposition. In addition these periportal lesions show widespread angiogenesis, biliary duct hyperplasia and thickening of the portal arteries. PD represents portal ducts, (A) artery and (V) vein.

t w o distinct s y n d r o m e s (71). After p a s s i n g t h r o u g h an acute p h a s e of infection, s o m e a n i m a l s progress to d e v e l o p a h y p e r s p l e n o m e g a l y s y n d r o m e (HSS) characterized b y a m a s s i v e spleen, liver fibrosis, ascites, a n d a n e m i a that resembles hepatosplenic h u m a n schistosomiasis along with portal hypertension and shunting. Other animals develop moderate splenomegally (MSS) s y n d r o m e w i t h less s e v e r e p a t h o l o g y t h a t p a r a l l e l s m o s t c h r o n i c infection. A n i m a l s that d e v e l o p HSS h a v e e x p a n d e d p o p u l a t i o n s of activated T a n d B cells, increased levels of m R N A for T N F a , a n d d i m i n i s h e d IL-10 p r o d u c t i o n c o m p a r e d to MSS a n i m a l s . This suggests a d i v e r g e n c e in the i m m u n o r e g u l a t o r y p a t h w a y s in these t w o g r o u p s of mice. Because the t w o g r o u p s of mice are genetically identical, h a v e similar b u r d e n s of infection a n d d u r a t i o n of disease, o t h e r factors t h a t are n o t fully a p p r e c i a t e d can d e t e r m i n e the o u t c o m e of disease. Differences in p a r a s i t e strains or variation

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Schistosomiasis

in location of the adult worms within the host may affect the expression of this morbidity (72).

Intestinal

Disease

Intestinal schistosomiasis can be a prominent feature of this infection that can produce abdominal cramps, dysentery and possibly abnormal absorption of fluids, electrolytes formation of vitamins (62). The pathology associated with intestinal schistosomiasis can produce nodular inflammation, areas of hyperemia, villous atrophy, and punctate hemorrhagic lesions in the intestinal mucosa (73-76). These lesions are most pronounced with acute infections, or among previously unexposed adults without prior infection, but may also be a prominent symptom among heavily infected individuals. The contribution of schistosomiasis to gastrointestinal complaints has been difficult to unravel in endemic populations, however, where other intestinal parasites are common.

Other

Disease

Many other clinical aspects of schistosomiasis have been observed and are reviewed in other chapters. These conditions include nephropathies, cor pulmonale, involvement of the central nervous system, colonic pseudopolyposis, ectopic sites of adult worm residence, and complications involving schistosomiasis with a variety of other infections and neoplasias. Schistosomiasis also has the more pernicious effect of impairing the growth and development of children. The interrelationship with these illnesses seems inevitable because of the chronic and progressive nature of schistosomiasis.

Granulomas Description

and Function

Granulomas are inflammatory responses that form around ova deposited in tissues. They are CD4+, ccfJ, MHC class II-dependent T-cell responses to antigens secreted by viable parasite eggs (Figs. 4 and 5). The majority of ova


225

(a)

(b) Figure 4. Gross views of granuloma within the liver (A) and on the serosal surface of the large intestine (B) in a baboon infected with S. mansoni. In both specimens granuloma extended throughout the liver and wall of the intestine into the luminal surface of the intestine.

are released in the capillaries of the portal venous system in the large intestine (S. mansoni and S. japonicum) or in the venous plexus surrounding the bladder (S. haematobium). It has been hypothesized that the inflammatory responses induced by viable ova facilitate disruption of tissue barriers to permit the ova to migrate from the intravascular space into the lumen of the gut or bladder. This hypothesis is based on studies showing that T-cell-deficient

226 Schistosomiasis

Figure 5. A photomicrograph of an acute hepatic granuloma from a baboon six weeks after infection. At the center of the granuloma is a schistosome ova surrounded by a cellular infiltrate, x 400 magnification.

mice (77-79) and humans with advanced AIDS (80) have significantly reduced egg output. Host inflammatory responses may only partially account for the passage of ova from venulae into the parenchyma of the intestine or bladder. In vivo microscopy of single ova transiting through the venule wall has been shown to occur in the absence of inflammation and hemorrhage (81). This suggests molecules released by the egg itself facilitate its transit through the vascular endothelium. In contrast, clusters of eggs in the venules were observed to compromise blood flow, resulting in necrosis of the wall and thereby liberating the eggs into the parenchyma. This latter mechanism of egg release may be more associated with an inflammatory response. Many ova, however, fail to pass through the venule wall, become dislodged and are swept by venous blood to the liver, the lung and other organs. The live miracidia within each egg secretes antigenic material through ultramicroscopic pores in the shell (82). Soluble egg antigen (SEA) is very heterogenous, containing multiple proteins, glycoproteins, carbohydrates and glycolipids. Partial purification of active components in SEA has been undertaken and some dominant antigens have been described that are responsible for humoral and cellular reactivity (83-87). These antigens, continually released for 2 - 4 weeks, induce host sensitization and recruitment of macrophages, lymphocytes,giant cells, fibroblasts and numerous eosinophils


227

to constitute the host granulomatous response (82). The antigenic secretion stops when the embryo within the egg dies; granulomas then undergo a healing process with deposition of fibrous tissue. This process of individual granuloma formation and resolution can take u p to 6 weeks. Since eggs are being continuously released, each granuloma formed around individual

ova has its own natural history. Thus multiple granuloma sampled at any one time point are likely to be of different size and composition. This lack of synchronization in hepatic granuloma formation makes its biology difficult to study. The continuous release of ova results in repeated boosting of antigen-specific immunity and may have an important role in shaping the immune response. Factors such as the antigen mass, the number and spacing of ova and the nature of the antigen can all affect the pattern of T-cell differentiation and the potential to modulate the inflammatory responses. Some of these factors have been studied in some detail. The presence of oligosaccharides on developing larvae and ova, for example, favors induction of Th2-type immune response (88,89). Adult worm antigens generate a predominantly Thl-type immune response while egg deposition is the major stimulus of a Th2 cytokine response in S. mansoni-iniected mice and may be accompanied by the down-modulation of the previous Thl-type response (90-92). Recently, it was suggested that the strength of the antigenic stimulus may affect T-cell subset differentiation (93). Highly immunogenic antigens that stimulate rapid cell cycling of naive T cells result in increased IL-4 production. The heavy and frequent release of ova in mice would favor a strong Th2-type immune response. By contrast, the release of few eggs, such as in many light human infections, may favor generation of a more mixed Thl- and Th2-type immune response. Exactly how these phenotypic changes result in down-modulation of granuloma responses remains unclear. Factors such as active suppression or loss of egg-specific lymphocytes because of tolerance or apoptosis have been proposed as possible mechanisms; however, their role in this process remains controversial and is discussed further below. The granulomatous response functions help destroy the ova a n d also prevent the release of potentially toxic molecules from the egg that may damage host tissues (54, 78, 94). The impaired release of soluble egg antigens by granuloma formation limits further priming of antigenspecific lymphocytes in draining lymph nodes and the spleen.

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Schistosomiasis

Animal Models of Granuloma

Formation

Research on the immunology of granulomatous inflammation has attracted considerable attention because of the accessibility of the murine model. Mice are susceptible to human parasites, particularly S. mansoni, and develop similar pathology to humans. It is difficult to study the immune responses and associated pathology in humans because of the inability to experimentally infect humans and sample granuloma except under exceptional conditions. In vitro correlates to granuloma formation have been developed in humans using beads coated with egg antigen to which peripheral blood mononuclear cells are added from sensitized individuals during various phases of their infections (95-97). This approach has provided valuable information, but has its obvious limitations. The study of granulomatous nonhuman primates (in particular the baboon), which can be natural hosts for S. mansoni in East Africa, has provided important insights into the mechanisms of disease that differ from murine schistosomiasis and may better reflect disease in humans (69, 98-100). The patterns of egg distribution, granuloma formation, fibrosis and immune responses in baboons parallel those observed humans (98-101, 101a, 101b). Therefore most of our understanding of the immunologic mechanisms of granuloma formation derives from work performed in mice. Extrapolation of these findings to human disease must be done with caution (101c), for several reasons. First, much of the work performed in mice has studied pulmonary granulomas, which may have significant differences in their mechanisms of regulation compared to hepatic granulomas. Second, even one pair of adult worms in mice is a heavy infection compared to most infections in humans (typically 3 - 5 worm pairs) and nonhuman primates (102,103). As a consequence, the comparatively heavy infection in mice may trigger a different immune response. Third, portal shunting occurs in virtually all experimentally infected mice (104). Fourth, the distribution of ova within mice differs from those observed in primates. In mice, approximately half of the ova are recovered from hepatic granuloma (105). Usually less than 10% of ova are found in the liver of primates (69, 99,106). Most granulomas are found in the large intestine of primates (Fig. 4). This difference in granuloma distribution may have profound effects on egg-antigen-specific immune response, as described in greater detail below. Fifth, most studies in mice have been performed with inbred strains with obvious genetic


differences from humans. Finally, most of our knowledge of granuloma formation and regulation derives from studies of S. mansoni; whether these same mechanisms apply to granulomatous disease in other species of human schistosomiasis has not been adequately explored. The following discussions of granuloma formation and regulation derive primarily from work performed on hepatic granuloma in mice. The mechanisms of pulmonary granuloma formation will not be discussed here and have been well reviewed elsewhere (107).

Mechanisms

of Granuloma

Formation

In S. mansoni infection of mice, egg deposition begins about 4 - 5 weeks after infection, with the first detectable granuloma present by about 6 weeks. Granuloma size peaks in size by 8-10 weeks post-infection (Fig. 6). Dense cellularity and maximum cytokine production characterize this acute stage. As the infection progresses, granuloma size, cellularity and cytokine production diminish with the development of progressive fibrosis by 500

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Figure 6. Association of granuloma size and corresponding hepatic portal vein pressures during acute and chronic infection with S. japonicum infection in mice. Data modified from Olds et al. (253).

230 Schistosomiasis

Figure 7. Periovular collagen deposition around a dead schistosome ova in the liver from a baboon nine weeks after infection. Collagen stains blue with Trichome. Note the loss of periovular cellularity and the amorphus center of the granuloma characteristic of a resolving granuloma.

16-20 weeks postinfection (Fig. 7) (102, 108, 109). Although the average volume of collagen around individual eggs continues to decrease after reaching its peak at 8 weeks, the highest total liver collagen levels were observed in animals at 52 weeks (67). In this chronic mouse model, it appeared that cirrhosis results from the formation of fibrotic bands derived from residual collagen originating from granulomas. The initiation of granuloma formation involves a complex series of events that bear similarity to other inflammatory processes. Cell recruitment to antigen-secreting ova begins by immigration of phagocytic and egg-antigenspecific T cells followed by granulocytes, predominantly eosinophils, and B cells (Fig. 8) (109-111). At the time of peak granuloma formation in S. mansoniinfected mice, the T cells constituted about 20% of the cells. As the granuloma began to modulate, B cells became a more significant component, constituting more than 10% of the granuloma cell population (110). Small numbers of mast cells have been described in the granuloma (112). This recruitment of egg-antigen(SEA)-specific lymphocytes has been demonstrated experimentally (113). SEA-specific lymphoblasts inoculated by the tail vein into acutely infected mice preferentially localize to granulomas induced by eggs. These same animals were also inoculated with KLH-latex-coated beads to form


^ y Egg depostion in liver

Fibrosis -^—

Egg Antigen released stimulates SEAT & B c e | s in l LN/spleen

^ . T cells Recruited to ova in the liver (activation/proliferation)

\ Increased integrin expression on vascular endothelial cells & chemokine release

extracellular matrix deposition w

eosinophils ^

/

\ \

,

Recruitment of -monocytes -neutrophils -lymphocytes -B cells

Th2 dominant Response

Figure 8. Molecular events associated with granuloma formation.

KLH-specific granulomas. SEA-specific lymphoblasts did not localize to these lesions. Similarly, few KLH-specific lymphocytes localized to egg-induced granulomas. This demonstrated Ag-specific lymphocyte recruitment to areas of antigen concentration. The mechanisms of cell recruitment to hepatic granulomas have not been well defined, but likely follow similar mechanisms for inflammatory cell responses of activated T cells to sites of inflammation (Figs. 8 and 9). T cells initially enter tissues surrounding the eggs, perhaps by adhesion to LFA-1 to ICAM-2, which are constitutively expressed on all endothelial cells (114, 115). If schistosome Ag-specific T cells recognize egg antigens, they become activated to produce cytokines such as TNF-alpha and IL-4, which activates endothelial cells to express E-selection, VCAM-1 and ICAM1, and to also produce chemokines such as RANTES, MlP-la and MIP-lfJ (116). These chemokines in turn activate T cells to further express adhesion molecules such as VLA-4 and LFA-1, which bind with greater avidity to endothelial cells and in the process recruit more activated or effector T cells to antigen-secreting ova. At the same time, monocytes are recruited to these sites by adhesion to E-selectin, activated to chemokines such as IL-8, eotaxin and MCP-4, which in turn favor recruitment of neutrophils and eosinophils (117-119). The TNF-alpha and IFN-y released by activated

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Schistosomiasis

GRANULOMA FORMATION Cytokines TNFa IL-1 IL-2 IL-4 IL-5

Neurokines somatostatin Substance P VIP

IFN-Y

Chemokines MCP-1 MIP-1a 7RANTES ?eotaxin

Antibodies Immune complexes C3a, C5a complement mediated chemotaxis

MODULATION Antibodies Cytokines TNFa (FAS espression) ?IFNg 7IL-12 ?TGFB 7IL-10

immune complex-induced cytokines immune complex-FcyRinduced APC inhibition anti-idiotypic Abs block Ag secretion kill ova

Figure 9. Proposed regulatory molecules associated with granuloma formation and its modulation. The upper part of the figure represents molecular signals associated with granuloma formation. Granuloma modulation (lower part of figure) is associated with diminished cell recruitment a n d / o r proliferation. At the same time granuloma cells become anergic, some die and others emigrate from the granuloma to result in a net decrease in cellularity. Some of the same molecular signals involved in granuloma formation may also be associated with its modulation.

T cells also act synergistically to shape endothelial cells, allowing increased blood flow, greater vascular permeability (120-122), and further emigration of leukocytes to form the initial granuloma. Coincident with this increase recruitment of cells into the granuloma, B and T lymphocytes may also show increased IL-4- and IL-2-dependent


233

proliferation, respectively. Cell proliferation probably results from recruitment of T cells activated in the spleen that show increased proliferation (personal observations (92)). However, this period of proliferation in the granuloma must be transient, because by seven weeks postinfection proliferation virtually ceases compared to egg Ag-specific splenocytes (92) (and personal observations). Initially, this is not a consequence of a loss of Ag-reactive lymphocytes since these cells continue to produce cytokines. Cell activation required for granuloma formation can involve costimulation by B7-1 and B7-2 (123-127). Similar to other acute inflammatory responses, these are modulated by antiinflammatory cytokines such as IL-10 and TGFp (125, 128, 129). In spite of these complex and overlapping events in granuloma formation, two, possibly three, factors have emerged that so far appear necessary for granuloma formation. The first is the essential role of T cells. This is based on development markedly smaller or the absence of hepatic granuloma in T-cell-depleted (130), nude (131) or severe combined immunodeficiency mice (SCID) infected with S. mansoni (132). It is the CD4 subset of T cells that appears most critical for granuloma formation (133). Secondly, IL-2, IL-4 a n d / o r IL-13 release by egg antigen-specific T cells appear necessary for granuloma formation based on studies that deplete these cytokines either by neutralizing anticytokine antibodies, or targeted deletion of these specific cytokines (134-140). The most definitive evidence for a primary role of Th2-associated cytokines in the development of hepatic granuloma derives from studies of STAT6deficient mice that completely lack Th2-associated cytokines (141). Hepatic granulomas in these mice are significantly smaller and have diminished fibrosis compared to wild-type animals. In contrast, STAT4-deficient mice, which fail to make IFN-y, have granulomas similar in size and composition to those observed in wild-type mice. Thirdly, the tumor necrosis factor has been postulated to play a central role in granuloma formation. The inability to form hepatic granuloma in SCID mice can be specifically reconstituted by administration of recombinant TNF-alpha (132). TNF-alpha administration can also mediate immune priming in immunologically intact but previously naive animals, to form granuloma with direct injection of ova into the liver (142). These observations are supported by the observation that administration of anti-TNF-alpha to acutely infected animals can suppress granuloma size (143,144). A subsequent study, however, failed to show that administration of TNF-alpha reconstituted granuloma formation in S. mansoni- or S. japonicum-miected SCID mice (145).

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Schistosomiasis

Fibrosis Fibrosis is a major component of chronic schistosomiasis, particularly in areas with repeated granuloma formation (67). The mechanisms that regulate hepatic fibrosis, however, have been less completely studied in schistosomiasis than has the induction of granuloma formation. Periovular fibrosis is most frequently observed in mice, while in humans periportal fibrosis is the most common manifestation. Periportal or Symmer's fibrosis was first described it in 1904 and develops only in a minority S. mansoni individuals with a high endemicity of infection (4-12%). These percentages increase slightly for S. japonicum infection. Variations in percentages probably reflect the degree of tranmissibility in a particular geographic area. The cofactors responsible for the development of periportal fibrosis remain poorly understood. Heavy and chronic infection are considered the major risk factors; however, most heavily infected individuals do not develop fibrosis. Other risk factors may include the repeated exposures, duration of infection, age, and failure of immunologic modulation (101b, 145a). Genetic factors may also contribute, as demonstrated by the large variation in the amount of fibrosis observed among different strains of mice (146). The evidence for or against each one of these cofactors is meager and the problem remains complex. The difficulties in measuring the intensity of infection, its duration, exposure and measurement of fibrosis in human patients have made these studies difficult. Therefore the need for an experimental models is crucial. N o n h u m a n primates may be particularly useful as experimental models. Chimpanzees, like humans, develop the gross and microscopic features of pipestem fibrosis with S. mansoni (73). These animals, however, are not practical for routine study. The olive baboon, Papio cynocephalus anubis, also develops microscopic features of periportal fibrosis in animals that had been treated and repeatedly infected (101b) and possibly in wild caught animals (147). Most infected baboons, however, fail to develop periportal fibrosis which is similar to that observed in humans (69, 98, 99). Baboons are much more accessible for experimental use and are natural hosts for S. mansoni (148). Chronically infected mice can also develop features of periportal fibrosis (see below); however, this model has important differences from human disease, which has been examined in detail elsewhere (149, 150).


235

The development of microscopic fibrosis associated with granulomatous inflammation and periportal fibrosis which lead to portal hypertension may be a continuum of the same process, or the development of fibrosis may be differentially regulated. The following discussion focuses on the process of the induction of schistosome-induced fibrosis, potential molecular mechanisms associated with the regulation of fibrosis and finally some of the factors that may be associated with the development of periportal fibrosis. Most knowledge of the mechanisms of schistosome-induced fibrosis derives from murine models.

Histologic Patterns of Hepatic Fibrosis and Its to Granulomas

Relationship

Fibrosis has been primarily studied in the liver, but can also occur in the bladder and to a much lesser extent in the intestine (111, 151, 152). In acutely infected mice the pattern of fibrosis is mainly periovular, surviving for up to 20 weeks (153). More prolonged (23-52 wks) mouse infections with S. mansoni caused fibrotic expansion from granulomas into normal-looking hepatic parenchyma that coalesced into fibrotic bands (67, 149, 153). This picture followed a pattern similar in character to that observed in chronic human fibrosis. Although the average volume of collagen around individual eggs gradually decreases after reaching its peak at 8 weeks in mice, the total amount of liver collagen progressively increases with longer duration of infection (67). Periportal fibrosis, however, occurs in only a certain percentage of chronically infected mice, even when inbred animals are used (149). Similar to observation of hepatosplenic disease, this suggests that factors other than the intensity, duration and genetic background contribute to this development of disease. The relationship of fibrosis to granulomas in humans and non-human primates is less clear. Periportal fibrosis often develops in the absence of observable granulomas (73, 75, 154). The lack of a spatial association of granulomas with fibrosis has been observed in a number of animal models of schistosomiasis, including chimpanzees, baboons, rabbits and occasionally mice (100, 101a, 155). This implies that soluble factors released from the granulomas can induce fibrosis removed from the granuloma itself. Granulomatous inflammation can also develop in the absence of significant amounts of fibrosis. In acute and chronically infected baboons, fibrosis is minimal or absent. This lack of fibrosis in many

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Schistosomiasis

chronically infected humans and chimpanzees has also been observed (73, 75). Mice invariably develop fibrosis with infection; however, the amount of fibrosis varies considerably among different strains of mice (146, 156). This presence of infection without fibrosis in primates compared to mice may be due to genetic differences, but more likely results from differences in relative intensities of infection and distribution of ova within the host. In primates, most ova remain in the intestine and comparatively few are trapped in the liver (73, 99, 106). This suggests that a critical level of ova must be trapped in the liver before the development of fibrosis. In mice, even a single worm pair may exceed this threshold. Experimental studies of murine schistosomiasis suggest that granulomatous inflammation and fibrosis are independently regulated (157). This is suggested in studies showing that show administration of anti-IL-2 or antiIL-4 antibodies to S. japonicum-infected mice produced a significant reduction in periovular fibrosis with little effect on granuloma size (158,159). Treatment with anti-IL-2 or anti-IL-4 also causes a reduction in fibrosis S. mansoniinfected mice, but with variable affects on acute granuloma size (134, 160). Overall these data suggest that pathways leading to fibrosis may be less redundant than those that produce granulomatous inflammation.

Biochemical

Events Involved

in Hepatic

Fibrosis

Fibrogenesis is a dynamic process primarily involving fibroblasts, and macrophages that respond to a series of molecular signals generated by a variety of cell types (Fig. 10). The process of hepatic fibrosis with schistosome infection has been divided into several stages: (a) the recruitment of fibroblasts a n d / o r differentiation of hepatic Ito cells; (b) their proliferation; (c) the secretion of the extracellular matrix molecules; and (d) tissue remodeling following the degradation of the newly formed matrix (161). The initial hyperplasia of the fibroblast population near the acute phase granuloma results in the increased deposition of extracellular matrix constituents, collagen, proteoglycans, fibronection and hyaluronic acid, and remodeling of the extracellular matrix by degradative enzymes (152, 162). As the infection becomes chronic, collagen biosynthesis diminishes, and increased collagenolytic activity results in a decrease rate of collagen accumulation. It has also been shown that the type of collagen production changes, from type I to type III, in some infected animals (162). This change to type III

Initiation and Regulation of Disease in Schistosomiasis 237 Schistosome Egg

Figure 10. Proposed mechanisms of cytokine regulation of hepatic fibrosis in response to schistosome ova. Fibroblast release matrix proteins that cause fibrosis and macrophages modulate fibrosis by release of collagenases. Cytokines are shown that appear to cross-regulate fibrogenesis by their differential effects on macrophages and fibrosis in granulomas or in the portal tracts.

collagen may reduce morbidity. Type I collagen is more highly cross-linked and resistant to degradation than type III collagen. Animals that develop progressive fibrosis may continue to generate more type I collagen relative to other types of collagen. Therefore, signals that regulate collagen formation, the types produced, a n d / o r its degradation are probably critical in understanding why some individuals develop severe disease and others not.

Molecular Regulation

of Fibrosis

The fibrogenic process results from a complex interaction between T cells, m a c r o p h a g e s and cells of m e s e n c h y m a l / f i b r o b l a s t lineage.

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Schistosomiasis

T-cell-derived cytokines participate in the regulation of fibro-genesis (134,135,160,163,164,164a, 164b). Interleukin-2, IL-4, IL-13, IL-1 and TGFp have been shown to stimulate hepatic fibroblasts to produce extracellular matrix molecules in vitro and augment fibrosis in vivo. In particular, TGFbl appears to play a key role in fibrosis (127,163,165-167). TGFpi production by peripheral blood lymphocytes and TGFpl gene expression in hepatic granuloma is significantly increased in baboons that develop hepatic periportal fibrosis compared to animals with minimal fibrosis independent of worm burdens (101b). The role of TGFp in schistosome-induced fibrosis deserves more careful study, however. For example, do different strains of mice with varying levels of fibrosis correlate with genetic differences in TGFp production? A novel fibroblast growth factor, fibrosin, has also been identified as a product of a subpopulation of CD4+ lymphocytes resident in the hepatic egg granulomas (168). Fibrosin stimulates fibroblast proliferation as well as collagen and fibronectin production (169-172). It also stimulates Ito cells, i.e. resident mesengial cells in the liver, which are important in hepatic fibrogenesis. A homologue to murine fibrosin has been identified in humans (173). Circulating fibrosin can be detected in serum of mice infected with schistosomiasis (174) and potentially in humans. It may prove to be a useful marker for detecting hepatic fibrogenesis in schistosomiasis in humans. Cytokines can also suppress fibrosis. Administration of recombinant IFN-y or IL-12 inhibits schistosome-induced hepatic fibrosis in vivo (175-177). This treatment had the effect of switching the phenotype of SEA-specific lymphocytes from IL-4 to IFN-y secreting cells. Interleukin-4 may act directly on proliferation and collagen synthesis of fibroblasts. It is unclear whether IFN-y acts directly on fibroblasts or indirectly by suppression of IL-4 or IL13 production of egg Ag-specific T cells. Interleukin-10 can inhibit fibroblasts in vitro (163); however, no increase in fibrosis was observed in either the acute or chronic granuloma in IL-10 deficient mice infected with S. mansoni (178). A regulatory role of various cytokines a n d / o r chemokines with fibrosis remains a ripe area for further investigation in experimental schistosomiasis.

Immunomodulation Immunomodulation of immunopathogenesis is proposed as a major contributory force in the dynamics of the overall host-parasite relationship


239

(179). As discussed above, other important factors, such as resistance to infection which influences the overall intensity of infection (180-183) and host genetic effects, can influence the pattern of immune response (184). It is possible that differences in the parasite strain may also contribute to the spectrum of disease (184a). The composite of all these factors determine whether a well-balanced host-parasite relationship develops or the chronic infection advances to a debilitating or potentially fatal clinical illness.

Human Schistosomiasis Immune

modulation

The spectrum of infection and disease appears to correlate with patterns of antiegg (SEA) or adult worm antigens (SWAP) specific immune responses by peripheral blood lymphocytes. Individuals that are exposed, but not infected, have higher levels of SEA- and SWAP-specific lymphocyte proliferation, IL-2, IFN-y, IL-4 and IL-5 production by PBMC compared to infected individuals (185-192). These observations indicate that active infection is capable of suppressing T cell immunity, based in part on observation that radical cure with treatment can partially reverse this impaired T cell responses (186, 190, 193-195). It is also possible that this increase in immune response after treatment may result from rapid release of large quantities of antigen a n d / o r recruitment of additional lymphocytes that react to new epitopes exposed from parasite death rather than a release from immune suppression. This increased lymphocyte reactivity, particularly T cell proliferation and IFN-y production, can persist for months or even years after treatment until reinfection. At this point, parasite-Ag-specific immune response declines (186, 191), indicating that this suppression results, in part, from active infection. This effect of active infection on the development of diminished lymphocyte responsiveness was highlighted in a prospective study of urbanites that became infected with schistosomiasis for the first time during a family gathering in the country in Puerto Rico (196, 197). These studies were performed before safe and effective antischistosomal therapy was available, and therefore infections were allowed to progress without treatment. Comparison of lymphocyte reactivity between the acute and chronic phases of infection in individuals shows that this loss of immune response develops as the infection progresses from the acute to the chronic

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Schistosomiasis

stage of infection (187). The immune hyporesponsiveness in chronically infected individuals was more pronounced to egg than adult worm antigens.

Relationship

of Antigen-Specific

Immune Hyporesponsiveness

with Disease

It has been hypothesized that failure to develop this egg-Ag-specific immune hyporesponsiveness with infection results in worse disease. This is based on cross-sectional studies showing that individuals with hepatosplenic disease or severe bladder pathology have increased lymphocyte proliferation, IFN-y and TNF-a production in response to egg antigens compared to individuals with similar intensities of infection without disease (Refs. 198201, personal observations). In some of these studies the ratio of IL-4 and IL-5 to IFN-y production was greater in those without hepatosplenic compared to those without disease (201). A similar mechanism has been hypothesized to participate in the regulation of expression of disease in human lymphatic filariasis (202, 203). It is still uncertain whether this failure to down-regulate the egg-Ag-specific immunity directly causes worse disease, or whether heavy, long-term infections with associated pathology actually lead to increased egg-Ag-specific cellular immunity in some individuals. Resolution of these alternative hypotheses would require long-term prospective studies that would be difficult to address, because it would unethical to follow untreated patients until they developed clinical disease. The study of human immune responses to helminth infection has relied almost exclusively on PBMC. The phenotype of egg-Ag-reactive lymphocytes in granulomas in the liver, gut or spleen may be different from that in PBMC and these local responses may be more critical in the regulation of the granulomatous response. Studies in mice, nonhuman primates and very limited studies in humans suggest that the immune response may not be that compartmentalized (Refs. 113, 204, 205 and personal observations).

Mechanisms

of Immune

Hyporesponsiveness

The mechanisms responsible for this immunosuppression in h u m a n schistosomiasis remain poorly understood. Several mechanisms have been proposed that included (a) development of immunologic tolerance as a result of deletion of Ag-reactive T cells during development (e.g. fetal exposure)


241

or by the periphery (206), (b) suppression by counter-regulatory cytokines (e.g. IL-10, TGFp), antibodies, or lack of appropriate growth factors such as IL-2, IL-4, IL-7 (207, 208), or (c) development of immunologic unresponsiveness by down-regulation of the T cell receptor (209), lack of costimulatory signals (210), altered peptide ligands (211), or high concentrations of soluble peptides (212). Of these potential mechanisms, only a few have been examined in any detail in human schistosomiasis. Interleukin-10 has been shown to be important in the regulation of Ag-specific T cell hyporesponsiveness during chronic schistosomiasis (192, 213-215). Addition of neutralizing anti-IL-10 can partially reverse both SEA- and SWAP-specific lymphocyte proliferation and IFN-Y production, but does not affect Ag-induced levels of IL-4 and IL-5. The increased production of IL-10 was found to develop in parasiteAg-specific CD4+ cells and may exert its immunosuppressory effect by a decrease in surface expression of MHC class II and costimulatory B7 molecules on antigen-presenting cells. This can impair the ability of APC to present antigen to T cells. Indeed, in schistosomiasis, IL-10 has been associated with antigen down-regulation of B7 expression (192). IL-10 also acts by inhibiting IL-12, a principal cytokine in the activation and intitiation of the T h l arm of the immune response. The development of a Thl-type immune response to schistosome antigens correlates with the increase morbidity with schistosome infection (198, 199). Subjects with hepatosplenic appear to produce less endogenous egg Ag- and adult worm Ag-specific IL-10 compared to individuals with a chronic intestinal form the disease (213), which suggests that this cytokine may be important in controlling morbidity in human schistosomiasis mansoni. In addition to active suppression by IL-10, APC in schistosomiasis may be further impaired in their function. This is reflected in the observation that a component of nonspecific immune suppression also develops in chronically infected patients (198) and that APC-dependent mitogen PHA stimulates less cytokine production compared to the APC-independent mitogens PMA and ionomyosin (192) in chronically infected patients. Depletion of adherent cells (monocytes) has been shown to partially reverse Ag-specific immunosuppression (187, 216). The possibility that APC are unable to deliver the appropriate activation or costimulatory signals for T cell activation has not been fully examined. Some studies have initially examined the CD28-B7 costimulatory pathway. Addition of antibodies that cross-linked CD28 on T cells to bypass a lack of B7 expression on APC,

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Schistosomiasis

however, has not reversed this hyporesponsiveness (personal observations), nor has there been any evidence that Ag-specific T cells have reduced CD28 expression (217). However, there are many other costimulatory pathways. It is also possible that increased serum levels of soluble intercellular adhesion molecule 1 (sICAM-1) that have been observed in patients with severe disease compared to individuals with mild or no illness may interrupt proper costimulation in the granuloma microenvironment (200). Recently it has been reported that APC, enriched for dendritric cells, when added to PBMC cultures from chronically infected patients with chronic urinary schistosomiasis can enhance Ag-specific proliferation and cytokine production (218). Overall, an important component of this i m m u n o suppression in chronic schistosomiasis appears to arise from a defect in APC function in their ability to process a n d / o r present Ag or deliver an appropriate costimulatory signal. How this defect in APC function develops, the precise mechanisms involved, and why it occurs in some infected individuals and not others remain poorly understood. Another mechanism that has been put forward that may influence whether individuals develop little or severe clinical disease is the expression of immunoregulatory ids, such as antibodies directed to variable regions of anti-SEA antibodies. Evidence that this idiotypic network participates in regulating the immune response and pathology is based on three fundamental observations. First, purified Ids from regulated patients without significant clinical disease can stimulate PBMC responses, while patients with overt clinical disease cannot (219). Second, the levels of immunoregulatory ids can be identified in the serum of patients with little or no clinical illness while they cannot be detected in patients with hepatosplenic disease (220). Finally, anti-idiotypic antibodies have been shown to down-modulate the granulomatous response in several experimental models (12a, 221). The specific molecular mechanisms by which ids are processed and presented by APC to T and B cells to elicit an antigen-specific immune response remain unclear, though their participation in the regulation of the immune response is very convincing.

Experimental Models of Schistosomiasis Immunomodulation has also been observed with murine schistosomiasis and provides a much more tractable system for studying the mechanisms


243

of immunoregulation. Acute infections are associated with a florid granulomatous response and increased lymphocyte proliferation and cytokine production in the spleen, mesenteric lymphnodes and hepatic granulomas. The granulomas begin to decrease in size and cellularity after about 10 weeks postinfection, such that by 16-20 weeks after infection the granulomas are about half the size of acute granulomas (Fig. 4). This diminished granuloma size is accompanied by a marked reduction in cytokine production by granuloma cells and lymphocyte proliferation, and has been termed "granuloma modulation." The mechanisms associated with granuloma modulation have been previously reviewed in the murine model (107, 144, 157, 222). The induction and modulation of hepatic granuloma and its associated immunological events remain poorly studied in nonhuman primates (69, 99, 223). Studies in nonhuman primates can help to bridge our limited knowledge of the process of immunoregulation in humans with our much better understanding of the immunologic events associated with immunoregulation in the murine model of schistosomiasis.

Model of

Immunoregulation

Proposed mechanisms of immunodulation are highlighted in Fig. 9. Down-modulation of the granuloma probably involves several interrelated pathways. First, this must involve either decreased recruitment of cells a n d / or increased emigration of cells, though trafficking of cells into and out of chronic granulomas has not been examined (113). Second, proliferation ceases early in hepatic granuloma formation, resulting in decreased expansion granuloma cell numbers (Refs. 113, 224 and 225, personal observations). This may occur by a loss of responsiveness to T cells, to egg antigens, or anergy, possibly by a mechanism of altered APC function as described above. Third, accelerated cell death by apoptosis develops in hepatic granulomas (92,113). The kinetics of cell death by apoptosis or lack of growth factors has not be examined and it also remains to be established whether this accelerated cell death accounts for maintaining small granulomas. Fourth, either cellular a n d / or humoral immunity may result in more rapid killing of the newly released ova (226). This would have the effect that newly released ova would quickly die and involute to prevent the formation of large granulomatous response. The mechanisms of increased cell death, diminished cell recruitment and impaired cell proliferation within hepatic granulomas are still unsettled and

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Schistosomiasis

remain an area of active investigation. However, a significant component of this immune modulation is a result of active suppression.

Cell-Mediated

Suppression

The role of T cells in mediating granuloma modulation was initially shown by adoptive transfer experiments. Spleen cells of 20- or 32-weekinfected mice with S. mansoni that represented chronically infected and immuno-modulated when transferred to 6-week-infected recipients (acute infection), suppressed the maximal egg-Ag-induced hepatic granulomatous response at 8 weeks (227, 228). Both CD4+ and CD8+ T cells mediated suppression (229). The specificity of this effect was demonstrated by the suppression of egg granulomas but not antigen-coated bead granulomas developing simultaneously in the lungs of cell recipients. Monocytes, macrophages, and B cells obtained from spleens of chronically infected mice failed to transfer suppression. Infected adult mice that have been thymectomized also have an impaired ability to down-modulate their granulomatous response (230, 231). This suggests that lymphocytes capable of down-modulating the immune response require replenishment with thymic precursors and implies a rapid turnover of Ag-specific lymphocytes. The molecular mechanisms of how T cells modulate granulomas are under active investigation. Similar to studies in humans, a leading molecule has been interleukin-10. IL-10 can inhibit lymphocyte proliferation and IFN-y production, in part by inhibiting expression of costimulatory molecules on APC such as B7-1 and/B7-2 molecules (129, 232). Administration of IL-10/Fc fusion protein (which prolonged the half-life of IL-10) was able suppress acute granuloma formation (125). Although IL-10 may be important in modulating the acute granulomatous response, its role in maintaining the down-modulation of chronic granulomatous response is unclear, for several reasons. Interleukin IL-10 production by splenocytes peaks at 8 weeks postinfection (232), but rapidly diminishes during the chronic phases of infection. If IL-10 participated in active suppression it would be expected that its levels would remain elevated during chronic infection. Furthermore, mice lacking the gene for IL-10 (IL-10 knockout mice) are still able to modulate granulomas (178). Other T-cell-derived cytokines characteristic of egg-Agspecific lymphocytes may participate in the initial induction of granuloma down-modulation, but not in its maintenance for similar reasons. Their


245

production by splenocytes and granuloma cells also diminishes as the infection becomes chronic (233, 234). Down-modulation of granulomas is also not affected in mice with targeted gene deletions of IL-4, IL-2, IL-5 and IFN-Y (139, 235). One possible cytokine that has not been fully investigated to participate in down-modulation of the granulomatous response is TGF(3.

TGFP is a potent inhibitor of T cell proliferation and IFN-y (IL-4 and IL-5) production (236, 237), and augments macrophage activity (238). This latter effect may accelerate the killing of viable ova trapped in the liver. Endogenous TGFP production has been shown to inhibit egg-Ag-specific IFN-y and IL10 production by hepatic-granuloma-derived T cells (239). Our own studies have shown that SEA-specific TGF(5 production by PBMC remains elevated, coincident with down-modulation of hepatic granulomas in baboons (101a). This contrasts with a marked decline in other SEA-specific IL-2, IL-4, IL5, IFN-y and IL-10. The source of continued TGFP production by egg-Agreactive lymphocytes may derive from granulomas formed in the intestinal lymphoid tissues, where the vast majority of granulomas are found in humans and other primates. This contrasts with the murine model of schistosomiasis, where the majority of ova are recovered in livers of chronically infected mice. Granulomas formed in the intestine may generate a different phenotype of SEA-specific lymphocytes compared to hepatic granulomas. The gutassociated lymphoid system favors differentiation of lymphocytes that produce TGF-p (208). Such lymphocytes have been shown to play a key role in oral tolerance. Gut-derived egg-Ag-specific T cells may migrate and populate hepatic granulomas and participate in their down-modulation.

Role of Gut-Associated

Immunity

in

Schistosomiasis

Compared to immune responses in splenocytes and hepatic and pulmonary granulomas, the phenotype of lymphocytes from intestinal granulomas has received little study. Granulomas recovered from the murine colon are smaller, but show a similar pattern in development and down-modulation to that observed in the liver (111). Intestinal granulomas differ in cell composition compared to hepatic granulomas and contain a greater proportion of macrophages (almost 50%) and proportionally fewer granulocytes and lymphocytes. In contrast to hepatic and colonic granulomas, granulomas that develop in the ileum are smaller, do not modulate (151) and differ in the dynamics and composition of connective tissue matrix deposition (152). These results

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Schistosomiasis

highlight that the local environment plays a critical role in shaping the granulomatous response and that egg-Ag-specific lymphocytes generated in such locations may participate in shaping the granulomatous response in other tissues. This possibility is highlighted by studies showing that enteric administration of S. mansoni eggs to mice stimulates splenic T cell populations capable of modulating liver granuloma size (241, 242). The gut immune response to schistosomiasis may differ from other enteric pathogens. Intestinal mucosal tissue from naturally infected mice failed to synthesize SEA-specific IgA, for example, which might be expected of a local gut immune responses (243). Other studies, however, have found that induction of SEA-specific IgA correlated with egg deposition in the intestinal mucosa (244) and parasite-specific IgA occurs at significant levels in humans infected with schistosomiasis (245). The role of gut-associated immunity in schistosomiasis remains poorly studied and the pattern of cytokine responses by intraepithelial lymphocytes or cells obtained in Peyer's patches has not been examined for murine or human schistosomiasis. Cytokine responses by schistosome specific lymphocytes located in the gut submucosa may not only participate in regulating granulomatous responses in other tissues, but also participate in the modulation of the local inflammatory response to schistosomiasis. This may be important in limiting the extent of intestinal disease associated with schistosomiasis. The possibility that down-modulation of inflammatory responses to intestinal granulomas can also protect against disease was recently highlighted in studies of IL-4-deficient mice (246). These knockout animals had a much higher mortality infected with schistosomiasis compared to wild-type animals with similar intensities of infection. These deaths were associated with severe gastrointestinal-hemorrhage-associated elevated levels of TNF-a and nitric oxide. Such damage to the mucosa may allow the resident gastrointestinal microflora to invade the host blood stream and cause sepsis. The production of IL-4 by egg-Ag-specific lymphocytes (or possibly mast cells) in the gut may protect against this pathology by down-modulation of macrophage activation to the intense proinflammatory responses to egg antigens. TGFp may serve the same role in modulating the intestinal granulomatous response as that hypothesized for IL-4. Although down-modulating the intestinal granulomatous responses may protect the host against excessive disease, the host inflammatory response to ova facilitates parasite transmission. This was initially described


in T-cell-deficient mice that had only a marginal reduction in ova deposited in the tissues, but a significant reduction in ova recovered in their stool (247). This ability to secrete ova was partially restored by the transfer of immune serum from chronically infected mice. Mice lacking B cells have also been reported to have decreased ova excretion (248). These results suggest that parasite-specific antibodies may have a proinflammatory role as well as an anti-inflammatory role (see below) in the granulomatous responses.

Role of Antibodies

in the Modulating

the Granulomatous

Response

Antibodies appear to play a key role in modulating the granulomatous response to S. mansoni infection of mice lacking B cells (248-250). B-celldeficient mice develop normal or slightly larger acute granulomas, but the animals failed to down-modulate the granulomatous response with chronic infection. This was accompanied by more severe hepatic fibrosis in B-cell-deficient mice. Whether B cells, antibodies or both participate in the down-modulation remains to be fully determined. A role for antibodies in modulating the granulomatous response has been suggested by the failure of mice that lack FcR gamma chain to down-modulate their granulomatous responses (248). The role of specific antibodies in granuloma formation and modulation was also suggested by observations that IgE-deficient mice produce smaller hepatic granulomas (251). This contrasts with observations that FceRI-deficient and SJL/J mice which have markedly reduced levels of IgE show enhanced granuloma size and fibrosis (146, 252). These studies implicating a role for antibodies in modulating the granulomatous response using inbred mice with targeted deletions of specific genes contrast with earlier studies in which serum from chronically infected mice failed to modulate the acute granulomatous response when administered prior to the onset of egg deposition (228). It is likely that both antibodies and T cells participate in granuloma modulation. In contrast to S. mansoni infection, passive transfer of serum from mice chronically infected with S. japonicum suppressed acute phase granuloma formation in recipient mice (253). The active component in immune serum was IgGl (254). Several mechanisms have been put forward as to how antibodies may regulate granulomatous responses. First, antibodies may accelerate the killing of ova. This would shorten the release of egg antigens from the viable ova

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Schistosomiasis

necessary to maintain the granulomatous responses. Similarly, antibodies may also block egg antigen release, thereby preventing its access to antigenspecific lymphocytes. However, the continued excretion of viable ova up to two months after praziquantel treatment in humans, which kills adult worms and not ova, suggests only partial antiegg immunity (255,256). Second, antibodies may interact with T cells to regulate granuloma formation through anti-idiotypic networks. For example, anti-idiotypic (anti-anti-SEA, anti-ids) antibodies have been shown to stimulate a population of putative suppressor T cells to produce the immunomodatatory cytokines IL-10 and IL-4 (257). Third, antibodies form immune complexes that cross-link FcyR. Many cells within granulomas express FcyR including lymphocytes, neutrophils and monocytes. FcyR cross-linking inhibits up-regulation of B7-1 (CD80) and B72 (CD86) expression on APCs which are necessary for costimulation of T cells (258). Engagement of FcyR may also induce apoptosis of APCs (259) and suppress IL-12, IL-1 and TNF production. Fourth, antibodies may also participate in chemoattraction of lymphocytes that modulate hepatic granulomas. Antibodies that activate complement release C5a and C3a, which are potent chemoattractants (260-262).

The Effect of Treatment on Modulating

the Granulomatous

Response

The increasingly widespread treatment of schistosomiasis raises the important question of how this may affect the capacity to modulate the granulomatous response. Killing adults worms may enhance the host's immune response and worsen the impact of disease with reinfection in some individuals. Studies of S. japonicum treatment have demonstrated significantly worsened hepatic pathology with reinfection after treatment (263). Treatment and reinfection with S. haematobium results in re-establishment of urinary tract morbidity with two years after a single dose of treatment but, overall, treatment did not appear to worsen subsequent urinary tract disease (264). Repeated chemotherapy for S. haematobium and subsequent reinfection during a hiatus of 8-13 years since stopping chemotherapy resulted in reduced morbidity (265). Treatment of mice that were subsequently reinfected modulated their granulomatous response, similar to that observed in untreated mice (266). Overall these studies indicate that treatment does not have an adverse effect on disease; however, only a few studies have addressed this issue in detail.


249

Conclusions and Unresolved Questions Like many chronic infections, schistosomiasis infection in endemic areas is common and yet clinical overt disease develops infrequently. A dynamic tension arises from the host's inflammatory response to eliminate a foreign organism and development of immune-mediated pathology. In some individuals the granulomatous inflammation and development of fibrosis produces clinically overt disease. Other infected individuals successfully modulate their immune response to the parasite to prevent severe disease. Why this difference remains one of the major conundrums in schistosomiasis. Part of this difficulty arises from trying to fully understand the granulomatous response, which is a complex process that involves the interplay and trafficking of multiple cell types with varying phenotypes determined by the pattern of cytokine and antibody isotypes they produce. An important conclusion from this review is that the phenotype of egg-Ag-specific lymphocytes may differ greatly, d e p e n d i n g on their tissue of origin. These granulomatous responses in different tissues likely result from a dynamic interaction in which lymphocytes circulating from granulomas formed in certain tissues (e.g. intestine) affect those in other tissues (e.g. liver). Whether disease develops or not may be determined by the tissue site in which the bulk of granulomas form. This is certainly consistent with observations that the pattern of inflammatory and fibrosis can differ greatly in inbred strains of mice infected with a similar number of cercariae and for a similar duration. It will be critical to think of the granulomatous response in the context of the whole animal rather than a focus of hepatic or pulmonary granulomas. The mechanisms that induce this host immune response and its modulation have been reviewed in detail in this chapter and appear to involve multiple overlapping pathways. The relative contribution of each of these pathways in regulating disease is unclear and may depend on the animal model studied, the intensity, and the duration of infection. Studies of schistosomiasis in baboons suggest that exposure and treatment may act as additional risk factors for disease, issues that have not be adequately addressed in murine models. Studies of schistosomiasis in nonhuman primates, aside from the obvious greater genetic similarity to humans compared to mice, provide an experimental model in which different intensities and duration of infection on pathology can be better assessed.

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Several unresolved issues about the mechanisms of pathogenesis stand out as being potentially important and have received comparatively little study. First, host genetic differences may affect susceptibility toward disease. New molecular methods for performing whole genome searches allow examination of many potential genes that could contribute to development and modulation of disease that might not be predicted a priori. Given the multiple stages of the parasite within the host, the multiple antigens involved and the duration of infection, it is predicted that genetic differences among individuals may be an important, although minor, determinant to account for the heterogeniety of disease. The second factor is the impact of coinfections on disease. Prior or concomitant infections with intestinal helminths, malaria, HIV or tuberculosis, for example, may have a profound effect on the nature of the immune responses that develops and whether this immunity modulates the host immune responses. Coinfections of mice with schistosomiasis have been shown to have a significant impact on the immune response and clinical outcomes to certain viral, bacterial and other helminth infections (267, 268). The effect of other infections on schistosomiasis has received comparatively little study. Third is the direct effect that the parasite themselves may have on host immune responses (268a). It is possible that the parasite elaborates mediators that directly affect the host immune responses. For example, the filarial parasite Brugia malayi has been shown to produce a functionally active human homologue of the macrophage inhibitory factor (MIF) (269), which has potent modulatory effects on human immune responses. Finally, research into the impact of various parasite strains on the pathology is just beginning to be investigated. It has been suggested that strains of S. haematobium in Egypt cause a different pattern of moribidity than those from East Africa (270). Differences in morbidity among villages in Kenya with otherwise similar intensities of infection have also been attributed to differences in strains of S. mansoni parasites (72). Molecular techniques are now available to better identify strain variability among individual worms.

Acknowledgment I thank Dr Abram Stavisky for his comments on the manuscript and the many stimulating discussions.


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266. Warren KS, Rosenthal MS and Domingo EO (1969). Bull. N. Y. Acad. Med. 45: 211. 267. Actor JK, Shirai M, Kullberg MC, Buller RM, Sher A and Berzofsky JA (1993). Proc. Natl. Acad. Sci. USA 90: 948. 268. Pastrana DV, Raghavan N, FitzGerald P, Eisinger SW, Metz C, Bucala R, Schleimer RP, Bickel C and Scott AL (1998). Infect. Immun. 66: 5955. 268a. Rao KV and Ramaswamy K (2000). Mol. Biochem. Parasitol. 108 (1): 101. 269. Webbe C and Webbe G (1973). /. Helminthol. 47: 49.

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Chapter 7 Disease in Schistosomiasis Haematobia Charles H King

Introduction The trematode parasite Schistosoma haematobium infects over 90 million people in 53 countries (1). Its geographic distribution includes large parts of northern, central, and southern Africa, as well as sections of Saudi Arabia, Madagascar, Mauritius, Syria, Turkey, Iraq, and Iran (2). Figure 1 details the location of S. haematobium-endemic areas. By the year 2000 CE, an estimated 200 million people will be living in transmission zones where they will be at risk for recurrent S. haematobium infection. S. haematobium is the cause of urinary schistosomiasis, a disease that severely inflames and then deforms the urinary bladder, the ureters, and the kidneys. The disease occurs when parasitic adult S. haematobium worms seek out the veins of the human bladder and ureters and take u p residence there. The subsequent local release of parasite eggs (and the consequent egginduced host inflammatory response) leads to the generation of acute and chronic injury to the urinary tract. Hematuria, anemia, and undernutrition are correlates of infection in childhood. Manifestations of long term infection can include scarring and deformity of the ureters and bladder, chronic bacterial superinfection, and kidney dysfunction or failure. In some endemic areas, S. haematobium-infected populations are significantly predisposed to develop bladder cancer. While S. haematobium infection is chronic by nature, and is unlikely to be directly lethal, in endemic areas its toll of morbidity is high. Urinary schistosomiasis represents a significant health burden for many developing countries.

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Figure 1. Distribution of S. haematobium infection. Endemic areas (based on WHO data (3)) are shown in red.

The Pathogen Schistosoma haematobium was first described by Bilharz in 1852. It is one of a related family of digenetic schistosome trematodes that primarily parasitize humans (4). The adult S. haematobium schistosomes are roundbodied blood flukes that establish themselves in the venules of their human hosts, where they reside for an average of 3-6 years. A distinguishing feature of the male S. haematobium parasite is the presence of small tubercles, visible to the unaided eye, on the surface of its tegument (5). Distinct from other schistosome species such as S. mansoni and S. japonicum, S. haematobium prefers to reside in the perivesical venous plexus of the urinary tract and not in the portal veins. In a typical infection, deposition of S. haematobium eggs into the bladder and ureteral tissues results in inflammation, mass effect due to

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granuloma formation, and ulceration of the lumenal wall of the lower urinary tract, with resulting symptoms of dysuria, hematuria, and bladder dysfunction (1). Penetration of eggs into the urinary space is essential to parasite transmission, as the eggs must be transported out of the body to continue the parasite's life cycle. Eggs that reach fresh water hatch into miracidia and

infect Bulinus spp. snails that act as intermediate hosts for S. haematobium, and these, in turn, release cercariae that infect other humans (5). It appears that host inflammation plays a significant role in facilitating egg penetration into the urinary space. Thus, urinary tract injury is an inherent part of established S. haematobium infection. Disease is common in parasite-endemic communities and urinary schistosomiasis remains the leading cause of hematuria in the world (1). The predilection for infection of the urinary tract is a species-specific trait of S. haematobium. The preference is maintained even when S. haematobium hybridizes with other species, such as S. intercalatum and S. mattheei (6, 7). However, the behavioral or physiological reasons for this preference are not clear. Among human schistosome parasites, S. haematobium has a unique sensitivity to the organophosphate cholinesterase inhibitor, metrifonate (Bilarcil). The species-specific differential sensitivity is related to a higher expression of acetylcholinesterase on the surface of S. haematobium adults (8). S. haematobium also synthesizes a unique serine-protease inhibitor (serpin) antigen, which may play a role in protecting blood flow in the hyperosmolar environment of urinary tract veins (9, 10). Human infection with S. haematobium begins when the susceptible host is exposed to water containing parasite cercariae. These infectious larva are released into ponds, streams, and rivers by intermediate aquatic snail hosts of the Bulinus species. Geographical variation does occur within the S. haematobium species in terms of infectivity, egg output, and maturation time, and also in terms of intermediate host specificity (4). Depending on location, S. haematobium may be transmitted by snails of the Bulinus africanus group (tropical Africa), the B. truncatus-tropicus group (Mediterranean, Middle East), or the B. forskalii group (Indian Ocean, Saudi Arabia). S. haematobium is almost an exclusive parasite of man, but natural infection has been found in vervet monkeys, baboons, chimpanzees, guenons, sheep, pigs, and Nile rats in various endemic areas of Africa (4). These zoonoses are not thought to play a significant role in the perpetuation of transmission in these areas.

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Cercariae are shed daily by infected snails into their freshwater habitat. Those that come into contact with human skin will penetrate the epidermis and dermis to reach local capillary venules, then travel with the systemic venous circulation to the lungs over the space of 7-10 days. Subsequent maturation takes place in the region of the liver, after which the mature worms migrate to the perivesical veins, where they will spend most of their lives. Worms may localize in other pelvic organs such as the prostate, seminal vesicles, uterus, cervix, or fallopian tubes (2, 11). Parasites may localize in the wall of the bowel, particularly the sigmoid colon or rectum, and parasite eggs may be seen in the stool, particularly if the intensity of infection is high. In rare cases, S. haematobium worms have been found in the skin, spinal canal, liver, and lungs, where disease occurs due to local egg deposition and subsequent inflammation (2,11). In an autopsy study of 46 patients in Cairo by Cheever et ah, quantitative egg recovery identified 42% of eggs in the bladder, 19% in the intestines, 12% in the genitalia, 16% in the lung, 7% in the ureters, and 5% in the liver. Female worm recovery was greatest in the bladder (48%), and the mesenteric and portal veins (47%), with 5% of worms in the ureters and genitals and 0.2% in the lungs (12). The worm localization findings may not be representative of premortem w o r m distribution, but measured egg burdens are likely to be representative of worm localization in the final years of life. Disease may occur at any stage of the infection. Cercarial dermatitis is an inflammatory response to cercarial skin penetration that occurs in presensitized individuals who are repeatedly exposed to infested waters (13, 14). Acute toxemic schistosomiasis is a systemic inflammatory condition involving fever, headache, arthralgia, myalgia, diarrhea, cough, weight loss, and urticaria, which occurs during early phases of parasite development in the circulation (2, 14). Hematuria is the first sign of a mature worm infection, typically occurring 10-12 weeks after exposure. Dysuria and urinary frequency are also early signs of infection (15). In established infection, the disease symptoms of chronic schistosomiasis are usually caused by granulomatous inflammation surrounding parasite eggs in the tissues. Ultimately, after years of infection and the resulting host inflammation, chronic fibrosis and tissue damage occur at sites of egg deposition, leading to dysfunction of affected organs. For schistosomiasis haematobia in particular, this focus of late disease is in the ureters and bladder. Common manifestations


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include hydronephrosis, bacterial superinfection and renal dysfunction (2, 11). The nature and extent of S. haematobium-associa.ted disease depends on the human host's prior experience with the parasite. Acute schistosomiasis is most frequently seen in "Northern" travelers to S. haematobium-endemic areas who acquire infection for the first time as adults. However, the prevalence of symptomatic acute schistosomiasis appears to be lower for S. haematobium infections than for S. mansoni infections (14, 15). Cercarial dermatitis affects some, but not all, local residents of endemic areas who are repeatedly exposed to infection. This skin reaction is more common among children. Granulomatous disease may be seen at any age, and disease is more severe with heavier S. haematobium infections (16). Chronic disease due to fibrosis is a result of long-term infection, and may take decades to develop — once established, it may be improved by therapy, but some of the late schistosomiasis-induced changes are irreversible (1, 2).

Immunopathology of Schistosomiasis Haematobia Human helminth infection represents a complex interplay between two multicellular species sharing the same space. During human infection, disease is caused by the human host's delayed-type hypersensitivity granulomatous response to parasite eggs that have been deposited in the venules of tissues where the S. haematobium worms reside. The parasite has long adapted to its human host, and adult worms themselves elicit very little inflammatory response, and cause little damage on their own (11). However, the parasite requires an effective means for propagation, and the strategy taken has been to release hundreds to thousands of eggs into the host tissues every day (17). Many eggs (over 50%) will become trapped in tissues, but the remainder will penetrate through the wall of the bladder, ureter, or colon to reach their internal lumena. Once mixed with urine or stool, the eggs will be passed out of the body to reach fresh water. In studying the host-parasite interaction, it is essential to recognize that parasite infection does not always imply human disease. Nevertheless, significant urinary tract disease occurs with sufficient frequency in S. haematobium infection that the disease prevalence is high in endemic areas (16, 18, 19). Multiple factors appear to contribute to the eventual expression

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of disease in any given patient. Environmental factors, such as level of economic development, coinfection with other pathogens, interaction with intermediate host habitat, and cultural factors associated with gender roles and occupations, all modify exposure risk and therefore may be associated with the development of heavier infections and more severe schistosomiasis (20-22). Yet, in S. haematobium-endemic areas, there is an uneven distribution of infection, with significant clustering of intense infection in a small subset of the population (23). Likewise, infection-related morbidity, which on a population basis is tied to the intensity and duration of exposure, is unevenly distributed among individuals having the same levels of exposure and the same intensity of infection (2, 24-26). It is generally accepted that pathology in the urinary tract is, in part, determined by the chance focal distribution of parasite eggs, and, in greater part, regulated by the host immune response (11). The antiegg granuloma is a cellular immune response comprising lymphocytes, macrophages, eosinophils, and fibroblasts regulated by CD4-positive, class II MHCdependent, T-helper cells. Their primary response is to antigens secreted by parasite eggs trapped in host tissue. Cellular immune responses are tightly regulated, and the role of specific cytokines in the induction and modulation of granulomatous response remains to be fully defined (27). Several subpopulations of T-helper cells (designated Th-1, Th-2, and Th-nuU) have been identified, based on the repertoire of their cytokine responses and the types of antigens that will drive their activation. Studies of granulomatous response in intestinal schistosomiasis have associated increased production of Th-1type cytokines, such as proinflammatory interleukin-2 (IL-2) and interferong (IFNg), accompanied by diminished production of Th-2 cytokines, with the development of antiegg granulomatous pathology (27). A key proinflammatory cytokine in this process is tumor necrosis factor-a (TNF-a), which has been established as an important mediator of murine granuloma formation and hepatic fibrosis in experimental S. mansoni infection. As the infection progresses, Th2-type responses, especially IL-10 and transforming growth factor-p (TGFp), replace the Th-1 response and down-regulate granulomatous inflammation (28). It should be pointed out that this clearcut polarization of T-cell subset and cross-regulation has not always been observed. Moreover, B-lymphocytes a n d / o r schistosome-specific antibody responses also participate in the modulation of the granulomatous response in some forms of experimental schistosome infection (29, 30).


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Less is known about human response to schistosomiasis, and, in particular, to S. haematobium infection. Despite its clinical significance, the S. haematobium life cycle has proven difficult to maintain in laboratory animals, and research on immunity and immunopathology in animal models has been limited. However, technical advances in the study of specific T- and B-cell responses

have now made direct study of human immunity to S. haematobium possible (31-34). For example, it has been observed that TNFa expression in response to parasite antigen varies between infected humans with the same level of infection. Among schoolchildren, elevated TNFa response has been associated with a greater risk of acute bladder wall disease. Significant differences in Th-2 cytokine production (IL-4 and IL-5) have been observed between Egyptian adolescents developing heavy versus light infections after equivalent exposure to infection (35). Greater production of IL-4, which stimulates IgE production, and of IL-5, which promotes eosinophilia, was associated with apparent reduction of the intensity of reinfection. Independent studies in Gabon indicate that higher antiworm IgE levels are correlated with lower levels of reinfection after drug-mediated cure of S. haematobium infection (36). Host genetic background has also been linked to susceptibility to infection and disease expression. In S. haematobium infection, different HLA markers (MHC DPA and DPB alleles) have been linked to risk for infection and reinfection, and for bladder pathology (37). Limited studies of human intestinal schistosomiasis caused by Schistosoma mansoni have shown familial aggregation of heavy infection in Brazilian patients and association between pathology and histocompatibility markers (38, 39). The Sml locus controlling intensity of S. mansoni infection has been mapped to the 5q31-q33 region (39). For now, the genetic role in regulation of S. haematobium infection and disease remains uncertain. In patients from endemic areas, prenatal exposure to schistosome antigens likely plays an important role in modeling the subsequent immune response to infection. For example, exposure in utero to schistosome antigens (due to maternal infection during pregnancy) might favor a predominant Th2-like immune response and possibly induce tolerance (31-33). This prenatal exposure may have the effect of reducing acute granulomatous inflammation or increasing modulation of later immune responses. Imprinting or biasing of fetal immunity might also favor development of partial protection to subsequent infection. Studies have demonstrated that the fetus becomes exposed to schistosome antigens in utero, and cord blood lymphocytes can

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express specific immune responses to parasite antigens at the time of birth. Little is known, however, of how immune responses to exogenous antigens acquired by the human fetus affects subsequent immunity upon primary exposure or re-exposure. For example, it is not known how long antiparasite memory may persist, or whether there is an appropriate booster effect with re-exposure to schistosome antigens. Moreover, it remains to be determined if the phenotype (Th-1-like vs. Th-2-like) of the memory T-cells is stable with time, or if it is altered with re-exposure to antigen.

Epidemiology of S. haematobium

Infection

One of the most striking features of the epidemiology of human S. haematobium infection is the linear rise (with increasing age) in both prevalence and intensity of infection up to the time of early adolescence, after which the average infection intensity drops markedly among older individuals (16, 18, 19, 40, 41). This age-specific pattern of infection has been highly reproducible, regardless of the overall level of infection within the population, or the geographic locality. Extensive studies of changes in water contact patterns with age have drawn conflicting conclusions regarding the role of water contact alone in determining age-specific patterns of infection intensity, and have led to the hypothesis (by several different groups) that acquired immunity develops with repeated exposure to the parasite (20, 22, 40, 4 2 46). Certainly, activities that involve more prolonged and more extensive immersion, such as swimming or bathing, are associated with greater risk of infection. However, our analysis of water contact and risk for infection in endemic Kenyan communities indicates that age and total duration of water exposure are independent predictors of S. haematobium infection (20, 45). More direct evidence of protective immunity in humans has been based on reinfection studies, in which infected individuals are cured of infection with chemotherapy, and subsequently observed for the development of reinfection (40, 47). The fact that some of these individuals fail to reacquire infection, despite continued moderate-to-heavy exposure, suggests that acquired resistance to S. haematobium infection does develop with continued exposure or with increasing age. Host genetic variation likely also plays a role in determining the relative intensity of S. haematobium infection and disease (37, 38).

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In endemic areas, S. haematobium is a cause of death in 1-2 per 1000 individuals per year, and is estimated to contribute, through renal disease, to 1-3% of deaths (48, 49).

Morbidity Due to S. haematobium Infection Stages of Disease Of all forms of h u m a n schistosomiasis, infection with Schistosoma haematobium is associated with the highest prevalence of clinical morbidity. As documented in population-based clinical studies, u p to 50-70% of S. haematobium-iniected individuals have symptomatic urinary tract findings, including either hematuria, dysuria, or ureteral or bladder dysfunction (16, 18). Studies of the immunopathology related to S. haematobium infection have established an association between acute granulomatous response to parasite eggs and certain active forms of urinary tract disease, such as urothelial ulceration and bladder polyposis. In contrast, the late pathology of urinary schistosomiasis, including fibrosis and tissue calcification, is more common in older individuals who may harbor only low-intensity infection or no infection at the time of study (1, 11). It is clear that the severity of parasitespecific morbidity and attendant complications are related both to the concurrent intensity of S. haematobium infection and to the total duration of infection. More specifically, because infection intensity increases during childhood, followed by a characteristic reduction in intensity in adult years, the integral or "area under the curve" of an individual's age/intensity history is most relevant in predicting his or her total accumulated S. haematobiumassociated injury, as well as the individual's risk for severe, life-threatening morbidity. Other factors, including genetic regulation of immune response to parasite antigens, and geographic variation in S. haematobium strains, may also be important determinants of disease expression (50).

Bladder Disease Infection-associated morbidity may manifest as microscopic or gross hematuria, proteinuria [from minimal u p to nephrotic range (> 3 gm/day)], bladder and ureteral granuloma formation, hydronephrosis, secondary

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bacterial infections, and significant urothelial metaplasia associated with increased risk for squamous cell cancer of the bladder (11, 16, 18, 19, 41, 51-53). Studies of the immunopathology related to S. haematobium infection have established an association between acute granulomatous response to parasite eggs and certain active forms of urinary tract disease, such as urothelial ulceration and bladder polyposis. This type of disease regresses rapidly after successful antiparasite drug therapy. In contrast, the late pathology of urinary schistosomiasis, including fibrosis and tissue calcification, is more common in older individuals, who may harbor only low-intensity infection or no infection at the time of study (11). This late disease may not regress with treatment, resulting in chronic urinary tract dysfunction. Lower egg counts in adult patients may be due to an age-related shift in susceptibility to repeat infection (35) or to a host immunity-mediated reduction in S. haematobium fecundity (54). However, both concurrent intensity of infection and cumulative exposure to infection (estimated by patient age for long-term residents of endemic villages) are significant correlates of disease expression in affected populations. It remains to be determined whether infection duration, intensity, or an individual's own immune response plays the greatest role in mediating late pathology. Typical bladder disease includes granulomatous polyp formation, ulceration, and epithelial hyperplasia or metaplasia (11). Polyp formation occurs as part of inflammatory response to localized egg deposition in the bladder wall. The polyps may be sufficient in size to cause superficial breakdown and hemorrhage, or to cause obstruction of urine flow into or out of the bladder. Chronic ulceration of the bladder may occur without polyp formation, and is a typical consequence of late, heavy infection (11). "Sandy patches," comprising yellow-gray, irregular areas of roughened bladder mucosa surrounding areas of heavy egg deposition, are a pathognomonic lesion of urinary schistosomiasis (1). Cystoscopy demonstrates hyperemia, sandy patches, granulomas, ulcers, nodules and polyps, in descending order of frequency (55). Extensive blood loss may lead to significant iron-deficiency anemia, which improves after antischistosome therapy (56-58). Later consequences of S. haematobium infection are bladder hypertrophy, and bladder wall fibrosis or calcification (2). Figure 2 demonstrates bladder pathology detected in a field study in coastal Kenya. Symptoms are usually minimal when infection intensity is light. However, in heavier infections, there is often a significant effect on the bladder neck


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Figure 2. Bladder wall granuloma (g) seen in the lumen on (a) transverse and (b) longitudinal ultrasound views.

Table 1. Frequency of bladder symptoms associated with S. haematobium infection. Symptom

Prevalence with light infection (< 100 eggs/10 mL urine)

Prevalence with heavy infection (> 100 eggs/10 mL urine)

Dysuria Hematuria Frequency Urinary colic Urinary incontinence Urinary retention

55% 60%

55-70% 80-95% 56% 37% 20% 13%

Summarized from Refs. 18 and 59.

leading to outlet obstruction or dysfunction. Complaints associated with active urinary schistosomiasis include frequency, burning, dysuria, suprapubic pain, and variable hematuria (1). Table 1 summarizes the typical prevalence of S. /zaematofo'um-associated symptoms in endemic areas. Late symptoms are rare and are most often caused by bacterial superinfection. Patients may report nocturia, urge incontinence or bladder retention, dribbling or painful micturition. Renal colic, hematuria, and passage of debris may also be reported (1).

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Ureters In terms of tissue egg burden, the ureters are less heavily involved than the bladder. However, because of the significant functional effects of focal ureteral granuloma formation and subsequent scarring, ureteral disease is the most important cause of patient morbidity (1). When egg burden in the ureter increases, the risk for significant obstruction is also increased (49). As happens in the bladder, the granulomatous inflammation in response to local parasite eggs produces granulomas, polyps, ulcers, sandy patches and calcification of the wall of the ureters (1). Hydronephrosis frequently occurs as a consequence of acute inflammation localized in the ureteral wall, or as a late consequence of cumulative injury to the ureters (11). The former is readily demonstrated by ultrasound examination (19, 24, 26, 60), while the latter is more clearly demonstrated on intravenous excretory urograms (IVP) (16). Ureteral stenosis is most common at the ureterovesicular junction (11). In some cases, the stenosis is anatomic, due to physical obstruction with granulomas, scar and/or calculi, while in others, the obstruction is functional. Functional obstruction occurs when the ureteral wall is made rigid and fibrotic, resulting in proximal dilation and disruption of normal urine flow (49, 59, 61). Hydroureter and hydronephrosis are typical sequelae of schistosomal obstructive uropathy. Ultrasound studies have shown urinary tract lesions to be more prevalent (43-80%) and more severe in early childhood than previously thought (60, 62-65). However, in childhood, structural lesions of the bladder and hydronephrosis have been shown to regress 6-24 months after treatment of S. haematobium infection (60, 64-66). Some controversy persists regarding the prevalence and severity of urinary tract abnormalities in adults. Heurtier et al. (67), working in Niger, examined 381 adults and children in areas both endemic and nonendemic for S. haematobium. They concluded that structural lesions were comparatively rare in adults, and not associated with S. haematobium endemicity. This contrasts with IVP studies in East Africa and Egypt, which have noted a significant risk of renal lesions in adults (particularly males) with S. haematobium infection (16, 48, 59). In studies from different geographic areas, results of ultrasound studies have been generally well correlated with findings from IVP and cystoscopic examinations (62, 67). However, some radiologists feel that there are significant differences in the information provided by IVP versus ultrasound examination: tissue calcification and ureteral dysfunction are likely to be more


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apparent on dye-contrast roentgenographic studies, while hydronephrosis and deformity of poorly functioning kidneys are better assessed on ultrasound examination. It appears from treatment studies that "acute" polypoidgranulomatous lesions, more common in younger individuals, are the first to regress after therapy (60,65). "Late" complications, such as hydronephrosis, may take a longer time to show improvement, particularly in young males (24,65). Elimination of infection does not terminate ongoing immune response to eggs trapped within tissues, and it is possible that fibrosis progresses for some time after successful cure of infection, resulting in persistent dysfunction of ureteral flow (11). Hydroureter may be either segmental, tonic, or atonic (11). Segmental dilation of the ureter is seen in approximately 25% of cases, and typically occurs in the lowest third of the ureter. Anatomically, there is concentric fibrosis of the muscle layers at the affected segment, and hypertrophy of the muscle above (11). This segmental form of hydroureter is only rarely associated with hydronephrosis. Tonic hydroureter occurs in another 25% of cases, manifesting as thick-walled, dilated and tortuous ureters that manifest active peristalsis above an area of stenosis. Atonic hydroureter shows a very dilated, thin-walled fibrotic ureter above an area of obstruction. In contrast to segmental disease, the tonic and atonic forms of hydroureter are frequently associated with hydronephrosis (11). Schistosomiasis-associated hydronephrosis is the final stage of urinary tract injury, progressing from renal pelvic dilation, to medullary atrophy, to renal cortical destruction (11). Along this pathway, tubular dysfunction typically precedes glomerular dysfunction, which is a late, very uncommon result of urinary schistosomiasis (59). Relief of hydronephrosis is usually associated with recovery of renal function — in early infection this may be obtained by anthelmintic therapy, whereas in late disease surgical repair of the ureter or diversion may be necessary to effect recovery of kidney function. Schistosomiasis has been associated with increased risk for pyelonephritis (49,61), most likely as a consequence of the combined risks of bladder outflow obstruction, chronic cystitis, stone formation, and ureteral obstruction (11). Organisms that are typically involved include Escherichia coli and other coliforms, Staphylococcus spp., Streptococcus faecalis, Pseudomonas aeruginosa, Proteus spp., Klebsiella spp., and Salmonella species (2). Invasive or recurrent Salmonella infection has been associated with S. haematobium infection. The Salmonella bacteria appear to adhere to the surface of the schistosome, which

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leads to persistence of bacterial infection (68). Effective therapy of the Salmonella involves successful eradication of both parasitic and bacterial infections (2).

Reproductive Tract A number of studies have documented the presence of S. haematobium eggs in other pelvic organs, including the male and female genitalia and reproductive tracts (2, 11, 69-73). Systematic autopsy studies indicate that average egg burdens in the epididymis, ovaries, and fallopian tubes are generally much lighter than for the organs of the urinary tract (11). However, occasional heavy egg deposition does occur in the reproductive organs, leading to epididymitis, oophoritis, pseudoelephantiasis, cervical stenosis, or vaginitis (1). When local egg burden is high, enlargement of the seminal vesicles has been reported in association with fibrosis, muscular hypertrophy, and calcification (49). Prostatic involvement is less common (72), as is involvement of the testis or penis (2). S. haematobium infection has been associated with ejaculatory pain, testicular pain, hematospermia, and alteration of semen consistency, with aggregated parasite eggs detected on semen analysis (74). S. haematobium involvement of the female genital tract, including the vulva, vagina, and cervix, is common, affecting about one-third of women who are infected (2). By contrast, involvement of the uterus, fallopian tubes and ovaries is more rare (10-20% prevalence). Pelvic involvement with S. haematobium can be present without egg excretion in the urine, making diagnosis of S. haematobium infection more difficult (75). Cutaneous disease of the vulva can be mistaken for venereal disease (see the subsection "Skin Disorders," p. 281). Ulceration due to granulomas in the vulva, vagina, or cervix is likely a risk factor for transmission of HIV (76). Polypoid or nodular lesions may also develop on the cervix or vaginal wall, with local symptoms such as dyspareunia and postcoital bleeding (1). Pelvic masses may develop in response to focal egg deposition in and around the uterus. Adhesions and ectopic pregnancy or infertility may result (77). Successful antischistosome therapy has been associated with reversal of infertility in most cases.


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Intestinal Tract and Liver Disease S. haematobium egg deposition in the liver and intestines is common (76% of infected schoolchildren in South Africa (78)) and is most significant in the lower intestinal tract. Egg counts per gram of tissue in the sigmoid and rectum correlate well with the overall intensity of infection, and these locations are considered typical and not ectopic sites of egg deposition in S. haematobium infection (11). Egg concentration in the appendix may be quite high (49). Heavy infection has been associated with appendicitis in some case reports, suggesting that local granulomatous disease contributes to appendiceal obstruction (79). Intestinal polyposis may develop in S. haematobium infection, as is common in S. mansoni infection, and may cause obstruction (80). Polyps may ulcerate and bleed, causing hematochezia, and may later scar or become calcified, causing bowel wall dysfunction. Intestinal metaplasia and adenocarcinoma have been associated with local S. haematobium infection in some case reports (2). S. haematobium eggs also accumulate in the liver in high numbers (11). Hepatomegaly is common, but unlike S. mansoni infection, periportal fibrosis is rare (1). Tissue egg burden and severity of urinary tract disease are not correlated with the intensity or severity of liver findings (49).

Lung and Chest Disease Schistosomiasis can produce lung disease in several ways, both in the early migratory phases of infection and during the later forms of established chronic infection. The first type of disease, acute larval pneumonitis, presents 1-2 weeks after exposure, during the lung phase of migration of developing schistosomules. Patients present with dry cough and eosinophilia, with what appears to represent an immediate hypersensitivity response to migrating larvae (81, 82). This disease is more common in newly endemic regions or areas with intermittent transmission. Pulmonary disease may also occur during acute schistosomiasis, 4 - 5 weeks after exposure. More common with S. japonicum and S. mansoni infections, acute schistosomiasis (Katayama fever or snail fever) is a systemic inflammatory condition occurring in previously unexposed adults traveling to endemic areas for the first time. Fever and cough are prominent, accompanied by myalgias, arthralgias, headache, and abdominal symptoms.

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Physical examination may show rales, and the chest radiographs may show diffuse mottled shadows or a miliary pattern. Eosinophilia is common, but parasite eggs may not yet be detectable in the urine or stool. Diagnosis is usually established by serology, and treatment consists of antiinflammatory agents a n d / o r corticosteroids, followed by antischistosome therapy. Developing parasites are less susceptible to praziquantel than adult worms, so retreatment is recommended 2-4 weeks after initial therapy (82). For established, patent infections, egg granulomas are common findings in the lung in both human and experimental S. haematobium infection (61, 82). However, symptomatic disease is rare when compared to the prevalence of pulmonary arteritis and cor pulmonale seen in S. mansoni infection (2, 82). Eggs likely travel to the lungs via microembolization from the perivesical plexus to the inferior vena cava, then to the right heart and the pulmonary circulation. Egg accumulation is likely to be gradual, with egg granulomas of varying age scattered throughout the lung parenchyma. This is in contrast to S. mansoni infection, where significant numbers of parasite eggs reach the lung only after liver fibrosis has induced portasystemic venous shunting. The difference in symptomatic pulmonary disease between S. haematobium and S. mansoni infections likely reflects the more rapid and intense embolization that occurs with S. mansoni (83). Pulmonary involvement is greater with higher intensity S. haematobium infections — the prevalence of pulmonary disease is low in areas such as Sudan, where infection levels are generally light, whereas in Egypt and South Africa, where infection is more frequent and more intense, lung involvement is common (82). In a study from Nigeria, 87% of heavily infected subjects had pulmonary eggs detected at autopsy, in contrast to only 17% of subjects with light infections (84). Clinically, pulmonary symptoms with moderate to severe urinary schistosomiasis are minimal. In one study, 77% of S. haematobium-infected patients had decreased FEV1 on spirometry, which was reversible with isoproterenol in 90% of those affected. Thirty percent had decreased total lung volume, suggesting a component of chronic, irreversible injury. Blood gases were normal at rest, but 30% had significant desaturation with exercise (85). In such patients, there is likely a component of hypersensitivity reaction mixed with chronic restrictive lung disease (82). Cor pulmonale is a known sequel to pulmonary disease from intestinal schistosomiasis (S. japonicum, S. mansoni), but has only rarely been reported with S. haematobium alone (2).


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Egg granulomas have been recorded in the pericardium in autopsy series (11), and a case of fibrous pericarditis with cardiac constriction has been reported (86). This complication remains extremely rare.

Skin Disorders Cutaneous involvement in S. haematobium infection may take one of three forms. Upon exposure to cercaria-infested waters, a localized eruption (called "swimmer's itch," dermatitis schistosomica, or cercarial dermatitis) may develop in response to antigens deposited in the epidermis and dermis during larval migration (87). During early, prepatent infection, acute schistosomiasis may develop, manifesting, in part, as an urticarial rash, known as schistosomides (88). Late cutaneous disease from S. haematobium infection is caused by ectopic migration of adult female worms within the subcutaneous tissues. This manifestation is most common in the genital and peri genital areas (2). Known as bilharziasis cutanea tarda (BCT), it develops as papular lesions surrounding parasite eggs in the affected skin (88). In Nigeria, BCT has been more common among European travelers than in indigenous populations (89). The lesions can become hypertrophic, erosive, nodular, fistulous, or wartlike, in some cases resembling those of secondary syphilis (condylomata lata) (90). Biopsy will show granulomatous infiltrate, and the presence of parasite eggs is pathognomonic. Cases of late, migratory BCT dermatitis have been reported in the periumbilical area, the abdominal wall, the chest, and the neck (2).

Central Nervous System Disease Disease of the central nervous system (CNS) is very rare in urinary schistosomiasis, but it is often severe. Spinal cord involvement is more common than brain involvement (11, 91). CNS disease may occur due to aberrant migration of adult S. haematobium female w o r m s leading to intrathecal oviposition or to release of eggs into the brain and spinal cord. Alternatively, eggs may cause microembolization in the spinal column via Batson's plexus (91). The majority of eggs reaching the CNS do not cause symptoms (2). However, granulomatous inflammation in response to eggs

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may cause "tumors" of the lower spinal canal (92,93), which may respond to antischistosomal therapy, but more often require neurosurgical decompression (94). A more severe transverse myelitis, caused by both inflammation and vascular compromise, may cause rapid and irreversible damage unresponsive to medical or surgical intervention (95, 95). The lower spinal cord granulomatous lesions typical of S. haematobium present as lumbar or sciatic pain, followed by rapid-onset incontinence, then flaccid paresis and sensory loss with areflexia in the lower extremities (92, 94, 97). Eosinophilia may be noted in the blood or CSF, and antischistosome antibody is usually detected in the CSF (91, 96). Myelography (or MRI) typically demonstrates blockage and compression of the cord or cauda equina. At surgery, there is cord or conus mass effect associated with dilation of the vessels of the dura. The lesions appear irregular and are rubbery in consistency, with gritty patches (92). Microscopically, multiple granulomas are seen, which comprise glial reaction and inflammatory leucocytes (macrophages, lymphocytes, plasma cells, and eosinophils). Endothelial changes may be seen in the wall of larger vessels, along with thickening and inflammatory infiltration of the media and, in some cases, lumenal obstruction by eggs (92, 98). Although deposition of S. haematobium eggs in the cerebral tissues is a common finding on autopsy (56%), related disease is very uncommon (91). In the few recorded cases of intracranial disease due to S. haematobium, both encephalitic and meningeal findings were present, including headache, visual disturbance, vertigo, confusion, limb pain, and seizures. Symptoms and neurological deficits have usually responded to antischistosomal and corticosteroid therapy (91).

Renal Parenchymal Disease Associated with S. haematobium Infection A controversial area of S. haematobium studies has been the debate as to whether immune-mediated glomerular and tubular disease can be attributed to S. haematobium infection in human populations (50). While there is general agreement regarding the existence of glomerulonephritis in a subset of patients infected with Schistosoma mansoni, and there is agreement on the significance of obstructive uropathy due to granulomatous


pathology in S. haematobium infection, there has been continued disagreement over the existence of S. haematobium infection-associated glomerulopathy and tubular dysfunction (11, 50). Although studies by Ezzat et al. (99) have demonstrated glomerulonephritic lesions (containing parasite antigens) in S. haematobium-iniected patients, a firm association between S. haematobium infection and disease could not be proven due to the failure to exclude potentially confounding variables such as secondary bacterial infection and concurrent S. mansoni infection. In the face of conflicting findings (i.e. no evidence for glomerulopathy) in other series, the likelihood of S. haematobium-speciiic glomerular disease appeared to be limited (49, 100). A clinical study of 101 young South African children infected with lightto-moderate S. haematobium infections failed to show clinical or chemical evidence of glomerular dysfunction. The study did, however, note that bimicroglobulinuria was present in 7% of infected children, suggesting the presence of significant proximal tubular dysfunction (101). In this study, the lack of concurrent uninfected local control subjects, and the relative insensitivity of the assays tested, make interpretation of these data uncertain with regard to the true prevalence of renal parenchymal disease in S. haematobium infection. A case series from Kuwait has continued the controversy (102), observing that nephrotic syndrome with biopsy-proven membranoproliferative glomerulonephritis is frequently associated with either S. mansoni or S. haematobium infection. Supportive evidence for S. haematobium glomerular disease has come from experimental animal studies. The observation by Sobh et al. (103) that hamsters infected with S. haematobium (but not control animals) develop significant glomerular damage related to the presence and intensity of S. haematobium infection, provides evidence in favor of specific S. haematobium infection-related glomerular injury. Their controlled experimental studies demonstrated glomerular accumulation of immune complexes containing specific parasite antigens, including the previously defined circulating anodic antigen (CAA) and circulating cathodic antigen (CCA). Such findings strengthen the possibility that a select proportion of S. haematobium-iniected humans do develop significant renal parenchymal disease as a consequence of infection (50). Early, subclinical renal tubular or glomerular injury may contribute to the burden of illness due to coexistent chronic diseases.

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Carcinoma of the Bladder Another controversial area of research has been the association of S. haematobium infection with b l a d d e r carcinoma. Several studies in West Africa have failed to demonstrate a consistent association between S. haematobium prevalence and bladder carcinoma (reviewed in Refs. 2 and 11). However, in several geographic regions, there appears to be a definite risk for s q u a m o u s cell carcinoma (in contrast to the more common transitional cell carcinoma) among S. haematobium-intected individuals. In Egypt, which is endemic for S. haematobium, bladder cancer has been one of the most common forms of cancer, accounting for 28% of cases at the Cairo Cancer Institute in the early 1970s (104). More recent studies (1993) in Alexandria show that bladder cancer remains the most frequent cause of cancer in men (17.5% of cases), with an incidence of 10.8 per 100,000 (105). Careful case-control analysis, allowing for other risk factors such as smoking and chemical exposure, indicates a multivariate bladder cancer odds ratio of 1.72 for patients with urinary schistosomiasis (105). The odds ratio was higher (3.3) for young patients with bladder cancer, and very much higher (15.8) for males who had ever smoked. Overall, the attributable risk of S. haematobium infection for bladder cancer among hospitalized Alexandria patients was 16%. S. haematobium infection is associated with bladder cancer in many other parts of Africa. Bladder cancer is common in the S. haematobium-endemic countries of Zambia (106), Malawi (107), and Kenya (108,109). A large-scale epidemiological survey of Zimbabwe has shown a strong correlation between regional S. haematobium prevalence and incidence of bladder cancer (110). In particular, the incidence of squamous cell bladder carcinoma was found to be over 4/100,000 in endemic areas (40-67% S. haematobium prevalence) and less than 1/100,000 in low prevalence areas. Several lines of evidence indicate a significant role for S. haematobium in cancer formation. It was noted in the Zimbabwe study (110) (and others (111, 112)) that squamous cell cancers and transitional cell tumors afflict different age groups, with squamous cell cancers more common among younger individuals (< 40 years of age), particularly among S. haematobium-iniected patients (110). Schistosomiasis-associated tumors may affect any portion of the bladder, unlike transitional cell carcinoma, which usually arises in the trigone (11). S. haematobium-associated squamous tumors tend to be well


285

differentiated, and to metastasize locally, in contrast to typical transitional cell tumors, which are often more aggressive and show bloodstream metastases (104). Arguments against an etiological role for S. haematobium in bladder cancer formation have been based on the lack of correlation between high S. haematobium prevalence and bladder cancer in Saudi Arabia, Nigeria, and parts of South Africa (2). Further, there is a high prevalence of squamous cell carcinoma in developing countries with very low or no prevalence of S. haematobium, such as Uganda and Jamaica (2). Large, consecutive autopsy studies in Cairo have also not shown a consistent pattern of bladder tumor type associated with urinary schistosomiasis (49, 61). It is likely that S. haematobium infection is part of a multifactorial cascade of events leading to cancer formation. A population-based survey in an endemic area of East Africa has shown that up to 25% of S. haematobium-iniected adults have definite urinary epithelial metaplasia on cytological examination (113). In this study, the prevalence of metaplasia was independent of other concurrent inflammation, increasing in frequency along with infection prevalence and intensity over the first three decades of life, but persisting among older adults, despite their decreased infection intensity. These findings, combined with in vitro evidence of (1) mutagenesis from granulocyte cell products (114, 115) and (b) increased excretion of active chemical and environmental mutagens in the urine of S. haematobium-iniected animals (116), favor the likelihood of S. /zaematobz'wm-infection-associated carcinogenesis in humans. Chronic bacterial infection may lead to the accumulation of nitrosamines, while loss of mucosal integrity and bladder stasis may significantly increase exposure to intraluminal carcinogens (2). Studies by Cheever et ah (117) of long-term S. haematobium infection in capuchin monkeys indicate that intense parasite infection is associated with the development of significant multifocal epithelial proliferative lesions that resemble low-grade carcinomas. Loss of infectious burden over several years is associated with regression of these lesions, indicating that they are noncancerous in nature. However, in humans, one could envisage that a combination of exposure to specific mutagens in the urine and a similar urothelial proliferative response could lead, through multiple-step mutagenesis, to cancer formation. Chronic bladder inflammation, whether from S. haematobium or chronic catheter drainage, has been specifically associated with chromosomal breakage and micronucleus formation (118). Schistosomiasis-associated bladder cancer has been associated

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with mutations in the tumor suppressor genes p53 (chromosome 17p) and CDKN2 (also known as MTS1, chromosome 9p) (119). The latter is particularly associated with squamous cell cancer formation. At present, sufficient evidence has accumulated in favor of an association between schistosomiasis haematobia and human bladder cancer that the International Agency for Research on Cancer (IARC) has formally determined that "Infection with S. haematobium is carcinogenic to humans" (52). S. haematobium is assigned to Group I, or "definite human carcinogen," status.

Other Manifestations of Infection Field research has identified the significant, negative impact of lightto-moderate S. haematobium infection on childhood growth and development. In a study in Kenya, Stephenson et al. (120) randomly assigned children with multiple parasitic infections to therapy with either metrifonate, praziquantel, or a placebo and monitored for improvement of hepatosplenomegaly, hemoglobin levels, weight for age, height for age, and skin-fold thickness, as a function of change in their S. haematobium, hookworm, and malarial infections. Hemoglobin level, which was negatively correlated with hookworm burden and S. haematobium burden before therapy, showed significant improvement after therapy only in the group given metrifonate, a drug which reduced both hookworm and S. haematobium infections. No improvement in anemia was noted in the groups treated with praziquantel or a placebo. By contrast, there was significant weight gain in both the metrifonate and praziquantel groups, and this event was significantly correlated with reduction in S. haematobium burden. These authors present limited evidence that S. haematobium infection is associated with anorexia. This, coupled with a modest loss of nutrients through urinary tract lesions, is most likely to be responsible for the weight-for-age deficit associated with S. haematobium infection. Advances in the knowledge of the systemic behavioral and metabolic effects of inflammatory cytokines [e.g. tumor necrosis factor-a (TNFa, cachectin) and interleukin-1 (IL-1)] suggest that the molecular mechanisms of S. haematobium-associated nutritional and exercise tolerance deficits in individuals with light-to-moderate S. haematobium infection are inflammation-based, and lend weight to arguments in favor of targeting control programs to treat both light and heavy S. haematobium infections for maximal control of infection-associated morbidity in childhood.


Figure 3. Egg of S. haematobium seen in a filtered urine sample. The distinctive terminal spine is at the upper left.

Diagnosis Direct diagnosis of S. haematobium infection may be made by detection of parasite eggs in the urine, by either urine sedimentation or filtration techniques (Fig. 3). While egg detection is close to 100% specific for the diagnosis of infection, the sensitivity of urine examination will vary, depending on the intensity of infection, urine sample size, circadian and day-to-day variation in egg excretion, and the skill of the observer performing the examination (121-123). Peak egg excretion occurs about noon, and midday is felt to be the best time to perform urine collection for diagnosis (124). Enumeration of egg output can be used to assess the relative intensity of infection, and serial examinations can be used to gauge the efficacy of therapy in suppressing infection intensity. Diagnosis may also be established by demonstrating S. haematobium eggs in tissues of the bladder or rectum obtained by endoscopic biopsy (15, 121). Urine filtration is performed by passing a well-stirred aliquot (10300 ml) of patient urine through a filter holder containing a polycarbonate (Nuclepore), polyamide (Nytrel), paper (Whatman), or fiber filter to trap S. haematobium eggs. Paper filters have to be stained to identify the parasite eggs (125). Polycarbonate allows immediate visualization and enumeration

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of the eggs, although Trypan Blue or other vital stains may be added to discriminate viable and dead eggs (121). Where equipment is available, urine may also be centrifuged and the sediment examined for S. haematobium eggs. Both filtration and centrifugation may be made less sensitive by red blood cells or other cells or crystals found in the urine (121). Cells may be lysed and crystals dissolved by the application of acetic or hydrochloric acid. Among women of child-bearing years, menstruation may obscure the detection of parasite eggs in both types of assays. Indirect evidence of infection is given by the presence of hematuria, proteinuria, or leukocyturia in urines obtained from residents in endemic areas. By means of dipstick reagent strips, the presence of these findings may be rapidly established in the field, and used as a surrogate for parasitological diagnosis. Field studies in highly endemic areas support the use of dipsticks for rapid diagnosis in large-scale treatment endeavors (122,126-129). However, specificity is lessened among older girls and women due to menses or prior circumcision (121). Sensitivity will be lowered if the proportion of heavy infections is low, as is seen in communities after widespread chemotherapy. Therefore, sensitivity, specificity, and positive predictive value should be assessed in pilot studies before widespread use of surrogate dipstick diagnosis in any given population. Antibody tests have been developed for serodiagnosis of S. haematobium infection. However, due to the complexity of parasite antigens and the variability in human antibody responses, serodiagnosis has not proven to be a useful tool for establishing the presence of active infection in endemic populations. Serological assays may prove sensitive but not specific due to cross-reactivity with other parasites, so that the diagnostic value often varies greatly from location to location. Such assays also have difficulty in distinguishing active infection or reinfection from prior, but inactive, infection. Antibody assays appear most appropriate for detecting recently acquired infection among expatriates with recent exposure who may have early, nonpatent infection or very light worm burdens not easily detected by urine testing (121). Detection of parasite antigens in serum or urine appears to be a more reliable method for detection of schistosome infection. Circulating cathodic antigen (CCA) and circulating anodic antigen (CAA) are two gut-associated antigens of adult worms that have been found to circulate during active


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infection with various schistosome species. Daily variation of antigen levels is less marked than for egg counts, and, in general, antigen levels correlate with intensity of infection (121). Further, antigen levels decline significantly following successful chemotherapy. Sensitivity for detection of light infections is high, making it a useful test for screening older age groups having limited infection intensity (130).

Imaging of Structural Urinary Tract Pathology A major advance in the understanding of the natural history of S. haematobium-related urinary tract morbidity has come through the introduction of the portable ultrasound machines for noninvasive examination of the kidneys and bladder. With the use of generators to supply power in nonclinical settings, and with the use of instant photography or miniaturized thermal printers to record permanent images, it is possible to examine scores or even hundreds of individuals in endemic communities each day. Whereas earlier studies based on autopsy findings or intravenous pyelography h a d defined structural morbidity in older children and adults, autopsy studies were restricted by unavoidable selection bias, and IVP studies were limited in size by the technical complexity of the procedure and by the risks of contrast injection and ionizing radiation. Such factors significantly limited the information that was obtainable on the natural history of diseases. In particular, little information was available on the effects of curative therapy on the progression of S. haematobium-associated urinary tract disease. In assessing the discrepancies between different studies, it is possible that, because standard guidelines were not available, differences in image interpretation can account for differences in measured prevalence or persistence of morbidity. Geographic differences in parasite strains, in endemicity of infection, or in host susceptibility may account for some of the differences in morbidity reported by different observers in Egypt and in East and West Africa. With the use of standardized WHO protocols for examination, the relative ease of ultrasonographic study (including a high degree of compliance with follow-up examination) should allow detailed analysis of these possible biological differences.

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Therapy Effective therapy for infection with S. haematobium may be obtained with either praziquantel (Biltricide, Bayer; and generic preparations) or metrifonate (Bilarcil, Bayer) therapy. With both drugs, therapy results in a substantial reduction in intensity of S. haematobium infection and morbidity among those treated (65). Metrifonate requires multiple 10 m g / k g doses in order to achieve its best effect, although a single dose may result in effective reduction of infection intensity (53). Metrifonate is less effective against S. haematobium in some endemic areas (131), and because of its effects on acetylcholinesterase activity, may have greater toxicity among rural patients exposed to agricultural insecticides. For these reasons, metrifonate use has been generally supplanted by praziquantel therapy, which can be given as a single 40 m g / k g dose. Neither drug is 100% effective in curing infection — follow-up parasitological testing at 6-12 weeks after therapy is recommended, with repeat treatment given as needed. Regression of morbidity is seen in some, but not all, patients with urinary schistosomiasis after anthelmintic therapy. The inflammation and ureteral dysfunction present during the early years of infection is likely to resolve with therapy (1). However, chronic scarring, fibrosis, and calcification, with resulting stricture formation and obstruction, are not likely to be reversible with drugs, and corrective surgery may be needed. For those with renal failure, renal transplantation has been successfully used to reverse end-stage renal dysfunction (132).

References 1. Farid Z (1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford, UK), pp. 159-194 2. Chen MG and Mott KE (1989). Trop. Dis. Bull. 86: Rl. 3. WHO Division of Control of Tropical Diseases (1998). Schistosomiasis Control, www.who.int/ ctd/html/schistogeo.html 4. Rollinson D and Southgate VR (1987). In The Biology of Schistosomes: From Genes to Latrines, eds. Rollinson D and Simpson AJG (Academic, New York), p p . 1-50. 5. Sturrock RF (1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford, UK), pp. 1-32.


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95. Cohen J, Capildeo R, Clifford Rose F and Pallis C (1977). Br. Med. J. i: 1258. 96. de S Quieroz L, Nucci A, Facure N O and Facure JJ (1979). Arch. Neurol. 36: 517. 97. Wood MG, Srolovitz H and Schetman D (1976). Arch. Dermatol. 112: 690. 98. Pittella JEH (1985). Am. } . Trop. Med. Hyg. 34: 898. 99. Ezzat E, et al. (1978). Proceedings of International Conference on Schistosomiasis 1: 625. 100. Sadigursky M, et al. (1976). Trans. R. Soc. Trop. Med. Hyg. 70: 322. 101. Cooppan RM, et al. (1987). Am. ]. Trop. Med. Hyg. 37: 556. 102. Abu-Romeh SH, et al. (1989). Int. Urol. Nephrol. 21: 25. 103. Sobh MA, et al. (1991). Nephron 57: 216. 104. Elsebai I (1977). CA—A cancer journal for clinicians 27: 100. 105. Bedwani R, et al. (1998). Br. }. Cancer 77: 1186. 106. Bhagwandeen SB (1976). S. Afr. Med. } . 50: 1616. 107. Lucas SB (1982). E. Afr. Med. } . 59: 345. 108. Anjarwalla KA (1971). E. Afr. Med. J. 48: 502. 109. Bowry TR (1975). E. Afr. Med. J. 52: 356. 110. Thomas JE, et al. (1990). Trans. R. Soc. Trop. Med. Hyg. 84: 551. 111. Groeneveld AE, Marszalek WW and Heyns CF (1996). Br. J. Urol. 78: 205. 112. Kitinya JN, et al. (1986). Trans. R. Soc. Trop. Med. Hyg. 1986: 935. 113. Hodder SL, et al, submitted. 114. Rosin MP, Anwar WA and Ward AJ (1994). Cancer Res. 54 (Suppl): 1929s. 115. Weitzman SA, et al. (1985). Science 227: 1231. 116. Gentile JM, et al. (1985). Arch. Environ. Health 40: 5. 117. Cheever AW, et al. (1988). Trans. R. Soc. Trop. Med. Hyg. 82: 107. 118. Rosin MP, El Din Zaki SS, Ward AJ and Anwar WA (1994). Mutation Res. 305: 283. 119. Gonzalez-Zulueta M, et al. (1995). /. Nat. Cancer Inst. 87: 1383. 120. Stephenson LS, Latham MC, Kurz KM and Kinoti SN (1989). Am. } . Trop. Med. Hyg. 41: 445. 121. Feldmeier H (1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford, UK), pp. 271-303. 122. Savioli S, et al. (1990). Am. } . Trop. Med. Hyg. 43: 289.


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297

Chapter 8 Disease in Schistosomiasis Mansoni in Brazil Aluizio Prata

History Schistosoma mansoni is believed to have been brought to America from Africa with the slave traffic. In America, the microorganisms became fixed in the Caribbean, Venezuela, Surinam and Brazil.

Epidemiological Considerations Brazil represents one of the most important zones of distribution of schistosomiasis in the world, not only in terms of the number of patients, but also in terms of the severity of the disease in some of them. The disease, first detected in the state of Bahia in 1908, spread through the northeast and east of the country until it reached a distance of 500 km from the Atlantic Ocean. The area of schistosomiasis has expanded to the north, west and south of the country, generally in the form of isolated focal points. In the Amazon region, which corresponds to about two-thirds of the country, there is no schistosomiasis except for some focuses in Para and Maranhao. Along the large rivers of Brazil where there is an abundance of water and no irrigation, schistosomiasis has no major impact. In addition to the Special Program for the Control of Schistosomiasis (PECE in Portuguese) set up by the government, many people have taken medication on their own and a marked migration has occurred from rural zones, where the disease is acquired, to the cities. A marked decrease in the number of patients with the more severe forms of the disease has occurred in parallel (5). The mortality rate due to schistosomiasis has decreased in Brazil from

Mortality/100,000 0,8-

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0,7-

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mortality

• • • • - • • admission

77

78

79

81

82

83

84

85

86

87

88

89

90

91

0,67

0,68

0,7

0,7

0,62

0,56

0,54

0,51

0,53

0,49

0,52

0,42

0,38

0,38

1,66

1,62

1,92

2,81

3,14

2,72

2,61

2,42

Figure 1. Mortality rate and hospital admission for schistosomiasis mans

Disease in Schistosomiasis Mansoni in Brazil 299

0.67 per 100,000 inhabitants in 1977 to 0.39 in 1995. The rate of hospitalization due to schistosomiasis has also been decreasing after 1988 (Fig. 1). Many factors may condition the onset and maintenance of the parasitosis in an area, among them vector species, environmental conditions and inhabitant life style.

Factors Related to the Snails and Cercariae In Brazil, the best transmitter of schistosomiasis is Biomphalaria glabrata, Biomphalaria straminea and Biomphalaria tenagophila also transmit schistosomiasis. The large water reservoirs of Brazil have not been reported as important focal points of schistosomiasis, possibly because they only serve as sources of electrical energy and do not attract people to their margins. In general, water bodies existing near dwellings in the rural area or near small villages and slightly or moderately polluted by organic substances or human feces are excellent breeding sites for the snails. These peridomiciliary focal points are very important in the transmission of schistosomiasis. Contact with water infested with cercariae is almost obligatory in some regions because of the absence of other water sources for human use. Excreta are also deposited in natura. In endemic areas, contact with water infested with cercariae starts soon after birth. Although schistosomiasis is the result of poverty and ignorance, it may also be aggravated by progress and development, as is the case for certain programs of irrigation and pisciculture. Although under seminatural conditions it is possible to maintain the cycle of S. mansoni between Nectomys squamipes or Holochilus brasiliensis and Biomphalaria glabrata, in Brazil there is no conclusive evidence of the epidemiological importance of this transmission and no maintained focus has been detected thus far without the presumed participation of man.

Infection and Reinfection with Schistosoma

mansoni

In many areas where schistosomiasis is endemic, contact with cercariae starts soon after birth and the number of young people who eliminate S. mansoni eggs in a single fecal examination progressively increases up to the age of 10-15 years. Based on the calculation that 16 eggs per gram of

300 Schistosomiasis

Table 1. Number of worm pairs in the initial infection of 36 children aged 1-3 years in Caatinga do Moura (Brazil). Worm pairs

Children

1 3 4 6 7 9 13 15 72

13 8 5 4 1 2 1 1 1

feces correspond to one pair of worms (19), early infection among children from Caatinga do Moura (Brazil) corresponded to a maximum of 15 worm pairs, except for one case (Table 1). Among these children submitted to a repeated fecal examination 5-6 years later, the number of worms was found to be less than 15 pairs only in 8, being 15-30 pairs in 13 and above 30 in 7. The quantity of worms increases progressively with age between 10 and 14 years, decreasing thereafter. This has been well demonstrated at least for individuals with portal hypertension from whom the worms were surgically removed (Table 2). Since youngsters aged 15-19 years continue to frequent the sites of transmission with the same assiduousness as youngsters aged 10-14 years (41), the reduction in parasite burden has been interpreted as the consequence of a slow and progressive

resistance to infection, whereby the worms that die cannot be replaced with new worms at the same rate. A long-lasting longitudinal study of the same population from Caatinga do Moura showed that all individuals eliminated S. mansoni eggs through the feces or presented a positive skin reaction at some time in life. Evolution studies of shorter duration (110) have shown that some individuals may show many negative fecal examinations even in the absence of specific treatment and with exposure to focuses of transmission of schistosomiasis. These patients


Table 2. S. mansoni removed by extracorporeal filtration from human portal blood. Age range (years)

5-9 10-14 15-19 20-24 25-29 30-34 35-39 40-44 45-49 >50

Number of patients

7 28 21 25 19 10 12 8 5 1

Worms removed per patient (mean)

(median)

1029 1219 716 539 677 753 409 408 584

673 1127 518 451 342 549 304 140 592

differ immunologically from those infected with S. mansoni by presenting strong cellular and humoral responses against certain schistosomiasis antigens (7, 29) and no circulating schistosomal antigens (110). The production of IFN-y may be associated with resistance to infection (28). Another type of reinfection that has motivated special studies is that occurring after specific treatment when individuals continue to be in contact with waters infested with cercariae. Re-infection in endemic areas after specific treatment occurs at higher frequency and intensity among younger individuals (88) and among those who have more contact with the focal points of infection (17, 41). The fact that after treatment (a) some individuals do not become reinfested, or (b) when they are reinfested the parasite burden is never as high as before treatment (31, 73, 103), (c) the peak of the curve of infection prevalence and of the number of eggs in the feces is reached in younger age groups, and that (d) hepatosplenomegaly does not develop among these individuals, suggests that treatment increases resistance to reinfection. Multiple drug treatment during a brief period does not induce resistance to reinfection (102). Attempts have been made to identify immunological characteristics that distinguish highly susceptible individuals from individuals resistant to reinfection. These studies have shown that the response to antiegg antigens

302

Schistosomiasis

declines more after treatment among resistant than among susceptible individuals (17) and that resistant individuals demonstrate stronger reactivity to the 37-kDa surface antigen of schistosomiasis (41). The higher susceptibility to reinfection of younger people may be related to antibodies that block protective immunity (17). Resistant individuals have higher IgE levels against adult worm (49) or larval (39) antigens, while IgG2 and IgG4 levels are higher in individuals more susceptible to reinfection (39). Some investigators believe that high infection intensity before treatment is a predictive factor of further reinfection (11, 41). This agrees with data reported by Abel et al. (4), who, using segregation analysis for the population of Caatinga do Moura, showed that resistance to reinfection is largely determined by the effects of a major codominant gene later referred to as SMI and mapped to the human chromosome 5q31-q33 by linkage analysis in the same population (75) and also in Sudan (78), i.e. in the genetic region that contains various genes encoding molecules that control T lymphocyte differentiation. These data, taken together with the observation that resistance is regulated by lymphokines characteristic of T helper (Th)2 subsets (32) and that resistant homozygotes show a ThO/2 response against schistosomes, as opposed to a Th0/1 response among susceptible homozygotes, strongly suggest that the differences in human susceptibility to schistosomiasis are influenced by the polymorphism of a gene controlling T lymphocyte subset differentiation (3). In this respect, segregation analysis in the same human population from Caatinga do Moura showed that interleukin-5 (IL-5) levels are also under the control of a major gene (98), raising the possibility that IL-5 may play a critical role in resistance. This agrees with its strong positive effect on the multiplication, differentiation and activation of eosinophils that are important for the destruction of helminths and for the synthesis of IgA, which is also thought to be involved in protection against schistosomes (3). Other immunology studies on man have shown the role of eosinophils and monocytes in larval destruction and the importance of cytokines for the e n h a n c e m e n t of these functions a n d also for the u n d e r s t a n d i n g of schistosomule vulnerability over the various days that follow infection. The contacts with the sources of infection, age (or, better, the duration of exposure) and the effects of the major gene represent more than 65% of the variance of infection intensity in the Caatinga do Moura study (40).

Disease in Schistosomiasis Mansoni in Brazil

303

Early Phase or Acute Schistosomiasis General Considerations This is the phase that starts with cercaria penetration and ends with the disappearance of symptoms. It is also called the acute or toxemic phase. The latter designation, however, should be reserved for the severe forms of acute schistosomiasis, i.e. according to the definition of Farley (50). The observation of newborn infants in endemic areas permitted us to conclude that the early phase of schistosomiasis presents under two forms, depending on age and on the conditions under which the first contacts with infectious focal points occurred.

Patients in Permanent Contact with Focal Points of Infection The early phase of schistosomiasis among children of inhabitants of hyperendemic areas progresses in a silent manner and therefore is not recognized. There is no liver enlargement or splenomegaly and the skin test with adult worm antigens is positive in 50% of cases. Eosinophilia is discrete, corresponding to less than 10% of all leucocytes in 78% of cases.

Patients in Sporadic Contact with Focal Points of Infection Patients with acute schistosomiasis are usually young males in sporadic contact with the sources of infection. They acquire the infection during their leisure activities when they bathe in lagoons, wells or pools contaminated with cercariae. Another risk group is represented by military men who come in contact with waters infested with cercariae during war or during military maneuvers.

Clinical Manifestations After cercaria penetration there may be pruritus lasting up to two hours, followed by urticaria or papulo-erythematous exanthema. Neves (79) reported that 13 of his patients (10%) showed immediate severe skin manifestations. There is evidence that pruritus and other skin manifestations are more frequent and intense in sensitized individuals.

304

Schistosomiasis

The period of incubation is almost always one to two months and is usually asymptomatic, although prodromic symptoms appear in some patients. Symptoms usually start in an abrupt manner, consisting of fever often accompanied by headache, shivering, sudoresis, asthenia, anorexia, myalgias, coughing, and diarrhea. Temperature reaches 39°C and is not continuous, often being reduced to less than 37°C for a few days, especially in the morning. Fever is long-lasting, often being present for more than one month and sometimes two months and later disappearing by lysis. Fever is occasionally accompanied by delirium. Shivering and sudoresis are constant and repeated and the intensity of the latter is remarkable. Cough is accompanied by bronchial spasm, with the possible occurrence of asthmatic attacks and areas of bronchopneumonia. Nausea and vomiting are common. Diarrhea and, not infrequently, dysentery also occur. Manifestations of hypersensitivity such as urticaria, generalized pruritus, facial edema, erythematous plaques, or purpuric lesions are almost always present. Weight loss is a rule. The liver is frequently enlarged and painful to palpation. The spleen is usually palpable. Hepatosplenomegaly usually disappears within a few months even without treatment. Micropolyadeny is present. The symptoms of the acute phase of schistosomiasis may be discrete or go unperceived. Blood tests always reveal leucocytosis reaching 50,000 leucocytes/ mm (3), and a 20 or 30% frequency of eosinophilia above 1000, at times exceeding 70% in specific counts. Globulins are increased due to the gamma fraction, and red cell sedimentation rate is increased. A myelogram shows hyperplasia of the eosinophil series. The Paul-Bunnell reaction may be positive.

Pathogenesis and Pathology The phase of cercarial penetration has been extensively studied in laboratory animals. The parasites penetrate the dermis, producing sulci and provoking an inflammatory infiltrate with a predominance of leucocytes, neutrophils and eosinophils. The products elaborated by the cercariae provoke the reactions, although from an antigenic viewpoint the cercariae of Schistosoma mansoni evoke a less intense response than other cercariae (8, 30).


305

In animals, the passage of metacercariae to the lungs may produce foci of arteriolitis, arteritis and necrosis (72). Acute focal intralobular hepatitis may occur, characterized by focal hepatocyte necrosis and infiltration with a predominance of neutrophils and lymphocytes. These changes may not be clinically exteriorized. The pathogenesis of the anatomoclinical manifestations found before the beginning of worm oviposition resides in phenomena of hypersensitivity to the juvenile forms of the schistosomes and in the eosinophilia that may arise as early as during this phase. The characteristic anatomopathologic changes of the symptomatic form of the acute phase of schistosomiasis arise with worm maturation and the beginning of oviposition. Liver biopsies (43, 79) and some autopsies performed on patients with a severe course or who died as a consequence of antimony treatment (81,92) have provided information about the anatomopathologic lesions, which we summarize below. Intense miliary dissemination of Schistosoma mansoni eggs was characteristic in deceased patients, with the consequent formation of granulomas in all organs and tissues, but especially in the liver, intestine, visceral peritoneum, abdominal and pulmonary hilus lymph nodes, lungs, pleura, and pancreas. These granulomas are characterized by a necrotic-exudative nature and by the fact that they enter a productive phase only after the 110th day of infection (91), a time when granuloma modulation occurs. Acute superficial ulcerative enterocolitis occurs in many cases. The spleen shows intense congestion and thickening of Billroth cords, with histiocyte hyperplasia and diffuse eosinophil infiltration. Obviously, the lesions must be much more discrete in patients with less severe clinical forms. Diaz-Rivera et al. (43) reported areas of diffuse acute eosinophil and lymphocyte infiltration which are more marked in the portal spaces and which disappear late, persisting only around the granulomas. During the initial phase of schistosomiasis observed in infants from a hyperdendemic area, there is no hypersensitivity reaction. Newborn infants have some immunity in the form of specific antibodies transmitted through the placenta (61). Might be probably desensitized in utero when in contact with the parasite antigens. The changes occurring in various organs should be considered as a reaction of the organism to the elimination of allergens by the worms and the eggs, producing a state of hypersensitivity. This hypersensitivity has been demonstrated by studying the immune response of patients (53). After some time,

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the response of the organism is modulated (92) and the disease rogresses to the chronic phase. Late skin manifestations such as urticaria, eyelid and facial edema, and purpuric manifestations can be explained by increased capillary fragility of allergic origin (43). Bronchospasm and pulmonary infiltrate are indistinguishable from the findings observed in infiltrative eosinophilia (42). Diarrhea is produced by irritation of the intestinal mucosa due to the presence of eggs, with edema and punctiform hemorrhagic spots detected by rectosigmoidoscopy. Hepatomegaly may result from the cell infiltrate and edema. Lymphadenopathy and splenomegaly may represent an immune reaction. Shivering, fever, prostration, and sudoresis are part of the manifestations of the toxemic state that sets in during this phase. The manifestations of the acute phase may also depend on parasite burden (97) and on the immune status of the organism (23) and may also appear as a consequence of reinfection (79). The acute phase ends without a reduction of the number of eggs eliminated in the feces.

Chronic Schistosomiasis It is usually the extent of liver involvement that conditions the clinical form under which chronic schistosomiasis will manifest.

Intestinal and Hepatointestinal Form The intestinal and hepatointestinal forms, with often ill-defined limits between them, are the habitual clinical forms presented by most schistosomiasis patients. It is even more difficult to determine their clinical picture with safety, since the results of questionnaires distributed to underprivileged populations parasitized by Schistosoma mansoni do not show appreciable differences compared to those obtained from nonparasitized populations. Diarrhea is the most common manifestation, reported by approximately half the patients. It is periodic and lasts a few days. It is often accompanied by blood, mucus, and tenesmus, and it alternates with constipation. Other patients report abdominal pain, especially in the hypocondria and in the left iliac fossa, difficulty in digesting certain foods, nausea and, rarely, vomiting and flatulence. Each of the following complaints is reported by


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a third of all patients: dizziness, nervousness, headache, shortness of breath, and loss of weight. Also frequent are insomnia, asthenia, somnolence (especially postprandially), myalgia and, more rarely, anorexia, cold extremities, heartburn, palpitations, sudoresis, impotence, anal pruritus, and rhinitis. Many patients are asymptomatic, but symptoms may arise any time, often abruptly. The liver is frequently enlarged and hardened. Ultrasound has shown a high frequency of patients habitually without portal hypertension and with typical Symmers fibrosis, with no splenomegaly and asymptomatic in endemic areas. Blood tests reveal blood eosinophilia that is at times quite elevated, but in general lower than during the acute phase. Among our patients, when anemia was present it was predominantly of the hypochromic macrocytic type. Liver function tests are normal. Rectosigmoidoscopy shows a normal mucosa in almost half the patients, while in others the mucosa has a granulose aspect and presents congestion, hyperemia, punctiform hemorrhage, and easy bleeding. Radiologic examination of the large bowel may demonstrate spasms, mucosal edema, or signs of intestinal atony.

Hepatosplenic Form In the presence of advanced hepatic lesions, portal hypertension may arise. When this is accompanied by splenomegaly, the form of the disease is called hepatosplenic schistosomiasis.

Importance The hepatosplenic form represents a divider in the evolution and prognosis of the disease. In the absence of hepatosplenomegaly in schistosomiasis there is no pulmonary hypertension, cyanotic syndrome, peri-intestinal fibrosis, or infantilism, the schistosomotic glomerulopathy is much less frequent, and severe and morbid associations with salmonellas are much less likely to occur. Some unusual localization of Schistosoma mansoni eggs may result in severe clinical forms in patients without hepatosplenomegaly, such as schistosomotic myelitis and the tumoral form of the disease. However, these are very rare

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occurrences. Without hepatosplenomegaly the importance of the disease would not differ much from that of the intestinal geo-helminthoses. From an epidemiological viewpoint, the severity of the endemy in an area is defined by the prevalence of the hepatosplenic form. On this basis, this form should be characterized in order to learn how it sets in and what factors are involved in its development.

Characterization The intense involvement of the liver is reflected on the onset of the typical hepatic fibrosis described by Symmers (104). The organ is usually enlarged, especially the left lobe, and becomes hardened. The surface presents nodules which anatomopathologists prefer to call pseudonodules. Bogliolo (15) described it as thickening of the portal spaces resulting from pylephlebitis and peripylephlebitis. No cirrhosis occurs since the hepatic structure is not disorganized and significant cell destruction is also absent. The basic lesion is fibrosis which produces intense neoformation of connective tissue associated with angiomatoid capillary neoformation forming a cuff around the intrahepatic portal branches (70) with no subversion of the lobular architecture. Periportal fibrosis is accompanied, to a greater or lesser extent, by obstruction of the portal ramifications (70), which, together with the loss of portal vessel elasticity, may lead to portal hypertension. As far as symptoms are concerned, patients with schistosomotic portal hypertension may present the same complaints as described for hepatointestinal involvement in addition to those inherent in the new situation created in the liver. Digestive hemorrhages, especially hematemesis, represent the most important clinical manifestations of the condition. At times they are due to the ingestion of aspirin tablets. They are somewhat proportional to the extent of portal hypertension and are often fatal, even during the first episode. Hematemesis may occur unexpectedly or be preceded by asthenia or epigastric discomfort, followed on the subsequent day by fever, elimination of coffee-dregs-like feces, and later by edema and ascites when blood loss is severe. The characteristic of ascites is that it promply responds to treatment. Melena is rare in the absence of hematemesis but some patients present it alone or in combination with enterohemorrhage, with a possibly lethal outcome. Superficial collateral circulation is discrete or absent except in the


presence of ascites, in contrast to the frequency of abundant deep collateral circulation. The liver is hard, has a cutting border and a frequently nodular surface, and its left lobe may be prominent. Liver volume apparently regresses with the course of the disease. Manifestations of severe hepatic insufficiency are rare and may arise during the terminal phase of the disease or as a consequence of surgeries for portocaval shunts. The spleen is palpable in the costal margin or beyond. Certain patients present infantilism. Amenorrhea is common. Low fever and not very high febrile peaks are frequent. Some patients with hepatosplenomegaly develop portal thrombosis which may go unperceived or may produce often intense symptoms such as pain, abdominal distention, and ascites, regressing after a few days. Varices of the esophagus or stomach can be observed by radiologic examination or by esophagoscopy. When the two methods are combined, varices are observed in about 80% of cases. Peritoneoscopy permits visualization of the aspect of the liver and of the intra-abdominal circulation. Splenoportography reveals the image of a large part of the extra- and intrahepatic portal system and of the hepatic circulation. Cholecystograms are almost always negative. Ultrasonography can provide information about the portal system, showing obstructions, vein caliber, thickening of vein walls, and collateral circulation, with the advantage of being a noninvasive and low-cost method. Portable equipment permits examinations in endemic areas. It is the best method for the demonstration of the pathognomonic portal thickening. Ultrasonography is imperative for a sure diagnosis of Symmers fibrosis, simultaneously providing information about liver and spleen size, gallbladder, pancreas, and other abdominal organs. Transparietal splenic pressure, which from a practical viewpoint reflects portal pressure, is almost always about 200 mm water (normal portal pressure: 5-10 mmHg). In contrast to what occurs in Laennec cirrhosis, the pressure of the occluded hepatic vein is normal or slightly elevated, reflecting the presence of a presinusoidal obstacle. In portal hypertension with splenomegaly, the hematologic picture is well defined and is essentially characterized by isolated or combined peripheral cytopenias, side by side with the corresponding medullary changes. Measurement of plasma proteins usually shows a reduction of albumin and an increase of globulins, especially of the gamma fraction. Liver function tests are rarely altered.

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Pathogeny of Simmers Fibrosis There is still a debate about the schistosomotic elements responsible for the lesion and the mechanisms that produce it. The debate is generally centered on the role of adult worms and of the eggs. Adult worms. Apparently these are well tolerated and have no mechanical, irritating or toxic effects while they live. Their excreted or secreted metabolic products are eliminated through the portal vein. These are definitely antigenic substances but their pathogenic importance is undefined. Hepatitis has been reported to occur before the beginning of oviposition both in acute human schistosomiasis (15) and in uni- or bisexual experimental infections (76), but there is no evidence that hepatitis produces fibrosis (77). The hepatitis that appears during the chronic phase may be related to the worms and may be important in the production of portal fibrosis. In chimpanzees, portal fibrosis in large and medium portal spaces precedes oviposition (100). Barreto et al. (9) reported that in Caatinga do Moura, wild guinea pigs (Cavia aperea aperea) become spontaneously infected on average with 259 schistosomes. In these animals, schistosoma eggs do not mature and do not produce granulomas. These animals do not develop hepatitis, fibrosis, or other vascular lesions of schistosomiasis. Even less likely is the possibility of lesion production by toxins, whose existence has not been proved even in experiments on parabiotic mice (90). The schistosomotic pigment, even though it stimulates hyperplasia of cells of the phagocytic system, is an inert substance that does not cause inflammation or fibrosis (94). When the worm dies spontaneously or as a consequence of specific treatment, it becomes stuck in one of the smaller branches of the portal vein and produces inflammation, obstruction, and necrosis, and eventually scar formation. However, the lesions are irregular and not systematic such as those of Symmers fibrosis (14), and are completely absorbed (115). Furthermore, millions of schistosomotic patients have been treated in Brazil, with no report of the development of hepatosplenic syndrome. In a few patients worm death has been associated with manifestations of worsening of the hepatic condition, elevation of portal pressure and hematemesis, or repercussions on the respiratory apparatus such as pneumonia, asthma, acute cor pulmonale, and even anaphylactic shock or generalized vasculitis and death (35). Eggs. Most of the investigators w h o study this topic believe that schistosomotic hepatic fibrosis is produced by schistosome eggs alone or in


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combination with other schistosomotic elements. Supporting this view are the systematization of the process, its nature, the type of vascular lesions, and the egg distribution. Bogliolo (15) stated that the process of vascular and connective tissue neoformation starts exclusively in the periportal vasculoconnective tissue, as demonstrated by the intravascular injection of stains and by plastic molds of the portal and arterial trees and of the suprahepatic veins. How the egg plays its pathogenic role has also been discussed. A few days after maturing, the myracidium starts to release histolytic substances that are also antigenic and that are eliminated through the egg shell. In the intestine, the purpose of this mechanism is to open a path to the outside environment. A cell infiltrate arises around the eggs that are not eliminated, such as those that go to the liver, and is transformed into fibrous tissue, forming the granuloma, which is the mechanism by which the host tries to localize, neutralize, and absorb the egg and its products. When present in large numbers, the eggs provoke granulomatous inflammation and vascular obstruction and act as fibrogenic agents. Fibrosis may be due to a chronic granulomatous inflammatory response to the eggs and perhaps to fibroblaststimulating factors. Many authors believe that the formation of granulomas around schistosome eggs is the major process responsible for the development of the severe forms of the disease, such as Symmers fibrosis and pulmonary arteritis. The granulomatous reaction to the egg is a cell-mediated immunologic response (113) that may be suppressed by drugs (47), neonatal thymectomy (45), antilymphocyte serum (46), antimacrophage serum (75), or be increased. The size of granulomas in animals may decrease in the more intense infections or with the course of infection (112). Since the aspect of human granulomas is similar to that of animal granulomas, suggesting a similar mechanism of formation (55), the study of granulomas has attracted the attention of investigators interested in the pathogeny of schistosomiasis. The granuloma is a source of fibroblast stimulation with an important effect on the regulation of hepatic fibrosis (118). Experimentally, the amount of collagen in the liver increases in proportion to the formation of a granuloma around the eggs (114). The schistosomotic granuloma may have the property of releasing substances capable of attracting to perisinusoidal spaces cells that synthesize the constituents of periportal fibrosis (56). In studies on humans, Raso (95) and Cheever (19) observed that not many eggs are present in the portal spaces before the onset of fibrosis, but that eggs concentrate there during the advanced stages of the disease.

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Symmers hepatic fibrosis does not seem to result from the simple fusion of granulomas since in man it is not related to the granulomas in terms of intensity or uniformity. Also, hepatic fibrosis may not be associated with granuloma size in mice (21). It has been postulated that the soluble antigens eliminated by eggs containing a live myracidium are the major factors responsible for the granulomatous reaction and periportal fibrosis of Symmers (94). Two types of soluble antigens were isolated from schistosome eggs (SEA), capable of stimulating the fibroblasts: one may act directly on these cells and the other on lymphocytes and monocytes, which may release fibroblast-stimulating factors. Side by side with the production of chemotactic and fibroblast-activating substances, there may also be the production of collagenase by macrophages responsible for the destruction of extracellular collagen. A disequilibrium between collagenosis and collagenolysis may occur in schistosomotic hepatic fibrosis, with increased collagen synthesis and tissue fibrosis. Host immune response. In spite of protection against schistosoma it is clear that the immune system is also related to immunopathologic side effects. Humoral hypersensitivity usually occurs. As for cellular immunity the results vary according to the authors and the duration of hepatosplenomegaly. Besides, cause and effect cannot be evaluated. For some authors (34) the cell immunity is depressed in hepatosplenic patients and, to a lesser extent, also in patients with hepatointestinal involvement, and is related to worm burden. The patients may recover their immune reactivity through specific treatment of schistosomiasis. Immunodepression may impair the elimination of pathogenic elements. For some others (27), only the late hepatosplenic patients are anergic while early hepatosplenic ones are hyperergic. Symmers fibrosis would be due to immune response lack of modulation. Some data suggest that 11-10 is an important cytokine in regulating the immune response and possibly controlling morbidity in h u m a n schistosomiasis mansoni (74). Animal model. Hepatic fibrosis that reproduces Symmers fibrosis to a greater or lesser extent may be induced in two animal species, the mouse (6, 111) and the chimpanzee (71). The absence of macroscopic similarity and the fact that Symmers hepatic fibrosis does not seem to be due to the simple fusion of granulomas in man, as is the case for mice, are two arguments raised by those who believe that the human lesion is not the same as the mouse lesion.


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Chimpanzees may present hepatic lesions quite similar to human Symmers fibrosis (100). The macroscopic aspect of the outer surface of the liver is nodular, and typical "clay pipe" lesions can be seen when the organ is cut. The animal may develop hepatosplenomegaly, collateral circulation, and esophageal varices even when portal pressure is within normal limits. Symmers fibrosis occurs in animals with a high parasite burden after two years of successive reinfections (100). Hepatic fibroplasia. In any organ, fibrosis results from the deposition of excessive connective tissue, mainly consisting of collagen, which represents the fibrillar proteins and the nonfibrillar components, in addition to other elements less frequently found and not yet fully characterized. Studies on connective tissue have shown that fibrotic processes are not always irreversible lesions (57), since the connective tissue they contain still retains its ability to form itself again (99). Even in installed fibrosis the activities of collagen synthesis and collagenolysis continue (48). Thus, we may propose that the fibrous process can be controlled (52). Collagen, the major component of connective tissue, is not a uniform substance and different types are found in man, distinguished by the precise amino acid sequence that forms the alpha chains. Five types of collagen are present in the liver and many of their characteristics in human schistosomiasis are known (12). Studies on cell cultures and enzymatic and biochemical determination have shown that collagen synthesis is not exclusively attributable to the fibroblastic cell line and that endothelial cells, Ito cells, Kupffer cells, myofibroblasts, smooth muscle cells, and even hepatocytes or other cells may form collagen as long as they are properly stimulated (58). The same cell may synthesize different collagent types and there is some evidence that the mechanism of collagen selection by the gene may depend on extracellular or environmental factors (12). These factors, which may operate on the cell to modulate the synthesis of a certain collagen type, are still unknown. Ultimately, hepatic fibrosis may result from the proliferation of cells that synthesize collagen, from the increased formation of collagen in existing cells, or from deficient degradation of continuously formed collagen (99). In a study in which we tried to determine whether urinary hydroxyproline would serve as a marker of hepatic fibrosis, we detected greater elimination in patients with hepatosplenic involvement, with a reduction after specific treatment (60).

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A lower reversal of fibrosis may occur in advanced phases of the disease (18). In the advanced phases of the hepatic fibrotic process there is a predominance of type I collagen, whose turnover is slower (117). However, treatment may reverse schistosomotic fibrosis regardless of the time when it became installed (36, 44). Focal areas of rarefaction, fragmentation and dispersal of collagen bundles, hyperplasia of elastic tissue, and dissociation of smooth muscle fibers seem to indicate the degradation of the extracellular matrix in schistosomotic portal fibrosis. Installation. The hepatosplenic form sets in slowly in patients older than five years, usually 5-15 days after the initial infection. In the exceptional case reported by Neves and Raso (80), Symmers fibrosis evolved 130 days after the initial infection. This form occurs in some individuals, usually less than 10%, among the populations living in certain hyperendemic areas. Sporadic reports of percentages higher than 13% in the literature are due to unacceptable diagnostic criteria. In our observations (86), three years are needed to characterize hepatosplenomegaly since it first starts to appear in 25% of cases, four years in 45% and more than four years in 30%. Enlargement and hardening of the left lobe of the liver are first observed, with the left lobe predominating over the right one. The appearance of nodules on the liver surface is not detected within less than two years. The initial changes are observed in the liver in a third of all cases, but enlargement of the spleen is simultaneously observed in two thirds of all cases. This initial enlargement of the spleen is not due to portal hypertension. When splenomegaly is recent there is a predominance of cell hyperplasia over dilatation of venous sinuses and thickening of the sinusoidal walls, which reflect spleen congestion (66). Periportal fibrosis probably begins around the small peripheral branches (54). We may state that in chronic schstosomiasis without advanced hepatic lesions the host and the parasite seem to be in equilibrium. Whatever the pathogenic elements may be, they are eliminated or neutralized by the organisms at an appropriate rate. The response of the host to the aggressions is contained and appears to be proportional to the stimulus. For still unknown reasons, this equilibrium may be broken either because the stimulus becomes more intense and the organism is unable to maintain the rhythm of neutralization of noxious elements or because there is a modification of the mode of reacting of the host, generatin tissue


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alterations. Thus, the lesions detected in Symmers fibrosis are installed little by little.

Factors That Affect the Installation of Symmers Fibrosis In addition to the mechanisms of installation of schistosomotic hepatic fibrosis and to the schistosomotic elements involved in its genesis, some other factors should be considered which may be important in the development of hepatosplenomegaly. These factors may be related to the parasite (parasite strain and parasite burden) or to the host. Schistosome strains. Some investigators found no differences among the strains studied by them, whereas others did. There is no evidence that Symmers fibrosis, and consequently the hepatosplenic form, are related to the strain of Schistosoma mansoni. Parasite burden. Although the worms can be counted by extracorporeal removal of portal blood or evaluated by the determination of circulating antigen levels in plasma or excreted in urine, egg counts in feces are the usual method for the evaluation of parasite burden. The daily variation in the number of eggs in the feces is quite extensive. However, for epidemiological purposes, egg counts have been frequently used and are of great usefulness in schistosomiasis despite individual discrepancies. The relationship between the quantity of eggs eliminated through the feces and the development of the hepatosplenic form of schistosomiasis is still a controversial matter. This correlation may not be observed in older individuals (64) and ceases to exist as spelnomegaly sets in. The hepatosplenic form is encountered more in areas with greater prevalence and egg elimination in the population. The questionable aspect is whether the individuals who eliminate eggs through the feces are those who develop the hepatosplenic form of the disease. However, on average, the worms extracted from hepatosplenic patients represent a high parasite burden, a condition that is definitely important for the installation of Symmers fibrosis. Exposure to the risk of reinfection. Only individuals who are in repeated contact with the focal points of infection develop the hepatosplenic form of the disease. Even if they remain in endemic areas but their opportunities for reinfection are reduced by the continued use of moUuscicides or by health

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education, they do not develop the severe forms. We may assume that exposure to the risk of infection may increase the parasite burden, breaking the apparent equilibrium between egg laying and destruction in tissues (22), or may alter the response of the host (25) regardless of the presence or absence of an increased parasite burden. The use of a serologic reaction with a specific antigen of Schistosoma mansoni cercariae may be of help as a marker of exposure (89).

Factors Related to the Host In some regions the disease appears to be more benign than in others. In the same area in which people are apparently exposed to the same conditions and even eliminate the same amount of eggs, only some develop the hepatosplenic form. Age. It has been discussed whether patient age or only the duration of the disease is important. In Nova Esperanga, where transmission of the disease seems to have been introduced only recently, we found only young hepatosplenic individuals, suggesting that adults may not develop the severe form of the disease in this location. However, Pessoa and Coutinho (85) detected severe forms in adults exposed to continuous reinfection. Malnutrition. Experimental studies indicate that nutritional deficiencies seem to protect against, rather than aggravating, infection with the schistosomes. Furthermore, we have seen many hepatosplenic patients among families of good socioeconomic level and in endemic areas of the state of Bahia — mainly white people, who are known to have the best living conditions — develop hepatosplenomegaly more frequently. Alcoholism. There is experimental evidence that alcoholism reduces the granulomatous immune response of the host to Schistosoma mansoni eggs (84). Kasanda (63) showed that alcoholism reduces the number of worms, oviposition, granuloma formation, and extent of hepatic fibrosis in mice. The prevalence of positive fecal exams for Schistosoma mansoni eggs was 68.9% in abstemious patients and 57.1% in alcoholic patients, but there was no difference in the amount of eggs eliminated through the feces. The hepatosplenic form of the disease was detected in 26 (10.2%) of abstemious subjects and in 6 (6.8%) alcoholic subjects. Among the 26 abstemious subjects, the hepatosplenic form was decompensated in 1 and among the 6 alcoholic patients it was decompensated in 2.


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Host Predisposition Genetic influence. Different mouse strains are known to produce different degrees of fibrosis when infected with Schistosoma mansoni (20), and collagen synthesis is known to be regulated by genes (58). Race. In some parts of Brazil, blacks are more resistant to the development of the hepatosplenic forms of schistosomiasis even though they acquire the infection at the same frequency and intensity (87) and live in worse socioeconomic conditions. In another region, blacks developed more hepatic fibrosis but less splenomegaly, suggesting that differences in the response to the infection with respect to the clinical forms of the disease exist even among blacks. This may be reflected in the surnames of people, with hepatosplenic subjects having surnames related to animals or plants (preferred by white racial groups) more than surnames with a religious connotation (adopted by blacks) (105). Specific treatment produced regression of hepatosplenomegaly in 10 (47.6%) nonwhite persons and only in 2 (8.3%) white persons (106). Familial occurrence. Several hepatosplenic subjects occur within the same nuclear family and this is not a random occurrence. Since in general the members of a family frequent the same focal points of contagion, at times it is difficult to discriminate what may occur due to the environment. Studies of the pedigrees of these families do not suggest simple Mendelian inheritance, but probably a multifactorial and possibly polygenic inheritance. Inbreeding coefficient. In certain hyperendemic areas of schistosomiasis, hepatosplenic patients have a higher inbreeding coefficient (26.8%) than hepatintestinal patients (12.5%) (107), raising the suspicion of genetic influence (10).

Genetic Markers Although not yet in a conclusive way, some genetic markers have been associated with the hepatosplenic form. Blood groups. Some studies have shown that the hepatosplenic form of schistosomiasis mansoni, a n d also the Japanese form, may be more frequent among persons with group A blood, a fact that was not confirmed by others.

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Major histocompatibility complex. The hepatosplenic form has been correlated with the A l and B5 antigens of the histocompatibility system (1). The presence of HLA-DQB*0201 in the patient major histocompatibility complex is associated with a higher risk of developing hepatosplenomegaly (101). Carriers of the BW44-DEN haplotype are predisposed to schistosomotic hepatic fibrosis (82). Glioaxalase I system. The incidence of patients with the hepatosplenic form of the disease is four-fold higher in GLO*l/GLO*l homozygotes and threefold higher in GLO*l/GLO*2 heterozygotes than in GLO*2/GLO*2 homozygotes (51).

Complications of Hepatosplenomegaly Patients with portal hypertension may have pulmonary hypertension, cyanotic syndrome, peri-intestinal fibrosis, and glomerular disease, in addition to morbid associations.

Pulmonary Hypertension Approximately 25% of hepatosplenic patients have a mean pulmonary artery pressure exceeding 20 mm mercury. The more severe the portal hypertension, the higher the frequency of pulmonary hypertension. The anatomical substrate of this form is schistosomotic pulmonary arteritis. Hypertension is localized in the pulmonary artery and is later followed by chronic cor pulmonale. The major complaint of the patients is dyspnea, which at the beginning occurs only after an effort but may later become continuous. Palpitations are frequent. Both conditions arise soon after hypertension reaches a certain degree. Many patients report chest pain attributed to distention of pulmonary vessels. Effort syncope appears in some cases during exercise and is preceded by dizziness, clouded vision, headache, and epigastric and precordial discomfort. Coughing and hemoptysis may rarely occur. Physical examination reveals abnormal beats of the second, third, and fourth left intercostal spaces, and the second cardiac bubble and diastolic fremitus are palpable. The most frequent finding is hyperphonesis of the second bubble in the pulmonary focus, which at times is doubled.


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Auscultation can also demonstrate the noise of the opening of the pulmonary valve and murmurs. Radiologic examination reveals dilatation of the cone and of the trunk of the pulmonary artery, an increased vascular network, and enlargement of the hilar shadow, especially on the right, which resembles a large comma and often reaches the dimensions of an aneurysm. The middle arch, represented by the cone of the pulmonary artery, is bulging or at least straightened. The right ventricle may be enlarged. Radioscopy shows the typical pulsativity of the dilated right pulmonary artery. Angiocardiography confirms the findings and reveals, in some cases, the tortuous and irregular aspect of some peripheral pulmonary vessels. Ultrasound may be useful for diagnosis. The pulmonary circulation time may be delayed. Arterial oxygen saturation is normal but does not always reach 100% after the inhalation of pure oxygen due to the shunts established from the right to the left. The electrocardiogram shows no abnormalities before the appearance of right ventricular hypertrophy and sometimes even after the beginning of this condition. The electrocardiogram is a good indicator of disease progression. There is no parenchymal or capillary injury and cyanosis is not an important clinical manifestation. When it arises, it is associated with severe heart failure.

Cyanotic Syndrome Faria et al. (51) pointed out the existence of a syndrome accompanied by cyanosis and drumstick-shaped fingers in schistosomiasis. Many patients complain of effort dyspnea and present increased cardiac output, alveolar hypertension, and reduced arterial saturation even after breathing pure oxygen for 15 minutes. After exercise there is no increase in systolic pressure in the right ventricle and arteriolar resistance is reduced in the lung. These are patients with hepatosplenomegaly with or without discrete pulmonary hypertension. In about half the cases, cyanosis appears after splenectomy. It has been debated whether cyanosis is due to pulmonary arteriovenous fistulas, to portal-mediastinal anastomoses (also observed in Laennec cirrhosis) definitely related to portal hypertension, or to the decrease in oxyhemoglobin affinity for oxygen.

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Peri-intestinal Fibrosis Approximately 11% of the hepatosplenic patients submitted to autopsy present fibrotic thickening of the adipose tissue of the large bowel, especially of the rectum, frequently extending to the retroperitoneum. In contrast to Brazil, in Egypt there is a frequent occurrence of abdominal masses in the epiplons, mesenteries, and ganglia and of subserosal pericolonic masses, commonly associated with polyps, drumstick fingers, hypoalbuminemia, and dysentery (2) even among patients without Symmers fibrosis.

Nephropathy In areas where schistosomiasis is endemic, proteinuria can be found in 24.6% of hepatosplenic patients and 4.6% of hepatintestinal patients. Renal changes occur in 12% of hepatosplenic patients with lesions in an advanced stage. These lesions are practically of all types, but are more commonly of the nephrotic syndrome type with persistent proteinuria and generalized membranoprolipherative glomerulonephritis (96). The mesangial area of the glomeruli is the major site of nephropathy. Glomerulopathy has been reproduced in animals, depending on parasite burden over a prolonged period of time. The lesions are related to the presence of circulating antigens originating from the adult worm, from eggs and cercariae and their antibodies, with deposition of immune complexes in the glomeruli. The clearance of these antigen-antibody complexes should occur in the liver and for this reason the importance of collateral circulation in the pathogeny of glomerulopathy has been discussed. The patients may present edema and at times ascites, proteinuria, and gammaglobulinemia, but cholesterol is not elevated in blood.

Other Clinical Forms Peudoneoplastic

Form

This form involves patients with and without hepatosplenomegaly. It is caused by localized accumulation of large amounts of eggs and by an intense and often disproportionate fibrotic reaction. The tumor may be very large and is a frequent finding at laparotomy. These tumors are frequently located


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in the intestine or peritoneum. In the intestine, the following order of frequency is observed: descending and sigmoid colon, terminal ileum, and small intestine. The patients may present intestinal transit disorders, and if the tumor grows into the intestinal lumen, occlusion may occur. If the tumor grows from the serosa, there is an increase in volume that compresses organs.

The intestine may be found to be hardened upon palpation. The tumor may be located outside the digestive apparatus at sites such as the female or male genital tract or in the nervous system.

Neuroschistosomiasis The cerebral localization of S. mansoni eggs is not so frequent as for the eggs of S. japonicum. Transverse myelitis is more common, and is being currently diagnosed in Brazil at much higher frequency. The condition results from egg accumulation forming a tumor, usually in the lumbar spinal cord. Paraplegia sets in rapidly, with sphincter disorders, sensory alterations, and increased numbers of cells and protein in the cerebrospinal fluid. Other clinical pictures occur, such as cauda equina syndrome. The patients usually are not hepatosplenic.

Morbid Associations Prolonged Septicemic

Salmonellosis

More than 20 salmonella species of human or animal origin may be associated with schistosomes. They adhere to the surface of the schistosomes, mainly male worms, emitting fibers that penetrate their tegument. They are also found in the intestine of the worm. They may remain sheltered at these sites for more than two years. This association between the worm and a bacterium produces a clinical picture mainly characterized by fever of insidious onset and of long duration, diarrhea, weight loss, abdominal pains, pallor, edema of the lower limbs, a marked hepatomegaly and splenomegaly. Contrary to what occurs in typhoid fever, in septicemic salmonellosis, even induced by Salmonella typhi, the patients do not present sensory manifestations or intestinal perforation. In approximately half the patients, petechiae appear in the lower limbs in outbreaks lasting on average 3-7 days and coinciding with pain in the joints.

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Mild forms of the diseases have been reported to occur in endemic areas. With the reduction in the number of hepatosplenic patients in Brazil there has been a simultaneous decrease in the number of patients with prolonged septicemic salmonellosis. Laboratory tests show hypergammaglobulinemia, eosinophilia, and frequently moderate leucocytois and neutrophilia. Some patients with prolonged septicemic salmonellosis develop renal damage due to immune complexes in which the bacteria act as antigens (67). Recurrent infection caused by Staphylococcus aureus in the presence of high IgE levels in the serum has been reported in acute schistosomiasis (69). The association of S. mansoni with E. coli has also been reported.

Other Morbid Associations Association of schistosomiasis with hepatitis B and C has been observed in hepatosplenic patients. The patients with this association present a higher frequency of stellar angiomas, jaundice, and high aminotransferase levels. Chronic active hepatitis is more frequently detected in these patients, a fact that aggravates the evolution of portal hypertension. This association was observed in hospitalized patients or patients followed up at health services, where they are more exposed to the transmission of these viruses during diagnostic and therapeutic procedures. A well-conducted study in an endemic area did not confirm this association (108). Giant spleen follicular lymphoma has been detected in hepatosplenic schistosomotic patients.

Etiologic Diagnosis The diagnosis of schistosomiasis almost always needs to be confirmed by the detection of the parasite egg. Egg counts in feces, usually performed by the method of Kato modified by Katz et ah, are more often applied in epidemiological studies and in research. In Brazil, the examination most frequently used is spontaneous feces sedimentation, recommended by Lutz at the beginning of the century. The eggs are found more in rectal biopsies, which, being an invasive method, are performed only when repeated fecal examinations fail.


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The skin test with adult worm antigen is not frequently used because of the possibility of false-positive results and because it does not become negative when schistosomiasis becomes inactive. Serologic tests are rarely used in practice. The ELISA test using as antigen keyhole limpet hemocyanin, which shares a carbohydrate epitope with the surface of S. mansoni schistosomula (59), permits the distinction between acute and chronic schistosomiasis. A simple and rapid test that would identify the presence of worm antigens in blood or, better, in urine (37, 38), would be highly useful for the selection of patients for selective treatment in control programs (62, 109).

Clinical Diagnosis Acute schistosomiasis is suspected on the basis of clinical data, epidemiological history, and the presence of more than 1000 eosinophils per mm (3) of blood. Symmers fibrosis is suspected when the liver is found to be hard, with a nodular surface and almost always with a prominent left lobe. The diagnosis should be confirmed by ultrasound, which, in addition to revealing the extent of portal hypertension, shows the typical thickening of the portal space, which can also be demonstrated at autopsy or in surgical biopsies, but not by fine needle biopsy. The portal thickening observed by ultrasound is only pathognomonic for Symmers fibrosis when marked. Thickening classified as grade I may simply indicate a cellular infiltrate and not fibrosis (83). Symmers fibrosis characterizes the hepatosplenic form only when accompanied by splenomegaly of a certain size. The detection of a spleen palpable only during inspiration and usually flaccid is frequent even in non-endemic areas. Portal hypertension can be confirmed by the presence of esophageal varices demonstrated by radiography or esophagoscopy, by ultrasound or by splenoportography. The most frequent complication is pulmonary hypertension suspected upon clinical examination and demonstrated by a chest X-ray and by cardiac catheterization. Prolonged septicemic salmonellosis is suspected in hepatosplenic patients from endemic areas with fever of long duration. Salmonella is easily isolated from blood cultures and, to a lesser extent, from bone marrow, urine, bile, and feces.

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It is often difficult to determine the role of Schistosoma mansoni or Salmonella in glomerulonephritis among hepatosplenic schistosomotic patients. Decompensation of hepatosplenomegaly may occur, usually after repeated hemorrhages or in the presence of viral hepatitis, alcoholism, after portosystemic shunt surgery, or, more rarely, for no apparent reason. Under these circumstances, patients develop liver insufficiency with jaundice, ascites, superficial collateral circulation, portosystemic encephalopathy, and altered hepatic function tests.

Treatment Treatment of schistosomiasis without advanced lesions consists of cure of the parasitosis using specific medications. When the severe forms of the disease arise, they acquire an individual and prioritary characteristic at times depending and at others not depending on parasite activity. For these and other reasons, in the treatment of schistosomiasis it is importanmt to establish two diagnoses at the very beginning: the diagnosis of parasite activity and the diagnosis of the clinical form of the disease. In the treatment of clinical forms, auxiliary medications are used in addition to specific drugs. Treatment is also important in terms of the control and prophylaxis of the transmission of schistosomiasis.

Specific Treatment Only oxamniquine and praziquantel are used for the treatment of schistosomiasis in Brazil. Oxamniquine is effective by the oral route in a single dose of 15 m g / k g administered in capsules. The dose for children, of 20 m g / k g , in the form of a syrup, will be better accepted if divided into two administrations per day. Medication by the oral route produces somnolence and dizziness in some patients and, rarely, hallucinations, but in general is well tolerated, curing approximately 80% of chronic schistosomotic patients. Oxamniquine is widely used in Brazil in programs of disease control with no immediate medical supervision or special care. Praziquantel is administered orally at the dose of about 50-60 m g / k g body weight divided into two administrations. The major toxic reactions are abdominal pain, diarrhea, asthenia, and headache. The percentage of cure


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produced by praziquantel and its tolerance by the patients are the same as those obtained with oxamniquine. Resistance to treatment has been observed in some patients with both drugs (26). This is a source of concern although, in practice, it is a fact observed only exceptionally.

Surgical Treatment Surgical treatment of schistosomiasis is mainly indicated in the presence of hemorrhage caused by portal hypertension and for the extirpation of tumors caused by S. mansoni. The major types of surgery recommended are splenectomy, portosystemic shunts, and direct interventions on the varices. Splenectomy. The procedure has the following advantages: it eliminates the abdominal tumor, immediately corrects hypersplenism, cures infantilism and other endocrine disorders, improves the general condition and at times the liver function tests, and reduces portal pressure by about 40%. However, it does not provide sufficient relief of the basic alteration, which is portal hypertension, and therefore splenectomized patients may have digestive hemorrhage, although at low frequency. Splenectomy prevents the later performance of certain anastomoses. Portosystemic shunts. Theoretically these would be the ideal type of surgery for the treatment of schistosomotic portal hypertension, because they may facilitate blood drainage, thus attenuating the basic disorder of this clinical form. Portocaval anastomosis. Portal hypertension is not always substantially reduced. Erythrocyte half-life is shortened and there is an increase in indirect bilirubin. Tolerance to ammonia decreases and some of these patients develop portosystemic encephalopathy. The operation is contraindicated in the treatment of schistosomotic portal hypertension. Splenorenal anastomosis. A selective shunt has been preferred to a total shunt. Some of the problems mentioned about portocaval anastomosis are observed during patient follow-up, although at lower frequency. For this reason, many surgeons contraindicate this operation for schistosomotic patients (24). Other operations, such as ligation of the hepatic and splenic arteries, are less indicated.

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Direct operations on the varices. Disconnection surgeries such as transesophageal ligation, esophagogastric resection, etc., tend to replace shunt operations in schistosomiasis. Esophagogastric devascularization in combination with splenectomy has been frequently indicated (24). By producing sclerosis of the varices by endoscopy, it is often possible to eliminate them in many patients, but hematemesis will eventually recur in some patients. Treatment is slow and requires several applications. Complications such as ulcerations and narrowing occur in some patients after sclerotherapy.

Therapeutic

Conduct

The association of prednisone at the dose of 1 m g / k g body weight for one week may be recommended in acute schistosomiasis with intense symptoms, starting one day before the use of oxamniquine, followed by half the dose during the second week and by 0.25% m g / k g body weight for an additional week (68). The combination of the steroids increases the percentage of cure, improves the symptoms, and reduces the duration of the disease. In chronic schistosomiasis, when parasite activity is present, specific treatment should be instituted to relieve symptoms and to prevent an asymptomatic form of the disease from becoming symptomatic or even severe. The contraindications of specific treatment with oxamniquine or praziquantel are few. Obviously, treatment of children younger than two years, of pregnant women and of patients in the advanced stages of the disease with marked malnutrition, severe cardiac or renal failure, ascites, acute infections, or similar situations should be avoided. In the presence of portal hypertension with no history of digestive hemorrhage, regardless of patient age, specific treatment should be immediately instituted. This treatment can lead to reversal of the hepatosplenic form of the disease in 40% of cases within 6-24 months, and to improvement in other patients. Only after this period will it be possible to think about the possibility of surgery. Splenectomy, selective portal decompression, and esophagogastric devascularization reduce the probability of digestive hemorrhage, although they do not prevent them with safety. This does not mean that surgery should be systematically indicated for patients with portal hypertension in the absence of hemorrhage, since other factors, including surgical risk and the fact that some patients never


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present hematemesis, should be considered. Spleen volume may rarely be an indication for splenectomy, which also improves the general conditions of the patient and accelerates the cure of infantilism. In endemic areas of Brazil, there is no evidence that splenectomized patients will have more infections than nonsplenectomized patients. In portal hypertension with a history of hematemesis, the risks of new hemorrhages are higher and there is a more frequent surgical indication of intraesophageal ligation of varices associated or not with splenectomy or of portal-selective decompression by distal splenorenal anastomosis (Warren's operation). In the presence of hematemesis, patients with schistosomotic portal hypertension should be immediately hospitalized and kept under observation. The measures to be adopted are correction of volemia and anemia, gastric lavage, the use of coagulants, intestinal lavage, prevention of hepatic coma, and treatment of hepatic insufficiency. If repeated hemorrhages occur, an esophageal tamponade is immediately applied with a Sangstaken-Blakemore balloon. Pitressin may be included in the treatment. If hemorrhage is not inhibited, direct operations on the varices should be performed. Some physicians prefer to indicate them before using the balloon. Beta-adrenergic blockers (Propranolol) reduce cardiac output and splenic blood flow, reducing portal pressure. Some authors recommend their continued use to prevent the recurrence of hematemesis. The cyanotic syndrome does not benefit from removal of the spleen, but may be aggravated by it. In the pseudoneoplastic forms and in myelopathy, the use of corticosteroids should be combined with specific treatment and surgery is indicated only when clinical treatment fails. Specific treatment does not reverse the lesions of schistosomotic glomerulopathy but has a beneficial effect on the renal damage caused by prolonged septicemic salmonellosis. Starting in 1975, a vigorous Special Program for the Control of Schistosomiasis (PECE in Portuguese) was implemented in Brazil for the mass treatment of millions of people with oxamniquine. Applications of niclosamide (Bayluscide) have also been performed at some snail breeding sites and the program of piped water supply has been expanded. The result was a reduction of the prevalence of schistosomiasis. Today, the major objective of PECE is to control morbidity and to prevent expansion of the disease. The number of schistosomotic patients with the hepatosplenic form of the

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disease has been progressively declining in Brazil even in areas not submitted to special control programs or before such programs were implemented.

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Chapter 9 Disease in Schistosomiasis Mansoni in Africa John Ouma, Taha El-Khoby, A l a n Fenwick and Ronald Blanton

African Genesis Africa is a place central to human evolution and the likely birthplace of the host-parasite relationship in schistosomiasis mansoni. Natural infection in nonhuman primates indicates that S. mansoni is adapted to a variety of our relatives and suggests that this parasite family may have been part of the hominid microflora for eons here (1-6). There is at the very least a long relationship between schistosomes, the land, vectors and people of the continent. The eggs of Schistosoma haematobium can be found in Egyptian mummies from 3000 years ago (7-9), and while there are no estimates for the earliest human infection with S. mansoni, it is possible that a significant amount of human-parasite coevolution has occurred in Africa. In addition to innate human biology, culture and commerce also influence the transmission and expression of disease through cultural practices, social organization, forms of agriculture, fishing and water development projects. In Africa, schistosomiasis has some aspects of transmission, morbidity and response to therapy which are unique or accentuated compared to other regions. The size of the continent, the variety of environments and differences in levels of development influence infection dynamics and disease presentation. Complicating the picture, in Africa there are multiple schistosome species, overlapping infections with nonhuman parasites, viral hepatitis and the world's worst malaria. The major challenge for control will be to overcome the problems of development, population pressures, lack of infrastructure, lack of funding and drug resistance.

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Distribution and Changing Patterns of Infection Schistosomiasis mansoni is now endemic in at least 36 countries in Africa, with only the northwest and the southwest of the continent being free of transmission. Infection in any one region of the continent, let alone any one country, is a patchwork of distributions and intensities not well described by comparing the raw numbers of those infected to the total population. Within the borders of a country, the distribution of S. mansoni can be quite focal. In Kenya, for example, the parasite is completely absent from the coast and the northern third of the country, though it is endemic to the rest. Another characteristic of the epidemiology of schistosomiasis in Africa is that it is not static, and changes in transmission are continually being reported. Frequently, increases in prevalence occur whenever manmade water management schemes are implemented, or when political instability or wars lead to a breakdown in utilities, in health service delivery and disease and vector control measures. On the other hand in many countries, reductions in prevalence and morbidity have been reported as control measures and social development decrease transmission potential. Finally, accurate assessments of infection rates are difficult to find. On the whole, the distribution of S. mansoni infection appears to be advancing on the continent. This is clearly aided by increased human-water contact via new man-made bodies of water which provide an ideal habitat for the snail intermediate host. Irrigation schemes, which attract human communities to settle in less than sanitary conditions, are criss-crossed with open, mud-lined, grassy, snail-infected canals. Communities using these canals for irrigation, domestic washing and leisure use will quickly become heavily infected. Thus, economic development, which is key to the control of some aspects of transmission, may also promote infection. The greatest increases in infection in recent years have occurred as a result of the man-made environmental changes, for example the large scale construction of dams creating huge man-made lakes in Ghana (Lake Volta), Egypt (Lake Nasser) and between Senegal and Mauritania (the Diama Dam on the Senegal River). The resulting environmental changes from these water resource developments have been implicated in contributing to a huge increase of schistosomiasis transmission. In Mauritania, the first ever transmission of S. mansoni was reported in 1996 after the construction of Diama Dam on the Senegal river in 1988. On the opposite side of the

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project, transmission of S. mansoni in Northern Senegal was extraordinary — an epidemic with very high prevalence rates exceeding 90% in some areas, associated with high intensity of infection, but low morbidity as measured by ultrasonographic evidence of fibrosis (10). These examples represent the largest constructions, but the effect of smaller village dams seems to have equally significant consequences for transmission and distribution of schistosomiasis on a local scale, due to increased water-contact sites. A different phenomenon has been the marked change in the distribution of the two species of schistosomiasis in the Nile Delta in Egypt, where S. mansoni has virtually replaced S. haematobium after the construction, in 1968, of the Aswan High Dam. Many factors have contributed to this phenomenon, including changed irrigation patterns, increased salinity of water, and local ecological conditions. A similar increase in the prevalence and distribution of S. mansoni, and associated reduction in S. haematobium infection, has been reported from the Sudan. Again, a change in irrigation patterns contributed to an extension of S. mansoni's range. Other factors influencing the distribution of S. mansoni in different areas of Africa are the problems of rapid urbanization and refugee movements. The introduction of schistosomiasis into periurban areas through r u r a l urban migration is a major factor, because these new urban areas are often lacking in safe water supply and sewage disposal. The wars and lesser conflicts in Angola, Mozambique, Somalia, Southern Sudan and Burundi have resulted in the breakdown of health care delivery, disease prevention and vector control programs. In contrast to Sudan, the conflict in the Democratic Republic of the Congo may have little impact on a health infrastructure already severely strained. Although data are scarce at present, increases in the prevalence of various diseases, including schistosomiasis, can be expected to occur. Further, refugee movements as a consequence of war have also contributed to the spread of schistosomiasis to areas previously free of the parasite, such as in parts of Somalia. A summary of the current status of some of the African countries follows:

North Africa: Egypt and Sudan In general the Arab countries in North Africa (except Sudan) are enjoying a relatively peaceful period of economic development, and the result is an increase in the standard of living. As the rural population is increasingly

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provided with electricity, safe water supplies and improved sanitation, their dependence on unsafe water decreases. Health education and availability of praziquantel for treatment mean that those with infection can be treated and transmission is slowly but surely reducing. Egypt is the country in Africa whose situation has been best investigated recently and merits a more detailed review. There have been many reports of the prevalence and intensity of infection in Egypt dating back to 1935, but unfortunately it is impossible to directly compare the results, because of the variability in the quality, accuracy and sensitivity of the different examination techniques used for stool samples. In the early survey work, all examinations were direct stool smear. In later studies, ether concentration methods were used, and more recent surveys used the modified Kato-Katz method for examination. Another variable is in the population sample itself. While some surveys examined all inhabitants of randomly selected households, others surveyed schoolchildren, hospital inpatients, hospital outpatients, or those turning up at rural health centers for whatever reason. The definitive baseline data were collected in 1935 when Scott (11) estimated that 3.2 million people were infected with S. mansoni in Egypt. Of the infected people, 90% were living in the Northern and Eastern Delta, where the prevalence was over 60%, and the remainder in the Southern Delta, where the prevalence was only about 4% (in Menofia). Scott also estimated that 6.4 million people were infected with S. haematobium in Egypt, with a high prevalence all over the country, except in the oases. In 1983, a survey in the Nile Delta mirroring that carried out by Scott, demonstrated a prevalence of about 40% for S. mansoni; however, these high prevalence rates were found also in the Southern Delta (12). To the south of the Delta, and to the south of Cairo, there are a few oases and a strip of irrigation stretching about 1000 km along the banks of the Nile from Cairo to Aswan that have enough water to support transmission. In a few localities, S. mansoni was found during the 1990's, for example in Faiyum, Sohag and Quena Governorates, but to date the prevalence has usually been low. Schistosomiasis has not been found in oases in Egypt except for the special situation in the Faiyum area. Faiyum is a fertile depression around an oasis, and a huge irrigated area has been created there. However, the land is irrigated by canals taking water from the Nile, and it is these canals which are the source of schistosomiasis.


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A completely different situation is found in Sudan, where to the north of the country there are vast irrigation schemes, and to the south it is but a country torn by war. In the north, an extensive S. mansoni problem was created in the 1970's by the expansion of the Gezira and Managil irrigation schemes. The combined area of about 2 million feddan (1 feddan = 1.04 acre) supports a population of over 2 million residents and about 0.5 million migrant workers who spend the months of March-August there picking cotton and eking out a living on the land with their livestock. As with the Nile Delta in Egypt, the prevalence of S. mansoni increased and that of S. haematobium decreased in the period 1940 through 1980. A research and control project (The Blue Nile Health Project) in the years 1978-88 defined the extent of the problem and implemented a control strategy based on praziquantel, snail control and health education. Attempts were made to provide even the poorest villages with safer water supplies and latrines. The result was a marked reduction in S. mansoni from 1978 through 1988 (from over 50% to below 10%). With the political and financial upheaval in Sudan, however, an upsurge in infection in the irrigated area is always a danger if health services break down. A recent report from the fringes of the irrigated areas suggested that b o t h species were p r e s e n t in schoolchildren along the White Nile. Over 6000 children from 27 schools were examined, and prevalence rates had risen to 20% (13). In Southern Sudan and in the contiguous White Nile District in Uganda, S. mansoni is very prevalent, and causes morbidity from hematemesis. Unfortunately, hard data are impossible to collect, due to the political situation and an upsurge in diseases more acute than schistosomiasis, e.g. visceral leishmaniasis, malaria and sleeping sickness.

East Africa: Ethiopia, Kenya, Uganda and Tanzania With the abundance of lakes and rivers in East Africa, S. mansoni is, and has been, a prevalent and serious infection. The shores of Lake Victoria, for example, provide perfect transmission conditions, but prevalence, intensity of infection and morbidity vary from district to district. On Ukerewe Island, Tanzania, S. mansoni prevalence of 86% and median egg output of 176 eggs/ g of stool were reported, with associated morbidity (14). In Tanzania, Kenya and Uganda, S. mansoni prevalence in the lakeside communities is often

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over 60%. Recent reports suggest similar high prevalence rates near the Mozambique border. Beside Lake Tana in Ethiopia, 29% of 472 students were infected, and the intensity of infection was high, with over 20% of those infected having an egg count in excess of 800 eggs/g of stool (15). Other areas of extremely high prevalence are associated with water development projects, such as the Awash Valley in Ethiopia, and the rich agricultural land in areas like Machakos in Kenya where small local dams abound (16). In recent years, the improved primary health care systems in the east African countries have provided diagnosis and treatment, which has led to a decrease in prevalence and morbidity.

West Africa: Togo, Nigeria, Cameroon, Benin, Sierra Leone, Liberia, Ghana, Cote d'lvoire, Senegal, Mali, Gambia, Guinea, Burkino Faso and Niger In most countries in tropical West Africa, S. haematobium has traditionally been the more prevalent of the two species. Until recently, S. mansoni has been virtually unknown in Mauritania, Senegal and Niger, and in Ghana and Benin infection rates are usually below 10%. There are, however, many foci of S. mansoni in West African countries where the prevalence can be high (around 60%). This is the case in the southwest of Burkino Faso, near the capital in The Gambia, in the north of Sierra Leone, and in isolated foci in Guinea, Liberia and Cote d'lvoire. Other areas with high prevalence include the Niger flood plain in Nigeria, in Cameroon, and in many foci in Mali. In general, S. mansoni was not considered a major health problem in West Africa; however, the completion of the Diama Dam changed the situation very quickly after 1988. The resulting ecological changes favored S. mansoni transmission and during the 1990's a massive epidemic of S. mansoni took place, affecting naive populations in Senegal and Mauritania. Throughout the affected areas, every individual over the age of five years was infected, and the intensity of infection was extremely high. Praziquantel, which normally has a cure rate of over 80%, was reported to be effective in only 37% of those treated, giving rise to fears of praziquantel-resistant strains of the parasite (17). Potential drug resistance associated with this outbreak will be discussed later in more detail.


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Southern Africa: South Africa, Zimbabwe, Zambia, Botswana, Mozambique, Swaziland, Angola and Namibia There is a dearth of recent information from South Africa, where schistosomiasis mansoni is known to exist but has not previously been considered an important public health problem. However, recent estimates (Appleton personal communication) suggest that over one million people may be infected with S. mansoni in KwaZulu-Natal and another million in infected areas in Mpumalanga and the Northern Province. The prevalence probably has not changed over the last 10 years, although a new dam on the Komati River in Mpumalanga may increase the intensity of infection in an area where the prevalence is already high (over 50%). There is scant knowledge about morbidity, and no national control program in South Africa; however, the availability of drugs through the primary health care system probably prevents serious morbidity. Recent information from Zimbabwe (Blair Research Laboratory, Causeway, Harare) suggests that the overall prevalence in the rural areas, is less than 10%, but in selected areas there are foci where prevalence of up to 20% can be found. In Zambia, S. mansoni is the less widespread of the two species, and the overall prevalence is probably not more than 2% of the population. Near Lake Kariba, however, recent studies have identified infection rates as high as 63% in isolated communities and 77% in schools (18). Hospitalbased studies have suggested that hepatosplenomegaly due to schistosomiasis can be found in 10-20% of cases in some areas. A recent study around Lake Malawi (19) showed that among expatriates whose only exposure was in the Lake, 32% were serologically positive for schistosomiasis. However, 96% of the positive cases were S. haematobium, and only a small number (6/440) were for S. mansoni.

Central Africa: Chad, Central African Republic, Democratic Republic of the Congo, Burundi and Rwanda S. mansoni is endemic in all these Central African countries, but again the prevalence is usually quite focal. As long as favorable conditions for transmission exist, i.e. a stable water body supporting intermediate host snails, and close human-water contact, then the prevalence in surrounding

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communities will be higher than 50%. In Chad S. mansoni is found only in the south of the country, and reported prevalence rates are relatively low. As production of cotton and rice is improved, however, the transmission could be increased. Rwanda is considered to have relatively low infection rates, but in neighboring Burundi there is much more S. mansoni. The main conditions for high prevalence exist in fisherman communities near Bujumbura and around Lake Cohoha (20). Both the Democratic Republic of the Congo and the Central African Republic support three human schistosomes (S. intercalatum as well as S. mansoni and S. haematobium), and again distribution is varied and focal.

Morbidity Schistosomiasis mansoni is a complex of morbidities resulting from the human immune response to any stage of the parasite, especially the granulomatous response to eggs. The parasites reside and lay their eggs in the mesenteric venous system. The majority of the 300 eggs per day produced by each worm pair either lodge in the wall of the large or small intestine, or pass out in the stool. A percentage, however, are swept back into the systemic circulation, lodging first in the liver or, more rarely, the lungs, spinal cord or brain. The greatest degree of dysfunction occurs in the liver, where hepatic inflammation, periovular granulomas and the resultant fibrosis progressively occlude portions of the intrahepatic portal system, causing portal hypertension and bleeding esophageal varices. The increasing pressure in the main portal vein is transmitted to the splenic vein, producing splenic congestion, varices in collateral veins and shunting of eggs to other tissues. It is difficult to measure the attributable morbidity for any disease, but it is especially difficult for chronic multisystem diseases like schistosomiasis. Mortality rates are seemingly the most tangible way to measure morbidity. Attributing death wholly or in part to a chronic infection, however, is always problematic. Relative to the prevalence of infection, mortality rates are low in schistosomiasis mansoni and difficult to measure. The WHO estimates that 20,000 people die each year as a result of hemorrhage caused by bleeding esophageal varices due to schistosomiasis (21). This estimate excludes all conditions for which schistosomiasis was a contributing factor; it excludes extrahepatic disease and it excludes infant mortality, and peripartum


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mortality, about which little is known but for which several studies include schistosomiasis as a factor (22, 23). A great deal of the data are indirect, but they point to overall mortality rates due to all forms of schistosomiasis of 0.4-2% (24-26). For those already presenting signs of severe disease, the risk of dying from schistosomiasis was examined prospectively by Kloetzel in Brazil (24). Kloetzel followed 105 individuals in one community with chronic splenomegaly due to S. mansoni infection for 11 years and recorded their outcomes. In those less than 20 years old the mortality rate was 8.8/1000 man-years, while in the group over the age of 20 the rate was 47.7/1000 man-years. Overall, the death rate for the community was 13.7/1000 man-years, while for those with schistosomiasis and splenomegaly the rate was 27.3/1000 man-years. In the entire group 7 of 19 deaths (37%) were due to hematemesis, and of the 13 who developed gastrointestinal hemorrhage, 7 died (54%). The cumulative risk to those presenting with evidence of severe disease, therefore, was high. In Africa, there has been only one cross-sectional hospital-based study that directly assessed mortality as an outcome. All the admission and mortality records for a mission hospital in the West Nile District of Uganda were collected and analyzed over a 27-year period (25). Schistosomiasis mansoni was the fifth most common admission diagnosis (1742 cases), and it was recorded as the primary cause of death for 104 (6%) deaths recorded among the patients with schistosomiasis mansoni as their primary diagnosis. The mortality rate in this hospital-based study was clearly higher than would be found in one that is community-based, but it does emphasize that mortality due to schistosomiasis is not negligible in some endemic areas. The concept of what constitutes morbidity in schistosomiasis is currently undergoing revision. The most easily identified sequela is the development of hepatic fibrosis and resultant portal hypertension and bleeding esophageal varices. The full scope of attributable illness in addition to hepatosplenic disease involves the digestive tract, renal, pulmonary and neurologic systems. More generalized morbidities include Katayama fever (acute schistosomiasis) and malnutrition. The impact of schistosome infection on host response to other pathogens and disease manifestations in Africa is only now being investigated. In one case, no effect on response to hepatitis B immunization was observed for children born to S. mansora'-infected mothers (27). Another study, however, found that children with prenatal exposure to S. mansoni antigens have significantly more Th2-type responses to BCG immunization

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rather than the expected protective Thl response (Chris King, personal communication). Other infections, host genetics, culture and environmental toxins may all influence local or regional manifestations of disease. How malaria might affect the presentation of schistosomiasis, for example, has not been formally studied, but the range of these two infections overlaps extensively in Africa, as does that of other helminthic infections. The effect of HIV infection on morbidity has yet to be determined, but HIV does appear to influence egg excretion. HIV-infected individuals excrete only one third the number of eggs found in the stool of seronegative individuals despite equivalent levels of the circulating antigen (a quantitative measure of adult worm burden) (28). This is unlikely to significantly reduce transmission, however. HIV infection or AIDS was not found to affect chemotherapy for S. mansoni. Not all infections influence S. mansoni morbidity even among those that might be expected to. Concurrent hepatitis B and S. mansoni infection has not been associated with worse disease in Africa (29, 30), and one study in Egypt found no synergistic effect of hepatitis C and schistosome infection (31). Despite the difficulties of any rigorous definition of race, race has been put forward as a factor to account for observed differences in morbidity (32, 33). Though the infection originated in Africa, it is doubtful that schistosomiasis exerted much of a selective pressure against human reproduction. There is also ample severe morbidity found in Sudan, Egypt and elsewhere to argue against selection on the continent. Further, in areas where populations have been described as racially mixed, there is not the intermediate severity that would be predicted for some inheritance patterns. There is, nonetheless, good evidence for a contribution of host genetics and in a few studies parasite genetics to morbidity. Multiple studies have identified an association between HLA types and schistosomal hepatic fibrosis or colonic polyposis (34-38), suggesting that there is a genetic component in susceptibility to morbidity. Recently both segregation (39) and linkage analysis (40) identified a major gene (designated SMI) that contributes to susceptibility to infection and thereby may contribute to risk for morbidity. The identified genetic locus for this putative gene is on the long arm of chromosome 5 (5q31-q33). This is a locus implicated in the pathogenesis of other immunologically mediated diseases and is rich in immune response genes (IL-3, IL-4, IL-5, IL-9, IL13, IRF1, CSF1 receptor, GMCSF). Though originally identified in a study

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of Brazilian pedigrees, the same locus was also most strongly associated with infection intensity in a Senegalese population (41). Parasite strain differences have also been identified in drug susceptibility (42), egg production (43), and may indicate differences in virulence (43-45).

Acute Schistosomiasis Acute schistosomiasis occurs 2-10 weeks following initial infection, in contrast to the majority of morbidities, which develop and extend over years and decades. Acute schistosomiasis is a systemic allergic reaction to the early stages of infection and the onset of egg laying. Since this syndrome is generally associated with the first exposure to schistosome infection, it is rarely observed in endemic populations, or it is confused with other causes of fever in children. By contrast, it may be recognized in over 50% of nonimmune visitors to an endemic area who become infected (46). The disease is reported most commonly in returning travelers from Africa, while many more endemic cases are reported from Brazil. Most commonly there is fever, sweating, joint a n d / o r muscle pain and urticaria. Allergic reactions in the intestinal or pulmonary mucosa lead to diarrhea, mild cough, bronchospasm, bronchitis or pneumonitis. Weight loss, hepatomegaly and splenomegaly can also be observed as part of a generalized reaction. Neurologic complications at this stage (headache, visual impairment, encephalopathy or hemiplegia) can be serious though uncommon. Antihistamines and even steroids have been used symptomatically. The acute syndrome tends to resolve spontaneously once egg laying is established, and eggs appear in the stool (47). While antischistosomal therapy is indicated for parasitologic cure, it is unclear that it hastens resolution of the acute syndrome. Further, antischistosomal drugs are ineffective against immature developmental stages, and therefore treatment is usually delayed 8-10 weeks, by which time the acute symptoms are resolving.

Hepatosplenic Disease and Periportal Fibrosis Both organ enlargement and portal hypertension form part of what traditionally has been described as hepatosplenic disease, and they have been generally recognized as the major complication resulting from S. mansoni

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infection. While both are associated with S. mansoni infection and correlate with higher intensity infections, it now seems clear that hepatomegaly and portal hypertension are not necessarily due to the same disease process (48). Much of hepatosplenomegaly appears to be a consequence of the active inflammatory response to infection, while fibrosis is one way in which the inflammation resolves. Still, hepatomegaly and hepatic fibrosis were sometimes treated as though they were equivalent. In the past, many studies defined schistosomiasis primarily by hepatosplenomegaly in children (49-53). Other studies focused on clinical and endoscopic evidence of portal hypertension due to hepatic fibrosis (54, 55). The advent of portable ultrasonography has significantly improved our understanding of hepatosplenic disease. Since the apparatus and an electrical generator can be carried to very remote areas, community-based surveys can be conducted. Further, the sampling error diminishes, since the entire liver can be examined. Mild forms of fibrosis by ultrasound criteria can be quite common in children and may resolve relatively quickly, while resolution of hepatic fibrosis is much slower (48). Epidemiologically, enlargement of the liver and especially of the spleen are more often observed in childhood infections and portal hypertension and severe periportal fibrosis is more often, but not exclusively, found in adults. Prior to extensive use of ultrasound, a study based in Kenyatta Hospital, Nairobi, examined stool, rectal snips and needle liver biopsy to diagnose portal hypertension due to S. mansoni. Thirty percent of esophageal varices were attributed to schistosomiasis (54), but the authors noted that the selection bias of a hospital population and the sample bias of a needle liver biopsy tend to underestimate the true prevalence of schistosomal disease. The concord between ultrasound and other measures of portal hypertension is high. By both endoscopic examination for esophageal varices (55) and hepatic ultrasound (14), the prevalence of frank portal hypertension in several African populations is 2-3%. Using both forms of diagnosis, Saad et al observed that 50-60% of

those with marked fibrosis by ultrasound had esophageal varices (55). More subtle morbidity due to hepatosplenomegaly or portal hypertension has been difficult to measure, though bacteremia or thrombocytopenia from splenic congestion is likely to be associated with the severe splenomegaly. In Africa, focal differences in the degree of morbidity have commonly been noted (56). Some investigators have observed that hepatosplenic disease is more severe north of the Sahara, but there are no published series


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documenting this directly (10). Preliminary ultrasound studies in the endemic areas of the Nile Delta and Machakos, Kenya, appear to support this observation, however. Using the same machines and the same criteria, the prevalence of all grades of hepatic fibrosis was 25% in Egypt, but only 2.5% in Kenya, though the prevalence of infection at these sites has historically been similar

or lower in Egypt (Cline et al. (12) and Ourna, Abdel-Salam and Blanton, unpublished). Similar population-based studies in Sudan in areas with 45-60% prevalence found that 13-18% had ultrasound evidence of periportal fibrosis (57), while in a recent area of infection in Senegal where the prevalence was nearly 100%, no serious fibrosis was observed. Given the degree of variation observed within any one country, definitive statements about the prevalence of morbidity cannot be based on studies conducted in only one area. Guyatt et al. (56) compared intensity of infection to prevalence in multiple countries in Africa and concluded that prevalence does not predict distribution of infection intensity in the population. Differences in the numbers of intense versus light infections may account for some of the observed difference in morbidity between specific sites despite similar prevalences. What factors control aggregation of disease, however, have not been defined.

Intestinal Schistosomiasis and Colonic Polyposis The gastrointestinal tract is one of the most important sites for schistosomiasis. Most of the eggs actually end up trapped in the wall of the large intestine rather than the liver (58). However, diarrhea is often attributed to S. mansoni infection. Diarrhea is such a common problem in endemic areas that it is hard to credit cross-sectional studies alone and the association they suggest with schistosome infections (59). The number and quality of the studies, however, begin to provide a significant weight of evidence in favor of bloody diarrhea, in particular as a consequence of schistosomiasis. In a case-control study of patients presenting to an outpatient clinic in Malawi with bloody diarrhea, 132 cases were compared to 73 outpatient controls being evaluated for tuberculosis. Shigellosis accounted for 53% of cases and 34% had some intestinal parasitic infection compared to 2% of controls. The most common parasite was S. mansoni (60). In Burundi, another case-control population-based study found that 35% of cases of bloody diarrhea could be attributed to S. mansoni, in contrast to only 9% of cases of simple diarrhea (61). The evidence for bloody diarrhea as a consequence of schistosomiasis

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is further strengthened by prospective intervention studies. In multiple studies in Africa from locations as diverse as the Sudan, Burundi, Cote d'lvoire and Senegal, rates of bloody diarrhea fell significantly following specific therapy for S. mansoni alone (62-65). As a sign of schistosomiasis mansoni, bloody diarrhea is one of the best clinical indicators, especially in school-aged children. Nevertheless, the majority of those infected do not complain of diarrhea and only 50% of bloody diarrhea could be attributed to schistosomiasis, so the sensitivity and specificity of this symptom as an indicator of schistosome infection are low. While diarrhea can be a common outcome wherever there is transmission, colonic polyposis is found almost exclusively in Egypt and countries of the Arabian Peninsula (66). Polyposis is otherwise rare, even in neighboring Sudan (67). Polyps may be sessile or pedunculated and present in either the colon or rectosigmoid. Microscopically, they show multiple granulomas formed around S. mansoni and there is degeneration of the muscularis mucosa of the colon (68). The development of this complication seems to require massive, rapid and focal deposition of eggs in the colon. The uneven distribution of this complication in Africa and the rest of the world remains unexplained.

Pulmonary Hypertension Pulmonary manifestations of schistosomiasis occur both early and late in the course of infection. Early after infection there is endothelial proliferation and eosinophilic infiltration associated with the migration of schistosomula. An associated pneumonitis with asthma-like symptoms occurs in some individuals. Inflammatory changes in the absence of eggs can also appear late in the course of infection, leading some to postulate that there is deposition of antigen-antibody complexes in the lung throughout infection (69). Clinically there may be cough, hemoptysis and fatigue at any stage. Pulmonary hypertension (cor pulmonale) may occur as a result of the same process occurring in the liver. Eggs lodge in the pulmonary vasculature and provoke a granulomatous occlusive arteriolitis. Shortness of breath with exertion or at rest is a common complaint with pulmonary hypertension from any source and is not specific for pulmonary schistosomiasis. This condition is rare, since 50% of the vascular bed must be obliterated to raise pulmonary arterial pressure and since the lung is not a preferred site of embolization.


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In Egypt, Cheever et al. demonstrated that out of 98 consecutive autopsy cases at the Cairo University Hospital, only 6% of all the eggs in tissues localized to the lungs (70). The diagnosis is made more frequently at autopsy than clinically. Portal hypertension is a predisposing factor, thus the distribution of pulmonary disease follows that of severe hepatic morbidity and heavy infection (71, 72). One autopsy series in Brazil of 1863 consecutive cases found a very small number with pulmonary involvement and those with hepatosplenic and pulmonary disease were more likely to have further ectopic locations for eggs (73). Deposition of S. mansoni eggs in the brain is usually preceded by cor pulmonale and pulmonary arteriovenous shunts (74). Another autopsy series reported from Sudan rarely found pulmonary schistosomiasis (67). Clinically significant pulmonary disease is occasionally present with S. japonicum infection, rare with S. mansoni and not a part of S. haematobium infections.

Nephropathy Nephropathy associated with S. mansoni infection has been difficult to document consistently. The major feature is proteinuria in the presence of infection. It is thus a diagnosis of exclusion. In areas where malaria is tranmitted, where there is S. haematobium or streptococcal glomerulonephritis is prevalent, it is difficult to attribute unexplained proteinuria to schistosomiasis without further investigation. In Brazil, renal disease is rare (75, 76) except in hepatosplenic patients (77). In contrast to South America, most studies in Africa find little or no evidence of S. mansoni nephropathy (78-80) except in Egypt (81, 82).

Neuroschistosomiasis Neurologic disease is a rare complication more commonly due to S. mansoni than any other species (83, 84). The disease has a variable presentation with acute or subacute symptomatology, but generally involves progressive paresis in the lower extremities and urinary incontinence or retention. With symptomatic schistosomiasis of the brain generalized seizures and focal paresis are common, although mass effect from an exuberant granulomatous response can produce focal signs or a patchy asymmetrical lesions (85). The

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pathologic lesion may consist of well-defined granulomas, frank necrosis or a combination of the two (84, 86). It is the impression of some investigators that individuals who are newly arrived in an endemic area are at greater risk for neuroschistosomiasis. Many of the reports have been in expatriates or returning tourists (87), but this may be a reporting bias. Neurologic complications are so sporadic that it is impossible to say whether it is more common in Africa than elsewhere.

Dermatologic Lesions The skin can be affected in two ways by schistosome infection. Penetrating cercariae produce an immediate dermatitis that is usually very mild in human schistosomiasis and marked by transient itchy areas that were in contact with surface water from which cercariae penetrated. This syndrome (swimmer's itch) is most associated with invasion of the skin by nonhuman schistosome species that develop no further. S. mansoni and the other species that affect humans are far less likely to cause skin lesions at this stage of infection. However, in a simultaneous infection of 30 European travelers to Mali, 36% had cercarial dermatitis (46). In conjunction with egg laying (6-8 weeks after infection), some individuals develop acute schistosomiasis that is associated with urticaria and periorbital edema (88). Eggs can also be deposited directly in the skin and cause a characteristic allergic response with a pruritic papular rash located in perigenital or periumbilical areas. This is cutaneous schistosomiasis and appears to be a complication of S. haematobium infection. It is not reported for S. mansoni. After reaching the portal system, most S. haematobium adults continue migrating to the vesicular veins around the bladder. They may thus migrate to areas where eggs become deposited near the genitalia or umbilicus (47).

Nutritional Deficits The nutritional cost of schistosome infections has been studied by several groups. While the nutritional cost of S. haematobium infection has been documented in Africa (89), the only prospective study of S. mansoni infection was conducted in Brazil and showed growth deficits that began to reverse with treatment (90). Boys were more malnourished at the start of this study


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than girls, and the boys' response to treatment was correspondingly greater. Malnutrition may be the most widespread morbidity/consequence of schistosome infection. Hepatosplenic disease only affects approximately 10% of those infected. The growth deficits due to schistosomiasis are generally mild in the short term, and they may be compounded by other concurrent infections. The mechanisms for these growth effects are still under investigation. Unlike geohelminths, schistosomes are invasive and stimulate a more chronic systemic immune response to infection. For example, tumor necrosis factor a is associated with wasting syndromes and has been found to be elevated in cases of S. mansoni infection (91), but not for geohelminths (92). How the nutritional impact of infection should be weighed against the cost of mass treatment will depend on the resources available to any given nation's control program.

Control of Morbidity Within the continent, the major area of challenge for combating schistosomiasis remains sub-Saharan Africa. To the north, Egypt has in existence a strong control program, while in the other Arab countries, S. mansoni, which was never highly prevalent, now seems to be controlled due to socioeconomic development and improved social status. Historically, emphasis has been placed on snail control, but developments in the fields of chemotherapy, epidemiology and diagnosis have changed the whole emphasis in control from preventing transmission to reducing morbidity (93). Specific measures such as chemotherapy and snail control have been developed alongside nonspecific measures aimed at the general improvement of health conditions and the provision of safe water supplies.

Chemotherapy Over the past 20 years, the treatment of schistosomiasis has been considerably improved by the use of praziquantel, a drug that is effective, generally as a single dose, against all schistosome species. Chemotherapy was the recommended strategy for morbidity control by the Expert Committee on the Control of Schistosomiasis at their meeting in November 1984 and was further endorsed at a second meeting in November 1991. Pilot control

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projects using chemotherapy as their main strategy have taken place in many countries. Though chemotherapy is not a perfect tool, there is good evidence that it can reduce infection intensity and morbidity. The effect on bloody diarrhea has already been mentioned. Autopsy series in Sudan and Brazil suggest that severe disease has declined where mass or targeted chemotherapy campaigns have been instituted (67, 73, 94). There is evidence that treatment can reverse some fibrosis from cross-sectional comparisons of treated and untreated communities in Egypt (95), from prospective studies using ultrasound in the Sudan (64, 96, 97), and from biochemical analysis pre- and post-therapy in hepatosplenic cases from the Democratic Republic of the Congo (98). There are three main strategies for chemotherapy delivery: mass treatment, selective population chemotherapy and targeted therapy. Mass chemotherapy implies treatment of the entire population without prior individual diagnosis. Success with this approach primarily requires adequate resources, ability to deliver the drug and good population compliance. In Africa, this approach may be most appropriate in highly endemic areas, particularly where cultural, religious or other factors do not allow collection or examination of stools (99). In the Sudan 2 - 3 years of mass chemotherapy significantly reduced the prevalence of all grades of fibrosis in all individuals for one area but not another and for all children regardless of the site (64). In addition, this approach significantly reduces the cost of diagnosis and may have the greatest effect as a single measure on transmission. In most instances, however, changes in transmission have been difficult to detect immediately after mass chemotherapy. In Brazil, mass therapy four times over five years resulted in an excellent initial response, but was followed by rapid reinfection (100). More than chemotherapy alone will be needed to hold down reinfection rates in some areas. It is best to combine this approach with other measures such as improved sanitation, education on personal hygiene and snail control, since no single measure seems to be capable of breaking the cycle of transmission.

Selective population chemotherapy, the most widely used approach, involves testing the entire community followed by identification and treatment of all infected individuals. Success with this approach depends on compliance and the sensitivity of the diagnostic method used. In rural Burundi, Gryseels et al. (101) treated all infected persons in a cluster of villages with a single 40 m g kg dose of praziquantel once each year for three years. By the end of the three-year period, the prevalence of infection was reduced from


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60-35% (depending on the village) to 25% overall. They attributed the failure to further reduce the prevalence to a high false-negative rate using a single stool examination for diagnosis and concluded that the sensitivity of the screening method was a major determinant of the outcome of repeated selective treatment. Failure to identify some infected individuals ensured continued transmission. Those treated, however, had lower intensities of infection and lower prevalence of abdominal pain and diarrhea. No change was noted in the rates of hepatosplenomegaly in the community within the three years of follow-up. Targeted chemotherapy combines elements of the other two strategies, but centers on treatment of the individual rather than the population. With targeted therapy, only the most heavily infected individuals in the community are treated. Since heavy schistosome infections are usually concentrated in a segment of children aged 8-15, this strategy can be further reduced to examination of cohorts of school-aged children and treatment of all children in the identified age group when the prevalence is high. The rationale, as argued by Warren and Mahmoud (102), is that most infections are light, produce few eggs and rarely result in major morbidity, while those few with high worms burdens are both more likely to have disease and to contribute to transmission. Targeting treatment to the most intensely infected segment should interrupt transmission and halt the development of disease. In 1982, a trial of targeted chemotherapy was carried out in Machakos District, Kenya, where the prevalence was 82% and the overall geometric mean of egg output 500 eggs per gram. Some reduction in organomegaly was observed at the end of three years, but nearly 100% of those with heavy infections who were treated had a return to high levels within a year (103). In a nearby area where the prevalence of S. mansoni was similar and 16% were heavily infected (> 400 eggs per gram), treatment was again targeted over three successive years to those with high egg counts. Targeting this group reduced morbidity as measured by hepatosplenomegaly but failed to affect transmission (104). Under some conditions, transmission has been affected by targeted chemotherapy in S. haematobium infection in Kenya (105). For S. mansoni, the ability to dramatically reduce prevalence and intensity in the face of continuing transmission, as occurred during control programs in Egypt, suggests that transmission could be affected with repeated treatments. The differences in response are more likely due to environmental factors and sampling rather than intrinsic differences in the two species.

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Emergence of Drug Resistance A potential threat to chemotherapy-based control programs is the emergence of drug resistance to the only two practical agents, oxamniquine and praziquantel. Oxamiquine is an S. mansoni-specific drug, whereas praziquantel is also effective against all other species of schistosomes as well as some cestodes. Both drugs have been used extensively, although oxamniquine was found to require higher doses for cure in some regions of Africa than the Americas, probably due to intrinsic resistance in those strains (106). Other studies, however, show S. mansoni in Africa to be fully sensitive to oxamniquine (107), even when the parasite is relatively resistant to praziquantel (42). Praziquantel is more widely used, because it is less expensive, can be given as a single dose and will treat all species of schistosome as well as some tapeworms. Resistance and/or tolerance of S. mansoni to oxamiquine is known to occur in Brazil and Kenya (108,109). The appearance of isolated resistance in Brazil, however, has not diminished the overall efficacy of oxamniquine there and the drug is not very much used in Africa. Widespread use of praziquantel in endemic areas over the past 20 years has led to concern about development of resistance to this drug as well. Recently, Cioli (110) thoroughly reviewed the available studies on the potential emergence of praziquantel-resistant or -tolerant schistosomes. He concluded that resistance is likely to exist. In field studies, the failure of repeated doses of praziquantel and the ability of a modest dose of oxamniquine to effect a cure in these instances support this conclusion. Studies using Egyptian (111) or Senegalese isolates (112) of putatively resistant strains showed increased ED50s to praziquantel, further supporting the impression of praziquantel resistance. How important this is at present is not clear. Interpretation of some of the human cases is confounded by extreme worm burdens or high transmission rates. Where worm burdens are extreme, cure rates will appear low because a 95% reduction in worm burden still leaves a significant number of viable worms. Where transmission is very high, a large number of individuals will harbor immature worms that are innately resistant to the drug at this stage of their development (113). In other instances of apparent resistance, repeat treatment after 40 days produces the expected cure rate. As with oxamniquine, there is no immediate need to modify recommendations for chemotherapy in general, but resistance should be monitored, production of all available drugs


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maintained and new drugs developed. This may require development of the regional capability to produce these drugs, as occurs in Egypt and Sudan. Oxamniquine even in Africa may serve as an alternative when praziquantel resistance is encountered.

Snail Control Past efforts to control schistosomiasis were concerned mainly with snail control but this was much reduced after recent developments in the field of chemotherapy which ensured the availability of safer, effective and relatively inexpensive drugs. The interest in snail control renewed when, in 1993, the WHO raised the alarm for a return to snail control in association with chemotherapy (26). Existing snail control methods are aimed at the management of snail populations that inhabit endemic foci. Where feasible, environmental management including elimination of natural water bodies and regulation of human settlement in areas of significant risk, has proven to be effective. Modification of the environment through good engineering practices in areas concerned with irrigation development and regulation of the human population have been effective in reducing S. haematobium transmission (26). The role of molluscicides depends very much on the local epidemiological and ecological situation and the human and financial resources available. Niclosamide, which is the synthetic molluscicide of choice, is expensive and its use has to be restricted focally to important transmission sites. The natural molluscicidal compounds from plants such as Phytolacca dodecandra are available (114), but problems encountered with their large scale production have considerably limited their exploitation. Alternative methods of snail control have been developed using different biological control agents. Field trials largely employing competitor snails, crustaceans or fish have been carried out, and the results have been equivocal.

Obstacles to Control Efforts to control schistosomiasis in Africa have been overtaken to some extent by emerging problems. For example, the discovery of HIV/AID in the late 70's/early 80's and more recently the decision to put more effort

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and resources into malaria control have made schistosomiasis and other parasitic diseases lower priorities for control by donor nations and others in the affected endemic countries. This is happening in spite of the spread of schistosomiasis resulting from water and agricultural development projects which are crucial to ensuring the availability of food for the fast-growing African population.

Topics for Further Research It seems odd to say that a major area for further research remains the definition of morbidity in schistosomiasis mansoni. Since it is clear that infection is not tantamount to disease in schistosomiasis, efforts continue to define what constitutes an early sentinel lesion requiring therapy or monitoring and what is an adverse outcome. What conditions, signs or symptoms are a direct result of S. mansoni infection and for which does S. mansoni act as a cofactor? Part of the confusion in definition stems from technological advances that disconnect findings on physical exam (hepatosplenomegaly) from what is clearly recognized as the pathogenesis of schistosomal portal hypertension (periportal fibrosis). Both hepatosplenomegaly and hepatic fibrosis are clearly associated with schistosome infection, but they appear to be less closely correlated with each other. One current paradigm considers that hepatosplenomegaly represents the early lesion that does not necessarily progress to fibrosis as seen on ultrasound (48). Understanding the sequence of events that results in severe debilitating disease, the genetics of who it will affect, the time frame in which it occurs and the stage in which the lesion is reversible are all critical questions for meaningful planning. Tremendous variability in the development of disease within Africa has been observed, but the basis for these differences has not been investigated. The frequency and importance of extrahepatic manifestations of schistosomiasis mansoni are still being measured and evaluated. Finally, as treatment has become more widespread in other areas of the world, the effect has been to convert a prevalent infection with high intensity to a prevalent disease with low intensity. The consequences of this process have not been well explored anywhere and least of all in Africa. Along with better definition of disease, there is a need for standardized ultrasound diagnosis (115). At present, several grading systems are circulating,


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Figure 1. Hepatic ultrasound of mild (left panel) and moderate fibrosis. In mild pattern fibrosis there is thickening of the portal segmental branches (PSB's) and some smaller ones are completely occluded. In the right panel, at the primary branching of the portal vein, pipestems appear along the portal segmental branches and ring echoes in cross sections.

making comparisons difficult. Furthermore, some findings, especially of early lesions, have poor specificity. Minimal hepatic periportal thickening, for example, is frequently associated with febrile illnesses including typhoid fever (116). The WHO recently convened experts from around the world to establish guidelines for ultrasound staging and diagnosis of morbidities due to schistosome infections. At the 1996 meeting in Niamey, Niger, a consensus was reached that combines the image pattern of liver texture (Fig. 1) with objective measurements of the two wall thickness of a peripheral segmental portal vein and main portal vein diameter. These guidelines still need to be field-tested to demonstrate practicality and determine which elements are most specific and useful. Chemotherapy alone is a stopgap control measure that only occasionally influences transmission. Sanitation, improved water quality and snail control are important, but are expensive adjuncts to chemotherapy. Chemotherapy itself is threatened due to the probable development of resistance to praziquantel and oxaminquine. Resistance has been slow to develop and slow to spread, but this does not provide much confidence for the long term prospects. Apart from development of new drugs and economic development in the region, one of the most important control measures would be a vaccine

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for schistosomiasis. In combination with chemotherapy and other measures, an inexpensive, long-acting vaccine would reduce the need for frequent administration of drugs, would circumvent the problem of resistance and would hold out the prospect of significantly reducing transmission on a broad front. A useful vaccine, however, does not appear to be on the horizon in the short term. Where resources are available, recognition of the problem at national level and national commitment to control go a very long way toward providing relief from S. mansoni infection and its attendant morbidities.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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Chapter 10 Disease in Schistosomiasis Japonica Remigio M O l v e d a

Introduction Schistosomiasis japonica is associated with several unique features that distinguish it from schistosomiasis mansoni and hematobia. In contrast to other schistosome species affecting man (S. mansoni and S. hematobium), S. japonicum also affects a wide range of mammalian hosts, which include different species of wild, domestic or feral animals (1). This feature of S. japonicum may make it the most difficult schistosome species to control. The clinical manifestations of schistosomiasis japonica are similar to those produced by S. mansoni infection; severer manifestations are generally ascribed to S. japonicum infection. This is attributed to the latter's higher egg output and pattern of laying eggs in large aggregates resulting in more florid and presumably destructive tissue reaction. For example, compared to schistosomiasis mansoni, serum-sickness-like syndrome or Katayama fever, growth retardation in children, acute cerebral manifestations and hepatosplenic disease are more pronounced in S. japonicum (2). In addition to the above features, the mechanism of granuloma formation in S. japonicum infection seems to be an immediate manifestation rather than a manifestation of delayed hypersensitivity reaction to egg antigen (3). Modulation of this granulomatous reaction around eggs deposited in the liver tissue can be transferred by serum but not cells from chronically infected animals (4). Furthermore, immunity to S. japonicum seems to be species-specific (5). Thus, vaccine preparations against S. mansoni or hematobium may not be protective against S. japonicum.

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Epidemiology Historically, the different geographic strains classified as S. japonicum include the anthrophilic strains from China, the Philippines and Japan and the zoophilic strain from Formosa. In recent decades, new foci of endemicity have been uncovered in Indonesia, Malaysia, Thailand, Laos and Cambodia. Subsequent investigations resulted in the identification of the new geographic strain of S. japonicum-like flukes in Thailand and Malaysia, while a new species, a close relative of S. japonicum, was recognized as S. mekongi, recovered from Laos, Cambodia and Thailand (6). Whether the strains in these endemic regions represent a single species with similarities in morphology and biopathology to the classical S. japonicum, is the subject of current research. S. mekongi, however, may actually be a species distinct from S. japonicum. China represents the largest endemic area of S. japonicum infection. In the 1950's, 10 million persons were estimated to be infected (7). In China, infection is endemic mainly in areas along the Yangtze River and to the south of the river basin. These areas include 348 countries in 10 provinces, the Shanghai Municipality and the Guangxi Autonomous Region (8). In recent years, the prevalence, transmission and intensity of the infection have significantly decreased to a low level in most of the endemic areas of China. In 1981, after 25 years of extensive control efforts, the number of infected individuals has dropped to 705,000 (7). However, in some swamps, lake regions, and a few mountainous areas, transmission remains uncontrolled and acute cases are not infrequent (9). In the Philippines, despite control efforts, S. japonicum infection remains a serious health problem affecting 167 towns of 24 provinces, with the more prominent foci existing in Sorsogon in the northern part of the country, in Oriental Mindoro, Samar, Leyte, Bohol in the central part, and in Mindanao in the south. In the island of Samar and Leyte alone, there are over 500,000 infected individuals, thus placing approximately 10 million Filipinos at risk (10). In Indonesia, schistosomiasis is localized in two limited foci in Lake Lindu valley and Napu valley in Central Sulawesi. It was estimated in 1984 that around 7000 persons were at risk of infection and about 4000 persons had an active infection (11). In Japan, there were formerly five endemic areas: four in Honshu and one in Kyushu. As a result of an intensive control program, only a few small snail colonies now exist and no new human infections have been reported since 1978 (12). It was estimated that in 1986 about 100,000 patients with chronic schistosomiasis remained in Japan (12).

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Life Cycle Cercariae, the infective larvae of S. japonicum, gain entry into a vertebrate host system (either man or animal) by penetrating the skin (13) and eventually settle in the mesenteric vasculature, where they grow into adult worms. Subsequently, the female worm lays fertilized eggs numbering from 300 to 3000 eggs per day. The majority of the eggs are intended to be passed out to complete the life cycle. Some eggs remain within the host tissues; they are either trapped in the liver or may be carried through the circulatory system to remote areas, such as the brain (13). Aberrant adult worms in these remote areas may also directly deposit the eggs.

Pathology The schistosome egg is the primary inducer of pathogenesis in the infected host. In schistosomiasis japonica, the major pathologic lesion is the granulomatous inflammation, which forms around the parasite eggs trapped in the host tissues. Eggs trapped in presinusoidal spaces of the liver release soluble egg antigens (SEA's), which stimulate a vigorous hypersensitivity reaction that ultimately destroys the trapped ova (14). This inflammatory process also results in the extracellular deposition of fibrotic macromolecules, including collagen and glycosaminoglycans, along the portal tracts (15). Fibrosis and inflammation obstruct the portal blood flow and lead ultimately to complications of portal hypertension. Severe complications can lead to bleeding from ruptured esophageal varices, the leading cause of death in schistosomiasis japonica (16). Eggs carried by the bloodstream or directly deposited by aberrant adult worms particularly in the brains can significantly induce granulomatous lesions, producing symptoms similar to spaceoccupying lesions in the central nervous system. Pathologic lesions due to egg deposition are also found in the lungs, the intestine and the reproductive organs. Eggs may lodge in other sites such as the lungs, gastrointestinal tract and reproductive organs. In rare instances, eggs may be carried to the skin, causing dermal tissue ulcerations. While host reactions to eggs are pathogenic, host response to cercarial skin penetration, schistosomula migration, and to the adult worms in the mesenteric vasculature, although immunogenic, is less significant.

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Schistosomiasis at Community Level In S. japonicum-endemic areas, the majority of the population remain uninfected based on stool examination. Among the infected residents, the majority are lightly infected and only a very small percentage harbor a heavy parasite burden. Of the infected group, a very small segment develop the hepatosplenic form of the disease affecting 11-15% of the entire community in the Philippines (10, 17). A higher prevalence of liver lesions in other endemic areas of the country has also been observed (18). The endemicity in the Philippines for the hepatitis B virus (HBV) may contribute to the liver disease observed in S. japonicum-endemic areas. A study in this respect showed that HBV exposure and HBsAg carrier rates among S. japonicum-iniected and -uninfected subjects were not significantly different (19). These data suggest that the observed hepatomegaly in this region is mainly secondary to schistosomiasis. Generally, the size of liver enlargement correlates with the intensity of infection. In our recent study (10), hepatomegaly was seen more frequently among moderately infected individuals (101-400 egg/gm), but further increases in the intensity of infection do not necessarily increase the frequency of liver enlargement. Liver enlargement is more frequently seen along the midsternal line than along the midclavicular line. Likewise, hepatomegaly is more commo among males than females. Splenomegaly is more frequently seen among the infected population but there is no correlation with the intensity of infection. Symptoms attributable to chronic schistosomiasis, like inability to work, weakness, abdominal pain and diarrhea, are present to the same extent between those with and without infection. Except for diarrhea, the same symptoms showed no correlation with the intensity of infection.

Clinical Stages Three clinical stages are recognized in schistosomiasis japonica. The first and second stages consist of the early and the acute phase respectively. They are discussed together because of the overlap in the time-frame. The third or chronic stage is discussed separately because of the different clinical features. The early phase of infection starts from the time of host penetration by the cercaria until the initial stage of larval migration, while the acute or toxemic phase occurs at the time of maturation and initial period of

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oviposition by the gravid female fluke. Exposure to cercaria via water contact immediately but not necessarily results in itching with erythema and papular rashes usually known as swimmer's itch. This is seldom observed in an indigenous population and when present is usually associated with primary infection. Exposed individuals may have chills, fever, headache and pulmonary involvement manifested by unproductive cough and abdominal cramps during the period corresponding to larval migration. There is wide variation, however, in the time of onset and intensity of the above manifestation (20). The maturation of the worm with the onset of oviposition 4 2 70 days postexposure paves the way for the toxemic phase marked by prominent eosinophilia, which mainly accounts for the high level leukocytosis observed. This serum-sickness-like syndrome (Katayama fever) is usually associated with malaise, lymphadenopathy and hepatomegaly. Diarrhea or dysentery, seen in about 25% of cases during this stage, is usually referable to colonic involvement. The more severe syndrome, seen more frequently among nonresident individuals of endemic areas, is not related to the intensity of infection. The toxemic phase is most severe in S. japonicum infection compared with those of other human schistosomes, and the disease may prove fatal for heavily infected individuals. At the chronic stage, a variety of clinical manifestations may result from infection, depending on the organ involved, and these may range from mild to severe, with several gradations in between. For purposes of discussion, the sequelae of chronic schistosomiasis japonica are presently categorized into hepatosplenic, hepatointestinal, pulmonary and cerebral forms. Cardiac and renal localization of lesions are so very rarely encountered as to merit inclusion.

Chronic Schistosomiasis Japonica Hepatosplenic Form The hepatosplenic form is the predominant clinical manifestation of chronic schistosomiasis japonica. Two types have been recognized. The more common is the compensated type, seen in the earlier phase of chronic infection. The rarer type represents the decompensated stage, seen in the later stage of chronicity.

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The compensated hepatosplenic disease is prominently characterized by portal hypertension as manifested by ascites, palpable liver and enlarged spleen. The block to portal blood flow has been traced to the classical pipe-stem fibrosis (PSF) around the intrahepatic radicles of the portal vein, which is presinusoidal. Thus, splenic pulp and portal pressures are markedly elevated while wedged hepatic vein pressure is almost always normal. Varying degrees of liver fibrosis are distinct grossly as well as microscopically. In a coronal section, the characteristic PSF is evident (21). There is fibrous thickening of the portal tracts diminishing progressively from the hilus to the smaller portal radicles, and resulting in some degree of course and irregular distortions of liver architecture. Thick collars to fibrous tissue surround intrahepatic radicles of the portal vein. Microscopically, ova are present in the portal areas with periportal fibrosis. Some ova may be seen as emboli inside the lumina of the small portal branches. Thickening of the walls of the portal radicles may be present. Increased vascularization may be present in areas of fibrosis which have not undergone collagenization. Other earlier lesions in the form of pseudoabscess and pseudotubercles around the ova may be found. The main portal vein is usually dilated. Sclerotic thrombosis may or may not be present. Granulomas in varying stages, irrespective of calcification, may be found. The early stage acute granuloma consisted of eosinophils, lymphocytes and a few histiocytes (22). At a later stage, histiocytes become more prominent together with formation of multinucleate giant cells which engulf or entrap the eggs. Sometimes only the egg shells could be found in the giant cells. The more chronic stage of the granuloma reaction is the healed granuloma, which shows fibrosis and hyaline degeneration. At this stage, remnants of eggs are seldom found. Sometimes, only fibrosis and widening of the portal trials are seen with preservation of the hepatic artery and bile duct. Occasionally, numerous capillaries are seen in the fibrous portal tissue, which had an angiomatoid or hemagioma-like appearance. This was probably similar to those seen in autopsied cases in the Philippines (21), which showed cavernous transformation of the portal vein from an antecedent thrombosis. Correlation of this with the clinical and hemodynamic picture is uncertain. This is probably due to the limited amount of the sample in the biopsy, which may not be representative of the whole liver. However, in another study (20) a correlation between the number of eggs in feces and the degree of fibrosis in liver biopsies was observed. Similarly, there also was noted a correlation between liver


size and degree of fibrosis, and thus, the higher the degree of fibrosis, the smaller the size of the liver. Despite the clinical and morphologic evidence of portal hypertension, the liver retains its lobular architecture and parenchymal hepatic function. A study (23) of 69 patients showed that there was no disturbance in bile flow in almost all of the patients, especially those in the acute and early chronic stages. Even among those with advanced liver pathology, only 3 out of 20 revealed interference in bile flow without evidence of jaundice. In advanced fibrosis, liver function may be compromised. Cirrhosis when present may reflect a superimposed pathologic process. Jaundice in this case may be due to hepatic cell failure. In schistosomal hepatofibrosis the stigmata of chronic liver disease, such as jaundice, spider angioma, intolerance to dietary protein, palmar erythema, testicular atrophy and/or gynecomastia, are inconspicuous or absent. The study by Sulit et al. (21) has ruled out obstruction of the hepatic portal venous system by splenoportography. The changes observed were mainly of three types: (a) the existence of numerous collaterals arising from the splenic a n d / o r portal veins, (b) dilated portal veins which end abruptly at the hilus or with its branches pinched off, and (c) the existence of a retrograde flow with filling of the mesenteric vein. Hypersplenism consisting of pencytopenia in the presence of hypercellular bone marrow is common. Spleen size does not correlate with the degree of liver disease and is usually of a congestive nature due to portal hypertension (24). However, it is also possible that parasitism with S. japonicum may cause direct or indirect hyperlasia of the spleen. It was observed by Tsutsumi and Nakashima (1972) that splenomegaly does not always run parallel with the degree of hepatic lesion. Except for anorexia, pallor, ascites and a loss of flesh, the patient generally has a feeling of physical well-being and may be able to carry out normal activities. The course of the disease in some patients, however, may be punctuated by hematemesis, and unless portal hypertension is corrected, the patient may die of exanguination due to ruptured esophageal varices. Portal shunting operations seem to offer the best remedy for this condition but sclerotherapy may be the preferred treatment at present. Splenectomy is also being done to remove as much as 20% of the load in the portal system (20). It has been demonstrated that the frequency of hematemesis or upper gastrointestinal bleeding was not dependent on the age and sex of the patient, duration of ascites, prothrombin time activity, serum albumin and globulin levels, hepatic wedge and splenic pulp pressures, splenic-hepatic wedge

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pressure, gradients and degree of hepatic fibrosis, as ascertained from biopsy specimens (21). The decompensated form of hepatosplenic disease presents physical and laboratory evidence of hepatic parechymal damage, failure and insufficiency with stigmata of portal hypertension. This usually occurs following a protracted duration of chronic disease. Since the majority of hepatosplenic patients may die of gastrointestinal bleeding or intercurrent disease, patients who reach this stage are quite rare. The continued poor nutritional state of the liver would no doubt lead to changes akin to portal cirrhosis (21). These changes may resemble those of decompensated micro- or macronodular cirrhosis except that schistosome eggs are found in the liver. In a few patients, elevation of portal, splenic pulp pressure and intrahepatic venous pressure suggestive of classical cirrhosis may be seen. Obstruction to hepatic blood flow can be at the intrahepatic or postsinusoidal level. Superimposed nutritional cirrhosis as a consequence of prolonged duration of illness may also be seen. These are the patients with secondary hepatic encephalopathy and hepatic coma as the terminal event. Lately (Yokogawa, 1976), it has been observed that in Japan the incidence of hepatoma among chronic schistosomiasis was four times higher than that in the control patients. It was argued, however, that in Japan it is not feasible to directly relate schistosomiasis etiologically due to complicating viral hepatitis infection (22). It is believed that alcohol intake cannot be ruled out as a factor contributing to the frequency of cirrhosis among residents of endemic areas in the Philippines (25). Occasionally, there are reports of calcification of eggs and areas around the parasite eggs. The pathogenetic significance of the presence of calcified eggs in S. japonicum infection is unknown, but they are generally surrounded by only a slight cellular reaction (26). It is felt by some workers in the Philippines that wherever calcification in suspected organs is seen in a patient coming from an endemic area, the possibility of schistosomiasis should be considered (Villegas-Cinco et ah, 1961). Furthermore, an association between susceptibility to postschistosomal hepatic fibrosis and HLA-DR-DQ alleles has been demonstrated (27), reflecting the possible genetic basis of the disease.

Hepatointestinal Form Organs or tissues whose supply is drained by the portal vein are the ones frequently showing pathologic changes in schistosomiasis japonica. This is


shown by the deposition of the parasite eggs particularly in the rectum, appendix, colon and liver. The eggs deposited in these tissues act as foci for granuloma formation in the submucosa, causing irritation, and lead to round cell infiltration, or giant cell formation around a cluster or nest of schistosoma eggs (27). Submucosal fibrosis may be an attempt to eliminate the eggs but results instead in ulceration. Intensification of submucosal fibrosis toward the chronic stage may render an infected person stool-negative. However, this may not be the general rule inasmuch as there are no data correlating the diminution of egg excretion with the advancing age of infected individuals in S. japonicum-endemic areas. While there is a tendency to be asymptomatic toward the chronic stage, eggs may continue to be passed in stools and the pathologic processes induced by oviposition in the bowels may or may not be associated with diarrhea or dysentery. In the majority of stool-negative chronic individuals, our experience with patients seen in Metro Manila in whom rectal mucosa tissue was obtained by biopsy forceps, consistently reveals eggs when pressed between two glass slides. It will be remembered that the rectum is one of the organs whose tissues contain eggs most frequently and in the largest number (28), which will justify the use of rectal biopsy as a diagnostic tool. Recio and Cruz (1950) observed that eggs found on some rectal biopsy of S. japonicum-iniected patients usually show no cellular reaction. The unique reaction of the rectal mucosa is in contrast to those of other tissues such as the liver and intestinal submucosa where fibroblasts usually proliferate. Nonimmune Americans, 14-16 months postexposure in S. japonicumendemic areas in the Philippines, were examined sigmoidoscopically by Holands and Palmer (28), who observed aphemeral clusters of 2-4 bright l x l mm dry petechiae in an otherwise normal mucosa. Other pathology observed consisted of dull patches with sand-paper-like appearance. An important complication of the hepatointestinal form of schistosomiasis japonica is appendicitis caused by the extension of lesions into the vermiform appendix, in the form of nodular tubercles. In a study of 127 schistosomiasis cases in Manila (30), about 64% involved the appendix, suggesting an important etiology of appendicitis in S. japonicum-endemic areas of the Philippines. Histopathological examination of the removed appendices from some patients admitted to the Philippine General Hospital (31), showed S. japonicum ova. The lesions were those of a chronic inflammatory process with the presence of typical tubercles including giant cells,

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necrosis and infiltration of round cells, eosinophils and polymorphs in the early stages. The tubercles were quite similar to those of tuberculosis, so much so that if whole or broken parasite eggs were absent, the disease is likely to be confused for tuberculosis. Colorectal lesions, on the other hand, have been classified according to the stage of pathologic change (27), as follows: (1) Catarrhal coloproctitis. The earlier indication of colonic irritation is excessive secretion of mucus. Clinically, there may be no symptoms, or occasional attacks of mucus diarrhea with the passage of dysenteric stools. Grossly, the mucosa becomes reddened and glistens, highlighting some areas. A coat of mucus is present and close examination may show punctiform hemorrhages. Microscopically, there are numerous egg nests with the formation of giant cells, a typical tuberculoid reaction resulting therefrom. The tissue reaction may in some cases be more marked; there may even be areas of hemorhage and beginning ulceration is evident in some portions. (2) Suppurative coloproctitis. As the eggs escape from the submucosal venous plexuses, they form minute abscesses. Clinically, the attacks of dysentery become more exaggerated; the mucoid stools acquire a sanguinopurulent character, and bowel movement is accompanied by gripping lower abdominal pains. Grossly, the mucosa appears definitely inflamed and the mucus coating is replaced by tiny flecks of pus. Submucosal abscesses can be seen microscopically. (3) Ulcerative coloproctitis. The submucosal abscesses rupture, causing minute ulcerations. The dysenteric attacks become worse and the lower abdominal pain is exaggerated. The ulcers are half a millimeter or so in diameter and appear as small elevated nodulations with undermined edges. Microscopically, mucosal ulceration is seen and the giant cells are replaced by round cell infiltration in most areas, though there may still be evidences of minute submucosal abscesses. (4) Polypoid coloprotitis. Due to the continuous irritation produced by the egg nests, more fibrosis occurs at the base of the ulcerated area, accompanied by hyperplasia and proliferation in the mucosa, causing polyps to form. The stools may become more blood-streaked and the patient may in time present with the symptoms of irritable rectum. Patients may even waste just like those with malignancy. Grossly those polyps appear


as finger-like buds or present a coraliform picture. Microscopically, these polyps are structurally similar to the polyps caused by an irritation with chronic inflammatory reaction, except that one can discern the egg nests embedded in the scar tissue at the base. (5) Granulomatous coloproctitis. Granulomata result from prolonged and persistent irritation. If the process is of some duration, there may be an intermediate ulceronecrotic stage evident on the surface of the granuloma. This is caused by superimposed secondary infection on the surface. Irritative symptoms of the neighboring viscera, such as the urinary bladder and the uterus, may be set u p and the clinical picture becomes complicated, simulating more closely that of rectal neoplasm. Fibrosis becomes more marked. There is infiltration of the surrounding tissue and pseudofixation may occur very much like a tumor. The symptoms of low colonic obstruction with an irritable rectum become more marked and the clinical picture often leads to the diagnosis of rectal carcinoma rather than rectal schistosomiasis. (6) Carcinomatous degeneration. Following the constant irritation set up by the deposited ova, it would not be surprising to find actual neoplastic changes at the terminal stage of rectal schistosomiasis. Should ulceronecrotic changes supervene at this stage, and portions of the granuloma slough off, the proctosigmoidoscopic similarity to carcinoma becomes more marked. However, neoplastic changes in the colon or rectum together with hepatointestinal schistosomiasis are quite rare. There is scanty evidence to indicate that the association is causal. In all the above-mentioned pathologic stages, there is secondary lymphadenitis and pariadenitis. The mesenteric or other regional lymph nodes may become hard, enlarged and darkened. The pigmentation is due to hemoglobin deposition. In some instances the gland itself may be invaded by the parasite. Stool negativity in patients with nodular lesion of schistosomiasis in the rectum indicates a preulcerative stage or relative tolerance of the rectum to the parasite ova (27). But the actual changes of finding eggs in stools are governed by a host of other factors, namely the stage of infection, worm load or intensity or infection, organ involvement, stool sample size, frequency of stool examination, chemotherapy and sensitivity of the stool examination technique used.

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Pulmonary Form There are presently no available data from population-based studies on the frequency of pulmonary schistosomiasis japonica, but it definitely cannot parallel the incidence of portal hypertension. It is quite probable, though, that the frequency of lung lesions follows those of the liver and intestines. It has been observed in a hospital-based study (32) that chronic cor pulmonale secondary to schistosomiasis may be observed immediately after the detection of hepatic involvement since it is possible that deposition of ova in the liver and lungs may take place almost immediately. But, in the other cases seen, pulmonary involvement developed long after hepatic involvement. It was the conclusion that the chances of developing pulmonary schistosomiasis and later chronic cor pulmonale are greater if liver involvement has taken place. A study of 122 autopsy cases in the Philippine General Hospital (25) revealed that there were ova in the lungs in more than 50% of the cases but signs or symptoms indicating lung involvement were usually present only in those with a very heavy worm load. The shunting of ova into the pulmonary circulation may be achieved during severe portal hypertension (26) via portosystemic collaterals as emboli (Olveda et ah, 1986). It must be mentioned, however, that pulmonary lesion due to parasite eggs, in all likelihood, occurs even in the early chronic phase of infection in the absence of portal hypertension. Furthermore, the pulmonary signs and symptoms observed during the early stage must be delineated from those of the chronic stage. The former is more a case of hypersensitivity or an allergic phenomenon induced by the schistosomulum as it traverses the pulmonary capillaries (33), while the latter is due to parasite ova arrested in the pulmonary vasculature. There may be two types of lesions at this stage of the pulmonary form: (1) parenchymatous, consisting of chronic bronchitis, bronchiectasis and emphysema; and (2) vascular, prominently characterized by necrotizing arteriolitis and obliterating endarteritis. This severe form which results in pulmonary hypertension has been reported to occur in China and is a more common complication of pulmonary schistosomiasis in the Philippines. The chest X-ray of patients with pulmonary schistosomiasis may or may not reveal cardiomegaly. When present, a markedly bulging pulmonary artery conus segment may be compatible with pulmonary hypertension. The lung field may appear hypervascular although it may not be easy to


distinguish it from fibrotic strands. The right venticle may be enlarged, especially in the oblique projections. In uncomplicated cases, the lung findings may not suggest any active parenchymal disease (34). Cases with early pulmonary schistosomiasis seen at the University of Santo Tomas Hospital in Manila (Salazar et ah, 1982) showed hazed densities over the right lung

base or both perihilar regions and residual fibrosis of both upper lung fields. A semitriangular density in the right paratracheal area with suggestive narrowing of the intercostal space over the right was also observed. Other early stage pulmonary schistosomiasis cases presented normal radiographic features. A pathologic study of pulmonary schistosomiasis japonica conducted by Ostrea and Marcelo (33) revealed essential information on this clinical entity. They found out that ova deposition was of a diffuse, scattered configuration rather than a localized clumping as seen when ova are laid locally, which underscores the role of embolism of the ova rather than local egg deposition by aberrant worms. The vascular changes observed were as follows: atheriosclerosis, cellular hyperplasia, endateritis, thrombosis, elastosis or diffuse intimal fibrosis, glomoid formation, medial hyperplasia and arteriovenous communication. These lesions were attributed, either directly or indirectly, to whether or not viable or nonviable (calcified) ova were demonstrated in the vicinity of tissue cellular reaction. With the arrest of ova in the smaller branches of pulmonary capillaries, the authors noted that granuloma formation with or without thrombus was possible intraluminally. A diffuse and local necrosis of the blood vessel wall may then take place around the site, with resultant injury to the vessel wall. This is followed by endothelial cell proliferation while the media is converted into a swollen, hyaline mass of necrotic debris. Here, the ovum temporarily lies until it erupts into the lung parancyma. This vascular lesion heals by fibrocellular thickening, which may narrow the lumen of the blood vessel. Thus, if the pulmonary vasculature is extensively and repetitively damaged by the ova, this process of necrotizing arteriolitis results in an obliterating endarteritis with consequent narrowing of the lumina of the blood vessels. Such vascular changes were observed to be minimal in the non-cor-pulmonale cases and striking in those with cor pulmonale. It was further observed that once the ovum has penetrated the wall of the pulmonary arterioles, there are three possible consequences: (1) it may penetrate an adjacent vein, producing an arteriovenous communication;

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this communication is not direct but rather formed by new vessels; (2) a granulomatous reaction occurs at various stages of resolution or healing; (3) occasionally, the ova may penetrate into the alveolar lumen and therefore be present in the sputum. It was the conclusion of the authors that, pathologically, pulmonary endarteritis with diffuse initial fibrosis and media hyperplasia secondary to schistosomiasis is similar morphologically and pathologically to the endarteritis described in rheumatic fever, periarteritis nodosa and primary pulmonary arteriosclerosis. Hyaline thrombus or fibrinous enderteritis, claimed to be evidence of the allergic mechanism seen in schistosomiasis mansoni, was not observed. A study of early pulmonary alterations in schistosomiasis conducted by Salazar et ah (1982) using noninvasive techniques such as arterial blood gas analysis, pulmonary function tests and perfusion lung scan. Five out of eight patients had a restrictive ventilatory defect. A combined restrictive and obstructive ventilatory defect was seen in one case, indicating an interstitial involvement. Respiratory alkalosis with mild hypoxia was seen in five of the eight patients, an evidence that alveolar membrane fibrosis has taken place.

Cerebral Form It is estimated that the frequency of the cerebral form of schistosomiasis japonica is about 1 case per 1000 infected individuals (26). It is considered to be one of the important causes of focal epilepsy in the Far East. Earlier autopsy records of the Philippine General Hospital revealed the presence of brain lesions in 8 out of 122 (6.5%) schistosomiasis cases examined from 1953 to 1960 (35). Inasmuch as some cerebral involvement may be asymptomatic, it may be that the actual frequency of brain schistosomiasis would be higher if autopsy records were to be followed. Speculations as to the probable route of migration of eggs from the site of oviposition to the brain were varied. Evidence for local or direct oviposition by aberrant worms has been presented based on the finding of nests or clusters of eggs in the brain (36), but mature worms have never been found in the brain in any case of human cerebral schistosomiasis. Another possibility may be the maturation of circulating carciriae in the brain itself, but this appears to be less likely. The more probable route, however, may be via venous


anastomoses between the systemic and hemorrhoidal view, which is believed to facilitate access to the dural sinus by the adult worms (37). Blankfein and Chirico (38) have proposed that the parent worms in the mesenteric veins may enter Batson's intercommunicating plexus during periods of increased intra-abdominal pressure, travel via the internal jugular vein upward into the dural sinuses draining the brain. Thus the mature worms or their ova may reach the brain directly from the abdominal cavity without going by way of the heart or lungs. However, it was noted (39) that the presence of severe intra-abdominal infection with marked portal collaterals and portal hypertension does not necessarily favor the formation of ectopic cerebral lesions. Evidently, other mechanisms still unknown may be at play in ectopic deposition of ova and consequent development of cerebral lesion. The cerebral form of schistosomiasis japonica has been classified into acute, subacute and chronic forms (40). The acute phase may be accompanied with fever, urticeria, absolute eosinophilia and angioneurotic edema suggesting an allergic encephalopathy. There may be delirium, confusion, personality changes, incontinence, coma, nuchal rigidity and pyramidal tract signs. Cerebrospinal fluid findings are unremarkable at this stage and stool examination will usually reveal the ova of the parasite (39). This phase occurs within 6 months postexposure. The acute phase may merge into a chronic phase, which either could be asymptomatic or may mimic the picture caused by intracranial neoplasm. Occasionally, a subacute form of cerebral involvement occurs, usually 3-4 months after initial infection, with characteristics of both acute and late onset disease (38). The chronic phase or late onset disease manifests symptoms of cerebral involvement 6 months or more after systemic disease or exposure. The shortest period noted from the time of infection to the onset of cerebral symptoms in a series of 42 patients studied by Torres (41), was 6 months, while the longest was 9 years. The average incubation period was 2.3 years. Seizures secondary to brain schistosomiasis may be motor, sensory or psychomotor. Psychomotor and motor involvement are the more dominant forms of neurologic disturbances. The majority of the motor disturbances were of the grand mal or Jacksonian type of epileptic seizures. The most common site of the lesion in those with Jacksonian seizures was the parietal lobe. Other neurological signs and symptoms that may be observed are headache, speech difficulties, visual disturbances, papilledema and cerebellar symptoms. The eosinophil count is increased in most cases of infection with

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schistosomiasis. Stools and a rectal biopsy may or may not be positive for parasite ova (41). The cerebral fluid (CSF) circumoval precipitin test (COPT) has been used to distinguish cerebral from noncerebral schistosomiasis japonica inasmuch as the serum precipitins do not diffuse through the blood brain barrier (Reyes et ah, 1964). CSF examination may, in some instances, reveal increased pressure, cell count or protein level (41). Clinically and pathologically, two types of lesions have been recognized in cerebral form of schistosomiasis japonica (42). They are: (1) A diffuse lesion of the brain that may be asymptomatic or may present with cerebral seizures. On contrast roentgenography, this group of cases may not present discrete mass but on autopsy will show a diffuse small lesions of the gray and white matter. This is supportive of the embolic mechanism of egg dissemination. (2) A predominantly solitary lesion consisting of large irregular granulomata containing schistosoma ova in fairly large aggregations. It is believed that his type of lesion suggests local oviposition by aberrant worms. While this may possibly occur together with both hematogenous embolizations, recovery of the adult worm in the brain has never been reported. The greater number of eggs laid by S. japonicum compared to S. mansoni is considered to be responsible for the greater incidence of ectopic deposits of S. japonicum ova in the brain as well as for the more rapid development of acute cerebral and systemic symptomatology (43). S. japonicum likewise shows a tendency toward the development of larger intracranial granulomata which may cause so much pressure as to necessitate surgical intervention, in contrast to small granulomata produced by S. mansoni or hematobium in the spinal cord (37). Histologically, the lesion in cerebral schistosomiasis is essentially that of a chronic granulomatous infiltration with or without caseation (42). Accordingly, it is not easily distinguishable morphologically from other granulomata of the brain, especially a tuberculoma, which is another common brain granuloma in S. japonicumendemic areas in the Philippines. Reyes et al. (42) observed that the eggs in brain tissues are of various forms due to distortion. They may not be readily evident on cursory examination, especially when the distribution is diffused. It has also been observed that the ova are not always located at the center of the granuloma (41). It is easier to identify eggs by teasing a tubercle removed at surgery into a slide in a fresh state than to look


for them in a frozen or paraffin section (42). Lymphocytes, plasma cells and eosinophils are scattered around the periphery of the granulomata but eosinophilic infiltration may not be marked. The ova may be immediately surrounded by either giant cells or collagenous fibers (41, 42). Both the small size veins and arteries may show perivascular round cell

cuffing involving plasma cells, eosonophils and lymphocytes (41, 44). Electroencephalographic (EEG) studies conducted on a series of 70 chronic schistosomiasis japonica patients with subjective symptoms of fatigue, mild headache and dizziness revealed abnormal EEG's in about half of the subjects (Hayashi et ah, 1968, cited by Yokogawa, 1976). Another study was conducted on individuals with a past history of S. japonicum infection but stool-negative for the parasite egg. The field survey revealed 28% of 76 cases showing abnormal or borderline EEG's. There was noted a significant difference in the occurrence of abnormal EEG's between the two groups with and without subjective symptoms such as fatigue, mild headache and dizziness. Yokogawa (40), citing other workers, noted that the appearance of seizure discharges in EEG's in schistosomiasis is relatively infrequent in the Philippines compared with those in Japan. Treatment of cerebral cases using antischistosomal drugs has been reported to result in improvement with disappearance of seizures in 100% of patients (24 in one series studied), all of whom previously manifested proxysmal disease before treatment (44).

Hematologic Garcia (20), reviewing previous studies, observed that the more common hematologic findings among Filipinos with schistosomiasis japonica are: (a) anemia is present, the severity increasing with the seriousness of hepatosplenic disease; (b) usually moderate leukocytosis is observed, with a tendency to be lower in more advanced hepatosplenic disease; and (c) eosinophils are present in all cases. Anemia tends to improve with treatment. There is gradual but significant reduction of the red blood cell count from the acute stage to the late stage of the disease (45). This may stem either from hypersplenism due to a shortened life span of red blood cells or from massive hematesis. In like manner, hemoglobin values fall with the advancing stage of infection. The study by Medado (45) further showed that leukopenia was present only among severe cases in the stage of chronicity (probably the start of the

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decompensated stage) while apparently the total leukocyte count was within the normal limit in the acute and the early chronic stage. Eosinophilia was higher among cases in the earlier stage compared to those in the late chronic stage. Morphologically, there was a slight macrocytic anemia in the early stage and normocytic anemia in the later stages. The sedimentation rate was definitely high and found to be more so as the disease progressed. In contrast, there was apparently normal hematocrit among acute patients compared to different degrees of low hematocrit in the chronic stage, which tends to go lower toward the late stage of the disease.

Ectopic Usually localization of lesions in schistosomiasis japonica other than the lungs and brain has drawn the least attention among investigators in this field. This is probably due to the mild or asymptomatic nature of most of the resulting morbidity arising from ectopic lesions requiring no treatment, their relative infrequency of occurrence or the presence of more important concurrent organ involvement such as the liver and intestines which usually necessitates more attention. An interesting case reported was the finding of eggs in the interventricular septum of a patient who died of generalized schistosomiasis (46). This patient had eggs in the liver, intestines, lungs, brain, kidney and heart. Cases of cutaneous schistosomiasis japonica were reported by Garcia et al. (20) and Fishbon (47). The former case yielded parasite eggs in the skin lesion while the latter resembled that of larva migrans involving probably an adult female. Most et al. (48) reported a S. japonicum-mfected soldier with chorioretinitis possibly parasitic in origin which returned to normal after a period of antischistosomal therapy. Mendozas-Guazon (49) reported a finding of parasite eggs in the mesenteric and hemolymph nodes, omentum, and eosinophilic infiltration of the heart and spleen among a series of cases. Jongco and Lee (50) reported involvement of the pancreas, spleen and lymph nodes in addition to the usual sites in a single case. VillegasCinco (30) found eggs or lesions in the spleen, mesenteric lymph node, visceral peritoneum, pancreas, fallopian tube and ovary aside from the usual sites in a series of 127 cases. Other ectopic sites reported in the literature include the hernial sac, stomach and spinal cord (51). In Japan, examination of the egg distribution in each organ revealed that eggs were most frequent in the liver, their number diminishing in the following order: sigmoid colon, cecum,

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descending colon, ascending colon, appendix, ileum, jejunum, duodenum, pancreas, lungs stomach, urinary bladder, kidneys, spleen and brain (52). The role of worm burden related to repetitive infection, presence of cardiac septal defects or certain hemodynamic factors are possibilities that may underlie these ectopic foci, but conclusive proofs are lacking. It will be noted that reported cases of multiorgan localization involving ectopic foci are rare.

Child Growth and Nutrition in Schistosomiasis Japonica Stunting of growth and delay in the pubertal development of children have been described as another aspect of schistosomiasis-japonica-related morbidity (53,54). The underlying mechanism is not yet clear; chronic anemia, anorexia and decreased nutrient absorption have been suggested as causative factors (53). After schistosomicidal therapy, an improvement of haemoglobin levels was noted which was considered significant; therapy might improve growth conditions as well (55), but long term follow-up data on growth after praziquantel treatment are not available. In a recent prestudy of schistosomiasis-related morbidity in Leyte, the Philippines, stunting appeared to be especially pronounced in children with sonographically detectable liver abnormalities (unpublished). Liver dysfunction can be an important contributor to growth disorders due to impaired synthesis of obligatory growth factors like IGF 1 and IGFBP 3. No data are available in the literature on the relation of schistosomal growth impairment with hepatic morbidity including endocrine dysfunction of the liver.

Diagnosis of Schistosomiasis Japonica The diagnosis of schistosomiasis japonica in endemic areas is at present mainly based on the detection of the schistosome eggs in the stools of infected individuals. Often used for large scale field application is the Kato thick smear (56) or the "quick" Kato technique (57). This assay forms the basis of community-based data in the Philippines. Other quantitative stool examinations, like the merthiolate-iodine-formaldehyde concentration technique (MIFC), the merthiolate-formaldehyde concentration technique (MFCT) and the formaldehyde-ether concentration technique (FECT), have also been used in the Philippines, but these tests require more time and labor

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to perform, thus making them impractical for wide scale application. In China, the Kato thick smear for detecting S. japonicum infection has not been used extensively. The Chinese used miracidial hatching instead. Although this technique is more sensitive than the Kato thick smear, the procedure is nonquantitative, laborious, difficult to perform and cannot be used under field conditions. Since a single stool examination can miss up to 30% of lightly infected individuals (58) and repeated stool examinations are not practical in wide scale field activities, other diagnostic tests were developed. Serologic tests to detect in the blood of infected individuals antibodies against different stages of S. japonicum or the circulating antigen of the parasite were established. Historically, detection of antibodies using indirect hemagglutin assay (IHA) and circumoval precipitin tests (COPT's) were extensively adapted in China and the Philippines respectively. These tests, however, cannot distinguish between past and present infection, and are thus useless in following changes in ova prevalence in longitudinal studies. In addition, serologic tests are expensive and less accepted by individuals in the community. A major advance in immuno diagnosis has been an adaptation of a system developed for S. mansoni (59). In this system, gut-associated antigens (GAA's) are detected in the serum and urine using the murine monoclonal anti-GAA antibody. Using similar technology, Chinese scientists have raised their own S. japonicum mouse monoclonal anti-GAA which can detect circulating antigens in the serum of S. japonicum-intected individuals (60). A field trial done on this test showed encouraging results (61). The Chinese have also developed a murine monoclonal anti-idiotypic antibody, which is an antigen mimic to an as-yet-unknown epitope of GAA. This molecule was used instead of worm-derived GAA in a standard antibody-based ELISA (personal communication). Although the above-mentioned tests adapted in China showed encouraging results, they have not been used for nationwide application. Philippine scientists, on the other hand, have

produced mouse monoclonal anti-GAA, which can detect S. japonicum circulating antigens excreted in the urine (manuscript in preparation). The monoclonal antibody is directed to the low molecular weight circulating cathodic antigen (CCA) filtered in the kidney and passed out with the urine. The test appears to be more sensitive than the single Kato thick smear. The procedure is u n d e r g o i n g field-testing for sensitivity, specificity and reproducibility in preparation for field application by the Philippine National


Schistosomiasis Control Service. Since compliance for urine submission is better than stool sample collection, detection of S. japonicum CCA in the urine will be ideal for diagnosis of active infection and will be valuable in monitoring the success of chemotherapy. In the diagnosis of cerebral schistosomiasis detection of antibodies or antigen in the cerebrospinal fluid

(CSF) has been demonstrated (62). Assessment of organ morbidity in schistosomiasis japonica, particularly the presence of hepatomegaly or splenomegally, and detection of hepatic fibrosis at the community level have also been the subject of investigations. As screening tool for organ morbidity, abdominal palpation has been used (10), but the clinical relevance of hepatomegaly in this context is actually unknown and prognostically important features like portal hypertention, particularly in the early stage of the disease, may not be detected. Since the classic lesion is periportal fibrosis or PSF, detection of this lesion in the liver by a noninvasive procedure will differentiate schistosome-induced hepatic fibrosis from other causes of fibrotic liver diseases like chronic viral hepatitis, postnecrotic cirrhosis and excessive intake of alcohol. The introduction of ultrasonography paved the way for the detection of the classic pipe-stem schistosome-induced liver fibrosis. Portable ultrasound scanners were first used to detect and quantify hepatosplenic schistosomiasis mansoni under field conditions (63, 64). Several patterns differentiating S. mansonz-induced fibrosis from other causes of fibrotic diseases were described. Ultrasonographic pictures have been shown to correlate with histology (65, 66), presence and grade of esophageal varices (67) and with the risk of upper gastrointestinal bleeding (68, 69). Since the ultrasonographic findings may be different in S. japonicum compared to S. mansoni infection, the approach has also been adapted to investigate S. japonicum-Telated morbidity in the Philippines, Japan and China (70-78). In China, more investigations have been performed earlier but have not been published in the internationally available literature. Several sonographical features of hepatic schistosomiasis japonica have been described, like periportal fibrosis, and echodense lesions similar to that seen in S. mansoni infection were mentioned. They observed different sizes of echodense nodules, sieve, mottled and mixed pattern of the liver texture, features not described in S. mansoni. The peculiar echogenic lines scattered throughout the liver parenchyma, which was called a "network" pattern unique only to S. japonicum infection, were also reported. This finding was also seen on computerized tomography (CT) scanning (79).

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Schistosomiasis

The clinical relevance of this finding is not yet clear from the published articles, which are mostly cross-sectional and merely descriptive. Overall, as far as the diagnosis of hepatic fibrosis in schistosomiasis japonica is concerned, the studies published have been too unsystematic to derive a general description of the sonographical aspect of hepatic involvement in schistosomiasis japonica. The WHO workshops on ultrasound in schistosomiasis held in 1996 were thus unable to decide on recommendations for the practice of sonography in schistosomiasis japonica. More descriptive and analytic studies in this area were urgently demanded (80). Recently, Doppler sonography has become the standard method used to investigate portal hypertension in industrialized countries. The procedures were attempted in the study of schistosome-induced hepatic fibrosis with concomitant presence of portal hypertension. The single study on the late stages of S. mansoni cases from Brazil demonstrated that the technique is generally feasible for field application (personal communication). A preliminary study in the Philippines done by us and our partners (81) indicates that Doppler sonographic signs suspicious of portal hypertension are rather frequent in an endemic setting, around 20%, and that there is a correlation of these signs with portal fibrosis, but with a "network" pattern. It appears that the Doppler technique will give important information that will correlate the qualitative features of hepatic sonography with the future risk of gastrointestinal bleeding. Diagnosis of schistosomiasis japonica in the hospital is practically the same as in the endemic community except for additional examinations, which cannot be performed in the field. While the history of exposure is important, the egg is the confirmatory unit of diagnosis and it is the only stage of the parasite to be seen except where miracidial hatching tests are done. These procedures include demonstration of eggs from rectal mucosa obtained by rectal imprint or biopsy from liver tissue by needle biopsy or specimens from the brain or other organs. An ultrasound or CT scan of the liver may reveal the classic "pipe-stem" hepatic fibrosis. Mixed lesions in the liver are, however, difficult to demonstrate. A CT scan of the brain for suspected cerebral lesions may demonstrate mass lesions, but this will not completely differentiate S. japonicum granuloma from other causes of spaceoccupying lesions in the brain. An electroencephalogram (EEG) in the diagnosis of cerebral schistosomiasis may give a abnormal patterns in the areas affected by S. japonicum-induced pathology but the procedure cannot give a definite


diagnosis. A CT scan and EEG, however, can be valuable in assessing regression of cerebral pathology following treatment (82).

Chemotherapy In the past, antischistomal drugs effective against S. hematobium or S. mansoni infection were either not effective (hyacanthone, metrifonate, oxamiquin, etc.) or less effective (niridazole, antimonials) against S. japonicum infection. This was partly due to the development of toxicity before the therapeutic levels of the drugs were reached. The situation has changed dramatically since praziquantel was developed in the late 1970's. The drug has been shown to be effective against the three species of human schistosomes. For S. japonicum infection, the drug was given orally u p to the total doses of 40, 50, 60 or 70 m g / k g in divided doses in one day or two days. In China, up to 120 m g / k g was used in acute schistosomiasis with heavy infections. Single-dose treatment with 50 or 60 mg per kg was also used (83). Parasitological cure rates measured at 3 and 6 months after treatment ranged from 80 to 97%. Side effects like abdominal pain or discomfort, fever, sweating and giddiness are minimal and transient (83). Community studies in S. japonicum endemic (83) using praziquantel to determine its effect on the prevalence, incidence and intensity of infection have been clearly established (82). In the Philippines, for example, the long term impact of chemotherapy was examined in three endemic villages in Leyte from 1981 to 1988 in an eight-year longitudinal study, which followed 5122 individuals (84). The three villages used were representatives of communities with low, moderate and high prevalence of S. japonicum. Infection was determined by Kato-Katz smears on a single sample. More than 85% of the population were examined and more than 90% of the infected population were treated in each year of the study. The prevalence of infection in year 1 was 26%, 39% and 44% for villages A, B and C, respectively. Treatment of all individuals over three years was associated with a steady decrease in the prevalence of infection which stabilized after two years at 4 - 5 % , 7-9% and 11-13% for villages A, B and C, respectively. The incidence of infection during this eight-year period paralleled the changes in the prevalence of infection. In addition, the geographic mean egg count of infected individuals decreased significantly

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Schistosomiasis

from 85 eggs per gram of stool in year 1 to 25 eggs per gram by year 2 and remained at this level for the rest of the study. These data suggest that case finding and treatment with praziquantel is associated with a significant decrease in the prevalence, incidence and intensity of infection within three years of the initiation of selective treatment of the infected population. The prevalence and incidence of infection did not further decrease despite continuous annual screening and treatment. These observations, together with the results from endemic areas in China and Indonesia, suggest the significant contribution of nonhuman reservoirs in the transmission of S. japonicum. Regression of clinical symptoms, liver and spleen size after treatment has been reported from different endemic areas (77). The study in the Philippines (84) also looked at the impact of chemotherapy on liver enlargement. The prevalence of hepatomegaly defined as > 3 cm in the misternal line or > 2 cm in the mid-clavicular line as determined by physical examination was 17.7% in infected individuals and 8.3% in uninfected individuals in year 1. The age-specific prevalence of hepatomegaly associated with infection increased in the 10-20-year age group and then decreased to 6-7% over 5 years, suggesting that treatment stimulated reversal of schistosoma-induced hepatomegaly. This persistence of hepatomegaly was not due to failure to respond to chemotherapy, since > 85% of individuals of all ages resolved their hepatic enlargement following treatment within 2 years. The effects of praziquantel treatment on hepatic fibrosis, as assessed by ultrasound were also investigated. A study {77) followed up for 1.5 years 52 newly infected patients with different grades of pathology who had been treated with praziquantel. Patients were examined u p to 1.5 years after treatment. The study showed regression of mild to moderate fibrotic changes, but not of advanced hepatic fibrosis, and concomitant collateral vein formation. Similar studies performed in China demonstrated no significant decrease in hepatic fibrosis, as shown by ultrasonography in a community

subjected to population-based chemotherapy for 3 years. Cohort analysis of infected individuals treated with praziquantel showed, however, a significant decrease in advanced hepatic fibrosis. Since data available showed contrasting results and few studies have been done, further studies on this aspect are needed in order to make clear statements on the effects of the praziquantel on hepatic fibrosis reversal.


In the past, treatment of cerebral schistosomiasis has been disappointing. In the study of 99 individuals with symptoms of epileptic seizures, who were treated with trivalent antimonials and followed up for 4-19 years, 51% had disappearance of seizures, while 22% continued to have seizures, although of reduced frequency. In another study, 70 cases were treated with either

stibopen or praziquantel and followed up for 6 years (83). Seventeen percent and 75% of individuals treated with stibipen and praziquantel, respectively, had disappearance of seizures. Praziquantel seemed to be more effective than the other drugs in reducing the occurrence of seizures. Another study in the Philippines (85) examined 60 patients with impression of cerebral schistosoma japonica who were followed up for 7 years after treatment with praziquantel. Seventy percent of the treated individuals became seizure free. EEG abnormalities in these patients decreased from 43% to 23%. Cerebral schistosomiasis has also been successfully treated with praziquantel in eight out of nine proven cases (82). However, one patient who did not respond completely had signs of improvement as shown by clinical manifestations and dissipation of cerebral edema and near-complete resolution of mass lesions as measured by a CT scan. Whether the resolution of a cerebral lesion was secondary to the effect of praziquantel or antiinflammatory agents coadministered with the drug or natural course of a S. japonicum egg induced cerebral granuloma is not known. Treatment with praziquantel, however, can decrease the chance of recurrent deposition of ova in the brain. Control of schistosomiasis japonica through the development of vaccine candidates has progressed recently but not to the extent of practical applications (86).

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53. McGarvey ST, Wu G, Zhang S, et al. (1992). Am. J. Trop. Med. Hyg. 46: 571. 54. McGarvey ST, Wu G, Zhang S, Wang Y, Peters P, Olds GR, Wiest PM (1993). Am. /. Trop. Med. Hyg. 48: 547. 55. McGarvey ST, Aligui G dL, Graham KK, Peters P, Olds GR and Olveda RM (1996). Am. /. Trop. Med. Hyg. 54 (5): 498. 56. Mott KE and Cline BL (1982). Bull. WHO 60: 729. 57. Peters PA, El Alamy M, Warren KS and Mahmoud AAF (1980). Am. }. Trop. Med. Hyg. 29: 217. 58. Walter SD and Irwig LM (1988). /. Clin. Epidemiol. 41 (9): 923. 59. Deelder AM, Qian ZL, Kresmner PG, Acosta LP, Rabello ALT, Enyong P, Simaro PP, van Etten EECM, Krijger FW, Rotmans JP, Fillie YE, de Jonge N, Agnew AM and van Lieshout LE (1994). Trop. Geog. Med. 46 (4): 233. 60. Yan Zizhu, et al. (1990). Chin. Acad. Prev. Med. 2 (2): 39. 61. Yan Zizhu, et al. (1992). Chin. Acad. Prev. Med. 4 (6): 275. 62. Reyes VA, Yogore MG Jr and Pardo LP (1964). Acta Medica Philippina 6: 365. 63. Jenkins JM and Hatz C (eds.), the Cairo Working Group (1992). Acta Trop. 51: 45-63. 64. Doehring-Schwerdtfeger E and Kardorff R (1995). Mem. Inst. Oswaldo Cruz 90 (2): 141. 65. Abdel-Wahab MF, Esmat G, Milad M, Abdel-Razek S and Strickland GT (1989). Am. J. Trop. Med. Hyg. 40: 72. 66. Homeida M, Abdel-Gadir AF, Cheever AW, et al. (1988). Am. }. Trop. Med. Hyg. 38: 86. 67. Ravera M, Reggiori A, Cocozza E, Cianta F and Riccioni G (1996). Eur. }. Gastroenterol. Hepatol. 8: 693. 68. Davidson RN, Houston S and Kiire CF (1991). Trans. R. Soc. Trop. Med. Hyg. 85: 380. 69. Richter J, Zwingenberger K, Ali QM, et al. (1992). Radiology 184: 711. 70. Tanaka M (1992). Mem. Inst. Oswaldo Cruz 87 (Suppl 4): 277. 71. Mott KE, Chen MG, Abdel Wahab F, et al. (1992). Acta Trop. 51: 65. 72. Wei-min C, Dong-chuan Q and Hatz C (1992). Acta Trop. 51: 37. 73. Ohmae H, Tanaka M, Hayashi M, et al. (1992). Am. J. Trop. Med. Hyg. 46: 89.

74. Ohmae H, Tanaka M, Hayashi M, et al. (1992). Am. /. Trop. Med. Hyg. 46: 99.


75. Wiest PM, Wu G, Zhang S, et al. (1992). Trans. R. Soc. Trop. Med. Hyg. 86: 47. 76. Wiest PM, Wu G, Zhong S, et al. (1993). Trans. R. Soc. Trop. Med. Hyg. 87: 290. 77. Hatz C, Murakami H and Jenkins JM (1992). Acta Trop. 51: 29. 78. Wiest PM, Wu G, Zhong S, et al (1994). Am. ]. Trop. Med. Hyg. 51: 162. 79. Cheung H, Lai YM, Loke TKL, et al. (1996). Clin. Radiol. 51: 51. 80. Campagne G (1998). 2nd WHO expert meeting in ultrasonography in schistosomiasis. WHO White Paper Report, in preparation. 81. Kardorff R, Olveda RM, Acosta L, Duebbelde U, Aligui E, Gryseels B and Doehring E (1999). Am. J. Trop. Med. Hyg. 60: 954. 82. Watt G, Long G, Ranoa C, Adapon B, Fernando M and Cross J (1986). The Lancet, Sept. 6, 1986, pp. 529-532. 83. McGarvey ST, Wu G, Zhang S, et al. (1992). Am. } . Trop. Med. Hyg. 46: 571. 84. Olveda RM, Daniel BL, Ramirez B dL, Aligui G dL, Acosta LP, Fevidal P, Tiu E, De Veyra F, Peters PA, Romulo R, Domingo EO, Wiest PW and Olds GR (1996). /. Infect. Dis. 174: 163. 85. Hayashi M, Matsuda H, Tormis LC, Nosenas JS and Bias BL (1984). SE Asian J. Trop. Med. Pub. Health 15: 502. 86. McManus DP (2000). Int. } . Parasitol. 30: 265.

391

Chapter 11 Disease Due to Schistosoma mekongi, S. intercalatutn and Other Schistosome Species Charles H King

Introduction Nineteen schistosome species parasitize mammals (1). Of these, seven develop mature, patent infections in humans. They are: Schistosoma haematobium, S. mansoni, S. japonicum, S. intercalatum, S. mekongi, S. mattheei and S. malayensis (see Table 1) (2,3). For each of the seven species, it has been shown that deposition of parasite eggs into the host circulation (with the consequent granulomatous immune reaction) produces chronic disease in those patients who are infected. In addition, various case reports have claimed patent human infection with other schistosome species such as S. bovis, S. curassoni and S. margrebowiei, which are normally parasites of cattle and wild ruminants. However, evidence for human infection with these species is not as substantial or clear-cut. There are schistosome parasites of birds, such as Trichobilharzia and Bilharziella species, that are known to infect human skin, causing an acute, superficial infection that is associated with cutaneous eruption at the site of cercarial penetration ("swimmer's itch"). These do not cause chronic disease, but may seasonally affect a large proportion of the human population in an enzootic area. This chapter reviews current knowledge of S. intercalatum, S. mattheei, S. mekongi and S. malayensis infection, and summarizes data on the pathogenesis of cercarial dermatitis. The previous chapters (7 through 10) have described Schistosoma haematobium, S. mansoni and S. japonicum, which are responsible for the more prevalent forms human schistosomiasis. Those three species are distributed in tropical and subtropical Africa, South America and

392 Schistosomiasis

Table 1. Other schistosome species that may infect humans. Group and species

Location

Vector snail

Host range

Human disease

Africa

Bulinus

Primates

S. mattheei

Southern Africa

Bulinus

Cattle, sheep, goats, primates

S. bovis

Africa, Southern Europe, Middle East

Bulinus, Planorbarius

Cattle, horses, sheep, goats, camels, pigs

Intestinal schistosomiasis Urinary and intestinal schistosomiasis Uncertain

Laos, Cambodia, Thailand Peninsular Malaysia

Tricula

Primates, carnivores Primates, rats

Intestinal schistosomiasis Intestinal schistosomiasis

Cattle, goats, sheep, equines, rodents Birds

Cutaneous schistosomiasis

S. haematobium group S. intercalatum

S. japonicum group S. mekongi S. malayensis Other species S. spindale

Trichobilharzia, Gigantobilharzia, Ornithobilarizia, Bilharziella, Microbilharzia

Robertsiella

India, Sri Lanka, Southeast Asia

Indoplanorbis

Temperate northern and southern regions

hymnaea, Physa, Planorbis, Polyplis, Chilina

Cutaneous schistosomiasis

Asia according to the distribution of their vector snails — Bulinus spp. snails for S. haematobium, Biomphalaria spp. snails for S. mansoni, and Oncomelania spp. snails for S. japonicum (2). The schistosome species described in this

chapter are less common causes of human disease and are less well studied (4). S. intercalatum and S. mattheei are closely related to S. haematobium, and are found in central and southern Africa. S. mekongi and S. malayensis are related to S. japonicum, but are transmitted in limited areas of Southeast Asia. Surveys of infected communities generally show lower prevalence, with lower intensity of infection and lower levels of parasite-associated morbidity as compared to infection with the major schistosome parasites, S. haematobium,

Disease Due to Schistosoma mekongi, S. intercalatum and... 393

S. mansoni and S. japonicum. On a regional basis, the geographic distributions of S. intercalatum and S. haematobium and of S. mattheei and S. haematobium may be said to overlap. However, each species of parasite favors transmission by distinct species of snails within the Bulinus genus, such that, on a much more local scale, snail ecology favors transmission of only one schistosome

species. It is likely that enzootic transmission among wild and domestic mammals perpetuates the transmission of many of the species discussed here. Hybridization between schistosome species has occurred during concurrent natural human infection (5, 6). Evidence suggests, though, that a single species will predominate, typically the more aggressive species, S. haematobium (7). For example, experimental studies of mixed S. haematobium and S. mattheei infections in animals indicate predominant pairing of S. haematobium males with females of both species, to the detriment of S. mattheei males, if there is a scarcity of S. haematobium female worms. Many reports of h u m a n infection with S. intercalatum and S. mattheei have probably described mixed S. haematobium and hybrid infections, resulting in confusion over the clinical syndromes related to each species. It is important for researchers and clinicians to be aware of the additional species that cause human schistosomiasis, as their clinical and laboratory presentations are likely to be atypical.

Schistosoma

intercalatum

The Pathogen S. intercalatum is an uncommon schistosome parasite of humans. It is found only in Central Africa. Initially reported in an isolated section of the Congo River Basin, it is referred to in some early reports as Schistosoma chestermani (4). Infections have since been reported in Cameroon (8), Equatorial Guinea (9), Gabon (10), Sao Tome (11) and the Republic of Congo (Zaire) (12). Sporadic cases have been reported from Angola, Burkina Faso, Senegal, Chad, Nigeria, Congo, the Central African Republic (4), Mali (13) and N o r t h e r n Uganda (14). The parasite infects primarily the mesenteric veins, resulting in bowel inflammation and passage of eggs in the stool. S. intercalatum infection is typically lighter and associated with less morbidity than other forms of human intestinal schistosomiasis (e.g. S. mansoni and S. japonicum). It is possible for S. haematobium and S. intercalatum to hybridize

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Schistosomiasis

(15). These hybrid forms are infectious for humans, and their polymorphic eggs may be found either in the urine (as with S. haematobium) or in the stool (12). Like S. haematobium, S. intercalatum is transmitted by Bulinus species snails, which are found throughout Africa. However, S. intercalatum strains have a specific preference for either Bulinus forskalii or B. africanus snails, which limits their geographic distribution relative to that of S. haematobium, which is transmitted by many different Bulinus species (8). Two distinct strains of S. intercalatum are recognized. These differ in their preferred snail host and in their prepatent periods. The dominant Lower Guinea strain favors B. forskalii as a snail host. The other, Congo strain favors B. africanus group snails and will not infect B. forskalii. Hybridization of S. intercalatum with S. haematobium appears to be introgressive. That is, S. haematobium-like parasites gradually supplant S. intercalatum-like worms in the human population, although some features of S. intercalatum, such as isoenzyme markers and Ziehl-Neelsen staining characteristics of parasite eggs, may persist in clinical samples (6, 8). While S. haematobium x S. intercalatum hybrids can infect a wider range of host snails for transmission, their clinical distribution has been, in fact, limited, perhaps due to a low fitness of S. intercalatum for human infection (5, 16).

Epidemiology S. intercalatum transmission occurs by exposure to freshwater inhabited by vector Bulinus snails. The extent and duration of the water contact are important determinants of infection risk (10). In terms of the age profile, infection is usually more prevalent and more intense in younger individuals (peaking between 8 and 17 years of age). The average infection intensity is typically light [< 50 eggs per gram (epg) feces], although the intensity may range u p to 3200 epg in some patients (10, 14). Concurrent infection with S. mansoni or S. haematobium is possible (14). S. intercalatum prevalence is typically low (1-30%) in an endemic community (4). Recent population-based surveys in the Republic of Sao Tome, where S. intercalatum is the only schistosome species infecting humans (17), have identified an S. intercalatum prevalence of 1-28% (with an overall rate of 11%) in a systematic random survey of schools nationwide (11). A high community prevalence (43%) was identified in only one village, San Marcal, with 14 cases of heavy infection


(> 100 epg), all of which were in subjects over 40 years old (11). This age profile is atypical for endemic S. intercalatum (18), suggesting recent introduction of S. intercalatum into this part of Sao Tome. Nevertheless, the observation contradicts the previously held belief that persons over the age of 35-40 are not infected (4).

Pathology In human infection, S. intercalatum inhabits the mesenteric plexus, predominantly the veins of the colon, and eggs are most commonly found in the stool (4). On examination, the rectal mucosa has a granular appearance, and may have bloody exudate with mucosal petechiae and excess mucus (19). Viable or calcified eggs may be found in the wall of the bowel on rectal biopsy. Ulceration and g r a n u l o m a t o u s polyp formation are typical. Granulomas may also be seen on liver biopsy, although they are smaller in size than those seen with S. mansoni (20). Exudative inflammation is found in 50% of liver samples, but is confined to portal areas. No evidence of portal hypertension has been observed in a case series of 49 liver biopsies (19). Infection with S. haematobium x S. intercalatum hybrids will result in egg deposition in the bladder and ureters, and will result in hematuria and localized granuloma formation. This is not usually seen with S. intercalatum infection alone (11).

Disease In population surveys, chronic morbidity has been rare. 75% of individuals with documented infection are asymptomatic (4). In endemic areas, individuals passing S. intercalatum eggs are significantly more likely to report blood or mucus in the stool, and are more likely to have mild anemia than those without eggs (10, 11). Hepatomegaly is not more common with S. intercalatum infection. 50% of patients in one series had splenomegaly on examination, but concurrent ultrasound evaluation showed no liver morbidity or portal enlargement (14). Dysuria may be reported in heavy infection, and hemospermia has been reported (21). During acute S. intercalatum infection, minimal pruritis may be noted on exposure, but cercarial dermatitis is rarely seen in endemic populations (20).

396

Schistosomiasis

Febrile illness with nausea, vomiting, diarrhea, hepatosplenomegaly and eosinophilia has been reported during acute infection, but such acute illness is considered rare in endemic foci. Disease is more severe in previously unexposed nonimmune adults. In a recent outbreak of mixed S. mansoni, S. haematobium, and S. intercalatum infections among European travelers visiting the Dogon region of Mali, 36% had cercarial dermatitis and 54% had an acute febrile illness ("Katayama fever"). In the single patient from this outbreak who had S. intercalatum infection alone, both dermatitis and acute febrile illness were observed (13). S. intercalatum infection, like S. haematobium and S. mansoni infections, may predispose one to relapsing bacterial superinfection with Salmonella (22, 23). These Salmonella infections may be life-threatening, with typhoid, paratyphoid or nontyphoidal enteric fevers, especially in children (22). Salmonella infections often recur after antibacterial therapy unless the S. intercalatum infection is also treated. In Gabon, the prevalence of S. intercalatum infection is significantly higher among patients with invasive salmonellosis (91% S. intercalatum prevalence for patients with nontyphoidal Salmonella strains versus 38% for local hospital controls, P < 0.001) (23). Salmonella adhere to and colonize the schistosome surface, leading to prolongation of bacteremic infection in the S. intercalatum-iniected human host.

Table 2. Egg features of schistosome species that infect humans. Group and species

Spine

S. haematobium group Terminal, may S. intercalatum be curved S. mattheei Terminal, may be curved Terminal S. bovis S. japonicum group S. mekongi S. malayensis

Lateral knob Lateral knob

Shape

Egg size (length by width, in |im) a

Usual location

Long oval with central bulge Spindle

175-190 by 59 (Zaire) 160 by 58 (Gabon) 173 by 53

Stool

Spindle

202 by 58

Urine and stool Stool

Round oval Round oval

66 by 58 67 by 54

Stool Stool

"Adapted from Refs. 2, 17, 24 and 25. See also Ref. 26.

Disease Due to Schistosoma mekongi, S. intercalatum and...

397

Diagnosis S. intercalatum eggs are found in the stool, and may be detected by KatoKatz smears or by iodine-formaldehyde or formalin-ether concentration techniques. The eggs are spindle-shaped with a terminal spine, and average 175-190 by 59 mm in size (17). The eggshell stains red with Ziehl-Neelsen stain (3). Table 2 summarizes the size and shape characteristics of eggs from the various schistosome species infecting humans. Figure 1 demonstrates the morphology of S. intercalatum eggs compared with the related species S. mattheei and S. bovis. Egg excretion fluctuates from day to day, and repeat daily stool samples should be tested to exclude infection (9). Serological testing has been performed with various antigens (10). Seroreactivity to standard S. mansoni and S. haematobium antigens may be low

Figure 1. Eggs of Schistosoma intercalatum (A), S. mattheei (B), S. bovis (C) and S. mekongi (D).

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Schistosomiasis

(4). Perhaps the most useful tests for detecting infection are the circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) described by Deelder and coworkers (9). CCA detection in blood has been found to be 100% sensitive, while CAA antigen levels, although less sensitive, were directly correlated with infection intensity. S. intercalatum eggs may also be detected on rectal biopsy or liver biopsy, as described earlier. Rectal biopsy is more sensitive than stool examination for detection of infection. However, recovery of dead or calcified eggs may not resolve the issue of whether the S. intercalatum infection is presently active (4). Ziehl-Neelsen staining of eggs is helpful in species identification of S. intercalatum eggs.

Therapy Effective treatment of S. intercalatum has been obtained with praziquantel 40 m g / k g given as a single dose (4, 11). Efficacy for cure is over 92%, with 90% reduction in mean egg output. Older drugs such as antimonials and niridazole have been used to treat S. intercalatum, but are significantly less effective than praziquantel (4).

Prevention Risk for S. intercalatum infection depends on contact with cercaria-infested waters. Reductions in parasite transmission in S. intercalatum-endemic areas may occur with sufficiently broad-based chemotherapy to reduce egg excretion (and thus transmission) on a population-wide basis. Focal snail control and provision of safe washing and bathing facilities also have potential to reduce transmission (11). On an individual basis, only avoidance of transmission-related water sources, i.e. freshwater rivers, streams, lakes and ponds, or use of barrier clothing (boots, gloves) will prevent infection.

Schistosoma

mattheei

The Pathogen S. mattheei is member of the S. haematobium group of schistosome species (1, 2). It is a common parasite of cattle, sheep and goats in Southern Africa.


It is frequently found as far north as Tanzania and Zambia, where it overlaps the distribution of S. bovis (2). Sporadic cases have been reported as far north as Chad and Nigeria (27). Wild primates and wild ruminants, including the water buck, sable antelope, wildebeest and cape buffalo, also harbor S. mattheei, providing an enzootic reservoir of transmission. The vector host is limited to Bulinus species snails of the B. africanus group (2). Human infection occurs in the same regional distribution as the vector snail. Infection results in both intestinal and urinary tract colonization, with eggs detected in both the urine and feces (28). Human prevalence may range up to 40%, and cattle prevalence u p to 90% in areas where the parasite is transmitted (29). Notably, studies indicate that coinfection with S. haematobium or S. mansoni is common or universal when S. mattheei infection is present in humans. This suggests that, within the human host, S. mattheei females are unable to produce eggs (or, alternatively, to successfully oviposit) except in the presence of males of other schistosome species (30-32). Both structural and biochemical studies indicate frequent hybridization between S. mattheei females and S. haematobium males during human infection. Experimental studies of combined S. haematobium and S. mattheei infections in mice indicate that S. haematobium males pair more effectively with either S. haematobium or S. mattheei females than do S. mattheei males (7). The S. haematobium x S. mattheei hybrid eggs are fertile and infectious for snails, and the resultant cercariae can produce patent experimental infections in rodents (33,34). These factors likely account for the continued prevalence of S. mattheei infection in human populations despite its poor transmission potential to the human host.

Epidemiology and Disease S. mattheei is most prevalent in the low veld region of the Transvaal in South Africa, where the prevalence in cattle may range u p to 90% (35). Human infection has been detected in eastern and western Transvaal and in Swaziland. Peak prevalence was 40% on one farm in eastern Transvaal (29). Because they share the same intermediate snail host, S. mattheei and S. haematobium share the same geographic distribution in South Africa. Coinfection is the rule in human S. mattheei cases, with S. haematobium having greater intensity of egg output in coinfected patients (32). S. haematobium x S. mattheei hybridization occurs, and egg morphology from these hybrids

400

Schistosomiasis

may vary from very similar to S. haematobium to very similar to S. mattheei (33). Because of the presence of coinfecting schistosome species, the specific consequences of S. mattheei infection for humans are unclear. Urinary tract, intestinal and gynecological infections have been reported (28, 29, 36). Infections are typically low intensity and short-lived (35), and it appears that the health impact of S. mattheei is limited. However, patients exposed to S. mattheei may fail develop infection but have persistently positive antischistosome serology (37) reducing or nullifying the positive predictive value of blood tests for detecting low intensity S. haematobium or S. mansoni infections.

Diagnosis Diagnosis of S. mattheei infection is made by detection of large, terminally spined, spindle-shaped schistosome eggs in the stool or urine (see Fig. 1) (28). The eggs are on average 173 by 53 mm in size, larger than those of S. haematobium (2). Eggs of the hybrid S. haematobium x S. mattheei are likely intermediate in size between those of S. haematobium and S. mattheei (29). Rectal or bladder biopsy is also diagnostic if egg morphology is preserved. There is no specific anti-S. mattheei serological test at present.

Therapy S. mattheei infections in humans have responded poorly to oxamniquine (38). This suggests that hybridization with S. haematobium in human infection is common, resulting in resistance to this drug. Praziquantel is the most effective anthelmintic treatment for all hybrid infections and mixed S. haematobium/ S. mattheei infections in humans (39).

Schistosoma

bovis

The Pathogen

S. bovis is a parasite of cattle, sheep and other ruminants and is found in the Mediterranean basin (except Egypt), the Middle East, and throughout


central Africa, including areas of Kenya, Tanzania, Sudan, Niger, Senegal, Angola, Zaire and northern Zambia (2). Across Africa, S. bovis is transmitted by a broad range of Bulinus species snails, while in Spain it is transmitted by Planorbarius snails. The prevalence among animals may be extremely high (up to 90% among Sudanese cattle (40)). Human infection, if it occurs, is rare. Epidemiology and Disease Controversy exists as to whether human infection actually occurs with S. bovis. Spurious stool excretion of S. bovis eggs can occur after patient ingestion of cow or sheep organs, such as liver or intestines, which contain parasite eggs. This phenomenon would account for sporadic case reports of human infection. Spurious excretion of S. bovis eggs has been demonstrated experimentally after liver ingestion in two clinical studies (41, 42). The prevalence of S. bovis eggs in human stool samples has ranged as high as 1-4% during surveys for S. mansoni in Sudan and Niger (41, 43). Passage of S. bovis eggs is typically intermittent, suggesting that it is variation in the dietary intake of egg-containing meats that is the cause of infrequent egg excretion, and not patent infection. However, one third of subjects have S. bovis eggs in the stool repeatedly over 3-4 months (43, 44), and this prolonged excretion has been credited as patent infection by some authors (18). Egg counts are typically less than 40-80 eggs per gram of feces, and egg excretion is generally not associated with symptoms or clinical signs. S. bovis infection of the urinary tract, with haematuria and polyposis, has been described in a case report from South Africa (45). In fact, this probably represented infection with S. mattheei (37). Serology studies performed in Sardinia, which was enzootic for S. bovis but not other schistosome species, found antischistosome antibodies in subjects exposed to S. bovis cercariae, but no patent infections (46). In summary, if S. bovis does cause human schistosomiasis, its attack rate is low and its clinical morbidity is limited.

Diagnosis Eggs of S. bovis can be detected on standard stool screening tests or urine filtration assays. The eggs are on average 202 by 58 mm in size, spindleshaped with a terminal spine, and somewhat larger than the eggs of

402

Schistosomiasis

S. mattheei (average 173 by 53 mm), which have a similar shape (Fig. 1) (2). S. bovis eggs are most often found in patients who also have either S. haematobium or S. mansoni infection. Tissue biopsy would therefore be unreliable in specifically establishing S. bovis infection, unless species-specific tissue labeling techniques become available. Patients exposed to S. bovis eggs or cercariae may have false-positive serology for other schistosome infections unless highly specific antigens are used for testing. As with other schistosome species, seropositivity to S. bovis indicates exposure to the parasite, but not necessarily patent infection.

Therapy Therapy with standard doses of anthelmintics, including praziquantel, has been associated with clearance of S. bovis egg excretion from the stool (44).

Other Zoonotic Schistosome Species in Africa That May Infect Humans Case reports have described human infection in Africa with the antelope parasite S. margrebowiei, which has an egg resembling that of S. japonicum (3). A single case has been reported of S. rodhaini, a rodent parasite that has a lateral spine like that of S. mansoni. The ruminant parasite S. curassoni has also been reported to cause human infection, although the data are limited (2).

Schistosoma

mekongi

The Pathogen S. mekongi is an S. japonicum-like human parasite found along the Mekong and Mun rivers in Southeast Asia. Cases have been reported from Laos, Cambodia and Thailand. Humans and dogs appear to be the only natural definitive hosts. S. mekongi is transmitted by Tricula species snails, in contrast to S. japonicum, which is transmitted by Oncomelania snails. S. mekongi is smaller in size and has a longer prepatent period (35 days as compared to


26 days for S. japonicum) in the mammalian host (47). Studies of alloenzyme variation as well as subsequent RNA and DNA studies have confirmed the identity of S. mekongi as a distinct species (48).

Epidemiology Schistosoma mekongi is transmitted only in Indochina, in the distribution of its intermediate host, Tricula aperta, along the Mekong River and its tributaries. This includes the Mekong above Kratie, Cambodia to Luang Prabang, Laos, and up the Mun River into Thailand. Humans are the primary mammalian host, and the prevalence may be as high as 25% in some endemic regions (49). Transmission occurs predominantly during the dry season, as waters recede, snails mature, and the vector and host populations are concentrated together. Population studies of Cambodian refugees relocated in camps in Thailand indicated a 9.3% skin reactivity to schistosome antigen, and a 3.8% prevalence of S. mekongi eggs in stool samples (50). Infected subjects were predominantly male (11 : 6 sex ratio) and ranged in age from 13 to 50 years, with the peak prevalence between 31 and 40 years.

Pathology S. mekongi inhabit mesenteric vessels and release eggs into the portal circulation. While many eggs are deposited in intestinal tissues, others are swept to the liver with the flow of portal blood. Eggs induce a delayedtype granulomatous reaction, which progresses from inflammation to localized fibrosis. The net result of chronic infection is liver enlargement, portal hypertension, splenomegaly and esophageal varices (51, 52). Assessment of disease-related morbidity is complicated by frequent coinfection with other parasites, particularly the liver fluke Opisthorchis viverrini, and with malaria, which also causes significant hepatosplenomegaly in S. mekongi-endemic areas (53).

Disease Patients from endemic areas have been profiled in a number of case series. Physical examination is typically normal, but hepatomegaly may be present

404

Schistosomiasis

in up to 55% of patients, being more common in children. Splenomegaly was present in 9-13% (49, 54). Eosinophilia (> 500 per (iL) was present in two-thirds of patients. As with other forms of intestinal schistosomiasis, colonic polyps may develop, and these typically contain multiple parasite eggs (49). Liver enzyme abnormalities, hematemesis and esophageal varices were not seen among the 12 Laotian patients described by Hofstetter et al. (49), although anemia and hypergammaglobulinemia were frequent. Portal hypertension and hepatic fibrosis do occur with S. mekongi (52, 55), although the rate of progression to severe disease is unknown.

Diagnosis Clinical diagnosis requires an exposure history of travel or residence in the endemic area of Southeast Asia (Laos, Cambodia or Thailand along the Mekong River and its tributaries). In North America and Europe, S. mekongi infections are most common among Cambodian and Laotian refugees (49, 52,55). Asymptomatic cases are often detected among relatives of a primary, symptomatic case. It is possible that S. mekongi is transmitted within Southest Asian refugee camps (50). Because of this, a patient risk of infection depends both on his or her village of origin and on the duration of his or her residence in refugee camps. Laboratory diagnosis of S. mekongi infection is readily made by detection of eggs on stool examination. Although similar in appearance to eggs of S. japonicum, S. mekongi eggs are smaller and more round (see Fig. 1). A quantitative test such as the Kato-Katz smear will provide an estimate of infection intensity. In very light infection, concentration techniques such as formalin-ether sedimentation may be needed to detect infection (49). Diagnosis may also be made by rectal biopsy with identification of characteristic schistosome eggs. However, in order to establish the presence of active infection, the viability of eggs recovered on biopsy should be determined. Liver biopsy may reveal periportal fibrosis with relative preservation of the parenchymal cord pattern (52). If present, schistosome eggs in the liver are likely to be surrounded by granulomatous infiltration. Patients with S. mekongi infection have significant titers to S. japonicum egg antigens, although their serum response to S. japonicum adult worm antigen may be limited (49). Immediate hypersensitivity to schistosome antigen is present in over 90% of patients. Detailed study of antibody and


lymphocyte responses of S. mekongi patients to parasite antigens has indicated that there is cross-reactive immunity to other schistosome species (S. mansoni, S. japonicum). The strongest IgG, IgE, and lymphocyte proliferation responses were to S. mekongi antigens, however (56). These results suggest the possibility of developing species-specific clinical tests to identify S. mekongi exposure.

Therapy Praziquantel has proven safe and effective in treating patients with S. mekongi (54, 57). The effective dose, as for S. japonicum, is 60 m g / k g body weight given in two or three divided doses spaced over one day (39). Side effects occurred in 45% of patients, including chills, fever, abdominal pain, malaise, headaches, diarrhea, anorexia, dizziness, hives, nausea and vomiting, in decreasing order of frequency. These resolved within 24-48 hours. Treatment was followed by clearance of parasite eggs from the stool in 91-100% of patients, and a decrease in anti-S. mekongi IgG in subsequent serological assays over 6—12 months (57).

Schistosoma

malayensis

S. malayensis is an S. japonicum-\ike parasite of humans and rats that is found only in limited areas of peninsular Malaysia (58,59). It is distinguished from S. japonicum by the smaller size of its eggs, and from both S. japonicum and S. mekongi by its use of Robertsiella spp. snails as an intermediate host (25). These Tricula-like snails are aquatic, and have a narrow habitat range in the streams of foothills and mountains of Malaysia. Isoenzyme analysis has further established the distinct species identity of S. malayensis, although it appears to be closely related to S. mekongi (48). Zoonotic S. malayensis infection is found among rats in this region. Human infection appears to be of low intensity and exceptionally rare, either because transmission is nocturnal or because the parasite is not well adapted to the human host (25). The extent of human exposure to S. malayensis has been demonstrated by serology (60), indicating 4-27% seroprevalence among aboriginal Orang Asli villagers in the central part of the Malay peninsula. However, patent infection (with passage of eggs in the stool) has not been observed — the only clinical cases have been established by autopsy or liver

406

Schistosomiasis

biopsy (58, 59). Infection involves the liver, pancreas and mesentery near the ileocecal junction. Mild to moderate liver fibrosis has been reported in association with infection, but no specific symptoms have been linked to S. malayensis. Due to the rarity of S. malayensis infection, the extent of infection-associated disease is not known. If antemortem diagnosis is established in a symptomatic patient, then praziquantel therapy, as for S. japonicum (39), should prove efficacious in eliminating infection and reducing morbidity.

Other Rare Forms of Schistosomiasis in Asia Sporadic cases of S. japonicum-like infection have been reported in patients from Java and northern and southern Thailand (2). The latter focus appears to have died out by the mid-1980s. In addition, a single case of infection with a schistosome having lateral-spined eggs like S. mansoni has been reported from northern Thailand. Urinary schistosomiasis has been reported in one village in Ratnagiri, India. The eggs appear similar to those of S. haematobium, but the intermediate host has yet to be identified (2).

Cercarial Dermatitis — "Swimmer's Itch" Schistosome dermatitis or cercarial dermatitis (dermatitis schistosomica) is caused by localized skin inflammation provoked by intraepidermal penetration of invading schistosome cercariae (61, 62). The condition can be caused by human schistosome parasites such as S. haematobium and S. mansoni when a previously exposed/infected individual comes into contact with cercariae-infested water (62). Cercarial dermatitis more typically occurs when human skin is penetrated by cercariae of bird, rodent or cattle schistosomes, such as Trichobilharzia, Gigantobilharzia, Schistosomatium and Schistosoma spindale, for which humans are dead-end hosts (63).

Epidemiology In the English literature, cercarial dermatitis has been variously referred to as "swimmer's itch," "bather's itch" and "clam digger's itch." However,


"sawah itch" has been reported in Malaysian rice farmers who come in contact with the cattle schistosome, S. spindale. In Japan, "kabure" dermatitis has been associated with exposure to rice paddies. A more severe form, "koganbyo" (lakeside disease), has been associated with exposure to cercariae of Gigantobilharzia parasites of starlings and sparrows (64). Cercarial dermatitis

also occurs along the Murray River in New South Wales, Australia, and in various lakes in New Zealand. Scattered reports have come after exposure to lakes and streams in Wales, France, Germany and Switzerland. Reports of dermatitis from bird schistosomes have also come from Haiti, Cuba, El Salvador and Mexico. More frequent cases have been reported from Canada, in the Province of Manitoba, along the Great Lakes, and along the St. Lawrence River Basin (63). Attack rates range up to 50% among bathers at some beaches and lakes. The parasites that are usually involved are Trichobilharzia ocellata or T. stagnicolae, which are schistosome parasites of ducks and other water birds, although finches, cardinals and blackbirds may also serve as their definitive hosts. In the United States, disease has been reported from the Washington State and Oregon, in the Rocky Mountains, the Great Plains (along the Mississippi flyway migration route for wildfowl), in the Great Lakes Region and along the East Coast. Cercariae of many different bird schistosome species are responsible, and their individual vector snails may be found in either fresh or salt water (63, 65). Transmission is seasonal, depending on the life cycle of the growth and maturity of the vector snail, and the time to maturation of cercarial forms within the snail (66). The time of day may also be critical in determining exposure, as cercariae of some species emerge at mid-day, while some emerge only in the evening. In the most common forms of cercarial dermatitis in North America due to Trichobilharzia, cercariae reach maturity in late June through early July, and continue to be shed by vector snails through their demise in late summer or early fall. Warm weather speeds the cercarial development and bright light increases the numbers of cercariae released. Shallow water is more likely to harbor cercariae than deep water, so that nonswimmers and children are more likely to be exposed (63). In Asia, infections are most common in hotter months when rice paddies are flooded and farmers are engaged in weeding the rice crop. Once the paddies are drained for harvest, infection significantly declines (64). In Central America and Caribbean countries, cercarial dermatitis may be seasonal,

408

Schistosomiasis

according to the resident bird population. Typically, rashes occur a number of weeks after the yearly arrival of migrating ducks from North America.

Disease Cercarial dermatitis may be quite severe. It begins, following exposure, as a prickling or itching sensation reminiscent of insect bites. Over the next hour, a 1-2 mm macule appears at the site of cercarial penetration. This may disappear, or progress over the next 10-15 hours to form a 3-5 mm red papule, which is intensely pruritic. Urticaria may also be noted during the first few hours after exposure (61-63). Rubbing or scratching worsens the erythema and edema associated with the lesions. After two or three days, vesicles may form. These will crust if broken by scratching. The rash resolves over 1-2 weeks, leaving small pigmented spots at the site of penetration. Secondary bacterial infection may prolong disease if infection is introduced by scratching (63). The differential diagnosis includes chigger and mosquito bites, algae dermatitis, or sting from coelenterates (61). Severe cases may mimic varicella, impetigo or scabies (62).

Pathology Reactions are rare on first exposure to cercarial infested waters, and become more p r o n o u n c e d after repeat exposure. This suggests that sensitization plays a significant role in producing the inflammatory reaction. Biopsy studies of experimental infections show minimal reaction on first exposure. The cercaria penetrates the epidermis but causes little damage with mild edema and minimal cellular response. The dead cercaria is eventually sloughed with dead skin cells. On second or subsequent exposure, there is more edema, with lymphocyte, neutrophil, eosinophil and macrophage cellular response. Skin sloughing may be extensive if the exposure is heavy or the extent of the reaction is severe. The targets of immune response are presumed to be proteases excreted by the penetration glands of the cercaria, and later the cell-associated proteins and polysaccharides released as dead cercariae break down (63). Sensitization may last for over 12 years. Cross-sensitization may occur to other schistosome species, and North Americans and Europeans who have only been exposed


to bird schistosomes have had false-positive reactions to S. mansoni or S. japonicum on serological or skin testing. However, it should be noted that not all individuals become sensitized or develop dermatitis after cercarial exposure.

Treatment Therapy of cercarial dermatitis is symptomatic. Relief may be obtained with antihistamines and antipruritic lotion (61). Antibiotic therapy should be added if bacterial superinfection occurs (62).

Prevention Cercarial dermatitis may be prevented by avoiding bathing or bare skin immersion in lakes, streams or marshes known to harbor schistosome species. Toweling off after exposure appears to significantly reduce the risk of infection, as does rinsing the skin with alcohol (63). Removal of vegetation near bathing sites may reduce the local vector snail population. MoUusciciding with agents such as copper sulfate has also been used (63), although this may cause damage to other aquatic plants and animals. Recently, praziquantel has been used to treat local wildfowl to eradicate their infection with Trichobilharzia. This can reduce infection levels of local snails and cercarial release by at least 90% (67).

References 1. Barker SC and Blair D (1996). /. Parasitol. 82: 292. 2. Rollinson D and Southgate VR (1987). In The Biology of Schistosomes. From Genes to Latrines, eds. Rollinson D and Simpson AJG (Academic, New York), pp. 1-50. 3. Sturrock RF (1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford UK), pp. 1-32. 4. Chen MG and Mott KE (1989). Trop. Dis. Bull. 86: Rl. 5. Southgate VR (1978). Zeitschrift fur Parasitenkunde 56: 183. 6. Ratard RC and Greer GJ (1991). Am. }. Trop. Med. Hyg. 45: 332.

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7. Southgate VR, Tchuem Tchuente LA, Vercruysse J and Jourdane J (1995). Parasitol. Res. 81: 651. 8. Tchuem Tchuente LA, et al. (1997). Irons. R. Soc. Trap. Med. Hyg. 91: 664. 9. Kremsner PG, et al. (1993). Trans. R. Soc. Trop. Med. Hyg. 87: 167. 10. Martin-Prevel Y, et al. (1992). Trans. R. Soc. Trop. Med. Hyg. 86: 401. 11. Almeda J, et al. (1994). Trans. R. Soc. Trop. Med. Hyg. 88: 406. 12. Tchuem Tchuente LA, et al. (1997). Trans. R. Soc. Trop. Med. Hyg. 91: 263. 13. Visser LG, Polderman AM and Stuiver PC (1995). Clin. Infect. Dis. 20: 280. 14. Odongo-Aginya EI, et al. (1994). Am. }. Trop. Med. Hyg. 50: 723. 15. Wright CA, Southgate VR, Van Wijk HB and Moore PJ (1974). Trans. R. Soc. Trop. Med. Hyg. 68: 413. 16. Tchuem Tchuente LA, et al. (1996). Parasitology 113: 129. 17. Southgate VR, et al. (1994). Trans. R. Soc. Trop. Med. Hyg. 88: 479. 18. Jordan P and Webbe G 1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford UK), pp. 8 7 158. 19. Van Wijk HB and Elias EA (1975). Trop. Geog. Med. 27: 237. 20. Farid Z (1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford UK), pp. 159-194. 21. Corachan M, et al. (1994). Am. J. Trop. Med. Hyg. 50: 580. 22. Gendrel D, et al. (1984). Am. ]. Trop. Med. Hyg. 33: 1166. 23. Gendrel D, Kombila M, Beaudoin-Leblevec G and Richard-Lenoble D (1994). Clin. Infect. Dis. 18: 103. 24. Pitchford RJ (1959). Trans. R. Soc. Trop. Med. Hyg. 53: 213. 25. Greer GJ, Ow-Yang CK and Yong H-S (1988). /. Parasitol. 74: 471. 26. Ash LR and Orihel TC 1990). Atlas of Human Parasitology, third edition (ASCP, Chicago). 27. Pitchford RJ (1977). /. Helminthol. 51: 229. 28. Blair DM (1966). Cent. Afr. ]. Med. 12: 103. 29. Pitchford RJ (1959). Trans. R. Soc. Trop. Med. Hyg. 53: 285. 30. Kruger FJ and Hamilton-Attwell VL (1988). /. Helminthol. 62: 141. 31. Kruger FJ and Evans AC (1990). /. Helminthol. 64: 330. 32. Kruger FJ (1990). /. Helminthol. 64: 333. 33. Wright CA and Ross GC (1980). Trans. R. Soc. Trop. Med. Hyg. 74: 326.


34. Southgate VR and Rollinson D (1987). In The Biology of Schistosomes from Genes to Latrines, eds. Rollinson D and Simpson AJG (Academic, New York), pp. 347-378. 35. Van Wyk JA (1983). S. Afr. Med. J. 63: 201. 36. Berry A (1974). Trans. R. Soc. Trap. Med. Hyg. 68: 263. 37. Schutte CHJ, et al. (1983). S. Afr. Med. }. 64: 239. 38. Pitchford RJ and Lewis M (1978). S. Afr. Med. J. 53: 677. 39. King CH and Mahmoud AA (1989). Ann. Intern. Med. 110: 290. 40. Majid AA, et al. (1980). Am. J. Trap. Med. Hyg. 29: 435. 41. Teesdale CH (1976). Trans. R. Soc. Trop. Med. Hyg. 70: 165. 42. Kinoti GK and Mumo JM (1988). Trans. R. Soc. Trop. Med. Hyg. 82: 589. 43. Mouchet F, Develoux M and Magasa MB (1988). Trans. R. Soc. Trop. Med. Hyg. 82: 257. 44. Chunge R, et al. (1986). Trans. R. Soc. Trop. Med. Hyg. 80: 849. 45. Kisner CD, Stoffberg N and De Meillon B (1953). S. Afr. Med. }. 27: 357. 46. Sadun EH and Biocca E (1962). Bull. WHO 27: 810. 47. Voge M, Bruckner D and Bruce JI (1978). /. Parasitol. 64: 577. 48. Woodruff DS, Merelender AM, Upatham ES and Viyanant V (1987). Am. J. Trop. Med. Hyg. 36: 345. 49. Hofstetter M, et al. (1981). /. Infect. Dis. 144: 420. 50. Keittivuti B, D'Agnes T, Keittivuti A and Viravaidya M (1982). Am. }. Trop. Med. Hyg. 31: 988. 51. Sornmani S, Vivatanasesth P and Thirachantra S (1976). SE Asian }. Trop. Med. Pub. Health 7: 270. 52. Wittes R, MacLean JD, Law C and Lough JO (1984). Am. ]. Trop. Med. Hyg. 33: 1159. 53. Gang CM 1993). In Human Schistosomiasis, eds. Jordan P, Webbe G and Sturrock RF (CAB International, Wallingford, UK), pp. 237-270. 54. Keittivuti B, Keittivuti A, O'Rourke T and D'Agnes T (1984). Trans. R. Soc. Trop. Med. Hyg. 78: 477. 55. Bruet A, et al. (1983). Am. } . Gastroenterol. 78: 346. 56. Barral-Netto M, Hofstetter M, Cheever AW and Ottesen EA (1983). Am. }. Trop. Med. Hyg. 32: 106. 57. Nash TE, Hofstetter M, Cheever AW and Ottesen EA (1982). Am. ]. Trop. Med. Hyg. 31: 977. 58. Murugasu R and Dissanaike AS (1973). Trans. R. Soc. Trop. Med. Hyg. 67: 880.

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59. Murugasu R, Wang F and Dissanaike AS (1978). Trans. R. Soc. Trop. Med. Hyg. 72: 389. 60. Greer GJ and Anuar H (1984). SE Asian }. Trop. Med. Pub. Health 15: 303. 61. Mulvihill CA and Burnett JW (1990). Cutis 46: 211. 62. Gonzalez E (1989). Dermatol. Clin. 7: 291. 63. Cort WW (1950). Am. }. Hyg. 52: 251. 64. Hunter GW, Ritchie LS and Tanabe H (1951). Trans. R. Soc. Trop. Med. Hyg. 45: 103. 65. Stunkard HW and Hinchliffe, MC (1952). /. Parasitol. 38: 248. 66. Lindblade KA (1988). /. Parasitol. 84: 19. 67. Reimink RL, DeGoede JA and Blankespoor HD (1995). /. Parasitol. 81: 1027.

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Chapter 12 Strategies for Control of Infection and Disease: Current Practice and Future Potential Robert Bergquist

Introduction Control deals with transmission of infection and a spectrum of disease manifestations spanning from cases with variably pronounced symptoms to the relatively large proportion of people with low-grade infection without clinical signs. The differentiation between infection and disease is an important one, considering that schistosomiasis is chronic with symptoms which often persist after the infection has been cured (posttransmission schistosomiasis). Interruption of the parasite life cycle through activities directed against the snail intermediate host has historically been the corner-stone of control programs and, although the last few decades of the 20th century have seen a shift in favor of chemotherapy, the former is still important and continues to be used in conjunction with drug treatment. Operations cover communities or populations and can, in the case of national control programs, be extended to the country level. Activities go on at many different levels and, naturally, the more tactics used together, the better the prospects of success. Lasting results, however, can only be achieved by consistent access to safe water, sanitation and health education; in short, through social and economic development leading to improved infrastructures. Epidemiology and transmission dynamics vary considerably from area to area, making it necessary to adapt a flexible stand, taking the particulars of the local situation into account when plans of action are drawn up. In addition, methods requiring a high degree of community participation are more difficult to introduce and sustain than other approaches, in part

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because they demand a level of social and economic standards which most endemic communities do not yet possess. Obviously, sanitation and health education can only make so much impact as the basic infrastructure permits. In the absence of safe water and adequate sewage systems, the former is not applicable and the objectives of the latter cannot be achieved, a situation which can only be slowly rectified by the process of basic economic development. Although this may seem a pessimistic view, it should also be said that enormous progress has been made and, even if eradication is still a distant goal in most parts of the world, morbidity has been reduced in large areas. Successful implementation depends upon the chosen strategy, the quality of basic health services, their distribution, logistic support and management. Integration and sectoral cooperation from the government level and down makes a difference, since social and economic progress is intrinsically bound to the general health of the population. Although longterm, integrated interventions are costly and require discipline, progress can be expected even if complete interruption of transmission is seldom achieved. There are, however, certain sine quo non elements without which set goals are difficult to achieve and results impossible to maintain: • reliable background data regarding the prevalence and burden of infection and disease in the area • a will on the part of the government/community and a demand on the part of those living there • a sustainable, coherent strategy acceptable to the people and the different layers of government • some level of infrastructure and long-term access to the resources and manpower needed • built-in resources for evaluation, monitoring and feedback A sound strategy cannot be developed without good knowledge of epidemiology and a clear understanding of how morbidity develops in the patient, including its age-specific distribution, while policy-makers will not be convinced without an assessment of the extent of the disease burden and its impact on populations and the economy in general. It is important to develop both a political will and a demand to do something about schistosomiasis and this requires the popularization of information so the implications are understood by a wider audience. The community should understand the natural history of schistosomiasis and the need for action early on to prevent

Strategies for Control of Infection and Disease: Current Practice and...

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damage in the future, the main message being that the disease works its damage during childhood well before symptoms appear. Although a vertical orientation, focussing on the specifics of the infection, might be needed in the attack phase in heavily infected areas, means must be found to ensure continuation through a horizontal approach using, for example, the primary health care system. Programs must take into account some of the factors that made vertical campaigns successful: a real sense of mission with feasible objectives, a clear strategy and continuous monitoring. However, without a public health infrastructure endowed with a management system able to handle resources and personnel, progress is not assured. Past control projects have all too often been short on basic training needs, vehicles and supplies, and it is clear from the experience in, for example, Sudan, Egypt and the Philippines, that the key to success lies in sustained distribution of safe and effective drugs. In addition, unless those who are actually carrying out control work in the field get to know the success or failure of their actions, goals are unlikely to be met. It is equally important that policy-makers have access to a summation of results on a regular basis to ensure continuing support for the control effort.

Retrospect Although schistosomiasis must have accompanied hominids and humans for millions of years, we can only trace the disease for the last 10,000 years with any certainty (1). Papyrus scrolls and carvings in stone bear witness that the ancient Egyptians acknowledged the distinct signs of the disease but there is no evidence that they elaborated any further on the subject. This is of course hardly surprising since, prior to the modern concept of microbial connection, pests were regarded as being no different from the weather or any other general aspects of the environment and, consequently, in the hands of the gods. The problem could not be approached in a logical, consistent way until the 19th century, when the currently reigning paradigm of the cause of infectious diseases took hold and schistosomes were linked to the specific clinical pictures we now know as urinary (caused by Schistosoma haematobium) and intestinal schistosomiasis (caused by S. mansoni, S. intercalatum, S. japonicum or S. mekongi). It should further be noted that the distribution and intensity of the disease have increased with the construction

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of dams, man-made lakes and large-scale irrigation systems, which, ironically, also form the economic base for the logarithmic increase of human populations living in these areas. In spite of the advent of new effective tools for the control of schistosomiasis during the last century, the prevailing situation has deteriorated when seen from a global, long-term perspective. Historically, fewer people were infected with generally lower worm burdens, while today the disease is a very real hazard for more than 600 million people, about a third of whom are chronically infected and 20 million are seriously ill for most of their lives. Schistosomiasis is, in fact, the second most prevalent serious parasitic infection of humans, after malaria, and its impact on public health and the economy should not be underestimated. Already in its first year of existence the World Health Organization (WHO) convened a meeting entitled "Bilharziasis in Africa" (2) and it has since held numerous meetings on various aspects of the disease, the most important of which are reported on in the WHO Technical Reports Series (2-17). National health policies are frequently based on information contained in these publications regarding morbidity and health with reference to important diseases. Despite access to a variety of effective, uncomplicated means bearing on the problems caused by schistosomiasis, the translation of theory into reality has proved less than straightforward and attempts to reduce the problem to a manageable level were not, with very few exceptions, particularly successful initially. The Expert Committee on Schistosomiasis convened by WHO in 1984 realized that a radical change of approach was needed and a new strategy was launched (15). Defeat turned into victory when a revised program of morbidity control through chemotherapy replaced attempts at eradication or reduction of prevalence through interruption of transmission. The introduction and immediate success of praziquantel in the 1970s (18, 19) played a crucial part in this redirection to a more realistic, achievable target, and the understanding that morbidity could be controlled in the face of ongoing transmission also contributed. Encouraging results were not long in coming: within less than a decade, serious implications of urinary tract infection and signs of hepatosplenic involvement due to intestinal schistosomiasis were on the decrease in most endemic areas, and certainly in those where resources and infrastructure permitted access to chemotherapy for a majority of people living there. The most recent meeting of the WHO Expert Committee on the Control of Schistosomiasis took place in 1991 (16) and the next one is planned for 2001. Meanwhile, although continued progress in some areas


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of the world was reported at the WHO consultation on schistosomiasis control held in 1998, it was clear that lack of financial resources too often prevents implementation, particularly in sub-Saharan Africa, which harbors the majority of the world's burden of schistosomiasis.

Assessing the Situation Even with access to appropriate and effective tools, efforts to control an endemic infectious disease are doomed to failure if not relying on a careful analysis of all the parameters that play a role in maintaining infection in the community. Although a decision to conduct mass chemotherapy may be defended as an immediate response to widespread, high-intensity schistosomiasis, it would be more useful to spend a sizeable proportion of available funds to first assess the situation at hand. There will never be sufficient funds to do everything and it is crucial for ultimate success that decisions are based on a reliable assessment of the key variables. In judging the extent of the problem, the prevalence and intensity of infection in the human host, the distribution and rate of infection in the snail intermediate host and the infrastructure in the community, all play an important role. A distribution of available financial means between baseline and follow-up studies on the one hand, and the application of control tools on the other, is therefore well justified. While relatively good qualitative markers of infection exist, there is no direct way of revealing the actual numbers of worms in affected individuals and, apart from clinical examination and its extension in the form of ultrasonography, the pathological sequelae cannot be unequivocally assessed by laboratory methods. The intensity of infection largely determines the severity of morbidity but one should preferably assess the latter independently by identifying and evaluating indicators which measure morbidity more directly. A proper analysis of the status of populations living in endemic areas requires different markers, one set for infection and quite another for morbidity.

Markers of Infection Reliable estimates of the prevalence and intensity of disease can be obtained by the application of individual diagnosis and statistical evaluation.

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Parasitological examination, using the cellophane fecal thick-smear tech-nique of Kato and Miura, modified by Katz et ah, (20) continues to be recommended with respect to S. mansoni, S. intercalatum and S. japonicum, while S. haematobium is diagnosed by urine filtration. Confidence is improved by increasing the number of samples processed but this practice is labor-intensive and tends to unduly restrict large-scale field studies. From the practical point of view, the examination of duplicate preparations from three separate samples is probably the limit. Compounding the sensitivity issue and adding to the uncertainty, pronounced variations with regard to egg counts are increasingly reported, both within and between samples (21, 22). Although depending to some extent on the species of the schistosome, this principle holds for all forms of schistosomiasis. The perceived accuracy of parasitological diagnosis is further undermined by the fact that egg counts provide only an indirect estimate of the worm burden which is influenced by the degree and consistency with which eggs are excreted by the host and also by the fecundity of the female worm contributing to a less than direct relationship between egg counts and worm burdens. Variation in egg counts becomes particularly disturbing in the case of low-intensity infection as swings in egg excretion can reach levels below the sensitivity of the diagnostic technique applied. In such instances, the problem would not be confined to simply providing a false impression of low intensity of infection but could produce a falsely negative result, something which becomes a very real possibility in areas of low endemicity where a majority of cases would escape detection with all currently available methods. These facts are not purely of academic interest, since there are regions, e.g. in North Africa and the Middle East, where transmission and prevalence have reached levels which cannot be satisfactorily monitored without access to more sensitive techniques. Egg counts are generally regarded as a more direct measure of the intensity of infection than can be provided by serology. While this is obviously true for antibody detection, circulating antigens are no less direct in detecting infection than egg counts. Nonspecific results and cross-reactions with epitopes, common with other pathogens, contribute to the uncertainty of antibody detection but the overriding problem with this approach is that it does not recognize cure as antibody titers decrease only slowly. Apart from the role played in the diagnosis of affections arising from tourism and military interventions (originating in nonendemic countries), antibody detection is only useful with regard to population movements between nonendemic and


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endemic areas and as a surveillance tool in areas with negligible or discontinued transmission. Antigen detection, on the other hand, provides an estimate of worm burdens and can confidently be used for the assessment of cure. In addition, this technique produces a more stable return of results, minimizing the risk of missing individuals excreting few eggs due to day-to-day variation of egg excretion (23). The use of monoclonal antibodies specific for the circulating anodic (CAA) and cathodic (CCA) antigens has proved beyond doubt that both these antigens reach stable levels in the blood of the host when adult worms have developed (23, 24). They can also be detected in the urine, in particular CCA (25). Production of the reagent monoclonal antibodies has now been scaled u p and tested in various parts of the world — with excellent results (23-25). However, although there is less variation from sample to sample, it must be admitted that the general sensitivity of antigen detection is no better than repeated egg counts. In contrast to parasitological diagnosis, where samples are manually prepared and examined in the microscope one by one, serological techniques lend themselves to standardization. Once the methodology is properly set u p and subjected to quality control, all samples can be tested with the same degree of accuracy and these assays have now reached the level of sensitivity, specificity and reliability where it is warranted to contemplate incorporating them into the diagnostic arsenal. The disadvantages of parasitological techniques become accentuated in large-scale applications and followup studies aimed at the identification of communities in need of treatment. The challenge to develop much-needed practical diagnostic methods has led to the recognition of the possibility of rapidly screening populations in areas endemic for urinary schistosomiasis for microscopic hematuria using reagent strips (26, 27). The blood emanating from the bladder wall damage caused by the passage of schistosome eggs is an indirect demonstration of infection, but since the associated pathology is not progressive (as it is when eggs are trapped in the bladder wall) they provide an estimate of the intensity of infection rather than of morbidity. These reagent strips can be used by unskilled personnel and, although the technique has limitations in terms of sensitivity and specificity, they are useful for mass screening in the field. The simultaneous detection of hematuria, proteinuria and leucocyturia (28) might be better but, on the other hand, this approach is more expensive. Dipstick-based systems for blood detection in the stools are, however, less straightforward and

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interpretations are not clear-cut. When almost 5000 schoolchildren, aged 7 17 years, were screened for Ascaris lumbricoides, Trichuris trichiura, hookworm, S. mansoni and S. haematobium, taking bloody stools, spleen and liver enlargement into account, no signs or symptoms were significantly associated with any geohelminth infection, not even hookworm (29). These results indicate that further work lies ahead before we can make a decision regarding the potential for using the presence of blood in the stools as the base for rapid assessment and identification of communities with a high prevalence of S. mansoni infection. The introduction of self-diagnosis using simple questionnaires constitutes one more step towards simplicity. This approach has been investigated in collaboration with national scientists and local schools and the results clearly show that it is feasible, less labor-intensive and less demanding for personnel and communities in screening for infection due to S. haematobium (27, 29). The findings have been validated in seven African countries and the approach has since been recommended for the screening of urinary schistosomiasis by district health management teams (26). The questionnaire approach seems to be better suited for screening for intestinal schistosomiasis diagnosis than dipstick-based systems for blood detection, at least for now. However, the questionnaires must be more elaborate than those used for urinary infection, including a list of signs and symptoms.

Markers of Morbidity Even if the intensity of infection is the key determinant of morbidity, it cannot be used for quantitative pathology estimates since the lesions produced are also proportional to the length of time that this level of intensity has been manifest. While the clinician easily spots the gross pathology, minor changes are only revealed by imaging techniques such as X-ray, ultrasound, etc. These are powerful tools but need careful standardization of protocols

and records so that results from different settings, and different investigators, can be compared. Although attempts in this direction have been successful, particularly with reference to ultrasonography of urinary schistosomiasis (30), it should be realized that the methodological standardization of imaging techniques has not yet reached the level where morbidity can be precisely graded.


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Portable ultrasound machines have improved morbidity assessment by permitting advanced investigations in the field. Although the value of ultrasonography varies according to the species of schistosome involved and, like other imaging techniques, presents only a snapshot of the dynamic process, it is sensitive and specific, useful in all endemic areas, and acceptable to the community and patients alike. Recorded changes in the bladder wall, ureters and kidneys, in schoolchildren with S. haematobium infection, correlate well with both intensity of infection and markers of morbidity and ultrasound is routinely utilized for the evaluation of regression of morbidity following cure (31). However, although lesions of all grades of severity in both bladder and kidneys decline after cure, they do so more slowly (up to six months) than the excretion of eggs which disappears within two months. Reinfection leads to the reverse development, resulting in the reappearance of eggs within months of cure, while morbidity takes a year or two to return (31). Interpretation of ultrasonography is less straightforward in patients with S. mansoni infection and the identification and grading of early or minor specific lesions have not reached the level of sophistication of that described for S. haematobium. The relative lack of early ultrasonographic changes highlights the need for other more sensitive morbidity markers in S. mansoni infections. The technique has, however, proved useful in identifying periportal fibrosis before esophageal varices have become established. A statistically highly significant correlation has been shown between ultrasound scores and the occurrence of variceal hemorrhage (32). The question of reversibility has been addressed by clinical studies in children with severe hepatosplenomegaly, and a slow decline in the prevalence and severity of hepatosplenomegaly after repeated annual treatment with praziquantel has been observed (33). Two types of lesion stand out in S. japonicum infections, the first being a typical periportal fibrosis associated with reduced portal venous blood flow velocity, and the second being a fibrous network, anatomically separate from the portal branches and not associated with changes in portal venous blood flow but instead with cholestasis and enzyme changes indicative of liver damage (30, 34). Both types of lesion have been observed in adults coming from areas where annual treatment of infected individuals is the norm suggesting that S. japonicum infection causes a more "resistant" form of fibrosis than that of other species. In the absence of indicators that can be easily read and standardized, physicians grade morbidity by formulating a consolidated view of the results

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provided by egg counts, bedside findings and imaging techniques. However, only standard measurements will permit proper comparisons of expressions of the disease in different geographical areas. The development of a noninvasive and sensitive assay, relying on the eosinophilic cationic protein (ECP) excreted in the urine in individuals with urinary schistosomiasis, is a step in this direction. The test is specific for eosinophil responses against eggs in the bladder and has produced some promising results; for example, it was shown that ECP in the urine remains stable after storage and that it shows little circadian or diurnal variation (35,36). A study of schoolchildren in Kenya and Tanzania indicated that urinary ECP levels correlate with both the presence and the severity of bladder lesions before treatment and declines more slowly than egg counts after cure (36, 37). A study of Kenyan schoolchildren in an area endemic for S. haematobium has shown that excretion of soluble egg antigens (SEA) in the urine correlates well both with egg counts and morbidity before treatment, and that egg counts fall rapidly after cure, while urinary SEA levels abate more slowly, in parallel with reduction of lesions of the bladder and kidneys (38). The results discussed here underline the potential usefulness of quantitative determinations of ECP and SEA but there is yet no similar approach that can be used for intestinal schistosomiasis.

The Snail Intermediate Host The rate of transmission of schistosomiasis to humans is directly proportional to the distribution of infected, compatible snails in the area in question but, since the principal epidemiological character of transmission is focal and changing, extrapolation of data from one area to another is seldom valid even for closely adjacent areas. A reliable picture, permitting the application of appropriate measures at relevant times, therefore requires continuous surveying and identification of snails in every area surrounding the communities selected for intervention. The rates of infection are determined by sampling and examining a sufficiently large number of individual snails and calculating the overall distribution of infection. Traditionally, snails are tested by microscopic examination of crushed specimens or by initiating and observing cercarial shedding, but these methods cannot detect prepatent infection and they are unable to determine the species of schistosome. On the other hand, this information can be extracted by immunological detection of schistosomal antigens in the snail hemolymph (39) or by hybridization


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of snail extracts by a DNA probe (40). A comparative evaluation of these tests, taking into account diagnostic qualities and costs, underscores the advantage this approach for large-scale detection of infected snails. It is potentially useful for providing extended information on schistosome-snail epidemiology that may facilitate rapid evaluation of the danger of postcontrol reinfection, and help make decisions on the time and place of supplementary control measures.

Chemotherapy Before currently used drugs became generally available, health problems caused by schistosomiasis were almost intractable, seriously reducing the quality of life for millions of people in the endemic areas, in particular for those with increased water contact due to habitats near dams and irrigation systems. Available drugs were expensive, not sufficiently effective and the side effects did not facilitate compliance. Since the early part of this century, various drugs have succeeded one another but acceptable results were not achieved until metrifonate (effective against S. haematobium) and oxaminquine (effective against S. mansoni) were introduced. However, the former has now been taken out of the WHO list of essential drugs and oxamniquine is only used on a large scale in Brazil and will soon be replaced with praziquantel. The removal of metrifonate can be defended since, even if it was very inexpensive, it had to be taken twice and was only effective against S. haematobium, but the demise of oxamniquine is of greater concern since, without it, there would be only one drug available to treat schistosomiasis. When praziquantel was introduced (18, 19), it immediately proved vastly superior and soon became the drug of choice in the great majority of endemic countries. After more than two decades of chemotherapy using praziquantel, it is obvious that its great advantage lies in the reduction (and reversal) of morbidity, while it does not influence transmission significantly. Thus, even if the therapeutic effect and the cost-benefit ratio of drug treatment are excellent, it is not sufficiently potent to terminate or significantly reduce prevalence. This was not fully appreciated at first but it is now fully accepted that reinfection is swift in most areas and that the preservation of advances made requires continuous surveillance, follow-up and repeated treatment. The design of realistic treatment schedules is currently seen as crucial for the maintenance of low-level morbidity and has become an integral part of

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control activities. As a further consequence, despite the decrease of treatment costs to currently below US$0.25 per person, the initial success has proved expensive to maintain. It is also increasingly clear that the elimination of morbidity as a public health problem cannot be achieved with chemotherapy alone and, as a consequence, few countries have reached stated targets of reduction and fewer still have achieved eradication. There are, however, reasons to be concerned that essentially, only one effective drug is available, in particular since the triumph of chemotherapy has reduced interest in research on new drugs and led to a virtual standstill in this field in the face of the threat that the widespread use of praziquantel may eventually induce drug resistance. Although induction of resistance to praziquantel has yet to be proven, scientists in the field are increasingly worried that this could become a reality (41). No new drugs with effect against adult worms are available, and even if the pharmaceutical industry allocated funds for research in that direction now, we would be a long way from an alternative drug. A detailed knowledge of the overall morbidity, the intensity of infection, and the predominant level of transmission, including its seasonal changes, must precede any intervention. Chemotherapy can be used in various ways, depending on the level of endemicity or, if control has been enacted, what stage has been reached (initiation, maintenance, elimination, etc.). At the present time, three delivery systems are recognized: mass treatment, selective population treatment and targeted chemotherapy. The choice between them is based on preliminary diagnostic data. Although serological tests are starting to provide reliable results that can be useful in determining these parameters, parasitological assays continue to be the accepted basis, for both individual diagnosis and assessment of endemicity. The qualitative and quantitative detection of schistosome eggs is a widely accepted standard technique for estimating worm loads. Naturally, the choice between alternative methods for drug distribution must also be based on the predominant social and economic level, existing drug distribution programs, diagnostic equipment, and the availability of trained personnel.

Mass Chemotherapy As the name implies, everybody in an endemic area would be treated, avoiding the costs of individual diagnosis. However, this approach can only be advised where endemicity is high, but as the cost of drug procurement


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has steadily diminished over the years it follows that mass therapy can now be given with less strict justification. The caveat here is compliance and everything must be done to ensure that as many people as possible actually take the drugs. Studies in various countries indicate that numbers above 70% of the targeted populations are seldom reached, which prompts the question how often the term mass chemotherapy is adequate. Follow-up diagnostic surveys would add confidence to the level of compliance achieved.

Targeted Chemotherapy This concept rests on observed differences in individual worm loads in endemic populations and prescribes that treatment should be reserved for those most in need. Most infected people in the endemic areas excrete few eggs as a sign of light worm loads, while the number of heavily infected individuals (those with > 816 eggs per gram of feces or > 50 eggs per 10 ml urine according to WHO (15)) is generally very low. This approach was more important in the old days when the side effects of the drugs used could be severe, but as therapy is becoming safer and less expensive, while the cost of diagnosis is edging higher, the value of this approach has declined. The safety of today's drugs makes it ethically questionable to withhold chemotherapy from anybody, regardless of the degree of infection.

Selective Population Treatment Here, various sectors of the population are treated, for example those with a pattern of particularly high water contact or certain age groups. The most common choice is to treat children and teenagers, as they harbor the highest worm burdens (Fig. 1) and probably also have the highest incidence of new infection. The lower-age percentiles of populations can probably be treated without prior diagnosis and with a high degree of confidence, but other groups should be subjected to individual diagnosis. The introduction of reagent strips for microscopic hematuria discussed above has improved the diagnostic score and it has also turned out be an excellent means of screening and "test and treat approaches" for urinary schistosomiasis. Blood in the stools may be detected in a similar fashion b u t specimen handling precludes the same success and compliance as

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Intensity of Schistosome Infection Related to Age 1000 S. mansoni S. haematobium

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Figure 1

experienced with S. haematobium. Not surprisingly, people are less prone to provide feces samples than urine samples, in particular if many follow-up examinations are required.

Retreatment Impressive reductions in schistosome infection rates and morbidity have been achieved with all the forms of chemotherapy distribution but, while the short-term results justify great optimism, these gains have proved difficult to maintain. There is ample evidence that transmission cannot be controlled with chemotherapy, and reinfection is all but inevitable in the endemic areas. Children in most communities commonly regain 30% or more of their pretreatment egg counts within a year. Although the question is not yet fully settled, the prevailing view is that the distribution depicted in Fig. 1


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is best explained by the development of partial immunity to reinfection with age under continued exposure, which leaves younger children susceptible and as a consequence rapidly reinfected. Thus, the initial fall in the prevalence and intensity of disease following treatment is always reversed and it is only a question of time when pretreatment levels are again regained in the community. However, the relationship between prevalence and intensity seen in uncontrolled situations does not apply in this situation as intensity rises more slowly, leading to a longer-lasting impact on morbidity than prevalence. Retreatment at some point is always necessary but the timing varies according to the characteristics of the endemic area in question. Ultrasonography has proved to be an excellent yardstick to gauge morbidity and follow its reversal after treatment. Retreatment should be scheduled according to the appearance, regression and reappearance of lesions, as recorded by ultrasound rather than inferred by egg counts. Experience shows that morbidity can be controlled by retreatment intervals of two years in most areas and longer where the prevailing endemicity is low (31).

Snail Control The application of chemical compounds, toxic to the snail intermediate host, remained the mainstay of control for the better part of this century and, before chemotherapy using praziquantel became a large-scale operation, it was not unusual to think of the control of schistosomiasis as essentially equal to snail control. However, partly due to the success of chemotherapy and partly due to the relative high cost of effective mollusciciding, snail control has come to occupy a diminishing niche in overall operations. Selective molluscicide treatment of snail-infested water bodies at the main contact points improves the economy and this approach is often used by control programs as a complement to chemotherapy. However, even at this reduced level, the cost of snail control would occupy a large proportion of allocated budgets and new ways of dealing with snail-related problems, including the development of control tools directed towards the miracidial and cercarial stages of the parasite, are urgently needed. Fluctuations in snail populations, the rate of infection and the production of cercariae are generally strongly influenced by the prevailing climate, which leads to a highly variable rate of transmission during the year. Snail control

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measures may, therefore, be required only at particular times and for limited periods. Although the season plays an important role, the presence of snails depends also on so many other variables that it is impossible to draw conclusions that hold always and everywhere. For example, the number of infected snails surviving the cold season or carrying infections from one wet period to the next following estivation will determine the relative risk of transmission at later times, as will the rate of infection of a new snail generation and the duration of incubation periods. On the other hand, in locations with stable hydrological and temperature conditions such as irrigated areas, snail populations and production of cercariae vary less, requiring that measures be applied regularly and / o r the snail habitats be permanently impaired. The focal nature of transmission is now better recognized and, in areas with adequate logistic support and management infrastructure, more cost-effective control measures can be implemented. Focal control utilizes less molluscicides since its use is limited to human water contact points, but it is, on the other hand, labor-intensive, with a need for appropriate transportation and supervision, including personnel trained in malacology as well as engineering. Meaningful results require that the form of transmission control desired be carefully defined and properly supported to define the foci of transmission to be addressed, its seasonality and delimitations. Certain countries, i.e. Morocco, Yemen, Saudi Arabia and Sudan, may be well served by transmission control based upon surveillance and focal mollusciciding, while the situation presents more of a challenge in areas where water bodies are large and human contacts with water are diverse.

Molluscicides Originally, different salts, mainly copper sulphate, were unloaded into streams and lakes in large quantities to control the snail, and although these compounds worked relatively well, their extensive use in natural habitats was precluded due to ecological considerations. This prompted the pharmaceutical industry to look for more environmentally acceptable products and, after a substantial research effort involving a number of newer substances (e.g. nicotinanilide, organotin, dibromo-nitroazo-benzene, sodium pentachlorophenate, tritylmorpholine, sodium dichloro-bromopheno and acetamide analogs), niclosamide could be registered for snail control by the


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Bayer company, and this substance is still the only commercially available molluscicide, most often sold under the trade name of Baylucide (42). Saltbased moUuscicides were largely abandoned in favor of niclosamide already in the 1950s but the cost is far too high for long-term use in most endemic situations. The agent is biodegradable but it is not completely without side effects, as many species of fish are more sensitive than snails. A large number of plants have been shown to contain substances capable of killing snails. Development of plant moUuscicides would permit local provision of the agent and lower the costs of snail control but, since plants are subject to biological variation due to local environmental factors such as weather and soils, ways and means of producing a standardized toxic principle have proved difficult. In addition, plant moUuscicides would have the same drawbacks as artificially produced chemical compounds. Even Phytolacca dodecandra (endod), the most well-researched plant moUuscicides, pioneered in Ethiopia by national scientists and shown to contain abundant amounts of saponins with antisnail effect (43), have yet to be embraced as a universally accepted tool for snail control. Plant moUuscicides were reviewed at a W H O meeting in 1983 and the updated and revised proceedings of the conference, published in 1987, stiU constitute the only comprehensive source of information on the subject (44). New antisnail plant compounds continue to be discovered but the above-mentioned issues have so far limited their development.

Biological Control Our experience of using predators or competitor snails has grown substantially during the last decade but the full ramifications, in terms of safety and impact on biodiversity, of this approach are still incompletely understood. Members of the Ampullariidae (Pomacea glauca and Marisa cornuarietis) and the Thiaridae [Thiara (Tarebia) granifera and Melanoides tuberculata] families have been successfuUy tried as competitors of Biomphalaria spp. in many of the West Indian islands, including Grenada, Guadeloupe, Martinique, Puerto Rico and St Lucia. Schistosomiasis transmission was interrupted in Martinique in the early 1970s, and when some of the sites were reactivated in the 1980s, the introduction of M. tuberculata rapidly displaced both B. glabrata and B. stramina (45, 46). Presently, thiarid snails have spread throughout the island's hydrographic system, effectively

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preventing its recolonization by planorbid snails for the foreseeable future. In St Lucia, on the other hand, M. tuberculata was not able to eliminate B. glabrata for good though it was very successful for a prolonged poeriod in the marshes and streams where it was introduced (47). The accidental introduction of Thiara (Tarebia) granifera in Puerto Rico seems to have wiped out the local snails and the whole island, which once was heavily endemic for schistosomiasis (48). Competitor snails have also been successfully utilized as a follow-up measure after mollusciciding in rivers of central Venezuela (49). In fact, the main future application of biological control may be to prevent recolonization after mollusciciding. Although further impressive results can be cited, they have not always been possible to repeat in other areas. For example, the experimental introduction of M. cornuarietis in Sudan had no measurable impact (50), while the elimination of Biomphalaria pfeifferi, Bulinus tropicus and Lymnaea natalensis was achieved in a small artificial lake in Tanzania two years after the introduction of M. cornuarietis (51). This discrepancy could possibly be explained by a less diverse ecology on these islands compared to larger land masses where the various snail species have had sufficient time to adapt to each other and create a balance which can only be altered very slowly if at all. However, regardless of the reasons for the promising results from the Caribbean area discussed here, the situation clearly justifies further research on the interrelationships between competitive snails, including follow-up field evaluations. Microbial pathogens and parasites have been considerably less investigated with regard to snail control, but it has been shown that the trematode Ribeiroia guadeloupensis is capable of sterilizing B. glabrata and a successful semi-field trial was carried out in a pond in Guadeloupe housing a natural population of the snail (52). In addition, although strictly speaking not an agent of snail control, Microsporidium spp. have been mentioned as a means of interfering with the sporocyst stage of the parasite. There are specific microsporidian species adapted to the majority of multicellular organisms, including

trematodes, but whether they would be useful in relation to the snail host stage of the particular schistosome species infective to humans remains to be investigated.

Environmental Management The creation of irrigation schemes, water reservoirs and canals greatly increases areas habitable for snails and this demands associated strategies


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to deal with the problem (53). Environmental management is probably the only measure that could guarantee a lasting effect. Large-scale changes of the environment in the Far East contributed to the eradication of human schistosomiasis in Japan (16) and led to an impressive improvement of the situation in China (54). The majority of canals and drainage systems in endemic provinces were restructured, and some dams subjected to intermittent drying out or letting the water level fluctuate, resulting in the eradication of schistosomiasis from large areas. However, success depends to a great part on the nature of the snail (Oncomelania is amphibious) and the results cannot be directly translated to regions, endemic for S. mansoni or S. haematobium, where the transmission of schistosomiasis relies on different intermediate snail hosts. Yet, alterations should make a noticeable impact on snail densities there as well, provided the ecological requirements of the species are known. Irrigation invariably translates into seepage and drainage problems, and intermittent drying out of small impoundments and irrigation canals, which could be instituted without too much difficulty in most places, would probably have a beneficial effect. Although, due to the capacity of aquatic planorbid and bulinid snails to survive desiccation, such measures cannot be expected to amount to total eradication, the long-term consequences of such treatment could conceivably still have a strong impact. The reduction of surface waters by tile drains a n d / o r other closed conduits helps control snail populations in irrigated areas and their drainage systems, as do alternating water levels in dams and reservoirs. Irrigation canals play an important role in snail breeding but the situation can be improved by straightening, deepening and lining them with cement as this contributes to higher lateral water velocity and less abundant aquatic vegetation. The limit water speed for Biomphalaria and Bulinus species is as low as 0.3 m / s , while velocities in excess of 1 m / s effectively dislodge almost all snails (55). There is now a vast experience of schistosomiasis control in irrigation schemes using environmental management measures. The conclusion is that only high-quality constructions can be advised, as otherwise, for example where rice is grown under periodically flooded conditions, snail habitats might develop in the drainage systems and result in the carryover of snail populations between wet seasons. It should, in this connection, be noted that the irrigated fields themselves usually do not provide good snail habitats because of the elevated water temperatures there. Schistosomiasis

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control is considerably facilitated if environmental considerations are included already at the design stage, which has been shown particularly well by a pilot project conducted at the Mushandike irrigation scheme in Zimbabwe (56). This design has been emulated elsewhere and most irrigation schemes which adopted it have a clearly lower incidence of schistosomiasis compared to schemes without schistosomiasis preventive measures as part of their design. Although environmental measures are useful, permanent results require far-reaching changes and provisions for maintenance, the costing and justification of which must be carefully considered. In summary, environmental changes do work relatively well in most settings but they must be well designed to fit each particular local characteristic and they must be maintained in good condition for the long term.

Water Resource Development The building of dams for the generation of electricity, irrigation for crop production and provision of drinking water has in general aggravated the schistosomiasis problem, particularly in Africa. For example, the exacerbation of urinary schistosomiasis in many West African countries, for example in Cote dTvoire (57) to take a recent example, and the introduction of S. mansoni to new areas, most spectacularly in Senegal (58), are directly due to the construction of dams. Ecological changes wrought by the Aswan High Dam have resulted in a change in the relative importance of S. mansoni vis-a-vis S. haematobium. While S. haematobium is still the predominant infection in Middle and Upper Egypt, S. mansoni is more prevalent in the Nile delta and the ecological changes and irrigation practices introduced have paved the way for a wider dispersion of its preferential vector, Biomphalaria alexandrina (59). Implementation of major water resource schemes in southern China and the Three Gorges dam in the central part of the country, scheduled for completion in 2003, may increase transmission of S. japonicum and again extend the endemic area (60). These developments derive largely from a common lack of a detailed knowledge of the potential risk factors leading to the inadvertent creation of snail-supporting habitats, health hazards which could be contained if engineers were trained to better identify the risks, a n d / or experts (e.g. medical ecologists or malacologists) could be regularly consulted (61). However, the human-behavioral factors effectively contribute


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to the development of the serious health risks commonly associated with water resources development projects and the community vulnerability is compounded by the many possibilities for the creation of hazardous habitats. Water bodies such as dams and canals normally attract people and habitation near such places is often coupled with lack of potable water and poor sanitation. The vicious circle of enlarged water resources translating into higher risks for increased populations can, however, be counteracted by avoiding: • reservoirs with shallow shores and little or no fluctuation in water levels; • storage dams for water conservation structures; • use of non-self-draining hydraulic structures; • irrigation and drainage canals with inadequate flow profiles; • inadequate irrigation water management, e.g. leaving pools of water in canal beds during drying; • drainage canals where water has a high fertilizer runoff content; and • seepage areas associated with defective structures. Leaking taps constitute a major risk, as resulting pools become breeding grounds for snails (as well as for mosquitoes) and effective drainage of waste or excessive water is essential at all water outlets (61). Pumps and connections are particularly weak parts of the distribution system, as they rely on spare parts and local maintenance, which is not always available. The Mushandike irrigation scheme, mentioned above, includes a relatively effective hydraulic design specifically aimed at reducing snail populations (56) but attempts to mechanically exclude snails have generally not met with success. However, irrigated areas, in e.g. Iraq, Kenya, Tanzania and the Philippines, serve as examples that it is possible to manage water resources to improve agriculture yields without attracting snails and exacerbating the schistosomiasis situation. The creation of snail habitats can to some extent be controlled by health impact assessment and subsequent health risk management measures during the planning and design phases of projects. This can be complemented by removal of aquatic weeds and other simple environmental manipulations that should be part of standard maintenance measures. Many safeguards of this kind are compatible with sound irrigation and drainage procedures and help save scarce water resources.

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Schistosomiasis

Water Supply and Sanitation Lack of access to safe drinking water and adequate sanitation are the driving forces behind the risk behavior of individual community members. The contamination of natural and man-made water bodies with human excreta is the main source of the spread of bacterial and viral agents causing diarrhea but, provided compatible snails are around, such human habits also support the schistosome life cycle. The risk for schistosomiasis is in fact greater than other microbiological contaminations, since the water contact is in this case not limited to drinking. Cercaria-free water can, in principle, only be guaranteed by a separate water source such as piped water, a well or some contraption for water storage. However, even these sources can come to contribute to the spread of the disease if the water source is not properly managed, permitting seepage and other spills to create new snail habitats. The health service built on education, hygiene and water supplies for the control of bilharzia and hookworm that was introduced in 1912 by Kitchener's administration in Sudan (62) is appropriate to this day. Water requirements vary from one area to another but, as a rule of thumb, allowance should be made for 20 liters per person per day from communal standpipes, about four times that figure if there is a tap in the house, while schools need about 15 liters per child (63). These figures are clearly higher than the bare minimum, as the amount of water normally carried from rivers and other water sources per day is much lower. While piped water or boreholes are usually provided by the government through a water development authority, many communities and households continue to rely on small man-made dams. Such dam constructions are legion in many parts of the developing world; less for domestic purposes but more for irrigation and breeding of fish, which does not make them safer. They are usually unauthorized and consequently outside any form of regulation or supervision and are no doubt responsible for increased transmission of schistosomiasis. Taken together, these dams are more of a hazard than any large dam construction, as it is difficult to prevent human access. Yet, with appropriate planning and little additional cost, safe water could be made available; for example, driving a pipe through the dam wall would provide uncontaminated water. Provision of safe water has always been advocated for schistosomiasis control by WHO, but it should be admitted that this issue should be (and is) part of the larger picture of social and economic development.


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Nevertheless, it would be useful if schistosomiasis was brought up when water provision is discussed and made part of the criteria when decisions are taken to expand coverage of clean drinking water supply. Not surprisingly, prevalence rates are lower where safe water is available than in otherwise similar areas, which was reported early on, for example from the Gezira, where well water is used (64), and in rural Egypt, where even partial use of protected water helped reduce the prevalence (65). Comprehensive studies to evaluate the role of water in the control of schistosomiasis have been carried out in many areas, for example in South Africa, where access to natural water bodies was prevented by wire fencing, while special pools were created for the children. Overall, the incidence of new infections fell dramatically in children and the prevalence and intensity of infection were reduced in all age groups (66). It should be remembered, though, that it is not enough to provide safe water without enforcing behavioral changes. Even if every home were fitted with piped water, many women would probably still wash clothes in the traditional way since they would otherwise be deprived of a reason for social gathering. For example, the international Drinking-Water Supply and Sanitation Decade was focused on diseases other than schistosomiasis but it could have had more of an impact on this disease than it actually had if the danger of exposure to most waters in Africa had been emphasized and simple laundry and shower units had been encouraged. Rain-water is an excellent source of clean water during the seasons when it is available, and all efforts should be made to make sure that as much as possible can be collected, for example by encouraging the use of corrugated iron roofs. Unsafe water can be cleaned by chemical treatment, filtration and even simple storage overnight. However, while storage is a simple method of killing cercariae, it is essential to ensure that snails are kept out of the storage tanks and one must also make sure that the water does not become a breeding place for mosquitos. It might be useful to know that temperatures of 45°C are sufficient to kill cercariae by 30 min exposure (67). The effect of chlorine is best at lower p H values, but even at the p H range of 7-9, cercariae are killed within 30 min by just 1 p p m chlorine residual (67). However, chlorination is not practical in rural areas, as the supply, storage and delivery of the compound is unreliable. Sand and diatomaceous earth filters remove cercariae, and results from Sudan show that a horizontal flow roughening filter combined with a slow sand filter enabled virtually stagnant water to be purified to the point of drinking standards (67).

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Most studies on the role of water and sanitation in control projects demonstrate that the intensity of infection is largely diminished when water and sanitation are applied. As early as 1898, Manson declared that "to kill the parasites is simply a matter of knowledge and the application of this knowledge — sanitary science and sanitation in fact" (68). Poor results reported from water projects are often due to negligence regarding sanitation, while improved water quality and sanitation strategies reduce waterassociated infections, including schistosomiasis (69). In Egypt, lower prevalence rates were found among those living in houses with latrines, but a direct causal relationship must be disentangled from the fact that the presence of a latrine is also an indicator of social status. Unfortunately, any number of latrines will not prevent human beings, particularly children, from urinating when they get into water, nor will it stop indiscriminate defecation. An alternative has been provided by the Blair Laboratory in Zimbabwe, which champions high-quality latrines in connection with housing and recommends that these constructions also be erected in the fields. A pilot study conducted in Madziwa, Zimbabwe, showed that the prevalence, intensity and incidence of schistosomiasis significantly decreased in an area where Blair latrines and safe water were provided (70). In other areas, e.g. Latin America and the Far East, pour-flush toilets are starting to become common (71). Based on there results and other past experience, water and sanitation have been made major components of control in Zimbabwe. For example, all new irrigation schemes are required to integrate Blair latrines in their designs and, according to the 1997/98 national sanitation inventory, the household coverage for Blair and flush toilets is 31.7%, while that of schools stands at 96% (72).

Health Education The strong emphasis on snail control and reduction of transmission in the past did not leave much room for education of the public regarding the risks of schistosomiasis. However, as the concept of morbidity control gained acceptance, the interest in and advantage of modifying people's behavior increased markedly. The major role of health education in countries with active national control programs may well be to encourage populations at risk to accept screening a n d / o r treatment. While diagnosis and treatment of school age children is a priority and their cooperation is essential, adults must be made aware of any ongoing control activities and how critical it


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is that they stimulate their children to also participate in activities outside the school such as particular television programs, talks and slide or video shows at the local health centre or other venues. Professionally trained health educators may be the best teachers but all personnel employed in the health services, such as doctors, nurses, midwives, assistants, technicians and other staff, can all play a part in educating the community. It is therefore important that everybody involved has a good understanding of the disease, the parasite life cycle, the benefits of treatment and the aims of control. Two types of health education are required. The first is appropriate to areas where treatment, with or without screening, is available and the aim is to reduce both morbidity and transmission by making people participate in health-related activities and to actually take the drugs provided. The second encourages those at risk to either keep away from water contact points harboring compatible snails or to stop contaminating these places. The aim of this approach is to impress on people that infection is linked to contact with unsafe water and to facilitate an understanding that humans, through undisciplined urination and defecation, play the role of vector as much as does the snail. The fact that human behavior has a profound effect on transmission should be stressed and no control project should be initiated without informing everybody why a special strategy was chosen, including the proposed ways and means of intervention. The objectives and long-term activities of health programs should be thoroughly explained and discussed in relation to prevailing epidemiological conditions with local people of authority such as local chiefs, village elders and religious leaders, stressing the need for community participation, while at the same time defining the role and objectives of education programs in relation to overall health. The channels for reaching the target populations require careful consideration and it must be recognized that teaching aids that are relevant to adults are often not understood by children. For this reason, special attention needs to be paid to developing and testing strategies specifically appropriate for children. These may consist of verbal instructions, slide shows, films, posters, radio and particularly television as there is no doubt about the incomparable power of the latter as it more and more penetrates society at large. With much of the general training increasingly being moved to formal schools, the best results can be expected by incorporating health education, including provision of stool and urine samples and drug treatment, as part of a continuing curriculum giving the responsibility to the teachers they already

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know and have learnt to respect. In spite of the advantages of health eduction, it receives a low priority compared to curative treatment, and job opportunities in this area are scarce. A major problem is that this approach must remain dubious if alternatives such as clean water cannot be supplied and there is thus little that can be done to prevent the continued exposure of high-risk occupational groups. Women are particularly vulnerable in view of their frequent clothes-washing activities and children cannot be expected to stop swimming and playing in unsafe water. This notwithstanding, bettersuited indicators are required for the monitoring and evaluation of health education and a research methodology appropriate to the special demands of this discipline is much needed.

Economic Aspects Schistosomiasis plays an important role in the economy of endemic countries. The high morbidity and mortality in, for example, Brazil, China, Egypt and Japan, was at times so serious that whole communities disintegrated, resulting in reduced agricultural production (73). The first reference to the socioeconomic benefits of schistosomiasis control goes back to the 1920s when successful campaigns in Egypt, using copper sulphate for mollusciciding and antimony for treatment, were shown to virtually wipe out the disease in selected areas for many years resulting in improved working capacity of the villagers and a substantial increase in the area of land cultivated (74). This r e p o r t serves to h i g h l i g h t the difference m a d e by treatment and control of a highly prevalent, incapacitating disease before the modern era of highly effective drugs and sophisticated means of control. Later studies focussing on the economical aspects of control have invariably concluded that the costs are inordinately high compared to the per capita health expenditure in sub-Saharan Africa (75-77). While the price of praziquantel has fallen sharply over the past twenty years, the total cost of treatment is still above the health budgets of most endemic countries. However, the incorporation of schistosomiasis control into the general health services would help since distribution costs are higher still. An integrated control p r o g r a m requires an infrastructure and / o r experience in public health interventions, and progress relies on the ability of policy-makers to recognize the disease as a major public health problem, and those w h o make the decisions at the government level usually


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demand proof of the benefits before committing funds. The key is to show an economic advantage but the real costs are difficult to pin down since control does not operate as an unaligned, separate activity. Salaries are a good illustration of this point, since tenured staff could either be fully accounted for in the control program or be treated as part of the overall government infrastructure. Moving from vertical to horizontal programs further complicates the situation, as personnel would then also carry out tasks other than those related to schistosomiasis. Logistic support and longterm resource provision of drugs also incur considerable expenditure, and it has always been difficult to convince governments in the endemic countries of the need for continuing financial support for this. In addition, the implementation of recommended strategies for schistosomiasis control may not succeed in eliminating the infection if other social sector investments fail to provide adequate water supplies and reduce the risk for contamination of the environment. Intersectoral planning and allocation of funds could accomplish this but, needless to say, this would further increase the overall costs of control. Although the time needed to realize safe water and sanitary installations and the capital investments needed have, in some cases, discouraged program managers eager to show positive achievements, an analysis of areas with sanitary installations clearly indicates that the returns outweigh the capital investment originally made. In fact, effective intersectoral consultation, resulting in the inclusion of recommended measures at the project design stage, lowers overall costs considerably. For example, the long-term benefits of such measures have reduced the frequency of chemotherapy and mollusciciding in the lowveld region of Zimbabwe over the years (70). The Mushandike project is an excellent example of a project where all parties were consulted at all stages of the project development, which resulted in diminished schistosomiasis transmission at a reasonable cost (56). There is ample evidence that ignoring potential problems or postponing intervention leads to uncontrollable situations. Epidemiological settings change rapidly and experience tells us that, once introduced, infectious agents establish transmission patterns which cannot easily be eradicated. The Senegal River Basin development has shown what lack of consultation and ignoring health aspects in favor of immediate economic returns can do (58, 78). Health benefits have a tendency to be viewed as a human rights issue which needs no justification in economic terms. This was certainly the case

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in the so-called "welfare countries" in the North until recently, but current economic reality has started to change this way of thinking and the less developed countries in the South could never espouse this luxury. The benefits of controlling schistosomiasis may well outweigh the costs, but since the mortality is low and its impact cannot be stringently measured in economic terms, decisions often rest on nonspecialist subjective perceptions. Mortality obviously falls short as a measure of the economical impact of chronic diseases, but research spent on finding time-based measures of health status that incorporate nonfatal health outcomes has produced the concept of the Disability-Adjusted Life Year (DALY), which is claimed to provide a better gauge (79,80). While mortality is simply the number of deaths directly caused by a specific disease, the DALY encompasses an estimate of the number of years of life lost by premature death due to a disease added to the number of years lived with a disability of specified severity due to that disease. The publication of the 1993 World Development Report by the World Bank (81) contributed to the wide dissemination of this concept, which can be further refined, for example by allowing different values for different ages. In principle, however, the DALY can be defined as one year of healthy life lost but, even if this is a much better indicator of the economic impact of a disease than mortality, the cost to society in dollars and cents is not obvious. Although it was evident before the introduction of the DALY concept that the sequelae of schistosomiasis have a stronger economic impact on society than has death, we now have tangible support for putting the economic impact of schistosomiasis on a better footing. While local authorities can improve the conditions for control, external organizations can facilitate development by providing health sector credits or donor funding. With the exception of Iran, Iraq, Morocco, Puerto Rico, Saudi Arabia and Venezuela, control interventions have invariably relied on outside support, most of which has been provided by the World Bank in the form of loans or multilateral or bilateral agreements and a number of other international agencies, such as WHO and UNESCO, have played a major role in improving the situation. Although governments in the industrialized world have not generally been interested in direct investments in this area, the German Agency for Technical Cooperation (GTZ) has supported long-term projects in Yemen and several sub-Saharan countries (82). Private foundations, in particular the Rockefeller Foundation and the Edna McConnell Clark Foundation, have in the past donated substantial sums towards both


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research and control of schistosomiasis, which has had a major impact. Credits for disease control are crucial for many areas, particularly in Africa, which now harbors more than 80% of all schistosomiasis in the world. Most of the countries affected are among the least developed in the world and their health systems face severe strains to provide even the most basic care at the primary level. In these cases, health sector credits of the normal type are not affordable and it might be necessary to facilitate access to them by making conditions more flexible. The only way these countries could undertake schistosomiasis control today is through specially designed grants, which ideally should be based on the presence of a functional health system and an expressed willingness to undertake long-term control. Unfortunately, in some countries which initiated control p r o g r a m s with d o n o r s u p p o r t , the price of antischistosomal drugs made these interventions untenable to continue indefinitely as required. The immediate way forward in control for these countries is through scheduled drug therapy schemes subsidized by affluent countries in the North or donations by the drug industry. The commitment of WHO could make a difference in these negotiations.

Monitoring All serious control activities necessitate the establishment of a system permitting continuous monitoring of the different control inputs implemented, and the task increases in complexity with the size of populations and regions involved. However, it is a sine quo non for progress and maintenance of achieved results. Research institutes or universities can provide valuable additional independent evaluations by carrying out random sample checks, but only as long as the control activities per se are not interfered with. For example, the concept of an index village could be useful and it is a reasonable probability that cross-sectional surveys in areas where population movement is high result in unreliable transmission data, while longitudinal studies of cohorts of the indigenous population would give more accurate information. The process of evaluation and monitoring in schistosomiasis control with special emphasis on the maintenance phase has been reviewed and a conceptual framework of evaluation suggested (83). By using specific indicators such as input, output, outcomes (in terms of effects and coverage) and impact, some important conclusions were drawn:

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• control and evaluation activities should be integrated into existing health care delivery services, once the maintenance phase has been achieved; • continuous evaluation concentrating on outcomes contributes to early recognition of problems, and facilitates reinforcement and fine-tuning of intervention strategies. Large-scale monitoring of diseases is one of the responsibilities of WHO. In 1987, after years of painstaking collection of data, an atlas of the global distribution of schistosomiasis was published (84). A new edition of this valuable map may not be needed, since we will soon have access to digitalized maps over the Internet. Support for this service will come from the modern-day ubiquity of satellites for communication, remote sensing and geographical positioning, the technological progress of which has been nothing less than astounding. The relatively new discipline, Geographical Information Systems (GIS), promises not only to considerably improve our knowledge regarding schistosomiasis distribution in the world but also to provide information on which areas are at risk for becoming endemic. Not only can data, collected the old-fashioned way by case-finding and snail surveys, be directly fed into computers, including the exact location given by small hand-held global positioning systems (GPS) devices, but the areas where the disease is most likely to be found can be identified by remote sensing technology. The reason for the latter is that distributions of vectorborne diseases are confined to areas where the spatial distribution of the parasite, host and vector coincide, and that these areas can be adequately monitored by satellites. If not known exactly, the probable distribution of human infection can be inferred since the required environmental conditions for vector-borne infections are as strict as, or more strict than, those characterizing the natural habitats of a species. The sensitivity of current satellitebased surveys has, for example, been evaluated and proved useful for the national control program in Egypt (85).

Current Control Strategies Due to differences in social and economic development, control activities vary according to region, but the bottom line is that, in spite of the increasing transmission and public health importance of schistosomiasis, the great majority of the currently 76 endemic countries are unable to extend consistent


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health policies across the whole of their jurisdiction. Only Brazil, China, the Dominican Republic, Egypt, Iran, Iraq, Morocco, Puerto Rico, St. Lucia, the Philippines, Tunisia and Venezuela have mounted coordinated efforts to control this disease. In general, countries without control programs are the least developed. China and Egypt are the only countries with a low GNP per capita income (1995 per capita incomes of US$620 and US$790, respectively) which have invested in control, while, with the exception of Gabon and Namibia, national control programs exist in all endemic countries with a per capita income above US$1000 (86). In contrast, it has been argued that special schistosomiasis control programs are not justified in certain regions and countries, e.g. Egypt and Sudan, since the relatively modest level of morbidity there can be controlled by ensuring the availability of appropriate schistosomicides in local health facilities (87). Recent research may, however, modify this perspective (37). Control programs rely on long-term political support and public health infrastructures and it should be remembered that some of the most successful programs, such as those in Brazil and China, followed political decisions and directives at the government level (88, 89). The current strategy is primarily aimed at preventing the development of chronic severe pathology, and it has been suggested that dispensaries and local health centres be upgraded by charging them with detection and treatment of symptomatic schistosomiasis (87). Supplementary measures may be required in areas characterized by relatively high incidences of morbidity, limiting short-term intensive chemotherapy interventions to areas with unusually high levels of morbidity and mortality. Any viable strategy must modify the goals of intervention according to a consolidated view of the problem, taking into account epidemiological conditions, infrastructure and available resources, addressing long-term maintenance requirements with logistic support. The "vertical" approach offers many obvious operational advantages, which explains why this type of structure is almost always adopted when national control programs are established. However, such programs depend on financial resources from the government and the budgets have proved difficult to maintain at the original level for long, in particular if initiated by a time-limited infusion of external funds and staff. Their integration into available horizontal services is not always a painless operation, as strategies agreed for one disease add expenses which cannot be met by the overall budget and cannot be handled by the staff employed for more general activities.

444

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It was expected that the new strategy for morbidity control, first recommended in 1984 by W H O (15), applying rapid, field-applicable diagnostic techniques and chemotherapy, would lead to more countries undertaking control. However, although many projects, successful in terms of reduction in the prevalence and intensity of infection, were initiated and operated in sub-Saharan Africa for several years with bilateral funding, few of them could be continued, let alone further developed into national programs, as the local authorities did not have the financial resources to meet the maintenance costs. However, it must be recognized that, with a few notable exceptions, proposed delivery systems are not optimal. They are not universally applicable and they have not been adequately evaluated in terms of the various constraints imposed by epidemiological and economic factors, and adequate financial resources for continuity have not been ensured. Since the methodology chosen for control of schistosomiasis depends on the level of prevalence in the locality in question, a comprehensive information system must be developed in which the areas of intervention can be mapped according to prevalence and ongoing activities. Once the level of infection has reached a sufficiently low level in a given municipality, the responsibility for screening of the population could revert to a horizontal approach, such as a primary health care system of active surveillance and notification of cases. The distribution of schistosomiasis in the world is nonrandom (Fig. 2). While the prevalence and intensity of disease is well controlled in Latin America, North Africa and the Far East, sub-Saharan Africa harbors most of the world's schistosomiasis. A brief discussion on control projects around the globe serves to highlight the achievements and remaining challenges.

North Africa A swathe of countries stretching from Morocco to the Middle East are characterized by low prevalence (Fig. 2). However, breaking this pattern, schistosomiasis is a priority in Egypt, which is geographically dominated by the Nile and has almost half its workforce engaged in irrigation-based agriculture. Both S. mansoni and S. haematobium are endemic, with the former increasing in relation to the latter (59, 90-93). With the adoption of praziquantel as the drug of choice and the strengthening of health education to ensure community compliance, the overall national prevalence fell to 10.5% from 16.8% between 1988 and 1992, accompanied by a reduction in the

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intensity of infection and morbidity (94). There is strong political commitment to the National Control Program; the current strategy involves selective population chemotherapy, focal moUusciciding and a health education program targeting school children in particular. From the onset, the program has been fully integrated within the Ministry of Health and managed centrally by the Endemic Diseases Control Department. At the peripheral district level, some 800 units specialize in snail control, while chemotherapy delivery is being integrated with the Public Health Care program, which relies on about 2600 rural health units and centers. Selective group chemotherapy is organized and implemented by rural health unit personnel focusing at the schools. Independent evaluation teams appointed by the Ministry are responsible for sample evaluations of index villages. The executive director at the governorate level is supported by a schistosomiasis committee responsible for all control activities and coordination of the delivery systems based on epidemiological intelligence. Rural health units are present in villages of more than 4000 people and undertake case-finding, treatment, follow-up, and health education. Control activities have been expanded in the delta of lower Egypt, where the same infrastructure exists to combat the high prevalence and intensity of S. mansoni (92, 93). The USAID-supported 10-year Schistosomiasis Research Program (SRP) was phased out in 1998 after a period of enormously positive influence on the Egyptian scene, not only upgrading research capabilities but also improving control. Control activities in Sudan began in 1979 in the irrigated areas under the Blue Nile Health Project and the strategy included health education, chemotherapy, provision of potable water, improved sanitation, and focal moUusciciding (94). In the Gezira, the efforts resulted in the reduction of the prevalence of infection from 50% to 6% between 1981 and 1989, while transmission and prevalence were unaffected in uncontrolled parts of the irrigation scheme (95). It was planned to incorporate control activities into the general health services by the end of the Blue Nile Health project but,

instead, due to the civil war, all control activities ceased and no new ones are currently planned. The prevalence can therefore be expected to rise over the next few years. In Morocco, activities geared towards the elimination of S. haematobium have been unusually successful, which is shown by the steady decline in the prevalence and the number of people at risk over the last 20 years. Transmission of the infection currently persists only in a few foci at very low


447

levels and total eradication is now the stated goal of the Ministry of Health (96). In Tunisia, a control program based on snail control, case-finding and treatment was begun in 1973 and, except for a focal outbreak between 1981 and 1982 in the governorates of Gafsa and Kebili, there have been no autochthonous cases there since 1984 (97). Transmission of S. haematobium continues in many foci in Algeria and Libya but, in the latter country, almost all reported cases have been foreigners (94). S. mansoni occurs in the northwest of Libya and the prevalence is 21% according to a 1993 survey (98). The authorities are dealing with the situation by focal mollusciciding for the present, while piped water is under construction to reduce the levels of infection in the households in the future.

Central and Southern Africa Harboring more than 80% of all schistosomiasis, including three of the five species adapted to man, sub-Saharan African is more in need of control programs than any other region. The German Agency for Technical Cooperation (GTZ) has supported schistosomiasis control in selected areas of Congo, Madagascar, Malawi and Mali (82). National surveys were conducted in Malawi and Mali and methods for community-based chemotherapy were elucidated. Reductions in the prevalence and intensity of infection were recorded in all these countries during the years that the programs were active but, with the exception of Mali, where national resources were able to maintain some control, improvements rapidly disappeared when activities terminated. Mali is endemic for S. mansoni as well as S. haematobium and has one of the longest-running schistosomiasis control programs in subSaharan Africa. In 1978, following a substantial increase in the prevalence and intensity of urinary schistosomiasis in the Dogon Plateau, GTZ added schistosomiasis control as part of its support for a dam project. A countrywide survey conducted between 1984 and 1989 showed that S. haematobium was common in many parts of the country, and a follow-up in the early 1990s, using various diagnostic approaches, including ultrasound, corroborated these findings, concluding that passive case detection would not cover early stages of the disease and recommending treatment based on surveys with reagent strips (99). The national Schistosomiasis Control Program, supervised by the National Institute for Research in Public Health with GTZ providing material support and expertise, adopted the objective of reducing prevalence below

448

Schistosomiasis

20% and heavy infections below 5% by means of mass chemotherapy and snail control. Operating the vertical program proved costly (100) and the government started to provide the running costs from 1989. When GTZ terminated its support in 1992, the state took over the main financial responsibility. A short-term strategy for control has now been adopted, with the aim of reducing the level of morbidity by providing mass treatment with praziquantel for the whole population in areas where the prevalence of hematuria in school age children exceeds 70%; at 50% prevalence or below, treatment is limited to infected children, while all children are treated when the prevalence stands between 50% and 70%. Less than 18 months after the Diama dam at the mouth of the Senegal river was completed in 1996, a sugar plantation 150 km u p the river because the scene for an S. mansoni "epidemic" (58). The dam provoked a profound ecological change permitting an explosive growth of the snail intermediate host Biomphalaria and, 10 years later, both urinary and intestinal schistosomiasis were increased in several countries in the river basin (101-103). In Ghana, construction of water resources contributed to the establishment of new foci throughout the country but, although the creation of lake Volta multiplied S. haematobium infections, this species already existed there, so the increase did not quite take on the epidemic characteristics seen in Senegal. An extensive project carried out in 1971-1978 (104) formed the basis of control activities but, in spite of intensive research and a successful pilot control project, a country-wide national program never evolved. The important components included selective population chemotherapy using metrifonate, focal mollusciciding and health education. The control activities were later absorbed into the primary health care structure, but financial constraints moved schistosomiasis down the priority list, which resulted in the virtual absence of effective control. In Burundi, selective chemotherapy and provision of clean water, laundry facilities and latrines were instituted in rural and urban foci, resulting in a reduction in the prevalence of infection in children from 23.3% to 6.4% over five years (105). The situation on the islands east of the African continent is generally encouraging. Control interventions carried out on the Unguja (1981-1983) and Pemba (1986-1991) islands of Zanzibar, the former based on selective population chemotherapy with metrifonate and health education and the latter using praziquantel, were highly successful. The prevalence of S. haematobium fell from about 50% before the interventions to about 10% on


449

both islands as judged by hematuria and urine filtration (94, 106). The prevalence of urinary schistosomiasis on the island of Mauritius was about 10% before 1988, when a program based on praziquantel and focal snail control was introduced, contributing to the steep decline to levels below 1% within two years (107). Madagascar is divided between a northwestern part where intestinal schistosomiasis dominates and a southeastern part where there is mainly urinary schistosomiasis. The situation on the island is serious, with high levels of morbidity reported by several authors using ultrasound (108, 109). A decision has been taken to re-establish a control program in four districts with World Bank funding. In South Africa, control is part of school health programs in certain areas, for example in the Kwazulu and Mpumalanga regions, but little information is available. However, a recent study in the Port St Johns district of the former Transkei noted an increase in overall infection rates in the region compared to previous studies (110). In Botswana S. mansoni is found in the Okavango delta, while there is urinary schistosomiasis in the southeast. A project to control the latter using praziquantel, instituted in 1985 and then integrated into the primary health care system, produced a drop of prevalence in schoolchildren from 80% to 6.7% within a few years (111). A control program in the northeast of Namibia, providing schoolchildren with praziquantel every six months and focal mollusciciding with niclosamide every month from 1987 to 1993, was carried out by the South African Research Institute for Diseases in the Tropical Environment with funds from the Theodor Bilharz Foundation. A review three years later showed an 82% reduction in prevalence and 99% reduction in egg output, and whereas hepatomegaly was a common sign at the beginning of the project, very few children had enlarged livers when it was completed (112, 113). The program confirmed that morbidity control is feasible through chemotherapy, even in the face of continued transmission.

Latin America All schistosomiasis in this region is due to S. mansoni and Brazil is the most affected country by far. The infection was limited to the northeastern states of Brazil up to the early 1900s but has since spread all along the coast to involve 19 of the 27 states. A five-year special program for schistosomiasis control (PECE) was launched in 1975 in seven northeastern states, with a population at risk of 5 million (88). The objective of halting transmission by

450

Schistosomiasis

reducing the prevalence to 4% or lower was adopted with the intention of expanding to all endemic areas in due course. Based on the prevalence of infection in school age children, operational components were defined with respect to how mass and selective chemotherapy were to be used. Although transmission proved difficult to interrupt, the goal of prevalence reduction was achieved in many of the states, leaving only few municipalities with a prevalence over 25%. The program was administratively placed under the Superintendent for Public Health Campaigns (SUCAM), an autonomous, vertically structured agency which also has responsibility for other endemic diseases, such as yellow fever and Chagas' disease. Fully staffed with federal employees, it had a headquarters support center and autonomous field units which allowed swift adjustment to local needs. Its overall performance was good, because its management structure and decision-making processes were closely related to the delivery system. Complete penetration was achieved at the local community level but difficulties regarding treatment compliance emerged since SUCAM had little demand-creating and motivational capacity within the communities and it suffered also from short-term changes in government priorities. In the 1980s, an attempt was made to decentralize activities to state and municipal levels as well as to reduce the role of mass chemotherapy. Realizing that the same control approaches or strategies may not be appropriate for areas of different endemicity, the program was reorganized, with operational areas stratified according endemicity and likelihood of transmission. Chemotherapy remained the main approach, with some backup by snail control. The latter was not, however, carried out systematically and improved sanitation and safe water supply were rarely provided. In the early 1990s, without change of control policies, SUCAM was incorporated into the National Health Foundation (FNS), which now has the overall responsibility for disease control in Brazil. The current objectives are to prevent mortality, reduce morbidity, and prevent expansion of the endemic area by using mass or selective chemotherapy, focal mollusciciding, health education and sanitary improvements (114). Oxamniquine has been the drug of choice in Brazil for 22 years, using 15 m g / k g of body weight for adults and 20 m g / k g for children under 15 years of age, but, from the end of 1997, praziquantel, provided at doses of 50 m g / k g for adults and at 60 m g / k g for children under 15 years, has replaced oxamniquine as the main drug (114). Activities are tailored to each area, for example mass or universal treatment is used where the


451

prevalence is over 50%, while treatment is limited to those infected and members of the same household in areas where the prevalence is between 25% and 49%, and when less than 25%, only infected cases are treated. The prevalence of infection is determined by carrying out parasitological surveys of the whole population of the endemic areas, every two or four years. It is expected that these treatment regimens will be accompanied by improvement in the provision of water and sanitation facilities. Snail control using molluscicides is done if the sanitation component is absent, and if (imported) cases are detected where transmission is not supposed to occur, control program managers should be notified. In Venezuela, integrated interventions with an emphasis on environmental modification, sustained since the 1940s, have kept the overall prevalence below 2% (115). In Surinam, a program begun in 1973 and integrated into the general health service in 1979 achieved a drop of the prevalence to less than 6% (116). The current strategy for elimination is based on identification of cases by serology, treatment with praziquantel, surveillance for new transmission foci and continued snail control. On the smaller islands of the Caribbean, transmission has been interrupted and there is now a real potential for eradication of schistosomiasis from this area (117). Low-level transmission continues in the Dominican Republic (118), while it may have ceased altogether in Puerto Rico (48, 119).

The Middle East A control program in the Khuzestan province of Iran which operated between 1970 and 1979 using selective population chemotherapy, snail control and health education reduced the prevalence of S. haematobium infection from 8.3% to less than 1% (120), which has since diminished further. In Iraq, records of the Institute of Endemic Diseases show that the prevalence of urinary schistosomiasis among children, 6-20 years old, was reduced from 21% in 1958 to 3.9% in 1978 (83). The control program consists of case-detection and treatment at the primary health care level in the three high-risk governorates, screening of foreigners for infection, and surveillance for snail vectors. It was relatively successful until the war with Iraq, but the number of cases has increased lately due to difficulties in procuring supplies (94). The potential for transmission exists in Jordan as many agricultural workers regularly come from endemic countries, but no autochthonous cases have

452

Schistosomiasis

been detected since an outbreak of 42 cases of S. haematobium infection in 1985 (94). In Saudi Arabia, both S. haematobium and S. mansoni are endemic and over a million people were infected in 1967 (94). Control centers were established in 1973 to carry out snail control, provide chemotherapy and conduct health education. These activities were integrated into the primary health care system in 1988 and the overall prevalence of infection is now less than 1% (121). Most of the detected cases are imported (122) and there is evidence that baboons act as a reservoir for S. mansoni infection (123). There is as yet no control program in Yemen due to lack of resources, but it can be estimated that about 3 million people are infected (with either S. haematobium or S. mansoni) out of the population of 15 million (94, 124).

The Far East In Japan, control efforts began shortly after the discovery of the parasite but large-scale environmental management was not possible until the 1950s. These activities resulted in the elimination of schistosomiasis and, even though vector snails are still present in one area, no human case of schistosomiasis has been detected since 1977 (86). There is no doubt that socioeconomic development and industrialization contributed to the rapid eradication of the disease from this country. Compared to Japan, the problem is of a greater magnitude in China, which also did not have the benefit of rapid economic development at the time. Estimates of the number of people infected in China in 1949 range from 5 million to almost 12 million, and more than 100 million people were at risk of infection in 400 counties (73). Faced with increasing transmission and widespread high levels of the intensity of infection, China initiated a control program relying on popular participation supervised by provincial institutions (125). Using available methods with a strong emphasis on environmental management directed at the snail intermediate host resulted in interruption of transmission in 5 of 12 provinces (73,125). The remaining endemic areas are the most difficult to control, as they are situated in swampy, lake, hilly or mountainous regions. Since 1992, a $71 million World Bank credit has been used to implement a control program emphasizing chemotherapy but including a research component, managed by a Joint Research Management Committee (JRMC), supporting operational projects and workshops focussed on health economy, diagnostics, health education and snail control.


453

Comparison of data from nation-wide surveys carried out in 1989 and 1995 indicates a prevalence reduction of close to 50%, including a 32% reduction in the prevalence of infection in cattle and buffaloes (54). The current situation requires some degree of reorientation, as further reductions of prevalence in the central endemic areas of the country have met with resistance. In addition, acute schistosomiasis is increasing in spite of good coverage of praziquantel, indicating that full control of schistosomiasis is far from being achieved. The ongoing building of the biggest dam in the world, aimed at creating an artificial lake west of the Three Gorges of the Yangtze river in the Sichuan Province, may lead to increased transmission (60). Schistosomiasis control in the Philippines began during World War II with environmental sanitation and snail control, then switched to chemotherapy as the major tool when the program was decentralized in 1984 (126). S. japonicum is still endemic in the central and southern areas, where 5 million are at risk and 5% actually infected in 183 municipalities of 24 provinces and, in the island of Mindanao, the prevalence remains above 10% (127). From 1990 to 1994, World Bank support strengthened the control program by increasing manpower, mobility and supplies focusing on high-risk populations such as schoolchildren, fanners and fishermen. The reduction of prevalence was impressive and consistent during this time but concern has been voiced that it may again increase if control activities are relaxed (127). A separate study found that the morbidity due to S. japonicum is more severe than that caused by S. mansoni (and parasitological diagnosis less sensitive), leading to the conclusion that mass or targeted treatment is more cost-effective than case-finding (128). There are currently no control activities in the minor schistosomiasis foci in India and Malaysia but about 12,000 people are at risk of infection in the Lindu and Napu valleys on the island of Sulawesi in Indonesia. However, the endemic areas are remote and difficult to reach, minimizing the risk for spread to other parts of the country. In 1981, a program using praziquantel and mollusciciding was started, and it resulted in a fall of the prevalence rate in Lindu from an initial range of 43-79% to about 2.5% (129), while a regress in the number of palpable livers in 46 children fell from 95.6% to 58.7% in Napu (130). Although studies are ongoing in both Cambodia and Laos, few published recent studies of the epidemiology of S. mekongi exist. However, the infection is endemic along the Mekong river, particularly on Khong and neighboring islands, in Laos, causing high morbidity and

454

Schistosomiasis

mortality (131). A control program was initiated in 1989 with support from the WHO Regional Office for the Western Pacific (WPRO), and a total of 60,000 people have been treated with praziquantel between 1995 and 1998, decreasing the overall prevalence from 70% to less than 20%. The German Pharma Health Fund has supported an integrated program for the control of schistosomiasis, opisthorchiasis and other intestinal nematodes in the Champasaak province, Laos, where it has been possible to keep the prevalence below 2% by means of repeated cycles of treatment and retreatment.

Role of Research Although schistosomiasis can be controlled with the tools available today, this should not obscure the fact that new approaches might achieve the goal more rapidly and at a lower cost. In fact, there has been steady progress on many fronts but, on the other hand, the way control is carried out has hardly changed during the last 20 years. Investments in research depend on convincing donors that allocation of funds is worthwhile but efforts can even be counterproductive if the progress achieved is not turned into practice without undue delay. However, research is seldom simple and straightforward as, for example, shown by the need for work on infection, transmission and morbidity including the parameters determining reinfection after treatment. Likewise, the development of improved sustainable and costeffective strategies requires a better understanding of the epidemiologic factors governing the dynamics of infection. A brief overview might help define current needs for study and pinpoint the priorities.

Chemotherapy and Biochemistry So far, only modest progress has been noted in the development of immortalized schistosome cell cultures (132,133) though this is an undisputed priority in schistosomiasis research which would revolutionize screening for new drugs, a well-known bottleneck in the pharmacological industry. Moreover, the mode of action is not fully known for any of the drugs used to control schistosomiasis, and further biochemical and immunological research on this subject is needed. Such studies should also facilitate the development of new effective drugs and elucidate how resistance develops.


455

Investigation of the susceptibility of S. mansoni to praziquantel, carried out with isolates from Senegal, Puerto Rico and Kenya, indicates that recent worm isolates have longer prepatent periods than those maintained for years in the laboratory (134). Apart from indicating the possibility that resistance may be evolving, this research highlights the variability of the parasites and the risk of relying on "standard" laboratory strains in research. Drug resistance may or may not become a real problem but it should be recognized that the larval stage has always been refractory to praziquantel (135). This selective effect with regard to the parasite developmental stages permits new worm burdens to develop after treatment even without reinfection, a phenomenon which cannot easily be distinguished from drug resistance, particularly in areas of high-intensity transmission. Interestingly, artemether, an antimalaria drug developed from the Artemisia plant, has been shown to be effective against this very stage, and experimental animals do not develop schistosomiasis if treated at least twice with artemether during the first month after challenge (136, 137). The usefulness of artemether as a prophylactic drug has been proved in China but further research, both in the laboratory and in the field, will be needed to confirm these findings and to know if susceptibility to this drug is a general trait of schistosomes. Prophylactic drug treatment for schistosomiasis would be a new application of potential interest for tentative eradication programs.

Diagnostics Research on diagnosis has been dealt with in the paragraphs above on markers of infection and morbidity. Suffice it to state here that very good progress has been made and that reliable serological techniques for the detection of circulating antigens as well as antibodies of different isotypes are now at our disposal. The level of sensitivity of antigen detection is similar to that of parasitological techniques, but it can be argued that the assessment of cure based on the presence or absence of circulating antigens is preferable since schistosome antigen levels in the blood vary less than egg excretion in stools and urine and, consequently, only one sample is needed for serology, in comparison to several for the parasitological techniques. In addition, the potential for standardization of reagents and assays makes immunodiagnosis well suited for base-line and follow-up surveys in endemic areas.

456

Schistosomiasis

Preliminary results using noninvasive and sensitive assays, relying on the excretion, in the urine, of soluble egg antigens (SEA) and the eosinophilic cationic protein (ECP) in individuals with urinary schistosomiasis, show that these compounds correlate well both with egg counts and with morbidity before treatment. More importantly, they retain their levels when the egg counts fall after cure and diminish only slowly, in parallel with the reduction of lesions of the bladder and kidneys as shown by ultrasonography (BBSS). These approaches are so far only available for S. haematobium infection and research is needed to see if the idea can be extended to morbidity caused by intestinal schistosomiasis.

Epidemiology Epidemiological research has benefited from the development of standard statistical software packages but Geographical Information Systems (GIS) constitute the perfect interphase between databases and the human brain. The display of data in the form of instantly intelligible maps, including the various levels of probability of vector distributions now and in the future, makes the overall picture mentally easy to grasp. Remote sensing is proving to be invaluable in prospective research on environmental changes such as the impact of major dam constructions and irrigation schemes. Another tool, mathematical modeling, facilitates decision-making with regard to control by extrapolating results from field trials that, for practical reasons, can only cover confined areas during limited periods of time. Models could become important in selecting and implementing available measures, but a good understanding of schistosome biology is necessary since the assumptions and mathematics used will only provide useful results if they reflect the reality. Practical contributions can be expected regarding the assessment of specific control tools vis-a-vis integrated approaches. Possible applications include the cost and impact of different chemotherapy strategies on transmission, infection and morbidity, the cost-effectiveness of snail control, water supply and health education, and the usefulness of rapid assessment methods capable of identifying communities and individuals at high risk. The "pocket chart" (138) which was developed for assessment of the real prevalence and intensity of schistosomiasis is an excellent example of how mathematical modeling can extrapolate from limited


457

measurements and deliver accurate data (except in extreme cases of very high or very low intensity of disease). Modeling may also be used to evaluate future scenarios; for example, vaccine design may be facilitated by mathematical prediction of the level and type of protection needed. Other types of rapid assessments are also much needed and some practical applications have been developed, such as the attributable risk analysis (139) for the estimation of public health impact (applied for S. japonicum in China).

Operational Research This is a large research area and suffice it here to underline that successful control programs should contain the capacity to undertake studies to answer questions that will remove barriers to a meaningful execution of a strategy. A prime example is the "red urine study group," established by WHO/TDR to rapidly determine the impact of schistosomiasis haematobia, which has now been validated in many different African locations (26). The new disciplines of GIS and modeling facilitate epidemiological research, while population genetics is growing rapidly along with advances being made in the identification and function analysis of genes. For example, the discovery that the level of infection is controlled by one major gene locus, Sml, serves as an explanation of the field observation that the distribution of hepatosplenomegaly is nonrandom (140). In addition, there are indications that another gene, Sm2, is involved in the development of fibrosis (141). These findings have encouraged research on p o l y m o r p h i s m s in the human genome and led to the localization of the genes encoding several cytokines to an area physically near the Sml gene. Here, field observations have led to the application of sophisticated laboratory research, and combined expertise from the two fields has delivered an explanation for the occurrence of serious morbidity in certain subjects and the diversity of immune responses in a given population, contributing to the elucidation of how the immunological responses are triggered. The emerging picture of schistosomiasis as the result of an interplay between opposing mechanisms has implications outside the epidemiological/genetic focus as the unraveling of the mechanisms of immune responses of prime importance and for vaccination strategies.

458

Schistosomiasis

Vaccine Research Parasite vaccine development in general is a daunting task but, in the case of schistosomiasis, several factors warrant optimism. The almost complete protection reached with irradiated cercariae in experimental animals provides the "proof of principle," while a wealth of epidemiological studies shows that humans express protective immune responses resulting in various degrees of resistance to infection. By the same token as gross pathology was successfully conquered by sustained, large-scale use of anthelmintic drugs, a vaccine targeting morbidity rather than aspiring to achieve sterile immunity would complement chemotherapy by inducing immunological protection, which would possibly result in a lowering, or translation, of the peak of the intensity of infection, as hypothesized in Fig. 3 (broken lines). Although experimental antipathology vaccination has been quite successful lately (142) and the final product might incorporate several targets, the first-generation vaccines will

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-


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no doubt be directed against infection a n d / o r worm fecundity. So far, only one antigen has reached the level of Phase I/II testing: S. haematobium Glutathione-S-Transferase (GST). However, S. mansoni paramyosin and S. mansoni Triose-Phosphatase Isomerase (TPI) are strong contenders (143). Continued research into other antigens, including those involved in cercarial penetration of the host skin, and the immune response thus elicited would be useful, as early obstruction could reduce the number of developing schistosomula and facilitate other preventive measures aimed at the later stages. Encouraging protection experiments in animal models and the accumulation of positive data from human correlate studies have moved the field forward and vaccine development must now be accepted as realistic. The current understanding is that nature has struck a balance, the outcome of which reflects the tempering of a strong antischistosome response by stimuli downregulating the granulomatous reaction against eggs lodged in the tissues. Research in this area promises to improve our understanding of cytokine interaction in the development of pathology and immunity, hopefully leading to a way to induce maximum levels of immunity without enhancing eggassociated reactions.

Snail Research Most experts agree that control cannot be sustained without control of the intermediate snail host, but financial constraints and the effects of the chemical agents on fish, shellfish and other ecologically important animals make this strategy difficult to maintain. On the other hand, the successful utilization of biological control in the Caribbean islands might contribute to a rethinking and possible revival of the area. However, so far, there have been few innovations and the current situation calls for research on several fronts: diagnostics, molluscicides and biological control.

Research Support How can valid research demands be met when the whole area of tropical diseases receives only a small fraction of the funds available for health research, and where schistosomiasis is not considered a priority, neither for donors nor for industry? Funding in this field has undergone a drastic

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reduction as a number of major, long-term research projects (primarily supported by the Rockefeller and Edna Mc Connell Clark Foundations and the World Bank) have come to an end. However, the need for research is still recognized and financial support, albeit on a smaller scale, is still available for work in key areas; for example, the broad-based Schistosomiasis Research Project (SRP) in Egypt has been continued under the Schistosomiasis Vaccine Development Program (SVDP), which started in 1998. National research councils also merit mention, such as those in Brazil, France and the USA, to mention some of the more important ones. In fact, the US program, executed by the National Institute of Allergy and Infectious Diseases (MAID), is now the largest regular contributor to schistosomiasis research in the world. The USA provides funding through its USAID program, and international organizations such as WHO/TDR and the European Union together spend between two and three million US dollars annually on various aspects of schistosomiasis research. The bottom line, however, is that this research has become almost exclusively dependent on national research funding agencies and that applications must compete with proposals covering an array of other diseases. Although schistosomiasis-related research applications have done well in this competition, there is no doubt that a new major, long-term project is needed to facilitate theoretical and practical work towards superior control strategies.

Novel Approaches, New Strategies Mathematical modeling, GIS and population genetics have improved epidemiological know-how and made control less "one-dimensional," while assessment of cure by the detection of circulating antigens is a new diagnostic tool ready for implementation. Antigen detection provides more standardized results than stool and urine examinations and contributes to making the assessment of infection levels and their geographical distribution simple, rapid and, above all, more reliable. Diagnostics have been the focus of intense research in the laboratory and the inclusion of serology in current control programs can now be seriously contemplated, first on a pilot basis and later, if its value is vindicated, more permanently. The questionnaire approach and the use of reagent strips for assessment of urinary infection have already been taken aboard in many African control projects, and the use of noninvasive morbidity markers such as ECP and SEA will contribute to improving


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the possibility of rapidly assessing the situation of urinary schistosomiasis in various settings. It is not beyond imagination or cost to combine all the serological techniques into one dipstick capable of assessing infection and morbidity simultaneously. There are reasons to be optimistic about the future of schistosomiasis control. For example, targeting schoolchildren and other high-risk groups with chemotherapy has resulted in a clear and significant reduction of morbidity in those countries able to institute the country-wide, large-scale treatment schedules, e.g. Brazil, China and the Philippines. In most areas, treatment every two years seems sufficient to keep the situation well under control. Elimination of the infection as a public health problem is within reach in Botswana, Iran, Iraq, Mauritius, Morocco and Saudi Arabia, and there are no new cases reported in Japan, Puerto Rico and Tunisia, even if transmission has not completely ceased in all areas. A few countries, notably Morocco, have even embarked on a program of eradication. On the other hand, the number of countries endemic for schistosomiasis has not changed significantly during the last few decades and the overall number of people infected remains around 200 million. Furthermore, it is disturbing that the current strong emphasis on chemotherapy relies on the sustained superiority of one drug and it is of constant concern that this approach could be jeopardized by emerging resistance. Oxamniquine would probably provide respite in some areas (103) but, since there are no new schistosomicides in the pipeline, it is of some urgency that the prophylactic use of a schistosomulucide be investigated with a view to developing a drug of last resort. In the absence of safe and effective drugs, activities during most of the century have been dominated by the use of various types of molluscicides, while current large-scale application of praziquantel has shown that morbidity can be controlled in the face of ongoing transmission even if reinfection requires repeated interventions at regular intervals. The transition from snail control to chemotherapy shifted the emphasis from eradication of the infection to the more practical goal of reduction of morbidity. However, an analysis of the situation shows that there need not be a dichotomy between transmission control and morbidity control. Instead, various strategies can be brought together to shape an approach consisting of partly overlapping components or levels. Public health experts would be required at the general level, while clinical considerations must rely on medical expertise, and the biology of

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the parasite and its life cycle are best dealt with by engineers, biologists, and social and economic experts. We are now in a good position to enlarge the morbidity control concept to a three-step procedure, moving the focus from morbidity via infection to transmission. This way of structuring control activities would require that we first concentrate on populations with disease and dispense drugs to reduce overall morbidity, then provide medical care for individuals developing gross pathology and, finally, embark on transmission control as the means of reducing the overall risk of infection. Operational research will be important in elucidating the best way forward, particularly by studying how to improve and sustain compliance, which is usually good at the start of a program but deteriorates with time, the role of people movement for the spread of infection to new areas and new populations, and which special risk factors are associated with schistosomiasis transmission and morbidity. There are clear signs that a consolidated, modernized view of how to control schistosomiasis with the tools currently at our disposal is emerging.

References 1. Deelder AM, Miller RL, de Jonge N and Kruger PW (1990). Lancet i, 525: 724. 2. Report on the First Session of a Joint O I H P / W H O Study-Group on Bilharziasis in Africa (Cairo, 1949). Technical Report Series, No. 17 (1950), 16 pp. 3. First Report of the Expert Committee on Bilharziasis (San Juan, Puerto Rico), No. 65 (1953), 45 pp. 4. Report of a Study-Group on Bilharzia Snail Vector Identification and Classification in Equatorial and South Africa (Paris, 1954). Technical Report Series, No. 90 (1954), 22 pp. 5. Report of a Study Group on the Ecology of Intermediate Snail Hosts of Bilharziasis (Paris, 1956). Technical Report Series, No. 120 (1957), 38 pp. 6. Report on an African Conference on Bilharziasis (Brazzaville, 1956). Technical Report Series, No. 139 (1957), 43 pp. 7. Report on the Second WHO/CCTA African Conference on Bilharziasis (Lourenco Marques, 1960). Technical Report Series, No. 204 (1960), 37 pp. 8. Molluscicides — Second Report of the Expert Committee on Bilharziasis (Geneva, 1960). Technical Report Series, No. 214 (1961), 50 pp.


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9. Third Report of the WHO Expert Committee on Bilharziasis (Geneva, 1964). Technical Report Series, No. 299 (1965), 56 pp. 10. Report of a WHO Scientific Group on Chemotherapy of Bilharziasis (Geneva, 1964). Technical Report Series, No. 317 (1966), 71 pp. 11. Report of a WHO Scientific Group on Measurement of the Public Health Importance of Bilharziasis (Geneva, 1964). Technical Report Series, No. 349 (1967), 93 pp. 12. Report of a WHO Expert Committee on Epidemiology and Control of Schistosomiasis (Geneva, 1966). Technical Report Series, No. 372 (1967), 35 pp. 13. Report of a WHO Expert Committee on Schistosomiasis Control (Geneva, 1972). Technical Report Series, No. 515 (1973), 47 pp. 14. Report of a WHO Expert Committee on Epidemiology and Control of Schistosomiasis (Geneva, 1978). Technical Report Series, No. 643 (1980), 65 pp. 15. Report of a WHO Expert Committee on the Control of Schistosomiasis (Geneva, 1984). Technical Report Series, No. 728 (1985), 113 pp. 16. Second Report of the W H O Expert Committee on the Control of Schistosomiasis (Geneva, 1991). Technical Report Series, No. 830 (1993), 86 pp. 17. Report of a WHO Study Group on Control of Foodborne Trematode Infections (Geneva 1995). Technical Report Series, No. 849 (1995), 157 pp. 18. Davis A and Wegner DHG (1979). Bull. WHO 57: 767. 19. Wegner DHG (1984). Arzneimittel Forchung 34: 1132. 20. Katz N, el al. (1970). Rev. Inst. Med. Trop. Sao Paulo 14: 397. 21. Barreto ML, et al. (1990). Trans. R. Soc. Trop. Med. Hyg. 84: 554. 22. Engels D, et al. (1997). Am. J. Trop. Med. Hyg. 57: 571. 23. Van Etten L, et al. (1993). Trans. R. Soc. Trop. Med. Hyg. 87: 311. 24. Van Lieshout L, et al. (1995). /. Infect. Dis. 172: 1336. 25. Van Lieshout L, et al. (1993). Trans. R. Soc. Trop. Med. Hyg. 87: 311. 26. Lengeler C, et al. (1993). Acta Trop. 53: 39. 27. Social and Economic Research project report No. 15 (Geneva, 1995), 83 pp. 28. Doehring E, et al. (1984). /. Infect. Dis. 149: 615. 29. Booth M, et al. (1998). Trans. R. Soc. Trop. Med. Hyg. 92: 484. 30. The Cairo Working Group: the use of ultrasound in schistosomiasis — attempts at standardization of methodology (eds. Jenkins JM and Hatz C) (1992). Acta Trop. 51: 769.

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Hatz CF, et al. (1998). Am. J. Trop. Med. Hyg. 59: 775. Richter J, et al. (1998). Trop. Med. Int. Health 3: 728. Homeida MA, et al. (1998). Am. J. Trop. Med. Hyg. 55: 360. Ohmae H, et al. (1992). Am. J. Trop. Med. Hyg. 46: 89. Reimert CM, et al. (2000). Am. }. Trop. Med. Hyg. 62: 19. Wennervald BJ, et al. (1994). Trop. Geog. Med. 46: 239. Vennervald BJ, et al. (1998). Parasitol. Today 14: 385. Kahama Al, et al. (1998). Am. ]. Trop. Med. Hyg. 59: 769. Hamburger J, et al. (1989). Am. } . Trop. Med. Hyg. 40: 605. Hamburger J et al. (1992). Mem. Inst. Oswaldo Cruz 8 (Suppl. 4): 243. Cioli D (1998). Parasitolol. Today 14: 418. Andrews P, et al. (1982). Pharmacol. Ther. 19: 245. Lemma A and Yau P (1974). Ethiop. Med. J. 12: 109. Plant MoUuscicides (ed. Mott KE). Published by John Wiley & Sons (1987) on behalf of the UNDP/ World Bank/WHO Special Program for Research and Training in Tropical Diseases, 326 pp. Pointier JP, et al. (1989). Ann. Trop. Med. Parasitol. 83: 263. Pointier JP and Guyard A (1992). Trop. Med. Parasitol. 43: 98. Pointier JP (1993). Acta Trop. 54: 13. Giboda M, et al. (1997). Am. }. Trop. Med. Hyg. 57: 564. Pointier JP, et al. (1994). The Nautilus 107: 124. Haridi AA, et al. (1985). /. Trop. Med. Hyg. 88: 145. Nguma JF, et al. (1982). Acta Trop. 39: 85. Pointier JP (1989). /. Med. Appl. Malacol. 1: 83. Ault SK (1994). Am. J. Trop. Med. Hyg. 50 (Suppl. 6): 35. Epidemic Status of Schistosomiasis in China — A Nation-Wide Sampling Survey in 1995. Office of the Endemic Disease Control, Ministry of Health, People's Republic of China (Beijing, 1998), 80 pp. Webbe G (1986). Ann. Trop. Med. Parasitol. 60: 78. Chimbari MJ, et al. (1998). In press. N'Goran EK, et al. (1997). Bull. WHO 75: 541. Talla I, et al. (1990). Ann. Soc. Belg. Med. Trop. 70: 173. Wilmott S (1987). Trans. R. Soc. Trop Med. Hyg. 81 (Suppl.): 1. Ross AG, et al. (1997). Parasitol. Today 13: 152. Tamein O, et al. (1985). /. Trop. Med. Hyg. 88: 115. King WG (1914). Trop. Dis. Bull. 4: 452.

45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

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63. Cairncross S and Feachem R (1986). The Ross Institute Information Bulletin, No. 10 (Small Water Supplies). London School of Tropical Medicine and Hygiene. 64. Greany WH (1952). Ann. Trop. Med. Parasitol. 46: 250. 65. Farooq M and Mallah MG (1966). Bull. WHO 35: 377. 66. Pitchford RJ (1966). S. Afr. Med. J. 40 (Suppl.): 3. 67. In Human Schistosomiasis, p. 430. Jordan P, Webbe G and Sturrock RF (eds.). CAB International, 1993. 68. Manson P (1898). Br. Med. J. I: 1167. 69. Esrey SA, et al. (1966). Bull. WHO 69: 609. 70. Chandiwana SK, et al. (1993). Cent. Afr. f. Med. 37: 69. 71. Cairncross S (1988). The Ross Institute Information Bulletin, No. 8 (Small Scale Sanitation). London School of Tropical Medicine and Hygiene. 72. The 1997/98 Zimbabwe National Sanitation Inventory. Department of Environmental Health Services, Harare, Zimbabwe, 1998. 73. Khalil M (1927). Lancet ii: 1235. 74. Korte R, et al. (1986). Trop. Med. Parasitol. 37: 149. 75. Gryseels B (1989). Trop. Med. Parasitol. 40: 134. 76. Guyatt H, et al. (1994). Health Pol. Plan. 9: 385. 77. Southgate VR (1997). /. Helminthol. 71: 125. 78. Murray CJ (1994). Bull. WHO 72: 429. 79. The Global Burden of Disease, Vol. I, 990 pp., and Vol. II, 906 pp. (eds. Murray CJL and Lopez AD). Harvard University Press, 1996. 80. World Development Report, 1993. Investing in Health. World Development Indicators. Published for the World Bank by Oxford University Press, 1993. 81. Proceedings of the G T Z / W H O / A F R O Seminar on Maintenance of Schistosomiasis Control (1989). Trop. Med. Parasitol. 40 (Suppl II): 121. 82. Tanner M (1989). Trop. Med. Parasitol. 40 (Suppl II): 207. 83. Centre d'Etude de Geographie Tropicale/WHO atlas of the global distribution of schistosomiasis. Doumenge J, et al. (1987). Presses Universitaires de Bordeaux, Bordeaux, pp. 400. 84. Abdel-Rahman MS, et al. (1997). /. Eg. Soc. Parasitol. 27: 299. 85. World Development Report 1997. Investing in Health. World Development Indicators. Published for the World Bank by Oxford University Press, 1997. Table 1 — Basic indicators. 86. Tanaka H and Tsuji M (1997). Int. ]. Parasitol. 17: 1465.

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Gryseels B and Polderman A (1991). Parasitol. Today 7: 244. Machado PA (1982). Am. J. Trop. Med. Hyg. 31: 76. Mao S and Shao B (1982). Am. } . Trop. Med. Hyg. 31: 92. Cline, et al. (1983). Am. }. Trop. Med. Hyg. 41: 56. Barakat R, et al. (1995). Trop. Geog. Med. 47: 266. Barakat R, et al. (1995). Trop. Geog. Med. 47: 271. W H O Eastern Mediterranean Regional Office (EMRO) Regional Workshop on Comprehensive Approaches for Schistosomiasis Control (Alexandria, 1993), EM/PDP/29-E/L. Elias E, et al. (1994). Acta Trop. 58: 115. Ministry of Health, Morocco, Program de lutte contre la bilharziose, 1995. Evaluation du program contre schistosomiase en Tunisie 1-30 Juin, 1994 (L Rey). WHO report EM/PDP/35/F/09.94/24. Baccush MM, et al. (1993). /. Eg. Soc. Parasitol. 23: 527. Traore M, et al. (1998). Am. J. Trop. Med. Hyg. 59: 407. Brinkmann UK, et al. (1988). Trop. Med. Parasitol. 39: 157. Picquet M, et al. (1996). Trans. R. Soc. Trop. Med. Hyg. 90: 340. Urbani C, et al. (1997). Med. Trop. 57: 157. Stelma FF, et al. (1997). /. Infect. Dis. 179: 304. Scott D, et al. (1982). Bull. WHO 60: 89. Engels D, et al. (1994). Acta Trop. 58: 127. Savioli L, et al. (1989). Trans. R. Soc. Trop. Med. Hyg. 83: 803. Dhunputh J (1994). Trans. R. Soc. Trop. Med. Hyg. 88: 507. Boisier P, et al. (1995). Trans. R. Soc. Trop. Med. Hyg. 89: 208. Serieye J, et al. (1996). Trans. R. Soc. Trop. Med. Hyg. 90: 398. Mqoqi NP, et al. (1996). S. Afr. Med. }. 86: 76. Ali MI, et al. (1989). Trop. Med. Parasitol. 40: 195. Schutte CHJ, et al. (1995). Ann. Trop. Med. Parasitol. 89: 631. Schutte CHJ. Report for the Ministry of Health and Social Services

(Namibia, 1998), unpublished. 114. Schistosomiasis in Brazil. Internal Fundacao Nacional do Saude (FNS) document (Brasilia, 1997), unpublished. 115. Incani RN (1987). Mem. Inst. Oswaldo Cruz 82 (Suppl. 4): 89. 116. Ruiz-Tiben E, PAHO Consultation, Ministry of Health, Republic of Surinam, June 1986, internal document.


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Chapter 13 Progress in Vaccine Development Stephanie L James and Daniel G Colley

Rationale for Vaccine Development The Seventh Programme Report of the UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, covering the period 1983-84, declared that schistosomiasis was a disease "that is still spreading, in association with water and agricultural development projects, in the 74 countries where it is endemic and where over 600 million people are exposed to the risk of infection and an estimated 200 million more are actually infected." With the widespread introduction of the safe, effective, and reasonably priced anthelmintic drug praziquantel in the mid-1980's, there was great hope that this ancient disease would finally be conquered. The WHO developed a strategy for schistosomiasis control, endorsed by expert committees in 1985 and 1991 (1), based on provision of diagnosis and treatment at the primary health care level, selective or mass chemotherapy, health education, control of the snail intermediate hosts, safe water supplies, and environmental management, which was expected to result in a 40% decline in the prevalence of disease by 1995 (2). Nevertheless, while the distribution may have altered somewhat, the estimated prevalence of schistosomiasis remains almost unchanged today (2, 3). Thus, while good tools are available in principle, control of this parasitic infection has proven difficult to sustain, especially in Africa (3). In addition, man-made environmental changes adjunct to water resource development have resulted in increased transmission in several areas of Africa (3), and pose a threat for other regions where schistosomiasis remains endemic (4).

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To effectively control morbidity, chemotherapy must be repeated at regular intervals. Because the impact of chemotherapy on transmission appears limited (5), reinfection often follows treatment. Annual chemotherapy, even when targeted at the most vulnerable population, can be costly to maintain in terms of both drug cost and human resources. In schistosomiasis japonica, rapid rebound in morbidity following discontinuation of chemotherapy has been reported (6). Furthermore, as selective mass chemotherapy programs are being extensively implemented in more regions, reports of parasite resistance or tolerance to praziquantel (7, 8) are raising a warning signal against complete reliance on this control method. It is generally agreed that integration of complementary strategies is the best means of achieving disease control (3). There are several reasons to believe that vaccination could be an important component of a future integrated control strategy. Partial immunity against schistosomiasis develops naturally in individuals living in endemic areas (9) and can be readily induced in experimental animal models by prior exposure to radiation-attenuated parasites (10). Because morbidity is believed to be related to egg production (11), even a vaccine that is only partially protective could still be beneficial if it reduces the number of eggs retained in the tissues a n d / o r the deleterious immune responses mounted by the host against those eggs.

Potential Vaccination Approaches Because of the relationship between egg production, egg-directed immune response, and disease, a vaccine against schistosomiasis might take several forms. The most obvious, because it would work similarly to most vaccines currently on the market, would be an anti-infection vaccine. This type would target protective immune response(s) against the infective or migratory stages of parasite larvae, which would reduce their numbers before they were able to mature into egg-laying adult worms. A second type, termed an antifecundity vaccine, would work to prevent adult parasites from producing eggs. A third possibility, called an antipathology vaccine, would redirect the host immune response in such a way that it would fail to stimulate production of the harmful fibrous lesions leading to disease, while still protecting the host against toxins released from eggs trapped in the tissue. Recent research in animal models of schistosomiasis has provided examples of each of these

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potential vaccine types. Another somewhat different concept, which is being explored in S. japonicum infection because of the role of domestic livestock in transmitting this infection to man, is a veterinary vaccine that would decrease egg excretion by infected animals and thus reduce human exposure.

Anti-infection Vaccines In experimental animal models of schistosomiasis, anti-infective vaccines are typically assessed by portal perfusion and counting of the actual number of adult parasites developing from a quantitative challenge infection. Thus, in this case, a finding of 50% protection indicates that on average the vaccinated group harbors half as many parasites as the appropriate unvaccinated control group. "Sterile immunity," i.e. absolute protection against challenge infection, has been achieved only rarely and sporadically, and it is usually difficult to verify whether such a result in an individual animal reflects a technical problem with the infection. It is believed, however, that any substantial reduction in worm numbers will be reflected in a reduction in the numbers of eggs trapped in the tissues, and thus a reduction in morbidity.

Attenuated

Vaccines

The best-studied animal model of protective immunity against schistosomiasis is the radiation-attenuated cercariae vaccine in the mouse. Mice are permissive hosts for S. mansoni and S. japonicum (12), meaning that they allow full maturation of the parasite from infective cercariae to egg-laying adults. Much as live attenuated polio vaccine stimulates strong immunity against subsequent polio infection, infection with attenuated cercariae induces high levels of resistance to subsequent schistosome infection. In some mouse strains, the level of protection against S. mansoni reaches 60-75%, as assessed by reduction in the number of adult worms developing from a challenge infection (10). Somewhat lower levels are reported in mice with an attenuated S. japonicum vaccine (13). The mouse is not a permissive host for S. hematobium, but immunization with irradiated cercariae has also been reported to protect in this, as well as other, experimental hosts (14, 15). Interestingly, despite antigenic homologies that will be discussed below, cross-protection between schistosome species is inconsistent. Immunization with irradiated S. mansoni

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cercariae does not protect mice against subsequent S. japonicum challenge (16). Immunization with attenuated S. mansoni cercariae has been reported to protect mice against S. hematobium, but not vice versa (14). Unfortunately, unlike attenuated viral vaccines, an attenuated schistosome vaccine is not practical for human use. Because schistosome parasites cannot be grown in vitro, it is impossible to achieve either the quantity or the level of sterility that would be required for this purpose. Maintaining the viability of attenuated parasites, as would be necessary for wide-scale vaccine distribution, would also present a virtually insurmountable obstacle. Nevertheless, a great deal has been learned about mechanisms of protective immunity using the irradiated cercariae vaccine, although much still remains imperfectly understood. Research on the S. mansoni model has led to the conclusion that elimination of challenge infection in mice that have been immunized with irradiated cercariae is largely manifested as larval parasites migrate through the lungs on their way to the liver (10). In mice that have been vaccinated by a single exposure to attenuated cercariae, protective immunity is IFNydependent (17, 18). Treatment with IL-12, a cytokine that stimulates production of IFN7 and other Thl-type immune responses, at the time of vaccination enhances the level of protection against challenge infection achieved in this model (19,20). Indeed, this is one of the rare situations where complete protection has been reported. Several lines of evidence point to a role for nitric oxide, produced by IFNy-activated macrophages a n d / o r endothelial cells, as an effector molecule of protective immunity (21). Nitric oxide kills both skin-stage and late lungstage larvae by the inhibition of vital metabolic processes (22), and is produced in the lungs of vaccinated mice within the cellular inflammatory foci that form around challenge larvae (23). Nevertheless, recent results showing only approximately 30% abrogation of resistance in mice that are genetically deficient in the inducible nitric oxide synthase indicate that this is not the only potential mechanism of resistance (24, 25). Interpretation of results in nitric-oxide-deficient mice is complicated, however, by the extraordinarily high levels of IFNy and TNFa that these animals produce in the absence of normal nitric oxide feedback regulation (25). Such high cytokine levels could participate in additional resistance mechanisms not normally invoked by vaccination. In this regard, a direct role for IFNy in resistance to challenge infection has also been hypothesized, such as through up-regulation of the expression of cellular adhesion molecules that might contribute to entrapment


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of challenge larvae within the lungs of vaccinated mice (18). Results in nitricoxide-deficient animals, showing decreased resistance in the presence of greatly increased IFNy as well as TNFa levels, argue strongly against a direct role for these cytokines. A more recent study has revealed that mice which are both IFNy- and B-cell-deficient fail to demonstrate resistance to challenge infection after a single exposure to irradiated cercariae (26), indicating that antibodies as well as cell-mediated immune mechanisms participate in protection in this model. The mechanism through which the antibody functions is unclear, as these same studies revealed no requirement for cell signaling through the Fc receptor. One possibility is that the antibody serves to increase the binding of effector cells to the larval target, thus maximizing exposure to toxic effector molecules. Multiple exposures to attenuated S. mansoni cercariae only slightly increase the level of resistance to challenge infection. Interestingly, however, hyperimmunization alters the balance of cytokine production such that Th2-type response becomes more prominent (27). Although IFNy production and macrophage activation can still be demonstrated in these mice, antibody reactivity is substantially increased and serum IgGl can transfer resistanc (as assessed by decrease in challenge worm burden) to naive animals (27-29). IL-12 treatment also augments resistance in this model, enhancing both Thl and antibody responses (19). Multiple immunization fails to increase resistance in mice deficient in B cells, while the absence of IFNy production inhibits resistance to a lesser, although still significant, degree (26). Thus, it appears that both cellular and humoral immunity are involved in the resistance induced by multiple vaccination with attenuated cercariae, with antibody-mediated responses playing a larger role than in single immunization.

Defined

Vaccines

Several groups are working on the discovery of schistosome antigens that might serve as vaccine candidates. At this point, however, few have been shown to be protective in more than one laboratory. Insights gained in the vaccine model based upon exposure to irradiated cercariae have stimulated attempts to activate protective cell-mediated immune mechanisms using nonliving parasite antigens. Significant levels of resistance, up to 50% reduction in numbers of adult worms developing from challenge infection, are obtained

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following one or two intradermal injections of dead S. mansoni schistosomula, or the saline-soluble components of larval or adult worms, together with BCG as an adjuvant (30). As in irradiated cercariae-vaccinated mice, resistance in this model correlates with IFNy production and macrophage activation (21). Within the complex mix of parasite components used in this non-living vaccine model, a protective defined antigen, schistosome paramyosin, has been identified (31, 32). Paramyosin, a 97 kDa myofibrillar protein, appears localized to regions just below the tegumental and gut syncitia of adult S. mansoni worms (33). Immunization with S. mansoni paramyosin induces IFNy production and macrophage activation. In this vaccine model, it has been postulated that paramyosin induces secondary immune response in vaccinated animals as a result of its release from migrating larvae of a challenge infection (31). In considering the concept of vaccination based on Thl cell-mediated immunity, it is important to recognize that activated macrophage effector cells do not need to recognize antigens on the surface of the challenge parasites. Rather, IFNy produced locally by T lymphocytes in response to antigens released from challenge parasites could stimulate nonspecific macrophage cytotoxicity through the release of nitric oxide or other effector molecules. Paramyosin has also been identified as a candidate vaccine antigen for S. japonicum (34). Interestingly, in this species, paramyosin has been localized in the postacetabular gland as well as the tegument and muscle layers of larvae, leading to speculation that paramyosin is actually secreted (35). One study has also reported finding paramyosin on the surface of lung-stage S. japonicum schistosomulae (36), in which case a role for antibody-mediated protective mechanisms might also be envisioned. More recently, another potential vaccine antigen has been identified in the S. mansoni model employing BCG plus larval antigens. A T cell clone, characterized as Thl-like based on its cytokine production profile, was shown to transfer protection against challenge infection to naive mice. This clone recognizes schistosome calpain, a Ca +2 -activated neutral proteinase (37). Vaccination with

a recombinant vaccine, composed of the large subunit of calpain expressed in baculovirus, results in substantial reduction of the challenge worm burden in mice (38). Another candidate for an S. mansoni anti-infective vaccine antigen that has been postulated to function at least in part via Thl-cell-mediated immune mechanisms is the glycolytic/gluconeogenic enzyme triose phosphate isomerase (TPI). This antigen was originally identified by a monoclonal antibody


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that could transfer resistance to naive mice and inhibit the enzymatic activity of the antigen (39). Because the parasite molecule has high (79-87%) sequence homology to the mammalian enzyme, a vaccination approach based on multiple antigenic peptides (MAPs) constructed of T and B cell epitopes that do not cross-react with the host enzyme is being pursued for TPI (40).

Several investigators have used antibodies from mice hyperimmunized with irradiated S. mansoni cercariae to identify candidate vaccine antigens (41, 42). An early study noted the unique or enhanced immunogenicity of a series of adult worm glycoproteins, ranging from 43 to 200 kDa (41), of which the 200 kDa antigen is of particular interest. A recombinant protein of 62 kDa, representing a portion of the native 200 kDa antigen, stimulates significant resistance to challenge infection in mice, as measured by reduced worm burden (43). This antigen, designated rIrV-5, has been defined as schistosome myosin and shares homology with myosins of other species (43). Recombinant IrV-5 also demonstrated some protective potential in baboons, when used in micelle or proteosome formulations, but the range of protective activity was quite wide in this outbred population (44). Another antigen, designated IrV-1 and found to have similarity to the molecular chaperone calnexin, was identified in the same manner (45), but its protective potential has not been documented. An integral membrane protein, termed Sm23, is also recognized by antibodies from mice vaccinated with irradiated cercariae (42). This antigen, detected in all stages of the parasite found in the mammalian host, is a member of a superfamily of membrane antigens found primarily in hemopoietic and malignant cells (46). An S. japonicum homologue of Sm23 has also been cloned (46). DNA vaccine approaches are being explored for both S. mansoni myosin and Sm23 (47). Another vaccine candidate antigen, Sml4, originally recognized in saline extracts of adult worms, has been found to partially protect mice against challenge infection (reduction in adult worm burden) when administered -with complete Freund's adjuvant (48). In addition, this immunization procedure was reported to decrease liver damage due to eggs produced by surviving parasites. The mechanism of action of this vaccine is not yet clear. Sml4 is a fatty-acid-binding protein, with homology to a Fasciola hepatica antigen that has also been proposed as a vaccine antigen. Indeed, Sml4 has been reported to protect against F. hepatica infection in a mouse model, and is being proposed as a dual purpose anti-helminth vaccine (48). The homologous S. japonicum fatty-acid-binding protein has been localized within lipid droplets

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below the subtegumental region of the male worm, and within the vitelline glands of the female (49).

Antifecundity Vaccines The concept of an antifecundity vaccine arose indirectly from early research in a rat model of schistosomiasis mansoni. The rat is a nonpermissive host for this parasite, in which most infective cercariae fail to survive and surviving parasites develop into stunted forms incapable of egg-laying (12). Prior vaccination with irradiated cercariae speeds up the elimination of a challenge infection (50). The consensus of several studies is that immune elimination of challenge parasites in the rat-attenuated vaccine model occurs in the lungs (10). Sera from rats vaccinated with irradiated cercariae transfer strong protection against challenge infection, implicating antibody-mediated immunity in this model (51). Early studies suggested that antibody-dependent cell-mediated cytotoxicity is central to the immune mechanism of resistance to S. mansoni in the rat (52). Eosinophils, macrophages and platelets were identified as effector cells of in vitro larval killing, acting under the direction of IgE and also, in the case of eosinophils, IgG2a antibodies (52). Later studies aimed at identifying protective antigens reported that immunization of rats with an antigenic fraction purified from schistosomula, containing 25-30 kDa molecules, conferred protection against challenge infection through a mechanism requiring IgE (53). Ultimately, these studies led to the identification of Sm28, glutathione S-transferase (GST), as a candidate vaccine antigen. The 28 kDa GST is localized in the parenchyma of the schistosomulum and adult worm, including the dorsal spines, and is present in S. mansoni, S. japonicum, S. hematobium and species of bovine schistosomes (54). Depending on the method of immunization, purified native or recombinant Sm28 protects rats against challenge infection, measured as reduction in worm burden, while inducing high levels of IgG, IgE and IgA production (54, 55). Interestingly, in permissive hosts such as mice and cattle, immunization with 28 kDa GST was also found to reduce egg production by surviving parasites from a challenge infection (56, 57). Immunity in cattle is believed to involve antibody-dependent mechanisms (56), while experiments in mice suggested that IFNy plays a role (57). Further studies revealed that antibody inhibition of GST enzymatic activity correlates with reduction of female w o r m fecundity and egg viability. In mouse


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immunization experiments using various peptide epitopes of Sm28, the carboxyl terminus of the molecule was associated with decreased worm burden, while both the amino and carboxyl terminal portions reduced tissue egg deposition and egg viability (58).

Antipathology Vaccine This unusual vaccination approach is aimed not at the parasite, but at the host. The idea of an antipathology vaccine against schistosomiasis was first broached when the immunologic nature of the disease became recognized through studies in the mouse model of S. mansoni infection (59). The inflammatory cellular reaction (granuloma) forming around schistosome eggs deposited in the mouse liver resolves into a fibrotic lesion causing impaired hepatic portal blood flow and bleeding. While the exact relationship of the murine lesion to the extensive fibrotic lesions observed in severe human hepatosplenic disease remains controversial (12), the mouse model has provided significant insights into the immunopathology of schistosomiasis. Early studies, performed at a time when the existence of various subsets of T lymphocytes with different biological functions was not fully appreciated, likened the schistosome egg granuloma to the cellular reaction against tubercle bacilli and thus reported that it represented a delayed hypersensitivity response (60). Nevertheless, the analogy to other delayed hypersensitivity responses was always recognized to be less than perfect because of the unusual numbers of eosinophils present in the antiegg lesions. It is now understood that the egg granuloma is a complex and constantly evolving immunologic entity which is largely controlled by CD4+ T cells (61). Egg deposition by the adult worm pairs provides the major stimulus for induction of Th2-type immune response in murine schistosomiasis (62), and the U n related cytokine IL-4 is abundant in granulomatous livers (63). Inhibition of the Th2-related cytokines IL-4 and IL-5 inhibits hepatic pathology and eosinophilia, respectively (64,65). Finally, it has been shown that mice which lack Th2 cell function (Stat6-deficient mice) produce only small egg granulomas, almost entirely devoid of eosinophils and with greatly reduced fibrotic potential (66). Together, these observations support the concept that diversion of the antiegg immune response away from Th2 reactivity and toward more Thl-like reactivity would result in the abrogation of disease even in the face of ongoing infection. This concept has been brought to fruition,

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at least in the mouse model of schistosomiasis, using the approach of vaccination with egg antigens plus IL-12. Prior exposure to S. mansoni eggs together with IL-12, which favors induction of Thl-type immunity, partially inhibits granuloma formation and dramatically reduces the tissue fibrosis accompanying subsequent natural infection (67). While this represents a dramatic experimental breakthrough, further development of this vaccine model depends on identification of defined egg antigens that can reproduce the activity of the complex mixture of egg antigens used in these studies. A recent study of B-cell-deficient mice also implicates B lymphocytes in the regulation of granulomatous pathology (68). Animals lacking either B cells or Fc receptors for antibodies display exacerbated egg pathology following S. mansoni infection and fail to develop the spontaneous modulation of granuloma size normally observed in chronic infection. A better understanding of the immune process which normally down-regulates granuloma production in late-stage infection (59) might offer another approach to development of an antipathology vaccine.

Transmission-Blocking Vaccine All of the major human forms of schistosome can complete their life cycles in other animals, particularly rodents (12). It is in schistosomiasis japonica, however, that the role of reservoir hosts seems to most influence human infection. S. japonicum can infect cattle, water buffalo, pigs and dogs — all of which often live in close proximity to man. Some current estimates in China suggest that most of the water contamination by schistosome eggcontaining feces in that country is now due to animal defecation (69-71). Thus, even highly efficient chemotherapy programs might be unable to interrupt the transmission of this parasite in endemic regions. For this reason, development of a veterinary vaccine against S. japonicum is being considered (69). Demonstration of the efficacy of attenuated cercarial vaccines in domestic livestock suggests the feasibility of this approach (70-72), although systematic evaluation of the transmission blocking effects on human infection has not been attempted. In addition to several of the vaccine candidates listed above, many other S. japonicum antigens have been identified and are being tested in various animal models (69). S. japonicum GST (26 kDa) has been reported to stimulate antifecundity immunity, as well as some reduction in worm


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burden, in both pigs and sheep (73, 74). Efforts are also well underway to examine the efficacy of vaccination with S. japonicum paramyosin in domestic animals (75). Further modeling and field studies need to be pursued to more fully determine the utility of transmission-blocking vaccination in reservoir hosts, but it is clearly an exciting possibility that these approaches may be quite useful in S. japonicum control programs.

Insights from Clinical Studies Vaccination studies in animal models offer the advantage of wellcharacterized experimental conditions, including quantifiable parasite exposure as well as hosts that are relatively genetically homogeneous. It remains uncertain, however, to what extent observations of immunity in rodent or even non-human primate models can be extrapolated to man. For this reason, a number of investigators are attempting to identify potential vaccine antigens in the context of human immunity. This tactic presents a series of complexities of its own. Because prepatent or early patent schistosome infections can lead to serious sequelae such as transverse myelitis, especially in persons from nonendemic areas (76), it is generally agreed that ethical considerations preclude experimental human infection. Unfortunately, natural parasite exposure under field conditions is difficult to accurately assess. Moreover, multiple variables related to genetic predisposition a n d / or prior immunologic history play a difficult-to-define role in such populationbased studies. Consequently, our actual understanding of the mechanisms involved in the resistance to schistosomiasis seen in people is much less rigorous than it is for experimental rodent model systems.

Natural and Naturally Acquired Resistance to Infection Even in a highly permissive experimental host, such as the mouse, it is clear that only some of the cercariae to which the host is exposed manage to develop into adult worms, and that this percentage differs among genetically different strains of mice (77). Furthermore, physiologic characteristics of the host, such as gender and hormonal status, can influence the proportion of cercariae that reach maturity (78, 79). Whether these or other such inherent traits play a role in determining "innate" resistance, or its inverse, i.e. susceptibility,

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in people remains entirely unknown. Resistance in humans could theoretically be influenced in a variety of additional ways. In persons from nonendemic areas who have never been exposed to schistosomes, relative resistance to initial exposure could be affected by constituitive factors such as: unique dermal qualities; vascular differences not conducive to worm maturation following cercarial penetration, as seen in some 129/J mice (80); and innate differences in the speed or magnitude in which different types of immune responses are expressed. For persons from schistosome-endemic areas, where frequent water contact and parasite exposure are expected, additional possible influences on how a person responds to infection must be considered. These "endemic-arearelated" factors might include: prior exposure in utero or while nursing to immune mediators a n d / o r parasite antigens because one's mother was infected (81); active immunity due to previous exposure to parasites that did not develop to maturity; prior patent infection which "self-cured" in the manner observed upon initial experimental infection in rats (12, 82). All of these scenarios could impart some degree of acquired resistance. Thus, for persons in any of these categories, it is prudent to use the term "putatively resistant," since previous exposure or lack of exposure by contact with cercariae-containing water is quite difficult to prove. Within this category of putatively resistant persons is a group that has been termed "endemic normals." In the strictest sense, this term should apply only to individuals who: (1) reside in endemic areas; (2) are documented to frequent sites of active transmission; (3) have never been treated for schistosomiasis; and (4) are egg-negative upon multiple stool examinations (83, 84). It is, of course, always possible that such individuals carry nonpatent single sex schistosome infections, have forgotten a previous infection and treatment, or are passing eggs in their stools in such low numbers and so intermittently that they cannot be detected. In spite of these qualifying concerns, it remains of interest that such endemic normal individuals form a rather well-defined, distinct group based on their unique immunologic profile. As a group, they display elevated levels of antibodies to purified schistosome paramyosin compared to patients with patent infections (83). Also, their peripheral blood mononuclear cell proliferative and IFNy production responses to crude schistosomal antigenic preparations from eggs, adult worms or cercariae are considerably higher than those of patently infected patients (84). In addition, endemic normals exhibit higher mean levels of antischistosomular extract


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IgE than IgG4 antibodies, which is completely opposite to the pattern seen in the sera of patients with patent infections (85). As discussed earlier with regard to animal models, human resistance to schistosome infections is likely to be partial or relative, rather than absolute, and may be manifested in different ways. One example would be a resistance that prevents heavy infection, i.e. high worm burdens that may result in high egg production a n d / o r excretion. Another would be an antifecundity resistance that does not interfere with infection, but rather decreases egg production. The latter would be seen as resistance based on stool examinations for eggs, but not when based on levels of circulating, worm-derived antigens. Evidence for what is thought to be the former situation has been provided in an interesting series of studies that demonstrate a genetic predisposition to the development of less intense infections (86). This phenotype maps to the chromosome 5q31-q33 gene region, which is also known to be involved in the coordinate regulation of IL-4, IL-5, IL-13 and IgE expression (87).

Resistance to Reinfection Following Treatment Since the advent and implementation of efficacious, safe treatment for schistosomiasis in the early 1980s, several different groups of investigators have carried out studies in endemic regions to identify immune correlates of resistance to reinfection in patients following treatment. These studies have involved S. mansoni (88-90), S. haematobium (91) and S. japonicum (92) infections and reinfections. In adults, analyses of immune responses to crude schistosome antigenic preparations from eggs, worms, schistosomula or cercariae generally seem to indicate that elevated peripheral blood lymphocyte proliferative responses and eosinophilia correlate with lower reinfection levels (88, 93), as do higher levels of IL-5 and IgE (primarily, but not only, against worm extracts) (93-101). Furthermore, a rise in IgE posttreatment that appears to correlate with resistance often occurs in the face of a decline in IgG4 levels (and sometimes IgG2 and IgM levels) (102-104). It has further been observed in several studies that IgE levels to certain crude (92, 95, 100, 104) as well as defined schistosome antigens such as a 22.6 kDa antigen from S. mansoni adult worms (105,106), correlate with lower intensities of infection or of reinfection after treatment. Also, resistance to reinfection with S. mansoni has been associated with higher levels of IgG reactivity to a 37 kDa larval surface antigen (107).

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Preliminary Immune Correlate Studies with Defined Antigens As described above, several candidate vaccine antigens have been identified through the use of experimental animal models. In an effort to understand human responsiveness to these candidates, humoral and in vitro cellular immune responses to highly purified or recombinant antigens are being evaluated in patients either before and after treatment, relative to reinfection, or in endemic normals (47, 101, 108, 109). In Egypt, Al-Sherbiny, Barakat et al. (47, 108) evaluated responses to six candidate S. mansoni vaccine antigens initially being advanced for further study by the Special Programme for Research and Training in Tropical Diseases at the World Health Organization. Based on preliminary analyses, those patients who were considered to be resistant, because they either did not get reinfected after treatment or were classified as endemic normals, appeared to have high IgA antibody levels against GST, high IL-2, IFNy and IL-5 production against paramyosin, high circulating levels of IgE and IgA antibodies and IFNy production against IrV-5, high IFNy production against a synthetic TPI peptide (MAP-4), high IgG2 levels against a synthetic Sm23 peptide (MAP-3), and high IgG2 and IFNy production against Sml4. Additionally, these initial studies have allowed some estimates of the level of genetic restriction seen in the Egyptian population with regard to these candidates. These types of estimates have also been made for S. mansoni and S. haematobium 42-kDa proteins (glyceraldehyde 3-P dehydrogenase) in Egyptian populations considered to be resistant or susceptible, showing some substantial differences in the proportion of each group which were considered as responders in peripheral blood mononuclear cell proliferation assays (101). Similar studies in China, using several recombinant S. japonkum candidate vaccines (109), showed that some, such as GST, might stimulate proliferation by a high proportion of individuals in a given geographically defined population, but a low proportion of those residing in a different locale. Other such studies are proceeding in various endemic areas, from which it is hoped that a reasonable body of knowledge about the recognition of the leading candidate vaccines will be derived. The resultant genetic restriction information, as well as identification of responses that putatively correlate with resistance, could greatly assist vaccine development programs.


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Points to Consider for Vaccine Trials In his 1993 review, Basch has provided an excellent starting point for consideration of the many realistic aspects of vaccine testing that must be taken into account as this field moves forward (110). He points out major sticking points regarding actual field trials, such as case definition and case ascertainment. He also considers several of the ethical problems to be faced, and identifies the more practical research needs that still await answers, such as immune response definitions and sensitive, quantitative assays for worm burdens and egg outputs. Some of these points are reconsidered below in light of current knowledge.

Measures of Safety and Efficacy All experimental schistosome vaccines have been evaluated for efficacy in animals using immunization, challenge infections and perfusions to determine worm burden differences between immunized and unimmunized groups. It should be reiterated here that currently such studies all presuppose partial protection, i.e. lower worm burdens, to be a reasonable end point. Absolute protection has been difficult to achieve, even under controlled experimental conditions. Clinical efficacy testing cannot be done under such controlled circumstances. Because prepatent or early patent schistosome infections can lead to serious sequelae in previously unexposed individuals, challenge infections that are allowed to proceed beyond a few weeks are generally believed to expose volunteers to unreasonable risk and would therefore not be ethical. Practically, this translates into a need to terminate infection in the study population before it reaches a dangerous stage, which many would regard as being before or just at the point of egg production. There are currently no totally reliable diagnostic tests that would allow an infection to be detected and quantified prior to reaching patency. The necessity to terminate infections based on initial demonstration of infection (i.e. the production of a single egg) means that the clinical end point would be "allor-none" protection. Thus, the partial protection that it is assumed will occur based on the preclinical development of candidate vaccines would go completely undetected. Likewise, it would not be possible to evaluate an antifecundity effect. These concepts challenge the very manner in which Phase 2 and Phase 3 clinical trials can be designed. Whereas safety can be

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preliminarily assessed in previously unexposed individuals, efficacy cannot. How then can a schistosome candidate vaccine, brought through development to Good Manufacturing Practice (GMP) production, and shown to be nontoxigenic and safe, be evaluated for efficacy in people? The answer is — with considerable difficulty and a degree of imprecision. Assuming that efficacy trials must be done in endemic regions, assessing resistance in the face of natural exposure, let us further consider their design. The current tools for determining active infection are parasitologic (demonstration of eggs in the feces or urine) and immunologic (demonstration of circulating worm or egg antigens in the serum, urine or saliva) (111-114). At this time, the "gold standard" remains the demonstration of eggs, but it is clear that this standard suffers from several problems. First, there is considerable intrafecal and day-to-day egg count variation during S. mansoni infection (115). Some recent studies indicate that determination of circulating worm antigens might provide a more stable measure of the level of infection (116), but there remains the question of whether the sensitivity and specificity of these types of assays will be adequate for this purpose when low worm burdens are to be anticipated. A further potential difficulty with egg counts has been uncovered in studies of egg counts vs. circulating worm antigens in immunocompromised patients (117). These studies show that with comparable worm burdens, based on circulating antigen levels, patients' egg outputs vary depending on their peripheral blood CD4+ lymphocyte levels, i.e. efficacious egg output appears to be dependent upon immune response capabilities, as suggested in studies with experimental animals (118, 119). While this problem might be controlled for in a Phase 2 or Phase 3 trial by excluding individuals who are immunocompromised, it might also be necessary to determine whether variable prior exposure and/or immunization with vaccine candidates influences the "egg-excretion-related" immune response. Furthermore, since effective treatment with praziquantel is "immunedependent" (120-122), it will also be important to know if the vaccine induces

this capability as well as protection against infection. Even with the above-mentioned difficulties of determining accurate egg outputs and verifying their relationships to actual worm burdens, egg production is still likely to be one of the major measures of vaccine efficacy applied in the setting being discussed. It is equally likely that worm burden determination by circulating antigen levels will be used, but investigators should plan ahead for how they will deal with the potential discrepancies


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that may occur using these two measures. If partial protection is to be detected, both measures must be able to compare the levels of infection that develop with or without vaccine exposure. However, this can only be done if the time from vaccination until testing (during which natural exposure and worm maturation will have occurred) is sufficient to allow the stable development of infection to measurable levels in those being potentially exposed. The length of time for this to occur will depend on the transmission rate (incidence of infection) in the area where the extended Phase 2 or Phase 3 studies are being done. Due to factors such as seasonality of transmission, this will most likely require at least a year between evaluations of infections. Investigators and public health officials familiar with schistosomiasis seem to agree that since this is the minimal routine timing between administration of chemotherapy in any regional control programs in endemic areas, such a period between evaluations would not pose added health risks to those in the clinical trial. Nevertheless, this facet of such trials will need careful consideration, and may differ for the different species, or even different strains of parasite (123), as well as different locales. Because of the chronic nature of schistosome infection, serious, recognizable clinical disease takes years to manifest itself. It therefore seems unrealistic to expect that clinical end points of morbidity would be useful in the design of an efficacy trial for this infection. Nonetheless, reinfection is likely in areas where the vaccine would be tested. Moreover, the partial immunity that is expected to be engendered by any schistosome vaccine would leave open the possibility of future clinical disease. Because the vaccine could conceivably influence subsequent development of clinical disease (positively or negatively) in a way not directly correlated with infection (for example by altering the immunologic milieu), it will be important that any vaccine trials done on people in endemic areas be linked to long-term followup studies of morbidity. Thus, formal safety studies might need to extend through multiple reinfection cycles.

Use of Vaccines in Control Programs While it is obvious that the more efficacious an eventual vaccine is the more impact it may have on limiting transmission, infection and disease, the actual efficacy levels required to make a useful impact are unknown. The same is true in regard to the duration of protection provided by eventual

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vaccines. In part, both vaccine efficacy and the duration of this efficacy will depend on how, and in what setting, a given vaccine will be used. It is assumed by most investigators that a schistosome vaccine will initially be tested in populations, including both adults and children, in endemic areas where transmission is still occurring. This means, in practical terms, that most of those who will receive the candidate vaccine will have been previously infected and treated for their infection. It must be acknowledged that essentially nothing is known regarding the consequences of vaccinating infected, or previously infected and recently treated, persons. In fact, only one set of studies has even tried to look at this situation in regard to irradiated cercarial vaccination in infected and treated mice (124). In these studies, which were limited in terms of both duration of infection and time periods after treatment, it was seen that prior infection and treatment with praziquantel neither interfered with nor augmented eventual resistance levels following vaccination with irradiated cercariae. In a recent study it was reported that chemotherapy for S. haematobium infection leads to a switch from a mainly IgA-specific antibody response to a predominantly IgGl response within 12 weeks of treatment (125). This is the same type of immunologic switch that occurs in infected persons as they reach adulthood, and that correlates with the development of resistance. It is hoped that such an occurrence upon treatment might poise those to be vaccinated for a more rigorous, protective immune response. Whether prior treatment and the resultant massive systemic release of parasite antigens have an inhibitory or potentiating effect on vaccine-induced resistance may, however, depend on the nature of the protective response which the vaccine endeavors to elicit. For example, it has been shown in mice that the route of antigen exposure substantially influences the balance of cytokine response (38). The most appropriate time to wait between treatment and vaccination is totally unknown and will most likely be decided pragmatically. The effect of prior infection on monitoring vaccine immunogenicity may also prove to be a scientific challenge, given pre-existing immune reactivity in these individuals.

Testing an Antipathology Vaccine Some of the most elegant immunologic studies done in experimental and clinical schistosomiasis have been focused on the pathogenesis of the disease (126, 127). It appears that most of the morbidity and mortality associated


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with schistosomiasis is due to unregulated immune responses against the parasite, particularly the egg stage (128, 129). An often-stated rationale for studies to better understand the immunopathogenesis of schistosomiasis is the development of an antipathology vaccine. There are many reasons to study the immunopathogenesis of this fascinating infection, which is the epitome of chronic antigenic exposure. The past, current and future studies of immunopathogenesis and immunoregulation of immune responses involved in morbidity and mortality in schistosomiasis are both immunologically interesting and useful. Such studies have already taught us much about the interactions between immune responses and the changing nature of immune response profiles during chronic antigenic exposure. This type of information is likely to be essential in furthering our understanding of a multitude of immunologically focused chronic diseases, from chronic microbial infections to autoimmune diseases, to graft-vs.-host disease and transplantation, to neoplastic disease. Upon close inspection, however, the development of an antipathology vaccine would pose a practical nightmare. Clinical testing of the efficacy of an antipathology vaccine cannot be ethically undertaken when a good, safe, curative drug exists. It would be totally unacceptable to immunize certain people but not others, allow the groups to become infected, and then wait the decades needed to determine if the vaccine really prevented the development of morbidity. The only possible hope for the future clinical testing of such a vaccine would be by definition of a correlate to morbidity that becomes elevated early in infection and is reliably predictive of the subsequent development of clinical disease. The search for such correlates of morbidity and mortality may well have an additional payoff in schistosomiasis patient management. Since only a relatively small percentage of schistosome-infected persons progress to lifethreatening disease, an ability to predict who those individuals might be would allow focused case management efforts. Furthermore, with regard to vaccine development, if such morbidity correlates were known, it might be most useful to be able to include their measurement as a component of the safety/immunogenicity testing of all vaccine candidates.

Practical Issues in Vaccine Development Taking an experimental candidate vaccine forward from the research stage, through development (scale-up, standardization and production), to even the

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Phase 1 clinical trial phase (safety and limited immunogenicity testing) is a very expensive and challenging process. Moving from multiple Phase 1 clinical trials to Phase 2 (widespread safety and immunogenicity and limited efficacy) and Phase 3 (population-based efficacy) clinical trials with one or more promising candidates is a truly daunting task, both financially and logistically. The biopharmaceutical industry usually approaches such ventures expecting to invest $250,000,000-450,000,000 before marketing a vaccine. Given the current, nonexistent state of "market impact survey" information concerning the potential use of a schistosome vaccine, it is totally unrealistic to expect large-scale industrial interest in funding such a vaccine development program. It seems clear that if this process is to be pursued it must be done on a much lesser scale and through "patchwork" funding efforts from multiple concerned organizations. Two such efforts are currently moving forward. Through a bilateral agreement the Ministry of Health and Population (MOHP) of the Government of Egypt and the United States Agency for International Development (USAID) have agreed to begin the process of scaleup, standardization, production, and safety and immunogenicity evaluation. The initial focus is on two current candidates, paramyosin and TPI MAP4 of S. mansoni. This effort, called the Schistosome Vaccine Development Program (SVDP) (47, 130), will be done with the help of multiple partners, and is expected to take at least three years before Phase 1 trials are undertaken. The early phases will hopefully be done for between $4,000,000 and $8,000,000, which will depend on annual progress and the availability of funds. Other goals of the SVDP include: development of plans for Phase 2 and Phase 3 clinical trials in Egypt, and establishment of an Egyptian clinical vaccine testing and evaluation unit patterned on those currently funded by NIAID/ NIH. A second vaccine development effort, focusing on S. haematobium GST, is being actively pursued by Dr. Andre Capron and his colleagues, through the combined efforts and funding of the European Union and the Institute Pasteur (131). This program has progressed through initial successful Phase I studies in France, and is in the midst of Phase I trials in Senegal. It has been estimated that a vaccine with high efficacy and long duration of protection could compete favorably with chemotherapy as a general control method (132). However, based on the static model used for this prediction, this would require that the vaccine be effective following a single immunization and be available for a cost of approximately US$5. Through mathematical modeling it has also been pointed out (M. Woohouse in Ref. 47) that even


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with the availability of a good vaccine, it would be best used in conjunction with mass chemotherapy, and would take many years before its full impact could be realized. Based on the models, the best overall results could be expected from combining the initial impact of chemotherapy campaigns with the long-term impact of an efficacious vaccine.

Caveats and Conclusions In May 1997 a series of meetings was held in Cairo, Egypt, to address questions regarding the status of the development of vaccines against schistosomes and to discuss the suitability of potential avenues of attack (47). The discussions at that meeting, and those among investigators working on schistosomiasis around the world, have weighed the pros and cons of moving forward from vaccine research to vaccine development. The arguments center on whether it is time to move forward in a directed manner with some of the candidates we now have at hand, or whether to continue to put strong emphasis on discovering and manipulating new candidates. Perhaps the question is best framed by determining what is lost versus what is gained in terms of the goal of developing a vaccine for schistosomiasis if the field moves ahead to development now, or continues to emphasize research on this topic. What might be lost by pushing ahead to develop current candidates through GMP, toxigenicity testing, Phase 1, and possibly Phase 2, clinical trials? If the selected candidates prove very difficult to obtain in GMP form, if they are found to be toxic, or if they fail to induce reasonable immune responses in Phase 1 clinical trials, the vaccine effort on schistosomiasis could stand to lose credibility. While this might be true, the history of vaccine development surely tells us that many candidates must go through such trialand-error assessments before a suitable vaccine is developed. Therefore, it would seem that this argument will always be present, but should not hold sway in decision making. Given the tenuous funding situation for vaccine development and our current lack of understanding of the basis of protective immunity in humans, it is possible that those candidates which fail upon initial assessment will lose credibility and not be pursued even though they might have been effective in another formulation. This would surely be a blow to those scientists who have "championed" those particular antigens.

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Probably uppermost on most scientists' minds, however, is the possibility that the community stands to lose money if the chosen candidates fail. Even well-focused efforts to push current candidates forward through to Phase 1 clinical trials will require substantial sums of money, and it can be credibly argued that if the current candidates are not truly appropriate, this would be better spent on discovery-based research on other candidates. What might be gained by pushing ahead to develop current candidates through to Phase 1 and Phase 2 clinical trials? Obviously the best possible outcome would be a vaccine. Nevertheless, the history of vaccine development tells us that the schistosome community would be very fortunate, indeed, were it to stumble upon a successful vaccine with its first or second candidate to go forward. Thus, it would seem that the question that should actually be put forward might rather be, What might we gain (if anything) if we move forward and the candidates chosen fail to become reasonable vaccines? (1) At this time, very few of those involved in schistosome vaccine research know anything about vaccine development. If a focused vaccine development effort is carefully done and well controlled, the community will almost certainly learn a great deal about vaccine development. This will include finding out where the pitfalls are and how to anticipate and avoid them. It will involve learning a great deal from our industrial and biotechnology colleagues, as well as those in the regulatory business. (2) In addition to those things the community will learn through the process, pushing ahead will require that many of those in the community pull together and determine the things outlined above about how one would actually test a schistosome vaccine. Certainly, without the impetus of a vaccine trial these critical questions continually fail to be addressed. Are there ways to evaluate antipathology vaccines? Can we use egg excretion data as a realistic end point for reinfection? Will natural exposure yield the type of evidence needed to convincingly show the way forward? If so, how long after natural exposure can one wait to develop reasonable reinfection data? Are the types of vaccines we have been developing suitable for moving forward, or must the community begin to think of how a vaccine will be used before trying to discover one? (3) It is quite likely that the field of schistosomiasis will gain important insights into human immunity which will inform future research efforts. Is it not wise at this point, after decades of speculation on the applicability


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of observations in various rodent models, to attempt to determine directly whether they are predictive of human response? Would it not be beneficial, not just in schistosomiasis but for vaccine development more generally, to determine whether vaccination can alter or even redirect an already established pattern of immune responsiveness? (4) Lastly, pursuit of GMP candidate vaccines and their appropriate collaborative clinical testing with those in endemic countries could have a very positive impact on those countries. Endemic countries and their scientists stand to gain considerably in terms of developing the infrastructures and skills to have both GMP capable facilities and the personnel and means to pursue high quality clinical research. The discussion could go on and on. When is it ever time to shift some degree of emphasis from discovery research to developmental research? For vaccines with an attractive commercial market, this decision is usually made by industry. In the case of schistosomiasis, the question has been posed to a research community that is almost completely naive about clinical development and, truthfully, somewhat self-invested in mamtaining the status quo. The answer is not that one is better than the other, but rather that both approaches are needed. Clearly, more candidate discovery is a good thing, and will most certainly follow in this age of genomics and proteomics. For no one can know whether any current candidate vaccine will prove to be producible in realistic quality or amounts, or be safe, or be immunogenic, or be all of those yet not be efficacious in inducing protective resistance. Nevertheless, while we have no suitable in vitro correlates or in vivo models known to faithfully reproduce the immune setting found in people, the need for a vaccine exists and a strong case can be made for now moving to the next phase with some of the current candidates. This needs to be done in a solid, careful manner, so that we can learn as much as possible along the way. We propose that it is time to forge ahead toward schistosome vaccine development under a banner of "cautious enthusiasm" in the context of a "steep learning curve."

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Chapter 14 Immunology of Schistosomiasis: Towards a Consensus Viewpoint Alan Sher and Adel AF Mahmoud

Introduction The chronic nature of most helminthic infections as exemplified by the schistosomes offers an opportunity to understand several unique features of the pathogen and host responses. As the schistosome establishes itself in the definitive host, it presents a complex array of antigens produced by several stages of the parasite as well as the prolonged persistence of two stages of its life cycle: adult worms and eggs. As an example of the complexity of host immune responses, contrast the immunologic recognition of parasite adult worms and ova with the lack of local expression of these responses to the former but not the latter stage of the pathogen. Finally, the outcome of this protracted association with the host is a set of clinical manifestations that relate directly or indirectly to the nature of host-parasite interplay. Indeed, the ultimate clinical manifestation of infection and disease in human schistosomiasis is multifactorial in etiology. This dynamic appreciation of schistosomiasis is an outcome of remarkable progress in our understanding of the infection and its disease sequelae. In this chapter, we will focus on issues that significantly impact our current scientific appreciation to the perceived need for a quantum leap towards the ultimate goal of control.

Acquired Immunity in Humans The debate on acquired immunity to schistosomiasis in humans has been a constant but confusing feature over the past several decades. Part of the

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confusion stems from the attempt to appreciate a chronic helminthic infection in the same way a bacterial, viral or protozoan infection is appreciated. Furthermore, the 1970's brought about two fundamental quantitative approaches to studies of schistosomiasis, including egg counting and evaluation of water contact (2, 27, 42, 43). By the 1990's the genetics of the host and of the schistosomes have been regarded as fundamental parameters regulating infection and disease (5, 29). Few fundamental concepts have to be agreed upon before examining the issue of acquired immunity. Infection with any of the five schistosome species in humans is usually asymptomatic, the exception being S. haematobium. Indeed, disease sequelae of schistosome infection occur in a relatively small subset of infected individuals. Acquired immunity in humans may, therefore, be defined at the susceptibility-to-infection level — who gets infected, and with what intensity? — or at the clinical level — who gets the disease? Is it the strain or species of parasites? Is it the intensity of infection? Is it host genetics? Is it other associated infections or conditions? Or, is it all of the above? Most of the studies prior to introduction of quantitative methodologies for evaluation of infection and disease are difficult to interpret. Over the past two decades, the value of examining the pattern of reacquisition following successful chemotherapy has been appreciated (32). Although the answer is equivocal, the evidence tilts on the side of demonstrating acquired immunity to reinfection in several communities in endemic areas (14, 32, 38). Our equivocation is warranted because of the difficulties involved in these studies and the absence of comprehensive assessment of other factors in all of these studies. Susceptibility to disease is another complex phenomenon. Whether there is acquired immunity on repeated exposure to infection is unknown. It is the chronic nature of infection and the prolonged duration necessary for the expression of disease that prevent an accepted experimental protocol. Data over the past several decades, however, have indicated possible susceptibility or resistance patterns influencing the expression of disease in schistosomiasis. The evidence was originally based on clinical population-based observations, e.g. race or blood groups. Then epidemiological and histocompatibility antigen evaluation indicated certain associations (1, 34, 39); this is now being confirmed by detailed genetic analysis. The issue of proving or disproving acquired immunity to disease in schistosomiasis can become frustrating because of the lack of scientific evidence. Fortunately, experimental observations

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in multiple species of hosts provide the basis for our current understanding of acquired immunity and afford the opportunity to examine surrogates for human protective responses (33, 47).

Can Human Immunity Be Modeled in Experimental Animals? A great advantage of studying immunity to schistosomes as opposed to many other human pathogens is the broad vertebrate host range of two of the major species, Schistosoma mansoni and japonicum. This property of the parasite has allowed the development of a series of excellent animal models for studying immunity to challenge infection as well as immunopathology. The host species employed are diverse and include nonhuman primates, rabbits, woodchucks and inbred rodents. Although the schistosome infections produced in these experimental hosts in some cases closely mirror those observed in humans, in almost every instance there is at least one aspect in which the animal model differs from the clinical infection. An important example is Symmer's pipe stem fibrosis, a key pathologic parameter of human schistosomiasis, which can only be accurately and reproducibly modeled for S. mansoni and S. japonicum in chimpanzees (8). In the case of acquired resistance to infection, there has been considerable debate as to which animal models most closely reflect this process in man (40). Nonpermissive animal hosts such as the rat or rhesus monkey in which adult worms are spontaneously expelled obviously present infections quite distinct from those seen in humans, although the effector mechanisms generated against larval stages could nevertheless be similar. On the other hand, infections in permissive hosts such as the mouse or baboon, while closely reflecting human schistosome infestations in their chronicity, may not develop the same mechanisms of resistance generated in man. This debate over relevance is particularly intense amongst proponents of the rat vs. mouse models of immunity against S. mansoni. Rats develop a strong immune response against schistosomes of this species and expel their worms before patent infections are achieved. Conversely, laboratory mice develop infections which closely resemble those occurring in humans in terms of their persistence and granulomatous pathology. Nevetheless, the permissive mouse has several limitations as a model of human immunity. Firstly, acquired

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immunity is difficult to study in this model since circulatory changes develop in infected mice which cause nonimmunologically based reductions in challenge worm recovery (45). For this reason, most studies of acquired resistance to S. mansoni performed in the mouse now utilize the irradiated vaccine model, in which protection against challenge is completely dependent on the host immune response to schistosomula (13).The second difficulty with the mouse model is that certain effector mechanisms present in humans may be lacking from this rodent species. In particular, the high affinity receptor for IgE (FceRI) which is thought to be the major trigger for activation and degranulation of human eosinophils (19) is absent on eosinophils in the mouse. This distinction may explain why eosinophils appear to have no antischistosomal activity in the mouse as opposed to man and the rat. The latter hypothesis can now be tested, since transgenic mice have been constructed which incorporate the human FceRI gene and express the receptor on the rodent host's eosinophils ((15); D. Dombrowicz, personal communication). Regardless of the outcome of this debate concerning the nature of acquired immunity induced by natural infection, any mechanism of protection against schistosomes induced in an animal is potentially operative when stimulated against the parasite in humans, although care must be taken in considering mechanisms only documented to exist in nonpermissive hosts. Although it is not feasible to systematically induce and test the efficacy of different effector mechanisms in human vaccine trials, nonhuman primate models (e.g. the baboon) can offer at least a close approximation of applicability. Moreover, the continued development of genetically engineered mice expressing human immune system elements also offers the opportunity to pre-screen vaccine presentation strategies before testing in humans. Thus, the real issue is not whether a protective immune mechanism exists in man but whether it can be successfully induced artificially by vaccination.

Nature of Protective Immune Mechanisms As alluded to above, the divergent findings in different experimental hosts and man have led to a vigorous debate about the nature of protective immunity against schistosomes. A central issue in this debate concerns the functional significance of the Th2-associated immune responses (elevated IgE, eosinophilia) occurring in schistosome as well as other helminth infections.


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The traditional view is that these responses are a primary manifestation of acquired resistance, a concept which is now supported by the evidence associating high IgE levels with decreased reinfection rates in humans (7, 20). The alternative hypothesis is that because schistosome infections persist despite their elicitation of Th2- associated responses, the latter are not

inherently protective and are induced to down-regulate Thl responses potentially harmful to the parasite (23) a n d / o r host (6). The proponents of this view argue therefore that vaccination strategies not based on Th2 induction may be more fruitful and cite the highly effective Thl-associated immunity induced in mice by irradiated cercariae as evidence of this principle (45). Also supporting this viewpoint is the failure to convincingly demonstrate a Th2-dependent mechanism of protection in a fully susceptible animal model (see above). In the absence of such a direct demonstration the association between IgE and immunity in man can be viewed as merely correlative (45). As is the case with most polarized debates, both sides are likely to be partially correct and the "truth" lies somewhere in the middle. Thus, in terms of vaccination, schistosomes may be susceptible to either the Thl- or the Th2-dependent mechanism, depending on the specific antigenic target. Again, as noted above, the critical issue is not which mechanism operates during natural acquired resistance but which effectively protects when artificially induced. Finally, it may be naive to attempt to explain a process as complex as worm elimination in terms of a single arm of the immune system. Recent findings in the murine irradiated vaccine model underscore this caveat. Because of the importance of the T h l response in mice given a single immunizing dose of attenuated cercariae and the failure to transfer protection from these donors to naive recipients with immune sera, it has been assumed that the effector mechanism of resistance in this model is purely cell-mediated (13, 23, 45). Nevertheless, B-cell-deficient (mMT) mice develop only about half the resistance generated by wild-type animals. Moreover, immune sera from vaccinated wild-type mice (which, as expected, fail to protect naive wild-type mice upon transfer) restore immunity in vaccinated B-cell-deficient mice to the wild-type level (25). The above findings combined with earlier studies of Thl-deficient hosts (13) indicate that the high level protection achieved by attenuated vaccination depends on both cell-mediated and humoral immunity. Thus, effective immunization in humans may require the concurrent induction of two distinct arms of the immune system not typically associated with each other during natural helminth infection (22).

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Pathogenesis in Experimental Models The S. mansoni-infected mouse, while not consistently developing the pipestem fibrosis seen in humans, has provided a powerful model for studying the pathogenesis of both granuloma formation and hepatic collagen deposition. Nevertheless, over the years the work on this model has produced its own set of controversies. These have focussed largely on the following issues:

Mechanistic Relationship Between Granuloma Formation and Fibrosis It is largely assumed that the collagen deposition responsible for disease in schistosomiasis is a direct outcome of the granulomatous response to eggs in tissues. Based on this assumption the measurement of granuloma size has been used by most investigators as an index of pathology. Nevertheless, marked dissociations have been observed between these two disease processes in infected mice. For example, while granuloma volumes are unaffected by the intensity of infection, fibrosis per egg actually decreases with increasing worm burden (9). Similarly, whereas granuloma sizes in mice become markedly reduced in chronic infection, hepatic collagen levels are only marginally affected (17). Moreover, no quantitative correlation between granuloma size and fibrosis was observed in the progeny of crosses between mouse strains exhibiting high or low pathology (9). A dissociation between these two parameters has also been revealed in experiments involving immunologic interventions. For example, after infection with S. mansoni mice immunized with eggs plus IL-12 develop granulomas which are marginally reduced in size with respect to those in control animals, yet show a much greater suppression of fibrosis (46, 49). While such findings point to the potential inappropriateness of granuloma measurements for quantitating disease in schistosomiasis, they do not negate a role for the granulomatous response in the pathogenesis of fibrosis. Indeed, it would appear that the two pathologic parameters are causally connected but that each is subject to independent physiologic and immunologic influences. Clearly, in designing interventions, the key target should be fibrosis and not granuloma formation, a conclusion which is perhaps the most important take-home lesson from this debate.

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Role of Thl vs. Th2 Cells in Pathogenesis Egg pathology in schistosomiasis is clearly dependent on CD4+ T cells (30). The question of which CD4+ subset (Thl vs. Th2) mediates the granulomatous response has provided one of the major controversies of the last decade in the field of schistosomiasis. Based on Kenneth Warren's initial description of the granuloma as a delayed-type hypersensitivity (DTH) reaction, one would predict that it would depend on IL-2, IFN-y producing Thl cells for its pathogenesis. This argument has been supported by a variety of evidences. Thus, Thl cytokines can be detected during the early stages of acute egg pathology in mice (4), lung granulomas can be triggered by adoptively transferred Thl clones (11), and anti-IL-2 treatment reduces acute granuloma formation while IL-2 therapy reverses down-modulated pathology during chronic infection (31). Nevertheless, Warren's original immunologic description of the granuloma failed to take into consideration aspects of the reaction not typically associated with DTH responses. These include the presence of numerous eosinophils (up to 40% of the total number of cells in the lesion) and B cells. Indeed, cytokine measurements performed at the peak of granuloma formation in both the lungs and the liver reveal the dominant synthesis of the Th2 cytokines IL-4, 5 and 13 (21, 48). Moreover, in the lung model treatment with either anti-IL-4 or rIL-12 protein, each of which abolishes Th2 cytokine production also inhibits the tissue response to eggs (48, 49). On the basis of this and related data, it has been proposed that egg pathology is the manifestation of a Th2 rather than a Thl response. Nevertheless, a number of observations are at odds with a Th2 model of granuloma formation. Thus, down-modulation of granuloma size has been correlated with an IL-10-dependent induction of anergy in egg-induced Thl responses (41). A more glaring contradiction is the largely unimpaired egg pathology seen in IL-4 KO mice (36). Indeed, it is only very recently that formal proof of the role of the Th2 response in granulomatous inflammation has been obtained. In direct contrast to the results obtained with IL-4 KO animals, infected STAT-6- (28) and IL-4R-deficient mice show dramatically reduced granuloma formation and fibrosis comparable to that seen in Tdeficient RAG-1 KO animals (26). Both STAT-6 and IL-4 R deficiencies affect signaling by IL-4 and IL-13. That IL-13 signaling is the critical function absent in these animals is suggested by recent experiments in which granuloma formation has been shown to be blocked by a specific IL-13 antagonist (10).

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Thus, egg pathology is indeed dependent on the generation of a Th2 cytokine response but does not require IL-4 for either the generation of the response or its function. It would nevertheless be improper to conclude on the basis of the above evidence that Thl cytokines play no role in granulomatous inflammation. Indeed, there is fairly universal agreement that IL-2 participates in the response (30, 41, 48) and may be important in feeding Th2 development. IFN-y is also expressed at the early stages of granuloma formation and could play a role in the initial inflammatory response although its synthesis becomes rapidly down-regulated. Clearly, there are winners on both sides of the controversy over the function of Th responses in egg pathology, and this debate has fueled major advances in our understanding of the immunologic basis of disease in schistosomiasis.

What Causes the Down-modulation of Egg Pathology in Chronic Infection? One of the most fascinating immunologic features of schistosome infections is the diminished granulomatous inflammation occurring in the chronic phase of the disease. This phenomenon, which has been documented both in infected humans (3) and in experimental animals (16), is temporally associated with decreased T cell responsiveness to eggAg (35), although, as noted above, it need not result in decreased fibrosis. The mechanism underlying granulomatous down-modulation has been hotly debated and nearly every major immunoregulatory paradigm of the last 25 years has been invoked as an explanation. Probably the most actively pursued was the CD8 suppressor hypothesis of the late 1970's and early 80's, in which the induction of this now largely discredited regulatory cell was postulated to suppress the effector CD4 T cell responses causing egg granuloma formation (37). With the discovery by Colley of idiotypic network interactions in schistosomeinfected humans and mice, a new hypothesis emerged in which modulation of granuloma formation was postulated to be governed by anti-idiotypic T cells (12). Finally, after the emergence of the Thl/Th2 paradigm it was argued that changes in CD4 subsets and their production of cross-regulatory cytokines (e.g. IL-10, IFN-y) might account for the decreased egg pathology seen in later infections (23, 41). Inherent in all of the proposed mechanisms is the


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concept that immunoregulation of the T cell response directly controls the extent of granuloma formation. Recent experiments suggest that this key assumption may need to be reexamined. S. mansom'-infected B-cell-deficient (mMT) mice have been shown independently by two groups (18, 24) to display enhanced egg pathology and, importantly, to fail to undergo the down-modulation in granuloma size observed in WT animals (24). Interestingly, in the latter study the same animals which failed to down-modulate their granulomas displayed normal down-regulation in T cell responses during the same period, thus demonstrating that the two processes need not be causally related. In contrast to these findings, S. mansoni mice deficient in CD8 T cells or the cross-regulatory cytokines IFN-y and IL-10 were found to undergo normal down-regulation (50, 51), arguing against a requirement for either the CD8 suppression or T h l / T h 2 cross-regulation mechanisms proposed previously. Since FcR KO mice behave identically to B cell KO mice in failing to undergo modulation of granuloma formation, it would appear that the critical process deleted is the interaction of Ab with receptors on an unknown regulatory cell involved in dampening of the host tissue response (24). While it is also possible that this B cell dependence reflects the role of idiotypic regulation in egg pathology, it is important to emphasize that no effects on down-modulation of T cell responses were observed in the same B- or FcR-deficient hosts. Thus, an important new question has emerged from these recent studies: Is the suppression of CD4+ function observed in chronic schistosome infection functionally irrelevant in terms of the modulation of immunopathology, or can granulomatous inflammation be regulated independently by either the T or B cell arms of the immune response?

What Stages of the Schistosome Life Cycle Should Be Targeted in a Human Vaccine? The last and most important debated issue in the immunobiology of schistosomiasis concerns the form that an effective vaccine against schistosomiasis should take. This subject has already been discussed in depth in the previous chapter (by James and Colley) and therefore will be dealt with only briefly here. Clearly, the ideal vaccine would simultaneously affect infection, pathology and transmission. Because of the remarkable evolutionary

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adaptations of schistosomes to the vertebrate immune system, an effective vaccine which targets infection (i.e. worm invasion or development) is likely to be extremely difficult to achieve. For this reason immunologic interventions which target pathogenesis or transmission to the invertebrate host, processes which have no selective advantage for the adult worm within the vertebrate host, may ultimately be more feasible and could significantly reduce the level of morbidity in an endemically exposed population. Yet even these targets present their own new set of problems, such as the existence of a reservoir host for some species of schistosomes which could maintain transmission when it is reduced in human populations, or the possibility of inducing generalized immune defects with a vaccination method which modulates the immunopathologic response to eggs. These arguments should not discourage the attempt to develop such vaccines, since ultimately every reasonable approach may be worth the investment in both cost and effort. Nevertheless, the ideal vaccine would be one that targets an as-yetundiscovered Achilles' heel of the parasite. As argued previously (40), such a molecule would most likely not be recognized normally by the immune system during infection and therefore would not exist amongst the group of candidate vaccine antigens already identified. The schistosome genome project now offers an extremely powerful tool for identifying such a molecule given some information as to its critical function for the parasite. Unfortunately, the development of this new technology has occurred simultaneously and often at the expense of work on the basic physiology of schistosome parasites. For the latter reason we suggest that continued and perhaps increased funding of research aimed at the identification of parasitespecific physiological targets in this helminth be given a high priority and be directly coupled to both genomic and immunologic studies on the same molecules. Such a concerted "Achilles' heel" approach could directly and rapidly lead to an effective vaccine unencumbered by the complications inherent in our current approaches discussed above.

The Challenge Schistosomiasis is a major public health problem in many parts of the world, including Asia, Africa, Central and South America. The total population living in endemic areas exceeds half of the world population.

Immunology of Schistosomiasis: Towards a Consensus Viewpoint 507

Endemicity of infection is dependent not only on the presence of infection in humans — or, in some geographic areas, in other mammalian hosts — but also on the availability of susceptible intermediate hosts. Attempts at controlling infection and disease have been based on chemotherapy mollusciciding and other environmental measures. Successes are hard to find, since the long term impact of any of these measures is limited. The focus must be better appreciation of the multiplicity of variables contributing to expression of infection and disease and tilting the balance to the advantage of the host. The search for vaccines, therefore, must be accelerated; the effectiveness of our current chemotherapy-based strategy is limited and the appearance of resistance is only a matter of time. This is not a pessimistic assessment; rather, it is intended to be realistic and motivating. The target for antischistosome vaccines need not be absolute resistance to infection. With the clear-cut relationship between intensity of infection and disease there is ample opportunity for a vaccine that significantly reduces worm burden. Furthermore, other targets should be considered, such as vaccines that will reduce or inhibit egg production or decrease granuloma formation. At this stage what is clearly needed is to cast a wide net for multiple targets.

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TROPICAL MEDICINE Science and Practice

Schistosomiasis edited by Add AF Mahmoud

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This volume brings together updated fundamental knowledge of the schistosomes, their biology and epidemiology, the mechanism of disease and a full description of the pathological sequelae and clinical syndromes in humans. It concludes with chapters on diagnosis and treatment prospects of vaccine development and the most significant controversies regarding immunology and epidemiology.

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World Class Parasites: Volume 10 - Schistosomiasis (World Class Parasites)

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