Emerging Infections 6

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Author: W. Michael Scheld | James M. Hughes | Barbara E. Murray

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EMERGING INFECTIONS 6

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EMERGING INFECTIONS 6 Edited by

W. Michael Scheld Division of Infectious Diseases University of Virginia Health System Charlottesville, Virginia

Barbara E. Murray Division of Infectious Diseases Center for the Study of Emerging and Re-Emerging Pathogens University of Texas Medical School Houston, Texas

James M. Hughes National Center for Infectious Diseases Centers for Disease Control and Prevention Department of Health and Human Services Atlanta, Georgia

Washington, D.C.

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Address editorial correspondence to ASM Press, 1752 N Street NW, Washington, DC 20036-2904, USA Send orders to ASM Press, P.O. Box 605, Herndon, VA 20172, USA Phone: (800) 546-2416 or (703) 661-1593 Fax: (703) 661-1501 E-mail: [email protected] Online: www.asmpress.org Copyright © 2004 ASM Press American Society for Microbiology 1752 N Street NW Washington, DC 20036-2904 ISBN 1-55581-242-2 All Rights Reserved Printed in the United States of America 10

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Cover photos Larger photo: Immunolocalization of yellow fever virus antigens within mononuclear phagocytic cells in lung. Smaller photo: Immunolocalization of Ebola virus antigens in the skin. (Both images courtesy of Sherif R. Zaki, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention.)

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To Peggy Hamburg, Stacey Knobler, Joshua Lederberg, Stan Lemon, Adel Mahmoud, Steve Morse, Stan Oaks, Bob Shope, and Mark Smolinski for their efforts in highlighting the challenges posed by emerging microbial threats in the United States and worldwide

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CONTENTS Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii 1. Hepatitis C Virus and Chronic Liver Disease • Beth P. Bell . . . . . . . . . . . . . . . 1 2. Polio Reemerges in the Americas: a Vaccine-Derived Outbreak in Hispaniola in 2000 and 2001 • Ciro A. de Quadros, Mauricio Landaverde, Olen Kew, Zacarias Garib, Jean André, Cristina Pedreira, and Fernando Laender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3. Yellow Fever Vaccine-Associated Disease • Rachel S. Barwick, Anthony A. Marfin, and Martin S. Cetron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 4. Responding to Epidemics of Ebola Hemorrhagic Fever: Progress and Lessons Learned from Recent Outbreaks in Uganda, Gabon, and Congo • Daniel G. Bausch and Pierre E. Rollin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 5. The Global Epidemiology of AIDS • Thomas C. Quinn . . . . . . . . . . . . . . . . . 59 6. One Multidisciplinary Approach to AIDS in Africa: the Academic Alliance for AIDS Care and Prevention in Africa • W. Michael Scheld . . . . . . . . . . . . . . 83 7. Enteroaggregative Escherichia coli • James P. Nataro . . . . . . . . . . . . . . . . . 101 8. Wound Botulism, Tetanus, and Necrotizing Fasciitis among Injecting Drug Users in California: the Clostridial Connection • Duc J. Vugia, S. Benson Werner, and Celia J. I. Woodfill . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 9. Unexplained Severe Illness and Death among Injecting Drug Users in Scotland, Ireland, and England from April to August 2000 • Marc Fischer, Anne Whitney, Syed Ahmed, Joe Barry, and Jane Jones . . . . . . . . . . . . . . . . . . 121 10. Travel-Associated Fungal Infections • Rana A. Hajjeh and David W. Warnock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 11. Human African Trypanosomiasis: a Reemerging Public Health Threat • Anne C. Moore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 12. Raccoon Roundworm Infection (Baylisascariasis): a Zoonosis of Pediatric and Public Health Concern • William J. Murray . . . . . . . . . . . . . . . . . . . . 159 13. Can Surveillance for Unexplained Deaths Be Used as a Public Health Approach for Early Recognition of New Pathogens? • Marc Fischer, Rana A. Hajjeh, and Andre N. Sofair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 14. The International Emerging Infections Program in Thailand: an Early Report • Scott F. Dowell, Supamit Chunsuttiwat, Sonja J. Olsen, Pathom Sawanpranyalert, J. Mark Simmerman, Tamara L. Fisk, and Kumnuan Ungchusak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 vii

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CONTRIBUTORS Syed Ahmed • Public Health Protection Unit, Greater Glasgow NHS Board, Glasgow G3 8YU, Scotland Jean André • Ministry of Health, Port-au-Prince, Haiti Joe Barry • Department of Public Health, Eastern Regional Health Authority, Dublin 8, Ireland Rachel S. Barwick • Division of Global Migration and Quarantine, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Daniel G. Bausch • Department of Tropical Medicine, SL-17, Tulane School of Public Health and Tropical Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112 Beth P. Bell • Division of Viral Hepatitis, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Martin S. Cetron • Division of Global Migration and Quarantine, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Supamit Chunsuttiwat • Department of Disease Control, Ministry of Public Health, Tivanon Road, Nonthaburi 11000, Thailand Ciro A. de Quadros • Sabin Vaccine Institute, 1718 Connecticut Avenue NW, Suite 700, Washington, DC 20009 Scott F. Dowell • International Emerging Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, and Department of Disease Control, Ministry of Public Health, Building 7, 3rd floor, Tivanon Road, Nonthaburi 11000, Thailand Marc Fischer • Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Tamara L. Fisk • International Emerging Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, and Department of Disease Control, Ministry of Public Health, Building 7, 3rd floor, Tivanon Road, Nonthaburi 11000, Thailand Zacarias Garib • Ministry of Health, Santo Domingo, Dominican Republic Rana A. Hajjeh • Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Jane Jones • Public Health Laboratory Service, London NW9 5EQ, England Olen Kew • Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

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Fernando Laender • Division of Vaccines and Immunization, Pan American Health Organization, 525 23rd Street NW, Washington, DC 20037 Mauricio Landaverde • Division of Vaccines and Immunization, Pan American Health Organization, 525 23rd Street NW, Washington, DC 20037 Anthony A. Marfin • Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado 80521 Anne C. Moore • Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mail Stop F-22, 4770 Buford Highway, Atlanta, Georgia 30333 William J. Murray • Department of Biological Sciences, San Jose State University, San Jose, California 95192-0100 James P. Nataro • Center for Vaccine Development, Departments of Pediatrics, Medicine, and Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore Street, Baltimore, Maryland 21201 Sonja J. Olsen • International Emerging Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, and Department of Disease Control, Ministry of Public Health, Building 7, 3rd floor, Tivanon Road, Nonthaburi 11000, Thailand Cristina Pedreira • Division of Vaccines and Immunization, Pan American Health Organization, 525 23rd Street NW, Washington, DC 20037 Thomas C. Quinn • Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Pierre E. Rollin • Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Pathom Sawanpanyalert • Department of Medical Sciences, Ministry of Public Health, Tivanon Road, Nonthaburi 11000, Thailand W. Michael Scheld • Department of Internal Medicine, Division of Infectious Diseases, University of Virginia, P.O. Box 801342, MR 4, Room 2118B, Charlottesville, Virginia 22908 J. Mark Simmerman • International Emerging Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, and Department of Disease Control, Ministry of Public Health, Building 7, 3rd floor, Tivanon Road, Nonthaburi 11000, Thailand Andre N. Sofair • Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520 Kumnuan Ungchusak • Bureau of Epidemiology, Department of Disease Control, Ministry of Public Health, Tivanon Road, Nonthaburi 11000, Thailand Duc J. Vugia • Division of Communicable Disease Control, California Department of Health Services, Berkeley, California 94704 David W. Warnock • Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

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S. Benson Werner • Division of Communicable Disease Control, California Department of Health Services, Berkeley, California 94704 Anne Whitney • Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333 Celia J. I. Woodfill • Division of Communicable Disease Control, California Department of Health Services, Berkeley, California 94704

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FOREWORD In 1967, psychologist Stanley Milgram conducted the first recognized study of the smallworld phenomenon, now commonly known as “six degrees of separation” based on John Guare’s 1990 play by the same name. In Milgram’s study, packets of letters were distributed to people throughout the Midwest who were asked to forward them to acquaintances most likely to know the eventual recipients, a couple in Boston. Milgram’s study found that it took an average of five steps, separating the individuals by six acquaintances, to reach the intended couple. While his study introduced the concept of highly connected networks, it also identified key links between disparate groups, including individuals capable of singly bridging expansive networks, as the essential elements in this process. Although a social science theory for years, this small-world phenomenon was mathematically mapped by using both random and deliberate interactions in 1998 by Steven Strogatz and Duncan Watts, Cornell University researchers at the time. Since then, the idea of small-world networks has been applied to numerous fields, including the epidemiology of infectious diseases. Historically, such links and networks have been used to determine the cause and track the spread of infectious diseases. Infections spread throughout clusters of individuals with recognized associations; additional clusters develop because of less obvious links to others outside the initial cluster, giving rise to explosive outbreaks. The human immunodeficiency virus pandemic, described in two chapters in this volume, represents a worldwide network of more than 70 million interconnected individuals that is still expanding at an unrelenting pace. Today’s highly connected and accessible world has resulted in increases in both the number of these links and the speed at which they occur. Widespread travel, the globalization of the food supply, and the increasing overlap between the environments of humans and animals have afforded microbes rapid and easy access to new populations. Newly recognized pathogens are accelerating in distribution, spreading faster and further, and affecting national security and the global economy—the new normal in emerging infectious diseases. In addition to their role in disease transmission, and perhaps most importantly, such links also provide windows of opportunity to prevent disease by interrupting transmission patterns. Moreover, the same highly connected world that facilitates global disease transmission has also brought new opportunities for widespread sharing of information and resources among fairly compartmentalized disciplines such as the clinical, research, public health, and veterinary communities. The networks resulting from these collaborations and partnerships are essential for effective responses to global emerging infectious diseases in the future. The worldwide effort undertaken in 2003 to identify the cause and stop the spread of severe acute respiratory syndrome (SARS) should serve as a model for responding to global infectious threats. Coordinated by the World Health Organization (WHO), this response xiii

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involved unprecedented collaborations and unconventional exchange of evolving information among scientists and health officials throughout the world. Within days of WHO’s first global alert on the new illness, investigative teams were mobilized and dispatched to assist health officials in affected areas and prevention guidelines were developed. Within a few weeks, the causative agent, a novel coronavirus, was identified and sequenced and efforts were under way to develop and distribute appropriate diagnostics. The important lessons learned from the heroic response to SARS can serve us well as we prepare to address the next global microbial threat. Julie Louise Gerberding Director Centers for Disease Control and Prevention Atlanta, Georgia

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PREFACE During the early and mid-20th century, tremendous advances were made in prevention and control of infectious diseases. Improvements in sanitation and overall living conditions were followed by fast-paced development of effective vaccines and antibiotics directed against major health threats. Along with vast improvements in public health, these successes also brought a false sense of security regarding the threat of infectious diseases, especially for persons in the developed world. In recent years, there has been renewed public interest and concern surrounding infectious diseases. The catastrophic spread of human immunodeficiency virus, the emergence of West Nile virus infection in the United States, and the increasing problem of antimicrobial resistance are clear indications of the continued ability of microbes to emerge, spread globally, adapt, and survive. The World Health Organization estimates that nearly 15 million (26%) of the 56.5 million deaths that occurred throughout the world in 2001 were caused by microbial agents. In the United States, infectious diseases are the third leading cause of death. In March 2003, the Institute of Medicine (IOM) published an update to its landmark 1992 report on emerging infections. The new report, Microbial Threats to Health: Emergence, Detection, and Response, describes the current spectrum of global microbial threats, factors affecting their emergence or resurgence, and measures needed to effectively address them. In defining the spectrum of microbial threats, the report cites the global burden of AIDS, tuberculosis, and malaria and the resurgence of other endemic diseases, the emergence of new infectious diseases, the development of antimicrobial resistance, chronic diseases caused by infectious agents, and the intentional use of biologic agents for harm. The report also describes many factors that alone or in combination can affect the emergence and spread of infectious diseases. These factors can be broadly categorized as genetic and biological factors; physical environmental factors; ecological factors; and social, political, and economic factors. Among its conclusions and recommendations, the IOM report calls for improved global response capacity and strengthened domestic and global infectious disease surveillance. In the weeks after the release of the new report, the importance of such measures became strikingly evident as an unprecedented worldwide effort was launched to determine the cause and contain the spread of a new illness that would soon become known as severe acute respiratory syndrome or SARS. Since 1995, the program committee of the Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) and the officers of the Infectious Diseases Society of America (IDSA) have organized joint sessions on new and emerging pathogens during ICAAC and the IDSA annual meetings. These sessions have been carefully planned to include the spectrum of new and emerging bacteria, viruses, fungi, and parasites of recognized or potential scientific and public health importance, with appropriate discussions on strategies for their control. xv

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The chapters in Emerging Infections 6 focus on a variety of infections currently causing major challenges for the clinical, laboratory, and public health communities. Some of these are newly recognized diseases, while others are previously known pathogens with persistent or new obstacles. Some are described as domestic threats, while others affect populations elsewhere. However, as has been clearly demonstrated, the globalized world in which we live provides rapid and easy access to previously unaffected areas and populations for emerging infections. Effectively responding to infectious diseases requires strong national and international public health systems and health service infrastructures. These entities must include a highly trained and diverse workforce comprising dedicated individuals who can create and sustain strong partnerships and collaborations both domestically and globally. Future challenges are difficult to predict but certainly include continued problems with antimicrobial-resistant infections, influenza and other respiratory diseases, food-borne diseases, and new threats for immunocompromised populations. Additional links between chronic diseases and infectious agents will likely be discovered, providing new opportunities for disease prevention and treatment. Protecting the safety of the blood supply will also continue to present new challenges, as recent experiences with West Nile virus have shown. In addition to naturally occurring infectious disease outbreaks, we must continue to strengthen our ability to detect and respond to potential acts of bioterrorism. This volume, the sixth in the series, should serve as a valuable source of current information for those responsible for addressing current and future infectious threats to health. W. Michael Scheld Barbara E. Murray James M. Hughes

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ACKNOWLEDGMENTS We thank everyone who has helped us in preparing this volume. Most importantly, we thank all of the authors for their outstanding contributions. As editors, we are particularly grateful to those members of the Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) Program Committee who assisted us in coordinating topic and speaker selection for and/or moderating the joint symposia on emerging infections during the 2001 and 2002 ICAACs. Numerous other colleagues provided helpful discussion, advice, and criticisms. We are also grateful to our assistants, Eva Barnett, Susan Waisner, Sheila Marlow, and Robin Moseley. We thank Ken April of ASM Press for coordinating production of the book. And, finally, we thank our families for their understanding and support during this undertaking.

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Emerging Infections 6 Edited by W. M. Scheld, B. E. Murray, and J. M. Hughes © 2004 ASM Press, Washington, D.C.

Chapter 1

Hepatitis C Virus and Chronic Liver Disease Beth P. Bell

In the United States, hepatitis C virus (HCV) is the most common blood-borne pathogen, accounts for a large proportion of chronic liver disease cases, and is the leading indication for liver transplantation. This review focuses on disease burden and the natural history of chronic liver disease due to HCV infection and highlights recent advances in therapy. DISEASE BURDEN In 1998, there were over 25,000 deaths from chronic liver disease and cirrhosis, defined in the International Classification of Diseases, ninth revision (ICD-9), as code 571, in the United States (9). The age-adjusted death rate was 7.1 per 100,000 population, making chronic liver disease the tenth leading cause of death and the eighth leading disease-related cause of death (Table 1). However, this mortality rate most likely represents an underestimate of deaths from chronic liver disease and its sequelae, which could be recorded on the death certificate by other ICD-9 codes, such as viral hepatitis, liver cancer, or specific sequelae of chronic liver disease (21). If chronic liver disease ICD-9 codes are expanded in this manner, chronic liver disease becomes the eighth leading cause of death in 1998 (21). Most hepatitis C-related mortality results from complications of chronic liver disease, but mortality statistics do not provide an accurate assessment of the number or proportion of chronic liver disease deaths attributable to hepatitis C. Hepatitis C is listed on less than 20% of death certificates for those dying of chronic liver disease in 1998, and no etiology is recorded on about half of them (Centers for Disease Control and Prevention [CDC], unpublished data). A 1997 New York study comparing mortality data with medical records estimated the completeness of recording hepatitis C on death certificates at 17.7% (C. Wu, H. Chang, L. A. McNutt, P. F. Smith, Abstr. 128th Annu. Meet. Am. Public Health Assoc., abstr. 11681, 2000). Extrapolations from population-based studies indicate that an estimated 8,000 to 10,000 deaths per year from hepatitis C-related chronic liver disease occur (8). Hepatitis C is the indication for an increasing proportion of liver transplants in the United States, accounting for 36% of all transplants in 1999 (22). The few populationBeth P. Bell • Division of Viral Hepatitis, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333. 1


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Bell Table 1. Chronic liver disease and cirrhosis mortality, United States, 1998 Mortality characteristic

Value

No. of deaths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25,192 Rank (total)a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Rank (disease-related)b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Age-adjusted death rate/100,000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Rank (total)a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Rank (disease-related)b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 a b

Based on ranking of all causes of death. Based on ranking of all disease-related causes of death.

based studies suggest that hepatitis C accounts for 40 to 60% of chronic liver disease [15; CDC, unpublished data; B. P. Bell, V. J. Navarro, M. M. Manos, R. C. Murphy, W. A. Leyden, T. E. St Louis, K. Kunze, M. Taccariello, H. Wurtzel, W. Bower, and N. A. Terrault, abstract, Hepatology 34(Part 2):468A, 2001]. In a CDC-sponsored, population-based surveillance pilot project that includes patients from two defined populations meeting standard criteria for newly diagnosed chronic liver disease and who have visited a referral practice, the proportion of newly recognized chronic liver disease cases attributable to hepatitis C can be assessed, and the incidence of newly diagnosed chronic liver disease and hepatitis C can be estimated [Bell et al., Hepatology 34(Part 2):468A, 2001]. A standard case definition based on laboratory pathologic or radiographic test results is used to ascertain cases. Preliminary results indicate an estimated incidence of newly diagnosed chronic liver disease in referral practices in 1999 of 72.3 cases per 100,000. The incidence was higher for men (95.4 cases per 100,000) than for women (50.2 cases per 100,000). The overall incidence of newly diagnosed chronic hepatitis C was 31.6 cases per 100,000. More-detailed information was collected for a subset of patients. Of the 342 patients studied in 1999, the primary etiology of chronic liver disease was hepatitis C for 60% of the patients; excessive alcohol consumption also played a role for 16% of those diagnosed with hepatitis C. Excessive alcohol consumption alone was responsible for chronic liver disease in 8% of the patients studied (Fig. 1). Despite its limitations, the result of applying this pilot project’s chronic liver disease and chronic hepatitis C incidence rates to the U.S. population is interesting to consider. This study’s results suggest that an estimated 130,000 patients were newly diagnosed with chronic liver disease in referral physicians’ offices in 1999, about 80,000 of whom had chronic hepatitis C. The usefulness of this kind of information can be seen by comparing these findings to results of a similarly designed population-based study conducted by the CDC in one county in 1989 (CDC, unpublished data). In this study, the estimated incidence of chronic liver disease, 12 patients per 100,000, was lower than current estimates by more than fivefold; only 40% of the cases were attributable to hepatitis C alone or in combination with excessive alcohol consumption. The economic burden associated with chronic hepatitis C was recently estimated in a report sponsored by the American Gastroenterological Association (AGA), using publicly available data and proprietary information from third-party payers (21). In 1998, direct and indirect costs associated with chronic hepatitis C were estimated at $744 million.


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Chapter 1 • Hepatitis C

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Figure 1. Primary etiology of patients newly diagnosed with chronic liver disease: New Haven, Conn., and Kaiser, Alameda, Calif., 1999 (n ⫽ 342). HBV, hepatitis B virus; NAFLD, nonalcoholic fatty liver disease; alcohol, excessive alcohol intake. Other etiologies include primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis, granulomatous hepatitis, hemochromatosis, sarcoidosis, drug-induced hepatitis, cryptogenic, alpha-1-antitrypsin deficiency, and hepatocellular carcinoma. Findings are preliminary.

However, as indicated in the AGA report, the true costs are most likely underestimated because the effect of premature deaths from liver disease was not accounted for fully. Overall, the total estimated economic impact of liver disease related to hepatitis C in the late 1990s has been estimated at $1 billion to $1.3 billion per year (21). Consideration of trends in the incidence and prevalence of HCV infection provides a sense of the potential disease burden for the future. During the peak incidence years of the late 1980s, an estimated 242,000 new infections per year occurred; however, in the 1990s the incidence of acute infection dropped precipitously to an estimated 25,000 to 40,000 new infections per year in recent years (Table 2) (CDC, “Disease Burden from Viral Hepatitis A, B, and C in the United States,” accessible at http://www.cdc.gov/ncidod/ diseases/hepatitis/resource/PDFs/disease_burden2002.pdf). However, HCV infection is Table 2. Burden associated with HCV infection, United Statesa Measure

No.

Estimated new infections/yr 1985–1989 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242,000 (42,000)b 2001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25,000 (4,000)b Persons ever infected (1.8%) . . . . . . . . . . . . . . . . . . . . . 3.9 million (3.1 million–4.8 million)c Persons with chronic infection . . . . . . . . . . . . . . . . . . . . 2.7 million (2.4 million–3.0 million)c Estimated deaths from chronic disease/yr . . . . . . . . . . . . 8,000–10,000 a

Data from reference 2 and www.cdc.gov/ncidod/diseases/hepatitis/resource/PDFs/disease_burden2002.pdf. Numbers in parentheses are numbers of cases. c Numbers in parentheses are 95% confidence intervals. b


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prevalent in the United States. Results of the Third National Health and Nutrition Examination Survey (NHANES III), a nationwide representative sample of the noninstitutionalized, civilian U.S. population, indicate that 1.8%, corresponding to approximately 3.9 million people, have ever been infected with HCV, and an estimated 2.7 million people show evidence of chronic infection, as measured by the presence of HCV RNA in serum (Table 2) (2). This means that HCV is the most prevalent blood-borne infection in the United States. Further examination of the results of NHANES III suggests that despite a recent decline in new infections, the disease burden associated with hepatitis C may increase over the next several decades, because of the age-specific prevalence of chronic HCV infection and the many years that may pass before the complications of cirrhosis and liver cancer become clinically evident. In NHANES III, a peak in the prevalence of anti-HCV was evident among those aged 30 to 39, reaching a high of 5.3% among men in this age group (2). Assuming that these individuals would likely have acquired their infections in their early 20s, this large cohort will manifest the complications of chronic hepatitis C in the coming years. Estimates derived from mathematical models support the view that the burden from chronic hepatitis C will increase over the next several decades. The results of one such study estimate that the overall prevalence of chronic HCV infection reached a peak of just over 2% in the mid-1990s and would decline thereafter to approximately 1% by 2030 (Fig. 2) (3). However, the model also predicts that the prevalence of infections of longer duration would increase through the mid-2010s, projecting a fourfold rise in the number of persons whose infection was of at least 20 years’ duration between 1990 and 2015. Those who have had chronic hepatitis C for many years are most likely to develop symptomatic chronic liver disease and its outcomes of cirrhosis and liver cancer.

Figure 2. Effect of HCV infection incidence on future prevalence of infection. Reprinted in part from reference 3, with permission from Elsevier.


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NATURAL HISTORY AND FACTORS RELATED TO PROGRESSION Acute HCV infection is usually silent, but chronic infection develops in the majority of cases (1). Chronic hepatitis is also silent for many years, with a slow evolution, over 20 to 40 years, to symptomatic end-stage liver disease, but only in some patients. Despite a decade of research, there is still considerable disagreement about some aspects of the natural history, including the proportion of persons with chronic hepatitis C that will develop cirrhosis or hepatocellular carcinoma. Prospective studies of patients with transfusion-associated non-A, non-B hepatitis and patients seen in referral practices have shown relatively high rates of progression, with 8 to 17% of participants progressing to cirrhosis after 8 to 25 years (12, 19, 23, 31, 34, 39, 40). Studies of young adults and children infected by contaminated blood products have shown less evidence of severe disease. In two 20-year follow-up studies of women infected by contaminated anti-D immunoglobulin in the late 1970s, 15% no longer tested positive for antibody, and 45% of infected women were HCV RNA negative after 20 years (20, 42). Half of the chronically infected women showed no evidence of fibrosis when biopsied, only 10% had severe inflammation or fibrosis, and only 0.4 to 2% had progressed to cirrhosis. Two studies of young adults with community-acquired HCV infection also have shown slow rates of progression to severe chronic liver disease (33, 38). In one of these follow-up studies, eight individuals known to be HCV RNA positive 50 years earlier and available at the time of the study were contacted. Five had no liver-related illness; two were asymptomatic but had biochemical evidence of cirrhosis; and one died with radiological evidence of cancer of the liver, but the primary source (lung or liver) was not determined (33). A study of anti-HCV-positive adolescents and young adults (mean age of 19.8 years) who had received blood transfusions as infants or young children showed that less than 60% had persistent infection. Of 17 patients who underwent liver biopsies, only two had portal fibrosis; both patients also had congestive heart failure (41). Part of the explanation for the divergence in views on the frequency with which hepatitis C results in end-stage liver disease and cirrhosis can be found in differences in study populations, method of ascertainment, and length of follow-up. One recent review of published studies that evaluated progression to cirrhosis in cohorts of hepatitis C patients defined four categories based on how patients were recruited: cross-sectional studies of patients seen in referral practices; longitudinal studies of patients with posttransfusion hepatitis; case series of blood donors found at the time of screening to be anti-HCV positive; and community-based cohorts (14). At 20 years after infection, the proportion of patients with cirrhosis varied widely across categories, ranging from 4% among blood donor series to 24% in the posttransfusion studies. After accounting for host factors using regression methods, the estimate of the proportion progressing to cirrhosis remained considerably higher in studies of patients seen in referral practices, suggesting that selection bias played a role. Despite these differences in findings among natural history studies, the preponderance of evidence indicates a less than 20% incidence of severe liver disease during the first 20 years after infection (1). However, few data are available with respect to disease course during subsequent decades, which could follow several different patterns. If liver disease progression continued to be linear, approximately 50% of infected patients would develop severe liver disease after 60 years (1). However, the rate of fibrosis could accelerate after


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20 years or, conversely, could plateau. The highly variable outcomes observed among cohorts of HCV-infected patients suggest that progression will not be linear in all patients. Most likely, as some authors have suggested, the rate of fibrosis is highly variable, is determined by many factors, and will be rapid in some patients, slower in others, and may not occur at all in some (1, 31). A number of factors contribute to the observed differences in natural history by promoting progression or increasing severity. Older age at the time of infection has been consistently shown to be an important determinant of progression, which can be appreciated when cohort studies of natural history are stratified according to the age at the time of infection (Fig. 3). This effect appears to be independent of the duration of infection, as was demonstrated in a French study involving a large number of patients with hepatitis C from a number of established cohorts (31). After adjusting for the estimated duration of infection, patients infected with HCV at 40 years of age or older had a significantly higher grade for the stage of fibrosis than those infected at a younger age. Although methods differ widely, many studies now consistently demonstrate worse outcomes with excessive alcohol consumption. In a large cohort study, excessive alcohol consumption, defined as at least 50 g/day, was an independent risk factor for fibrosis progression, regardless of age or duration of infection (31). The adjusted odds ratio of progression among patients who drank at this level was 2.36 (95% confidence interval, 1.62 to 3.45). Although there was a suggestion of a dose-response effect, the association between lower alcohol consumption and progression was not statistically significant. In a study from Italy, lifetime teetotalers were compared with people who drank excessively, according to their HCV status (10). Those who did not drink and were anti-HCV negative served as the reference population. The odds ratio for cirrhosis was 15 times higher for anti-HCV-negative excessive drinkers than for nondrinkers. Among persons with HCV infection, the odds ratio for cirrhosis was 9 among nondrinkers and reached 147 among anti-HCV-positive excessive drinkers. In a population-based study also from Italy, among HCV-infected people

Figure 3. Natural history of HCV infection related to age at infection from cohort studies (12, 20, 33, 34, 38, 41, 42).


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who drank more than 30 g of alcohol per day, 32% had cirrhosis, compared to 10% of those who drank less than 30 g/day (4). Several studies from the United States have also showed that excessive alcohol intake, defined in different ways, is associated with cirrhosis among hepatitis C patients (17, 38, 43). Among the many unanswered questions are whether there is a threshold quantity of alcohol below which disease progression is not affected, whether the pattern of drinking is a factor, and what the relative importance is of total lifetime compared to more recent quantities of alcohol consumed. A number of studies have documented that the progression of hepatitis C is more rapid among those who also have human immunodeficiency virus (HIV) infections (5, 11, 24, 26, 30, 37). In a recent meta-analysis, the combined adjusted relative risk for cirrhosis or decompensated liver disease was 2.92 (95% confidence interval, 1.70 to 5.01) (16). In one case-control study from France, the association between coinfection and fibrosis was examined by comparing 122 patients coinfected with HIV and HCV with a control group of 122 HIV-negative, HCV-infected subjects. They were matched by age, sex, daily alcohol consumption, age at HCV infection, and duration and route of infection (5). The prevalence of liver fibrosis was higher (60%) among HIV-infected patients than HIV-negative patients (47%), and the rate of fibrosis progression among HIV-coinfected patients was significantly higher than among HIV-negative patients. Of interest, among HIV-coinfected patients, CD4 counts of less than 200, drinking at least 50 g of alcohol a day, and age greater than 25 at the time of HCV infection were all significantly associated with fibrosis progression. Similar routes of transmission lead to high rates of coinfection in certain groups, particularly persons with hemophilia and injection drug users. However, coinfection rates are not high in groups in which transmission routes do not overlap: the most notable group is men who have sex with men. Because HCV is not transmitted efficiently by the sexual route, the prevalence of HCV infection among HIV-infected men who have sex with men is low. The overall estimated prevalence of HIV infection among those with HCV infection is 10%, and an estimated 25% of HIV-infected individuals also are infected with HCV (35). With better survival rates of those with HIV infection, the morbidity and mortality associated with HCV infection have been demonstrated in a number of recent studies using different outcome measures, including hospital admissions and mortality (6, 28, 36). At one institution, among HIV-positive patients who died, the proportion of deaths attributable to end-stage liver disease increased from 11.5% in 1991 and 13.9% in 1996 to 50% in 1998 and 1999, while the prevalence of anti-HCV remained unchanged (6). End-stage liver disease has become the leading cause of death among hospitalized HIV-infected patients at this institution. In a number of studies, male sex also has been associated with a higher rate of progression of liver disease (7, 14, 32). Many other factors, such as viral characteristics and genetic factors, have not been consistently found to be associated with progression. TREATMENT Considerable advances in the treatment of hepatitis C have been made over the past several years. At the time of the 1997 National Institutes of Health Consensus Development Conference, the recommended therapy of a 48-week course of interferon alfa three times a week resulted in a sustained virological response (SVR) of 12 to 16% (29). Since then, combination therapy with interferon alfa plus the nucleoside analogue ribavirin improved


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the SVR to approximately 35 to 45%. More recently, the novel chemically modified form of interferon alfa, pegylated interferon, has been evaluated in comparison with interferon alfa as a monotherapy and in combination with ribavirin. Pegylation is a process by which a polyethylene glycol molecule is attached to a therapeutic protein, thereby increasing serum half-life. Early trials showed that pegylated interferons could be administered once a week without an increase in severe adverse events and demonstrated significantly higher SVRs with monotherapy with pegylated interferons than with treatment with standard interferons, ranging from 15 to 39%, depending on the dose (18, 25, 44). Combination therapies were compared in a trial including 1,530 patients with chronic hepatitis C. The patients were randomized into three groups and treated for 48 weeks with standard interferon and ribavirin or pegylated interferon alfa-2b at two different doses with ribavirin (27). Overall, the SVR, defined as no detectable HCV RNA at 24 weeks after the completion of treatment, was 54% in patients who received the higher dose of pegylated interferon and 47% among patients who received regular interferon or the lower dose of pegylated interferon. However, SVRs were similar across treatment groups for patients infected with genotype 2 or 3. With optimal therapy, SVRs were higher among patients infected with genotype 2 or 3 (82%) than with patients infected with genotype 1 (42%). A critical determinant of response to therapy has consistently been the HCV genotype. With optimal therapy of pegylated interferon and ribavirin, patients infected with genotype 1 have lower response rates (40 to 45%) than patients infected with genotype 2 or 3 (approximately 80%) (13). Published and unpublished studies have shown that patients infected with genotype 1 have higher response rates when treated for 48 weeks than for 24 weeks, whereas patients infected with genotype 2 or 3 need only 24 weeks of treatment. Further, it appears that patients infected with genotype 1 require higher doses of ribavirin than patients infected with genotype 2 or 3 (13). CONCLUSIONS In summary, hepatitis C currently accounts for 40 to 60% of patients newly diagnosed with chronic liver disease, and excessive alcohol consumption is a common cofactor. The disease burden associated with chronic hepatitis C is likely to increase in the coming years. The clinical course of chronic hepatitis C is variable, with slow or no progression in many patients and rapid progression in a minority of others. Factors consistently associated with disease progression include older age at infection, male sex, HIV coinfection, and excessive alcohol use. In clinical trials, the best current treatment, pegylated interferon and ribavirin, results in a sustained response in 50 to 60% of all patients, including 40 to 45% of patients infected with genotype 1 and close to 80% of patients infected with genotype 2 or 3. Important directions for the future include generating better estimates of disease burden through population-based studies, improving our understanding of disease progression and the many factors that affect the clinical course, and developing more effective primary and secondary prevention and treatment methods. REFERENCES 1. Alter, H. J., and L. B. Seeff. 2000. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin. Liver Dis. 20:17–35.


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2. Alter, M. J., D. Kruszon-Moran, O. V. Nainan, G. M. McQuillan, F. Gao, L. A. Moyer, R. A. Kaslow, and H. S. Margolis. 1999. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N. Engl. J. Med. 341:556–562. 3. Armstrong, G. L., M. J. Alter, G. M. McQuillan, and H. S. Margolis. 2000. The past incidence of hepatitis C virus infection: implications for the future burden of chronic liver disease in the United States. Hepatology 31:777–782. 4. Bellentani, S., G. Pozzato, G. Saccoccio, M. Crovatto, L. S. Croce, L. Mazzoran, F. Masutti, G. Cristianini, and C. Tiribelli. 1999. Clinical course and risk factors of hepatitis C virus related liver disease in the general population: report from the Dionysos study. Gut 44:874–880. 5. Benhamou, Y., M. Bochet, V. Di Martino, F. Charlotte, F. Azria, A. Coutellier, M. Vidaud, F. Bricaire, P. Opolon, C. Katlama, and T. Poynard for the MULTIVIRC Group. 1999. Liver fibrosis progression in human immunodeficiency virus and hepatitis C virus coinfected patients. Hepatology 30:1054–1058. 6. Bica, I., B. McGovern, R. Dhar, D. Stone, K. McGowan, R. Scheib, and D. R. Snydman. 2001. Increasing mortality due to end-stage liver disease in patients with human immunodeficiency virus infection. Clin. Infect. Dis. 32:492–497. 7. Bissell, D. M. 1999. Sex and hepatic fibrosis. Hepatology 29:988–989. 8. Centers for Disease Control and Prevention. 1998. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. Morb. Mortal. Wkly. Rep. 47(RR-19):1–39. 9. Centers for Disease Control and Prevention. 1999. Births and deaths: preliminary data for 1998. Nat. Vital Stat. Rep. 47:1–48. 10. Corrao, G., and S. Arico. 1998. Independent and combined action of hepatitis C virus infection and alcohol consumption on the risk of symptomatic liver cirrhosis. Hepatology 27:914–919. 11. Darby, S. C., D. W. Ewart, P. L. Giangrande, R. J. Spooner, C. R. Rizza, G. M. Dusheiko, C. A. Lee, C. A. Ludlam, and F. E. Preston. 1997. Mortality from liver cancer and liver disease in haemophilic men and boys in the UK given blood products contaminated with hepatitis C. Lancet 350:1425–1431. 12. Di Bisceglie, A. M., Z. D. Goodman, K. G. Ishak, J. H. Hoofnagle, J. J. Melpolder, and H. J. Alter. 1991. Long-term clinical and histopathological follow-up of chronic posttransfusion hepatitis. Hepatology 14:969–974. 13. Di Bisceglie, A. M., and J. H. Hoofnagle. 2002. Optimal therapy of hepatitis C. Hepatology 36:S121–S127. 14. Freeman, A. J., G. J. Dore, M. G. Law, M. Thorpe, J. Von Overbeck, A. R. Lloyd, G. Marinos, and J. M. Kaldor. 2001. Estimating progression to cirrhosis in chronic hepatitis C virus infection. Hepatology 34:809–816. 15. Frieden, T. R., L. Ozick, C. McCord, O. V. Nainan, S. Workman, G. Comer, T. P. Lee, K.-S. Byun, D. Patel, and K. J. Henning. 1999. Chronic liver disease in central Harlem: the role of alcohol and viral hepatitis. Hepatology 29:883–888. 16. Graham, C. S., L. R. Baden, E. Yu, J. M. Mrus, J. Carnie, T. Heeren, and M. J. Koziel. 2001. Influence of human immunodeficiency virus infection on the course of hepatitis C virus infection: a meta-analysis. Clin. Infect. Dis. 33:562–569. 17. Harris, D. R., R. Gonin, H. J. Alter, E. C. Wright, Z. J. Buskell, F. B. Hollinger, and L. B. Seeff. 2001. The relationship of acute transfusion-associated hepatitis to the development of cirrhosis in the presence of alcohol abuse. Ann. Intern. Med. 134:120–124. 18. Heathcote, E. J., M. L. Shiffman, W. G. E. Cooksley, G. M. Dusheiko, S. S. Lee, L. Balart, R. Reindollar, R. K. Reddy, T. L. Wright, A. Lin, J. Hoffman, and J. De Pamphilis. 2000. Peginterferon alfa-2a in patients with chronic hepatitis C and cirrhosis. N. Engl. J. Med. 343:1673–1680. 19. Hopf, U., B. Moller, D. Kuther, R. Stemerowicz, H. Lobeck, A. Ludtke-Handjery, E. Walter, H. E. Blum, M. Roggendorf, and F. Deinhardt. 1990. Long-term follow-up of posttransfusion and sporadic chronic hepatitis non-A, non-B and frequency of circulating antibodies to hepatitis C virus. J. Hepatol. 10:69–76. 20. Kenny-Walsh, E., and the Irish Hepatology Research Group. 1999. Clinical outcomes after hepatitis C infection from contaminated anti-D immune globulin. N. Engl. J. Med. 340:1228–1233. 21. Kim, W. R., R. S. Brown, N. A. Terrault, and H. El-Serag. 2002. Burden of liver disease in the United States: summary of a workshop. Hepatology 36:227–242. 22. Kim, W. R. 2002. The burden of hepatitis C in the United States. Hepatology 36:S30–S34. 23. Koretz, R. L., H. Abbey, E. Coleman, and G. Gitnick. 1993. Non-A, non-B post-transfusion hepatitis. Looking back on the second decade. Ann. Intern. Med. 119:110–115.


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24. Lesens, O., M. Deschenes, M. Steben, G. Belanger, and C. M. Tsoukas. 1999. Hepatitis C virus is related to progressive liver disease in human immunodeficiency virus-positive hemophiliacs and should be treated as an opportunistic infection. J. Infect. Dis. 179:1254–1258. 25. Lindsay, K. L., C. Trepo, T. Heintges, M. L. Shiffman, S. C. Gordon, J. C. Hoefs, E. R. Schiff, Z. D. Goodman, M. Laughlin, R. Yao, J. K. Albrecht, and The Hepatitis Interventional Therapy Group. 2001. A randomized, double blind trial comparing pegylated interferon alfa-2b to interferon alfa-2b as initial treatment for chronic hepatitis C. Hepatology 34:395–403. 26. Makris, M., F. E. Preston, F. R. Rosendaal, J. C. Underwood, K. M. Rice, and D. R. Triger. 1996. The natural history of chronic hepatitis C in haemophiliacs. Br. J. Haematol. 94:746–752. 27. Manns, M. P., J. G. McHutchison, S. C. Gordon, V. K. Rustgi, M. Shiffman, R. Reindollar, Z. D. Goodman, K. Koury, M. Ling, and J. K. Albrecht. 2001. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 358:958–965. 28. Monga, H. K., M. C. Rodriguez-Barradas, K. Breaux, K. Khattack, C. L. Troisi, M. Velez, and B. Yoffe. 2001. Hepatitis C virus infection-related morbidity and mortality among patients with human immunodeficiency virus infection. Clin. Infect. Dis. 33:240–247. 29. National Institutes of Health. 1997. National Institutes of Health Consensus Development Conference Panel Statement: Management of Hepatitis C. Hepatology 26(Suppl. 1):2S–10S. 30. Pol, S., B. Lamorthe, N. T. Thi, V. Thiers, F. Carnot, H. Zylberberg, P. Berthelot, C. Brechot, and B. Nalpas. 1998. Retrospective analysis of the impact of HIV infection and alcohol use on chronic hepatitis C in a large cohort of drug users. J. Hepatol. 28:945–950. 31. Poynard, T., P. Bedossa, and P. Opolon for the OBSVIRC, METAVIR, CLINIVIR and DOSVIRC Groups. 1997. Natural history of liver fibrosis progression in patients with chronic hepatitis C. Lancet 349:825–832. 32. Poynard, T., V. Ratziu, F. Charlotte, Z. Goodman, J. McHutchison, and J. Albrecht. 2001. Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis C. J. Hepatol. 34:730–739. 33. Seeff, L. B., R. N. Miller, C. S. Rabkin, Z. Buskell-Bales, K. D. Straley-Eason, B. L. Smoak, L. D. Johnson, S. R. Lee, and E. L. Kaplan. 2000. 45-year follow-up of hepatitis C virus infection in healthy young adults. Ann. Intern. Med. 132:105–111. 34. Seeff, L. B., F. B. Hollinger, H. J. Alter, E. C. Wright, C. M. B. Cain, Z. J. Buskell, K. G. Ishak, F. L. Iber, D. Toro, A. Samanta, R. L. Koretz, R. P. Perillo, Z. D. Goodman, R. G. Knodell, G. Gitnick, T. R. Morgan, E. R. Schiff, S. Lasky, C. Stevens, R. Z. Vlahcevic, E. Weinshel, T. Tanwandee, H. J. Lin, and L. Barbosa. 2001. Long-term mortality and morbidity of transfusion-associated non-A, non-B, and type C hepatitis: a National Heart, Lung, and Blood Institute collaborative study. Hepatology 33:455–463. 35. Sherman, K. E., S. D. Rouster, R. T. Chung, and N. Rajicic. 2002. Hepatitis C virus prevalence among patients infected with human immunodeficiency virus: a cross-sectional analysis of the U.S. adult AIDS Clinical Trials Group. Clin. Infect. Dis. 34:831–837. 36. Soriano, V., J. García-Samaniego, E. Valencia, R. Rodríguez-Rosado, F. Muñoz, and J. GonzálezLahoz. 1999. Impact of chronic liver disease due to hepatitis viruses as cause of hospital admission and death in HIV-infected drug users. Eur. J. Epidemiol. 15:1–4. 37. Soto, B., A. Sánchez-Quijano, L. Rodrigo, J. A. Del Olmo, M. García-Bengoechea, J. HernándezQuero, C. Rey, M. A. Abad, M. Rodriguez, M. Sales Gilabert, F. González, P. Mirón, A. Caruz, F. Relimpio, R. Torronteras, M. Leal, and E. Lissen. 1997. Human immunodeficiency virus infection modifies the natural history of chronic parenterally-acquired hepatitis C with an unusually rapid progression to cirrhosis. J. Hepatol. 26:1–5. 38. Thomas, D. L., J. Astemborski, R. M. Rai, F. A. Anania, M. Schaeffer, N. Galai, K. Nolt, K. E. Nelson, S. A. Strathdee, L. Johnson, O. Laeyendecker, J. Boitnott, L. E. Wilson, and D. Vlahov. 2000. The natural history of hepatitis C virus infection: host, viral and environmental factors. JAMA 284:450–456. 39. Tong, M. J., N. S. El-Farra, A. R. Reikes, and R. L. Co. 1995. Clinical outcomes after transfusion-associated hepatitis C. N. Engl. J. Med. 332:1463–1466. 40. Tremolada, F., C. Casarin, A. Alberti, C. Drago, A. Tagger, M. L. Ribero, and G. Realdi. 1992. Longterm follow-up of non-A, non-B (type C) post-transfusion hepatitis. J. Hepatol. 16:273–281. 41. Vogt, M., T. Lang, G. Frosner, C. Klingler, A. F. Sendl, A. Zeller, B. Wiebecke, B. Langer, H. Meisner, and J. Hess. 1999. Prevalence and clinical outcome of hepatitis C infection in children who underwent cardiac surgery before the implementation of blood-donor screening. N. Engl. J. Med. 341:866–870.


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42. Wiese, M., F. Berr, M. Lafrenz, H. Porst, and U. Oesen. 2000. Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20-year multicenter study. Hepatology 32:91–96. 43. Wiley, T. E., M. McCarthy, L. Breidi, and T. J. Layden. 1998. Impact of alcohol on the histological and clinical progression of hepatitis C infection. Hepatology 28:805–809. 44. Zeuzem, S., S. V. Feinman, J. Rasenack, E. J. Heathcote, M.-Y. Lai, E. Gane, J. O’Grady, J. Reichen, M. Diago, A. Lin, J. Hoffman, and M. J. Brunda. 2000. Peginterferon alfa-2a in patients with chronic hepatitis C. N. Engl. J. Med. 343:1666–1672.


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Chapter 2

Polio Reemerges in the Americas: a Vaccine-Derived Outbreak in Hispaniola in 2000 and 2001 Ciro A. de Quadros, Mauricio Landaverde, Olen Kew, Zacarias Garib, Jean André, Cristina Pedreira, and Fernando Laender

In October 2000, the Ministries of Health of the Dominican Republic and Haiti reported two cases of acute flaccid paralysis (AFP). These cases were identified with the aid of the national AFP surveillance systems, which require that each AFP case suspected of being poliomyelitis be reported. Under these circumstances, it is customary to obtain stool samples from the patients and to send them to one of the laboratories in the regional laboratory network for poliomyelitis detection to determine whether the paralysis is due to the wild poliovirus. The case reported in the Dominican Republic was that of a 9-month-old girl from a village in the province of Monseñor Nouel; the case reported in Haiti involved a 2-year-old girl from a town in the Northwest Department. The symptoms began on 12 July and 28 August 2000, respectively. The stool samples of both patients were sent to the Pan American Health Organization (PAHO) Laboratory for Poliovirus Detection at the Caribbean Epidemiology Center in Port of Spain, Trinidad and Tobago, where poliovirus type 1 was isolated in both samples. Subsequently, the isolates were sent to the Poliovirus Laboratory of the Centers for Disease Control and Prevention in Atlanta, Ga., for identification. The sequential analysis revealed that the virus associated with this outbreak was atypical because it is derived from the oral polio vaccine (OPV) but showed a genetic divergence of 3% with respect to the reproducing strain used in the OPV. The last case of poliomyelitis due to the wild poliovirus in the Dominican Republic was reported in 1985; in Haiti, the last case was reported in 1989 (4).

Ciro A. de Quadros • Sabin Vaccine Institute, 1718 Connecticut Ave. NW, Suite 700, Washington, DC 20009. Mauricio Landaverde, Cristina Pedreira, and Fernando Laender • Division of Vaccines and Immunization, Pan American Health Organization, 525 23rd St. NW, Washington, DC 20037. Olen Kew • Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333. Zacarias Garib • Ministry of Health, Santo Domingo, Dominican Republic. Jean André • Ministry of Health, Port-au-Prince, Haiti. 13


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POLIO ERADICATION IN THE AMERICAS The Western Hemisphere has not experienced wild poliovirus circulation since 1991, when the last confirmed case of paralytic poliomyelitis caused by the wild poliovirus was detected in the town of Pichinaki, Department of Junín, in Peru. Since then, nearly 40,000 stool samples from cases identified by the AFP surveillance systems have been analyzed in the region’s laboratories without any other wild poliovirus being detected until now. Intense efforts on both continents were required to implement strategies to eradicate the indigenous circulation of the wild poliovirus. The strategies for polio eradication used in the Americas relied on the achievement and maintenance of high immunization levels in the population at risk (children under 5 years of age) and disease surveillance (2). The basic issue that needed to be addressed at the launch of the polio eradication initiative in the Americas was to determine the optimal strategy for achieving and maintaining high vaccination coverage. In Brazil, national immunization days (NIDs) to administer live OPV resulted in a dramatic decline in the number of cases within a short time (Fig. 1). These NIDs are generally held twice a year and are conducted 1 or 2 months apart. During each NID, 20 million children under 5 years of age in Brazil are vaccinated with OPV3. NIDs were first used during the introduction of OPV in the early 1960s in Cuba, where transmission is believed to have been interrupted in the early 1960s (Fig. 2). Live oral polio vaccine has never been used routinely in Cuba but is given only during NIDs, which are held in January and March of every year. The success of NIDs in Brazil is a reminder of the effectiveness of this strategy to interrupt transmission of the wild poliovirus. This vaccination program also demonstrates that it is logistically possible to implement such a strategy in a large developing country like Brazil.

Figure 1. Polio cases by 4-week period, in Brazil, from 1975 to 1984. Sources: Departamento Nacional de Epidemiologia-Secretaria Nacional de Ações Básicas de Saúde, MS, and PAHO.


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Figure 2. Poliomyelitis in Cuba, from 1946 to 1992. Source: country reports. NVD, national vaccination days.

The results of this program were critical to the development of the approaches to be used in the different countries in the Americas. The countries were divided into two groups: those in which polio is endemic or in which at least one case was reported during the past 3 years, and those free of polio or not reporting any cases during the past 3 years. To facilitate surveillance, uniform case definitions for poliomyelitis were adopted: a suspected case was any case of acute paralytic illness in a child less than 15 years of age or in any individual for which the attending physician strongly suspected poliomyelitis. All patients were to be seen within 48 h by a trained epidemiologist for a thorough examination. If the case was AFP, it would be classified as probable poliomyelitis, and two stool specimens would be collected 24 h apart and sent to one of the laboratories included in a network of diagnostic laboratories that had been established. After 10 weeks, the case would be classified as confirmed poliomyelitis if the wild poliovirus had been isolated from the stools or if the patient had no adequate stool specimens and died within 60 days of the onset of symptoms or was not available for follow-up. During the later stages of the program, another category, compatible poliomyelitis, was added to the patient classification. Compatible poliomyelitis cases met other criteria for diagnosis, but two stool specimens had not been collected within 2 weeks of the onset of paralysis. Such cases represented failures of the surveillance system and could not be definitely classified as paralytic disease due to other causes. CERTIFICATION OF POLIO ERADICATION IN THE AMERICAS The International Certification Commission on Polio Eradication (ICCPE) established by PAHO in 1990 defined four basic components that were critical for the purposes of certification (9): (i) surveillance of AFP, (ii) surveillance of wild poliovirus, (iii) active searches for AFP cases, and (iv) mop-up vaccination campaigns in high-risk areas.


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Countries were considered for certification only if they had been free of poliomyelitis for at least 3 years with adequate surveillance. For surveillance of AFP, the ICCPE established the following five mandatory indicators: 1. At least 80% of all the health units included in the reporting network should regularly report the absence or presence of AFP each week. 2. The annual expected rate of AFP should be at least one case per 100,000 children less than 15 years of age in any country. 3. At least 80% of all reported AFP cases should be investigated within 48 h of reporting. 4. At least 80% of all reported AFP cases should have two stool specimens taken for virus culture within 2 weeks of the onset of paralysis. 5. At least 80% of all reported AFP cases should have stool specimens cultured for at least five contacts. During its third meeting held in Washington, D.C., on 24 and 25 August 1994, the ICCPE reviewed the data presented by the presidents of the National Certification Commission. The Commission had analyzed in depth the ICCPE’s country data on poliomyelitis and poliovirus surveillance over the previous 3 years and came to the following conclusions. 1. During the past 6 years, most countries in the Americas region have achieved and maintained OPV3 vaccination coverage levels of over 80%. Since the last confirmed case was reported from Peru in August 1991, over 6,000 AFP cases have been thoroughly investigated, and none has been confirmed as paralytic poliomyelitis due to the wild poliovirus. Furthermore, over 25,000 stool specimens obtained from patients and their contacts were negative for wild poliovirus. Finally, there has been constant monitoring of key surveillance indicators, which were, with few exceptions, at acceptable levels in all countries during the past 3 years. 2. After carefully reviewing these data, all National Certification Commissions of the region have recommended that their countries be certified as being polio free. 3. The ICCPE has carefully reviewed data that were presented by the presidents of the National Certification Commissions. The ICCPE recognizes and applauds the extraordinary accomplishments which have been achieved by the countries of the region. Overall, the quality of the National Certification Commission reports has been excellent: the members have taken their responsibilities seriously and responded in exemplary fashion. OUTBREAK OF POLIO IN THE DOMINICAN REPUBLIC AND HAITI The unusual outbreak of poliomyelitis caused by Sabin type 1-derived poliovirus was detected in the Dominican Republic in October 2000 (5). As of January 2001, which is the date of the last confirmed case in the Dominican Republic, there have been 13 confirmed


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cases due to vaccine-derived poliovirus type 1 that were reported between July 2000 and January 2001. Most of the cases involved unvaccinated children ⬍5 years of age in areas with very low coverage with oral polio vaccine. Investigations also revealed poor sanitation in these areas. The first confirmed patient was a 9-month-old girl in the province of Monseñor Nouel, county of Bonao. The onset of paralysis occurred on 12 July 2000. In Haiti, the first confirmed patient was a 2-year-old girl from the town of Nan Citron. For her, the onset of paralysis occurred on 30 August 2000. As of July 2001, the date of the last confirmed case in Haiti, there have been eight confirmed cases due to Sabin type 1-derived virus. These cases were identified between August 2000 and July 2001 (Fig. 3 and 4). Of the 21 persons with confirmed cases of poliomyelitis, 9 (42%) were unvaccinated, 6 (29%) had unknown vaccination status, 5 (24%) had received a single dose of OPV, and 1 reported having received three doses of OPV. Twelve patients were children under 5 years old, five patients were between 5 and 9 years old, and four patients were between 10 and 14 years old. The median age of the patients was 2 years, with a range of 9 months to 14 years (6). Analysis of the genomic sequence showed that the virus associated with the outbreak in both countries was unusual, because the virus was derived from OPV and had a divergence of 3% from the parental OPV strain (usually, vaccine-derived poliovirus does not diverge more than 0.5% from the parental strain). Normally, wild poliovirus has a genetic similarity to OPV of ⬍82%. The poliovirus associated with this outbreak appears to have recovered the neurovirulence and transmissibility characteristics of wild poliovirus type 1. The

Figure 3. Confirmed vaccine-derived polio cases in the Dominican (Dom) Republic (DOR) and Haiti (HAI) from 2000 and 2001. Source: country reports (data as of 15 May 2002).


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Figure 4. Areas with confirmed polio cases on the island of Hispaniola, from 2000 and 2001. Source: Polio Eradication Surveillance System/Division of Vaccines and Immunization (data as of 14 December 2001).

differences in nucleotide sequences among the outbreak isolates suggested that the virus had been circulating for approximately 2 years (Fig. 5) in an area where vaccination coverage was low and that the virus had accumulated genetic changes which restored its essential properties of wild poliovirus (3). Prolonged circulation of vaccine-derived poliovirus in areas with very low OPV coverage had only been documented in one other setting—a type 2 OPV-derived virus that circulated in Egypt for an estimated 10 years (1983 to 1993) and that was associated with more than 30 reported cases. Vaccination coverage was quite low, and circulation of the vaccine-derived poliovirus ceased rapidly once vaccination coverage increased (1). Besides low OPV vaccination coverage, epidemiological investigations have identified poor sanitation as a risk factor for transmission of the virus. In the Dominican Republic’s Constanza County of La Vega Province, where most of the outbreak-related cases have been identified, coverage with three doses of OPV has ranged between 20 and 30% in children ⬍5 years of age. National vaccination coverage with three doses of OPV in children ⬍1 year of age in the Dominican Republic has consistently been about 80% for each of the past 5 years; 20% of districts have had coverage above 80%. NIDs were discontinued in that country 5 years ago. In 1999, Haiti’s Northwest Department reported OPV coverage of 40%. During the last 10 years, national vaccination coverage with three doses of OPV in children ⬍1 year of age has ranged between 30 and 50%. NIDs were also discontinued 5 years ago (3). In the Dominican Republic, the AFP rate per a population of 100,000 aged ⬍15 years had been below 1 during six of the past 10 years, and the rate in Haiti had been about 0.1 per 100,000 since 1995 (Fig. 6).


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Figure 5. Rate of fixation of synonymous substitutions in complete capsid. Source: Centers for Disease Control and Prevention.

The proportion of AFP cases with adequate specimens in the Dominican Republic was approximately 80% from 1993 to 1998, 30% in 1999, and 36% in 2000. In Haiti, stool specimens were not collected for any of the AFP cases reported during the past 5 years. The proportion of notification sites reporting weekly during the past 10 years has been above 80% in the Dominican Republic, except in 1999 when it was 50%. In Haiti, the proportion of notification sites reporting weekly has been below 50% for the past 10 years, except in 1998 when it was 95%. Enterovirus isolation has been above 15% in the Dominican Republic for the past 10 years, except in 1996 and 1999 when it was zero and in 1997 when it was 9% (7). In response to low OPV coverage and limited surveillance, the Ministries of Health of the Dominican Republic and Haiti, with the support of PAHO and the Centers for Disease

Figure 6. AFP rate per 100,000 persons under 15 years of age, Dominican Republic (DOR) and Haiti (HAI) from 1988 to 2001. Source: PAHO (data as of 30 June 2001).


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Control and Prevention, undertook activities to determine the extent of the virus circulation, and to control and eradicate the virus by introducing house-to-house vaccination and NIDs. Intense public education and social mobilization activities were also instituted. Intensive case-finding activities were initiated to determine the extent and magnitude of the outbreak, to identify foci of disease activity, and to organize areas for vaccination activities. Teams of national and international epidemiologists were established to conduct active searches for AFP cases in both countries. Active searches were conducted in health centers, hospitals, emergency care centers, physical therapy clinics, orphanages, and day care centers and in the community. All suspected cases underwent a complete epidemiological investigation, with stool specimens being taken (8). In December 2000, a vaccination campaign with OPV was carried out in the Dominican Republic. At that time, 1.2 million children ⬍5 years of age received OPV (this represents coverage of close to 100%). A second mass vaccination campaign was carried out in January 2001, and one more was conducted in May 2001, with coverage close to 100% in each case. The patient with the last confirmed case had a date of paralysis onset of 25 January 2001. As of May 2002, 15 months after the last confirmed case, 65 more AFP cases have been reported through the surveillance systems and discarded after careful investigation, with all stool specimens being negative. In Haiti, the strategy of rolling campaigns carried out in February, May and June, and September 2001 using vaccines for polio and other antigens was successfully used to control the outbreak (1, 3, 6, 7, 8). Approximately 2.9 million children (coverage of ⬃85%) under the age of 10 years received OPV. The patient with the last confirmed case in Haiti had a date of onset of paralysis of 12 July 2001. As of May 2002, 10 months after the last case was confirmed, 17 more cases of suspected AFP had been detected through the surveillance system but were discarded after careful investigations (stool specimens were negative). Both countries have strengthened their surveillance systems and routine vaccination services, and both have programs to carry out at least 2 NIDs every year, using OPV and the other antigens. Because there is no sign of OPV-derived poliovirus circulation in the areas with high vaccination coverage against poliomyelitis, the current outbreak shows the need to maintain high polio vaccine coverage in disease-free areas until global eradication is achieved. At the same time, it is crucial to maintain good monitoring of AFP and poliovirus cases. Among the actions taken were the determination of the real extent of the outbreak as well as the risk factors and potential for future outbreaks. Currently, all Sabin poliovirus isolates from AFP cases in the Americas since 1995 have been sequenced in conjunction with epidemiological analysis of those high-risk areas where new Sabin-derived poliovirus had been identified. Given this new development, additional studies will be required before deciding whether to discontinue polio vaccination once global eradication has been achieved. A special call was issued to countries in the Americas and worldwide to remain highly vigilant by maintaining high levels of OPV coverage and active surveillance throughout each country. Specific recommendations include the following: • Carrying out within each country a risk analysis, both for potential circulation of the virus (imported wild poliovirus or vaccine derived) and for the AFP surveillance system’s capacity to detect circulation on a timely basis. Review level of OPV coverage.


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• Taking advantage of all mass vaccination activities to vaccinate with OPV, especially in risk areas. • Complying with the indicators of AFP surveillance, both at the national and at the departmental or provincial levels. • Maintaining 95% vaccination coverage with OPV in all municipalities or equivalent geopolitical areas. Countries that fail to reach this level of coverage in more than 80% of their municipalities should carry out at least two national immunization campaigns. GLOBAL IMPLICATIONS In 1998, the World Health Assembly issued Resolution WHA 41.28, in which it established the target of global poliomyelitis eradication by the end of 2000. Since then, major efforts have been made and, as can be observed in Fig. 7 and below, poliomyelitis cases due to wild poliovirus have been confined to a small number of countries. The annual total of cases shows a dramatic reduction, thanks to actions that have been based on reaching and maintaining high OPV vaccination coverage as well as maintaining adequate AFP epidemiological surveillance. As of the first half of 2002, the progress reported by countries in which there are still cases of poliomyelitis due to the wild poliovirus is so apparent that the eradication target is considered to be definitively reachable in the very near future. Such progress will require decision makers in the field of public health to consider different scenarios for posteradication immunization policies, namely, the development of vaccination strategies against poliomyelitis once circulation of the wild poliovirus has been completely interrupted globally.

Figure 7. Polio eradication progress, from 1988 to 2001.


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During a recent meeting convened by the World Health Organization in Geneva to discuss the scientific bases for interruption of poliomyelitis immunization (March 1998), the advisory group (11) concluded that immunization must meet the following requirements: (i) certification that the wild poliovirus has been eradicated at the global level, (ii) a guarantee that the wild polioviruses that still exist in laboratories are kept under adequate confinement conditions, and (iii) evidence that the OPV-derived poliovirus has circulated only over a limited period during the postvaccination era. The countries of the world have been grouped into regions and have been progressively meeting the requirements proposed by the Global Commission for the Certification of Polio Eradication. Currently, three of the five health regions (America, Europe, and the Western Pacific) have had no reported endemic cases of poliomyelitis caused by wild poliovirus during the past 3 years and, with adequate AFP monitoring, have been certified free from indigenous circulation of wild poliovirus (12). At the same time, the Global Plan of Action for Laboratory Confinement of the Wild Poliovirus (13) has been implemented. Its basic strategy consists of creating in each country an independent commission that conducts a national laboratory census and assembles an inventory to facilitate the identification of laboratories storing wild poliovirus or any potentially infectious material related to the wild poliovirus. Those laboratories with wild poliovirus or potentially infectious material will be asked to destroy it, maintain it under adequate biosafety conditions, or store it in specialized laboratories that will be specifically designated for such purpose and where adequate biosafety conditions will be guaranteed so as to prevent an accidental release of wild poliovirus into the environment or disease-free communities.

CONCLUSIONS The outbreak of poliomyelitis in the Dominican Republic and Haiti, coupled with the history of other outbreaks caused by OPV-derived poliovirus that has reversed and become neurovirulent (Egypt [1988 to 1992] and the Philippines [1991]), has revived a long-standing debate on the possibility that when vaccination is interrupted or when the immunity of the groups at risk is reduced, the vaccine-derived virus could be reintroduced, circulate in the environment, and cause epidemics. These outbreaks have occurred while epidemics caused by the vaccine-derived virus have never been reported and after such vaccine has been used for more than 40 years and billions of doses have been administered throughout the world without resulting in more than occasional cases of vaccine-associated paralysis, whether among the vaccinated or their contacts. The dilemmas presented in light of this new evidence provide an opportunity to consider different scenarios (10) of a time when poliomyelitis is eradicated from the world. The options range from interrupting OPV vaccination (following mass campaigns on a global level) to continuing the use of OPV indefinitely, using it alone or in combination with injectable vaccines, replacing it with inactivated poliomyelitis vaccine, or attempting to discover a new vaccine. Meanwhile, after intensive immunization efforts using OPV, the polio outbreak in the Dominican Republic and Haiti has been effectively interrupted.


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REFERENCES 1. Centers for Disease Control and Prevention. 2001. Circulation of a type 2 vaccine-derived poliovirus— Egypt, 1982–1993. Morb. Mortal. Wkly. Rep. 50:41–42. 2. de Quadros, C. A., J. K. Andrus, J. M. Olivé, C. G. de Macedo, and D. A. Henderson. 1992. Polio eradication from the Western Hemisphere. Annu. Rev. Public Health 12:239–252. 3. Kew, O., V. Morris-Glasgow, M. Landaverde, C. Burns, J. Shaw, Z. Garib, J. Andre, E. Blackman, C. J. Freeman, J. Jorba, R. Sutter, G. Tambini, L. Venczel, C. Pedreira, F. Laender, H. Shimizu, T. Yoneyama, T. Miyamura, H. van der Avoort, M. S. Oberste, D. Kilpatrick, S. Cochi, M. Pallansch, and C. de Quadros. 2002. Outbreak of poliomyelitis in Hispaniola associated with circulating type 1 vaccine-derived poliovirus. Science 296:356–359. 4. Landaverde, M., L. Venczel, and C. A. de Quadros. 2001. Outbreak of poliomyelitis in Dominican Republic and Haiti, due to a vaccine-derived poliovirus. Pan Am. J. Public Health 9:272–274. 5. Pan American Health Organization, Division of Vaccines and Immunization. 2000. Outbreak of poliomyelitis in Dominican Republic and Haiti. EPI Newsl. 22(6):1–2. 6. Pan American Health Organization, Division of Vaccines and Immunization. 2001. Update: OPVderived poliomyelitis outbreak in the Dominican Republic and Haiti. EPI Newsl. 23(1):4–5. 7. Pan American Health Organization, Division of Vaccines and Immunization. 2001. Haiti begins all out effort to halt measles and OPV-derived polio outbreaks. EPI Newsl. 23(2):1–2. 8. Pan American Health Organization, Division of Vaccines and Immunization. 2001. Haiti’s polio campaign: a success story. EPI Newsl. 23(4):2–3. 9. Robbins, F. C., and C. A. de Quadros. 1997. Certification of the eradication of indigenous transmission of wild poliovirus in the Americas. J. Infect. Dis. 175(Suppl. 1):281–285. 10. Wood, D. J., R. W. Sutter, and W. R. Dowdle. 2001. Stopping poliovirus vaccination after eradication: issues and challenges. Bull. W. H. O. 78:347–363. 11. World Health Organization. 1998. Report of the Meeting on the Scientific Basis for Stopping Polio Immunization. World Health Organization, Geneva, Switzerland. 12. World Health Organization. 2001. Report of the Sixth Meeting of the Global Commission for the Certification of the Eradication of Poliomyelitis. World Health Organization, Geneva, Switzerland. 13. World Health Organization. 2000. Global Action Plan for Laboratory Containment of Wild Polioviruses. WHO/V&B/99.32. 2000. World Health Organization, Geneva, Switzerland.


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Chapter 3

Yellow Fever Vaccine-Associated Disease Rachel S. Barwick, Anthony A. Marfin, and Martin S. Cetron

Yellow fever is the first described and prototypic viral hemorrhagic fever and is caused by yellow fever virus, a flavivirus. Currently, yellow fever only occurs in Africa and South America, where an estimated 200,000 cases occur each year (36) (Fig. 1). During human outbreaks, yellow fever is spread by the mosquito Aedes aegypti. The incubation period is 3 to 6 days, and the infection often results in an abrupt onset of fever, headache, photophobia, lumbosacral pain, epigastric pain, anorexia, and vomiting. Infection may result in subclinical illness, but it often progresses to moderate or severe illness in 15% of cases, including overwhelming systemic illness (22). The systemic illness is characterized by liver and renal failure and by hemorrhage caused by thrombocytopenia and an acquired coagulopathy. The fatality rate of severe systemic illness is approximately 20%. Treatment includes supportive care only because there is no specific antiviral treatment. Vaccination and eradication of A. aegypti are the only two effective strategies to reduce yellow fever morbidity and mortality in affected areas. Yellow fever vaccine, under the standardized seed lot system, has been available since 1945 and has been widely used in outbreaks, by the military, and by travelers to areas where yellow fever is endemic. The Centers for Disease Control and Prevention (CDC) and the Advisory Committee on Immunization Practices currently recommend vaccination for travelers aged 9 months and older going to areas at risk for yellow fever (Fig. 1) (7, 8). In addition, more than 125 countries legally require vaccination among international travelers for entry; some African countries require evidence of vaccination from all entering travelers; and other countries require vaccination from travelers coming from areas where there is a risk of yellow fever. Generally, countries that have a competent mosquito vector require proof of vaccination among travelers to prevent establishment of domestic transmission areas and essentially make up the potential range of the A. aegypti mosquito. In the 1930s A. aegypti was present throughout much of Central and South America. When mosquito eradication campaigns in the Southern Hemisphere ended in 1970, the geRachel S. Barwick and Martin S. Cetron • Division of Global Migration and Quarantine, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333. Anthony A. Marfin • Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521. 25

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Figure 1. Zones where yellow fever is endemic.


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ographic range of A. aegypti had been greatly reduced and was hardly present in South America. Since 1970, A. aegypti has spread across South America and reestablished itself in areas that it occupied before eradication (12). The reinfestation of A. aegypti is strongly suspected as having played a role in transmission in outbreaks occurring in South America in the past decade (33, 34; N. Troillet and F. Laurencet, Letter, Lancet 358:1908–1909, 2001). In Africa, A. aegypti eradication never occurred, and yellow fever epidemics continue to occur in urban and rural settings (23, 25). The number of travelers going to areas of yellow fever endemicity is increasing, and an estimated 500,000 to 1,000,000 unvaccinated travelers from the United States annually enter areas where yellow fever is endemic. As the range of A. aegypti has reexpanded and the number of travelers to areas of yellow fever endemicity has increased, there has also been an increase in the reported number of imported cases in the United States and Europe (5, 6, 37). In 1996, there was a report of an imported case of yellow fever in a U.S. traveler, the first U.S. case in 72 years (21). Since 1996, there have been two additional cases in the United States (5, 6). All three cases were fatal. Although the need for yellow fever vaccination remains and the vaccine has been considered to be one of the safest vaccines, recent reports have raised concerns about vaccine safety (2, 22). The purpose of this chapter is to give an overview of recently described vaccine-associated adverse events and give an estimate of the rate of such events. ADVERSE EVENTS Yellow fever vaccine is a live, attenuated virus preparation made from the 17D yellow fever virus strain and has given rise to the development of three distinct but closely related lineages, 17D-204, 17DD, and 17D-213 (22, 32). Vaccine type 17D-204 is manufactured in the United States, Europe, and Australia, vaccine type 17DD is manufactured in Brazil, and 17D-213 is manufactured by the World Health Organization in Germany. The three vaccine types share 99.9% nucleic acid sequence homology (26). General Adverse Events Reactions to 17D yellow fever vaccine are typically mild. After vaccination, vaccinees often report mild headaches, myalgia, low-grade fevers, or other minor symptoms for 5 to 10 days. In clinical trials, where symptoms are actively elicited, the incidence of mild adverse events has been 25% and up to 1% of vaccinees curtail regular activities (18, 24). Vaccine strain viremia after primary vaccination with yellow fever vaccine frequently occurs among healthy persons but is usually waning or absent after the first week (22, 27). Immediate hypersensitivity reactions, characterized by rash, urticaria, or asthma, are uncommon, occur principally among persons with histories of allergies to egg or other substances, and are estimated to occur in 1 out of 131,000 vaccinations (14). Gelatin, used as a stabilizer in yellow fever vaccine, has been implicated as a cause of allergic reactions to other vaccines and may play a role in reactions to yellow fever vaccine (13, 18, 30, 31). Yellow Fever Vaccine-Associated Neurotropic Disease In the early development stages of the vaccine, neurotropic disease (also known as postvaccinal encephalitis) among children was the most common serious adverse event associated with yellow fever vaccines. After the 17D seed lot system was standardized in


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1945, the frequency of those events decreased to 1 case per 8 million doses administered (22). From 1945 to 2001, only 23 encephalitis cases associated with or definitively caused by 17D vaccine have been reported worldwide (1, 15, 20, 22); of these cases, 16 occurred among children aged 9 months or less and 7 among persons aged 3 to 76 years. From 1965 through 2001, only one U.S. case was reported, in a 76-year-old male who had encephalitis and vaccine-associated viscerotropic disease (20). Since 2001, four U.S. encephalitis cases have been reported to the Vaccine Adverse Event Reporting System (VAERS). The patients in all of these cases were adults (aged 16, 36, 41, and 71 years) with illness onset 4 to 23 days after receiving a 17D-204 vaccine (4). In addition, five cases of Guillain-Barré-like syndrome in U.S. civilians have been reported to VAERS since 1995 (unpublished VAERS data) and currently are being investigated. Historically, postvaccinal encephalitis associated with yellow fever vaccine has a case fatality rate of ⬍5%; all nine of these patients survived without sequelae (4). In 2002, a fatal case of yellow fever vaccine-associated neurotropic disease was reported from Thailand and involved a 53-year-old man with previously unrecognized human immunodeficiency virus infection (15). Like patients with vaccine-associated viscerotropic disease, all 10 of these patients with neurotropic disease were primary vaccinees, but the illness onset was more variable (i.e., 4 to 23 days postvaccination). Most had fever, headache, impaired cognition, and focal neurologic deficits. For those cases that had cerebrospinal fluid specimens available, most had cerebrospinal fluid pleocytosis and all contained yellow feverspecific immunoglobulin M. Yellow Fever Vaccine-Associated Viscerotropic Disease In 2001, a syndrome of fever and multisystem organ failure was first described among recipients of 17D-204 and 17DD vaccines (9, 20, 35). Between 1996 and 2002, eight U.S. citizens (aged 22, 25, 56, 63, 67, 70, 76, and 79 years) became ill 2 to 5 days after receiving 17D-204 (six cases have been reviewed in references 4 and 20). All eight persons required intensive care after they experienced fever, hypotension, respiratory failure, elevated hepatocellular enzymes, hyperbilirubinemia, lymphocytopenia, and thrombocytopenia. Of the eight patients, five had renal failure that required hemodialysis and five died. Vaccine-type yellow fever virus was isolated from the blood of two patients 7 and 8 days after vaccination (20). A third isolate was made from cerebrospinal fluid that was obtained when the patient developed encephalitis (20). In two fatal cases, yellow fever antigen was identified by immunohistochemistry in liver biopsies in one case and multiple tissue specimens in the other (see larger cover photo). Flavivirus-like particles consistent with yellow fever virus were identified by electron microscopy from tissue from one of the patients. All reported patients with viscerotropic disease were primary vaccinees. In addition to these cases, an Australian citizen (aged 56 years) became similarly ill after receiving a 17D-204 vaccination in 2001 (9) and in 1999 and 2000, two Brazilian citizens (aged 5 and 22 years) became ill 3 or 4 days after receiving 17DD vaccine (35). All died 8 to 11 days after vaccination. In these cases, histopathologic changes characteristic of wild-type yellow fever were noted in the liver (midzonal necrosis, microvesicular fatty change, and Councilman’s bodies). Yellow fever antigen was identified by immunohistochemistry in areas of midzonal necrosis in liver specimens from two recipients, and flavivirus-like particles consistent with yellow fever virus were identified by electron


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microscopy in areas of midzonal necrosis in the third patient. A 17DD or 17D-204 yellow fever vaccine strain was isolated from blood and autopsy material (i.e., brain, liver, kidney, spleen, lung, skeletal muscle, or skin) from these three persons. Because genomic changes associated with a reversion to virulence were not detected in the recovered viruses and because these viruses retained their vaccine-type phenotype when put into experimental animals, Brazilian authorities assumed that the occurrences were caused by undefined host factors (11). Since these initial reports, three additional cases of severe adverse events after yellow fever vaccination have been reported among persons aged 45, 50, and 71 years (V. Adhiyaman, A. Oke, and C. Cefai, Letter, Lancet 358:1907–1908, 2001; U. Werfel and W. Popp, Letter, Lancet 358:1909, 2001; Troillet and Laurencet, letter, 2001). All were hospitalized with fever and elevated hepatocellular enzymes and renal abnormalities; all recovered from their illness. Yellow fever virus was isolated from the blood of one person 13 days after vaccination (Troillet and Laurencet, letter, 2001). Although yellow fever vaccine strain viremia after primary vaccination frequently occurs among healthy persons, it is usually waning or absent after the first week (22, 27) and rarely occurs 13 days following vaccination. Of the two persons who were serologically tested, one had unusually high levels of yellow fever antibodies (1:10,240 titer) (Werfel and Popp, letter, 2001). Whether and in what way underlying genetic or acquired host factors may have contributed to the course or outcome of yellow fever vaccine-associated viscerotropic disease are unknown. AGE-ASSOCIATED RISK Accurately measuring the incidence of vaccine-associated viscerotropic and neurotropic disease is difficult because adequate prospective data are unavailable. Although VAERS solicits reports of adverse events after vaccination, VAERS is a passive surveillance system and most likely underestimates the true number of events and may have biased reporting (29). In addition, the true number of doses of yellow fever vaccine administered is unknown. Despite these limitations, by using available data on the number of cases reported to VAERS, doses sold in the United States, and the age distribution of travelers receiving vaccine at a sample of travel clinics, we are able to estimate the reporting rate of these two events. The annual number of single-dose vials purchased by civilian health care providers from 1990 through 2002 was provided by Aventis Pasteur, the sole manufacturer for the United States. Because U.S. health care providers indicated little or no wastage, it was assumed that the total net number of doses sold was a good estimate of the total number of doses administered (19). In 1998, 13 U.S.-based GeoSentinel clinics, which provide vaccines to international travelers, reviewed records of vaccine administration (19). GeoSentinel, an international network of travel/tropical medical clinics established by CDC and the International Society of Travel Medicine (10), monitors geographic and temporal trends in infectious diseases among international travelers to detect sentinel events through pre- and posttravel consultation. All vaccine recipients during the most recent 12-month period for which complete information was available were categorized by age group. Data for children ⬍15 years old may be underestimated, since this group is underrepresented at U.S. GeoSentinel clinics. Military personnel were excluded. Other than


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these exceptions, the age distribution for vaccine recipients and other data from GeoSentinel clinics are considered representative of national vaccine use and representative for the years 1990 to 2002. The number of doses administered to each age group (i.e., the denominator) was estimated by multiplying the total number of doses per year by the proportionate age group distribution estimated from the GeoSentinel clinics. Yellow fever vaccine-associated adverse events among U.S. citizens that were reported to VAERS from 1990 through 2002 were categorized as yellow fever vaccine-associated viscerotropic disease or yellow fever vaccine-associated neurotropic disease and classified by age group. Reporting rates for yellow fever vaccine-associated viscerotropic disease and neurotropic disease in the United States were calculated by dividing the events reported by an estimate of the number of yellow fever vaccinations in each age group. Overall, the U.S. reporting rate of yellow fever vaccine-associated neurotropic disease is estimated at 4 cases per million distributed civilian doses, and for viscerotropic disease it is estimated to be 3 cases per million doses (Fig. 2). With data from 1996 to 2002, when the syndrome was first recognized, the estimated reporting rate for yellow fever vaccineassociated viscerotropic disease increased to 5 cases per million distributed civilian doses. For vaccinees aged 60 years or older, the rate for neurotropic disease was 11 cases per million distributed civilian doses and more than four times higher than the rate for persons ⬍60 years old, which was 2.5 cases per million doses (Fig. 3). For yellow fever vaccineassociated viscerotropic disease, this increased rate with age is more pronounced. The estimated reporting rate for yellow fever vaccine-associated viscerotropic disease is approximately 1 case per million distributed civilian doses for persons ⬍60 years of age. For ages 60 to 69 years, the rate increases to 11 cases per million net civilian doses, and among vaccinees age 70 years or older, the rate is 34 cases per million doses. The rate of either yellow fever vaccine-associated syndrome among vaccinees aged 60 to 69 years and aged 70 years or older is 21 cases and 45 cases per million distributed civilian doses, respectively. RISK ASSESSMENT Despite these adverse events and because of the increasing potential for severe and fatal yellow fever among unvaccinated U.S. travelers to areas where yellow fever is endemic, vaccination is still recommended for persons over 9 months of age traveling to yellow fever-affected countries. Physicians should be careful to administer yellow fever vaccine only to persons truly at risk for exposure to yellow fever. Travelers over age 60 should discuss with their physicians the risks and benefits of vaccination in the context of their risk for exposure to yellow fever based on their destination. Even after careful review of travel itinerary and determining that there truly is a need for yellow fever vaccine, data presented here and in other recent articles strongly suggest there may be a new group of travelers who should not be vaccinated. Older persons, i.e., persons ⬎60 years old, appear to be at higher risk for both neurotropic and viscerotropic adverse events after yellow fever vaccination. How do these rates of adverse events after yellow fever vaccination compare with adverse events after administration of other vaccines? There are three comparable and recent examples where increased rates of vaccine-associated adverse events likely resulted in changes in vaccination policy. National surveillance for GuillainBarré syndrome following vaccination against swine influenza in 1976 revealed a rate of


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Figure 2. Annual reported rate of yellow fever vaccine-associated viscerotropic and neurotropic disease per 1,000,000 U.S. civilian doses distributed and cumulative risk, United States, 1990 to 2002 and 1996 to 2002.

Figure 3. Age-specific reported rate of yellow fever vaccine-associated viscerotropic and neurotropic disease per 1,000,000 U.S. civilian doses distributed, 1990 to 2002.

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roughly five adverse events per million doses; distribution of the vaccine was ended (16). In the 1970s, after a 10-year multinational study, the risk of vaccine-associated paralytic polio following receipt of a first dose of oral polio vaccine was estimated to be 2 cases per million doses distributed (28). Like the current analysis of the rate of yellow fever vaccine adverse events, this estimate was based on passive reporting and the number of vaccine doses distributed as provided by the manufacturer. As a result of these analyses, in the United States, inactivated poliovirus vaccine is now given initially instead of oral polio vaccine. As a third example, in 1968, national active surveillance showed that the risk of experiencing a life-threatening event or encephalitis after receipt of smallpox vaccine was 14.2 and 2.4 cases per million doses, respectively (17). The rates of yellow fever vaccine-associated viscerotropic and neurotropic disease are approximately 3 and 4 cases per million doses distributed, respectively, and are comparable to the rate of adverse events for swine flu, oral polio, and smallpox vaccines. In addition, the reported rate of either viscerotropic or neurotropic disease after yellow fever vaccination for persons over age 60 increases to 21 to 45 cases per million doses distributed, much higher than most of the risks discussed for the other vaccines. But unlike swine flu, polio, and smallpox in the above examples, the risk of naturally acquired yellow fever disease is increasing. Therefore, stopping vaccination is not an option and underscores the importance of identifying the molecular, viral, or host factors that have resulted in the vaccine-associated adverse events and even the possibility of developing a new, safer vaccine. Until that time, this situation underscores the importance of continued surveillance for vaccine-associated and naturally acquired disease so that risk and an appropriate vaccination strategy can be determined for travelers. CONCLUSIONS Because of the potential severity of yellow fever infection and recent reports of yellow fever deaths among unvaccinated travelers to areas of yellow fever endemicity, vaccination is recommended for persons over 9 months of age traveling to countries where yellow fever is endemic or epidemic. However, because of reports of vaccine-associated viscerotropic and neurotropic disease, physicians should be careful to administer yellow fever vaccine only to persons truly at risk for exposure to yellow fever. Travelers over age 60 should discuss with their physicians the risks and benefits of vaccination in the context of their risk for exposure to yellow fever based on their destination. Information concerning known or probable affected areas is available from the World Health Organization (http://www.who.int), the Pan American Health Organization (http://www.paho.org), and CDC (http://www.cdc.gov/travel). Additional surveillance to better monitor and quantify yellow fever vaccine-specific adverse outcomes should also be established. Studies are being conducted to clarify the cause and risk factors for the rare adverse events associated with the 17D yellow fever vaccines. Acknowledgments. We thank Sherif Zaki and his team in the Infectious Diseases Pathology Activity, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, CDC, for their work on histopathology and Bob Chen of the Immunization Safety Branch, National Immunization Program, CDC, for his guidance and expertise. REFERENCES 1. Anonymous. 1966. Fatal viral encephalitis following 17D yellow fever vaccine inoculation: report of a case in a 3-year-old child. JAMA 198:671–672. 2. Barrett, A. D. 1997. Yellow fever vaccines. Biologicals 25:17–25.


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3. Reference deleted. 4. Centers for Disease Control and Prevention. 2002. Adverse events associated with 17D-derived yellow fever vaccination—United States, 2001–2002. Morb. Mortal. Wkly. Rep. 51:989–993. 5. Centers for Disease Control and Prevention. 2002. Fatal yellow fever in a traveler returning from Amazonas, Brazil. Morb. Mortal. Wkly. Rep. 51:324–325. 6. Centers for Disease Control and Prevention. 2000. Fatal yellow fever in a traveler returning from Venezuela, 1999. Morb. Mortal. Wkly. Rep. 49:303–305. 7. Centers for Disease Control and Prevention. 2001. Health Information for International Travel 2001– 2002. U.S. Department of Health and Human Services, Atlanta, Ga. [Online.] http://www.cdc.gov/travel. 8. Centers for Disease Control and Prevention. 2002. Yellow fever vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP). Morb. Mortal. Wkly. Rep. 51(RR-17):1–11. 9. Chan, R. C., D. J. Penney, D. Little, I. W. Carter, J. A. Roberts, and W. D. Rawlinson. 2001. Hepatitis and death following vaccination with 17D-204 yellow fever vaccine. Lancet 358:121–122. 10. Freedman, D. O., P. E. Kozarsky, L. H. Weld, and M. S. Cetron. 1999. GeoSentinel: the global emerging infections sentinel network of the international society of travel medicine. J. Travel Med. 6:94–98. 11. Galler, R., K. V. Pugachev, C. L. Santos, S. W. Ochran, A. V. Jabor, S. G. Rodrigues, R. S. Marchevsky, M. S. Friere, L. F. C. Almeida, A. C. R. Cruz, A. M. Y. Yamamura, I. M. Rocco, E. S. Travassos da Rosa, L. T. M. Souza, P. F. C. Vasconcelos, R. Guirakhoo, and T. P. Monath. 2001. Phenotypic and molecular analyses of yellow fever 17 DD vaccine viruses associated with serious adverse events in Brazil. Virology 290:309–319. 12. Gubler, D. J. 1998. Dengue and dengue hemorrhagic fever. Clin. Microbiol. Rev. 11:480–496. 13. Kelso, J. M., R. T. Jones, and J. W. Yunginger. 1993. Anaphylaxis to measles, mumps, and rubella vaccine mediated by IgE to gelatin. J. Allergy Clin. Immunol. 91:867–872. 14. Kelso, J. M., G. T. Mootrey, and T. F. Tsai. 1999. Anaphylaxis from yellow fever vaccine. J. Allergy Clin. Immunol. 103:698–701. 15. Kengsakul, K., K. Sathirapongsasuti, and S. Punyagupta. 2002. Fatal myeloencephalitis following yellow fever vaccination in a case with HIV infection. J. Med. Assoc. Thai. 85:131–134. 16. Kilbourne, E. D., and N. H. Arden. 1999. Inactivated influenza vaccines, p. 531–551. In S. A. Plotkin and W. A. Orenstein (ed.), Vaccines, 3rd ed. W. B. Saunders, Philadelphia, Pa. 17. Lane, J. M., F. L. Ruben, J. M. Neff, and J. D. Millar. 1969. Complications of smallpox vaccinations, 1968: national surveillance in the United States. N. Engl. J. Med. 281:1201–1208. 18. Lang, J., J. Zuckerman, P. Clarke, P. Barrett, C. Kirkpatrick, and C. Blondeau. 1999. Comparison of the immunogenicity and safety of two 17D yellow fever vaccines. Am. J. Trop. Med. Hyg. 60:1045–1050. 19. Martin, M., L. H. Weld, T. F. Tsai, G. T. Mootrey, R. T. Chen, M. Niu, M. S. Cetron, and the GeoSentinel Yellow Fever Working Group. 2001. Advanced age a risk factor for adverse events temporally associated with yellow fever vaccination. Emerg. Infect. Dis. 6:945–951. 20. Martin, M., T. F. Tsai, B. Cropp, G. J. Chang, D. A. Holmes, J. Tseng, W. Shieh, S. R. Zaki, I. Al-Sanouri, A. F. Cutrona, G. Ray, L. H. Weld, and M. S. Cetron. 2001. Fever and multisystem organ failure associated with 17D-204 yellow fever vaccination: a report of four cases. Lancet 358:98–104. 21. McFarland, J. M., L. M. Baddour, J. E. Nelson, S. K. Elkins, R. B. Craven, B. C. Cropp, G. J. Chang, A. D. Grindstaff, A. S. Craig, and R. J. Smith. 1997. Imported yellow fever in a United States citizen. Clin. Infect. Dis. 25:1143–1147. 22. Monath, T. P. 1999. Yellow fever, p. 815–879. In S. A. Plotkin and W. A. Orenstein (ed.), Vaccines, 3rd ed. W. B. Saunders, Philadelphia, Pa. 23. Monath, T. P. 1999. Facing up to re-emergence of urban yellow fever. Lancet 353:1541. 24. Monath, T. P., R. Nichols, W. T. Archambault, L. Moore, R. Marchesani, J. Tian, R. E. Shope, N. Thomas, R. Schrader, D. Furby, and P. Bedford. 2002. Comparative safety and immunogenicity of two yellow fever 17D vaccines (ARILVAX and YF-VAX) in a phase III multicenter, double-blind clinical trial. Am. J. Trop. Med. Hyg. 66:533–541. 25. Nasidi, A., T. P. Monath, K. DeCock, O. Tomori, R. Cordellier, O. D. Olaleye, T. O. Harry, J. A. Adeniyi, A. O. Sorungbe, A. O. Ajose-Coker, G. VanDer Lann, and A. B. O. Oyediran. 1989. Urban yellow fever epidemic in western Nigeria, 1987. Trans. R. Soc. Trop. Med. Hyg. 83:401–406. 26. Pugachev, K. V., S. W. Ocran, F. Guirakhoo, D. Furby, and T. P. Monath. 2002. Heterogeneous nature of the genome of the ARILVAX yellow fever 17D vaccine revealed by consensus sequencing. Vaccine 20:996–999.


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27. Reinhardt, B., R. Jaspert, M. Niedrig, C. Kostner, and J. L’age-Stehr. 1996. Development of viremia and humoral and cellular parameters of immune activation after vaccination with yellow fever virus strain 17D: a model of human flavivirus infection. J. Med. Virol. 56:159–167. 28. Robbins, F. C. 1999. The history of polio vaccine development, p. 13–27. In S. A. Plotkin and W. A. Orenstein (ed.), Vaccines, 3rd ed. W. B. Saunders, Philadelphia, Pa. 29. Rosenthal, S., and R. Chen. 1995. The reporting sensitivities of two passive surveillance systems for vaccine adverse events. Am. J. Public Health 85:1706–1709. 30. Sakaguchi, M., T. Nakayama, and S. Inouye. 1996. Food allergy to gelatin in children with systemic immediate-type reactions, including anaphylaxis, to vaccines. J. Allergy Clin. Immunol. 98(6 Pt 1):1058– 1061. 31. Sakaguchi, M., T. Yamanaka, K. Ikeda, S. Yasuko, F. Hiroyuki, M. Takuma, and I. Sakae. 1997. IgEmediated systemic reactions to gelatin included in the varicella vaccine. J. Allergy Clin. Immunol. 99:263– 264. 32. Smithburn, K. C., C. Durieux, R. Koerber, et al. 1956. Yellow Fever Vaccination. WHO Monograph Series No. 30. World Health Organization, Geneva, Switzerland. 33. Vainio, J., and F. Cutts. 1998. Yellow Fever. Publication No. (WHO/EPI/GEN) 98.11. Global Programme for Vaccines and Immunization, World Health Organization, Geneva, Switzerland. [Online.] http://www. who.int/emc-documents/yellow_fever/whoepigen9811c.html. 34. Van der Stuyft, P., A. Gianella, M. Pirard, L. Cespedes, L. Lora, C. Peredo, J. L. Pelegrino, V. Vorndam, and M. Boelaert. 1999. Urbanisation of yellow fever in Santa Cruz, Bolivia. Lancet 353:1558–1562. 35. Vasconcelos, P. F. C., E. J. Luna, R. Galler, L. J. Silva, T. L. Coimbra, V. L. R. S. Barros, T. P. Monath, S. G. Rodrigues, C. Laval, Z. G. Costa, M. F. G. Vilela, C. L. S. Santos, C. M. O. Papaiordanou, V. A. F. Alves, L. D. Andrade, H. K. Sato, E. S. T. Rosa, G. B. Froguas, E. Lacava, L. M. R. Almeida, A. C. R. Cruz, I. M. Rocco, R. T. M. Santos, O. F. P. Oliva, and the Brazilian Yellow Fever Vaccine Evaluation Group. 2001. Serious adverse events associated with yellow fever 17DD vaccine in Brazil: report of two cases. Lancet 358:91–97. 36. World Health Organization. 1998. District guidelines for yellow fever surveillance. Publication No. (WHO/EPI/GEN) 98.09. World Health Organization, Geneva, Switzerland. [Online.] http://www.who.int/ emc-documents/yellow_fever/whoepigen9809c.html. 37. World Health Organization. 2001. Outbreak news: imported case of yellow fever, Belgium (update). Wkly. Epidemiol. Rec. 76:365.


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Chapter 4

Responding to Epidemics of Ebola Hemorrhagic Fever: Progress and Lessons Learned from Recent Outbreaks in Uganda, Gabon, and Congo Daniel G. Bausch and Pierre E. Rollin

Ebola hemorrhagic fever (EHF) is caused by viruses of the genus Ebolavirus of the Filoviridae family. Four species of Ebola virus are known, Zaire, Sudan, Côte d’Ivoire, and Reston, each with considerable genetic heterogeneity compared with the others (Fig. 1). EHF is a febrile, often hemorrhagic illness with a high case-fatality ratio. Ebola viruses are presumed to have a zoonotic reservoir or reservoirs (unknown at present) found in tropical rain forests in sub-Saharan Africa, where large outbreaks occasionally occur. Epidemics are thought to be initiated by a single introduction of a virus into humans from the animal reservoir, with subsequent human-to-human transmission, often with amplification in a nosocomial setting (43). Transmission occurs via direct contact with infected body fluids, usually during the later stages of illness (18). Funeral rituals, which entail the touching of the corpse prior to burial, have also played a significant role in transmission (13). Laboratory diagnosis is available only in a few specialized laboratories, mostly in industrialized countries. Other than supportive care, there is no known treatment or approved vaccine for EHF. HISTORY OF EHF OUTBREAKS Except for a laboratory-acquired case in England in 1976, human cases of EHF have been noted exclusively in sub-Saharan Africa (Fig. 2) (20). The first recorded outbreaks occurred concurrently in the Democratic Republic of the Congo (DRC; formerly Zaire) and Sudan in 1976 due to Ebola virus species Zaire (Ebola-Zaire virus) and Ebola-Sudan virus, respectively (81, 82). A single case due to Ebola-Zaire virus was identified retrospectively in Tandala, DRC, in 1977, followed by a small outbreak again due to EbolaSudan virus in southern Sudan in 1979 (2, 30). Daniel G. Bausch • Department of Tropical Medicine, SL-17, Tulane School of Public Health and Tropical Medicine, 1430 Tulane Ave., New Orleans, LA 70112. Pierre E. Rollin • Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333. 35


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Figure 1. Phylogenetic tree and map showing the evolutionary relationships and geographic distributions of the different species of Ebola virus. The genetic sequences of viruses from recent outbreaks beginning in 2000 in Uganda and 2001 in Gabon and Congo have not yet been published and are therefore not included, although they have been identified as Sudan and Zaire species, respectively (13, 48). Marburg virus is shown as an outgroup reference. Imported cases or data based on antibody surveys are not included. Names listed on the phylogenetic tree indicate the geographic location of the outbreak from which the virus was obtained. Ebola virus species are depicted by the following shades: , Zaire; , Sudan; , Côte d’Ivoire; and , Reston. [Phylogenetic tree modified and used with permission from A. Sanchez, J. Infect. Dis. 179(Suppl. 1):iii, 1999.]

Following the 1979 outbreak, 15 years passed without a recognized case of human EHF, until 1994, when a single case due to Ebola-Côte d’Ivoire virus occurred after a researcher performed an autopsy on a dead chimpanzee found in the Taï Forest of Côte d’Ivoire (22). Three separate epidemics due to Ebola-Zaire virus occurred in Gabon from 1994 to 1996, one of which was clearly related to contact with an infected chimpanzee (23). During the last of these outbreaks, the disease was exported to South Africa by an infected physician, with a subsequent tertiary case in a South African nurse (66). A large outbreak due to Ebola-Zaire virus also occurred in Kikwit, DRC, in 1995 (43). After 1996 there was a brief hiatus until 2000, when an outbreak due to Ebola-Sudan virus struck northern Uganda (13). Less than a year later, in late 2001, EHF due to Ebola-Zaire virus again broke out in eastern Gabon and western Congo (48). From 1989 to 1996, numerous outbreaks of disease caused by Ebola-Reston virus were reported among monkeys imported into the United States and Europe (34, 61, 68). Although experience is very limited, this Ebola virus species does not appear to cause human disease, despite evidence of human infection (12, 21). The origin of each outbreak was

Figure 2. Temporal progression of outbreaks of Ebola hemorrhagic fever. There is a break in the timeline between 1979 and 1989, when no outbreaks were recorded. The year of onset is shown beneath the country involved in each outbreak. Ebola virus species are depicted by the following shades: , Zaire; , Sudan; , Côte d’Ivoire; and , Reston. Arrows indicate importations of Ebola virus from one country to another.


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traced to a single primate-exporting facility in the Philippines. Investigations in that country have not revealed the origins of these infected animals, and given the possibility that animals from Africa were trafficked through the site, even the continental origin of EbolaReston remains uncertain (12, 21). WHAT PROGRESS HAS BEEN MADE IN OUR UNDERSTANDING OF EHF FROM RECENT OUTBREAKS IN UGANDA, GABON, AND CONGO? In many ways, the 1995 outbreak in Kikwit, DRC, represented a benchmark in our understanding of how to respond to epidemics of EHF. It was the first large outbreak for which the investigation was not mostly retrospective. Intense scientific as well as public attention resulted in the development and indoctrination of certain organizational responses and control measures and the signaling of the strengths and loopholes in our knowledge about EHF. Most of the scientific findings were documented in a 1999 supplement to the Journal of Infectious Diseases (60). Among other measures, commonly signaled needs were (i) stronger local infectious disease surveillance and control, (ii) improved international preparedness, (iii) better media relations, (iv) improved patient management, (v) improved diagnostic tools, and (vi) research aimed at identifying the reservoir or reservoirs of the filoviruses (29, 41, 59). Five years after the Kikwit outbreak, the 2000–2001 outbreak in Gulu, Uganda, was the largest outbreak of EHF recorded to date. Analysis of this epidemic, as well as the more recent events in Gabon and Congo, will no doubt further our understanding of EHF. While we await more formal presentations of the scientific data, these outbreaks provide us the opportunity to reflect on our progress in the domain of EHF since 1995, to speculate on the future, and to reassess needs. Furthermore, these outbreaks present contrasting epidemiologic, virologic, and sociologic characteristics which are themselves illustrative (Table 1). Lastly, the bioterrorist attacks in the United States in late 2001 have highlighted the need for a better understanding of the potential that the Ebola viruses pose as agents of bioterrorism (69). HOW EFFECTIVE ARE LOCAL SURVEILLANCE AND CONTROL MEASURES FOR EHF? Outbreaks of EHF appear to be either increasing in frequency or more readily detected, or perhaps both. In the 25 years since its discovery, at least 11 distinct primary introductions of Ebola viruses into humans have been documented (excluding Ebola-Reston virus, laboratory accidents, and the possibility of multiple simultaneous primary introductions during a given outbreak) (Fig. 2). Seven (64%) of these have occurred in the last 8 years. Less than 1 year separated the end of the outbreak in Uganda and the beginning of the most recent outbreak in Gabon and Congo. Logical arguments can be made that there is a true increase in frequency. Although interpretation is sometimes difficult due to the nonspecificity of some older laboratory techniques and the possibility of cross-reacting, nonpathogenic filoviruses, evidence from serologic surveys nevertheless supports the existence of some degree of endemic transmission of Ebola viruses in various sites across sub-Saharan Africa (2, 6, 7, 10, 25, 26, 37, 39, 40, 50, 52, 67, 77, 79). However, because transmission appears to require direct physical

39

1 Rainy Savanna or farmland Rare Significant factor 29 (7) ⱖ13 (62)

1 Rainy Cleared rain forest or farmland Rare Major factor 80 (25) 1 (1)

ⱖ5 Rainy Tropical rain forest Abundant Minimal 3 (3) 0c

Centre International de Recherche Médicale de Franceville, Franceville, Gabon Yes (⬃5)

Mekambo Zaire 122 96 (79) 19/26 (42) 21 yr (3 days–72 yr) 13 of 45 (29) 47 (25 October–11 December 2001) ⱖ145 (25 October 2001–19 March 2002)

Gabon and Congo, 2001–2002a

This outbreak was still in progress at the time of the preparation of the manuscript. The data presented are preliminary. In many instances, absolute numbers were not available for all case patients, and percentages were calculated on the basis of subtotals. b In most cases, the identification of the first case was retrospective. When unknown, the date of onset was assumed to be 1 week prior to the date of death. c Does not include one case of a nurse working at a local clinic whose staff was not specifically equipped or trained to manage patients with EHF.

Yes (1)

National Institute for Virology, Johannesburg, South Africa

CDC, Atlanta, Ga.

No

Gulu Sudan 426 224 (53) 269/156 (63) 21 yr (15 days–65 yr) 106 (25) 46 (30 August–15 October 2000) 142 (30 August 2000–19 January 2001)

Uganda, 2000–2001

Kikwit Zaire 315 255 (81) 165/145 (53) 37 yr (2 mo–71 yr) 22 (7) 123 (6 January–9 May 1995) 187 (6 January–12 July 1995)

DRC, 1995

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a

Availability and turnaround time (days) of on-site diagnostics No. of apparent primary introductions of virus Season of onset Topography Prevalence of nonhuman primates Effect of nosocomial amplification No. of health care workers infected (% of all cases) No. of health care workers infected after the establishment of isolation wards (% of all health care workers infected)

Epicenter Virus species No. of cases No. of fatalities (case-fatality ratio [%]) No. of females/no. of males (% female) Mean age (age range) No. of children or adolescents (age, ⱕ16 yr) Time (days from onset of first case to official declaration of outbreakb) Duration of epidemic (days from date of onset of first to date of onset of last case) Site of first laboratory confirmation

Characteristic

Table 1. Characteristics of recent outbreaks of EHF in DRC, Uganda, Gabon, and Congo

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contact with an infected individual during the acute stage of illness, attack rates are generally low (18, 57, 81). Thus, although perhaps relatively common in some rural areas, primary infection usually results in only a small number of secondary cases, all of which go largely undetected or perhaps are attributed to malaria, typhoid, or other common tropical illnesses (9, 37). In most instances, what likely converts low-level endemic transmission to an epidemic is nosocomial amplification in the context of the incomplete health care infrastructure commonly found in tropical Africa in recent decades. The construction and seeking of care in “Western” hospitals often outstrip the maintenance of consumable goods, such as drugs, syringes, masks, and gloves, creating a fertile ground for epidemic EHF (73). Ever increasing population densities send humans continually deeper into tropical rain forests for economic purposes, such as logging, hunting, or farming; and presumably increase their exposure to the primary reservoir of the Ebola viruses. Upon their return to more populated areas, individuals with EHF seeking care at inadequately equipped hospitals may initiate epidemics. Furthermore, increasing human movement and trade may lead to the introduction of Ebola viruses into new areas with more susceptible populations. A case can also be made that surveillance for EHF has improved in recent years. This heightened surveillance is driven by intense interest in the disease among the scientific community and public alike, often fueled by the release of popular books and films such as The Hot Zone (63) and Outbreak. This interest is reflected in the marked increase in published research articles on EHF in the last decade (Fig. 3). A rough measure of both interest and surveillance might be gleaned from the listings on the widely read Internet surveillance service Program for Monitoring Emerging Diseases, or ProMED (http://www. promedmail.org), which shows a similar steady increase in listings concerning viral hemorrhagic fevers, with peaks for EHF in years when EHF outbreaks occurred (Fig. 4). Specific surveillance tools for EHF and other viral hemorrhagic fevers have recently been developed. The World Health Organization (WHO), the Centers for Disease Control and Prevention (CDC), and Médecins Sans Frontières have produced guidelines on the establishment of surveillance systems, case definitions, and specimen collection for EHF

Figure 3. Scientific publications on Ebola hemorrhagic fever, 1976 to 2000. Source: National Library of Medicine.


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Chapter 4 • Ebola Hemorrhagic Fever

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Figure 4. Listings of “viral hemorrhagic fever” or “Ebola hemorrhagic fever” on the Program for Monitoring Emerging Diseases (ProMED) website (http://www.promedmail.org) since the beginning of its service in 1994 through 2001.

(14, 51, 83). CDC has also provided a surveillance tool consisting of skin biopsy kits, complete with biopsy punch, decontamination materials, personal protective gear, and a mailing tube, for postmortem sampling of suspicious deaths, with subsequent immunohistochemistry testing in its laboratories in Atlanta, Ga. (49). Whereas surveillance for EHF may have improved, progress still needs to be made in the effectiveness of control efforts. Although the dynamics of each epidemic differ and control efforts certainly do temper their severity, there have been no drastic reductions in the number of cases, the case-fatality ratio, or the duration of recent outbreaks relative to those of the 1970s (Table 1 and Fig. 5). What are the impediments to the more effective control of EHF? The first bottleneck is usually at the epicenter of the epidemic. Interestingly, the time from the first case to official declaration of the outbreak appears to be diminishing, yet this earlier notification has not necessarily resulted in more rapid control (Table 1 and Fig. 5). Most likely, this response time, still over 6 weeks, is still too slow to effectively extinguish an outbreak of EHF in its earliest phases. Furthermore, because EHF is rare and alerts most often prove to be false, the participation of outside experts, along with their technical expertise and donation of protective materials, is generally contingent upon laboratory confirmation, still available in only a few select laboratories worldwide (see below). Once a specimen is received in a reference laboratory, a result is usually provided within 24 h. What happens during this interval between suspicion and confirmation of EHF represents the first challenge to effective control. Although often of intense public interest, EHF is a rare disease. Most health care workers will never see a case and have little familiarity with EHF. Furthermore, patients with EHF initially present with nonspecific signs and symptoms that are difficult to distinguish


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Figure 5. Duration, cases, and case-fatality ratios associated with major outbreaks of EHF, 1976 to 2001. Data for the outbreak in Gabon and Congo in 2001 are preliminary. Outbreaks associated with Ebola-Reston virus, which has not been noted to cause human disease, are not included.

from those of many other tropical illnesses (11). Bleeding is seen in a minority of cases. Thus, EHF is usually not suspected until clusters of suspicious cases occur. As the reservoir for the Ebola viruses appears to inhabit tropical rain forests, outbreaks of EHF usually occur in remote areas, where disease surveillance efforts and the health care infrastructure are often inadequate. Even when a pattern of disease suspicious for EHF is recognized, inadequate communication and transportation, along with the unavailability of diagnostic capabilities for EHF as well as other more common febrile illnesses, typically result in delays in response on the part of regional health officials. This delay may be exacerbated when the disease is recognized by one of the many unofficial or alternative sources of health care delivery without formal ties to government, such as private clinics and traditional healers. Further delays are often associated with the logistical difficulties of shipping specimens to a laboratory capable of performing the diagnosis, compounded often by significant national and international regulations as well as the reluctance of some shippers and airlines to handle such specimens even when appropriately packaged. Geographic isolation and underdeveloped infrastructure proved to be especially challenging aspects to the control efforts for the most recent EHF outbreak in Gabon and Congo, which occurred in sparsely populated, remote areas of rain forest where roads are often impassable, if they exist at all.


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Civil unrest may also exacerbate the delay in both the local and the international responses to outbreaks of EHF. For example, although the infrastructure in the area of northern Uganda afflicted by EHF was relatively good, case finding and contact tracing were hampered by the presence of local rebels, often necessitating military escorts for the movement of surveillance teams between villages in affected areas. During the period between initial suspicion and confirmation of EHF, health care workers and community members alike may continue to be exposed to Ebola virus because of insufficient stocks of protective materials and a lack of both clinical experience and technical expertise regarding appropriate public health messages. Even patients suspected of having EHF may not be managed safely. Typically, by the time the international community responds, multiple chains of virus transmission are under way in both the hospital and the community. Indeed, EHF in health care workers is usually one of the factors resulting in recognition of an outbreak. HOW PREPARED ARE WE TO RESPOND TO OUTBREAKS OF EHF? Significant gains have been made in our preparedness for outbreaks of EHF once they are declared. The international community usually reacts rapidly and donates generously. Within days, teams of experts and loads of supplies arrive on-site. Increasingly, the response to EHF is in real time rather than retrospectively, which was largely the case for studies of the pre-Kikwit outbreaks (2, 30, 81, 82). The recently created WHO Global Alert and Response Network seeks to establish a framework for the detection and rapid dispatch of capable international partners to respond to infectious diseases, such as EHF, which are deemed to be of global importance on the basis of humanitarian need and the possibility of international spread and/or significant interruption of travel or trade (28). This network is enhanced by the existence of a small group of trained experts from various centers around the world who stand ready to assist. Standardized materials have been prepared in both English and French on the establishment of isolation wards, maintenance of barrier nursing, and safe burial practices in the setting of a viral hemorrhagic fever (14, 51, 83). Organizations such as Médecins Sans Frontières have produced large, prepackaged kits of protective material and items necessary for the establishment of isolation wards, and these kits can be readily deployed once an outbreak is signaled. Again we must ask, why does this heightened preparation not translate into greater impact on the control of epidemics of EHF? Multiple challenges still exist. (i) As described above, Ebola virus is often already widely disseminated in the community and/or hospital by the time that an epidemic is recognized and full-fledged control efforts are mobilized. (ii) The WHO Global Alert and Response Network is in its infancy and working to address several challenges, such as definition of the roles and responsibilities of the response teams and coordination between host countries and regional and international WHO representation. In some fields, most notably patient care, there is a shortage of experts who are trained in the viral hemorrhagic fevers and who are available for rapid deployment. (iii) Gaps still exist in our basic understanding of Ebola virus transmission. Although we know that the viruses are transmitted through direct contact and that blood is infectious, what are the specific risk factors for transmission? How dangerous is exposure to other body fluids, such as urine, feces, sweat, vomitus, and breast milk? Is transmission via


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fomites a concern? If so, how long is the virus viable on inanimate objects? (iv) We still lack specific intervention tools, namely, treatments and vaccines (see the discussion below under patient management). Our present control mechanisms based on case finding, contact tracing, and isolation can limit the damage done but have not usually resulted in the swift abatement of an ongoing epidemic. (v) We are still learning how to properly use some of the tools that we have developed. (vi) Efforts at community education and social mobilization to improve participation with control efforts have often been underemphasized. Examples of the last two points can be seen in the use of case definitions, for which perhaps too much reliance on prepackaged algorithms developed from the Kikwit and Uganda outbreaks led to initial difficulties in Gabon (Table 2). The 2000 outbreak in Gulu was a large, community-based epidemic that was widely disseminated in the district. As such, broad case definitions that emphasized sensitivity over specificity were merited in order to detect EHF in virtually any geographic or social sector of the region. Initially, similar case definitions were used in 2001 in Gabon and Congo. However, it soon became evident that, in contrast to Uganda, the chains of transmission in Gabon were usually well delineated, commencing with hunters or others exposed in the forest, presumably from primary introductions, and continuing with secondary transmission between family members, other close contacts in the community, and, occasionally, nosocomially. In this context, the use of the case definition from Uganda proved disadvantageous, as its low specificity and positive predictive value resulted in the designation of a large number of possible case patients who obviously did not fit within the recognized chains of transmission and who were invariably excluded when laboratory results became available. Not only did this approach pose a logistical burden on the outbreak control team, but it also often contributed to a loss of confidence by the local population, with the international team sometimes perceived as overreactive in their insistence on isolation of suspected case patients. This was compounded by a delay in laboratory diagnosis, usually about 5 days, as Table 2. Case definitions for EHF used in outbreaks in Uganda, Gabon, and Congo Outbreak

Case definitions

Uganda, 2000–2001 . . . . . . . . . . . Fever and contact with a person with EHF Fever and unexplained bleeding of any kind Unexplained death in a person having presented with an acute onset of fever Fever and three or more of the following symptoms: headache, vomiting, anorexia, diarrhea, weakness or severe fatigue, abdominal pain, body aches or joint pains, difficulty swallowing, difficulty breathing, hiccups Gabon and Congo 2001–2002 . . . Fever and contact with a person with EHF Fever and unexplained bleeding of any kind Unexplained death in a person having presented with an acute onset of fever Fever in a hunter having entered into the forest or other person having contact with wild animals, living or dead, in the 3 wk before onset of illness New onset of fever within 3 wk of having been hospitalized in Ogooué-Ivindo Province For patients who do not meet any of the above criteria, acute febrile illness that persists despite at least 48 h of appropriate antimalarial and/or antibiotic therapy


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the diagnostic laboratory was distant from the epicenter of the epidemic. Subsequent revision resulted in an equally sensitive but more specific case definition; however, much of the damage to the community’s faith in the abilities of the outbreak response team was already done. A similar problem presented itself in regard to the establishment of isolation wards in remote areas of Gabon, a process that often proved cumbersome and slow. In some instances, no health center in which to establish an isolation ward existed. Guidelines were again established on the basis of the experiences in Kikwit and Gulu, emphasizing large, logistically complex wards designed to provide care for as many as 30 patients in the setting of large, nosocomially amplified outbreaks. While functional in these situations, the smaller, more circumscribed nature of the outbreak in remote and underdeveloped areas of Gabon and Congo called for smaller, rapidly established wards adequate for two or three patients at a time. Although grand-scale guidelines were eventually adapted to hospitals on a smaller scale, delays sometimes resulted in less-than-ideal isolation conditions for short periods, theoretically putting patients, family members, and health care workers at risk. To date, no guidelines on how to handle isolation of those with EHF in remote areas without basic health services are available. Isolation at home with care provided by family members has been proposed, but there are doubts as to whether this can be safely done, especially considering that transmission sometimes occurs even in nosocomial settings among those with considerably more training and protective materials. MEDIA RELATIONS The sometimes-strained relationship between the international media and public health specialists in Kikwit proved to be more amicable in Uganda, Gabon, and Congo. Although this improvement may simply represent a certain degree of familiarity with the situation on behalf of all parties, the designation of a specific media spokesperson has aided in satisfying the need for information on the part of journalists and the public while allowing the outbreak response team to concentrate on their activities. Furthermore, the media, especially local radio, has come to play a crucial role in transmitting health messages that help the public to better understand EHF and the responses to it, potentially leading to altered practices that could prevent transmission and curtail the outbreak. Unfortunately, local press reports linking EHF outbreaks to intentional releases by government or international sources still occasionally surface and complicate control efforts. WHAT PROGRESS HAS BEEN MADE IN PATIENT MANAGEMENT AND THE SAFE DELIVERY OF HEALTH CARE? When outbreaks due to the same Ebola virus species are appropriately compared, little progress can be claimed on our impact on case-fatality ratios related to EHF (Fig. 5). To date, only supportive care is available, and even this is often lacking because of inadequate infrastructure and supplies and the dangers posed to health care workers. The case-fatality ratio in Gulu was 53%, not significantly different from that noted in outbreaks of EHF due to Ebola-Sudan virus 25 years earlier (with case-fatality ratios of 53 and 65% in outbreaks in 1976 and 1979, respectively). Waning of the case-fatality ratio in the later stages of the Kikwit outbreak, interpreted by some as a response to improved organization and therapy


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as the epidemic progressed, was not statistically significant and could equally be interpreted as increased detection of those with milder disease as case-finding mechanisms improved (43, 72). Slight encouragement can perhaps be found in the fact that the casefatality ratio for those treated at a well-supported mission hospital in Gulu, where supportive therapy was able to be more aggressively and consistently implemented, was 43% (D. Bausch and M. Lukwiya, unpublished data). However, this difference is not statistically significant. In addition, even a patient for whom vasopressor agents and mechanical ventilation were available succumbed to EHF at that hospital (3). It appears that the effect of even aggressive supportive care is at best moderate. No specific treatments for EHF are available. Although the S-adenosylhomocysteine hydrolase inhibitors have shown some promise, they have not been investigated beyond the mouse model (8, 31). Despite positive reports, careful analysis of data on the response to infusion of convalescent-phase whole blood raises doubt about the conclusion regarding the efficacy of this therapy (55, 72). High-titer hyperimmune animal serum has shown mixed results (33, 36, 46). Even if efficacious, these products would unlikely be available in sufficient quantities to be of use in outbreaks, although they might prove helpful in the event of laboratory accidents. Monoclonal antibodies against Ebola viruses have been produced but not extensively tested (78, 80). A small trial of a DNA vaccine for EHF in nonhuman primates showed that it offers protection, although the prime-boost strategy of the vaccine, with consequent delayed immunogenicity, would limit its utility in outbreaks to all but the prevaccinated international team (75). Other vaccine candidates have been shown to be efficacious in guinea pigs (64, 65). No trials of steroids or recently developed antisepsis compounds, such as activated protein C, have been undertaken (5). A further impediment to effective treatment of EHF is the lack of understanding of the pathogenesis and clinical laboratory parameters associated with the disease. How important is disseminated intravascular coagulopathy? Do derangements in electrolyte balance and liver, cardiac, renal, and/or adrenal function significantly contribute to the high case-fatality ratio? What are the effects of concurrent human immunodeficiency virus or malaria parasite infection? Studies related to these issues, either in the field or in the laboratory, are limited, resulting in knowledge gaps that impair our ability to rationally design specific clinical management strategies. Effective treatment of EHF is necessary not only for the benefit of the sick individual but also for the success of the community control measures. In lieu of effective treatment, the emphasis in outbreaks of EHF is placed upon active case finding and contact tracing, with immediate isolation of suspected cases. Thus, community confidence in the benefits of the isolation wards is imperative. Nevertheless, it can be difficult to maintain. Ironically, although patient isolation may be effective on a community scale, it is purely preventive, offering little to the sick individual. In fact, given the limited treatment options, other than protecting one’s family from infection, isolation could be legitimately viewed as detrimental to the individual. It entails immediate physical isolation from loved ones, with a high degree of resulting emotional stress and stress over the prospect of burial outside the traditional funeral practices of the community should the patient die. Furthermore, the possibility of contracting Ebola virus in the ward cannot be excluded and has occurred, albeit rarely, in both Uganda and DRC (S. Mardel and P. Rollin, unpublished data). The high case-fatality ratios of EHF may also serve to create a causal association between entrance into the isolation ward and death in the minds of community members. As fear often


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causes health care workers to abandon their posts during outbreaks of EHF, patients may be cared for by health care workers with less experience, further undermining patient confidence. Fear may also induce some health care workers to inappropriately modify supportive care to minimize patient contact. This is especially the case when no health care worker with previous experience with EHF is available to serve as a positive model to show that duties can be safely performed. Negative views and resistance to admittance to isolations wards have proved to be particularly difficult obstacles to control of the outbreak in Gabon and Congo. The consequences of the loss of trust of the local population in the benefits of the isolation ward impair not only individual care but also case and contact tracing, as individuals and families often learn to hide their illness or exposure risks to avoid being isolated. Thus, the failure to isolate suspected cases may undermine all facets of the control effort. Good patient management is predicated on the ability to implement sound barrier nursing procedures, which ensure the safety of the medical staff (42, 56). In fact, without this assurance, and given the minimal impact of present treatments on mortality and the history of nosocomial infections in EHF, the risk-benefit of offering patient care becomes debatable. The proportion of cases of EHF occurring among health care workers has diminished in recent outbreaks (Table 1). In Kikwit, only 1 of 80 cases of EHF among health care workers was the result of contamination after the isolation ward was established, and even this case had evidence of other, nonnosocomial exposures (43). This success prompted the development and publication of specific guidelines on barrier nursing care in the setting of outbreaks of viral hemorrhagic fevers in Africa (14, 51). Identical measures were used in Gulu, but with less successful results, as at least 13 health care workers were infected after the implementation of isolation wards (M. Lamunu, unpublished data). However, attempts to offer curative care were much more aggressive in Gulu than in Kikwit, which may have resulted in more high-risk close contact between health care workers and patients (P. Rollin, unpublished observation). Furthermore, health care workers may have been exposed to Ebola virus by patients with initially undiagnosed EHF who were inadvertently hospitalized outside the isolation wards (M. Lamunu, personal communication). Nevertheless, none of the health care worker infections in Gulu was associated with an obvious exposure, such as a needle-stick injury or obvious splashing of infected body fluids directly onto mucous membranes. Concern over the nosocomial infections that occurred in Gulu prompted a reevaluation of the adequacy of the protection measures. Although speculative, the conclusion of a specific investigation as well as the consensus of those working in the isolation wards is that most infections occurred because of a lack of strict adherence to established protocols rather than some novel method of virus transmission (M. Bell, unpublished data). One factor that contributed to the lack of compliance in Gulu was the practical difficulties of wearing protective gear, including a gown, rubber boots, a plastic apron, double gloves, a mask, and goggles, for prolonged periods in a hot tropical climate (3). Goggles, which readily fogged over in the humid environment, presented a particular problem, which has been noted previously (27). One theory is that nosocomial infections occurred through inadvertent droplet deposition or autoinoculation from the hands to the mucous membranes of the eye (D. Bausch, unpublished observation). New recommendations are being developed to attempt to account for these possibilities, such as the use of full-face shields. The sporadic


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nature of EHF cases and the difficulty in isolating them when they occur limited the health care worker exposures in the most recent outbreak in Gabon and Congo. Nevertheless, no nosocomial infections have been reported since the establishment of isolation wards there, with the exception of one case of a nurse working in a local clinic whose staff was not specifically equipped or trained to manage patients with EHF (Table 1). WHAT DIAGNOSTIC TOOLS ARE AVAILABLE FOR EHF? The large outbreaks in Kikwit and Gulu, as well as those in Gabon in the mid-1990s, provided the opportunity to validate previously developed enzyme-linked immunosorbent assays (ELISAs) and reverse transcription-PCR tests for EHF (44, 45, 47, 74). In addition, the Gulu outbreak was the first occasion in which a laboratory capable of performing such tests was established in the field. That laboratory was able to provide an accurate diagnosis in approximately 24 h (Table 1). Whether this approach can be replicated in other sites that lack the relatively developed infrastructure necessary for the laboratory in Gulu remains to be seen. Although the utilities of the presently available laboratory tests have been borne out by these outbreaks, diagnostic testing for EHF and most other viral hemorrhagic fevers continues to be limited to a few reference laboratories with appropriate containment facilities that allow the safe production of reagents and manipulation of specimens. The outbreaks of EHF since the Kikwit outbreak have all been detected by preexisting laboratories. South Africa and Gabon remain the only sites on the African continent capable of routinely performing tests for acute EHF (Table 1). Equally problematic is the absence of diagnostic capabilities for the wide array of diseases comprising the differential diagnosis of EHF, which could often render the need for a test for the Ebola viruses unnecessary. Despite the frequent call for the development of new, safe, and rapid assays for the diagnosis of acute viral hemorrhagic fevers, none have been reported, with the exception of a few ELISAs with recombinant antigens or monoclonal antibodies (58, 62). These methods await more extensive validation. Aside from the purely scientific aspects, there are numerous financial and logistic impediments to the development of new tests for EHF. The rarity of the disease and the poverty of the populations typically afflicted certainly provide little economic incentive for companies focusing on research and development. Institutions wishing to develop or use new assays would need collaborative agreements for access to reagents and the exchange of specimen panels for quality assurance. The relative rarity of EHF would likely result in the infrequent performance of any technique in most laboratories, which often poses a challenge to the maintenance of diagnostic competence and up-to-date reagents. Furthermore, the potential ramifications of both false-positive and false-negative results should be considered. The former might especially pose a problem, as, with the inevitable use of the test in areas with low incidences of EHF, positive predictive values would be low. HOW CLOSE ARE WE TO FINDING THE ANIMAL RESERVOIR OF THE EBOLA VIRUSES? The mystery of the reservoir for the Ebola viruses persists, although with perhaps a few recent clues. The few field surveys undertaken since the Kikwit outbreak have yielded


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either negative or controversial results, with the latter consisting of a report of the identification of Ebola virus sequences by PCR and the observation of virus-like nucleocapsids by electron microscopy in rodents and shrews in the Central African Republic, but without the actual isolation of Ebola virus (54). Laboratory investigations have established the competency of certain possible reservoir animals, most notably bats, to permit Ebola virus replication (76). Epidemiologic data from an outbreak of Marburg virus, a related filovirus, suggest that the reservoir for this virus inhabits mines and caves, but the pertinence of this finding to the reservoir of the Ebola viruses is unclear (D. Bausch et al., unpublished data). Understanding the relationship between the different filoviruses is perhaps the first challenge in discovering their reservoirs. Even the different species of Ebola virus are genetically quite dissimilar, with over 40% difference at the nucleotide level in the nucleoprotein gene, usually considered to be the most conserved, between the Zaire and Reston or Sudan species (Fig. 1). Although we often assume a tight virus-host pairing and taxonomic proximity between the reservoirs of hemorrhagic fever viruses of the same family, do these assumptions hold given the genetic diversity of the filoviruses? There are genera, such as Lyssavirus, which include multiple viruses pathogenic for humans with diverse animal reservoirs. Does the apparent absence of nonhuman primate involvement in the Uganda outbreak relative to the primate involvement in the Gabon outbreaks simply reflect the present-day consequences of human alteration of habitat and the elimination of these animals, or does it reveal more fundamental differences in the maintenance strategies of Ebola-Sudan viruses relative to those of Ebola-Zaire viruses? In other words, would finding the reservoir of one species of Ebola virus give us clues to the reservoirs of the others, or does each species need to be considered independently? If the former is true, comprehensive reviews of the spectrum of environments in which EHF has occurred to search for common elements, such as characteristic flora and fauna, could be illustrative (53). Where should we look for the Ebola viruses? Certainly, the multiple outbreaks in Gabon suggest that the reservoir for Ebola-Zaire virus exists in tropical rain forests in central and West Africa, where nonhuman primates become infected as dead-end hosts, perhaps playing an amplifying role in the process. The clearing of the rain forest and the apparent absence of nonhuman primates in the areas of the Kikwit and Gulu outbreaks do not necessarily signify a different epizootiology, since Ebola viruses may have been imported by individuals exposed in surrounding forests or even more distant areas. Indeed, the Kikwit outbreak appeared to be initiated by a charcoal maker who worked in a forest near the city and who was in search of wood (43). Although it is clear that nonhuman primates contract Ebola viruses and can, through direct contact, transmit them to humans, it is unknown whether they are otherwise inconsequential dead-end hosts such as humans or play a role in amplification strategies that ultimately facilitate human infection (22, 23, 48). Practical impediments frequently hinder our search for the reservoir of the Ebola viruses. To make progress in this quest, it is necessary to have a notion of where the early primary case(s) contracted infection. However, delayed recognition and high case-fatality ratios often make this location difficult to ascertain. By the time one arrives to ask questions, those who might have been able to provide answers are often dead or have moved out of the area, leaving the trail cold. Civil insecurity in rural areas may also impede the process, since a precondition of this work is unhindered access to the bush to collect specimens. The lack of safe access precluded any meaningful reservoir hunt in relation to the Gulu outbreak.


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The most recent outbreak in Gabon offers perhaps the best opportunity to date to find the reservoir. Large-scale die-offs of nonhuman primates have occurred and have been best documented for gorillas and chimps, with die-offs preceding and continuing concurrently with the human outbreak (48). As this has involved numerous species over a fairly broad range of forest in Gabon and neighboring Congo, it likely represents multiple independent introductions rather than a single introduction with subsequent secondary spread between animals. Human epidemiologic data are equally suggestive of multiple independent chains of transmission, which typically begin with one or more hunters without obvious contacts with cases of EHF who fall sick after returning from a hunting expedition in the forest (Fig. 6). In some cases, the hunters recount contact with nonhuman primates, sometimes found dead in the forest and consumed. Whether these actually represent the infectious events or are simply surrogate markers for exposure to the true reservoir is unknown. The sex ratio of cases in the most recent and previous outbreaks in Gabon also supports the conclusion of multiple primary introductions (Table 1). In previous outbreaks of EHF in Uganda and DRC, females were infected more frequently than males (63 and 53% of all cases, respectively), presumably due to secondary spread related to their roles as primary caretakers in the household. However, in all four of the Gabon outbreaks, the majority of cases were seen in males (53, 55, 70, and 58%, respectively), presumably due to the overrepresentation of hunters, who are almost exclusively male. Ecologic studies presently under way, as well as phylogenetic data from isolates obtained from humans and animals, may significantly further our knowledge regarding the reservoir of the Ebola viruses. NEW PERSPECTIVES: WHAT THREAT DO THE EBOLA VIRUSES POSE AS AGENTS OF BIOTERRORISM? The recent use of anthrax spores as agents of bioterrorism in the United States has prompted an evaluation of our preparedness in the event of the use of microbes deemed likely candidates for those with ill intent, including the Ebola viruses (69). How likely is the intentional dissemination of an Ebola virus, and how vulnerable are we? On one hand, Ebola viruses might seem to be a poor choice for would-be terrorists. Their rarity in nature, high public profile, and virulence for those performing unprotected manipulation might make it difficult to obtain and manipulate Ebola viruses, at least in the absence of the most well-organized and well-funded network. Unlike viruses such as variola virus, Ebola viruses were never widely disseminated in research laboratories. Ebola virus transmission requires direct physical contact, and secondary attack rates are low, especially when barrier nursing precautions are well maintained, as they generally are in most hospitals in industrialized countries (18, 57, 81). The stability of the Ebola viruses after removal from the buffering effects of blood or serum appears to be limited, and they can easily be inactivated by a multitude of methods, including the use of UV light, heating, detergents, and gamma irradiation (15, 19). On the other hand, case-fatality ratios due to EHF caused by Ebola-Zaire virus are among the highest for any known communicable disease, with death generally occurring very rapidly (within 10 days of infection), and no therapy or vaccine that can be used as an intervention is known (11, 13). Although the epidemiologic data do not suggest aerosol transmission of Ebola viruses between humans in natural settings, infectious and moderately


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Figure 6. Typical transmission chain for EHF during an outbreak in Gabon and Congo, 2001 and 2002. A total of 13 cases, marked A through M, were noted in this chain. For each case, the relationship with others in the transmission chain, age (y/o, years old), sex (么, male; 乆, female), location where the illness began, outcome with the date of death if the illness was fatal, and whether the case was suspected or confirmed is provided. Transmission began with a group of hunters who reportedly became ill after contact with a gorilla found dead in the rain forest during a hunting expedition (cases A to C). Secondary transmission was then noted among friends and family members (cases D, E, and G to J) and in a health care worker (case F). The two villages involved, Oloba, Congo, and Ekata, Gabon, are on contiguous sides of the international border. Crossings by local villagers are frequent.

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stable aerosols have been artificially produced in the laboratory (4, 15, 18, 32, 35, 38, 81, 82). Aerosolized transmission of Ebola-Reston virus appears to have occurred between caged monkeys, although infection through the deposition of large droplets on mucous membranes cannot be excluded (32). Infection with Ebola-Zaire virus through oral and conjunctival exposures has also been documented, which could invoke fears over the vulnerability of our food supply (32). Lastly, infection through fomites cannot be ruled out (15). The manipulation of Ebola viruses and the delivery mechanisms required to invoke large-scale dissemination and fatalities would likely require a degree of knowledge and technology available in only a handful of sites worldwide. However, it is obviously difficult to predict the clandestine capacities of those with devious intentions. Laboratory manipulation of filoviruses with a design to increase their potential as biological weapons has apparently occurred (1). Regardless of the actual biological risk presently attributable to the use of Ebola viruses for bioterrorism, the potential for widespread panic and diversion of resources due to the fear that EHF invokes cannot be overlooked. CONCLUSIONS Outbreaks of EHF have been occurring with increasing frequency in the last decade. Significant gains have been made in our understanding of EHF, as well as our capacity to respond. Most notable are heightened surveillance, including the development of preformulated surveillance guidelines and case definitions; increased international preparedness, including a rapidly deployable network of technical experts as well as prepackaged manuals and kits for the establishment of isolations wards; improved media relations; and the field validation of existing diagnostic methods. Despite increased preparedness, the impact of control measures remains muted. Inadequacies exist at both the local and the international levels. Public health infrastructures at the community level in much of Africa are not sufficiently developed to allow early recognition of EHF or the independent implementation of measures to curtail transmission. International preparedness has improved, but the delay in detection of outbreaks and the absence of effective tools, such as antiviral therapies and vaccines, ultimately relegate the response to one of damage control rather than rapid termination of outbreaks. Difficulties related to the underdeveloped public health infrastructure in Africa are not unique to EHF. However, EHF poses a unique problem—too rare to merit a large investment in each African community, yet too virulent to completely ignore. The international community is faced with a similar predicament, attempting to balance the inevitable preponderance of false alarms across the African continent with readiness for rapid action when the real emergency occurs. What reasonable steps can be taken to incorporate appropriate surveillance and control of EHF into the day-to-day health priorities of sub-Saharan Africa? Even independent of the probability of EHF, maintenance of universal precautions should be emphasized in all health care facilities worldwide. Provision of protective materials, such as masks and gloves, should be a priority and would likely prove cost-effective in relation to a host of communicable illnesses. Given the rarity of EHF, the establishment of biosafety level 4 laboratories or local vertical programs focused on viral hemorrhagic fevers across the African continent is unwarranted and unrealistic, nor would they be cost-effective. Rather, mechanisms for the


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detection of EHF and other viral hemorrhagic fevers need to be integrated into more comprehensive health programs which encourage response-oriented, early-warning surveillance for viral hemorrhagic fevers and other priority epidemic-prone diseases. Even such programs will likely succeed only if diagnostic support is offered for common febrile tropical diseases in addition to EHF. The often-signaled development of safe, new, reliable diagnostic tools for the viral hemorrhagic fevers which could be used in the field is an imperative. A reasonable approach would be to develop and disseminate a diagnostic tool for EHF with a high negative predictive value that would allow the disease to be confidently ruled out by a negative test. An established laboratory network could then be created, with all positive specimens being sent to predesignated regional reference laboratories for confirmation of the result. Although the threshold for extensive international participation might still be confirmation from the reference laboratory, the provision of protective materials and implementation of surveillance guidelines should be contingent upon a positive field test result in order to eliminate continued transmission while awaiting a definitive diagnosis. The WHO Global Alert and Response Network provides a valuable framework for rapid intervention in outbreaks of international importance. However, the coordination of the network needs to be improved, with criteria for inclusion in the network more clearly defined. Technical meetings that assemble competent field partners from all concerned countries and institutions on a semiregular basis outside the setting of an epidemic to discuss approaches and organizational roles would enhance performance when outbreaks do occur. However, it should be remembered that it is local, not international, epidemic preparedness that is the most vital component to our response and that is often the most in need. Heretofore somewhat neglected, the importance of the comprehension and support of the local population in the success of control efforts cannot be overestimated. An effective response requires the involvement of not only the national and local health officials but also local religious and political leaders, such as village chiefs, to effectively communicate public health messages to their constituents and to provide feedback to the outbreak control team. Experts in health education, social mobilization, medical anthropology, and the behavioral sciences will have to play a bigger role in future outbreaks of EHF. The absence of effective therapies or vaccines for EHF severely limits our attempts at control of the disease. Furthermore, although the presently prescribed measures centered on isolation of suspected case patients may have a utilitarian value for the community, the lack of individual benefit from these measures may result in community resistance, which undermines the entire control effort. Research on compounds that have shown initial promise, such as the S-adenosylhomocysteine hydrolase inhibitors, should now proceed to testing with nonhuman primates. In addition to the development of antivirals, it is reasonable to explore the use of ancillary therapies for EHF. New antisepsis compounds have already been shown to be efficacious in other diseases (5). Steroid therapy, although not proven efficacious as an antishock therapy in most trials, could prove useful in EHF on the basis of steroid replacement for acute adrenal failure, which is suggested by both the clinical presentation of human EHF and the frequent finding of adrenal hemorrhage and necrosis in numerous animal models (16, 17, 24, 70, 71). Ancillary therapies such as these could be tested in nonhuman primates. In addition, given the high case-fatality ratio of EHF, it would seem ethical to


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readily consider compassionate use of novel therapies that have passed phase II safety trials. To better assess efficacy, research protocols could be developed and approved in advance by international investigators and then immediately submitted to the ethics committees and advisory boards of the countries in question when an outbreak occurs. The identification of the reservoir of the Ebola viruses represents more than just a curiosity. Only with this knowledge can we tailor specific public health messages on how to avoid infection. Furthermore, if the reservoir is not prevalent throughout Africa, this knowledge could allow us to quantify the risk for EHF, targeting areas where the disease is endemic for focused preparation. The potential for the deliberate release of Ebola viruses as agents of bioterrorism brings concerns to industrialized nations that had previously troubled primarily only those in subSaharan Africa. An integrated approach is required. A thorough understanding of the epidemiology and clinical presentation will occur only through continued international commitment to investigation and control of EHF with African partners in areas where the disease is endemic. Simultaneously, concerns about bioterrorism call for a renewed and accelerated dedication to basic science research in industrialized nations, including studies aimed at obtaining a better understanding of the pathogenesis of EHF as well as the development of treatments and vaccines. Only such an integrated approach can lead to the diminishment of the threat that EHF poses to industrialized and developing countries alike. Acknowledgments. We thank Kent Wagoner, Jonathan Towner, and John O’Connor for technical assistance in preparation of the manuscript and Ray Arthur, Bruno Baert, Pierre Formenty, Scott Harper, Thomas Ksiazek, Margaret Lamunu, Mary Reynolds, and Cathy Roth for helpful review. REFERENCES 1. Alibek, K. 1999. Biohazard. Dell Publishing, New York, N.Y. 2. Baron, R. C., J. B. McCormick, and O. A. Zubeir. 1983. Ebola virus disease in southern Sudan: hospital dissemination and intrafamilial spread. Bull. W. H. O. 61:997–1003. 3. Bausch, D. G. 2001. The Ebola virus . . . and the challenges to health research in Africa. U.N. Chron. 38:6–13. 4. Bazhutin, N. B., E. F. Belanov, V. A. Spiridonov, A. V. Voltenko, N. A. Krivenchuk, S. A. Krotov, N. I. Omelchenko, A. Y. Tereschenko, and V. V. Khomichev. 1992. The influence of the methods of experimental infection with Marburg virus on the course of illness in green monkeys. Voprosy Virusologii 37:153–156. 5. Bernard, G. R., J. L. Vincent, P. F. Laterre, S. P. LaRosa, J. F. Dhainaut, A. Lopez-Rodriguez, J. S. Steingrub, G. E. Garber, J. D. Helterbrand, E. W. Ely, C. J. Fisher, Jr., and the Recombinant Human Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) Study Group. 2001. Efficacy and safety of recombinant human activated protein C for severe sepsis. N. Engl. J. Med. 344:759–762. 6. Bourée, P., and J. F. Bergmann. 1983. Ebola virus infection in man: a serological and epidemiological survey in the Cameroons. Am. J. Trop. Med. Hyg. 32:1465–1466. 7. Bowen, E. T. W., G. Platt, G. Lloyd, L. B. McArdell, D. I. H. Simpson, D. H. Smith, D. P. Francis, R. B. Highton, M. Cornet, C. C. Draper, B. El Tahir, I. Mayom Deng, P. Lolik, and O. Duku. 1978. Viral haemorrhagic fever in the Sudan 1976: human virological and serological studies, p. 143–151. In S. R. Pattyn (ed.), Ebola Virus Hemorrhagic Fever. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands. 8. Bray, M., J. Driscoll, and J. W. Huggins. 2000. Treatment of lethal Ebola virus infection in mice with a single dose of an S-adenosyl-L-homocysteine hydrolase inhibitor. Antivir. Res. 45:135–147. 9. Breman, J. G., P. Piot, K. M. Johnson, M. K. White, M. Mbuyi, P. Sureau, D. L. Heymann, S. Van Nieuwenhove, J. B. McCormick, J. F. Ruppol, V. Kintoki, M. Isaacson, G. van der Groen, P. A. Webb, and K. Ngvete. 1978. The epidemiology of Ebola haemorrhagic fever in Zaire, 1976, p. 103–121. In S. R. Pattyn (ed.), Ebola Virus Hemorrhagic Fever. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands.


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10. Busico, K., K. L. Marshall, T. G. Ksiazek, T. H. Roels, Y. Fleerackers, H. Feldmann, A. S. Khan, and C. J. Peters. 1999. Prevalence of IgG antibodies to Ebola virus in individuals during an Ebola outbreak, Democratic Republic of the Congo, 1995. J. Infect. Dis. 179(Suppl. 1):S102–S107. 11. Bwaka, M. A., M. J. Bonnet, P. Calain, R. Colebunders, A. De Roo, Y. Guimard, K. R. Katwiki, K. Kibadi, M. A. Kipasa, K. J. Kuvula, B. B. Mapanda, M. Massamba, K. D. Mupapa, J. J. TamfunMuyembe, E. Ndaberey, C. J. Peters, P. E. Rollin, and E. van den Enden. 1999. Ebola hemorrhagic fever in Kikwit, Democratic Republic of Congo: clinical observations in 103 patients. J. Infect. Dis. 179(Suppl. 1):S1–S7. 12. Centers for Disease Control and Prevention. 1990. Update: filovirus infection in animal handlers. Morb. Mortal. Wkly. Rep. 39:221. 13. Centers for Disease Control and Prevention. 2001. Outbreak of Ebola hemorrhagic fever-Uganda, August 2000–January 2001. Morb. Mortal. Wkly. Rep. 50:73–77. 14. Centers for Disease Control and Prevention and World Health Organization. 1998. Infection Control for Viral Haemorrhagic Fevers in the African Health Care Setting. Centers for Disease Control and Prevention, Atlanta, Ga. 15. Chepurnov, A. A., Y. P. Chuyev, O. V. Pyankov, and I. V. Yefimova. 1995. Effects of some physical and chemical factors on inactivations of Ebola virus. Voprosy Virusologii 40:74–76. 16. Dalgard, D. W., R. J. Hardy, S. L. Pearson, G. J. Pucak, R. V. Quander, P. M. Zack, C. J. Peters, and P. B. Jahrling. 1992. Combined simian hemorrhagic fever and Ebola virus infection in cynomolgus monkeys. Lab. Anim. Care 42:152–157. 17. Davis, K. J., A. O. Anderson, T. W. Geisbert, K. E. Steele, J. B. Geisbert, P. Vogel, B. M. Connolly, J. W. Huggins, P. B. Jahrling, and N. K. Jaax. 1997. Pathology of experimental Ebola virus infection in African green monkeys: involvement of fibroblastic reticular cells. Arch. Pathol. Lab. Med. 121:805–819. 18. Dowell, S. F., R. Mukunu, T. G. Ksiazek, A. S. Khan, P. E. Rollin, and C. J. Peters for the Commission de Lutte contre les Epidémies à Kikwit. 1999. Transmission of Ebola hemorrhagic fever: a study of risk factors in family members, Kikwit, Democratic Republic of the Congo, 1995. J. Infect. Dis. 179(Suppl. 1):S87–S91. 19. Elliott, L. H., J. B. McCormick, and K. M. Johnson. 1982. Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation. J. Clin. Microbiol. 16:704–708. 20. Emond, R. T., B. Evans, E. T. Bowen, and G. Lloyd. 1977. A case of Ebola virus infection. Br. Med. J. 2:541–544. 21. Fisher-Hoch, S. P., T. L. Brammer, S. G. Trappier, L. C. Hutwagner, B. B. Farrar, S. L. Ruo, B. G. Brown, L. M. Hermann, G. I. Perez-Oronoz, C. S. Goldsmith, M. A. Hanes, and J. B. McCormick. 1992. Pathogenic potential of filoviruses: role of geographic origin of primate host and virus strain. J. Infect. Dis. 166:753–763. 22. Formenty, P., C. Hatz, A. Stoll, P. Rogenmoser, A. Widmer, and B. Le Guenno. 1999. Human infection due to Ebola Côte d’Ivoire: clinical and biological presentation. J. Infect. Dis. 179(Suppl. 1):S48–S53. 23. Georges, A. J., E. B. Leroy, A. A. Renaut, C. Tevi-Benissan, R. J. Nabias, M. T. Ngoc, P. I. Obiang, S. Mba-Bekale, E. J. Bertherat, D. D. Benoni, L. J. T. White, E. J. Wickings, J. P. Amblard, J. M. Lansoud-Soukate, S. P. Baize, and M. C. Georges-Courbot. 1999. Ebola hemorrhagic fever outbreaks in Gabon, 1994–1997: epidemiologic and health control issues. J. Infect. Dis. 179(Suppl. 1):S65–S75. 24. Gibb, T. R., M. Bray, T. W. Geisbert, K. E. Steele, W. M. Kell, K. J. Davis, and N. K. Jaax. 2001. Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice. J. Comp. Pathol. 125:233–242. 25. Gonzalez, J. P., R. Josse, E. D. Johnson, M. Merlin, A. J. Georges, J. Abandja, M. Danyod, E. Delaporte, A. Dupont, A. Ghogomu, et al. 1989. Antibody prevalence against haemorrhagic fever viruses in randomized representative Central African populations. Res. Virol. 140:319–331. 26. Gonzalez, J. P., E. Nakoune, W. Slenczka, P. Vidal, and J. M. Morvan. 2000. Ebola and Marburg virus antibody prevalence in selected populations of the Central African Republic. Microbes Infect. 2:39–44. 27. Guimard, Y., M. A. Bwaka, R. Colebunders, P. Calain, M. Massamba, A. De Roo, K. D. Mupapa, K. Kibadi, K. J. Kuvula, D. E. Ndaberey, K. R. Katwiki, B. B. Mapanda, O. B. Nkuku, Y. Fleerackers, E. Van den Enden, and M. A. Kipasa. 1999. Organization of patient care during the Ebola hemorrhagic fever epidemic in Kikwit, Democratic Republic of the Congo, 1995. J. Infect. Dis. 179(Suppl. 1):S268–S273. 28. Heymann, D. L., G. Rodier, and the WHO Operational Support Team to the Global Outbreak Alert and Response Network. 2001. Hot spots in a wired world: WHO surveillance of emerging and re-emerging infectious diseases. Lancet Infect. Dis. 1:345–353.


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Chapter 5

The Global Epidemiology of AIDS Thomas C. Quinn

In 1981, the first cases of AIDS were recognized in previously healthy homosexual men residing in the United States (12, 42, 67, 103). Within just a few years, thousands of cases were recognized in members of other high-risk groups, including injecting drug users (IDUs), hemophiliacs, and recipients of blood transfusions (11). By the mid-1980s, it was evident that AIDS was already widespread throughout North America, the Caribbean, subSaharan Africa, Europe, and Oceania (6–10). During this time, the causative agent, the human immunodeficiency virus (HIV), was identified, and classic serologic and virologic techniques were used to help define the spread of this new disease (4, 36). By the end of the first decade, it was clear that HIV had disseminated throughout the world and that AIDS had become a global pandemic. Two decades later, over 65 million people had become infected with HIV and over 25 million people had died from AIDS (Fig. 1) (17, 112). Although public health officials recognized very early how HIV was transmitted, not all countries recognized the potential of the speed with which it could spread throughout selected populations. Even today the disease continues to escalate in many areas of the world, and it continues to devastate communities and societies to an extent that was not predicted back in the 1980s when the disease was first recognized. Currently, 42 million people are infected with HIV, 5 million of whom were newly infected last year (Table 1; Fig. 2) (112). In sub-Saharan Africa, 3.5 million people became newly infected, resulting in nearly 30 million infected individuals on the continent. Of those newly infected, 50% worldwide are women, the first time that global statistics have indicated this marked increase in HIV infection among women. Half of all the new infections occurred in young individuals between the ages of 15 and 24. In 2002, 3.1 million people died from AIDS due to lack of access to treatment and care, including 610,000 children. As in previous years, more than 95% of all new infections and fatalities occurred in developing countries where access to antiretroviral drugs still remains limited and in some cases these drugs are not available at all (Fig. 3) (30, 98, 105, 112, 117).

Thomas C. Quinn • Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. 59


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Figure 1. Historical perspective on the timeline of HIV/AIDS as shown by the epidemic increase in the number of people living with HIV/AIDS worldwide. (Source: reference 111.)

The major mode of transmission of HIV continues to be sexual transmission, although injecting drug use remains the second leading mode of transmission responsible for the rapid spread of HIV throughout Asia and eastern Europe and in other developed countries (18, 30, 105). Perinatal transmission continues to increase in developing countries, although attempts are being made to provide antiretroviral drugs near the time of delivery to Table 1. Global summary of the HIV/AIDS epidemic, December 2002a Parameter

Group

No. of people

No. of people living with HIV/AIDS

Total Adults Women Children under 15 yr

42 million 38.6 million 19.2 million 3.2 million

People newly infected with HIV in 2002


5 million 4.2 million 2 million 800,000

AIDS deaths in 2002


3.1 million 2.5 million 1.2 million 610,000

a

Data from UNAIDS (112).


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Chapter 5 • Global Epidemiology of AIDS

Figure 2. According to UNAIDS estimates, 42 million adults and children were living with HIV/AIDS in 2002. Distribution of infected people is shown by region. (Source: reference 112.)

Figure 3. Distribution of HIV/AIDS deaths in 2001 and the number of people using antiretroviral drugs during the same time period by region. Note the extraordinarily large number of deaths in sub-Saharan Africa in contrast to the small number of people using antiretroviral drugs. Data from UNAIDS (111).

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prevent mother-to-infant transmission (13). Where these programs have been implemented, perinatal transmission has been noted to markedly decline, providing further impetus to provide access to prophylaxis and treatment (13, 21). In 2002, 800,000 children became newly infected (Table 1), and currently 14 million children have been orphaned by the premature death of their parents from AIDS (112). It is projected that if the epidemic continues at its current rate, this number will more than double in the next 5 years. MOLECULAR EPIDEMIOLOGY AND EVOLUTION OF HIV The diversity of the global AIDS pandemic is also reflected in the heterogeneity of the viral subtypes of HIV (3, 106, 109). Recent studies have suggested that HIV entered the human population through multiple zoonotic infections from simian immunodeficiency virus (SIV)-infected nonhuman primates sometime in the early to mid-20th century (37, 44, 77). The simian reservoirs for the human viruses have been defined on the basis of genetic relatedness to known SIVs and the close coincidence between the natural primate host range and geographic region of the greatest diversity of HIV-1 and HIV-2. HIV-1 is most closely related to SIVcpz isolated from the chimpanzee Pan troglodytes troglodytes (37, 44, 77). The most diverse forms of HIV-1 are all found in the geographic region corresponding to the range of P. t. troglodytes in western equatorial Africa, and the HIV-1 groups and SIVcpz sequences are interspersed in phylogenetic trees, suggesting that there are shared viral lineages in human and chimpanzee. HIV-1 is further divided into three groups based on their genetic diversity from independent cross-species transmission events, i.e., from three different chimpanzee viruses. The three groups are the main (M) group, an explosive, globally dispersed epidemic; the outlier group (O), confined mostly to western equatorial Africa; and the non-M, non-O (N) group, limited to a few cases in Cameroon (38, 57). HIV-1 group M is itself divided into at least nine subtypes, which are genetically differentiated by 15 to 30% variation in the env and gag regions of the viral genome. Analogously, HIV-2 is most closely related to SIVsm from sooty mangabeys, the natural range of sooty mangabeys overlaps the geographic region where the most diverse forms of HIV-2 have been identified, and HIV-2 and SIVsm sequences are interspersed in phylogenetic trees (3, 38, 44, 57). Korber et al. (57) reconstructed the evolutionary history of HIV-1 to estimate the age of the last common ancestor of the HIV-1 M group. This work was based on phylogenetic trees, the molecular clock of HIV-1, and an assumption of a constant rate of evolution. Full-length gene alignments were selected for primary data because longer sequences yield more accurate phylogenetic reconstructions. Multiple analyses suggested that the last common ancestor of the HIV group M was near 1931, with 95% confidence intervals roughly spanning the first half of the 20th century. Sequences from several other strains with known time periods of occurrences and which were not used in the original estimate seem to validate this time estimate. Thus, although the epidemic in Africa was not clinically recognized until the early 1980s (32, 82, 91, 92, 96), it is highly probable that the virus infected humans sometime in the first half of the 20th century. Then, due to population migrations, social upheavals, and urbanization, HIV-1 moved into more densely populated populations, providing further dissemination of the virus to other humans (32, 96). The timing of the epidemic in the United States and Europe was also estimated from these molecular sequences. Korber and colleagues’ study (57) estimated that the founder


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of the B subtype in the United States probably originated in 1967, with 95% confidence intervals ranging from 1960 to 1971. This would be consistent with the first recognition of AIDS cases in 1981 as a clinical syndrome, considering that HIV may remain latent for up to 10 years before causing clinical immunodeficiency. In both Haiti and the United States, where subtype B predominates, scattered cases of HIV infection or AIDS were identified in the late 1970s, with a handful of possible or probable cases noted between 1972 and 1976 (78, 85, 86). Thus, the timing estimates for an ancestral sequence are plausible. These analyses suggest that it is probable that there was a 5- to 15-year “pre-epidemic” period of subtype B evolution, in which a small number of HIV-1 subtype B infections were present but clinically unrecognized (57). Since the HIV-1 M group began its expansion in humans approximately 70 years ago, it has subsequently diversified rapidly, comprising a number of subtypes and circulating recombinant forms (CRFs). Recombination now appears to be occurring frequently and CRFs carry sections of two or more subtypes in a mosaic genome (38, 83, 87). A recombinant lineage is designated as CRF when related forms are found in multiple epidemiologically unlinked individuals. Currently, strains belonging to the same subtype can differ by up to 20% in their Env proteins and between-subtype differences can vary by 35%. Moreover, this diversity is continuing to increase. This poses a major challenge for the development of an efficacious vaccine against HIV. For comparison, a 2% amino acid change in influenza virus can cause a failure in the cross-reactivity of the polyclonal response to the influenza vaccine and thereby necessitates changing the vaccine strain (38, 109). Although there is some cross-clade protection for HIV, vaccine experts and epidemiologists are working closely to look at multiple clade vaccine development. To aid in our understanding of this viral diversity globally, more than 8,504 samples were analyzed between 1998 and 2000, representing all global affected areas (83). Due to the massive epidemic in southern Africa, 48% of global infections are estimated to be caused by subtype C strains. The highest incidence of subtype C strains was observed in southern African countries, Ethiopia, and India, countries with large absolute numbers of new HIV-1 infections. These epidemiologic trends, coupled with the fact that these countries are home to large, vulnerable populations, suggest that subtype C will continue to dominate the HIV pandemic in the coming years. The geographic spread of subtype C strains also is very diverse, being present in regions of Africa, Latin America, and Asia. The transmission route of subtype C strains is predominantly through heterosexual contact, although subtype C viruses have been reported among IDUs in Latin America and Asia. The second most prevalent genetic variant of HIV-1 is subtype A. This subtype has caused close to 27% of all global HIV infections in 2002, mostly in countries along the central belt of Africa. The infection rate due to the subtype follows a pattern of increasing gradient from east to west, with a proportion of subtype A infections of around 30 to 40% in the east and up to 80% in west Africa. The leading mode of transmission of subtype A also is predominantly heterosexual contact. More recently, subtype A has spread outside Africa and has been reported in eastern Europe, east Asia, and the Pacific. The global proportion of subtype A strains may increase as a consequence of the rapid increase of infections occurring among IDUs in eastern Europe, such as in Russia and Ukraine. Subtype B is the third subtype accounting for a significant number of global infections, estimated at 12.3% of global cases. Subtype B infections are predominant in the Americas,


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western Europe, and Australia. None of the other subtypes accounted for more than 6% of the global HIV incidence, although some of them showed regional importance. In this survey (83), as well as in other more recent studies (3, 26, 38, 70, 73, 76, 84, 87, 108), HIV-1 recombinant forms were identified as an important factor in the global pandemic. CRF01 AE caused up to 62.5% of infections in southeast Asia. However, despite some predictions in the past, the spread of this recombinant has remained limited to countries in the same region, with rare imported cases being reported elsewhere (69, 83, 114). CRF02 AG plays an important role in the epidemic in west Africa, where it has caused up to 31% of all new HIV infections (2, 60, 63, 83). These epidemiologic trends indicate that this recombinant will probably continue to grow in numbers and proportions with an expanding geographic spread. CRF03 AB is just starting its spread, fueling a recent HIV-1 epidemic among IDUs in eastern Europe (79, 80). Initially, the epidemic in this region was started in the mid-1990s with two parental pure subtypes, A and B, but very quickly the AB recombinant emerged and began a rapid spread in diverse locations of this region. The explosive spread of this regional epidemic was confirmed by molecular data showing close genetic relationships and low heterogeneity between strains isolated in widely separated geographic locales of the region. These three major CRFs constituted more than 50% of all recombinant forms detected in the World Health Organization survey (83). Nevertheless, an increasing number of other recombinant forms are being reported for some countries where multiple subtypes cocirculate, and the estimated prevalence of recombinant forms could be as high as 25%. Use of molecular subtyping also has helped define the epidemiology of transmission of HIV. For example, in more recent explosive epidemics among IDUs, there is a very small amount of genetic diversity between individuals within the epidemic, with approximately 1% variation compared to 10 times that amount for epidemics among heterosexuals. In Kaliningrad and Guangxi, an AB recombinant and an equally novel BC recombinant, respectively, have exploded among IDUs within these regions (54, 79, 80, 91, 107, 122, 123). Both of these CRFs show almost no variability across their respective genomes. Every single structural and regulatory gene of the virus has been extraordinarily conserved in thousands of infections. Molecular epidemiologists suggest that when specific strains fall into the right social network, or people become coinfected with two HIV subtypes in the right social circumstances, new strains may develop and consequently spread to hundreds of thousands of people in a matter of months to years after the initial recombination. These epidemics provide examples of the viral potential for genetic preservation amid great amplification. In contrast, when viral strains from heterosexually transmitted epidemics among African populations are measured, there is extraordinary diversity of the recombinants, with a range of 5 to 14% diversity between individuals. Instead of producing copies of identical CRF strains, the recombinants fill in the gaps between subtypes, thus creating broader epidemics of recombinants (38, 69, 83). In some countries in Africa, which either were among the earliest centers of infection or had significant population migration and transmission, at least five viral subtypes are known to be present (73, 75, 83, 107). Similarly, diversity is now being documented in India and Brazil, where there is documentation of recombination, such as BF recombinants (61, 74, 78, 88). In some cases the degree of viral divergence within a given region may also reflect the duration of the epidemic. For example, in Africa there is already 20 to 30% genetic diversity within a genotype,


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whereas in Thailand this diversity is much more limited (119). The immunologic importance of this genetic heterogeneity is not fully understood, but it is clear that any globally effective vaccine will have to induce protective immunity through a broad range of genetic and potentially antigenic subtypes. The low levels of genetic diversity seen in some regions of HIV-1 spread present rapidly closing windows of opportunity for vaccination against still relatively homologous viral challenge. The diversity in HIV subtypes also may have important biologic and immunologic implications. Intrinsic biologic properties of these viruses include infectivity and replication capacities that contribute to different epidemic curves (49). In 1985, a second human retrovirus associated with AIDS, HIV-2, was identified in west Africa (22, 50). This virus has been found to have relatively high prevalence rates among hospitalized patients, female prostitutes, and patients who attend sexually transmitted disease (STD) clinics in several countries of west Africa (21, 23, 27, 66). Because this virus is spread by the same modes of transmission as HIV-1 (21, 27), it is likely that similar epidemiologic patterns may be observed for HIV-2 in the near future. Currently, HIV2 is spread predominantly by heterosexual transmission, like HIV-1 (46, 62, 63). Systematic surveys for HIV-2 outside west Africa have documented cases in parts of central Africa, Europe, North America, and Brazil. In comparison with HIV-1, however, HIV-2 is characterized by lower rates of sexual and perinatal transmission, lesser cell killing, lower viral burdens, more gradual CD4+ cell loss, slower progression to AIDS and death, and relative geographic confinement (1, 29, 49, 51, 52, 53, 65). Travers and colleagues (110) demonstrated that HIV-2-infected women in Senegal had a lower incidence of HIV-1 than did seronegative women and close to a 70% reduction in risk for HIV-1 infection, despite similar high-risk sexual behavior and the same frequency of STDs. These data suggested that the protection observed may be a result of cross-reactive immunity to epitopes conserved between HIV-1 and HIV-2. However, the study also demonstrated for the first time sequential infection with two different HIV subtypes, first with HIV-2 and then with HIV1. Further studies are urgently needed to determine whether cross-reactive immunity can be induced to epitopes conserved between different strains of the virus.

REGIONAL EPIDEMICS Sub-Saharan Africa Sub-Saharan Africa represents the epicenter of the global HIV/AIDS pandemic. Although AIDS has had an enormous medical, cultural, and economic impact on all countries, this disease has taken its greatest toll in the countries of sub-Saharan Africa (30, 40, 64, 111, 112). More than 28 million individuals (7% of the population of the subcontinent) have been infected with HIV (Table 2) (112). Approximately 3.5 million new infections occurred in 2002 and 2 million people died. Ten million young people (age 15 to 24) and almost 3 million children under age 15 are living with HIV. In addition, 11 million children have been orphaned by the premature death of a parent due to AIDS. More than half of the infected adults are women, and as many as 6 million African children are estimated to have been infected as a result of mother-to-infant transmission. In some geographic areas, specific population groups are disproportionately affected by the epidemic. Men and women between 15 and 40 years old, people with STDs, and people in certain occupational groups


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Region

Sub-Saharan Africa North Africa and Middle East South and southeast Asia East Asia and Pacific Latin America Caribbean Eastern Europe and central Asia Western Europe North America Australia and New Zealand Total

Main mode(s) of transmission for adults living with HIV/AIDS

Adults and children living with HIV/AIDS

Adults and children newly infected with HIV

Adult prevalence rate (%)b

29.4 million 550,000

3.5 million 83,000

8.8 0.3

Heterosexual Heterosexual, IDU

6 million 1.2 million 1.5 million 440,000 1.2 million

700,000 270,000 150,000 60,000 250,000

0.6 0.1 0.6 2.4 0.6

Heterosexual, IDU IDU, heterosexual, MSM MSM, IDU, heterosexual Heterosexual, MSM IDU

570,000 980,000 15,000

30,000 45,000 500

0.3 0.6 0.1

MSM, IDU MSM, IDU, heterosexual MSM

42 million

5 million

1.2

a

Data from UNAIDS (112). The proportion of adults (15 to 49 years of age) living with HIV/AIDS in 2002, using 2002 population numbers.

b

such as long-distance truck drivers, military personnel, and women employed in commercial sex usually have the highest prevalences of infection (32, 59, 89, 90, 94–96). HIV prevalences higher than 80% have been reported for female sex workers in east Africa and central Africa. In some areas, HIV infection has now been documented among members of the general population, as evidenced by the initially slow but accelerating spread among pregnant women. Seroprevalence of HIV among pregnant women ranges from 5 to 35%, with the highest rates in urban centers such as Malawi, Botswana, Zambia, Zimbabwe, and South Africa (72, 104, 120). In Nigeria, a country with more than 117 million inhabitants (20% of sub-Saharan Africa’s population), HIV is rapidly spreading among female sex workers and their clients, and now in the general population. In some urban populations, more than 10% of the adults are infected, and the annual incidence is estimated to be 3% (112, 113, 116). Most HIV transmissions in sub-Saharan Africa occur through sexual intercourse, with unsafe blood transfusions and unsafe injections accounting for an unknown fraction. Although sexual behavior is the most important factor influencing the spread of HIV in Africa, that behavior varies greatly across cultures, age groups, socioeconomic class, and gender. The interplay of multiple factors, both biological and behavioral, determines the spread of HIV. One study in four African cities (Cotonou, Kisumu, Ndola, and Yaounde) revealed that the most common behavioral and biological factors in those cities with the highest HIV prevalence were young age at women’s first sexual intercourse, young age at first marriage, age difference between spouses, the presence of herpes simplex type 2 infection and trichomoniasis, and lack of male circumcision (9). There is substantial evidence that STDs enhance the risk of sexual transmission of HIV (34), that the level of HIV viral load in an individual enhances the probability of infectiousness (41, 97), and that


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male circumcision may be associated with reduced risk of transmission (43). Whereas the complex interplay of factors makes it difficult to estimate the likely growth of the epidemic within the region, evidence from the past two decades demonstrates that HIV can spread rapidly and widely from very low seroprevalence levels. Approximately 50 to 65% of HIV infections in Africa have been in east and central Africa, an area that accounts for only 15% of the total population of sub-Saharan Africa (111, 112). Recent studies, however, indicate that the pandemic has continued to evolve, particularly in western and southern Africa. At least 10% of those aged 15 to 49 years are infected in 12 African countries. Seven countries, all in southern Africa, now have prevalence rates higher than 20%. Women account for the majority of persons living with HIV in sub-Saharan Africa (58%), primarily due to heterosexual transmission. With increasing rates among women, HIV prevalence rates among pregnant women also appear to be increasing across the continent. The highest HIV prevalence worldwide for pregnant women now stands at 45% in Botswana (112). In neighboring countries HIV prevalence rates continue to rise among pregnant women. In Zimbabwe, the prevalence rose from 29% in 1997 to 35% in 2000; in Namibia, it rose from 26% in 1998 to 30% in 2000. Prevalence rates are even higher among specific age groups. For example, in Botswana, among 25- to 29-year-old women attending antenatal clinics in urban areas, 55.6% of pregnant women were infected with HIV. In Swaziland the corresponding prevalence was 33.9%, and in Zimbabwe it was 40.1%. For the Republic of South Africa, HIV prevalence rapidly escalated during the 1990s to a current rate of 25%. Increasing HIV prevalence is also being reported from west Africa. The relatively low adult HIV prevalences in Senegal and Mali (⬍2%) are shadowed by the more ominous patterns of growth in neighboring countries. HIV prevalence is estimated to exceed 5% in eight countries of west and central Africa, including Cameroon (11.8%), Central African Republic (12.9%), and Ivory Coast (9.7%). In Nigeria, the most populous country in sub-Saharan Africa, an increase in prevalence in the general population was reported from 1.9% in 1993 to 6% in 2002. Already more than 3 million Nigerians are estimated to be living with HIV/AIDS. These statistics serve as reminders that no country or region is safe or protected from this epidemic. Over the past decade AIDS has become the leading cause of death and years of productive life lost throughout the continent. Excess deaths attributable to HIV are highest in 25to 34-year-olds, usually a group with low mortality (28, 31, 33, 102). Nearly 90% of deaths in this age group are in excess of background rates and were attributable to HIV. Because AIDS deaths are concentrated in childhood and young adult ages, their effects are substantial, reducing life expectancy by more than 20 years in several countries (Fig. 4) (111). Demographic projections have estimated that population growth will decline more rapidly than expected and that the size of the African population in 2005 will be smaller than it would have been without AIDS. Average life expectancy in sub-Saharan Africa is now 47 years, when it should have been 62 years of age without AIDS (111). Life expectancy at birth in Botswana has dropped to a level not seen in that country since the 1950s. Current HIV prevalence levels merely hint at the greater lifetime probability of being infected with AIDS. In Lesotho it is estimated that a person who is 15 has a 74% chance of being infected with HIV by his or her 50th birthday. Seven nations in sub-Saharan Africa now have life expectancies less than 40 years, and this number will increase to 11 countries over the next 7 years. The declining life expectancies will soon reach levels that have not existed


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Figure 4. Reduction in life expectancy compared to the “no AIDS” scenario in selected countries, 2000 to 2005. (Source: reference 111.)

since the 19th century. In Zimbabwe and South Africa the infant mortality rate is higher than it was in 1990. Five African nations will experience more deaths than births by 2010, with a resultant decrease in population size. Life expectancy will drop to just 27 years in Botswana and Mozambique in the next 8 years, while Swaziland will have an estimated life expectancy of 33 years. In Zimbabwe, Zambia, and Namibia, the expected life span will be 34 years (89, 101). In contrast, the life expectancy in an Africa without AIDS would be 70 years by 2010 (111). HIV/AIDS cases will put an increasing strain on health care systems, which are already overburdened, and on households that are trying to manage with limited economic resources. Care and support for children orphaned by AIDS will become growing concerns throughout the region (93), and the social, economic, and demographic impact of AIDS will be enormous. For a country with a current HIV prevalence of 8%, the expected increased demand for health care services ranges from 2.3 to 9.3%, depending on the state of development of its health care sector. The strong association of HIV with a burgeoning tuberculosis epidemic (31, 46), combined with the excess mortality associated with HIV infection, underscores the critical importance of the HIV epidemic in Africa. In contrast to the escalation of the epidemic in several areas of Africa, prevention efforts have had substantial dividends in some countries. Treatment and prevention programs in Ethiopia, Uganda, Senegal, Zambia, and Ivory Coast demonstrate that HIV incidence can be stabilized and in some cases can even be reduced over time (113). HIV prevalence levels among young women in Addis Ababa declined by more than one-third between 1995 and 2001 (112). In Uganda, HIV prevalence has declined by over 50% over the past 8 years (68). In South Africa, HIV prevalence levels decreased among teenagers by 25% between 1998 and 2001 (111, 112). Although prevalence remains unacceptably high in many countries, the above positive trends confirm the value of investing in prevention programs aimed at youth and the introduction of treatment programs.


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Asia After sub-Saharan Africa, Asia has the largest number of HIV-infected individuals in the world, estimated at 7.2 million people (Table 2) (112). With more than half of the world’s population, Asia is still in the early phases of an explosive HIV/AIDS epidemic (94). Although HIV was introduced much later in Asia than in the rest of the world, Asia is now home to more people living with HIV/AIDS than any other region besides sub-Saharan Africa. In India, an estimated 4 million people were living with HIV/AIDS at the end of 2002—more than any other country except South Africa. Moreover, this region’s epidemic is spreading into the general population. In 2002, 1 million adults and children became newly infected, in part due to the explosive growth of the HIV epidemic in China, India, and several other countries in southeast Asia. With the exception of Cambodia, Burma, and Thailand, national HIV prevalence levels remain comparatively low in most countries of Asia and the Pacific, in part due to their large population base. For example, in China, Indonesia, and India, where large numbers of people are infected, national HIV prevalence rates in these highly populous countries do not provide the full impact of the epidemic. India’s national adult HIV prevalence rate of 1% offers little indication of the serious situation facing the country. HIV prevalence among women attending antenatal clinics was higher than 1% in Andhra Pradesh, Karnataka, Maharashtra, Manipur, Nagaland, and Tamil Nadu (111). The pattern of HIV spread in Asia appears to be different from that described in other regions. HIV was initially noted among IDUs in Thailand, Burma, and India. HIV seroprevalence increased dramatically between 1988 and 1991 from 1.2 to 45% in Thailand (8, 56, 118), and in the northeastern state of Manipur, India, it rose from 55 to 80% (6, 47). In addition, the first evidence of HIV in Yunnan Province, People’s Republic of China, bordering Burma and Laos and considered part of the “golden triangle” of heroin exportation, demonstrated an alarming HIV prevalence of 43 to 82% among IDUs (20, 121). Data from Malaysia and Vietnam show similar increases in HIV levels among IDUs (16). During this rise of HIV infection among IDUs, HIV infection was noted among female sex workers. Although highly variable by region, HIV prevalences of 30 to 65% have been reported among female sex workers in various cities of Thailand and India (6, 47, 111). Successive waves of heterosexual transmission from these sex workers to their male clients and subsequently to other sexual partners including spouses occurred, resulting in rapid spread of HIV to segments of the general population. Among military recruits in Thailand in 1993, HIV prevalence was 4% overall and 12.4% among recruits from the northern province of Chiang Mai (7, 81). Among pregnant women, HIV prevalence rose to 8% in Chiang Mai and Chiang Rai in northern Thailand, and the overall prevalence for pregnant women in the country was estimated as 2%. From these data, it was estimated that more than 840,000 persons in Thailand, or 1% of the Thai population, were HIV infected by 1994 (112). If this rate of HIV transmission continued, there would have been 2 million to 4 million cumulative HIV infections in Thailand alone by 2000. In urban centers in India, the rise in HIV seroprevalence among female sex workers and their sexual contacts has been equally dramatic. In a study of 2,800 attendees of STD clinics in Pune, the overall HIV seroprevalence was 23.4% (99). Among initially seronegative persons, the subsequent HIV incidence was 26.1 per 100 person-years of observation for female sex workers, 9.4 for men, and 8.4 for women who were not sex workers (71). Recurrent genital ulcer disease and urethritis or cervicitis during the follow-up period were


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independently associated with an increased risk of seroconversion. Given the prevailing sexual practices, large populations of HIV-infected female sex workers, low social status of women, male patronage of sex workers, high rates of STDs, low rates of condom use, and high frequency of injecting drug use, a scenario similar to that described for India and Thailand is likely for many other populous countries of Asia. For the more developed areas, such as Japan, South Korea, Taiwan, Singapore, and Hong Kong, HIV epidemics may not be as explosive, but there will probably be a slow and steady increase in HIV infections. Throughout Asia the HIV incidence could exceed 1 million new infections per year within the next few years. Throughout the region, injecting drug use remains one of the most prominent modes of transmission of HIV. Over 50% of IDUs in Malaysia, Burma, Nepal, Thailand, Indonesia, Manipur, and southern China have already acquired HIV. Very high rates of needle sharing have been documented among users in Bangladesh and Vietnam along with evidence that a considerable proportion of sex workers in Vietnam also inject drugs. The People’s Republic of China, with a fifth of the world’s population, has also witnessed a dramatic escalation of the HIV epidemic in the past 3 years, with a 67% increase in reported HIV in 2001 (112). Currently, 1 million Chinese individuals are estimated to be living with HIV. The HIV epidemic is particularly severe among IDUs in at least seven provinces, with prevalence rates higher than 70% among IDUs in areas such as Yili Prefecture in Xinjiang and Ruili County in Yunnan Province. Another nine provinces in China are on the brink of similar HIV epidemics because of the very high rates of needle sharing. To compound the tragedy of the epidemic in China, recent reports in Henan Province in central China demonstrate that many rural villagers became infected by selling their blood to collecting centers that did not follow basic blood donation safety procedures. It has been estimated that 150,000 people have been infected through these practices. In addition, there are new signs of heterosexually transmitted HIV epidemics in at least three provinces, Guangdong, Guangxi, and Yunnan, where HIV prevalence rates have been documented at 11 to 15% among sentinel sex workers in 2000. Several other factors highlight the swift escalation of HIV in China. STDs have quadrupled between 1997 and 2002, suggesting that unprotected sex with nonmonogamous partners is increasing in China. In addition, there is massive population mobility. Approximately 100 million Chinese are temporarily or permanently away from their registered addresses, and increasing socioeconomic disparities add to the likelihood of HIV spread. Indonesia, the world’s fourth most populous country, is another example of how quickly the HIV/AIDS epidemic can emerge (111). After more than 10 years of negligible HIV prevalence, the infection rate among IDUs, sex workers, and blood donors in some regions is rapidly increasing. In one drug treatment center in Jakarta, HIV prevalence rose from 15.4% in 2000 to more than 40% by 2002. In some countries of southeast Asia where HIV rose rapidly in the 1990s, strong prevention programs have limited the spread more recently (112). In Thailand, the number of new HIV infections has declined from 143,000 in 1991 to 29,000 in 2001. Similarly, in Cambodia, HIV prevalence fell by more than 4% between 1999 and 2001 as a result of a multifaceted response that included a 100% condom use program and steps to counter stigma. Despite these advances, AIDS is still the leading cause of death in Thailand, and 1% of the country’s 63.6 million people are infected with HIV. Although STDs and heterosexual transmission have declined as a result of the government’s prevention programs, HIV continues to spread rapidly among IDUs and men who have sex with men (MSM).


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Oceania Approximately 50,000 cumulative adult HIV infections have occurred in this region, with nearly half in Australia and New Zealand (Table 2) (112). The annual reported number of HIV infections from these two countries seems to have reached its peak, and since 1987 there has been a downward trend for both countries in the number of HIV infections reported each year. Most of the infections have occurred among homosexual men, and the male-to-female ratio among infected individuals is 7:1, indicating a lower degree of heterosexual transmission than that observed in other regions. The frequency of HIV infection among IDUs in Australia and New Zealand also remains lower than that in western Europe or North America, probably because of the early availability of sterile injection equipment. For most other countries of the Pacific region, the cumulative numbers of reported AIDS cases and HIV infections have been too few to allow meaningful analysis by time or mode of transmission. However, despite the low number of cases, there is a great potential for HIV to spread rapidly within this region. Epidemiologic and behavioral studies indicate generally high rates of injecting drug use, unprotected sexual activity including commercial sex, and a high prevalence of STDs. Moreover, many parts of Asia and the Pacific are undergoing rapid development. As trade, tourism, and migration increase, so may the opportunities for HIV dissemination (111, 112). Papua New Guinea has reported the highest HIV infection rates among the Pacific Island countries and territories. Results of recent studies in the capital, Port Moresby, show high HIV prevalence levels among female sex workers (17%) and attendees of STD clinics (7%). The Philippines continues to maintain a low HIV prevalence, although high rates of other STDs among Filipino sex workers, their clients, and MSM indicate low levels of condom use and the potential for a rapid rise in HIV. Europe and Central Asia The HIV epidemic has increased faster in eastern Europe and central Asia from 2000 to 2003 than in any other area of the world. In 2002, there were an estimated 250,000 new infections, bringing to 1.2 million the number of people living with HIV (Table 2) (112). In recent years Russia has experienced an exceptionally steep rise in reported HIV infections, 90% of which have been attributed to injecting drug use (Fig. 5) (48). It is now estimated that nearly 1% of the young people in eastern European and central Asian countries are injecting drugs, which places these individuals and their sex partners at high risk of being infected with HIV (112). In one study in Moscow, secondary school students acknowledged that 4% injected drugs (48). In some countries such as Azerbaijan, Georgia, Tajikistan, and Uzbekistan, HIV has experienced explosive growth (112). In the first 6 months of 2002 there were as many new HIV infections in these countries as has been reported in the whole of the previous decade. In the Baltic countries, similar explosively high rates of HIV are being documented among IDUs and heterosexuals at risk for STDs. Similar to the situation in China, very high rates of STDs are being found in eastern Europe and central Asia, increasing the odds of HIV being transmitted through unprotected sex. In 2000, the number of newly reported cases of syphilis in Russia was 157 per 100,000 persons, dramatically higher than the 4.2 per 100,000 persons in 1987 (111, 112). Similar trends have been reported in other countries of the Commonwealth of Independent States, the Baltic states, and Romania. In Estonia, Latvia, and Lithuania, major HIV outbreaks are also occurring among selected populations such as prison inmates (45). In one prison in


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Figure 5. Cumulative reported HIV infections per million population in eastern European countries, 1993 to 2001. Data from UNAIDS (112).

Lithuania, 15% of inmates were HIV positive, confirming the role of prisons in the spread of HIV in many countries of the region. The concentration of large numbers of young people in overcrowded prisons or juvenile justice facilities, often marked by the abundance of drugs but a scarcity of HIV information, clean needles, or condoms, provides fertile ground for the rapid spread of HIV among inmates and, upon their eventual release, into the wider population. Initially driven by injecting drug use among young people, heterosexual transmission of HIV has now become the prominent mode of spread in Belarus and Ukraine (43). With an estimated adult HIV prevalence rate of 1%, Ukraine is the most affected country in the region and in all of Europe (45). Three-quarters of HIV infections in Ukraine and Russia are related to injecting drug use, but the proportion of sexually transmitted infection is increasing (62). New diagnoses of HIV and persons infected through heterosexual intercourse accounted for 28% of all new cases reported in the first 6 months of 2002, up from 15.3% in 1998. Although many of these infections may occur among sex partners of IDUs, the trend may also indicate spread into the wider population of these countries. Recent studies in Donetsk, Ukraine; Moscow; and St. Petersburg revealed HIV prevalence rates of 13 to 17% among sex workers (45, 62, 112). In Russia and Ukraine, up to 30% of female IDUs are also involved in commercial sex work. The public health efforts to stem the tide of these epidemics in these countries are limited and, in some cases, nonexistent. In contrast, prevalence for HIV remains low in Poland, the Czech Republic, Hungary, and Slovenia, where well-designed national HIV/AIDS programs are in operation. If effective interventions are not implemented in the more severely affected countries, it is likely that the situation will become dramatically worse over the next 5 years (45).


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Over half a million HIV-infected individuals reside in western Europe, and in 2002, 30,000 became newly infected, with trends similar to those witnessed in the United States, Australia, and New Zealand (112). The introduction of combination antiretroviral therapy in 1996 to 1998 has dramatically reduced HIV/AIDS-related mortality, although this trend has begun to level off in the past 2 years. Approximately 500,000 people received antiretroviral drugs in high-income countries by 2002. Longer survival of people living with HIV/AIDS has led to a steady increase in the number of people living with the virus in high-income countries. The HIV epidemic in western Europe is a result of a multitude of epidemics that differ in their timing, their scale, and their effects on populations (112). A larger proportion of new HIV diagnoses (59% overall between 1997 and 2001) in western European countries occurred through heterosexual intercourse. More than half of the new HIV infections in the United Kingdom resulted through heterosexual sex compared to 33% in 1998. In Ireland a similar trend is visible, with the numbers of heterosexually transmitted HIV infections increasing fourfold between 1998 and 2001. Injecting drug use remains the main mode of transmission in Spain, but about one-quarter of all HIV infections are now heterosexually transmitted. Reported HIV prevalence among IDUs in Spain in 2002 was 30% nationwide, whereas in France prevalence rates range from 10 to 23%. Portugal’s serious epidemic among IDUs accounted for more than half of the newly diagnosed infections in both 2000 and 2001, although the number of reported HIV infections declined in 2001. Similar to the United States, most countries of western Europe have witnessed a marked decrease in AIDS fatality rates and in the reporting of AIDS due to the introduction of antiretroviral drugs. Consequently, adult HIV prevalence has risen slightly in these countries, primarily due to antiretroviral therapy keeping HIV-positive people alive longer. This benefit of increased survival, though, has led to a concomitant increase in unsafe sex, triggering higher rates of STDs and in some cases higher HIV incidence among MSM. A rising incidence of STDs among MSM has been noted in Amsterdam, London, Madrid, Sydney, and several cities in the United States (13, 35, 112). This widespread risk-taking is eclipsing the safer-sex ethic promoted so effectively for much of the 1980s and 1990s. In a French study in 2000, 38% of surveyed HIV-positive MSM said they had recently practiced unsafe sex, compared to 26% in 1997 (112). Latin America and the Caribbean An estimated 1.9 million adults and children are living with HIV in this region (Table 2) (112). Twelve countries in this region have an estimated HIV prevalence of 1% or higher among pregnant women (10). In several Caribbean countries adult HIV prevalence rates are surpassed only by the rates experienced in sub-Saharan Africa, making this the second most affected region in the world (40). Haiti remains the worst affected, with an estimated national prevalence of over 6%, along with the Bahamas where the prevalence is 3.5%. HIV/AIDS is now the leading cause of death in some countries of the Caribbean basin. In Haiti, the Bahamas, and Guyana, the number of deaths among 15- to 34-year-olds is 2.5 times higher than it would have been in the absence of AIDS. The estimated 100,000 AIDS deaths in 2001 have further increased the ranks of children orphaned by epidemics in this region. Some 330,000 of the orphans are living in Latin America (130,000 of them in Brazil) and 250,000 in the Caribbean (200,000 in Haiti alone).


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Homosexual transmission and heterosexual transmission continue to be the major modes of transmission throughout the region, although there is evidence that spread of HIV is increasing through sharing of infected drug equipment (100). Population mobility, spurred by high rates of unemployment and poverty, is emerging as a significant factor in the epidemic’s growth in this region. Central America’s geographical position also makes it an important transit zone for people moving between the rest of the region and North American countries. Appropriately, protecting vulnerable populations on the move, including adolescent girls and young women, is now the focus of a regional prevention program in Central America. In Mexico, adult HIV prevalence in the wider population is still well under 1%, but prevalence rates are higher in specific population groups—6% among IDUs and 15% among MSM. There is significant overlap between IDUs and MSM, especially in Brazil and the southern cone countries where injecting drug use is a growing social phenomenon. Injecting drug use is also a major route of HIV transmission in Argentina, Chile, and Uruguay. Despite many constraints, the region has made progress in providing treatment and care. An estimated 170,000 people, primarily in Brazil, were receiving state-sponsored treatment by the end of 2001 (111, 112). By reducing HIV/AIDS-related morbidity through treatment, Brazil’s treatment and care program is estimated to have avoided 234,000 hospitalizations in 1996 through 2000, thereby demonstrating the cost-effective approach to care. Argentina, Costa Rica, Uruguay, and Cuba now guarantee free and universal access to drugs through the public sector and sharp reductions have recently been secured in Honduras and Panama. Unfortunately, access to antiretrovirals remains unequal across the region, due largely to discrepancies in drug prices in different countries. Unless overcome, economic difficulties within some countries will continue to plague the region, facilitating both further economic decline and the spread of HIV. United States By 2002, the United States had reported the highest number of AIDS cases in the world, with more than 800,000 cases and nearly 500,000 fatalities (13). Estimates from the Centers for Disease Control and Prevention suggest that 900,000 people were living with HIV in the United States in 2002, of whom 360,000 had AIDS (18). Since the use of antiretroviral drugs, sharp declines in AIDS incidence have occurred from 1996 through 1999 (Fig. 6). Since then, AIDS incidence has leveled off and essentially no changes have occurred in incidence through 2002. Similarly, from 1996 through 1997 the number of deaths among persons with AIDS also declined sharply. As a result, HIV/AIDS prevalence has increased steadily over time due to prolonged survival. In 2002, 39% of persons living with AIDS lived in the South, 29% in the Northeast, 19% in the West, 10% in the Midwest, and 3% in U.S. territories. Of persons living with AIDS, 42% were African American, 37% were white or Caucasian, 20% were Hispanic, and 1% were Asian or Pacific Islander, and ⬍1% were American Indian or Alaska Native. Of reported AIDS cases, 57% were MSM, 24% were IDUs, 9% were exposed through heterosexual contact, and 8% were MSM/IDU. Of the 76,000 women with AIDS reported in 2001, 59% were infected through heterosexual contact and 38% via injecting drug use. The number of AIDS cases in children has markedly declined since the advent of antiretroviral drug use during pregnancy to prevent HIV perinatal transmission (18, 25). In 2001, only 175 new cases of AIDS were diagnosed in children, of which 86% were attributed to perinatal exposure.


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Figure 6. Estimated AIDS incidence, deaths, and prevalence by quarter year of diagnosis or death for the United States, 1981–2000. Data from the Centers for Disease Control and Prevention (13).

The impact of HIV/AIDS in the African American community has been particularly severe (5, 16, 55, 115). Representing only an estimated 12% of the U.S. population, African Americans make up 38% of all AIDS cases reported in the country. It is estimated that almost 129,000 African Americans are living with AIDS. In 2000, more African Americans were reported with AIDS than any other racial/ethnic group. Nearly half of all reported AIDS cases in 2000 were among African Americans. The 2000 rate of reported AIDS cases among African Americans was 58.1 per 100,000 people, more than two times the rate for Hispanics and eight times the rate for whites. Almost two-thirds (63%) of all women reported with AIDS were African American, and African American children represented two-thirds of all reported pediatric cases. With an epidemic that is now nearly into its third decade, complacency has increased and prevention efforts have dwindled as a result of declining mortality. Multiple studies illustrate that prevention efforts are not reaching the large number of at-risk individuals who engage in unsafe sex. The rates of sexually transmitted infections among MSM have been documented not only in the United States but also in Australia, Great Britain, Canada, and other developed countries. Rates of gonorrhea, syphilis, and chlamydia have more than doubled in the past 5 years among MSM in selected U.S. and European cities (14, 15, 35). Renewed efforts to enhance prevention efforts, particularly in HIV care clinics, are being echoed throughout all of these countries. FUTURE PROJECTIONS The long-term dimensions of the HIV pandemic cannot yet be forecast with confidence. However, on the basis of available data the WHO/UNAIDS has projected that by 2010 there will be a cumulative total of more than 100 million infections in men, women, and


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children, of which more than 90% will be in developing countries. By 2010 the number of HIV-related deaths among adults is predicted to rise from its current total of 3.5 million per year to more than 8 million individuals per year, depending on availability and access to therapy. Unless our interventions are more effective, between 15 million and 20 million new HIV infections will occur in the next 5 years. In addition, more than 5 million children younger than 10 years will be orphaned as a result of AIDS-related fatalities. The number of orphans will increase further in the early years of this century as a result of the deaths of mothers who were infected with HIV in the 1990s. As the epidemic matures in some parts of the world, a large number of young people becoming sexually active will replenish the pool of susceptible people, especially in developing countries, where the base of the age pyramid is quite broad. Evidence for a high incidence in young populations compared with older cohorts is already emerging for various countries. In the United States, the number of 13- to 21-year-olds who had become infected with HIV rose by 77% in the 1990s. In subSaharan African countries the highest HIV seroprevalence rates currently are among women between 15 and 25 years old. Heterosexual spread of HIV is causing an epidemiologic shift of infection from high-risk populations such as homosexual men and IDUs to populations more reflective of the general population, especially adolescents and young women of childbearing age. As a consequence, the number of persons with AIDS will continue to increase, causing unprecedented personal suffering, high direct costs for medical care, reduced economic output, and a substantial indirect cost to society. The impact of HIV in many countries will be immense. In conservative projections the annual number of new AIDS cases in North America and western Europe during the next two decades is expected to remain fairly constant at close to 100,000. This is because HIV-infected persons will eventually develop AIDS, and the annual HIV incidence in these regions, projected to be close to 100,000, will nearly equal the number of fatalities. In sub-Saharan Africa, the peak occurrence of AIDS, estimated at 750,000 cases per year, will not be reached until the middle of this decade. However, in Asia, the annual number of AIDS cases will increase steadily in this decade and will not begin to level off until 2010 at about 850,000 cases per year. Although the absolute number of new HIV infections in Asia is equal to the high level found in Africa, the rate of new infections or new cases per year as a percentage of the adult population is still much lower in Asia than in sub-Saharan Africa. This is because the adult population of Asia is more than five times larger than the adult population of sub-Saharan Africa. If unchecked, the rising incidence in Asia will produce an unprecedented number of new cases in that region. On the basis of current data, cumulative HIV infections in Asia are conservatively projected to be more than 10 million (0.46% of the population) in 2005 and more than 18 million in 2010. In sub-Saharan Africa, the cumulative number of HIV infections is projected to reach about 60 million in 2005 and about 80 million in 2010. From these epidemiologic trends, it is projected that within the next 8 years an additional 45 million people will become infected with HIV in 126 low- and middle-income countries that currently have the most concentrated or generalized epidemics, unless the world succeeds in mounting a drastically expanded global prevention and treatment effort. It is anticipated that more than 40% of these new infections will occur in Asia and Pacific regions, although it is apparent that the epidemic will continue to devastate nearly all the countries of the African continent. Over the next decade, without treatment and care in


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many countries, millions of individuals will join the ranks of the more than 25 million people who have already died. CONCLUSIONS With the magnitude of the current AIDS epidemic and the continued escalation in spread of HIV infection, it is evident that control and prevention of AIDS will require a sustained long-term commitment (24, 39). Research is still urgently needed to define the size of the problem in different geographic areas through serosurveys of representative samples of the population. Research is needed to clarify the dynamics of transmission and the possible role of intercurrent infections or other cofactors in increasing the risk of infection or of disease. Additional research is also urgently needed to develop effective control programs, which will rely on new studies of patterns of sexual behavior and the evaluation of the efficacy of health education interventions. Access to therapy for the prevention and treatment of opportunistic infections and the provision of antiretroviral drugs will be critical to helping those already infected. The overall success of these national programs and the regional and global AIDS control and care efforts will depend on a unifying international political and societal commitment. REFERENCES 1. Adjorlolo-Johnson, G., K. M. De Cock, E. Ekpini, K. M. Vetter, T. Sibailly, K. Brattegaard, D. Yavo, R. Doorly, J. P. Whitaker, and L. Kestens. 1994. Prospective comparison of mother-to-child transmission of HIV-1 and HIV-2 in Abidjan, Ivory Coast. JAMA 272:462–466. 2. Agwale, S. M., C. Zeh, K. E. Robbins, L. Odama, A. Saekhou, A. Edubio, M. Njoku, N. Sani-Gwarzo, M. S. Gboun, F. Gao, M. Reitz, D. Hone, D. Pieniazek, C. Wambebe, and M. L. Kalish. 2002. Molecular surveillance of HIV-1 field strains in Nigeria in preparation for vaccine trials. Vaccine 20:2131–2139. 3. Alaeus, A. 2000. Significance of HIV-1 genetic subtypes. Scand. J. Infect. Dis. 32:455–463. 4. Barre-Sinoussi, F., J. C. Chermann, F. Rey, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868–871. 5. Blair, J. M., P. L. Fleming, and J. M. Karon. 2002. Trends in AIDS incidence and survival among racial/ethnic minority men who have sex with men, United States, 1990–1999. J. Acquir. Immune Defic. Syndr. 31:339–347. 6. Bollinger, R. C., S. P. Tripathy, and T. C. Quinn. 1995. The human immunodeficiency virus epidemic in India. Medicine (Baltimore) 74:97–106. 7. Brown, T., and W. Sittirai. 1995. The HIV/AIDS epidemic in Thailand: addressing the impact on children. In Asia-Pacific Population and Policy, No. 35. Honolulu, HI: East-West Center Program on Population. 8. Brown, T., W. Sittirai, S. Vanichseni, and U. Thisyakom. 1994. The recent epidemiology of HIV and AIDS in Thailand. AIDS 8(Suppl. 2):S131–S141. 9. Buve, A., K. Bishikwabo-Nsarhaza, and G. Mutangadura. 2002. The spread and effect of HIV-1 infection in sub-Saharan Africa. Lancet 359:2011–2017. 10. Calleja, J. M., N. Walker, P. Cuchi, S. Lazzari, P. D. Ghys, and F. Zacarias. 2002. Status of the HIV/AIDS epidemic and methods to monitor it in the Latin America and Caribbean region. AIDS 16(Suppl. 3):S3–S12. 11. Centers for Disease Control. 1983. Acquired immunodeficiency syndrome (AIDS) update—United States. Morb. Mortal. Wkly. Rep. 32:309. 12. Centers for Disease Control. 1981. Kaposi’s sarcoma and Pneumocystis pneumonia among homosexual men—New York City and California. Morb. Mortal. Wkly. Rep. 30:305.


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Chapter 6

One Multidisciplinary Approach to AIDS in Africa: the Academic Alliance for AIDS Care and Prevention in Africa W. Michael Scheld

Since the first descriptions of AIDS in 1981, nearly 67 million people have become infected with the human immunodeficiency virus (HIV) and about 25 million have died, eclipsing the “black death” of 14th-century Europe and the 1918–1919 influenza pandemic. The AIDS pandemic originated in sub-Saharan Africa, and this region has been hardest hit in its aftermath (see chapter 5). For example, according to estimates by UNAIDS, of the 42 million people living with HIV infection at the close of 2002, nearly 30 million reside in sub-Saharan Africa. AIDS is now the leading cause of death in this region. Life expectancy has plummeted such that seven nations in sub-Saharan Africa now have life expectancies of less than 40 years and this number will increase fully to 11 countries over the next 7 years. Such declines in life expectancies will soon reach levels that have not existed since the 19th century. In 2002, 2.4 million deaths in sub-Saharan Africa were attributed to AIDS and nearly 3.5 million people became newly infected. Although newer therapies, most prominently multidrug antiretroviral regimens such as highly active antiretroviral therapy (HAART), have been introduced in many developed countries, such is not the case in resource-limited settings. Only about 30,000 (0.1%) of the nearly 30 million infected Africans were receiving antiretroviral therapy in 2002, compared to at least 30% in some more developed regions. Furthermore, prevention and treatment of many opportunistic infections, as well as prevention of transmission of HIV from mother to infant, are woefully inadequate in many resource-limited settings. This inequity in care between Africa and more developed regions has been attributed to multiple factors, including a lack of infrastructure, poverty, lack of political will, insufficient laboratory support, political corruption, lack of financial support from developed countries, insufficient health care delivery systems, lack of training and knowledge among African physicians of the multiple complexities of the care of HIV/AIDS, and others. Like W. Michael Scheld • Department of Internal Medicine, Division of Infectious Diseases, University of Virginia, PO Box 801342, MR 4, Room 2118B, Charlottesville, VA 22908. W.M.S. wrote this chapter on behalf of the founding members of the Academic Alliance for AIDS Care and Prevention in Africa. 83


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many organizations, the Academic Alliance for AIDS Care and Prevention in Africa (Academic Alliance) was formed in response to these issues. The Academic Alliance is led by a group of outstanding physicians from Makerere University, the Infectious Diseases Society of America (IDSA), and HIV/AIDS care and prevention experts from Uganda and North America. THE GENESIS OF THE ACADEMIC ALLIANCE Two of the founding members of the Academic Alliance, Merle Sande and Michael Scheld, have been members of the Pfizer international advisory board on infectious diseases for at least a dozen years. The Pfizer advisory board is somewhat unusual among similar boards in that the highest-ranking executives of the company, including the chief executive officer, attend the sessions. This close interaction, as well as informal discussions, fosters collaborations and friendship between advisors and the Pfizer leadership. Over the years, the unfolding calamity of AIDS in Africa has been the focus of many discussions among this group. As a result, it became apparent that Pfizer leadership was committed to addressing the care and prevention of HIV/AIDS in resource-limited settings. Recognizing the importance of fungal infections in AIDS patients, particularly invasive candidiasis and cryptococcal meningitis, Pfizer attempted to donate fluconazole to the Republic of South Africa in the summer of 2000. This donation was initially refused. Somewhat confused by this rebuff, Pfizer leadership sought advice and opinion from the members of the international advisory board on infectious diseases. It was pointed out that drug donations alone are not sufficient. Without appropriate infrastructure for health care and drug distribution, as well as building capacity through training for the proper use of the donated materials, such a donation would fall far short of its potential effects. Over the next several months, it became apparent that an approach emphasizing infrastructure development and capacity building to foster care and prevention might garner Pfizer support. The vision of the Academic Alliance is justly credited to Merle Sande, in concert with Nelson Sewankambo. Both recognized the collective strength of working together to accomplish mutual goals as the initial program took shape. In addition, the IDSA was approached to assist in the development of an AIDS training program. In January 2001, Merle Sande, Jerrold Ellner, and Thomas Quinn visited Kampala, Uganda, on a fact-finding mission to assist in the development of the program and a proposal for submission to Pfizer. Why Uganda? There are multiple reasons, including the following: • Political will. President Yoweri Museveni of Uganda recognized the enormous problem of AIDS in his country shortly after taking office. He created an office of AIDS in 1989, an early far-reaching vision, with direct responsibilities to the office of the president. • Concerted prevention efforts. Through multiple interventions, the seroprevalence of HIV infection in Uganda has decreased from one of the highest in the world at one time (25 to 30% in the late 1980s) to around 6 to 8% currently. This is in direct contrast to the trends in many other areas of sub-Saharan Africa. • Many of the members of the Academic Alliance from North America had previous experience in Uganda working on AIDS and related


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issues such as cryptococcal meningitis, tuberculosis, and sexually transmitted diseases (STDs) other than HIV infection. • Many of the most accomplished researchers on HIV/AIDS and its complications are members of the faculty of Makerere University. It became apparent that the infrastructure at Makerere University was insufficient for the conduct of the multiple goals and objectives of the fledgling program. This required the construction of new facilities. Furthermore, an approach based on capacity building, prevention, and care, with state-of-the-art laboratory support, was endorsed by all parties. In March 2001, a proposal for the establishment and financing of the Academic Alliance was presented by the five founding North American members to the Pfizer leadership. This proposal was eventually accepted, with funding from Pfizer of nearly $11.5 million for the first 2.5 years of the program. This initial funding is referred to as a “challenge grant,” and much of the creative energies of the Academic Alliance members has been focused on obtaining additional funding to ensure sustainability of the various programs beyond the initial grant period. The founding members of the Academic Alliance are listed in Table 1. They include the following. Merle Sande is the former chair of the department of medicine at the San Francisco General Hospital and of the department of medicine at the University of Utah School of Medicine. Sande has been intimately involved with the AIDS epidemic since 1980 and developed a clinical research program in Kampala focusing on cryptococcal meningitis. Sande serves as codirector of the Academic Alliance with Nelson Sewankambo, dean of the faculty of medicine at the Makerere University School of Medicine. Sewankambo has been a pioneer in research on HIV and AIDS in Uganda. Other North American members of the Academic Alliance include Jerrold Ellner, currently chair of medicine at the New Jersey School of Medicine; Allan Ronald, former chair of medicine at the University of Manitoba School of Medicine and founder of the Nairobi STD/AIDS Research Center in Kenya; Thomas Quinn, senior investigator of the National Institute of Allergy and Infectious Diseases and professor of medicine at the Johns Hopkins School of Medicine; and Michael Scheld, professor of medicine at the University of Virginia School of Medicine and currently president of the IDSA. The other Ugandan founding members include Moses Kamya, senior lecturer of Makerere University Medical School; Elly Katabira, associate dean of the Makerere University Medical School; Edward Katongole-Mbidde, professor of medicine and director of the Ugandan Cancer Center; Harriet Mayanja-Kizza, Table 1. Founding members of the Academic Alliance for AIDS Care and Prevention in Africa North America

Uganda

Merle Sande (codirector) Jerrold Ellner Thomas Quinn Allan Ronald Michael Scheld

Nelson Sewankambo (codirector) Moses Kamya Elly Katabira Edward Katongole-Mbidde Harriet Mayanja-Kizza Roy Mugerwa Philippa Musoke David Serwadda Fred Wabwire-Mangen


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chair of the department of medicine at the Makerere University Medical School; Roy Mugerwa, former chair of medicine at the Makerere University Medical School; Philippa Musoke, chair of the department of pediatrics at the Makerere University Medical School; David Serwadda, associate professor in the Institute of Public Health at Makerere University; and Fred Wabwire-Mangen, director of the Institute of Public Health at Makerere University. Several other physicians have been invited to join the group since its founding, including Peter Mugyenyi of the Joint Clinical Research Center in Kampala, one of the few facilities administering antiretrovirals in this setting, and Catherine Wilfert, professor emeritus of Duke University and scientific director of the Elizabeth Glaser Pediatric AIDS Foundation. The Academic Alliance also has been influenced greatly in the early development of its programs by many others. Although it is not possible to recognize all of these important contributions in this chapter, it must be stressed that the financial aspects of the Academic Alliance are managed by the Pangaea Global AIDS Foundation based in San Francisco, with Julia Martin and Chuck Wilson as “on the ground” administrators for programs and personnel and finance and operations, respectively; Stephen Labkoff of Pfizer for information technology; multiple trainers identified through the HIV Medicine Association of the IDSA; and other consultants such as John Bartlett of the Johns Hopkins University School of Medicine, David Bangsberg and Tom Coates of the University of California at San Francisco, and many more. MISSION AND CORE VALUES OF THE ACADEMIC ALLIANCE Mission The mission of the Academic Alliance is “to build capacity for delivering sustainable, high quality HIV/AIDS care and prevention for Africa through training and research.” The Academic Alliance was launched in 2001 to build capacity to fight the ongoing HIV/AIDS crisis in Uganda and elsewhere in Africa. This collaboration represents a true partnership of senior academic physicians in North America and those at a renowned medical school in Africa (Makerere University) to improve care and prevent HIV/AIDS. The goal of the Alliance is to capitalize on the synergy between clinical scientists in North America and Uganda to create a clinical training and research center where patients with HIV/AIDS can receive high-quality, sustainable model care, and where clinically relevant scientific research can be used to answer important questions about HIV and AIDS in Africa. This work will be focused on designing clinical and prevention strategies that will be readily applicable to both rural and urban health centers throughout Africa. The Infectious Disease Institute (IDI) will facilitate attainment of these objectives. Core Values The creation of the Academic Alliance was predicated on the following: • Equity in health care: the conviction that effective, high-quality HIV/AIDS care and prevention can and should be available and accessible for everyone, in Africa as elsewhere. • Recognition of the inherent human quality of all people and that, as patients, they must be treated with dignity, respect, and sensitivity.


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• The value of bidirectional training with an exchange component so that young physicians from Africa and Western countries can be inspired and encouraged to commit to improving health in Africa. • Respect for the human rights for all patients and research subjects, e.g., voluntary participation in medical research, informed consent, privacy, and confidentiality. • Equal value and respect for the contributions and responsibilities of all partners from both North America and Africa. In addition, certain principles guide the Academic Alliance, including the following: • The Academic Alliance is unique among HIV/AIDS programs in Africa, in that it is designed primarily to strengthen the existing academic structure in Uganda. • The Alliance will work within and enhance the current national HIV/AIDS programs in Uganda, including those of the Ministry of Health, the Ugandan AIDS Commission, and multiple other programs as described below under “Training Program.” • The Alliance will coordinate with the World Health Organization, UNAIDS, and other HIV/AIDS programs in Africa to ensure that efforts among all agencies are compatible and conflict free. • The Alliance is committed to a sound, transparent, fiscal infrastructure. • The activities of the Alliance will be monitored by, among others, a community advisory board and a scientific advisory board. THE INFECTIOUS DISEASE INSTITUTE Mulago Hospital is the national hospital of Uganda and one of the finest teaching hospitals in Africa. Although the daily census exceeds 1,000 patients, the clinical facilities were considered inadequate by our Ugandan colleagues for undertaking an expansive clinical program such as envisioned by the Academic Alliance. Thus, a new facility, the IDI, is being constructed on property donated by the Makerere University School of Medicine adjacent to Mulago Hospital. This is the first new building at the faculty of medicine of Makerere University in 35 years, and it will be the “home” of the Academic Alliance. Given the complexity of the project (nearly $5 million total cost), it is perhaps not surprising that planning and construction of the facility have taken longer than originally envisioned by the founding members of the Academic Alliance. Nevertheless, the IDI is under construction and should open in early 2004. This new facility will be approximately 32,000 square feet on two floors. The first floor is dedicated to patient care, with examining rooms, counseling areas, pharmacy, treatment rooms, waiting areas, children’s play area, prevention services, clinical research data storage, staff preparation areas, and a computerized data collection system. The second floor encompasses a state-of-the-art laboratory, including hematology, flow cytometry, chemistry, microbiology, molecular diagnostic assays, a computerized data center, a freezer repository, and a P3 laboratory for specialized research projects. In addition, the second floor will contain a training center unique to Africa that includes telemedicine, satellite communication, faculty offices, trainee study


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areas, library, conference and classroom facilities, administration, and offices for visiting faculty and fellows undertaking clinical and research endeavors. CLINICAL CARE PROGRAM An integral part of the core mission of the Academic Alliance is to enhance HIV/AIDS care in resource-limited settings. This will include prevention and therapy of opportunistic infections and antiretroviral therapy. Before 2001, the Mulago AIDS Clinic was operational only 1 day a week. Through assistance from the members of the Academic Alliance and the funding from the challenge grant from Pfizer, new space was identified and renovated, and the AIDS clinic is now operational 3 days a week. Approximately 250 to 300 patients are seen weekly. Pharmacy services and laboratory infrastructure have been expanded dramatically. The Academic Alliance has devised a formulary, and basic antimicrobial agents are now available (e.g., trimethoprim-sulfamethoxazole). In addition, through the fluconazole donation program, more than 150 patients with cryptococcal meningitis are now receiving this drug. Without this intervention, all of these patients would have died by now. Within the next year, and with the opening of the IDI, the clinic will expand to 5 days a week and approximately 300 patients will be seen daily. At this time, only about 300 patients of the more than 2,000 evaluated in the past year through the AIDS clinic staff by the Academic Alliance and Ugandan physicians are receiving HAART. Some patients, but very few in number, can afford triple antiretroviral therapy and have been placed on treatment if they meet current World Health Organization criteria. Funds for the purchase of medicines have been made available at a very low level to date, through the Global Fund for AIDS, tuberculosis, and malaria and the Map 1 allocations from the World Bank. In the near future, many more patients will be placed on HAART with drugs obtained through these funds and as an integral component of clinical research protocols. Our goal is to have more than 3,000 patients on HAART within the next 12 months. Although the goal is modest, the reality is that pharmaceutical companies are unwilling to make donations of antiretroviral agents at this time for such an open-ended commitment (i.e., lifetime treatments for the affected individual). Nevertheless, the Academic Alliance clinical care program will serve as a model for the provision of HIV care within the context of prevention and overall care in a resource-limited setting such as is found in the complex milieu of urban Africa. The Academic Alliance has also partnered with several other settings, including the Joint Clinical Research Center in Kampala and the AIDS support organization (TASO), the largest nongovernment organization in Africa. TASO is headquartered in Kampala but also operates six other clinics throughout southern Uganda. Over 60,000 patients are seen and cared for in these seven clinics. TASO provides state-of-the-art counseling to patients with HIV and AIDS and operates an extensive home care program. The Academic Alliance will provide medicines and laboratory testing for patients throughout the TASO network. As with the other programs of the Academic Alliance, a committee has been formed to oversee all clinical operations. The cochairs of this committee are Elly Katabira and Allan Ronald. PREVENTION PROGRAM Prevention is an integral mission of the Academic Alliance. The Academic Alliance believes that treatment of HIV/AIDS, prevention, and overall care of the patient (e.g.,


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prevention and therapy of opportunistic infections) must be delivered in concert for optimal care. The prevention program is under development through the Public Health Institute of Makerere University in cooperation with the Academic Alliance and other partners. The primary objective of the prevention program until the present has been to provide intensive counseling to HIV-infected patients. TASO counselors are already working within the Mulago AIDS Clinic in concert with the Academic Alliance members and Ugandan physicians to provide this service. Eight to 10 counseling rooms are planned for the new IDI, and this practice will continue in the future. Uganda has made enormous progress in prevention efforts, with a reduction in seroprevalence unmatched in Africa over the past decade. The prevention program for the general public has focused on the “ABCs” as follows: A is for abstinence, B is for being faithful to your partner, and C is for condoms. These prevention efforts are being expanded to the Mulago AIDS cohort family members and others to prevent subsequent transmission, both through education and counseling and through reducing viral load on HAART. The Academic Alliance is also working closely with the Elizabeth Glaser Pediatric AIDS Foundation and other partners to enhance reduction in mother-to-child transmission of HIV, including optimal care of pregnant women. Nevirapine is provided at the time of delivery, and both the mother and the child are subsequently treated following delivery. As observed in several other settings (e.g., men who have sex with men in San Francisco), HAART therapy may lead to changes in behavior that may actually cause an increase in the incidence of STDs, including HIV infection. The perception that treatment either is better than prevention or removes the need for aggressive prevention efforts is a real threat to the control of the HIV epidemic. The Academic Alliance believes that the introduction of antiretrovirals in the developing world will mitigate a host of issues that are certain to cause a rise in HIV incidence if the new definition of prevention and a new prevention program are not implemented alongside improved access to antiretrovirals. To meet this need, the design and implementation of a complete prevention program by the Academic Alliance and the Institute of Public Health are a proactive step in ensuring that antiretrovirals and other HIV treatments do not supersede HIV prevention but rather become a part of an overall prevention strategy. This enhanced and more complete prevention program has received a considerable boost by funding of a feasibility grant from the Bill and Melinda Gates Foundation (approximately $3.5 million over 2 years) to the Institute of Public Health at Makerere University and the Academic Alliance. There are seven major overall objectives of this HIV prevention program of the Academic Alliance: HIV prevention messages and information • to develop HIV/AIDS messages and information campaigns in relation to increased availability of antiretrovirals based on the results of a Ugandan knowledge, attitude and behavior study • to determine the feasibility of the development, design, and operation of an AIDS treatment information center for physicians and other health care providers in resource-limited settings Interventions in the clinical setting • to develop a medical inpatient HIV voluntary counseling and testing program integrated into the clinical and support services delivered through the IDI and other partner nongovernment organizations


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• to establish the feasibility and validity of adherence measurement strategies for women enrolled in maternal-to-child HIV transmission plus programs • to develop clinical algorithms and innovative laboratory techniques appropriate for developing countries for the initiation and monitoring of antiretroviral therapy Evaluative measures • to establish and implement a behavioral surveillance system to evaluate HIV prevention initiatives in relation to the provision of enhanced care and treatment including antiretrovirals • to conduct serial, cross-sectional surveys of HIV/AIDS knowledge, attitudes, and behaviors in the context of the availability of enhanced HIV treatments including antiretrovirals. Through these endeavors, the Academic Alliance and partners will ensure that prevention remains paramount in the setting of enhanced care and treatment. The prevention committee of the Academic Alliance is chaired by Fred Wabwire-Mangen, who also serves as the principal investigator of the Gates Foundation project. TRAINING PROGRAM The mission of the Academic Alliance includes building capacity for enhanced care, treatment, and prevention of HIV/AIDS in the resource-limited setting, and this aspect received considerable attention in the early efforts of the Academic Alliance. The primary purpose of the overall training program is to provide educational modules that address the care, treatment, and prevention of HIV/AIDS, opportunistic infections, and related infectious diseases for physicians, nurses, physician assistants, other health care providers, pharmacists, behavioral scientists, and clinical investigators. This overall program will be the first continuously run training effort focusing on the training of health care providers in the care of HIV/AIDS in Africa. Several aspects of the overall training program are still under development, but two are already operational under the guidance of the Academic Alliance. These programs focus on the training of physicians; other programs designed for the training of nonphysicians are under development. The physician training program is described in more detail below. Goal The goal of the physician training program is to build the capacity of African medical doctors to provide comprehensive care and prevention services, including the effective delivery of antiretroviral therapy, to both adults and children living with HIV/AIDS. Objectives The major objectives of the physician training program are as follows: • to establish an HIV/AIDS care, treatment, and prevention curriculum for medical doctors that is tailored to the clinical setting in sub-Saharan Africa


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• to provide quality didactic and clinical instruction in the use of antiretroviral therapy and delivery of HIV/AIDS care and prevention • to provide a training environment and mentoring opportunities to promote the highest standards of ethical care • to develop a means of disseminating new scientific and clinical information to graduate trainees • to develop a means of evaluating the utility and applicability of the training program, including the curriculum, the program format, and the clinical experience. Trainee Selection Prospective trainees are required to have a degree in the medical field (M.B., Ch.B., M.D., M.Med., or an equivalent medical degree) and must be registered, or be eligible for registration, with the Uganda Medical and Dental Council or similar councils in other African countries. Calls for applications for this training program are made through newspaper advertisements and general postings in various universities, schools of medicine, national hospitals, ministries of health, and embassies in sub-Saharan Africa. In addition, the call for applications is posted on the Academic Alliance website where individuals can access the application form and other information pertaining to this training program. The application process requires applicants to write a personal statement of interest about the training offered, along with how they anticipate applying knowledge acquired through the training and a description of previous related experience in HIV/AIDS care. A curriculum vitae and two references must be provided. A multinational selection committee of representatives from Makerere University Medical School, the Ministry of Health, AIDS care organizations, the regional AIDS Training Network, other African universities, and the Academic Alliance training program committee reviews all applications. To date, approximately 10 to 20 applications have been received for every trainee position available. Trainer Selection Much of the training is conducted by Ugandan physicians, members of the Academic Alliance, and other consultants. Furthermore, some lectures are delivered by expatriates living in Kampala and the region. To supplement these considerable resources, additional trainers are identified through the ranks of the HIV Medicine Association of the IDSA. The application and related materials are posted on the AIDS training program section of the IDSA website. Calls for trainer applications are made periodically. The trainer application also requires personal statements on HIV/AIDS training experience and work in a resource-limited setting and how the prospective trainer views the impact of the training program on his or her professional life. A trainer selection committee, chaired by Michael Scheld, consists of four other North American physicians who are not members of the Academic Alliance and two Ugandan physicians. To date, nearly 10 applications have been received for every trainer position available. Previous trainers have come from the United States and Canada. Curriculum, Training Structure, and Teaching Methods Didactic training encompasses about 50% of this physician training curriculum and includes standard lectures, telemedicine presentations, small group discussions, seminars,


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case studies, journal club discussions, interactive sessions, and active web-based learning with actual cases through Internet access. Every trainee receives a course binder that includes all slides from the lectures delivered and copies of journal articles discussed. Trainees also receive books that have been donated to the Academic Alliance. Clinical experience comprises the other 50% of the training program and is conducted in both inpatient and outpatient settings; it includes bedside teaching rounds, an overview of a systematic approach to HIV/AIDS care and management, home visitation for AIDS palliation, and exposure to community outreach programs in both HIV/AIDS care and prevention. Trainees are also provided with opportunities to enhance their computer skills through tutorials and basic computer literacy and Internet searches. Students are also required to use newfound or enhanced computer skills in the completion of a project that each trainee presents at the end of the training program. This training program is comprehensive and includes topics such as global epidemiology of AIDS and the impact of AIDS in Africa; molecular biology and immunology; clinical epidemiology; HIV virology, including a discussion of receptors, coreceptors, genetics, and pathogenesis; the natural history of HIV and AIDS; classification of HIV and AIDS; manifestations of HIV by organ system; prevention and management of opportunistic infections; laboratory diagnosis of HIV and AIDS; syndromic approach to patients with HIV and AIDS; antiretroviral therapy; mental health, home-based care, hospice, and nutritional issues; mother-to-child transmission; pediatric issues; voluntary counseling and testing; research priorities in a resource-limited setting; ethical issues; interaction of HIV/AIDS with other infections, particularly tuberculosis and malaria; and various aspects of prevention not covered above. A sample outline of the topics covered in the first week of the 4-week curriculum is shown in Fig. 1. The curriculum is constantly updated and refined on the basis of evaluations of the trainees, the trainers, and consultants to the Academic Alliance. Partner Sites All trainees rotate through different clinical settings and community organizations to ensure a wide range of exposure to the clinical management of HIV/AIDS. These sites include the Mulago adult and pediatric infectious diseases clinics, the Mulago medical wards, the Joint Clinical Research Center, Mildmay International (a United Kingdomfunded facility providing care, including antiretroviral therapy, to children with HIV/AIDS), the Makerere University-Johns Hopkins laboratory, the AIDS information center, TASO clinics, Hospice Uganda, and Nsambya Home Care. Trainees to Date Due to space constraints, which will be alleviated considerably with the opening of the IDI, only about 10 to 12 trainees can be accommodated each month of this physician training program. Since April 2002, when the physician training program began operations, seven months of training have been offered. As of April 2003, 86 physicians have completed the program and have come from Uganda, Kenya, Tanzania, Nigeria, Zambia, South Africa, Namibia, Botswana, Zimbabwe, and Rwanda. This is a “train-the-trainers” program, and it is anticipated that these individuals will continue to train others.

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HIV/AIDS Impact in Africa & Global Epidemiology Introduction to Molecular Biology & Immunology (10:30-11:30) Introduction to Computers (11:30-1:00)

Lunch Pretest (2:00-3:00)

Clinical Epidemiology 1 (3:00-4:00) Welcome tea party in Dean’s Boardroom (4:30)

9:05-10:05

10:35-1:00

1:00-2:00 2:05-3:05

3:20-4:30

Lunch HIV Pathogenesis CD4, CD8, dendritic, cytokines, natural killer, B cells Clinical Epidemiology 2

Computers (11:30-1:00)

HIV: Receptors; co-receptors; genetics

HIV: Virology

Tuesday

Computer Literature Searches; Introduction to Scientific Articles & Group Health Care Projects

Lunch Clinical Presentation & Classification of HIV in Children

Mulago IDC (5 trainees) Medical Ward (6 trainees)

Natural History of HIV and HIV/AIDS Classification Introduction to Principles and Antiretroviral Therapy (ARVT) General Approach to Patients with HIV/AIDS

Wednesday

Case Studies & Discussions (Cancer and Neurology) (2:00-3:30)

Lunch HIV: Renal, cardio, endocrine and ophthalmology

Manifestations of HIV: AIDS-Related Malignancies Manifestations of HIV: Gastrointestinal; Hepatobiliary; Enteric Pathogens Clinical Teaching: Cancer Ward

Thursday

Review of weekly take-home exam (3:45-4:30)

Lunch Clinical Epidemiology 3

Dermatology Clinic (All trainees) (10:30-1:00)

Manifestations of HIV: Dermatologic

Manifestations of HIV: Pulmonary

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Figure 1. Example of a template for the first week of a 4-week AIDS training program in Kampala, a joint effort of the IDSA and the Academic Alliance. IDC, infectious disease clinic.

Introduction: Course and Logistics

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Course Examinations and Program Evaluation To evaluate the progress made by trainees at the completion of the session, a pretest is given to all trainees at the start of the session and is repeated on the final day of the session. The training committee uses the results of the posttest to measure whether the learning objectives have been met for each trainee. The trainees also are required to complete a weekly take-home examination of at least 45 questions drawn from specific topics covered during that week. For this exercise, trainees are encouraged to work together and to use resources provided during the session. Each trainee must complete a project through Internet searches and make a presentation of his or her findings in the last week of training. Finally, the trainees complete a 40-question proctored exam to establish individual knowledge levels of the course material at the conclusion of the session. All of these examinations are used by the training committee as tools for evaluating the overall training program. In addition to the examinations, weekly evaluations are distributed to all trainees for completion. All trainees also complete an overall evaluation, which provides greater insight into whether the objectives of the training program have been met. On the last day of the session, an interactive debriefing session is held between trainers and trainees to solicit additional feedback on the training program. All of these tools are used to evaluate and modify the program. Program Management The Academic Alliance training programs are codirected by Moses Kamya and Michael Scheld. In addition, the Academic Alliance has established and entrusted a training committee with planning, implementing, monitoring, and evaluating the training program. In addition to several members of the Academic Alliance, this training committee includes other Ugandan physicians, a programs and personnel administrator, and a training coordinator. Continuing Education and the AIDS Treatment Information Center (ATIC) Education is lifelong. Although the physician training program of the Academic Alliance provides trainees with considerable information and facilitates dissemination of this information to others, feedback on the course to refine development of future training sessions is essential. Trainees are invited back to Kampala on a regular basis to provide feedback on the course material relevant to their own experience in the field. In addition, shorter “refresher” courses extending over a weekend are planned for the future to accommodate former trainees on a regular basis. Furthermore, as HIV/AIDS care, treatment, and prevention are a rapidly changing area of clinical practice, the ATIC, a partnership among the Academic Alliance, Makerere University, Mulago Hospital, the Ugandan Ministry of Health, the Ugandan AIDS Commission, and the National Medicines Information Center based at Trinity College/St. James Hospital, Dublin, is currently under development. The ATIC is intended to be a facility linked by phone, e-mail, fax, and regular mail to health care providers throughout sub-Saharan Africa. The key purpose of the ATIC is to provide up-to-date comprehensive and relevant answers to treatment and information inquiries in a timely, efficient, and culturally sensitive manner. Questions can be submitted 24 h a day, 7 days a week, and the ATIC will be staffed by clinical pharmacists and physicians with expertise in HIV/AIDS care to respond to those inquiries. In addition, a quarterly news-


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letter will be developed and distributed to graduates of the Academic Alliance training programs, HIV/AIDS care providers, and health care facilities throughout Africa that focuses on recent advances in HIV/AIDS care, treatment, and prevention. The ATIC and the newsletter will be poised to collect and disseminate postmarketing drug surveillance data on adverse drug reactions and drug interactions that may arise in the African setting. Postdoctoral Fellowship in Infectious Diseases Only a small number of African physicians obtain fellowships in Europe or North America and receive quality training. The training is often inappropriate for the requirements of academic practice in African countries and applicability is limited. In addition, the majority of the individuals who receive advanced training outside Africa are recruited to positions in the countries in which they trained, thus contributing to the “brain drain.” As a result, their skills and leadership potential are lost to Africa. Although infectious diseases is not a recognized area of specialization in most African countries, this is particularly unfortunate in a setting in which HIV/AIDS has emerged as an overwhelmingly clinical and public health issue and in which at least 80% of illness and death are due to infections. To alter these statistics in the long term, clinicians and scientists must receive advanced Africa-appropriate training in infectious diseases. The Academic Alliance has developed a program to train the future leaders in infectious diseases in Africa. This fellowship includes the following elements over 3 years: clinical experience with infectious diseases with a particular emphasis on HIV/AIDS, laboratory skill development with an emphasis on microbiology, exposure to public health and preventive medicine, completion of a research project, and development of expertise in pharmacological and drug usage and information technology. Although a portion of this training may take place outside Africa, the majority of the clinical experience and research is conducted at Makerere University. One fellow has already been recruited into the existing Academic Alliance fellowship program. Other Training Programs As the above training programs become established and mature, members of the Academic Alliance are in the early stages of planning other training programs and have submitted proposals for support to multiple organizations, including the World Bank, the National Institute of Allergy and Infectious Diseases, the Fogarty International Center, and the Doris Duke Foundation, among others. These training programs include the following: • Health care implementer training program. This program is being developed for health care providers that include nurses, clinical officers, counselors, pharmacists, dispensers, and laboratory technicians. The purpose of the program will be to provide these groups of health care providers with an opportunity to strengthen their practical skills in the delivery of HIV/AIDS care, treatment, and prevention, including opportunistic infections. • Topical cluster training program. Health care is optimally provided through a team approach. The skill sets of physicians, nurses, laboratory technologists, and pharmacists/dispensers are all different and necessary


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to deliver comprehensive HIV/AIDS care. The purpose of the topical cluster training is to deliver short courses on specific topics to teams of health care providers. Although this may be developed in Kampala, ideally, curricula will be developed in key topical areas and training conducted in select regions of Uganda and neighboring countries. • Clinical pharmacy graduate program. The Academic Alliance proposes to collaborate with the school of pharmacy to develop a 2-year M.Sc. program at Makerere University for pharmacists from African countries, focusing on infectious diseases, particularly HIV/AIDS. The course subjects will include practice of clinical pharmacy, therapeutics with emphasis on the management of infectious diseases, pharmacokinetics and pharmacodynamics, aseptic drug delivery, patient counseling skills, pharmacoeconomics, and presentation and teaching skills. • Satellite training centers. Building on the experience of the physician training program described above, the Academic Alliance is exploring options for developing satellite training centers in neighboring countries. The Academic Alliance is hopeful that a proposal submitted to the World Bank to develop an African Regional Educational Center for HIV/AIDS care, treatment, and prevention will facilitate development of satellite training centers in Ethiopia, Eritrea, and Kenya. OPERATIONAL RESEARCH As with building capacity, the Academic Alliance has not waited for completion of the IDI to initiate operational research. Nevertheless, by integrating a new clinical care program with a computer database system, clinician researchers will be able to address a wide variety of research questions. The members of the Academic Alliance have interacted extensively with other clinician investigators and representatives from the pharmaceutical industry in the discussion of various protocols for implementation at the IDI, Makerere University, and the Institute of Public Health. Some of the questions being considered include the following: • When should one start therapy in an African setting? Can the clinician use clinical criteria such as wasting, diarrhea, and thrush for initiating, or should one use inexpensive laboratory tests such as a total lymphocyte count or a rapid simple CD4 count? • How does one identify and select individuals for therapy who are HIV infected? What parameters, including socioeconomic status, family support, previous history, behavioral characteristics, and others, allow one to identify patients who will achieve the best response? • Which antiretroviral regimens of the many now available are the best combinations to use as the “standard regimens” with the greatest effectiveness and lowest number of adverse events? • What measures can be used to identify issues relating to adherence and compliance? What is the role of directly observed therapy administered or supervised by family members?


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• What is the role of structured treatment interruption in this resourcelimited setting? • What is the specific role of antiretroviral therapy in preventing transmission to sexual partners or within high-risk groups such as sex workers? • What are the pharmacokinetics of antiretroviral agents in patients living in Uganda or the region? Are there interactions with illness, other infectious diseases, food, diarrhea, or customs that need to be investigated? • How commonly will resistance emerge, and how can it be most readily detected? Which regimens should be used when resistance occurs? • What is the role of various agents, currently just becoming available in Uganda, in the prevention and treatment of opportunistic infections? What treatment algorithms for opportunistic infections need to be developed for sub-Saharan Africa? Use of antimicrobials for the prevention and treatment of opportunistic infections may affect resistance of other endemic pathogens. Which ones would be so affected? • What are some of the interactions between HIV and malaria or tuberculosis, both of which are highly endemic in this region? • How do prevention and care interact in ways that ensure that the focus and priority of the country, its media, and its societies remain on prevention rather than care? How can we measure this effect? • What new aspects of prevention must be developed as antiretroviral therapy is introduced into a resource-limited setting? (For example, multiple prevention efforts are currently under way by the Academic Alliance with support from the Gates Foundation.) It is anticipated that the Academic Alliance will address some, but not all, of these questions and develop and validate the most clinically and cost-effective guidelines for the care of patients with HIV/AIDS in Africa; conduct clinical trials to adapt new drugs and protocols for Africa; define the critical factors that influence the spread of HIV/AIDS in Africa and develop effective prevention strategies; answer basic biologic questions that are applicable to HIV/AIDS in Africa; disseminate research findings through workshops, conferences, publications, and other scientific fora; and evaluate new and novel diagnostic and HIV monitoring assays that are appropriate and cost-effective for Africa, among other issues. The Academic Alliance has responded to a request for proposals for the program titled Comprehensive International Programs on Research on AIDS of the National Institute of Allergy and Infectious Diseases. This proposal contains five projects and three cores and is analogous to a “program project” or PO1 award. The projects include development and validation of an algorithm for the approach to central nervous system manifestations of HIV/AIDS, the development of treatment algorithms for other opportunistic infections in patients with HIV/AIDS, investigation of the causes and therapy of chronic diarrhea in children with AIDS, testing methods for validation of adherence to antiretroviral therapy in the resource-limited setting, and AIDS vaccine efficacy, among others. Other proposals have been submitted to the Doris Duke Foundation, Becton Dickinson, and other pharmaceutical companies to support operational research conducted by the


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Academic Alliance and its partners. For example, some angiogenesis factors appear to be down-regulated by HIV protease inhibitors. Thus, would a triple drug regimen including a protease inhibitor slow or arrest the progression of Kaposi’s sarcoma when compared to a triple drug regimen not containing a protease inhibitor? It is worth noting that over 600 new patients with Kaposi’s sarcoma are evaluated at the Makerere Cancer Center annually. The overall research program of the Academic Alliance is directed by Roy Mugerwa and Jerrold Ellner. Edward Mbidde is chair of the research committee. The research committee reviews all protocols prior to submission to the local investigational review board. In addition, the research committee has constructed a research credo on ethics of operational research conducted in a resource-poor setting. King Holmes of the University of Washington is chair of the Scientific Advisory Board of the Academic Alliance. This board reviews all protocols, research direction, and coordination of research efforts of the Academic Alliance and offers advice, criticism, and direction. LABORATORY PROGRAM The primary purpose of the laboratory is to provide virologic, immunologic, and microbiologic laboratory support for the care of HIV-infected patients. Once the IDI is completed, the currently functional Makerere University-Johns Hopkins University laboratory will be consolidated into the new facility. The laboratory will be responsible for assessing blood specimens and processing and performing laboratory tests, with reports and results sent directly to a central database and fully integrated into the clinical record of each patient. The laboratory will provide clinical laboratory and diagnostic assay training for visiting scientists, clinicians, and laboratory technologists from Uganda and the region. In addition, laboratory space has been dedicated to the performance of translational research, which has immediate clinical applications, such as antiretroviral resistance testing, clade/subtype identification, development of new rapid and inexpensive diagnostic tests, and molecular amplification assays for HIV and other STDs. The laboratory will serve as a critical asset for all of sub-Saharan Africa. Furthermore, various aspects of the laboratory operation have been facilitated by donations, e.g., instruments from Becton Dickinson, rapid HIV tests from Abbott, and PCR reagents for viral load determination at a fraction of the current costs in the developed world by Roche, as well as many others. FUTURE PLANS The Academic Alliance for AIDS Care and Prevention in Africa is a collaboration among North American physicians and Ugandan counterparts with a comprehensive mission as outlined above. Programs in building capacity (training), care, treatment, prevention, laboratory support, research, and outreach have been the focus of the Academic Alliance. The Academic Alliance enjoys the firm support from the highest levels of government and the Ministry of Health, including endorsement by President and First Lady Museveni of Uganda. The Academic Alliance also expects future support from Pfizer, after the initial 2.5 years of grant support, for operations described above in Kampala and elsewhere in the region. Multiple grant proposals have been submitted over the past 2 years by members of the Academic Alliance, including those to the Gates Foundation (described above under “Prevention Program”), the World Bank (see “Training Program”


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above), the Doris Duke Foundation, the National Institute of Allergy and Infectious Diseases, the Fogarty International Center, the Semnani Foundation, the National Cancer Institute, the United States Agency for International Development, the Health Resources and Services Administration, UNAIDS-Global Fund, the Bechtol Foundation, the Elizabeth Glaser Pediatric AIDS Foundation, the Rockefeller Foundation, the Thrasher Foundation, and multiple corporate entities supplying books, training materials, information technology (valued at over $1 million), diagnostics, laboratory equipment, Internet and website, architectural costs and development of the IDI, legal services, transportation vehicles, medical equipment (e.g., X-rays and a potential computed tomography scanner), software, used lab equipment and furniture, and medications, among many others. Thus, the initial Pfizer challenge grant has provided an enormous multiplier effect that will contribute to enhanced care, treatment, and prevention of HIV/AIDS in sub-Saharan Africa. Much remains to be done, and the Academic Alliance is only one of several noteworthy initiatives to combat the problem of HIV and AIDS in resource-limited settings. Nevertheless, there are ample opportunities to participate in this initiative. Members of the HIV Medicine Association of the IDSA are encouraged to apply for trainer positions in the AIDS training program in Kampala. Furthermore, on the basis of the extensive operational research agenda, many other clinician investigators should express interest in collaborative projects with the Academic Alliance. Several medical students, residents, and fellows have already visited the Mulago Hospital under the auspices of various members of the Academic Alliance and more are expected to do so in the future. Structured electives for medical students, residents, fellows, and faculty are in development. Conversely, African physicians have already completed training in the programs described above and many more will undoubtedly do so in the future. The fellowship training program is just beginning, but many African physicians, the future leaders of infectious diseases and academic medicine in Africa, will benefit from the program and complementary electives in institutions in Europe and North America. Partners will be invaluable in this exercise. Other examples of cross-disciplinary collaboration abound. The Academic Alliance is a multidisciplinary collaboration, but only a start. Individuals who may be interested in assisting the Academic Alliance in its mission can contact the author of this chapter.


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Chapter 7

Enteroaggregative Escherichia coli James P. Nataro

Escherichia coli has been recognized as an enteric pathogen for more than a century (17). However, recognition of enteric pathotypes has long been impeded by the difficulty in distinguishing pathogenic E. coli from the commensal flora. In 1979, Cravioto et al. (5) observed that most strains of E. coli belonging to the so-called enteropathogenic E. coli (EPEC) serotypes adhered to HEp-2 cells in cell culture. Subsequently, Scaletsky et al. (40) and Nataro et al. (27) showed that adherence to HEp-2 cells was widespread among E. coli strains and that three distinct adherence patterns could be discerned. The adherence pattern of EPEC isolates was described as “localized” adherence, denoting the presence of clusters or microcolonies on the surfaces of the HEp-2 cells (26). However, on the basis of the morphology of adherence, non-EPEC isolates could be divided into two further categories: aggregative and diffuse. Aggregative adherence (AA) was defined by prominent autoagglutination of the bacterial cells to each other; the defining characteristic of AA was, and remains, the characteristic layering of the bacteria in a “stacked brick” configuration (Fig. 1). In diffuse adherence, by contrast, bacteria are dispersed across the plasma membrane of the epithelial cells, with little aggregation and little adherence to the glass coverslip free from the cells. Thus, enteroaggregative E. coli (EAEC) is defined as E. coli which does not secrete heat-labile enterotoxin or heat-stable enterotoxin and which adheres to HEp-2 cells in an AA pattern (25). It is likely that this definition encompasses both pathogenic and nonpathogenic clones, which share a common phenotype. It remains controversial whether all EAEC isolates have any factors in common contributing to the shared phenotype, and as discussed below, recent evidence argues against the existence of any common factor. Much research on EAEC involves the identification of virulence factors that will define truly virulent strains. In view of these questions, one of the early priorities of research on EAEC was to identify truly pathogenic strains to serve as prototypes for virulence studies. Identification of such a strain, as well as description of the clinical features of EAEC infection, was provided by volunteer studies (20, 29). In a study conducted by Nataro et al. of five volunteers James P. Nataro • Center for Vaccine Development, Departments of Pediatrics, Medicine, and Microbiology and Immunology, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore MD 21201. 101


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Figure 1. AA in the HEp-2 assay. EAEC is defined by the characteristic pattern of adherence after 3 h of incubation in the presence of viable HEp-2 cells. Bacteria adhere to the surfaces of the cells as well as to the glass substratum in a characteristic stacked brick phenotype.

fed 1010 EAEC strain 042 (O44:H18) organisms, three had stools that met the study definition of diarrhea (200 ml of loose stool) (23). Diarrhea was mucoid and green or brown in color and did not have blood or fecal leukocytes. Volunteers did not complain of fever or significant abdominal cramps. One volunteer who was fed strain 042 and who did not have stools that met the study definition of diarrhea nonetheless developed enteric symptoms, including low-volume liquid stools and borborygmi. Notably, of 15 volunteers fed other EAEC strains, none had stools that met the study definition of diarrhea and only 1 of the 15 had stools looser than normal. That study supported the hypothesis that the pathogenicity of EAEC is not uniform. Although volunteer studies suggest that most individuals with EAEC infections do not manifest signficant signs of mucosal inflammation, some studies suggest that EAEC diarrhea may be mildly inflammatory in nature (43, 44). Steiner et al. (43, 44) have found that lactoferrin can commonly be demonstrated in the stools of patients with EAEC infection. Indeed, even asymptomatically infected children reveal fecal lactoferrin levels higher than those in controls, and such children may exhibit significant growth retardation. Thus, although most studies of EAEC focus on diarrhea as the relevant clinical outcome, some evidence suggests that colonization without diarrhea may be an important clinical entity. More such studies are needed to confirm this hypothesis.


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Chapter 7 • Enteroaggregative E. coli

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EAEC EPIDEMIOLOGY The first reported outbreak of EAEC diarrhea occurred in Serbia, where 19 newborns in a nursery developed watery diarrhea (4). Three infants developed persistent diarrhea, but none died. No specific vehicle was implicated in the outbreak. Several other outbreaks have subsequently been reported, the largest of which involved nearly 2,700 children in Japan. These children developed watery diarrhea linked to consumption of a contaminated school lunch (15). Thirty children came to medical attention for persistent diarrhea, and the stools of each of these patients yielded a common EAEC strain. Several studies have implicated EAEC in endemic diarrhea in developing populations, most prominently in association with persistent diarrhea (14 days). In the first such study, Bhan et al. (3) performed weekly household surveillance of a cohort of 452 children (3 years of age) who lived in the rural village of Anapur-Palla in northern India. Fecal specimens were obtained from 240 children with episodes of diarrhea and from agematched asymptomatic controls in the same population. EAEC (defined by AA in the HEp-2 assay) isolates were found in the stools of 10 and 13% of the controls and the children with acute (14 days) diarrhea, respectively. By contrast, the stools of 30% of those with persistent diarrhea yielded EAEC isolates (P 0.0052 versus children with acute diarrhea and P 0.0006 versus controls). Patients with EAEC diarrhea had a mean duration of illness of 17.0 days; fever was present in 12.2% of the patients, and grossly bloody stools were present in 12.2%. Cravioto et al. (6) subsequently monitored a cohort of 75 infants and children under 2 years of age born in the Mexican village of Lugar Sobre la Tierra Blanca. EAEC strains were found in 29 (51%) of the 57 infants and children with diarrhea that persisted for 14 days. In contrast, EAEC strains were found in 49 (8%) of 579 infants and children with acute cases of diarrhea but in only 5 (5%) of 100 nondiarrheal controls (P 0.001). In that study, EAEC was the pathogen most frequently associated with bloody diarrhea: 33% of those with EAEC in their stools manifested gross blood. Guerrant and coworkers have consistently isolated EAEC at a high rate from impoverished children in Fortaleza, Brazil. Among the individuals in that population, Wanke et al. (50) found EAEC strains in 2 (5%) of 28 asymptomatic controls and 4 (8%) of 50 patients with acute diarrhea, but they found that the acute diarrhea persisted in 8 (20%) of 40 of the children for more than 2 weeks (P 0.05 for patients with persistent diarrhea versus patients with acute diarrhea and controls). EAEC was found in 18% of patients with persistent diarrhea when stools were cultured during the first 14 days of illness and 27% of such patients after 14 days. Working with the same population, Lima et al. (18) found EAEC in the stools of 10 (53%) of 19 patients with persistent diarrhea. Subsequent studies have implicated EAEC as an important endemic pathogen of pediatric diarrhea in many other countries of Asia, Africa, and South America (28). In those studies, EAEC has generally been associated with acute or persistent watery diarrhea, usually without blood or mucus. Children of any age may be afflicted, although breast-fed infants may be at least partially protected (35). Recently, EAEC has been implicated as an important agent of diarrhea in industrialized countries as well. In a large prospective study of adults and children with diarrhea in the United Kingdom, Tompkins et al. (45) found that EAEC was strongly associated with diarrheal illness and was the second most commonly implicated bacterial pathogen after


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Campylobacter. Importantly, in that study, EAEC was defined by positivity by an assay with an empirically derived DNA probe (see below) rather than by the more inclusive HEp-2 adherence assay. Using the same probe, Cohen (M. Cohen, unpublished data) has found that EAEC can be isolated from the stools of approximately 10% of U.S. infants presenting to a hospital, whereas it was isolated from less than 2% of an asymptomatic control group (P 0.05). The fact that these studies used a molecular approach to diagnostics lends greater credence to the hypothesis that the investigators have implicated a specific E. coli pathotype. EAEC IN TRAVELER’S DIARRHEA Evidence of a role for EAEC in traveler’s diarrhea (TD) was demonstrated by Glandt et al. (11). Three hundred fifty-four U.S. travelers to Jamaica or Mexico who developed TD were screened for “conventional” pathogens (including enterotoxigenic E. coli [ETEC], Shigella, Giardia, Entamoeba histolytica, and Campylobacter). Among patients in whom none of these pathogens were detected by screening, 29 of 64 (45.3%) excreted EAEC. Patients infected with EAEC had abdominal pain or cramps (89.7%), watery diarrhea (72.4%), urgency to defecate (86.2%), flatulence and nausea (72.4%), vomiting (24.1%), and tenesmus (20.7%). Only one patient reported a fever. Of the stool samples obtained from 26 EAEC-infected patients, 9 had fecal leukocytes, 10 had visible mucus, 6 had occult blood, and 2 had visible blood. Despite having a higher incidence of fecal leukocytes (50 versus 7.7%), patients infected with EAEC who received ciprofloxacin had a significantly shorter duration of diarrhea (35.3 versus 55.5 h; P 0.049). Adachi et al. (J. Adachi, M. Glandt, Z.-D. Jiang, J. Mathewson, R. Steffen, C. Ericsson, and H. DuPont, 37th Annu. Meet. Infect. Dis. Soc. Am., p. 23, 1999) examined 636 patients with TD for the presence of EAEC by HEp-2 adherence. Among travelers to Jamaica, Guadalajara, Mexico, and Goa, India, EAEC and ETEC were found in similar numbers (25.5 and 30.3%, respectively), with all other bacteria combined accounting for only 24% of isolates. The authors of the study estimated that EAEC could be the cause of 16.3 to 53.3% of otherwise unexplained cases of TD. Together, these studies strongly implicate EAEC as a frequent cause of TD, with a prevalence rivaling that of ETEC. EAEC IN HIV-INFECTED PATIENTS In 1995, Mayer and Wanke (22) isolated EAEC from a patient with AIDS and chronic diarrhea but identified no other pathogen; the patient responded to antibiotic therapy. Those investigators subsequently isolated EAEC from 30 of 68 patients with AIDSassociated diarrhea and from 18 of 60 without diarrhea (P 0.05) (21). The patients with EAEC infection and diarrhea had significantly lower CD4 counts than those with EAEC infection but no diarrhea (P 0.02). A double-blind, placebo-controlled crossover trial of ciprofloxacin versus placebo in this study population demonstrated improvement in diarrhea and intestinal symptoms in patients with human immunodeficiency virus (HIV) and EAEC infections when they were treated with antibiotics (49). The largest reported study of HEp-2-adherent E. coli in HIV-associated diarrhea involved 114 patients in Zambia, of whom 80 were HIV positive (19). HEp-2-adherent bacteria were significantly associated with HIV infection and diarrhea in that cohort.


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EAEC, diffuse adherent E. coli, and EPEC were identified in 36, 36, and 26% of HIVpositive patients with diarrhea, respectively. Of the 80 HIV-infected patients infected with EAEC, 32 underwent endoscopy because of chronic diarrhea. Eight patients manifested adherent bacteria in the colon, which was associated with a mixed inflammatory infiltrate, epithelial disruption, and mucus depletion. PATHOGENESIS OF EAEC INFECTION The basic strategy of EAEC infection appears to comprise colonization of small-bowel and large-bowel mucosal surfaces, followed by elaboration of enterotoxins (28). Studies with human intestinal explants from the jejunum and colon suggest that EAEC induces mild but significant mucosal damage (14). Both adherence and mucosal damage appear to be the most severe in colonic sections. Mild inflammatory changes are observed in animal models (46, 47), and evidence suggests that at least some EAEC strains may be capable of limited invasion of the mucosal surface (2). Mucosal Adherence EAEC colonizes the mucosal surface abundantly, and the bacteria that adhere to the mucosal surface have been likened to a biofilm (41). Indeed, the most dramatic histopathologic finding in infected animal models is the presence of a thick layer of bacteria adhering loosely to the mucosal surface. EAEC prototype strains adhere to HEp-2 cells and the intestinal mucosa by virtue of fimbrial structures termed AA fimbriae (AAF) (7, 24, 30, 31, 39), which are related to the Dr family of adhesins. These organelles exhibit a semiflexible bundle-forming structure. At least four allelic variants of AAF exist, but importantly, each is present in only a minority of strains (8); doubtless, more remain to be discovered. It is notable that the major pilin subunits of AAF are highly divergent, whereas the accessory genes required for assembly are more conserved. The conservation of accessory genes may provide a greater opportunity for the development of diagnostic reagents based on AAF and may also facilitate the discovery of new variants of AAF. It should be noted, however, that not all EAEC strains adhere by virtue of AAF. My laboratory has recently implicated a plasmid-borne fimbrial adhesin of the type IV class in one EAEC outbreak-associated strain (J. Nataro, unpublished data). This strain did not hybridize with any other virulence-associated genes implicated in other EAEC strains, suggesting that the strain defines a new subset of EAEC. Given that the strain is a proven pathogen, it may pave the way for the discovery of a new diarrheagenic pathotype. Sheikh et al. (42) have recently described a protein layer loosely associated with the surfaces of EAEC strains. This protein, called dispersin (encoded by the aap gene), appears to loosen the character of AA to facilitate spread across the mucosa. The aap gene is under control of the AggR transcriptional activator. Toxins Most EAEC strains harbor a chromosomal locus which encodes an autotranporter mucinase called Pic and, on the overlapping strand, the oligomeric enterotoxin called the ShET1 toxin (Shigella enterotoxin 1) (8, 12). Interestingly, this locus is 99% conserved


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within most Shigella flexneri isolates of serotype 2a. The ShET1 toxin has been shown to elicit rises in the short-circuit current in rabbit tissue mounted in an Ussing chamber (10). The mode of action of the ShET1 toxin is not yet understood, but it is thought to contribute to secretory diarrhea caused by EAEC and Shigella infections. The Pic mucinase has yet to be fully characterized, but it may play a role in intestinal colonization. EAST1 is a 38-amino-acid homolog of the ETEC heat-labile toxin (38). EAST1 clones yield net increases in the short-circuit current in the model of rabbit mucosal tissue mounted in an Ussing chamber (36). It is conceivable that EAST1 could contribute to watery diarrhea in EAST1-positive strains; however, it has been observed that the EAST1encoding gene can also be found in a large percentage of commensal E. coli isolates (37). The role of EAST1 in diarrhea remains an open question. Many EAEC strains secrete an autotransporter toxin called Pet, which is encoded on the large virulence plasmid, near AAF. Pet has been shown to elicit rises in the short-circuit current on rat intestinal tissue mounted in an Ussing chamber (33). In addition, Pet induces rounding of epithelial cells in culture. This effect is dependent on the presence of functional protease activity (9, 33, 34). Surprisingly, Pet was shown to be internalized by epithelial cells, and blockage of the internalization process resulted in resistance to cytopathic effects (32). Pet induces dissolution of the actin cytoskeleton in affected cells, followed by cell elongation and sloughing from the monolayer. Although the full mode of action of Pet has not been elucidated, some evidence suggests that Pet is able to cleave spectrin, an important component of the membrane cytoskeleton (48). A role for Pet in EAEC infection is supported by the observation that an EAEC strain mutated in the Pet-encoding gene is unable to elicit damage to the colonic mucosa in the organ culture model (13). Inflammation in EAEC Pathogenesis As noted above, EAEC infection commonly manifests as signs of mild inflammation. Steiner et al. (43) have shown that EAEC strains induce the release of interleukin-8 (IL-8) from cultured intestinal epithelial cells. In a subsequent study (44), the investigators showed that IL-8 release was due to expression of a novel flagellin protein homologous to a flagellin encoded by Shigella dysenteriae. The clinical importance of proinflammatory flagellar expression remains to be determined. However, neutrophil transmigration across the epithelium in response to IL-8 can itself lead to tissue disruption and fluid secretion, both of which could contribute to the clinical features of EAEC infections. Association of EAEC Virulence Factors with Disease As described above, a large number of putative virulence factors have been identified in EAEC strains, yet no single factor appears to be consistently present in all pathogenic strains. Evidence from my laboratory points to the presence of a “package” of plasmidborne and chromosomal virulence factors under the control of a previously described transcriptional activator called AggR (31; Nataro, unpublished) (Fig. 2). AggR is a member of the AraC family of transcriptional activators, which control virulence factor expression in a large number of enteric pathogens. AggR was initially found to induce expression of AAF/I and AAF/II fimbriae, but my laboratory has noted that many strains carrying the aggR gene do not express any known adhesin of AAF. Recently, we have shown that AggR controls not only the plasmid-borne


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Figure 2. Genetics of EAEC virulence. Most EAEC strains harbor a ca. 60-MDa plasmid designated pAA, which encodes the adhesin of AAF in many isolates. The plasmid may also encode the toxins Pet and EAST1. pAA also encodes the transcriptional activator AggR, which induces the expression of genes encoding AAF, as well as additional as yet cryptic loci on pAA itself and on the bacterial chromosome. Three chromosomal loci have been identified in EAEC, including the yersiniabactin island (similar to that of Yersinia enterocolitica), the Pic-ShET1 island, and a cryptic island under AggR control.

AAF but also chromosomal genes located on an island located at 94 min on the EAEC chromosome (E. Dudley and J. Nataro, unpublished data). Additional genes are apparently also under the control of AggR. One such locus comprises the EAEC probe used in epidemiologic studies (1) (see above), suggesting that positivity with the probe serves as a surrogate for identification of the AggR regulon. Characterization of the AggR regulon could elucidate the fundamental shared mechanisms of EAEC pathogenesis. Interestingly, Jiang et al. (16) have recently shown that the presence of genes associated with the AggR regulon (aggA, aspU [also called aap], aafA, and aggR itself) was predictive of significantly increased concentrations of fecal IL-8 and IL-1 in patients with diarrhea excreting EAEC. This observation suggests for the first time the link between a molecular package of virulence factors (the AggR regulon) and a particular clinical presentation, inflammatory enteritis. This observation is potentially a major advance in the understanding of the pathogenesis of EAEC. Moreover, the representation of the pathogenesis of EAEC as the nexus of multiple virulence factors is reminiscent of the pathogenesis of EPEC, in which strains possessing both the LEE chromosomal island and the EAF plasmid are designated typical EPEC strains, whereas those harboring only the LEE chromosomal island are called atypical EPEC strains (25). Extending this to EAEC, I


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suggest that the term “typical EAEC” be reserved for strains carrying AggR and at least a subset of AggR-regulated genes (for which the traditional EAEC-specific probe is an adequate marker) and that the term “atypical EAEC” be used for strains lacking the AggR regulon. REFERENCES 1. Baudry, B., S. J. Savarino, P. Vial, J. B. Kaper, and M. M. Levine. 1990. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J. Infect. Dis. 161:1249–1251. 2. Benjamin, P., M. Federman, and C. A. Wanke. 1995. Characterization of an invasive phenotype associated with enteroaggregative Escherichia coli. Infect. Immun. 63:3417–3421. 3. Bhan, M. K., P. Raj, M. M. Levine, J. B. Kaper, N. Bhandari, R. Srivastava, R. Kumar, and S. Sazawal. 1989. Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J. Infect. Dis. 159:1061–1064. 4. Cobeljic, M., B. Miljkovic-Selimovic, D. Paunovic-Todosijevic, Z. Velickovic, Z. Lepsanovic, N. Zec, D. Savic, R. Ilic, S. Konstantinovic, B. Jovanovic, and V. Kostic. 1996. Enteroaggregative Escherichia coli associated with an outbreak of diarrhoea in a neonatal nursery ward. Epidemiol. Infect. 117:11–16. 5. Cravioto, A., R. J. Gross, S. M. Scotland, and B. Rowe. 1979. An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenic serotypes. Curr. Microbiol. 3:95-99. 6. Cravioto, A., A. Tello, A. Navarro, J. Ruiz, H. Villafan, F. Uribe, and C. Eslava. 1991. Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet 337:262–264. 7. Czeczulin, J. R., S. Balepur, S. Hicks, A. Phillips, R. Hall, M. H. Kothary, F. Navarro-Garcia, and J. P. Nataro. 1997. Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in enteroaggregative Escherichia coli. Infect. Immun. 65:4135–4145. 8. Czeczulin, J. R., T. S. Whittam, I. R. Henderson, F. Navarro-Garcia, and J. P. Nataro. 1999. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect. Immun. 67:2692–2699. 9. Eslava, C. E., F. Navarro-Garcia, J. R. Czeczulin, I. R. Henderson, A. Cravioto, and J. P. Nataro. 1998. Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli. Infect. Immun. 66:3155–3163. 10. Fasano, A., F. Noriega, J. Liao, W. Wang, and M. M. Levine. 1997. Effect of Shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo. Gut 40:505–511. 11. Glandt, M., J. A. Adachi, J. J. Mathewson, Z. D. Jiang, D. DiCesare, D. Ashley, C. D. Ericsson, and H. L. DuPont. 1999. Enteroaggregative Escherichia coli as a cause of traveler’s diarrhea: clinical response to ciprofloxacin. Clin. Infect. Dis. 29:335–338. 12. Henderson, I. R., J. Czeczulin, C. Eslava, F. Noriega, and J. P. Nataro. 1999. Characterization of Pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect. Immun. 67:5587–5596. 13. Henderson, I. R., S. Hicks, F. Navarro-Garcia, W. P. Elias, A. D. Philips, and J. P. Nataro. 1999. Involvement of the enteroaggregative Escherichia coli plasmid-encoded toxin in causing human intestinal damage. Infect. Immun. 67:5338–5344. 14. Hicks, S., D. Candy, and A. Phillips. 1996. Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro. Infect. Immun. 64:4751–4760. 15. Itoh, Y., I. Nagano, M. Kunishima, and T. Ezaki. 1997. Laboratory investigation of enteroaggregative Escherichia coli O untypeable:H10 associated with a massive outbreak of gastrointestinal illness. J. Clin. Microbiol. 35:2546–2550. 16. Jiang, Z. D., D. Greenberg, J. P. Nataro, R. Steffen, and H. L. DuPont. 2002. Rate of occurrence and pathogenic effect of enteroaggregative Escherichia coli virulence factors in international travelers. J. Clin. Microbiol. 40:4185–4190. 17. Levine, M. M. 1987. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J. Infect. Dis. 155:377–389. 18. Lima, A. A., G. Fang, J. B. Schorling, L. de Albuquerque, J. F. McAuliffe, S. Mota, R. Leite, and R. L. Guerrant. 1992. Persistent diarrhea in northeast Brazil: etiologies and interactions with malnutrition. Acta Paediatr. Suppl. 381:39–44. 19. Mathewson, J. J., Z. D. Jiang, A. Zumla, C. Chintu, N. Luo, S. R. Calamari, R. M. Genta, A. Steephen, P. Schwartz, and H. L. DuPont. 1995. HEp-2 cell-adherent Escherichia coli in patients with human immunodeficiency virus-associated diarrhea. J. Infect. Dis. 171:1636–1639.


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20. Mathewson, J. J., P. C. Johnson, H. L. DuPont, T. K. Satterwhite, and D. K. Winsor. 1986. Pathogenicity of enteroadherent Escherichia coli in adult volunteers. J. Infect. Dis. 154:524–527. 21. Mayer, H., D. Acheson, and C. Wanke. 1995. Enteroaggregative Escherichia coli Are a Potential Cause of Persistent Diarrhea in Adult HIV Patients in the United States. Joint United States-Japan Cooperative Medical Sciences Program: malnutrition and cholera, Kiawah Island, S.C. National Institutes of Health, Bethesda, Md. 22. Mayer, H. B., and C. A. Wanke. 1995. Enteroaggregative Escherichia coli as a possible cause of diarrhea in an HIV-infected patient. N. Engl. J. Med. 332:273–274. 23. Nataro, J., D. Yikang, S. Cookson, A. Cravioto, S. Savarino, L. Guers, M. Levine, and C. Tacket. 1995. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J. Infect. Dis. 171:465–468. 24. Nataro, J. P., Y. Deng, D. R. Maneval, A. L. German, W. C. Martin, and M. M. Levine. 1992. Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes. Infect. Immun. 60:2297–2304. 25. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142–201. 26. Nataro, J. P., J. B. Kaper, R. Robins Browne, V. Prado, P. Vial, and M. M. Levine. 1987. Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr. Infect. Dis. J. 6:829–831. 27. Nataro, J. P., I. C. Scaletsky, J. B. Kaper, M. M. Levine, and L. R. Trabulsi. 1985. Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichia coli. Infect. Immun. 48:378–383. 28. Nataro, J. P., T. S. Steiner, and R. L. Guerrant. 1998. Enteroaggregative Escherichia coli. Emerg. Infect. Dis. 4:251–261. 29. Nataro, J. P., D. Yikang, S. Cookson, A. Cravioto, S. J. Savarino, L. D. Guers, M. M. Levine, and C. O. Tacket. 1995. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J. Infect. Dis. 171:465–468. 30. Nataro, J. P., D. Yikang, J. A. Giron, S. J. Savarino, M. H. Kothary, and R. Hall. 1993. Aggregative adherence fimbria I expression in enteroaggregative Escherichia coli requires two unlinked plasmid regions. Infect. Immun. 61:1126–1131. 31. Nataro, J. P., D. Yikang, D. Yingkang, and K. Walker. 1994. AggR, a transcriptional activator of aggregative adherence fimbria I expression in enteroaggregative Escherichia coli. J. Bacteriol. 176:4691–4699. 32. Navarro-García, F., A. Canizalez-Roman, J. Luna, C. Sears, and J. P. Nataro. 2001. Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells. Infect. Immun. 69:1053–1060. 33. Navarro-García, F., C. Eslava, J. M. Villaseca, R. López-Revilla, J. R. Czeczulin, S. Srinivas, J. P. Nataro, and A. Cravioto. 1998. In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli. Infect. Immun. 66:3149–3154. 34. Navarro-García, F., C. Sears, C. Eslava, A. Cravioto, and J. P. Nataro. 1999. Cytoskeletal effects induced by Pet, the serine protease enterotoxin of enteroaggregative Escherichia coli. Infect. Immun. 67:2184–2192. 35. Okeke, I. N., A. Lamikanra, J. Czeczulin, F. Dubovsky, J. B. Kaper, and J. P. Nataro. 2000. Heterogeneous virulence of enteroaggregative Escherichia coli strains isolated from children in Southwest Nigeria. J. Infect. Dis. 181:252–260. 36. Savarino, S., A. Fasano, J. Watson, B. Martin, M. Levine, S. Guandalini, and P. Guerry. 1993. Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heatstable toxin. Proc. Natl. Acad. Sci. USA 90:3093–3097. 37. Savarino, S., A. McVeigh, J. Watson, A. Cravioto, J. Molina, P. Echeverria, M. Bhan, and A. Fasano. 1996. Enteroaggregative Escherichia coli heat-stable enterotoxin is not restricted to enteroaggregative E. coli. J. Infect. Dis. 173:1019–1022. 38. Savarino, S. J., A. Fasano, D. C. Robertson, and M. M. Levine. 1991. Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model. J. Clin. Investig. 87:1450–1455. 39. Savarino, S. J., P. Fox, D. Yikang, and J. P. Nataro. 1994. Identification and characterization of a gene cluster mediating enteroaggregative Escherichia coli aggregative adherence fimbria I biogenesis. J. Bacteriol. 176:4949–4957. 40. Scaletsky, I. C., M. L. Silva, and L. R. Trabulsi. 1984. Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect Immun. 45:534–536. 41. Sheikh, J., S. Hicks, M. Dall’Agnol, A. D. Phillips, and J. P. Nataro. 2001. Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol. Microbiol. 41:983–997.


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42. Sheikh, J., J. R. Czeczulin, S. Harrington, S. Hicks, I. R. Henderson, C. Le Bouguenec, P. Gounon, A. Phillips, and J. P. Nataro. 2002. A novel dispersin protein in enteroaggregative Escherichia coli. J. Clin. Investig. 110:1329–1337. 43. Steiner, T. S., A. A. Lima, J. P. Nataro, and R. L. Guerrant. 1998. Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. J. Infect. Dis. 177:88–96. 44. Steiner, T. S., J. P. Nataro, C. E. Poteet-Smith, J. A. Smith, and R. L. Guerrant. 2000. Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells. J. Clin. Investig. 105:1769–1777. 45. Tompkins, D. S., M. J. Hudson, H. R. Smith, R. P. Eglin, J. G. Wheeler, M. M. Brett, R. J. Owen, J. S. Brazier, P. Cumberland, V. King, and P. E. Cook. 1999. A study of infectious intestinal disease in England: microbiological findings in cases and controls. Commun. Dis. Public Health 2:108–113. 46. Tzipori, S., J. Montanaro, R. M. Robins-Browne, P. Vial, R. Gibson, and M. M. Levine. 1992. Studies with enteroaggregative Escherichia coli in the gnotobiotic piglet gastroenteritis model. Infect. Immun. 60:5302–5306. 47. Vial, P. A., R. Robins Browne, H. Lior, V. Prado, J. B. Kaper, J. P. Nataro, D. Maneval, A. Elsayed, and M. M. Levine. 1988. Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease. J. Infect. Dis. 158:70–79. 48. Villaseca, J. M., F. Navarro-García, G. Mendoza-Hernández, J. P. Nataro, A. Cravioto, and C. Eslava. 2000. Pet toxin from enteroaggregative Escherichia coli produces cellular damage associated with fodrin disruption. Infect. Immun. 68:5920–5927. 49. Wanke, C. A., J. Gerrior, V. Blais, H. Mayer, and D. Acheson. 1998. Successful treatment of diarrheal disease associated with enteroaggregative Escherichia coli in adults infected with human immunodeficiency virus. J. Infect. Dis. 178:1369–1372. 50. Wanke, C. A., J. B. Schorling, L. J. Barrett, M. A. Desouza, and R. L. Guerrant. 1991. Potential role of adherence traits of Escherichia coli in persistent diarrhea in an urban Brazilian slum. Pediatr. Infect. Dis. J. 10:746–751.


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Chapter 8

Wound Botulism, Tetanus, and Necrotizing Fasciitis among Injecting Drug Users in California: the Clostridial Connection Duc J. Vugia, S. Benson Werner, and Celia J. I. Woodfill

Clostridia are anaerobic, spore-forming, gram-positive bacilli. Clostridium species are ubiquitous in nature and are frequently isolated from soil and the intestinal tracts of animals and humans. Of the more than 130 known species, only a small proportion have been recovered from clinical specimens (1). Several species are clinically significant in humans and cause diverse diseases ranging from botulism to soft tissue infections (Table 1). Of these clinically significant species, Clostridium perfringens is the most commonly encountered. Diseases caused by many of these clostridia are often mediated via toxins. For botulism and tetanus, specific neurotoxins produced by Clostridium botulinum and Clostridium tetani affect different areas in the nervous system to cause weakness or spasticity (1, 3, 4). In C. perfringens food-borne disease, an enterotoxin injures intestinal epithelial cells, resulting in abdominal cramps and diarrhea (1, 14). In myonecrosis due to C. perfringens and several other clostridia, histotoxicity is primarily due to alpha-toxin or phospholipase C (1, 15). In 1995, the California Department of Health Services (CDHS) documented an increase in the reported number of cases of wound botulism (WB) that started in 1994 (7). All cases except one occurred in injecting drug users (IDUs). In 1997, 11 cases of tetanus were reported from California, six of which occurred among IDUs (8). At about the same time, clusters of necrotizing soft tissue infections (NSTIs), including necrotizing fasciitis, among IDUs were noted by some community hospitals in California. Warnings of “flesheating disease” among this population were circulated by some local health departments and the local media. However, since this disease entity (necrotizing fasciitis) is not a reportable condition in California (nor is it reportable in most other states), it could not be easily confirmed that these clusters represented unusual increases.

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Vugia et al. Table 1. Selected clostridia and associated clinical illnesses Species

Illness(es)

C. botulinum . . . . . . . . . . . . . . . . . . . . . . . . Botulism C. difficile . . . . . . . . . . . . . . . . . . . . . . . . . . Antibiotic-associated colitis C. novyi . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myonecrosis C. perfringens . . . . . . . . . . . . . . . . . . . . . . . Bacteremia, food-borne disease, myonecrosis C. septicum . . . . . . . . . . . . . . . . . . . . . . . . . Bacteremia, myonecrosis C. sordellii . . . . . . . . . . . . . . . . . . . . . . . . . . Bacteremia, myonecrosis C. tetani . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tetanus C. tertium . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacteremia

To determine the extent of these diseases among the IDU population in California, we reviewed cases of WB and tetanus reported to CDHS as required by regulations, and we reviewed published and unpublished reports of NSTIs that have occurred among IDUs in California. WB ASSOCIATED WITH IDUs IN CALIFORNIA WB is a form of botulism in which C. botulinum contaminates a wound, grows in the anaerobic wound environment, and produces sufficient botulinal toxin in situ to cause disease. While the clinical presentation is slightly different from that for food-borne botulism, in that there are no gastrointestinal symptoms, the presentation is otherwise typical, with acute, bilateral, flaccid, descending paralysis starting with cranial neuropathies (3). Fever, however, may be associated with the wound infection. In California, besides botulism being a reportable disease, CDHS, under agreement with the federal Centers for Disease Control and Prevention (CDC), releases botulinum antitoxin for treatment of suspected cases, facilitating case surveillance. Therefore, virtually all persons with suspected cases of botulism in California come to our attention once they seek medical care, the diagnosis of botulism is suspected by a clinician, and antitoxin is sought for treatment. For clinically suspected cases, antitoxin is released for treatment and sera are tested by mouse bioassays for the presence of botulinal toxin, while it is recommended that wound swabs, when wounds are present, be submitted for anaerobic culture. Only laboratory-confirmed cases are finally reported. From 1988 through 2000, a total of 163 cases of laboratory-confirmed WB were reported to CDHS. Of these, 156 (96%) were among IDUs, five (3%) were posttraumatic, and two (1%) were postoperative complications. The number of cases of WB associated with IDUs has increased dramatically since 1994 (Fig. 1). Of the 156 cases of WB associated with IDUs, 93 (60%) occurred among males, and the median age of the subjects was 40 years (age range, 20 to 68 years); 80 (51%) were Hispanic, 62 (40%) were white, and 9 (6%) were African American. Seven (5%) of these patients with WB died. By physical examination, 115 (79%) of the patients with WB had an infected wound, often as a result of “skin popping” (injecting a drug subcutaneously or intramuscularly) of the arms, shoulders, legs, abdomen, buttocks, vulva, or base of the tongue. One-hundred forty-two (91%) admitted to injecting heroin; of these, 106 (75%) specified that they injected black tar heroin (BTH). Two IDUs reported “shooting” only amphetamines. Five


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Figure 1. Number of IDUs with WB in California by year, 1988 to 2000. (Source: California Department of Health Services.)

couples developed WB, one member within days to months after the other, after sharing BTH but not drug equipment. The botulinal toxin detected was type A for 137 (88%) patients and type B for 16 (10%) patients. Of 62 wound specimens cultured, 36 (58%) yielded C. botulinum: 25 type A and 11 type B. Cases of WB were distributed in 24 (more populous) of the 58 California counties. In 1996, we implemented a case-control study to identify risk factors for WB among IDUs in California (17). Case patients were IDUs who developed laboratory-confirmed WB from 1 January 1994 through 1 March 1996. Controls were IDUs enrolled in a methadone detoxification program from March through May 1996. We enrolled 26 case patients and 110 controls. A total of 96% of case patients and 97% of controls used BTH. However, 92% of case patients but only 44% of controls injected drug subcutaneously or intramuscularly (skin popped) (odds ratio [OR], 15.5; 95% confidence interval [CI], 3.5 to 139.5). In the final regression model, skin popping of BTH was the only behavior associated with WB among IDUs (OR, 14; 95% CI, 3 to 63). While cleaning of the skin before injection was borderline protective against the development of abscesses, cleaning of the skin or cleaning of the injection equipment was not protective against WB. Also, the quantity of BTH used was a more important risk factor than the frequency of use. Therefore, besides skin popping, the risk of WB among these IDUs was most likely due to the use of BTH contaminated with C. botulinum than to skin contamination with C. botulinum. We hypothesized that BTH was contaminated with C. botulinum probably during dilution or “cutting” of the drug, for which soil and other unhygienic materials could have been used (17). Furthermore, since California had most cases of WB during these years, even though BTH was available in many western states, we also hypothesized that the implicated BTH was likely diluted and contaminated within California (19). TETANUS ASSOCIATED WITH IDUs IN CALIFORNIA A total of 96 cases of tetanus were reported to occur in California between 1988 and 2000. Among the patients with these cases, 43 (45%) reported a history of IDU. The number of IDUs with tetanus in California has increased since 1994 (Fig. 2). Of the 43 cases of


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Figure 2. Number of IDUs with tetanus in California by year of onset, 1988 to 2000. (Source: California Department of Health Services.)

tetanus associated with IDU, 36 (84%) occurred among males and the median age of the subjects was 39 years (age range, 26 to 60 years); 38 (88%) were Hispanic, 3 (7%) were white, and 2 (5%) were African American. Cases were distributed in 17 of the 58 California counties; 12 of these counties were also counties with WB cases during this period. Eight (19%) of these IDUs with tetanus died; all of them were Hispanic. Of the 19 patients with a known vaccination history, 14 (74%) had not received a dose of tetanus vaccine during the previous 10 years. Five of the eight deaths occurred among these IDUs with tetanus who had not received a recent tetanus vaccine. Although not asked systematically of all patients, 26 reported using either heroin or BTH, 22 had abscesses noted at presentation, and 18 reported skin popping. The descriptive epidemiology of these reported IDUs with tetanus is similar to that of the IDUs with WB during this period, with Hispanics being even more overrepresented among the patients with cases of tetanus. NSTIs ASSOCIATED WITH IDUs IN CALIFORNIA NSTIs and necrotizing fasciitis are not reportable conditions in California. Our review of the literature revealed only three publications describing cases of NSTIs in California (5, 6, 11). These three case series originated from three large community hospitals in northern California (in Oakland, San Francisco, and Sacramento). Additional informal reports from a few other California community hospitals, especially those serving indigent patients, provided epidemiologic and clinical features similar to those provided in the published reports (CDHS, unpublished data). In the first publication, in 1996, Bosshardt and colleagues (5) described cases of NSTIs from 1990 through 1995 at a community hospital in Oakland and noted that cases of NSTIs associated with IDUs increased significantly in 1994, while non-IDU-associated NSTI cases did not (Fig. 3). The other two publications (6, 11) from large community hospitals in California showed similar increases in the numbers of IDUs with NSTIs starting in 1994 or 1995 (Fig. 4 and 5). Selected characteristics of the NSTI cases involving IDUs at these three California hospitals are summarized in Table 2. Among the cases of NSTIs described from the Oakland


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Figure 3. Number of cases of necrotizing soft tissue infections by year at a hospital in Oakland, Calif., 1990 to 1995. (Reprinted from Archives of Surgery [5] with permission of the publisher. Copyrighted 1996, American Medical Association.)

hospital, 67% occurred among IDUs, while all cases from the other two hospitals that are described here occurred among IDUs. The patients were predominantly male, and the mean age was above 40 years in each case series. For the Sacramento hospital case series for which race and ethnicity information for the IDUs was reported, 64% of IDUs with NSTIs were white, 25% were Hispanic, 7% were African American, and 2% were Asian (11). The proportion of cases with necrotizing fasciitis was over 80% at two of the three hospitals, and the mortality rate ranged from 10 to 27%. Among IDUs, heroin use and skin popping were common features. Bacteriologic findings from these three case series are summarized in Table 3. In most of these cases, wound cultures yielded polymicrobial results, with a mixture of aerobic and anaerobic bacterial pathogens. The aerobic bacteria reported to have been isolated included

Figure 4. Number of IDUs with necrotizing soft tissue infections by year at a hospital in San Francisco, Calif., 1992 to 1997. (Reprinted from Archives of Surgery [6] with permission of the publisher. Copyrighted 1998, American Medical Association.)


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Figure 5. Number of IDUs with necrotizing soft tissue infections by year at a hospital in Sacramento, Calif., 1987 to 1999. (Reprinted from Clinical Infectious Diseases [11] with permission of the publisher.)

gram-positive organisms such as Staphylococcus aureus, coagulase-negative Staphylococcus, Streptococcus pyogenes, viridans group streptococci, and Enterococcus species and gram-negative organisms such as Pseudomonas aeruginosa, Enterobacter species, Klebsiella species, and Eikenella corrodens (5, 6, 11). The anaerobic bacteria reported to have been isolated included Clostridium species and Peptostreptococcus species. Where anaerobes were isolated, clostridia made up less than 25% of the total. The Clostridium species most commonly identified was C. perfringens; other clostridia isolated included C. sordellii, C. septicum, C. botulinum, and C. tetani (5, 6, 11). IDUs with NSTIs typically had a clinically recognizable infection at admission, underwent debridement within the first 24 h after admission, and underwent multiple subsequent surgical procedures, some requiring amputation (6, 11). The rate of mortality, as mentioned earlier, was substantial among these patients. Table 2. Characteristics of NSTIs involving IDUs at three California hospitals a

Characteristics

Oakland (n ⫽ 45 patients with NSTI)

San Francisco (n ⫽ 30 IDU)

Sacramento (n ⫽ 59 IDU)

No. (%) of male patients

27 (60)

18 (60)

45 (76)

Mean age (yr)

46

41

No. (%) of patients: Necrotizing fasciitis Requiring amputation Died IDU Used heroin Skin popped

38 (84) 0 12 (27) 30 (67) 27 (90) 19 (63)

11 (37) 5 (17) 6 (20) 30 (100)

a

Data are from references 5, 6, and 11.

⬎40

47 (82) 6 (10) 6 (10) 59 (100) 40 (68) 27 (46)


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Table 3. Bacteriologic findings of NSTIs among IDUs at three California hospitalsa Oakland (n ⫽ 45 patients with NSTI)

Bacteriologic finding

No. (%) of patients with: Polymicrobial Single organism No organism

35 (78) 9 (20) 1 (2)

Clostridium spp.

C. perfringens, C. sordellii, C. septicum, C. tetani, C. botulinum

San Francisco (n ⫽ 30 IDU)

17 (57) 11 (37)

C. perfringens, C. sordellii

Sacramento (n ⫽ 59 IDU)

27 (46) 23 (39) 9 (15) C. perfringens, other Clostridium species

a

Data are from references 5, 6, and 11.

Informal reports to CDHS showed similar increases in the number of NSTIs recognized among IDUs in some other community hospitals in California. These patients also had a median age of 40 years or older, many of them admitted to skin popping of heroin, and some died. Polymicrobial flora were recovered upon culture of specimens from the patients, and of the clostridia species isolated, C. perfringens and C. sordellii were the more common (CDHS, unpublished data). Overall, these published and unpublished reports give further support to a statewide problem with NSTIs among IDUs starting in the mid-1990s. CULTURES OF BTH Several laboratories have attempted to culture small samples of BTH from IDUs with WB or NSTIs for anaerobic bacteria. Most of these cultures were negative. One BTH sample from an IDU with a fatal case of NSTI in San Francisco yielded C. perfringens (2), and our culture of the inside of a used syringe yielded C. botulinum on one occasion (17). THE CLOSTRIDIAL CONNECTION The recent epidemic of WB, tetanus, and NSTIs among IDUs in California is likely due, in great part, to skin popping of BTH contaminated with Clostridium species. The simultaneous statewide increases in the rates of different clostridial diseases among IDUs in California in 1994 suggested that contaminated BTH had been available in that state at least since the mid-1990s. Other possible reasons such as increased rates of skin popping or decreased hygiene of skin or drug equipment among IDUs either are less likely or cannot explain why these cases are occurring primarily in California. Rare cases of WB associated with IDUs in California and other states have been described previously (16). However, the dramatic increase in the number of cases of WB associated with IDUs in California since 1994 is unprecedented and has still not been seen elsewhere. Our studies of these WB cases strongly implicated skin popping as the main risk factor for WB and pointed toward contaminated BTH as the source of the epidemic. Our case-control study discounted the importance of a skin source for the development of WB among California IDUs. The fact that several couples were affected after sharing BTH


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but not equipment further supports the idea that the source is more likely to be at the BTH level. Finally, one BTH sample did yield C. perfringens, supporting the notion that BTH in California can be contaminated with soil organisms such as other Clostridium species. On the basis of information gathered from drug enforcement agencies, BTH was illegally imported to the United States from south of the U.S. border and sold mostly in western states. In California, BTH became the predominant form of heroin sold by the early 1990s (18). This predominance, coupled with the common and profitable practice of diluting or “cutting” BTH with unknown but likely unhygienic adulterants, including possibly soil or dirt, sets the stage for this epidemic. Skin popping, in which the contaminated drug is directly injected into the subcutaneous tissue or muscle, provides both a nidus of infection stimulated by gross BTH residues as contaminated foreign bodies and a favorable anaerobic environment for germination of any clostridial spores introduced, leading to disease. This pathogenetic scenario may be the same for California IDUs with tetanus, except that the BTH was likely contaminated with C. tetani. Rare cases of tetanus among IDUs have previously been reported elsewhere (12), and other potential sources for this infection in this population include dirty skin or contaminated drug equipment. However, such a large increase in the number of cases in California cannot be explained by possible (but unlikely) recent and local increases in unhygienic drug injecting practices or the use of unhygienic equipment. An increased availability of BTH contaminated with C. tetani remains the more likely explanation for this increase in the number of cases of tetanus associated with IDUs seen statewide in California. The overrepresentation of Hispanics among IDU patients with tetanus may be due, in part, to a lower prevalence of immunity to tetanus in this population compared to that in the general population. A population-based serologic survey published in 1995 showed that 58% of Mexican Americans had protective levels of tetanus antibodies, whereas 73% of non-Hispanic whites had protective levels (13). The relatively older median age among these IDUs with clostridial disease may be due to the higher prevalence of skin popping in chronic IDUs who have “used up” or scarred most of their superficial veins. Like WB and tetanus, NSTIs have previously been recognized infrequently in the IDU population (5). We believe that the increase in the number of NSTI cases among IDUs seen at several hospitals in California starting in the mid-1990s was due, in part, to the increased availability of BTH contaminated with soil organisms, including clostridia. The IDU patients presenting with NSTIs at these hospitals are very similar in several demographic characteristics to the IDUs presenting with WB and tetanus, and BTH was commonly found to be the drug of choice among these IDUs. While Clostridium species were isolated from only a small proportion of patients and cultures more commonly yielded polymicrobial flora, including some skin and oral pathogens, it is still likely that an otherwise low baseline number of NSTIs among IDUs has been boosted since 1994 by clostridial contamination of BTH in California, especially when similar trends were seen for WB and tetanus. Also, while bacteria from the skin or mouth may contribute to some cases of NSTIs among IDUs (such as when the needle is licked before injection), such practices cannot explain why there has been a substantial increase in the number of NSTI cases since the mid-1990s. In 2000, an outbreak of severe unexplained illnesses and deaths was reported among IDUs in Scotland, Ireland, and England (9). Most of these cases had evident soft tissue infections at drug injection sites, and C. novyi and C. perfringens as well as other bacteria


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were isolated from some tissues (10). The finding of these clostridia suggested that “soil or fecal contamination of the drugs or other materials used by these IDUs” may have caused these illnesses (10). A case-control study found the risk factors for disease to be skin or muscle popping of heroin and sharing of a needle or filter (M. A. Fisher, Abstr. 41st Intersci. Cont. Antimicrob. Agents Chemother., abstr. 2009, 2001). Clearly, severe clostridial soft tissue infections among IDUs, probably due to contaminated drug, can be a clinical and public health problem anywhere. As in most illicit drug connections, the casualties of this clostridial connection are the IDUs, and since 1994, those in California have increasingly been suffering from WB, tetanus, and NSTIs, in addition to other blood-borne infections usually associated with injection drug use. Tracing back the implicated vehicles to where contamination might have occurred has been impossible, and some evidence (such as more microbiological evidence of contaminated BTH) is hard to come by. The sources of contamination of BTH in California are still undetermined. HOW CAN THE TIDE BE STEMMED? Injection drug use is a complicated issue, and IDUs are a difficult population to reach with prevention messages. These messages will have to address drug rehabilitation to decrease the risk of infection with blood-borne pathogens along with the risk for WB, tetanus, and NSTIs. Several local health departments in California have sent alerts to IDUs via drug rehabilitation centers and through local press releases. Increased awareness of these clostridial diseases associated with IDUs among clinicians in emergency rooms and surgical staff will decrease the time to diagnosis, leading to more rapid and appropriate treatment interventions, and thereby, it is hoped, decrease rates of morbidity and mortality in association with these diseases. Opportunities to update or complete tetanus vaccination should be taken whenever an IDU is seen in a clinical setting. CONCLUSIONS Clostridium species can cause diseases ranging from botulism to soft tissue infections. In recent years, California clinicians have seen increasing numbers of serious Clostridium infections among IDUs, including WB, tetanus, and necrotizing fasciitis. We reviewed cases of WB and tetanus reported to CDHS and publications of NSTIs in California. From 1988 through 2000, we documented 156 cases of WB in California IDUs, with an epidemic increase since 1994. Most had an infected wound, and C. botulinum was isolated from 65% of IDUs when a specimen from the wound was cultured anaerobically. A casecontrol study implicated skin popping of BTH as the risk factor for WB. From 1988 through 2000, 43 cases of tetanus have been reported among IDUs, with an increase in the number of cases since 1994. Abscesses were noted in 22 patients, and 18 reported skin popping. Publications from three California hospitals showed increased cases of NSTIs among IDUs since the mid-1990s. Cultures of wound specimens yielded mostly polymicrobial results. When isolated, the Clostridium species isolated included C. perfringens, C. sordellii, C. septicum, C. tetani, and C. botulinum. One sample of BTH obtained from an IDU with a fatal case of NSTI yielded C. perfringens. The recent epidemics of WB, tetanus, and NSTIs among IDUs in California are likely due, in great part, to skin popping


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of BTH contaminated with Clostridium species. Clinicians should have a heightened level of suspicion for these clostridial diseases among IDUs and should include anaerobic cultures in evaluations of infected wounds in this population. Acknowledgments. We are indebted to our public health partners at the local health departments for reporting cases of botulism and tetanus to CDHS. REFERENCES 1. Allen, S. D., C. L. Emery, and J. A. Siders. 1999. Clostridium, p. 654–671. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 7th ed. American Society for Microbiology, Washington, D.C. 2. Bangsberg, D. R., J. I. Rosen, T. Aragon, A. Campbell, L. Weir, and F. Perdreau-Remington. 2002. Clostridial myonecrosis cluster among injection drug users: a molecular epidemiology investigation. Arch. Intern. Med. 162:517–522. 3. Bleck, T. P. 2000. Clostridium botulinum (botulism), p. 2543–2548. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Disease, 5th ed. Churchill Livingstone, Philadelphia, Pa. 4. Bleck, T. P. 2000. Clostridium tetani (tetanus), p. 2537–2543. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Disease, 5th ed. Churchill Livingstone, Philadelphia, Pa. 5. Bosshardt, T. L., V. J. Henderson, and C. H. Organ. 1996. Necrotizing soft-tissue infections. Arch. Surg. 131:846–854. 6. Callahan, T. E., W. P. Schecter, and J. K. Horn. 1998. Necrotizing soft tissue infection masquerading as cutaneous abscess following illicit drug injection. Arch. Surg. 133:812–818. 7. Centers for Disease Control and Prevention. 1995. Wound botulism––California, 1995. Morb. Mortal. Wkly. Rep. 44:889–892. 8. Centers for Disease Control and Prevention. 1998. Tetanus among injecting-drug users—California, 1997. Morb. Mortal. Wkly. Rep. 47:149–151. 9. Centers for Disease Control and Prevention. 2000. Unexplained illness and death among injecting-drug users—Glasgow, Scotland; Dublin, Ireland; and England, April–June 2000. Morb. Mortal. Wkly. Rep. 49:489–492. 10. Centers for Disease Control and Prevention. 2000. Update: Clostridium novyi and unexplained illness among injecting-drug users—Scotland, Ireland, and England, April–June 2000. Morb. Mortal. Wkly. Rep. 49:543–545. 11. Chen, J. L., K. E. Fullerton, and N. M. Flynn. 2001. Necrotizing fasciitis associated with injection drug use. Clin. Infect. Dis. 33:6–15. 12. Cherubin, C. E., S. J. Millian, E. Palusci, and M. Fortunato. 1968. Investigations in tetanus in narcotics addicts in New York City. Am. J. Epidemiol. 88:215–223. 13. Gergen, P. J., G. M. McQuillan, M. Kiely, T. M. Ezzati-Rice, R. W. Sutter, and G. Virella. 1995. A population-based serologic survey of immunity to tetanus in the United States. N. Engl. J. Med. 332:761–766. 14. Kokai-Kun, J. F., and B. A. McClane. 1997. The Clostridium perfringens enterotoxin, p. 325–357. In J. I. Rood, B. A. McClane, J. G. Songer, and R. W. Titball (ed.), The Clostridia: Molecular Biology and Pathogenesis. Academic Press, Inc., New York, N.Y. 15. Lorber, B. 2000. Gas gangrene and other Clostridium-associated diseases, p. 2549–2561. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Disease, 5th ed. Churchill Livingstone, Philadelphia, Pa. 16. MacDonald, K. L., G. W. Rutherford, S. M. Friedman, J. R. Dietz, B. R. Kaye, G. F. McKinley, J. H. Tenney, and M. L. Cohen. 1985. Botulism and botulism-like illness in chronic drug abusers. Ann. Intern. Med. 102:616–618. 17. Passaro, D. J., S. B. Werner, J. McGee, W. R. MacKenzie, and D. J. Vugia. 1998. Wound botulism associated with black tar heroin among injecting drug users. JAMA 279:859–863. 18. U.S. Drug Enforcement Administration. 1994. Domestic Monitor Program: 1993 Annual Summary. U.S. Drug Enforcement Administration, Washington, D.C. 19. Werner, S. B., D. Passaro, J. McGee, R. Schechter, and D. J. Vugia. 2000. Wound botulism in California, 1951–1998: recent epidemic in heroin injectors. Clin. Infect. Dis. 31:1018–1024.


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Chapter 9

Unexplained Severe Illness and Death among Injecting Drug Users in Scotland, Ireland, and England from April to August 2000 Marc Fischer, Anne Whitney, Syed Ahmed, Joe Barry, and Jane Jones

On 5 May 2000, public health authorities in Glasgow, Scotland, learned that two injecting drug users (IDUs) who resided at the same hostel had died in the previous week following similar acute illnesses. By the end of May, active surveillance identified 31 cases of severe unexplained illness among IDUs in Glasgow, including 14 (45%) deaths (10, 21). Following public health alerts regarding the situation in Scotland, authorities in Ireland and England identified 29 similar cases among IDUs in April and May, 14 (48%) of whom died (7, 8, 21). In all locations, the illnesses were characterized by extensive soft tissue inflammation at an injection site, often followed by profound leukocytosis and cardiogenic shock. Postmortem examinations did not reveal a specific cause of death. Deaths among IDUs are not uncommon. However, at the peak of this outbreak, fatalities among IDUs in Glasgow and Dublin, Ireland, increased from an average of 19 deaths per month from January through April 2000 to 38 deaths in May. Of additional concern, in mid-May, Norwegian authorities reported that an IDU residing in Oslo had recently died due to Bacillus anthracis meningitis after initially presenting with a localized abscess and markedly elevated white blood cell (WBC) count (45,000 cells/mm3) (12). Coupled with the expanding number of cases and severity of illness among IDUs in the United Kingdom and Ireland, this report of anthrax heightened the sense of urgency to identify the cause of the outbreak. This chapter summarizes a collaborative effort among public health authorities in Scotland, Ireland, England, Wales, and the United States to (i) describe the clinical and epidemiologic features of the case patients, (ii) identify the cause of their unexplained illness, and (iii) define a potential source of the outbreak.

Marc Fischer and Anne Whitney • Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333. Syed Ahmed • Public Health Protection Unit, Greater Glasgow NHS Board, Glasgow G3 8YU, Scotland. Joe Barry • Department of Public Health, Eastern Regional Health Authority, Dublin 8, Ireland. Jane Jones • Public Health Laboratory Service, London NW9 5EQ, England. M.F., A.W., S.A., J.B., and J.J. wrote this chapter for the International Outbreak Investigation Team. 121


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SURVEILLANCE FOR POTENTIAL CASES For the purpose of this report, a case patient was defined as an IDU who presented to a hospital or who was found dead between 1 April and 31 August 2000 with (i) extensive soft tissue inflammation at an injection site; (ii) signs of severe systemic toxicity (i.e., sustained systolic blood pressure of ⬍90 mm Hg despite fluid resuscitation or a total peripheral WBC count of ⬎30,000 cells/mm3); or (iii) postmortem evidence of a diffuse toxic or infectious process. Active surveillance was conducted through inquiries to area emergency departments, intensive care units, and coroners’ offices. Passive surveillance was initiated in the form of public health alerts to all hospitals in the United Kingdom and Ireland. Information was also disseminated to IDUs through drug treatment and outreach services. To identify potential cases outside of the United Kingdom and Ireland, early in the investigation reports were published in the public health literature in both Europe and the United States (4, 5, 7). PATIENT DEMOGRAPHICS Between 1 April and 31 August 2000, investigators identified 108 IDUs meeting the case definition in Scotland (n ⫽ 60), Ireland (n ⫽ 22), and England (n ⫽ 26), including 44 (41%) deaths. The median age of the case patients was 30.5 years (age range, 19 to 51 years); 56 (52%) were male. Among the 71 case patients for whom data were available, there was a median of 4 days (range, 0 to 16 days) between symptom onset and hospitalization. Among the 36 persons who died while hospitalized, the median time from admission to death was 1.5 days (range, 0 to 13 days). The number of cases peaked in mid-May and had essentially resolved by early July (Fig. 1) (14, 17).

Figure 1. Cases of severe illness among IDUs by week of hospitalization or death, Scotland, Ireland, and England, 1 April to 31 August 2000.


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In Scotland, 55 (92%) case patients resided in the city of Glasgow or its neighboring districts, 3 (5%) resided in Aberdeen, and 2 (4%) resided in Kirkcaldy. Glasgow is a city of about 620,000 persons with an estimated 12,500 IDUs. No cases were identified in Edinburgh, Scotland’s second-largest city. In Ireland, all identified case patients resided in a small area of Dublin. Dublin has a population of 1.1 million persons, with an estimated 13,000 IDUs. In England, the majority of cases were clustered in the northwest part of the country, with several others scattered over a wide geographical area. CLINICAL CHARACTERISTICS Of the 108 patients identified, 17 (16%) presented to hospital in extremis or were found dead at home (17). For the remaining 91 case patients, the presenting complaint was infection of a local injection site, with one patient injecting through an indwelling catheter. The most common clinical finding was marked edema of the soft tissue (79%). Necrosis (36%), frank pus or abscess (22%), gas production (9%), and foul odor (1%) of the injection site were less common. Although 91 patients presented with only minimal or moderate systemic symptoms, 28 (31%) of them deteriorated rapidly, with hypotension, respiratory distress, and cardiac arrest, often despite surgical debridement and treatment with broadspectrum antibiotics. Among the 95 patients for whom a peripheral WBC count was measured, the median value was 18,500 cells/mm3 (range, 1,900 to 153,000 cells/mm3). However, 34 (92%) of the 37 patients who died had WBC counts over 35,000 cells/mm3, and the median WBC count among the patients with fatal cases was 53,700 cells/mm3, whereas it was 12,700 cells/mm3 among the patients with nonfatal cases (Kruskal-Wallis test, P ⬍ 0.001). Several important clinical features were not prominent findings among these patients. Few patients had a rash outside of the wound site. Thrombocytopenia, disseminated intravascular coagulation, liver failure, and renal failure were also uncommon. Although a number of patients developed altered mental status, none had focal neurologic changes indicative of botulism or tetanus. Finally, only three patients were known to be human immunodeficiency virus positive, including one fatal case and two nonfatal cases. Of the 44 case patients who died, autopsy data were available for review for 37 (84%). For these patients, postmortem examination of the injection-site wounds (i.e., skin, subcutaneous tissue, fascia, and muscle) revealed swelling or edema (68%), necrosis (65%), and frank pus or abscess formation (41%). In areas other than the injection site, large pleural and pericardial effusions were the most common findings (76%), followed by subendocardial hemorrhage (19%) and gas in the retroperitoneum (14%). There was no evidence of vasculitis, septic shock, or drug overdose. DIFFERENTIAL DIAGNOSIS The localized inflammatory process affecting skin or muscle combined with systemic toxicity characterized by a leukemoid reaction suggested a chemical contamination or toxin-mediated infection due to B. anthracis, Staphylococcus aureus, group A streptococci, or histotoxic clostridia. The several-day delays from symptom onset to hospitalization and hospitalization to death were most consistent with an infectious process as opposed to a premade chemical or endotoxin. Initial cultures of blood and tissue had


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yielded multiple organisms from several patients. However, early on, the variable and polymicrobial results and potential postmortem contamination complicated the interpretation of these findings and failed to reveal a definitive cause. At the outset of the investigation, the fatal case of anthrax in the Norwegian IDU focused substantial attention on B. anthracis. Although the initial symptoms of anthrax can be nondescript before the onset of circulatory collapse and death, with severe disease, B. anthracis is usually readily identifiable by blood culture and immunohistochemistry of the wound tissue (11, 13). Furthermore, despite its presence in soil throughout the world, B. anthracis has not been previously reported to cause a cluster of disease among IDUs. Streptococcal toxic shock syndrome and staphylococcal toxic shock syndrome are both characterized by the sudden onset of shock and organ failure, often associated with skin and soft tissue damage (6, 26). S. aureus and/or group A streptococci were isolated from the skin or blood of several patients evaluated early in the investigation. However, few of the IDUs identified in this cluster exhibited the renal, hepatic, and gastrointestinal manifestations that are prominent in toxic shock syndrome; group A streptococci were isolated from cultures of blood or wound specimens from only six patients (three with fatal cases, three with nonfatal cases); and none had a desquamating rash, a diagnostic criterion of staphylococcal toxic shock. Histotoxic clostridia can cause a distinctive, toxin-mediated infection characterized by soft tissue inflammation, sudden onset of shock, and leukemoid reaction in the absence of high fever or rash. In addition, because these organisms produce toxins that directly affect the cardiac muscle, rapid deterioration may be seen without signs of multiorgan failure or septic shock. Finally, the variable growth and staining requirements of several clostridial species may make them difficult to isolate or identify under routine conditions (22). EARLY INTERVENTION On the basis of the results of the clinical investigation, public health alerts recommended that drug users with injection-site wounds be evaluated and treated as early as possible. Surgical intervention was emphasized in the management of possible cases, including debridement and culture of samples from the area of any injection-site infection. Clinicians were urged to include antibiotics with good activities against anaerobic organisms as part of their empiric therapy. Microbiology laboratories optimized their procedures for the isolation and identification of anaerobic bacteria. For fatal cases, coroners were asked to perform autopsies as soon as possible after death. There is some evidence that these early interventions may have had an effect. The case-fatality ratio for IDUs presenting in April and May was 50% (35 of 70 patients), whereas it was 24% (9 of 38 patients) for patients identified from June to August (P ⬍ 0.01), perhaps reflecting a change in patient management or the improved identification of earlier, milder, or unrelated cases. LABORATORY EVALUATION OF CLINICAL SPECIMENS Diagnostic testing responsibilities were divided between local and reference laboratories. Tissue samples were evaluated by using enhanced culture for aerobic and anaerobic organisms, histopathology, immunohistochemistry against group A streptococci and B. anthracis, and broad-range bacterial 16S ribosomal DNA (rDNA) PCR. Enhanced cul-


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tures for aerobic and anaerobic organisms were performed by use of a standard protocol that included enriched thioglycolate, chopped meat glucose, and peptone yeast glucose media. The methods used for identification of isolates included conventional biochemical testing, gas-liquid chromatography, and 16S rDNA sequencing. Isolates identified as Clostridium species were screened for toxin production by Vero cell line and mouse biotoxin assays. Cultures that were toxic were neutralized with specific antitoxin and retested. Clostridium isolates were also subtyped and compared to a set of background strains by amplified fragment length polymorphism (AFLP) analysis. On the basis of the results of the diagnostic testing performed at all laboratory sites, one or more potential etiologies of soft tissue infection were identified for 55 (51%) of the 108 patients, including pathogenic clostridia (n ⫽ 24), S. aureus (n ⫽ 26), beta-hemolytic streptococci (n ⫽ 10), Streptococcus intermedius group (n ⫽ 3), and Bacillus cereus (n ⫽ 1) (Table 1) (14, 17). There was no evidence of B. anthracis in any of the specimens tested. Of the 24 (22%) patients in whom pathogenic clostridia were identified, 11 (10%) had Clostridium novyi infections, 7 (6%) had Clostridium perfringens infections, and 6 (6%) had both C. novyi and C. perfringens infections. Of these patients, one or more other organisms were also identified in 4 (17%) patients, including Clostridium sporogenes (n ⫽ 1), Clostridium scindens (n ⫽ 1), Clostridium argentinense (n ⫽ 1), and mixed flora (n ⫽ 1). The 26 (24%) patients with evidence of infection due to S. aureus and/or beta-hemolytic streptococci included 14 (13%) with S. aureus infections, 6 (6%) with both S. aureus and group A streptococcus infections, 2 (2%) with both S. aureus and group B streptococcus infections, and 3 (3%) with both S. aureus and group C streptococcus infections. Of the 26 patients infected with S. aureus and/or beta-hemolytic streptococci, 8 (31%) were also infected with one or more other organisms, including Escherichia coli (n ⫽ 1), Candida Table 1. Microbiologic findingsa among IDUs with severe illness or death, Scotland, Ireland and England, April to August 2000 No. (%) of patients positive for organism Organism Clostridium novyi type Ab Clostridium perfringensb,c Staphylococcus aureusc,d Group A streptococcid Group B streptococcid Group C streptococcid Streptococcus milleri Bacillus cereus Other organismse No organism identified a

Scotland (n ⫽ 60)

Ireland (n ⫽ 22)

England (n ⫽ 26)

13 (22) 10 (17) 12 (20) 3 (5) 1 (2) 1 (2) 2 (3) 1 (2) 5 (8) 23 (38)

2 (9) 0 (0) 7 (32) 0 (0) 1 (5) 2 (9) 1 (5) 0 (0) 1 (5) 11 (50)

2 (8) 3 (12) 7 (27) 3 (12) 0 (0) 0 (0) 0 (0) 0 (0) 7 (27) 7 (27)

Aerobic and anaerobic culture, immunohistochemistry, and 16S rDNA PCR. Six patients had evidence of both C. novyi and C. perfringens infections. c One patient had evidence of both C. perfringens and S. aureus infections. d Eleven patients had evidence of both S. aureus and beta-hemolytic streptococcus infections, including infections with group A streptococci (n ⫽ 6), group B streptococci (n ⫽ 2), and group C streptococci (n ⫽ 3). e Includes Clostridium sporogenes (n ⫽ 2), Clostridium innocuum (n ⫽ 1), Clostridium roseum (n ⫽ 1), Clostridium orbiscindens (n ⫽ 1), Candida species (n ⫽ 2), coagulase-negative staphylococcus (n ⫽ 1), and mixed flora (n ⫽ 5). b


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albicans (n ⫽ 1), coagulase-negative staphylococci (n ⫽ 1), Enterococcus species (n ⫽ 1), viridans group streptococci (n ⫽ 1), Candida species and Clostridium subterminale (n ⫽ 2), and mixed flora (n ⫽ 1). Although one patient was infected with both C. perfringens and S. aureus, none of the remaining patients infected with pathogenic clostridia had evidence of infection with S. aureus, beta-hemolytic streptococci, or any other likely pathogen. SUBTYPING AND TOXIN TESTING All of the 14 C. novyi isolates available for testing showed evidence of toxin production on the basis of mouse or Vero cell culture biotoxin assays. None of the four C. perfringens or five nonpathogenic clostridial isolates tested showed evidence of toxin production, and toxin could not be detected in four serum specimens evaluated by mouse biotoxin assay. One isolate of group A streptococci obtained from both blood and injection site tissue of a patient with a fatal case was identified as a toxin-producing strain that expressed M type 43. All C. novyi isolates were identified as type A by conventional biochemical testing and 16S rDNA sequencing. Of the 17 C. novyi isolates obtained from IDUs meeting the case definition between April and August 2000 and available for subtyping by AFLP analysis, all were designated type A and were indistinguishable from each other. By contrast, only 3 (12%) of 26 C. novyi isolates obtained from a comparison collection of human (n ⫽ 18), animal (n ⫽ 5), environmental (n ⫽ 1), or unknown (n ⫽ 2) sources were AFLP type A. In addition, the C. perfringens isolates obtained from 13 Scottish, Irish, or English IDUs during the outbreak period revealed 11 different AFLP types (18). There was no evidence that the pathogenic clostridia identified in tissue specimens could be attributed to postmortem contamination. For 14 (58%) of the 24 patients in whom C. novyi and/or C. perfringens was identified, the clostridia were obtained from premortem tissue, and for 23 (96%) of these patients there was no evidence of nonclostridial organisms (e.g., bowel or oral flora). In addition, although many Clostridium species can be found in the human gastrointestinal tract, C. novyi is not considered part of the normal human flora and would be an unlikely postmortem contaminant. 16S rDNA SEQUENCING Broad-range 16S rDNA PCR and sequencing performed with wound-site tissue specimens from 17 patients yielded a specific organism from 9 patients (53%), including C. novyi (7 of 17; 41%), C. perfringens (1 of 17; 6%), and C. subterminale (1 of 17; 6%) (Table 2). Of the eight patients from whom pathogenic clostridia were identified by 16S rDNA sequencing, the same organism was cultured from fresh tissue from seven (88%) patients. For one Irish patient from whom an organism could not be cultured, the only evidence of C. novyi infection was based on sequencing of multiple recombinant clones from repeated PCR amplifications with DNA extracted from paraffin-embedded tissue. Contrary to what might be expected, investigators had similar success amplifying clostridia from pre- versus postmortem and fixed versus frozen tissues. Broad-range 16S rDNA PCR was negative with tissue from 5 (29%) IDUs. For one of these patients, 16S rDNA PCR was negative with fixed tissue, while C. novyi was cultured from a frozen specimen that was not available for PCR. Amplification products were


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Table 2. Results of 16S rDNA PCR with wound-site tissue specimens from IDUs in Scotland and Ireland, by tissue type and outbreak site No. (%) of patients positive for organism Organism

Any (n ⫽ 17)

Premortem (n ⫽ 11)

Postmortem (n ⫽ 6)

Frozen (n ⫽ 12)

Fixed (n ⫽ 5)

Scotland (n ⫽ 11)

Ireland (n ⫽ 6)

C. novyi C. perfringens C. subterminale More than one organisma Negativeb

7 (41) 1 (6) 1 (6) 3 (18) 5 (28)

4 (36) 1 (9) 1 (9) 1 (9) 4 (36)

3 (50) 0 (0) 0 (0) 2 (33) 1 (17)

5 (42) 1 (8) 1 (8) 1 (8) 4 (33)

2 (40) 0 (0) 0 (0) 2 (40) 1 (20)

6 (55) 1 (9) 0 (0) 0 (0) 4 (36)

1 (17) 0 (0) 1 (17) 3 (50) 1 (17)

a

Amplification products were obtained but could not be sequenced, suggesting either the presence of more than one organism or degration of the nucleic acid due to formalin fixation. Organisms isolated from other tissue specimens obtained from these patients included S. aureus and a group C streptococcus (one patient) and mixed flora (one patient). For the third patient only fixed tissue was available for evaluation, precluding isolation by culture. b Organisms isolated from other tissue specimens obtained from these patients included C. novyi (one patient), C. perfringens (one patient), S. aureus (one patient), and S. aureus and a group C streptococcus (one patient).

obtained, but a unique sequence could not be resolved from another 3 (18%) patients, suggesting either the presence of more than one organism or degradation of the nucleic acid due to formalin fixation. Specimens from Ireland were more likely to result in products that could not be sequenced, potentially reflecting mixed infections that were not part of the outbreak or differences in tissue handling. Broad-range 16S rDNA PCR played an important role in directing the initial outbreak investigation. The first Clostridium species from this outbreak was isolated at a Glasgow microbiology laboratory on 10 May. However, that laboratory did not have the capacity to identify C. novyi, and the pathogenic significance of that isolate remained unclear. Thus, the first evidence of the role of C. novyi in the outbreak came from 16S rDNA PCR performed with postmortem tissue on 31 May. It was not until 13 June that typing of the initial isolate from Glasgow confirmed the presence of C. novyi from anaerobic culture. The early amplification of C. novyi from fixed tissue by broad-range 16S rDNA PCR highlighted the importance of (i) obtaining fresh tissue for anaerobic culture and (ii) providing adequate empiric coverage against anaerobic organisms for patients with potential cases. Given the difficulties in identifying C. novyi by phenotypic methods, 16S rDNA PCR also played an essential role in identifying the clostridia isolated during the investigation. LABORATORY EVALUATION OF ENVIRONMENTAL SPECIMENS FROM SCOTLAND AND IRELAND Heroin samples thought to be associated with the outbreak were evaluated by aerobic and anaerobic culture with various combinations of diluents, filters, media, and conditions. Several samples were also submitted to the U.S. Drug Enforcement Agency (DEA) for high-pressure liquid chromatography, mass spectrometry, and nuclear isotope analysis to assess whether they had originated from a common region and were derived from a similar batch. Although 20 heroin specimens were obtained for evaluation, only 6 contained sufficient quantities for analysis. Of these, four could be directly linked to a case patient, and


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none were linked to a patient with laboratory evidence of clostridial infection. Aerobic and anaerobic cultures of the six heroin specimens yielded four nonpathogenic Bacillus species and two C. perfringens isolates. No C. novyi strains were isolated from the heroin specimens. Only three heroin specimens had sufficient quantities for subsequent analysis at DEA, including one specimen from a case patient in Ireland and two specimens from Scottish IDUs not associated with this outbreak. These specimens had alkaloid purities ranging from 31 to 61%, and all were consistent with a DEA classification of Southwest Asia type A, the site of origination of the majority of heroin sold in Scotland and Ireland. However, on the basis of further analysis, it is highly probable that these three specimens were from different batches of heroin. Given the diversity of sources for the specimens, the significance of this finding is unknown. RISK FACTORS FOR DISEASE A case-control study was performed in Glasgow to identify specific behaviors or exposures that may have been associated with disease. All Scottish patients meeting the clinical case definition, regardless of laboratory findings, were eligible for enrollment. Three controls who injected drugs and lived in the same neighborhoods were enrolled for each case patient. Case patients and controls were interviewed in person regarding details of their drug use, including the types and source of drugs used, the mode of administration, the use of other substances and equipment (e.g., citric acid, solvents, cutting agents, filters, needles, and syringes), and recent changes in behavior. Questions were also asked about demographics, living conditions, and other recent illnesses or exposures. For deceased case patients, surrogates who were familiar with the routine habits and exposures of the patients were interviewed. Overall, 50 (83%) of the 60 case patients investigated in Scotland and 144 controls were enrolled in the study. Of note, only 46% of the enrolled patients were male compared to 75% of the controls (Fisher exact test, P ⬍ 0.01). Although exact numbers are not available, it is believed that the vast majority of Glasgow’s IDU population is male. There was no difference in the median ages of the cases and controls. After adjustment for all other significant factors and confounders, injecting drug into skin or muscle (i.e., “popping”) was the most important independent risk factor for disease during this outbreak (Table 3). Injecting drug with a used filter also independently increased the risk of disease. Despite its inverse relationship with heroin injection, the use of cocaine remained associated with a lower risk of disease. A case-control study performed in Dublin also found that injection of drugs into a muscle was the most significant risk factor for disease (L. Mullen, J. Barry, D. Igoe, E. Keenan, K. Murray, and M. Ward, Program Conf. Clostridium novyi and Co [http://www.iduoutbreak.abelgratis.com]). The association with injection into tissue is consistent with findings from previous outbreaks of clostridial infections among IDUs. For example, recent studies from California found that injection of drugs into skin or muscle significantly increased the overall risk of soft tissue infections (1) and was associated with specific outbreaks of botulism and tetanus (3, 19, 25). The investigators suggested that the subcutaneous space may provide a hospitable environment for pathogenic organisms that are introduced by nonsterile injection of drugs, contaminants, or fillers. Furthermore, several chemical agents used to dis-


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Table 3. Independent risk factors for severe illness or death among IDUs, Scotland, April to August 2000 Risk factor

ORa

95% confidence interval

Injects heroin into skin or muscle Injects with used filter Age ⱖ30 yr Female Uses cocaine

18.3 21.4 5.4 4.6 0.2

4.0–83.9 4.0–116.0 1.4–20.6 1.0–21.8 0.0–0.8

a

OR, odds ratio adjusted for all other variables included in the model.

solve the drugs may cause local tissue reactions (e.g., vasoconstriction or ischemia) that predispose the individual to infection with anaerobic organisms. Reuse of needles and other drug paraphernalia has also been associated with an increased risk of bacterial infections among IDUs, but not specifically infections due to clostridia (9). In this outbreak, one might speculate that reused and shared filters provided a potential source for the dissemination and germination of clostridial spores. ETIOLOGY AND SOURCE OF THE OUTBREAK Wound-site infections and deaths due to overdose are both relatively common events among IDUs, providing a constant background of activity from which it may be difficult to discern an unusual event. Up to 32% of IDUs, particularly those who inject drugs subcutaneously or intramuscularly, have soft tissue abscesses or cellulitis at any given time, with S. aureus and streptococcal species being the most commonly identified etiologies (1, 2, 24). Furthermore, because of the risk of polymicrobial contamination, defining a primary causative pathogen for wound infections among IDUs can be difficult (23). Given the prevalence of wound infections in this population, it is likely that some of the cases included in this investigation represent sporadic infections due to various pathogens. However, during the spring of 2000, the combination of a significant increase in overall deaths among IDUs and a unique cluster of illnesses within relatively small areas in Glasgow, Dublin, and northwest England suggested a possible point-source outbreak. The distinct clinical presentations and diagnostic laboratory findings for a subset of these patients provided evidence that histotoxic clostridia, and C. novyi in particular, played a primary role in this outbreak. HISTOTOXIC CLOSTRIDIA The members of the Clostridium genus are generally anaerobic, spore-forming, grampositive rods. However, some species are aerotolerant, and many appear as gram-negative rods on smears from gangrenous lesions or in vitro after overnight culture (22). Clostridium spores are widespread throughout nature and are readily found in the soil worldwide. Some species such as C. perfringens are also part of the normal intestinal flora. C. perfringens, C. histolyticum, and C. septicum are the classic causes of gas gangrene, which can result in fulminant shock through direct toxigenic effects on myocardial contractility. However, these organisms typically result in extensive tissue necrosis and gas production


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as well, features that were not as prominent in the current cases. C. sordellii can also cause a distinctive, toxin-mediated illness characterized by tissue edema, myonecrosis, leukemoid reaction, and sudden onset of shock and has been implicated in a recent outbreak among IDUs in California (15, 16; A. C. Kimura, J. I. Higa, R. M. Levin, and D. J. Vugia, Program 39th Annu. Meet. Infect. Dis. Soc. Am., 2001). Several other Clostridium species (e.g., C. tetani and C. botulinum) have been implicated in clusters of wound infections among IDUs, but their neurotoxic effects result in distinctive clinical presentations (3, 19, 25). Finally, the finding of several Clostridium species in the tissue of patients with injection-site wound infections is well described, and although several of these species (e.g., C. innocuum, C. scindens, and C. subterminale) are considered nonpathogenic, investigators have speculated that certain organisms (i.e., C. sporogenes) may contribute to an increased severity of illness when they are associated with pathogenic clostridia (11). Although C. novyi has been described as a significant pathogen in traumatic wound infections, perhaps because of its fastidious and strict anaerobic growth requirements, it is less commonly isolated than other clostridial species, especially in pure culture (11, 15). The attention paid to anaerobic culture technique and the use of 16S rDNA PCR during this investigation may account for the significant number of cases of infection associated with C. novyi and/or C. perfringens. However, clostridia are ubiquitous organisms that may be present in a large proportion of contaminated wounds, and it is the clinical presentation of individual cases that most directly suggests the role of C. novyi in this outbreak. The distinctive clinical presentation of this cluster is consistent with the unique case descriptions of C. novyi infections, including marked edema with minimal gas production or necrosis, leukemoid reaction, and dramatic toxemia out of proportion to the obvious local lesions (11). C. novyi was identified in 16% (17 of 108) of the case patients, whereas C. perfringens (without C. novyi) was identified in 6% (7 of 108) and S. aureus was identified in 24% (26 of 108). However, patients with C. novyi and/or C. perfringens infections had more severe clinical courses (Table 4), and only C. novyi isolates showed evidence of toxin production by Vero cell and mouse biotoxin assays and clonal distribution on the basis of AFLP analysis. The identification of C. novyi in clinical and pathologic specimens suggests soil contamination of the drugs or other materials used by these IDUs, but it provides only circumstantial evidence as to the true source of the outbreak. On the basis of the epidemiologic data, sharing of utensils and soft tissue injection of a common batch of heroin were the likely sources of this outbreak. Microbial contamination can occur at one of many steps, Table 4. Case-fatality ratio and WBC count for IDUs with evidence of infection due to C. novyi, C. perfringens, or S. aureus, Scotland and Ireland, April to August 2000 Organism

No. (%) of patients who died

Median (range) WBC count (no. of cells/mm3)a

Histotoxic clostridia (n ⫽ 24) S. aureus (n ⫽ 26)

12 (50)b 9 (35)

39,500 (6,000–82,000)c 13,200 (1,900–153,000)

a

Data were available for 20 (83%) patients infected with histotoxic clostridia and all patients infected with S. aureus. Mantel-Haenszel P value of 0.3 for difference in case-fatality ratio between patients with evidence of C. novyi and/or C. perfringens infection versus patients with evidence of S. aureus infection. c Kruskal-Wallis P value of 0.03 for difference in median WBC count between patients with evidence of C. novyi and/or C. perfringens infection versus patients with evidence of S. aureus infection. b


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including production, mixing, dilution, or preparation of the drugs, or at the time of injection through contaminated diluents, paraphernalia, or skin. Unfortunately, the small quantities of heroin available for evaluation make it difficult to prove a direct microbiologic link between the drugs and the illnesses. CONCLUSIONS Although outbreaks of clostridial infections among IDUs have been well described, to our knowledge this is the first such outbreak that has been associated with C. novyi. Furthermore, we believe that this is the first time that broad-based 16S rDNA PCR has helped identify a causative agent during an acute outbreak of unexplained infectious disease. 16S rDNA PCR played a key role both in the evaluation of clinical samples and in the genotypic typing of an organism that is otherwise difficult to identify. Finally, in addition to typical issues of data and specimen management and diagnostic testing, outbreaks among IDUs pose unique medical and legal challenges similar to those experienced following the recent acts of bioterrorism. This investigation required the rapid integration of a large amount of clinical, laboratory, and epidemiologic resources and extensive collaboration among local, regional, and international public health agencies and law enforcement authorities. Improving our ongoing capacity to evaluate cases of unexplained infectious disease will streamline and simplify the response to these public health emergencies when they occur. Acknowledgments. This investigation was a collaborative effort by the Greater Glasgow Health Board, Scottish Center for Infection and Environmental Health, Eastern Regional Health Authority (Ireland), National Disease Surveillance Center (Ireland), Public Health Laboratory Service (United Kingdom), Centers for Disease Control and Prevention (United States), and numerous public health agencies and clinical institutions. The members of the International Outbreak Investigation Team included J. Lingappa, K. Murray, S. Reagan, S. Zaki, W.-J. Shieh, J. Guarner, H. Holmes, D. Whaley, R. Weyant, R. Meyer, M. Bowen, T. Popovic, L. Mayer, S. Maslanka, J. Tappero, B. Perkins, L. Gruer, C. McGuigan, G. Penrice, K. Roberts, J. Hood, P. Redding, G. Edwards, M. Black, J. McFarlane, D. Cromie, H. Howie, A. Leonard, D. Goldberg, A. Taylor, S. Hutchinson, K. Roy, S. Wadd, R. Andraghetti, G. Sayers, M. Cronin, T. O’Connell, M. Ward, P. O’Sullivan, B. O’Herlihy, E. Keenan, J. O’Connor, L. Mullen, B. Sweeney, D. O’Flanagan, D. Igoe, C. Bergin, S. O’Briain, C. Keane, E. Mulvihill, P. Plunkett, G. McMahon, T. Boyle, S. Clarke, E. Leen, J. Connolly, M. Cassidy, T. Djuretic, N. Gill, V. Hope, G. Nichols, A. Weild, R. George, P. Borriello, J. Brazier, J. Salmon, N. Lightfoot, A. Roberts, J. McLauchlin, and M. Brett. REFERENCES 1. Binswanger, I. A., A. H. Kral, R. N. Bluthenthal, D. J. Rybold, and B. R. Edlin. 2000. High prevalence of abscesses and cellulitis among community-recruited injection drug users in San Francisco. Clin. Infect. Dis. 30:579–581. 2. Centers for Disease Control and Prevention. 2001. Soft tissue infections among injection drug users—San Francisco, California, 1996–2000. Morbid. Mortal. Wkly. Rep. 50:381–384. 3. Centers for Disease Control and Prevention. 1998. Tetanus among injecting-drug users—California, 1997. Morbid. Mortal. Wkly. Rep. 47:149–151. 4. Centers for Disease Control and Prevention. 2000. Unexplained illness and death among injecting-drug users-Glasgow, Scotland; Dublin, Ireland; and England, April–June 2000. Morbid. Mortal. Wkly. Rep. 49:489–492. 5. Centers for Disease Control and Prevention. 2000. Update: Clostridium novyi and unexplained illness among injecting-drug users—Scotland, Ireland, and England, April–June 2000. Morbid. Mortal. Wkly. Rep. 49:543.


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6. Centers for Disease Control and Prevention. 2001. Update: investigation of bioterrorism-related anthrax and interim guidelines for clinical evaluation of persons with possible anthrax. Morbid. Mortal. Wkly. Rep. 50:941–948. 7. Communicable Disease Surveillance Center. 2000. Serious unexplained illness among drug injectors. Communicable Dis. Rep. Wkly. 10:195. 8. Eastern Regional Health Board, National Diseases Surveillance Centre Dublin. 2000. Deaths from unexplained illness in heroin users in Dublin. Eurosurveillance Wkly. 4:000601. [Online.] http://www.eurosurv.org/ 2000/000601.htm. 9. Gershon, R. R. M. 1998. Infection control basis for recommending one-time use of sterile syringes and aseptic procedures for injection drug users. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 18(Suppl. 1):S20–S24. 10. Greater Glasgow Health Board. 2000. Unexplained illness among injecting drug injectors in Glasgow. Eurosurveillance Wkly. 4:000518. [Online.] http://www.eurosurv.org/2000/000518.htm. 11. Hill, G. B., and H. P. Willett. 1992. Clostridium, p. 636–656. In W. K. Joklik, H. P. Willett, D. B. Amos, and C. M. Wilfert (ed.), Zinsser Microbiology, 20th ed. Appleton & Lange, Norwalk, Conn. 12. Hoiby, E. A. 2000. Systemic anthrax in an injecting drug user: Oslo, Norway—April 2000. Eurosurveillance Wkly. 4:000511. [Online.] http://www.eurosurv.org/2000/000511.htm. 13. Jernigan, J. A., D. S. Stephens, D. A. Ashford, C. Omenaca, M. S. Topiel, M. Galbraith, M. Tapper, T. L. Fisk, S. Zaki, T. Popovic, R. F. Meyer, C. P. Quinn, S. A. Harper, S. K. Fridkin, J. J. Sejvar, C. W. Shepard, M. McConnell, J. Guarner, W.-J. Shieh, J. M. Malecki, J. L. Gerberding, J. M. Hughes, B. A. Perkins, and members of the Anthrax Bioterrorism Investigation Team. 2001. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg. Infect. Dis. 7:933–944. 14. Jones, J. A., J. E. Salmon, T. Djuretic, G. Nichols, R. C. George, O. N. Gill, J. S. Brazier, M. M. Brett, B. I. Duerden, N. K. Fry, V. Hall, V. Hope, A. Lieftucht, J. McLauchlin, P. G. Pitcher, and A. Wield. 2002. An outbreak of serious illness and death among injecting drug users in England during 2000. J. Med. Microbiol. 51:978–984. 15. MacLennan, J. D. 1962. The histotoxic clostridial infections of man. Bacteriol. Rev. 26:177–276. 16. McGregor, J. A., D. E. Soper, G. Lovell, and J. K. Todd. 1989. Maternal deaths associated with Clostridium sordellii infection. Am. J. Obstet. Gynecol. 161:987–995. 17. McGuigan, C. C., G. M. Penrice, L. Gruer, S. Ahmed, D. Goldberg, M. Black, J. E. Salmon, and J. Hood. 2002. Lethal outbreak of infection with Clostridium novyi type A and other spore-forming organisms in Scottish injecting drug users. J. Med. Microbiol. 51:971–977. 18. McLauchlin, J., J. E. Salmon, S. Ahmed, J. S. Brazier, M. M. Brett, R. C. George, and J. Hood. 2002. Amplified fragment length polymorphism (AFLP) analysis of Clostridium novyi, C. perfringens and Bacillus cereus isolated from injecting drug users during 2000. J. Med. Microbiol. 51:990–1000. 19. Passaro, D. J., S. B. Werner, J. McGee, W. R. MacKenzie, and D. J. Vugia. 1998. Wound botulism associated with black tar heroin among injecting drug users. JAMA 279:859–863. 20. Reingold, A. L., N. T. Hargrett, B. B. Dan, et al. 1982. Nonmenstrual toxic shock syndrome: a review of 130 cases. Ann. Intern. Med. 96:871. 21. Scottish Centre for Infection and Environmental Health. 2000. Serious unexplained illness among drug injectors in Scotland and Ireland: update as at 30th May 2000. SCIEH Wkly. Rep. 34(21):121. 22. Stevens, D. L., and J. I. Rood. 2000. Histotoxic clostridia, p. 563–572. In V. A. Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Rood (ed.), Gram-Positive Pathogens. American Society for Microbiology, Washington, D.C. 23. Summanen, P. H., D. A. Talan, C. Strong, M. McTeague, R. Bennion, et al. 1995. Bacteriology of skin and soft-tissue infections: comparison of infections in intravenous drug users and individuals with no history of intravenous drug use. Clin. Infect. Dis. 20(Suppl. 2):S279–S282. 24. Vlahov, D., M. Sullivan, J. Astemborski, and K. E. Nelson. 1992. Bacterial infections and skin cleaning prior to injection among intravenous drug users. Public Health Rep. 107:595–598. 25. Werner, S. B., D. Passaro, J. McGee, R. Schechter, and D. J. Vugia. 2000. Wound botulism in California, 1951–1998: recent epidemic in heroin injectors. Clin. Infect. Dis. 31:1018–1024. 26. The Working Group on Severe Streptococcal Infections. 1993. Defining the group A streptococcal toxic shock syndrome. JAMA 269:390–391.


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Chapter 10

Travel-Associated Fungal Infections Rana A. Hajjeh and David W. Warnock

Endemic mycoses have a restricted geographic distribution, being largely confined to areas of the world where the etiologic agents are found in nature. In recent years, however, increased domestic and international travel has led to a rise in the number of reported outbreaks and sporadic cases of histoplasmosis and coccidioidomycosis among individuals who normally reside in places far from the areas where these diseases are endemic. Travelers have developed these and other fungal diseases as a result of a wide range of recreational and work activities, many of which have involved well-recognized risk factors for these infections. In some instances, the infection was acquired during a short trip (travelrelated infection), but in others, the disease has followed a longer period of residence in an area where the disease is endemic with subsequent migration to a region where the disease is not endemic (“migration-related” infection). In many cases of travel-related fungal disease, individuals have presented with nonspecific symptoms and signs of acute respiratory illness shortly after their return. In others, however, the disease may remain silent until months or years later. Because they are usually easier to detect, more cases of travelrelated fungal disease have been identified in outbreak settings than in sporadic circumstances. Indeed, recent outbreak reports have served to heighten the awareness of fungal infections among returning travelers by health care providers. The most commonly reported travel-related mycoses have been histoplasmosis and coccidioidomycosis; but cases of penicilliosis have occurred among travelers returning from Southeast Asia, and occasional migration-related cases of other diseases, such as paracoccidioidomycosis, have been reported. The largest number of travel-related mycoses has been described among U.S. residents, many of whom have acquired an infection, such as histoplasmosis, during a visit to an area of endemicity within North or Central America or, less commonly, South America, Africa, or Asia. Travel-related mycoses have also been reported among international visitors to North America or to countries in Latin America, Africa, and Asia. Most of these infections have occurred among travelers returning to European countries, Australia, or Japan. However, with increasing numbers of visitors Rana A. Hajjeh and David W. Warnock • Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333. 133


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and immigrants to North America from other Asian countries, travel-related and migration-related endemic mycoses are now being described in countries such as India. In this chapter we focus on fungal infections acquired during recent travel abroad rather than migration-related infections. HISTOPLASMOSIS Histoplasmosis is caused by Histoplasma capsulatum var. capsulatum, a dimorphic saprobic fungus which grows best in soil enriched with bat or bird droppings. In the United States, the disease is most prevalent in the midwestern and central states. It is also endemic in many parts of Mexico, Central and South America (Fig. 1), as well as parts of southern and eastern Europe, Africa, eastern Asia, and Australia (7). Human infection follows inhalation of airborne spores that have been released during disturbance of contaminated soil or accumulation of droppings. Exposures may occur during activities such as building construction, renovation, and demolition; soil excavation; spelunking; and cleaning of sites harboring the fungus (7). The risk of infection depends on the activities performed and the duration and degree of exposure. Longer and more intense exposures usually result in more severe acute pulmonary disease. The spectrum of illness is wide. In some cases the infection is asymptomatic, but in returning travelers it usually presents as a flu-like illness characterized by high-grade fever, chills, headache, nonproductive cough, myalgia, pleuritic chest pain, loss of appetite, and fatigue. Chest radiographs often reveal hilar lymphadenopathy and diffuse reticulonodular infiltrates. Symptoms typically develop 1 to 3 weeks after exposure, and most individuals recover spontaneously within 2 to 3 weeks, although fatigue may persist for a longer duration. Disseminated infection is a rare complication but can occur in persons with severe immune deficits (e.g., persons with human immunodeficiency virus [HIV] infection). Individuals can be reinfected with H. capsulatum with intense exposure, in which case the incubation period may be as short as 3 days. Histoplasmosis has long been recognized as a common recreational disease among spelunkers (cavers) in North America, 60 to 64% of whom have been found to have a positive skin test reaction to histoplasmin (2, 20). Outbreaks of histoplasmosis have been reported among traveling U.S. residents after spelunking trips to bat-infested caves in Latin America (24, 31). Recently, with ecotourism and adventure tourism becoming more popular, histoplasmosis has also been recognized as a cause of respiratory illness among such travelers. A recently reported outbreak (32a) involved 15 U.S. travelers who went on a field trip to Nicaragua and engaged in various activities which included visiting a silver mine that was contaminated with bat guano. Within 3 days of returning home, 12 (80%) developed high fever, headache, and malaise, and 6 (40%) required hospitalization. A very high attack rate was noted during this outbreak, as 14 (93%) members of the group had laboratory-confirmed acute histoplasmosis. Not all outbreaks of travel-related histoplasmosis are related to obvious environmental exposures. In March 2001, a large outbreak of the disease occurred among U.S. college students who developed an acute respiratory febrile illness within 1 to 2 weeks after their return from Acapulco, Mexico, where they had spent spring break vacation (J. Morgan, M. V. Cano, D. R. Feikin, M. Phelan, O. Velazquez Monroy, P. Kuri Morales, J. Carpenter, A.


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Figure 1. Geographic distribution of H. capsulatum (light gray) and C. immitis (dark gray) infections in the Americas (subject to limitations of surveillance and reporting). Although C. immitis is restricted in distribution to the Americas, infections due to H. capsulatum have been reported from parts of southern and eastern Europe, parts of Africa, eastern Asia, and Australia (7); however, these reports are usually limited to single or a few cases, often with no clear source of exposure, and no outbreaks were reported outside the Americas, which makes it very difficult to accurately map the worldwide distribution of infection with this organism.

Weltman, P. G. Spitzer, H. H. Liu, S. A. Mirza, D. E. Bronstein, D. J. Morgan, L. A. Kirkman, M. E. Brandt, N. Iqbal, M. D. Lindsley, D. W. Warnock, and R. A. Hajjeh, submitted for publication). Students from 44 colleges in 24 states and Washington, D.C., as well as a few nonstudent travelers, developed acute pulmonary histoplasmosis. None of the travelers had exposures typically associated with acquisition of histoplasmosis, but most had resided


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in or visited one particular hotel that was undergoing internal construction work during the spring break period. Epidemiologic and environmental investigations suggested that many travelers were infected within the hotel building, with the most likely source of infection being exposure to dust generated during building renovation and construction activities. Tissue samples are rarely obtained from patients with acute pulmonary histoplasmosis for histopathologic examination and culture. Therefore, the diagnosis of acute disease in returning travelers is usually dependent on serologic tests, such as immunodiffusion (ID) and complement fixation (CF) tests, or on antigen detection. Because two serum samples (acute- and convalescent-phase serum samples) are required for unequivocal confirmation of acute infection, the ID and CF tests are not ideal for making an early diagnosis. The CF test is more sensitive but less specific than the ID test. It becomes positive between 2 and 6 weeks following infection; a titer of at least 1:32 or a fourfold increase in titer between paired acute- and convalescent-phase serum samples is considered strong presumptive evidence of infection (34). The ID test is more specific but less sensitive than the CF test. When histoplasmin is used as the antigen, two major precipitin bands can be detected by the ID test. The M band can be detected in up to 75% of cases of acute histoplasmosis but can persist for many months after the initial infection. The H band is specific for acute disease but occurs in only 10 to 20% of cases. Antigen detection in urine is sometimes useful for the diagnosis of acute pulmonary histoplasmosis, provided samples are obtained within 2 weeks after exposure (5); otherwise, the sensitivity of this test for diagnosis of acute primary histoplasmosis is low. The histoplasmin skin test has also been previously used in epidemiologic studies to detect past exposure to the fungus; however, the reagent is not available in the United States. All diagnostic tests for histoplasmosis require cautious interpretation, because cross-reactivity can occur with other endemic mycoses, such as blastomycosis and coccidioidomycosis, and prior infection or residence in an area of endemicity can result in the presence of low titers of antibody to H. capsulatum. The Infectious Diseases Society of America (IDSA) has developed guidelines for the management of histoplasmosis (33). Antifungal treatment is not usually indicated for persons with acute, localized pulmonary infection, because this form of the disease is selflimited and usually resolves within 3 weeks. For persons with severe symptoms who do not improve after 1 month of observation, itraconazole (200 mg once daily) can be given for 6 to 12 weeks. In our recent experience during outbreaks, more physicians are opting to treat acute pulmonary histoplasmosis at an earlier stage; however, no clinical trials have been conducted to assess whether such treatment is effective in reducing the severity or duration of symptoms or in preventing complications. For persons with underlying immune deficiency or chronic disease, itraconazole should be given for longer periods. All persons with severe disease, including diffuse pulmonary and disseminated histoplasmosis, should be treated with either amphotericin B or itraconazole. Prevention of sporadic exposure to sources of H. capsulatum in areas where it is endemic is difficult. Nonetheless, individuals who are at increased risk for severe infection, especially immunocompromised persons, should be advised to avoid situations in which contaminated material can become aerosolized. If, however, exposure cannot be avoided, travelers should be advised to wear masks and special protective equipment (7). After engaging in high-risk activities, such as cave exploration, it is prudent to hose off boots and place clothing in airtight plastic bags to be laundered. Transportation of soil,


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guano, and other potential fomites should be avoided. Public health authorities need to place warning signs at known high-risk locations. COCCIDIOIDOMYCOSIS Coccidioidomycosis is caused by Coccidioides immitis, a dimorphic saprobic fungus found in the soil of certain arid semidesert regions of the southwestern United States (southern Arizona, central and southern California, southern New Mexico, western Texas, and parts of Utah). The region of endemicity extends southwards into the desert regions of northern Mexico and parts of Central and South America (Fig. 1). Molecular analysis has shown that strains of C. immitis from California are distinct from those from other geographic areas. It has recently been proposed that the organism be separated into two species, C. immitis and Coccidioides posadasii, the former name now being restricted to isolates from California (18). Isolates of both species cause identical forms of clinical disease. Human infection follows inhalation of airborne arthrospores that have been dislodged from the soil following ground-disturbing activities, such as building construction, landscaping, farming, archaeological excavation, and numerous recreational pursuits (22). Natural events that result in the generation of large amounts of dust, such as earthquakes and windstorms, also increase the risk of infection and have resulted in large outbreaks (29). Approximately 40% of infected persons develop a flu-like illness, usually 1 to 3 weeks after exposure. This is typically characterized by fever, headache, chest pain, nonproductive cough, myalgia, arthralgia, weight loss, and malaise. Up to 50% of patients develop a mild diffuse erythematous or maculopapular rash covering the trunk and limbs within the first few days of the onset of symptoms. About 5% develop erythema nodosum or erythema multiforme. Most otherwise healthy persons recover without treatment, their symptoms disappearing in a few weeks. In rare instances, individuals have developed severe pulmonary disease (e.g., cavitary pneumonia) or dissemination to the central nervous system (e.g., the meninges), joints, bones, and skin (22). Those at increased risk for disease dissemination include African Americans, Asians and Filipinos, and persons with immunodeficiency (e.g., persons with HIV infection and those receiving immunosuppressive medications), as well as pregnant women in the third trimester. Persons with diabetes mellitus and other chronic medical conditions can be at increased risk for severe pulmonary disease (28). Unlike histoplasmosis, once individuals have been infected with C. immitis, they are usually immune to reinfection. Coccidioidomycosis has long been recognized as a travel-related mycosis associated with visits to areas of endemicity in southern California, Arizona, and other neighboring states. Several recent reports have begun to document the extent of the problem. Between 1992 and 1997, 161 individuals in New York State were discharged from hospitals with a diagnosis of coccidioidomycosis; all patients for whom travel information was available had reported travel to areas where the disease is endemic, most notably, the southwestern United States (12). Between 1980 and 1998, 23 cases of coccidioidomycosis were diagnosed among patients at the Cleveland Clinic in Ohio (13). With increasing domestic and international travel to areas of endemicity, as well as increased awareness of the disease among physicians and the public, an increase in the number of cases of coccidioidomycosis throughout the United States seems almost inevitable.


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Travelers from the United States to areas of endemicity in Latin America have also succumbed to coccidioidomycosis following activities that resulted in massive exposures to dust. In recent years, Mexico has become the country most frequently visited by Americans, and several outbreaks of the disease have been reported among returning travelers. In 1996, 21 (17%) cases of coccidioidomycosis occurred among a group of 126 church group members from Washington State who had recently returned from a 6-day stay at an orphanage near Tecate, Mexico (6). Members of the group had assisted with construction projects requiring excavation. More recently, 35 church group members from Pennsylvania traveled to Hermosillo, Mexico, to assist with a church construction project (8). Within 2 weeks of returning home, many of the travelers developed a flu-like illness and sought health care, and 8 (23%) had serologically confirmed coccidioidomycosis. In both of these outbreaks, the diagnosis of coccidioidomycosis was only suspected, once a good travel history had been obtained. These outbreaks illustrate the importance of alerting travelers to regions where C. immitis is endemic to the risk of developing the disease and to its clinical manifestations. It is also clear that health care providers, especially those in areas where the disease is not endemic, need to have increased awareness of the risk of coccidioidomycosis among travelers to areas where the disease is endemic and need to be more familiar with this disease and its diagnosis and management. Visitors from other countries have also developed coccidioidomycosis after trips to the southwestern United States. In the past, most of these infections occurred among travelers returning to European countries (30), Japan (25), or Australia (3). More recently, however, changing migration patterns have resulted in several cases of coccidioidomycosis among Indian citizens who had previously resided and worked in Arizona (4, 32). In addition to these sporadic cases, an international outbreak of the disease was recently reported (9). In October 2001, more than 300 individuals from 30 countries participated in the World Championship of Model Airplane Flying in Lost Hills, Calif., an area where coccidioidomycosis is highly endemic. One week after returning home, a participant from the United Kingdom developed acute pneumonia but was not diagnosed with coccidioidomycosis until culture of bronchoalveolar lavage specimens was performed. Cases among participants were subsequently reported from Finland, Australia, and New Zealand (9). Again, this illustrates the need for clinicians to have a high index of suspicion for travelrelated mycoses so that appropriate serologic tests, which may afford an earlier diagnosis than culture, may be performed. In addition, early suspicion of coccidioidomycosis might help to ensure that clinicians alert microbiologists to the possibility of isolating C. immitis from patient specimens, thereby reducing the risk of laboratory-acquired infection. The diagnosis of acute pulmonary coccidioidomycosis can be made by direct microscopic examination of lower respiratory tract specimens for C. immitis spherules or by culture of such specimens. Serologic tests are also invaluable in making the diagnosis. The immunodiffusion tube precipitin (IDTP) test, which uses heated coccidioidin as antigen, detects immunoglobulin M (IgM) antibodies to C. immitis and is most useful for the diagnosis of recent infections. These antibodies can be found within 1 to 3 weeks after the onset of symptoms but disappear within a few months among persons with acute pulmonary disease (26). The sensitivity of the IDTP test can be improved by first concentrating the serum. The CF test measures IgG titers, but these antibodies do not appear until 4 to 12 weeks after infection and may persist for long periods in patients with chronic pulmonary or disseminated disease. High (ⱖ1:32) or rising CF test titers are consistent with


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spread of the disease beyond the respiratory tract. A latex agglutination test, which is more rapid than the IDTP test, is also available but has a ⬎5% rate of false-positive results (23). IDSA has developed guidelines for the management of coccidioidomycosis (19). Most patients with acute symptomatic infection do not require antifungal treatment because the illness is self-limited. However, treatment may be indicated for persons who are at increased risk of dissemination. The drug of choice is fluconazole (400 mg daily for 3 to 6 months). Patients with bilateral reticulonodular or miliary infiltrates should be treated with amphotericin B, as should pregnant women. Prevention of exposure to sources of C. immitis in areas where it is endemic is difficult. Nevertheless, travelers should be advised to decrease their risk by limiting their exposure to outdoor dust. The wearing of well-fitted masks or the use of outdoor vehicles with enclosed air-conditioned cabs can provide some protection (17), although the benefit of these measures has not been well documented. Groups undertaking construction work or other dust-generating activities should be informed about dust-control measures, which include the wetting of soils before disturbing the earth. Travelers should also be warned to avoid transporting soil and other potentially contaminated fomites (e.g., geologic specimens). PENICILLIOSIS Penicilliosis is a disseminated and progressive infection caused by the dimorphic fungus Penicillium marneffei. Infections with this pathogen are becoming increasingly common in Southeast Asia, particularly in northern Thailand and the southern part of the People’s Republic of China (Guang Xi Province), where P. marneffei is now recognized as one of the most frequently encountered opportunistic infections among persons with AIDS. However, indigenous cases of penicilliosis in apparently immunocompetent individuals have also been reported. The reservoirs of P. marneffei in nature and the exposures that lead to human infection with this organism are still incompletely understood, but P. marneffei is thought to be a saprobic soil fungus that is endemic throughout Southeast Asia, and inhalation of spores is believed to be the most common mechanism of infection. The incubation period of disseminated P. marneffei infection is variable. In some cases, symptomatic disease develops within a few weeks of exposure (11). In others, long periods of asymptomatic infection prior to reactivation have been reported (21). The first case of natural human infection with P. marneffei was reported in 1973. The patient was a 61-year-old man, a U.S. citizen, who had traveled to Southeast Asia. The infection was diagnosed when the patient, who had Hodgkin’s disease, presented with a splenic abscess (14). More than a decade elapsed before a second case was reported (27). Again, the patient was a male U.S. citizen who had traveled throughout Southeast Asia. Between 1988 (when the first cases of AIDS-associated penicilliosis were described) and 1994, 14 cases of the disease were reported among HIV-infected individuals returning to their native countries after visits to Southeast Asia (15). Members of this group included residents of Australia, France, Italy, The Netherlands, the United Kingdom, and the United States. Although it is now clear that the greatest impact of penicilliosis has been felt by the populations of Southeast Asia, cases of the disease continue to be diagnosed among returning travelers. The most common clinical features of penicilliosis include fever, marked weight loss, nonproductive cough, lymphadenopathy, hepatosplenomegaly, and anemia. About 60 to


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70% of patients present with multiple papular skin lesions, some of which show a central necrotic umbilication resembling molluscum contagiosum. These are often found on the face, neck, trunk, and upper limbs (16). Histoplasmosis and cryptococcosis occur in the same regions as P. marneffei infection and can cause similar necrotic skin lesions in persons with AIDS. Chest radiographs may reveal cavitary lesions or infiltrates. The diagnosis can be made by direct microscopic examination or culture of specimens from the bone marrow, lymph nodes, skin, and other infected sites. Microscopic examination of Wright-stained bone marrow smears or touch smears of skin or lymph node biopsy specimens can permit a rapid diagnosis if the characteristic round or oval cells of P. marneffei, often with prominent cross-walls, are seen. Culture is important because other fungal infections, such as histoplasmosis and cryptococcosis, may have similar clinical manifestations in immunocompromised persons. P. marneffei colonies produce a distinctive red pigment which diffuses into the agar. There are no widely available serologic tests for this disease. Amphotericin B is the drug of choice for the treatment of severe cases of P. marneffei infection. The usual regimen is 1 mg/kg of body weight daily for 2 weeks, after which itraconazole (200 to 400 mg daily) or ketoconazole (400 mg daily) should be given for a further 6 weeks. In milder infections, an azole agent can be used from the outset. Fluconazole appears to be less effective than itraconazole, but it may be useful in patients who fail to absorb the latter drug. The new triazole agent voriconazole is active against P. marneffei in vitro. Prevention of exposure to sources of P. marneffei in the environment is difficult. There is evidence of seasonal variation in the incidence of the infection in Thailand, with the highest number of cases occurring during the rainy season, from May to October (10), which suggests that the infection may be acutely acquired. To date, however, no definite environmental source or route of transmission has been identified. Immunocompromised individuals should be advised about the risk of developing penicilliosis when traveling to areas of Southeast Asia (e.g., eastern India [Manipur State], Indonesia, Laos, Malaysia, Myanmar, Taiwan, Thailand, and Vietnam) and the southern part of the People’s Republic of China, where P. marneffei is known to be endemic. OTHER INFECTIONS In addition to the three mycotic diseases described so far, occasional travel- and migrationrelated cases of a number of other subcutaneous and systemic fungal infections have been reported from a number of countries. As an example, paracoccidioidomycosis has been diagnosed in patients in North America, Europe, and Asia (1). However, all the reported cases have been diagnosed in persons who had lived in Latin America at some point before the diagnosis. In most instances, symptomatic disease developed years after a period of residence in an area of endemicity. CONCLUSIONS With international travel becoming more commonplace, a number of mycotic diseases are now emerging as important health threats among visitors to regions of the world where these infections are endemic. Travelers, especially those who are immunocompromised, need to be made aware of the risks of acquiring mycotic diseases when visiting such regions and of the measures that they can take to minimize their risk of infection. Recently,


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histoplasmosis and coccidioidomycosis have been included as travel-related illnesses in the Health Information for International Travel Yellow Book (available at http://www. cdc.gov/travel/diseases.htm). Health care providers need to be familiar with the presenting features, diagnosis, and treatment of these diseases and with the patient’s travel history, as well as history of previous residence in areas of endemicity. REFERENCES 1. Ajello, L., and L. Polonelli. 1985. Imported paracoccidioidomycosis: a public health problem in nonendemic areas. Eur. J. Epidemiol. 1:160–165. 2. Ashford, D. A., R. A. Hajjeh, M. F. Kelley, L. Kaufman, L. Hutwagner, and M. M. McNeil. 1999. Outbreak of histoplasmosis among cavers attending the National Speleological Society annual convention, Texas, 1994. Am. J. Trop. Med. Hyg. 60:899–903. 3. Baird, R. W., H. Teichtahl, H. M. Ednie, A. Tasiopoulos, N. Ryan, and D. Gee. 1999. A fluffy white traveler: imported Coccidioides immitis infection in an Australian tourist. Pathology 31:47–50. 4. Bharucha, N. E., K. Ramamoorthy, J. Sorabjee, and T. Kuruvilla. 1996. All that caseates is not tuberculosis. Lancet 348:1313. 5. Buxton, J. A., M. Dawar, L. J. Wheat, W. A. Black, N. G. Ames, M. Mugford, and D. M. Patrick. 2002. Outbreak of histoplasmosis in a school party that visited a cave in Belize: role of antigen testing in diagnosis. J. Travel Med. 9:48–50. 6. Cairns, L., D. Blythe, A. Kao, D. Pappagianis, L. Kaufman, J. Kobayashi, and R. Hajjeh. 2000. Outbreak of coccidioidomycosis in Washington State residents returning from Mexico. Clin. Infect. Dis. 30:61–64. 7. Cano, M. V. C., and R. A. Hajjeh. 2001. The epidemiology of histoplasmosis: a review. Semin. Respir. Infect. 16:109–118. 8. Centers for Disease Control and Prevention. 2000. Coccidioidomycosis in travelers returning from Mexico—Pennsylvania, 2000. Morb. Mortal. Wkly. Rep. 49:1004–1006. 9. Centers for Disease Control and Prevention. 2001. Coccidioidomycosis among persons attending the World Championship of Model Airplane Flying—Kern County, California, October 2001. Morb. Mortal. Wkly. Rep. 50:1106–1107. 10. Chariyalertsak, S., T. Sirisanthana, K. Supparatpinyo, and K. E. Nelson. 1996. Seasonal variation of disseminated Penicillium marneffei infections in northern Thailand: a clue to the reservoir? J. Infect. Dis. 173:1490–1493. 11. Chariyalertsak, S., T. Sirisanthana, K. Supparatpinyo, J. Praparattanapan, and K. E. Nelson. 1997. Case-control study of risk factors for Penicillium marneffei infection in human immunodeficiency virusinfected patients in northern Thailand. Clin. Infect. Dis. 24:1080–1086. 12. Chaturvedi, V., R. Ramani, S. Gromadzki, B. Rodeghier, H. Chang, and D. L. Morse. 2000. Coccidoidomycosis in New York State. Emerg. Infect. Dis. 6:25–29. 13. Desai, S. A., O. A. Minai, S. M. Gordon, B. O’Neil, H. P. Wiedemann, and A. C. Arroliga. 2001. Coccidioidomycosis in non-endemic areas: a case series. Respir. Med. 95:305–309. 14. DiSalvo, A. F., A. M. Fickling, and L. Ajello. 1973. Infection caused by Penicillium marneffei: description of first natural infection in man. Am. J. Clin. Pathol. 60:259–263. 15. Drouhet, E., and B. Dupont. 1995. Infection a Penicillium marneffei: mycose systemique a manifestations cutanees associee au SIDA. J. Mycol. Med. 5(Suppl. 1):21–34. 16. Duong, T. A. 1996. Infection due to Penicillium marneffei, an emerging pathogen: review of 155 reported cases. Clin. Infect. Dis. 23:125–130. 17. Fisher, F. S., M. W. Bultman, and D. Pappagianis. 2000. Operational guidelines for geological fieldwork in areas endemic for coccidioidomycosis (Valley fever), p. 1–16. In U.S. Geological Survey Open-File Report. U.S. Geological Survey, Reston, Va. 18. Fisher, M. C., G. L. Koening, T. J. White, and J. W. Taylor. 2002. Molecular and phenotypic description of Coccidioides posadasii sp. nov., previously recognized as the non-California population of Coccidioides immitis. Mycologia 94:73–84. 19. Galgiani, J. N., N. M. Ampel, A. Catanzaro, R. H. Johnson, D. A. Stevens, and P. L. Williams. 2000. Practice guidelines for the treatment of coccidioidomycosis. Clin. Infect. Dis. 30:658–661.


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20. Johnson, J. E., G. Radimer, A. F. DiSalvo, L. Ajello, and W. Bigler. 1970. Histoplasmosis in Florida. I. Report of a case and epidemiologic studies. Am. Rev. Respir. Dis. 101:299–305. 21. Jones, P. D., and J. See. 1992. Penicillium marneffei infection in patients infected with human immunodeficiency virus: late presentation in an area of nonendemicity. Clin. Infect. Dis. 15:744. 22. Kirkland, T. N., and J. Fierer. 1996. Coccidioidomycosis: a reemerging infectious disease. Emerg. Infect. Dis. 3:192–199. 23. Morrison, C. J., and M. D. Lindsley. 2001. Serological approaches to the diagnosis of invasive fungal infections, p. 667–716. In R. A. Calderone and R. L. Cihlar (ed.), Fungal Pathogenesis: Principles and Clinical Applications. Marcel Dekker, Inc., New York, N.Y. 24. Nasta, P., A. Donisi, A. Cattane, A. Chiodera, and S. Casari. 1997. Acute histoplasmosis in spelunkers returning from Mato Grosso, Peru. J. Travel Med. 4:176–178. 25. Ogiso, A., M. Ito, M. Koyama, H. Yamaoka, M. Hotchi, and M. R. McGinnis. 1997. Pulmonary coccidioidomycosis in Japan: case report and review. Clin. Infect. Dis. 25:1260–1261. 26. Pappagianis, D., and B. L. Zimmer. 1990. Serology of coccidioidomycosis. Clin. Microbiol. Rev. 3:247– 268. 27. Pautler, K. B., A. A. Padhye, and L. Ajello. 1984. Imported penicilliosis marneffei in the United States: report of a second human infection. J. Med. Vet. Mycol. 22:433–438. 28. Rosenstein, N. E., K. W. Emery, S. B. Werner, A. Kao, R. Johnson, D. Rogers, D. Vugia, A. Reingold, R. Talbot, B. D. Plikaytis, B. A. Perkins, and R. A. Hajjeh. 2001. Risk factors for severe pulmonary and disseminated coccidioidomycosis: Kern County, California, 1995–1996. Clin. Infect. Dis. 32:708–715. 29. Schneider, E., R. A. Hajjeh, R. A. Spiegel, R. W. Jibson, E. L. Harp, G. A. Marshall, R. A. Gunn, M. M. McNeil, R. W. Pinner, R. C. Baron, R. C. Burger, L. C. Hutwagner, C. Crump, L. Kaufman, S. E. Reef, G. M. Feldman, D. Pappagianis, and S. B. Werner. 1997. A coccidioidomycosis outbreak following the Northridge, Calif, earthquake. JAMA 277:904–908. 30. Symmers, W. 1967. Cases of coccidioidomycosis seen in Britain, p. 301–308. In L. Ajello (ed.), Proceedings of Second Coccidioidomycosis Symposium. University of Arizona Press, Tucson. 31. Valdez, H., and R. A. Salata. 1999. Bat-associated histoplasmosis in returning travelers: case presentation and description of a cluster. J. Travel Med. 6:258–260. 32. Verghese, S., D. Arjundas, K. C. Krishnakumar, P. Padmaja, D. Elizabeth, A. A. Padhye, and D. W. Warnock. 2002. Coccidoidomycosis in India: report of a second imported case. Med. Mycol. 40:307–309. 32a.Weinberg, M., J. Weeks, S. Lance-Parker, M. Traeger, S. Wiersma, Q. Phan, D. Dennison, P. MacDonald, M. Lindsley, J. Guarner, P. Connolly, M. Cetron, and R. Hajjeh. 2003. Severe histoplasmosis in travelers to Nicaragua. Emerg. Infect. Dis. 9:1322–1325. 33. Wheat, J., G. Sarosi, D. McKinsey, R. Hamill, R. Bradsher, P. Johnson, J. Loyd, and C. Kauffman. 2000. Practice guidelines for the management of patients with histoplasmosis. Clin. Infect. Dis. 30:688–695. 34. Wheat, L. J. 2001. Laboratory diagnosis of histoplasmosis: update 2000. Semin. Respir. Infect. 16:131–140.


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Chapter 11

Human African Trypanosomiasis: a Reemerging Public Health Threat Anne C. Moore

The explosive increase in the prevalence of human African trypanosomiasis at the beginning of the 20th century is one of the best examples of an emerging infection. Written records from Arab traders suggest that the disease, commonly known as sleeping sickness, existed in discrete, limited foci in the Lake Chad and Congo basins for hundreds of years. However, when the era of colonial development began in earnest in the late 1800s, the geographic distribution of sleeping sickness expanded rapidly to engulf large tracts of subSaharan Africa (10). Massive epidemics at the turn of the century nearly decimated the population in some areas. Trypanosomiasis control was implemented widely in the 1920s. It was the first systematic, large-scale public health intervention in Africa. The approach rested on vertically structured vector control and the use of specialized mobile teams to screen the population regularly and to treat cases. This was an effective strategy, and although several decades of sustained effort were required, sleeping sickness was nearly eliminated by 1960. Trypanosomiasis control was a victim of its own success. By midcentury, the disease was no longer considered a serious public health threat. In the countries where the disease was endemic, diminishing resources for public health, and, in some areas, civil unrest and conflict led to curtailment or elimination of control measures. Unfortunately, during the period of neglect, a dramatic resurgence of sleeping sickness occurred in central Africa, and the disease is now one of the best examples of a reemerging infection. HUMAN AFRICAN TRYPANOSOMIASIS: BACKGROUND Transmission and Ecology Human African trypanosomiasis is caused by two subspecies of Trypanosoma brucei, an extracellular, flagellate protozoan parasite. It is a vector-borne disease transmitted by the bite of the tsetse fly (Glossina spp.), a bloodsucking insect found only on the African Anne C. Moore • Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop F-22, 4770 Buford Highway, Atlanta, GA 30341. 143


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continent. Although the two subspecies that infect humans, T. brucei gambiense and T. brucei rhodesiense, are morphologically indistinguishable, there are key differences in their abilities to infect nonhuman hosts. T. brucei rhodesiense is a zoonotic parasite. Domestic livestock and a variety of wild animals serve as the reservoirs of infection. In contrast, humans are the only epidemiologically important reservoir of T. brucei gambiense, although transient light infections occur in some animals. A third member of the T. brucei complex, T. brucei brucei, infects a wide variety of wild and domestic animals, but it is unable to establish infection in humans because it is destroyed by a lytic factor in human serum (44, 50, 65). Trypanosomes are transmitted cyclically between the tsetse fly and the mammalian host. When a tsetse fly ingests trypomastigotes from the mammalian bloodstream, the parasite undergoes several phases of development, maturation, and multiplication in the fly midgut and salivary gland to transform into infective metacyclic trypanosomes. Completion of the life cycle in the intermediate insect host varies with Glossina species and ambient temperature but, on average, takes about 1 month. Flies of both sexes can become infected, and flies remain infected for life (at most, a few months). Inoculation of another vertebrate host can occur in subsequent blood meals. Metacyclic trypanosomes at the inoculation site in the human host gain access to the bloodstream and, within about 5 days, transform into the trypomastigote bloodstream forms. These forms are pleomorphic, are 17 to 30 ␮m in length, and have a free flagellum and a small subterminal kinetoplast. A unique feature of bloodstream trypanosomes is their ability to evade host immune destruction by antigenic variation (for reviews, see references 2, 3, 9, 33, and 55). The parasite is covered with a variable surface glycoprotein (VSG), which protects invariant components from the host immune response. T. brucei nuclear DNA contains up to 1,000 genes encoding different VSGs, although a single trypanosome expresses only one VSG at a time. The host mounts an antibody response to the VSG; however, during infection, individual parasites spontaneously switch the VSG that they express. This allows the trypanosome population to stay one step ahead of the host immune system, and it explains the periodic fluctuations observed in the level of parasitemia. The large repertoire of trypomastigote VSG genes and the ability of the parasite to create new VSGs by recombination have thwarted the development of an effective vaccine directed at bloodstream forms. Transmission of trypanosomiasis to humans depends on ecologic factors that determine Glossina densities and infection rates and also on human behavioral factors that influence the frequency and extent of human-fly contact. The two subspecies of T. brucei that infect humans are epidemiologically distinct. T. brucei gambiense is distributed in foci throughout West and Central Africa, where it is transmitted by riverine Glossina species (palpalis group). These flies inhabit humid and dark areas of thick vegetation, particularly along rivers and streams. Human activities, such as growing and harvesting of crops, washing or fishing, or collecting water or firewood, bring potential hosts into contact with tsetse flies. Intense transmission is often associated with particular sites, such as water collection points or river crossings, because there is frequent contact between the fly and the human disease reservoir (48). T. brucei rhodesiense is transmitted by savannah species of Glossina (morsitans group). Morsitans flies inhabit the woodlands and thickets of the savannah areas of the Rift Valley and eastern and southern Africa. A wide range of wild animals on the African savannah


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serve as reservoir hosts for T. brucei rhodesiense (14). Most wild animal species are well adapted to the parasite and remain asymptomatic for extended periods of time, but domestic animals and humans develop acute illness. Sporadic human infection occurs when individuals, such as hunters, poachers, and tourists, intrude in the sylvatic cycle. T. brucei rhodesiense can also exist in epidemic form. In outbreaks, peridomestic transmission from a livestock reservoir occurs (12, 15). Clinical Features Human African trypanosomiasis is a progressive illness that is invariably fatal if untreated. The initial clinical sign of infection is an acute inflammatory lesion (chancre) that sometimes develops at the inoculation site within a few days of infection. In settings where insect bites are commonplace, the trypanosomal chancre is usually not recognized. As the parasites disseminate throughout the hemolymphatic system (stage I, early stage), a systemic, febrile illness develops. The spectrum of clinical manifestations in this phase is highly variable, but ultimately, the central nervous system is invaded (stage II, late stage), leading to meningoencephalitis and death. The time course of disease progression is determined by the infecting subspecies of trypanosome. T. brucei rhodesiense causes an acute, severe, and rapidly progressive illness with early central nervous system (CNS) involvement, while T. brucei gambiense causes a protracted illness lasting up to several years, reflecting the adaptation of this subspecies to its reservoir host. With early T. brucei gambiense infection (West African trypanosomiasis), patients are minimally symptomatic for weeks or months before developing intermittent fever and a variety of other nonspecific signs and symptoms, most commonly, malaise, headaches, arthralgias, pruritis, and lymphadenopathy of supraclavicular and posterior cervical groups. After months of nonspecific systemic illness, the insidious development of CNS involvement becomes apparent. Unrelenting headaches, mood changes, and increasing somnolence are common. Weight loss can be considerable. A wide range of clinical manifestations can accompany the progressive neurological deterioration, including ataxia or, occasionally, focal neurologic signs, psychosis, or endocrine disorders. Eventually, coma and death ensue. T. brucei rhodesiense infection (East African trypanosomiasis) follows a similar, but more acute course. The onset of illness generally occurs within a few days to about 3 weeks from the time of the infective bite and is associated with easily detectable parasitemia. The trypanosomal chancre is more common, and patients experience very high intermittent fevers and the development of thrombocytopenia early in infection. Cardiac involvement is also frequently noted with T. brucei rhodesiense infection. Rhythm disturbances or evidence of myocarditis (41) can be detected in 10 to 20% of patients and can cause death even before the CNS is invaded. Abnormalities of cerebrospinal fluid (CSF) are detectable within a few weeks of infection, and death occurs within weeks to a few months. Diagnosis and Stage Determination Diagnosis rests on detection of the parasite in blood, lymph node fluid, chancre, or CSF (56). Microscopic examination for motile trypanosomes is usually performed with wet films, although Giemsa-stained films can be used. The level of T. brucei rhodesiense parasitemia is generally high, and infection is readily diagnosed by examination of peripheral blood. In contrast, the parasite load in T. brucei gambiense infection fluctuates, and it


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is often low and hard to detect. Lymphadenopathy is common in this form of sleeping sickness, and examination of lymph node fluid is particularly helpful. However, the sensitivity of microscopy of lymph node aspirates, even if combined with examination of a blood film, rarely exceeds 75%; therefore, blood concentration techniques and repeated examinations may be needed. The miniature anion-exchange column technique is the most sensitive tool (22), with a detection limit of about 100 parasites/ml; however, it is not widely available. Microhematocrit capillary centrifugation, with examination of the zone just above the buffy coat, is an alternative (61). Molecular techniques or in vitro culture (1) has not replaced the traditional parasitologic methods. Detection of trypanosome DNA by PCR, although successfully used clinically for the detection of infections imported to other countries from Africa (18) or in research studies (16, 17, 35, 42, 54), is not practical for diagnosis in the field at present. A new fluorescence in situ hybridization test is promising but needs further evaluation (43). In areas where T. brucei gambiense is endemic, trypanosomiasis is often recognized clinically by the neurologic signs and symptoms of late-stage disease. However, because the clinical features of early infection are nonspecific, serologic testing is helpful for the screening of patients with suspected trypanosomiasis. A simple, rapid field test, the card agglutination trypanosomiasis test (CATT) (23), is available for the detection of T. brucei gambiense infection but not for the detection of T. brucei rhodesiense infection. The test detects antibody by using the variant antigen LiTat 1.3, a variable antigen expressed by most, but not all, T. brucei gambiense strains. The sensitivity of the test is high (92 to 100%) in most areas where the disease is endemic. The specificity of the test, particularly when it is performed with diluted serum, is also thought to be high (~95%) (27, 32), although it is not sufficient for a definitive diagnosis. Additional serologic tests for trypanosomiasis have been developed and are used in research settings, but CATT is the only one used at present in areas of endemicity. Its use by case-finding teams can greatly improve the efficiencies of control programs. Although the test has been available for two decades, national trypanosomiasis programs have been slow to implement CATT, largely because of cost ($0.35/test). In the past 2 years, its use has increased, and about half of all screening is done by CATT. After diagnosis, the disease stage (hemolymphatic or meningoencephalitic) is determined by examination of CSF. Accurate assessment of the stage is critical because it dictates the choice of therapy; however, existing tools are not optimal. When trypanosomes are not found in CSF, the most widely used indicator of CNS involvement is an elevated leukocyte count (more than five cells), with a sensitivity of roughly 85% and a specificity of 65% (28). The concentration of nonspecific immunoglobulin M in CSF is elevated in late-stage trypanosomiasis, and a simple latex agglutination test based on this marker has been developed and is under evaluation (21). Trypanosome-specific antibodies, autoantibodies directed against CNS components, and a wide variety of inflammation-related markers (e.g., nitric oxide, cytokines, and prostaglandins) are detected in the CSF of patients with trypanosomiasis but have not been used in new methods for stage assessment. Therapy and Prevention Chemotherapy for human African trypanosomiasis is far from ideal (for reviews, see references 36 and 57). Three of the four drugs in regular use were developed at least


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50 years ago. In general, efficacy has been satisfactory (92 to 100%); however, toxicity has been a serious problem. In addition, treatment courses are quite lengthy, and some of the drugs are difficult to administer, particularly in rural African settings. The choice of therapy is determined by the trypanosome subspecies and the presence or absence of CNS involvement. Two drugs, suramin and pentamidine, are used to treat hemolymphatic stage (early-stage) disease. For T. brucei gambiense infection, pentamidine is the most widely used agent, although suramin is also effective. The intramuscular injections are painful and may cause sterile abscesses. Other adverse effects are generally minor and include nausea, abdominal pain, hyposalivation, and mild, reversible nephrotoxicity. Hypoglycemia or hypotension can occur during treatment, particularly if the drug is given intravenously. Suramin sodium is the preferred drug for early-stage T. brucei rhodesiense infection. The most common adverse effects of suramin are fever, nephrotoxicity, and cutaneous reactions. Anaphylaxis is rare, but a test dose is usually administered. Suramin and pentamidine are among the least toxic drugs in use, and both are excellent trypanocides, but neither drug effectively penetrates the CNS. Melarsoprol, an organoarsenic compound, has remained the drug of choice for latestage infection with either T. brucei rhodesiense or T. brucei gambiense. It is able to cure the disease during both stages of infection, but it is used exclusively for late-stage trypanosomiasis because of its toxicity. Adverse effects are common and include cutaneous reactions, fever, vomiting, diarrhea, and peripheral polyneuropathy (possibly a direct effect of arsenic). The most serious adverse effect of melarsoprol is a frequently lethal encephalopathic reaction, which complicates treatment in 2 to 10% of patients (36). The reaction is thought to be immune mediated, but the mechanism and the potential role of trypanosomes are unknown. One study found that concurrent therapy with prednisolone reduced the incidence of encephalopathy in T. brucei gambiense infection by threefold (37, 38), but no comparable data are available on the use of steroids for the treatment of T. brucei rhodesiense infection. A recent advance has been the introduction of a short, simplified dosing schedule based on the drug’s pharmacokinetics (8). For anti-T. brucei gambiense therapy, it replaces the previous complex, empiric regimens without a decrease in safety or efficacy, but it has not been used to treat T. brucei rhodesiense infections. Eflornithine, originally developed as an antitumor agent, has been the only new drug brought to the market in decades. It is highly effective against T. brucei gambiense, although not against T. brucei rhodesiense. It is less toxic than melarsoprol, although it can cause diarrhea, anemia, thrombocytopenia, and seizures. It never achieved widespread use due to its high cost, limited availability, and difficulty of administration in rural, basic African health centers. However, it is very useful for treatment of patients with late-stage disease for whom melarsoprol therapy fails. Eflornithine is a trypanostatic drug, and limited data suggest that it may be less effective in patients infected with human immunodeficiency virus (HIV) (39). Nifurtimox, a drug indicated for therapy against Chagas’ disease, has shown some success in curing both stages of T. brucei gambiense infection (57). It has been used alone and in combination with other agents, primarily to treat disease refractory to melarsoprol. Adverse effects, especially gastrointestinal effects (anorexia, vomiting, abdominal pain) and neurologic effects (peripheral neuropathy, vertigo, tremors, seizures), can be problematic when nifurtimox is administered in combination with melarsoprol, which has similar toxicities.


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Chemoprophylaxis for African trypanosomiasis is not used, although in the past, mass campaigns consisting of single pentamidine injections at 6-month intervals were conducted (60). This approach was abandoned because it tended to mask infection and it caused concern about the induction of drug resistance. For travelers, chemoprophylaxis is not advised because of the toxicities of antitrypanosomal agents and the low risk of infection. The best way to reduce the risk is to avoid tsetse fly bites. The flies are day biters and are attracted to bright colors and to the dust and motion of vehicles. They can bite through thin fabric, and insect repellent provides only partial protection. Routine preventive measures should include avoidance of known areas of tsetse fly infestation, the use of insect repellent, and the wearing of wrist- and ankle-length clothing in neutral colors. Elements of Trypanosomiasis Control Two strategies are used for control of African trypanosomiasis: reduction of the disease reservoir through case detection and treatment and reduction of human-fly contact through vector control. Active case finding is the cornerstone of control for T. brucei gambiense infection (63). Because the only significant reservoir is human, periodic population screening by mobile teams, coupled with case treatment, will rapidly reduce disease prevalence if community participation is adequate. Participation rates today are lower than they were in the colonial era, when screening was compulsory, but the higher sensitivity of case detection by CATT generally can compensate for the lower turnout. Passive case detection alone is not sufficient to interrupt transmission, because infected persons may not seek care for many months during the early stage of infection, when symptoms are intermittent and often mild. To date, reduction of the disease reservoir through treatment has not been an important strategy against T. brucei rhodesiense trypanosomiasis. However, it is possible that treatment of cattle could be an effective control measure in T. brucei rhodesiense outbreaks, in view of the key role played by the livestock reservoir (12, 15). Tsetse fly control is the second approach for reducing African trypanosomiasis (for a review, see reference 47). Tsetse fly populations are a vulnerable target because they are slow to reproduce and very susceptible to insecticides. At present, vector control relies primarily on the use of visual or olfactory baits to attract tsetse flies to traps, impregnated screens, or live animals covered with insecticide. These bait technologies work well in reducing fly densities, and they have largely replaced insecticide application by aerial or ground spraying. In areas where T. brucei gambiense is endemic, vector control is not usually the sole strategy used, but trapping is often used as an adjunct approach to supplement control by case finding and treatment. Although a variety of tsetse fly control methods are effective, reinvasion occurs if areas are not protected, and sustainability of vector control has been a problem. CURRENT EPIDEMIOLOGY AND ISSUES Reemerging Trypanosomiasis in Africa In the past decade, trypanosomiasis has reemerged as a serious public health threat to rural Africans (51). Annual incidence data from the World Health Organization (WHO) (http://www.who.int/health-topics/afrtryps.htm) illustrate the remarkable success of trypanosomiasis control during the first half of the 20th century (Fig. 1). By 1964, fewer than 4,500 cases were reported. However, by 1997, the number of reported cases had returned


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Figure 1. Reported incidence of African trypanosomiasis, 1926 to 2000. Data are from the WHO. The countries reporting are shaded.

to levels not seen for more than 50 years. Despite the similarity in the annual incidence (40,000 cases), the past and present situations are not comparable. In the past, a high proportion of the population at risk was under regular surveillance, being screened by mobile teams. At present, of the 60 million people living in endemic foci in Africa, less than 8% are under surveillance, and most infections remain unrecognized and unreported. WHO estimates that the true prevalence of African trypanosomiasis is 300,000 to 500,000 cases (http://www.who.int/health-topics/afrtryps.htm), with an incidence of ⬎100,000 cases per year. More than 97% of reported cases, and probably a higher proportion of unreported cases, are caused by infection with T. brucei gambiense. The present epidemic is a consequence of the dismantling of trypanosomiasis control programs. After the disease was nearly eliminated in the 1960s, it was no longer viewed as a public health problem. Resources for health were limited, and trypanosomiasis control activities were judged to be not cost-effective. Furthermore, in many of the countries where the disease is endemic, war, civil unrest, and insecurity contributed to the resurgence by eroding the health infrastructure, by limiting access of mobile teams to the rural areas where transmission occurs, and by causing population movements which spread the disease. The trend away from vertically structured health programs toward decentralized, community-based care has also had an impact. Trypanosomiasis management is complex, and integration into primary health care has been difficult to implement and sustain. The disease is not easily recognized clinically, diagnosis is difficult, management requires frequent lumbar punctures and a long follow-up period, therapeutic drugs are toxic and difficult to administer, and transmission is not interrupted without active case finding. National health programs in Africa coping with the resurgence of the disease lack financial resources and sufficiently trained personnel for these activities.


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The case-fatality rate for untreated African trypanosomiasis is 100%. Because a high proportion of cases remain undiagnosed and untreated, the burden of disease in sub-Saharan Africa is substantial, but it is difficult to determine precisely. It was recently estimated at 2.05 million disability-adjusted life years (DALYs) lost (64). This calculation was based on very conservative assumptions about prevalence and mortality, and it probably underestimates the true burden by about 50%. Comparative values (in millions of DALYs) for sub-Saharan Africa are 36.8 for malaria, 8.7 for tuberculosis, 3.6 for meningitis, and 1.6 for schistosomiasis (62). Knowledge of the current geographic distribution of disease is incomplete; however, the resurgence of T. brucei gambiense has occurred in the central African region (Fig. 2). The Democratic Republic of Congo (DRC; formerly Zaire) bears the bulk of the global burden. The population at risk is estimated to be 12.6 million. The annual incidence rose from fewer than 100 cases in 1960 to more than 26,000 cases by 1998 (59). More than 150,000 new infections were detected in the past decade, despite inadequate coverage of areas where the disease is endemic. The overall rate of detection (by case-finding teams) during this period has been nearly 2%, but rates of more than 70% were found in some communities (11, 58). The rate of mortality from trypanosomiasis in DRC may be roughly comparable to that from AIDS (11). Although trypanosomiasis has reemerged primarily in rural areas, active transmission is also occurring in urban Kinshasa (4). Human trypanosomiasis is also creating a major public health problem in northern Angola, where 4 million people live in zones of endemicity. A highly effective Angolan control program had essentially eliminated the disease in the past. In 1974, only three cases

Figure 2. Geographic distribution of African trypanosomiasis, 2000. In each country, the spatial distribution is uneven and the disease occurs in foci.


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were detected among nearly a half million people screened (53). However, case finding plummeted during the decades of civil war. Limited population screening has resumed in recent years, and the incidence has been 6,000 to 8,000 cases annually; however, only 6% of the population at risk is under active surveillance (53). Peace in Angola presents an opportunity to better define the epidemiologic situation and to resume control programs; however, the basic health care infrastructure must be rebuilt, and in some areas of endemicity, land mines must be cleared before case-finding and vector control teams can function. The situation is similar in southern Sudan, where civil war caused the collapse of an effective treatment and control program. Several epidemic foci with prevalence levels of 5 to 30% have been identified, and several thousand cases have been diagnosed annually since 1998, but many areas of endemicity remain unexamined, and only about 5% of the 1 million to 2 million people at risk have access to screening (29). Although the problem of trypanosomiasis is most severe in DRC, Angola, and Sudan, high-prevalence foci (⬎5%) are also present in several other countries in the central African region, including the Central African Republic, the Republic of Congo, and Chad (J. Jannin, personal communication). In addition, T. brucei gambiense infections have been a chronic problem in northern Uganda, with 800 to 2,000 cases identified annually for more than a decade. The porous borders with Sudan and population movements caused by war and civil unrest have made it difficult to halt transmission in these foci. The resurgence of T. brucei gambiense trypanosomiasis appears to be confined to central Africa. There is no evidence of a similar increase in West Africa; however, very little active case detection is conducted there. Ivory Coast and Guinea still report modest numbers of cases. Ecologic changes have reduced human-fly contact in some areas, resulting in a decline or the disappearance of transmission. The incidence of human infections with T. brucei rhodesiense remains relatively low in most of southern and eastern Africa. An epidemic in Uganda in the mid-1970s caused 40,000 cases over 15 years (15). It is now under relatively good control. However, the spread of T. brucei rhodesiense beyond its historical focus in southeastern Uganda has recently been documented (11), and in Tanzania, the incidence of trypanosomiasis has increased 50% in the past 5 years (S. Kibona, personal communication). A factor contributing to the situation in Tanzania may be the expansion of livestock husbandry into trypanosomiasis foci. Other factors, e.g., changes in tsetse fly belts or fly densities, may also be operating, because a sharp increase in sporadic cases was recently noted in travelers. Imported Trypanosomiasis in the United States Since 1967, the Centers for Disease Control and Prevention (CDC) has conducted surveillance for African trypanosomiasis using data collected during the release of drugs for treatment. In the United States, CDC is the sole supplier of therapeutic agents for T. brucei rhodesiense infections. Imported African trypanosomiasis is seen infrequently (6). During the 35 years of surveillance, 39 cases have been reported to CDC (unpublished data). Most (31 cases) were Rhodesian trypanosomiasis (Fig. 3), commonly acquired by tourists during a photographic or hunting safari (6, 24, 26, 30, 49, 52). Between 1967 and 2000, T. brucei rhodesiense infection of a traveler occurred, on average, every 1.5 years. However, in 2001, there was


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Figure 3. Imported T. brucei rhodesiense trypanosomiasis in the United States, by year, 1967 to 2002.

a striking increase in the number of North American cases: seven travelers from the United States and one traveler from Canada contracted the disease. A similar increase was noted among European travelers (31, 45). Tanzania was the geographic site of exposure for almost all of the recent cases. Although the number of visitors to African game reserves is unknown, the predominance of cases acquired in Tanzania may reflect a tourist preference for that destination or a greater number of visits. However, the sharp rise in the number of cases suggests an elevated country-specific risk. Safari operators in Tanzania reported increases in Glossina densities in game-viewing areas in 2000 and 2001. Imported T. brucei gambiense trypanosomiasis rarely occurs in travelers, because they seldom visit rural areas of central and West Africa where the disease is transmitted. Since 1967, CDC has been notified of eight cases. Five of these were diagnosed in African nationals who were living in the United States (40, 46, 52). In view of the epidemic in central Africa, a slight increase in imported T. brucei gambiense trypanosomiasis might be expected in the future. Of the eight cases identified in the past 35 years, two occurred in 2001. Crisis of Chemotherapy Control efforts for reemerging African trypanosomiasis face huge challenges, among them, a looming crisis of chemotherapy. The proven strategies for control of T. brucei gambiense infection rely on case detection and effective treatment. However, the future availability of effective treatment is in question. The threat has two components: the lack of secure production of antitrypanosomal drugs and the decreasing efficacy of the most important agent, melarsoprol.


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There is little financial incentive for pharmaceutical manufacturers to produce drugs for a disease that affects predominantly rural African communities (34). This not only has resulted in a lack of research and development for new antitrypanosomal drugs, but it also has jeopardized the manufacture of existing drugs (13, 58). One by one, the agents used to treat trypanosomiasis and other tropical diseases are being withdrawn from the market because they are not sufficiently profitable. By the beginning of 2000, the production of virtually every therapeutic agent used to treat African trypanosomiasis was threatened or the agent was out of production. Although pentamidine manufacture was not in doubt, an impending price increase made the cost prohibitive for African patients. A halt of suramin production was imminent. Nifurtimox was no longer manufactured, and although melarsoprol was still being produced, its future was uncertain because of problems with its manufacture in Europe. Eflornithine had been out of production for several years. Its manufacture resumed in 2000 for use in a cosmetic cream for facial hair, but the formulation needed for sleeping sickness therapy was not available. Thus, despite the resurgence of trypanosomiasis and the enormous human impact in central Africa, there were fears that the disease would become untreatable. Fortunately, the WHO Sleeping Sickness Treatment and Drug Resistance Network, in an effort led by WHO and Médecins sans Frontières, was able to secure production of the existing drugs and negotiate drug donations for 5 years. This welcome development has undoubtedly staved off a potential catastrophe, but ways to secure the availability of these drugs in the long term must still be identified. Concerns about the availability of therapeutic drugs have been compounded by reports of declining melarsoprol efficacy in some areas. Melarsoprol has been the only drug widely available for the treatment of late-stage African trypanosomiasis. Although occasional treatment failures were observed, the failure rate remained at 5 to 8% for 5 decades (36). However, this situation changed recently when a much higher proportion of melarsoprolrefractory infection was noted in several epidemic T. brucei gambiense foci. The magnitude is alarming, with reported failure rates of 30% in Uganda (19, 20), 16 to 21% in southern Sudan (29), and 25% or more in northern Angola (53). The factors responsible for the decline in melarsoprol efficacy at these sites are not well understood and are under investigation. No evidence of deterioration of drug quality, handling, or administration has been found, and no differences in drug pharmacokinetics were noted between responders and nonresponders in Angola (7) and Uganda (5). Coinfection with HIV is also unlikely to be the primary cause of this phenomenon, because, in Sudan, elevated treatment failure rates are observed among HIV-negative patients as well as among those who are HIV infected (A. Moore, unpublished data). Drug resistance may be important. Few recent T. brucei gambiense isolates from patients with relapses have been tested for melarsoprol susceptibility because of technical barriers. Among the limited number of Ugandan isolates that have been examined, no correlation between treatment failure and reduced drug sensitivity was established (25). However, all the recent Ugandan isolates were less sensitive than historical specimens from Uganda and Ivory Coast; MICs for these isolates were less than, but in the range of, peak melarsoprol concentrations (5, 25). It is possible that some trypanosomes could survive during treatment. In Angola, parasites are detected in the CSF and blood of some patients immediately following a course of treatment with melarsoprol (F. Mayala, personal communication).


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The potential for the geographic spread of resistant organisms is considerable, in view of the insecurity and civil war in areas where treatment failure has been documented. In 1998, intensified conflict in northern Angola resulted in the movement of populations into DRC, where health centers diagnosed and treated Angolan patients with melarsoprolrefractory trypanosomiasis (C. M. M. Bilenge, personal communication). Sentinel surveillance for treatment failure in Africa, recently implemented by the WHO and CDC, will help to better define the current geographic distribution of infections refractory to melarsoprol and to monitor trends. CONCLUSIONS Human African trypanosomiasis has dramatically reemerged as a serious public health problem in the central African region. We face huge challenges in implementing previously successful strategies to regain control of the disease, in view of the weak or absent health systems in some of the most affected areas. The tools available for control are better than those used decades ago to bring trypanosomiasis to near elimination. However, effective therapy is essential. Increasing rates of treatment failure and the insecurity of future drug production threaten to undercut control efforts. During the past decade, the resurgence of trypanosomiasis attracted little international attention; however, a few recent developments allow hope. The declaration by the Organization of African Unity of the intent to eradicate tsetse flies on the African continent, while perhaps unrealistically ambitious, indicates that there is growing political will to address the problem of trypanosomiasis. Research and development of new antitrypanosomal agents has resumed through public-private partnerships. To reduce the human impact of the present epidemic, research aimed at optimizing the use of the currently available drugs, alone or in combination, should also be a priority. REFERENCES 1. Aerts, D., P. Truc, L. Penchenier, Y. Claes, and D. Le Ray. 1992. A kit for in vitro isolation of trypanosomes in the field: first trial with sleeping sickness patients in the Congo Republic. Trans. R. Soc. Trop. Med. Hyg. 86:394–395. 2. Barry, J. D. 1997. The biology of antigenic variation in African trypanosomes, p. 89–107. In G. Hide, J. C. Mottram, G. H. Coombs, and P. H. Holmes (ed.), Trypanosomiasis and Leishmaniasis: Biology and Control. CAB International, Oxford, United Kingdom. 3. Barry, J. D. 1997. The relative significance of mechanisms of antigenic variation in African trypanosomes. Parasitol. Today 13:212–218. 4. Bilenge, C. M. M., V. K. B. K. Meso, F. J. Louis, and P. Lucas. 2001. Trypanosomiase humaine Africaine en milieu urbain: l’exemple de Kinshasa, Republique du Congo en 1998 et 1999. Med. Trop. 61:445– 448. 5. Brun, R., R. Schumacher, C. Schmid, C. Kunz, and C. Burri. 2001. The phenomenon of treatment failures in human African trypanosomiasis. Trop. Med. Int. Health 6:906–914. 6. Bryan, R., H. Waskin, F. Richards, T. Bailey, and D. Juranek. 1989. African trypanosomiasis in American travelers: a 20-year review, p. 384–388. In R. Steffen, H. Lobel, J. Hawarth, and D. J. Brandley (ed.), Travel Medicine. Springer-Verlag, New York, N.Y. 7. Burri, C., and J. Keiser. 2001. Pharmacokinetic investigations in patients from northern Angola refractory to melarsoprol treatment. Trop. Med. Int. Health 6:412–420. 8. Burri, C., S. Nkunku, A. Merolle, T. Smith, J. Blum, and R. Brun. 2000. Efficacy of a new, concise schedule for melarsoprol in treatment of sleeping sickness caused by Trypanosoma brucei gambiense: a randomized trial. Lancet 355:1419–1425.


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9. Cross, G. A. M. 1996. Antigenic variation in trypanosomes—secrets surface slowly. Bioessays 18:283–291. 10. Duggan, A. J. 1970. An historical perspective, p. xli–lxxxviii. In H. W. Mulligan and W. H. Potts (ed.), The African Trypanosomiases. George Allen & Unwin, London, United Kingdom. 11. Ekwanzala, M., J. Pepin, N. Khonde, S. Molisho, H. Bruneel, and P. De Wais. 1996. In the heart of darkness: sleeping sickness in Zaire. Lancet 348:1427–1430. 12. Fèvre, E. M., P. G. Coleman, M. Odiit, J. W. Magona, S. C. Welburn, and M. E. J. Woolhouse. 2001. The origins of a new Trypanosoma brucei rhodesiense sleeping sickness outbreak in eastern Uganda. Lancet 358:625–628. 13. Gastellu Etchegorry, M., J. P. Helenport, B. Pecoul, J. Jannin, and D. Legros. 2001. Availability and affordability of treatment for human African trypanosomiasis. Trop. Med. Int. Health 6:957–959. 14. Geigy, R., P. M. Mwanbu, and M. Kaufmann. 1971. Sleeping sickness survey in Musoma district, Tanzania. IV. Examination of wild mammals as a potential reservoir for T. rhodesiense. Acta Trop. 30:49–56. 15. Hide, G. 1999. History of sleeping sickness in East Africa. Clin. Microbiol. Rev. 12:112–125. 16. Kabiri, M., J. R. Franco, P. P. Simarro, J. A. Ruiz, M. Sarsa, and D. Steverding. 1999. Detection of Trypanosoma bruce gambiense in sleeping sickness suspects by PCR amplification of expression-siteassociated genes 6 and 7. Trop. Med. Int. Health 4:658–661. 17. Kanmogne, G. D., T. Asonganye, and W. C. Gibson. 1996. Detection of Trypanosoma brucei gambiense, in serologically positive but aparasitemic sleeping-sickness suspects in Cameroon, by PCR. Ann. Trop. Med. Parasitol. 90:475–483. 18. Kirchhoff, L. V. 1988. Use of PCR assay for diagnosing African trypanosomiasis of the CNS: a case report. Cent. Afr. J. Med. 44:134 –136. 19. Legros, D., C. Fournier, M. Gastellu Etchegorry, F. Maiso, and E. Szumlin. 1999. Échecs thérapeutiques du mélarsoprol parmi des patients traites au stade tardif de trypanosomose humaine africaine à T. brucei gambiense en Ouganda. Bull. Soc. Pathol. Exot. 92:171–172. 20. Legros, D., S. Evans, F. Maiso, J. C. Enyaru, and D. Mbulamberi. 1999. Risk factors for treatment failure after melarsoprol for Trypanosoma brucei gambiense trypanosomiasis in Uganda. Trans. R. Soc. Trop. Med. Hyg. 93:439–442. 21. Lejon, V., P. Buscher, N. H. Sema, E. Magnus, and N. Van Meirvenne. 1998. Human African trypanosomiasis: a latex agglutination field test for quantifying IgM in cerebrospinal fluid. Bull. W. H. O. 76:553–558. 22. Lumsden, W. H. R., C. D. Kimber, D. A. Evans, and S. J. Doig. 1979. Trypanosoma brucei: miniature anion exchange centrifugation technique for detection of low parasitemias: adaptation to field use. Trans. R. Soc. Trop. Med. Hyg. 73:312–317. 23. Magnus, E., T. Vervoort, and N. Van Meirvenne. 1978. A card agglutination test with stained trypanosomes (CATT) for serological diagnosis of T. b. gambiense trypanosomiasis. Ann. Soc. Belge Med. Trop. 58:169–176. 24. Malesker, M. A., D. Boken, T. A. Ruma, P. J. Vuchetich, P. J. Murphy, and P. W. Smith. 1999. Rhodesian trypanosomiasis in a splenectomized patient. Am. J. Trop. Med. Hyg. 61:428–430. 25. Matovu, E., J. C. K. Enyaru, D. Legros, C. Schmid, T. Seebeck, and R. Kaminsky. 2001. Melarsoprol refractory T. b. gambiense from Omugo, northwestern Uganda. Trop. Med. Int. Health 6:407–411. 26. McGovern, T. W., W. Williams, J. E. Fitzpatrick, M. S. Cetron, B. C. Hepburn, and R. H. Gentry. 1995. Cutaneous manifestations of African trypanosomiasis. Arch. Dermatol. 131:1178–1182. 27. Miézan, T., F. Doua, P. Cattand, and P. de Raadt. 1991. Évaluation du Testryp CATT appliqué au sang préléve sur papier filtre and au sang dilué, dans le foyer de trypanosomiase à Trypanosoma brucei gambiense en Côte d’Ivoire. Bull. W. H. O. 69:603–606. 28. Miézan, T. W., H. A. Meda, F. Doua, F. B. Yapo, and T. Baltz. 1998. Assessment of central nervous system involvement in gambiense trypanosomiasis: value of the cerebro-spinal white cell count. Trop. Med. Int. Health 3:571–575. 29. Moore, A., and M. Richer. 2001. Re-emergence of epidemic sleeping sickness in southern Sudan. Trop. Med. Int. Health 6:342–347. 30. Moore, A., E. T. Ryan, and M. A. Waldron. 2002. A 37-year-old man with fever, hepatosplenomegaly, and a cutaneous foot lesion after a trip to Africa. N. Engl. J. Med. 346:2069–2076. 31. Moore, D. A. J., M. Edwards, R. Escombe, D. Agranoff, J. W. Bailey, S. B. Squire, and P. L. Chiodini. 2002. African trypanosomiasis in travelers returning to the United Kingdom. Emerg. Infect. Dis. 8:74–76. 32. Noireau, F., J. L. Lemesre, M. Y. Nzoukoudi, M. T. Louembet, J. P. Gouteau, and J. L. Frézil. 1988. Serodiagnosis of sleeping sickness in the Republic of Congo: comparison of indirect immunofluorescence antibody test and card agglutination test. Trans. R. Soc. Trop. Med. Hyg. 82:237–240.


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33. Pays, E., L. Vanhamme, and M. Berberof. 1994. Genetic controls for the expression of surface proteins in African trypanosomes. Annu. Rev. Microbiol. 47:521–534. 34. Pécoul, B., P. Chirac, P. Trouiller, and J. Pinel. 1999. Access to essential drugs in poor countries. A lost battle? JAMA 281:361–367. 35. Penchenier, L., G. Simo, P. Grebaut, S. Nkinin, C. Laveissiere, and S. Herder. 2000. Diagnosis of human trypanosomiasis, due to Trypanosoma brucei gambiense in central Africa, by the polymerase chain reaction. Trans. R. Soc. Trop. Med. Hyg. 94:392–394. 36. Pepin, J., and F. Milord. 1994. The treatment of human African trypanosomiasis. Adv. Parasitol. 33:1–49. 37. Pepin, J., F. Milord, C. Guern, B. Mpia, L. Ethier, and D. Mansinsa. 1989. Trial of prednisolone for prevention of melarsoprol-induced encephalopathy in gambiense sleeping sickness. Lancet 333:1246–1249. 38. Pepin, J., F. Milord, N. Khonde, T. Niyonsenga, L. Loko, B. Mpia, and P. De Wais. 1995. Risk factors for encephalopathy and mortality during melarsoprol treatment of Trypanosoma brucei gambiense sleeping sickness. Trans. R. Soc. Trop. Med. Hyg. 89:92–97. 39. Pepin, J., L. Ethier, C. Kazadi, F. Milord, and R. Ryder. 1992. The impact of HIV infection on the epidemiology and treatment of Trypanosoma brucei gambiense sleeping sickness in Nioki, Zaire. Am. J. Trop. Med. Hyg. 47:133–140. 40. Petru, A. M., P. H. Azimi, S. K. Cummins, and A. Sjoerdsma. 1988. African sleeping sickness in the United States. Am. J. Dis. Child. 142:224 –228. 41. Poltera, A. A., R. Owor, and J. N. Cox. 1973. Pathological aspects of human African trypanosomiasis in Uganda. A post-mortem survey of fourteen cases. Virchows Arch. Pathol. Histopathol. 373:249–265. 42. Radwanska, M., F. Claes, S. Magez, E. Magnus, D. Perez-Morga, E. Pays, and P. Buscher. 2002. Novel primer sequences for polymerase chain reaction-based detection of Trypanosoma brucei gambiense. Am. J. Trop. Med. Hyg. 67:289–295. 43. Radwanska, M., S. Magez, H. Perry-O’Keefe, H. Stender, J. Coull, M. Sternberg, P. Büscher, and J. J. Hyldig-Nielsen. 2002. Direct detection and identification of African trypanosomes by fluorescence in situ hybridization with peptide nucleic acid probes. J. Clin. Microbiol. 40:4295–4297. 44. Raper, J., M. P. M. Portela, E. Lugli, U. Frevert, and S. Tomlinson. 2001. Trypanosome lytic factors: novel mediators of human innate immunity. Curr. Opin. Microbiol. 4:402–408. 45. Ripamonti, D., M. Massari, C. Arici, E. Gabbi, C. Farina, M. Brini, C. Capatti, and F. Suter. 2002. African sleeping sickness in tourists returning from Tanzania: the first 2 Italian cases from a small outbreak among European travelers. Clin. Infect. Dis. 34:e18–e22. 46. Robinson, B., R. M. Clark, J. F. King, B. Hurt, and J. A. Mohr. 1980. Chronic Gambian trypanosomiasis. South. Med. J. 73:516–518. 47. Schofield, C. J., and I. Maudlin. 2001. Trypanosomiasis control. Int. J. Parasitol. 31:615– 620. 48. Scott, D. 1970. The epidemiology of Gambian sleeping sickness, p. 614–644. In H. W. Mulligan and W. H. Potts (ed.), The African Trypanosomiases. George Allen & Unwin, London, United Kingdom. 49. Sinha, A., C. Grace, W. K. Alston, F. Westenfeld, and J. H. Maguire. 1999. African trypanosomiasis in two travelers from the United States. Clin. Infect. Dis. 29:840–844. 50. Smith, A. B., J. D. Esko, and S. L. Hajduk. 1995. Killing of trypanosomes by the human haptoglobinrelated protein. Science 268:284–286. 51. Smith, D. H., J. Pepin, and A. H. Stich. 1998. Human African trypanosomiasis: an emerging public health crisis. Br. Med. Bull. 54:341–355. 52. Spencer, H. C., J. J. Gibson, R. E. Brodsky, and M. G. Schultz. 1975. Imported African trypanosomiasis in the United States. Ann. Intern. Med. 82:633–638. 53. Stanghellini, A., and T. Josenando. 2001. The situation of sleeping sickness in Angola: a calamity. Trop. Med. Int. Health 6:330–334. 54. Truc, P., G. Jamonneau, G. Cuny, and J. L. Frezil. 1999. Use of polymerase chain reaction in human African trypanosomosis stage determination and follow-up. Bull. W. H. O. 77:745–748. 55. Vanhamme, L., and E. Pays. 1995. Control of gene expression in trypanosomes. Microbiol. Rev. 59:223–240. 56. Van Meirvenne, N. 1999. Biological diagnosis of human African trypanosomiasis, p. 235–252. In M. Dumas, B. Bouteille, and A. Buguet (ed.), Progress in Human African Trypanosomiasis, Sleeping Sickness. Springer-Verlag, Paris, France. 57. Van Nieuwenhove, S. 1999. Present strategies in the treatment of human African trypanosomiasis, p. 253– 280. In M. Dumas, B. Bouteille, and A. Buguet (ed.), Progress in Human African Trypanosomiasis, Sleeping Sickness. Springer-Verlag, Paris, France.


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58. Van Nieuwenhove, S. 2000. Gambiense sleeping sickness: re-emerging and soon untreatable? Bull. W. H. O. 78:1283. 59. Van Nieuwenhove, S., V. K. Betu-Ku-Mesu, P. M. Diabakana, J. Declercq, and C. M. M. Bilenge. 2001. Sleeping sickness resurgence in the DRC: the past decade. Trop. Med. Int. Health 6:335–341. 60. Waddy, B. B. 1970. Chemoprophylaxis of human trypanosomiasis, p. 711–725. In H. W. Mulligan and W. H. Potts (ed.), The African Trypanosomiases. George Allen & Unwin, London, United Kingdom. 61. Woo, P. T. K. 1971. Evaluation of the haematocrit centrifuge and other techniques for the field diagnosis of human trypanosomiasis and filariasis. Acta Trop. 28:298–303. 62. World Bank. 1993. World Development Report: Investing in Health, p. 216–219. Oxford University Press, New York, N.Y. 63. World Health Organization. 1998. Control and surveillance of African trypanosomiasis. WHO Tech. Rep. Ser. 881:1–114. 64. World Health Organization. 2000. The World Health Report 2000: Health Systems Improving Performance. World Health Organization, Geneva, Switzerland. 65. Xong, H. V., L. Vanhamme, M. Chamekh, C. E. Chimfwembe, J. Van den Abbeele, E. Pays, N. Van Meirvenne, R. Hamers, P. De Baetseller, and E. Pays. 1998. A VSG expression site associated gene confers resistance to human serum in Trypanosoma brucei. Cell 95:893–896.


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Raccoon Roundworm Infection (Baylisascariasis): a Zoonosis of Pediatric and Public Health Concern William J. Murray Diseases are more easily prevented than cured, and the first step to their prevention is the discovery of their exciting causes. William Farr Few species of wild animal are so easily recognized by the public as the raccoon (Procyon lotor). These adaptable, clever, and mischievous animals make themselves conspicuous by their nocturnal pillaging of garbage cans, forays into pools and ornamental fishponds, and other depredations around urban and suburban homes. Few people, however, are aware of the potential raccoons have for disseminating zoonotic pathogens. Raccoons are wellrecognized carriers of rabies and other viral zoonoses as well as numerous bacterial, protozoal, and helminth zoonotic agents (1, 5, 34, 38). Notable among these is the common intestinal roundworm of raccoons, Baylisascaris procyonis, which is transmitted to other species by the fecal-oral route. The inadvertent ingestion of B. procyonis eggs containing infective larvae by nonraccoon hosts results in one of the most severe forms of visceral larva migrans (VLM) known (25, 26, 28, 31). VLM is characterized by the wandering of larval nematodes in somatic tissues of paratenic (advantageous but nonessential intermediate) hosts, resulting in structural and inflammatory damage. More so than other zoonotic nematode causes of VLM, the larvae of B. procyonis have a noted tendency to invade the ocular tissues and brain. This parasite’s remarkable ability to cause serious infections of the eye and frequently fatal central nervous system (CNS) disease in more than 90 other species of birds and mammals, including humans, has been well documented (28, 31, 42, 43). The extraordinary heteroxenosity (ability to use many hosts in its life cycle) of B. procyonis is of particular concern not only to wildlife biologists but also to public health professionals and medical practitioners. Populations of raccoons are substantially greater in urban and suburban areas than in rural or wilderness habitats (17, 32, 44, 45, 47; G. P. Roussere, W. J. Murray, M. J. Kutilek, and D. J. Levee, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. Y-9, p. 683, 2000). William J. Murray • Department of Biological Sciences, San Jose State University, San Jose, CA 95192-0100. 159


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Although a rare human infection, baylisascariasis is increasingly being recognized as a significant public health problem (11, 27, 28, 30, 31, 39). There is a frightening risk of infection to toddler-age children who, by their exploratory and unhygienic habits, are most likely to become infected by B. procyonis. This is especially so in urban and suburban environments and is exacerbated by parental lack of knowledge of this risk. Human CNS infections with B. procyonis invariably have a poor prognosis, and even among those who survive, permanent catastrophic neurologic impairment is the rule (8, 11, 25–28, 31, 44, 48). The first confirmed human cases of baylisascariasis came to light in the 1980s (8, 10, 19). Since that time, an increasing number of cases have been confirmed (11, 27, 28, 44, 48). As with all emerging zoonoses, the reasons for emergence are multifactorial. Nonetheless, in the case of baylisascariasis, several salient epidemiologic features are important. These include a reduction in hunting and trapping of raccoons, the movement of humans via housing expansion into previously rural or agricultural areas, the raccoon’s preference for and adaptability to living in suburban environments, lack of comprehensive wildlife control programs in many areas, and inadvertent or purposeful feeding of wildlife. It is not surprising under these circumstances that raccoons are among a growing list of problem wildlife in the United States (28, 31, 35, 51, 56). Raccoons, indigenous to North America, have been introduced to Germany and Japan, where they have also become a nuisance (12, 28, 33, 36). The potential for human infections with B. procyonis (then known as Ascaris columnaris and also as Ascaris procyonis) was predicted by Beaver in 1956 (3). Since the 1980s, building on the work of Beaver (3, 4), Tiner (58), and Sprent (53), Kevin R. Kazacos of Purdue University has established the importance of baylisascariasis as a zoonosis of significant public health concern (25–28). Concomitantly with ample opportunity for human contact with raccoons, a growing body of evidence suggests strongly that more cases of human baylisascariasis can be anticipated (27, 28, 31, 39). To understand why there is an increasing human risk for infection with B. procyonis, a discussion of the biology and behavior of raccoons is prerequisite to subsequent discussions of epidemiology, pathogenesis, clinical features, diagnosis, and treatment. THE BIOLOGY AND BEHAVIOR OF RACCOONS Raccoons (order Carnivora, family Procyonidae) are medium-sized mammals (average weight, 7 to 10 kg; weight range, 2 to 20 kg; maximum reported weight, 28.3 kg) of wide geographic distribution in most of North America, Central America, and the northern parts of South America, covering a range of habitat from Nearctic to Neotropical (6, 15, 20, 34, 40, 59). They are easily identified by their gray to charcoal coat, with a long, bushy tail with black rings encircling it, and a broad black stripe across their faces giving them the distinctive “masked-bandit” appearance so familiar to many (Fig. 1). Supremely adaptable to human environments, raccoons are aggressive, curious, and bold animals that have little fear of humans, especially in urbanized environments where they acclimate and thrive. They are excellent climbers with a keen sense of hearing, readily enter water, and generally are nocturnal creatures. Aside from humans, raccoons have few natural enemies, most of which (e.g., red foxes, coyotes, bobcats, and mountain lions) are not common in most urban and suburban locations (20, 34, 40).


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Figure 1. A young raccoon prowling around an urban house at night in northern California. Large numbers of raccoons thrive in suburbia throughout North America and in other countries where they have been introduced. Note the forepaws (see text). Image captured by infrared flash trigger. (Photograph © 1999 William J. Murray.)

Raccoons have a highly developed tactile sense, and when they are observed in the field, their propensity to handle and play with food and other objects of all kinds with their forepaws is apparent. The raccoon is the stuff of Native American legend, and it is interesting that the ability of raccoons to use their forepaws almost as hands is the basis for many Indian names for these animals (15, 18). Their primate-like forepaws make them unusually dexterous and enable them to manipulate things with ease. Because of this, raccoons have considerable skill in gaining access to buildings by removing vent screens, crawl space access covers, and the like, and even removing shingles from roofs and then clawing through the underlying subroof—an observation I have made on numerous occasions. In the wild, large social groups of raccoons are the exception, whereas in urban and suburban environments, large groups of raccoons of both sexes can be seen moving and foraging together. Presumably, the presence of abundant food, water, and shelter in urbanized habitats reduces intraspecies aggression among groups of raccoons. On the northern California coast, groups of raccoons have been observed raiding tide pools and prying open shellfish with their powerful forepaws (W. J. Murray, unpublished observation). In urbanized environments, raccoons make extensive use of storm drains, culverts, and sewers not only as shelter and den sites but to facilitate movement and foraging activities.


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It is not unusual to see raccoons emerge from storm drains in the early evening in many communities throughout the United States. They also use attics, crawl spaces, chimneys, and areas under decks and patios as den sites. Taken together, these combined traits enable raccoons not only to survive but to thrive in a human environment. Although classified as carnivores, raccoons are actually omnivorous and thus able to consume a wide range of potential food sources, varying only by season, geography, or perhaps, individual preferences. Fruits, nuts, berries, corn and other crops, insects and grubs, fish, eggs, crayfish, other small mammals, birds, carrion, and human refuse are all consumed. When food supplies are abundant, they tend to be more selective in food choice. Raccoons readily attack pet animals and eat them. The predation of house cats by hungry raccoons is well known to professional wildlife trappers (personal communication, Laurie Frazer, Vector Control Agency, Santa Clara County, Calif.). Frequently, an attack on a pet is what prompts a homeowner to call animal control agencies rather than the presence of latrines or depredating (i.e., property-damaging) raccoons on the property. On one memorable occasion, I witnessed a group of raccoons attack a dog—while the owner was walking it on a leash! Thus, from a dietary standpoint, raccoons are best considered opportunists, especially in suburban environments where their predilection for garbage can refuse is well known and their taste for household pets less so. Their cosmopolitan diet aside, it is the defecation habits of raccoons that contribute so significantly to the epidemiology of B. procyonis. Groups of raccoons instinctually defecate in communal areas called latrines, where large amounts of feces accumulate. Raccoons generally use latrines for long periods, resulting in substantial contamination of the surrounding environment. Although the motivation for this behavior is not clear, it is thought that it has to do with territorial marking or group socialization. Male raccoons can be observed urinating and rubbing their genitals on tree limbs, roofs, stumps, rocks, and other raised horizontal surfaces in the field and then defecating in those locations. When a raccoon of any age or gender happens upon a latrine, it seems compelled to contribute to it. In fact, social groups of raccoons have been observed going to difficult lengths to reach locations such as along the tops of fences or roofs to use an established latrine. In rural or wilderness areas, raccoon latrines are typically found off the ground in downed timber, rocky outcroppings, and tree cavities (21, 27, 28, 31, 41–43). In urban and suburban environments, latrines are commonly found directly on the ground, on lawns, at the bases of trees, along and on tops of fences, on roofs, in outbuildings, in attics, in crawl spaces, on stored firewood, and in other locations (39; G. P. Roussere, W. J. Murray, C. B. Raudenbush, M. J. Kutilek, D. J. Levee, and K. R. Kazacos, submitted for publication). Obviously, many of these sites are easily accessible and attractive to infants and young children. No matter where it may be found, the site of a latrine seems to be limited only by the ability of a raccoon to reach a new location out of curiosity or habit and to christen it with fresh droppings. Homeowners are frequently unaware that there are large latrines on roofs or hidden elsewhere on their property, thus increasing the risk of exposure to raccoon feces. Because latrines become foci of infection (see “Epidemiology and Pathogenesis of Human Infection” below), they are of particular concern as a primary risk factor for baylisascariasis. Raccoon populations and the density of raccoon latrines in suburban areas can be truly alarming (17, 21, 31, 44; Roussere et al., submitted). During a field investigation related to a near-fatal case of baylisascariasis in an 11-month-old child, 21 latrines were found on the


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parents’ 0.8-acre property (44). There is little doubt that such density significantly enhanced the likelihood of infection in that case. Because raccoons tend to be largely nocturnal, many people living in an area with a large population of raccoons may be unaware of their presence. It is a curious artifact of modern life that many property owners seldom thoroughly examine their property, including buildings and rooftops, on any regular basis. NATURAL HISTORY OF B. PROCYONIS INFECTIONS IN THE RACCOON B. procyonis is the large ascarid parasite of raccoons (Fig. 2). The biology, morphology, and ecology of B. procyonis are similar to those of other ascarid parasites of carnivores, such as the common canine roundworm, Toxocara canis (2, 13, 28, 46). First described as a separate genus within the family Ascarididae by Sprent in 1968 (54), other Baylisascaris species have been identified in mammalian hosts such as skunks (B. columnaris), badgers (B. melis), and bears (B. transfuga). The zoonotic disease significance of these other

Figure 2. Adult B. procyonis nematodes removed from the small intestine of a raccoon. Adult females (left) are about 24 cm long, whereas the smaller males (right) are about 12 cm long. (Reprinted from reference 39 with permission from Elsevier Science. © 2002 Elsevier Science.)


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Baylisascaris species is not known but presumably is possible based on experimental infections (28, 29). However, of the eight recognized species of Baylisascaris, B. procyonis is clearly associated with the most pathology in aberrant hosts (24, 28, 31). In North America, the prevalence of B. procyonis infections in raccoons reported in the literature ranges from 0 to 82%, depending on the geographic area examined (21, 28, 31, 32). In a 2-year study (1998 to 2000) conducted in northern California, the prevalence rate was 67% in 107 raccoons examined (W. J. Murray, unpublished data). The number of raccoons living in a particular area also has an impact on B. procyonis prevalence (28). In certain areas with high densities of raccoons, the prevalence of infection can be 100% (W. J. Murray, unpublished data). B. procyonis is similar to most other ascarid parasites of carnivores in that it causes little or no clinical disease in its definitive host unless there is an unusually heavy infection in juveniles (55). Like most other ascarids of mammals, B. procyonis has a typical direct life cycle, that is, it has no obligatory intermediate host but can use other animals as paratenic hosts. Raccoons become infected in one of two ways (Fig. 3). Generally, young raccoons become infected by ingesting eggs during feeding and grooming activities with other members of their social group. Alternatively, because a large variety of other species of mammals and birds can become infected with the larvae of B. procyonis, causing severe CNS disease (making them easy prey), adult raccoons can also acquire infection by ingesting the flesh of infected animals (28, 31). Humans become infected with B. procyonis just as these other hosts do and become accidental hosts following inadvertent ingestion of eggs containing infective larvae. On the other hand, the life cycle of B. procyonis differs somewhat from that of other common ascarids such as Ascaris lumbricoides of humans and Ascaris suum of swine in that there is no extraintestinal somatic (e.g., liver or lung) migration as part of the life cycle in the natural host. Instead, B. procyonis larvae emerge from ingested eggs in the raccoon small intestine and undergo further development in the mucosal tissues of the intestinal tract, finally reemerging into the intestinal lumen where they complete maturation into adult worms, a process taking about 60 days. The life cycle is completed when the adult worms mate and the females begin producing fertilized eggs that are expelled in the feces of infected raccoons. The total worm burden in an individual raccoon can be very high, especially in juveniles, but generally averages 48 to 62 worms (31). Adult female worms in the small intestine of raccoons collectively may produce millions of eggs per day, which are shed in the feces of infected raccoons (27, 28). Once outside the body, the eggs develop to the infective stage (Fig. 4) in about 2 to 4 weeks, depending on environmental conditions such as moisture and temperature (27, 28). Colder weather will delay egg development. Like other ascarid eggs, the eggs of B. procyonis are resistant to degradation in the environment and can survive for years under appropriate conditions (22, 28, 31, 46). This hardy longevity is a key component of the evolutionary survival strategy for ascarids along with a generally characteristic heteroxenous life cycle that further ensures survival of the parasite by allowing another means to sustain itself in paratenic hosts. The resistance of ascarid eggs to harsh chemicals is extraordinary (Fig. 5); eggs continue to embryonate even when placed in dilute solutions of acid or in formalin solutions (46, 50). The long-term viability of B. procyonis eggs and the tremendous numbers that are deposited in the environment by raccoons are key factors in understanding how this parasite is transmitted to other hosts, including humans. On average, infected raccoons shed over 20,000 eggs per g of feces and have been reported to produce as many as 256,700


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Figure 3. Life cycle of B. procyonis. (1) Ingestion of infective stage eggs by young raccoons occurs as a consequence of their close association with infected adults. The eggs hatch in the intestinal lumen, releasing larvae that continue to develop in the mucosa of the intestine; they then reenter the lumen and develop into adults that mate, and the females begin shedding fertilized eggs. This entire process requires about 60 days from the time eggs are ingested. (2) Although an intermediate host is not required for B. procyonis to complete its life cycle, older raccoons can become infected by consuming the flesh of a wide variety of host animals that become infected through feeding activities at latrine sites. The resulting visceral migration by B. procyonis larvae in the somatic tissues of these intermediate hosts frequently leads to neural larva migrans and severe CNS disease, making them easy prey for foraging raccoons. Once ingested by the raccoon, larvae encysted in the host’s tissues are released in the adult raccoon’s intestine where they develop into adults in about 35 days. (3) Humans become accidental hosts following inadvertent ingestion of eggs containing infective larvae. Young children are the most frequently affected due to poor hygienic habits, geophagia, and their well-known propensity to play with and mouth objects found in the environment. (Reprinted from reference 39 with permission from Elsevier Science. © 2002, Elsevier Science.)

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Figure 4. Photomicrograph of a B. procyonis egg containing a fully formed infective larva recovered from a latrine in a playground sandbox (magnification, ⫻32). B. procyonis eggs can be differentiated from other ascarid eggs commonly found in the environment, such as those of Toxocara, Ascaris, and Toxascaris species, by expert examination. Briefly, B. procyonis eggs are ellipsoid, approximately 75 ⫻ 60 ␮m in size with a brown, finely granular surface.

eggs per g of feces (27, 28). Thus, both actively used and abandoned latrines become focal accumulations of large numbers of the environmentally resistant eggs that remain viable and thus infective for years (46, 50). The eggs also possess a sticky proteinaceous surface coating that makes them adherent to objects with which they come into contact, including human hands, toys, garden tools, plant materials, and the like. Aside from incineration or soaking in volatile solvents such as mixtures of xylene and acetone, the eggs are very difficult to destroy or inactivate, as noted previously. Desiccation can also destroy infective eggs. Desiccation of eggs can occur naturally in locations exposed to high ambient temperatures and/or sunlight, such as what may occur in uninsulated lofts and attics or on rooftops exposed to direct sunlight. Such conditions can either retard further development of the embryo or kill larvae within infective eggs. For flammable objects or surfaces, mixtures of boiling water and/or bleach can be used to decontaminate them. However, it is important to realize that these methods may only remove the outer sticky coating of the egg, leaving a viable larva still protected within. Nonetheless, such action will reduce infectivity and moreover tends to have a dilutional and dispersal effect. The first step in reducing the risk of infection is to remove latrines and decontaminate the areas where they were located following guidelines that can found elsewhere (28, 31). Comprehensive management of the problem of raccoon latrines on a property requires determining what is attracting them to the location. Further information about methods that can be used to exclude raccoons from a property can be obtained by consulting appropriate


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Figure 5. Photomicrograph (magnification, ⫻32) of a living B. procyonis larva emerging from an egg that had been stored in 10% formalin for over 4 months, demonstrating the astounding resistance of ascarid eggs to destruction. During preparation of the specimen for photomicroscopy, manipulation of the cover slip cracked the egg open, freeing the larva. (Photograph © 1998 William J. Murray.)

government agencies (e.g., state fish and game departments, the U.S. Department of Agriculture, and local public health departments) or visiting associated Web sites. EPIDEMIOLOGY AND PATHOGENESIS OF HUMAN INFECTION A substantial risk factor for human infection with B. procyonis is the presence of raccoon latrines around areas of human habitation or recreation. Even in situations where a latrine may not be apparent, contamination of the surrounding soil (or other surfaces) can act as a source of infective eggs. An example is a roof latrine, where rain can wash feces off the roof and into gutters, thereby dispersing eggs at the downspout outflow location. Transmission of B. procyonis to humans occurs via geophagia, pica, or contaminated hands. Baylisascariasis cases in older children or adults typically involve developmentally delayed individuals who practice pica or geophagia. Young children (⬍4 years old), especially toddlers and crawling infants, are at greatest risk of infection because of their propensity to explore the environment, handling and mouthing objects they find interesting,


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and their poor hygienic habits. Most baylisascariasis cases reported in the literature involve young children. It is also important to remember that latrine sites may not be obvious, and unsupervised children can quickly consume a massive dose of eggs by playing in a latrine at the base of a tree or in another occult location in the environment. Once ingested, the eggs hatch in the intestinal tract, releasing the immature larvae that, instead of developing into adults as they do in raccoons, leave the intestinal lumen and migrate extensively via the bloodstream to various body tissues, causing VLM. In addition, for reasons that are not fully understood, compared to other carnivore ascarids, the larvae of B. procyonis have a marked propensity to invade the eye, causing ocular larva migrans (OLM), or the brain, causing neural larva migrans (NLM) (24–31). In heavy infections, both NLM and OLM may result (28, 44, 48). The biologic basis of this tissue tropism remains one of the most perplexing questions about baylisascariasis. Along with various other nematodes, B. procyonis has also been shown to be a cause of a variant of OLM called diffuse unilateral subacute neuroretinitis (DUSN). DUSN is characterized by visual loss secondary to nematode larva migrans in ocular tissues, resulting in postmigration inflammation of the retina and adnexa (14, 26, 30). DUSN caused by B. procyonis has been reported in Germany (33), indicating that baylisascariasis is not confined to North America. The migrating larvae produce damage to tissues directly and by an intense eosinophilic inflammatory reaction along the migration track (28, 29, 37). The inflammatory products of eosinophil degranulation along with toxic excretory products released by the migrating larvae contribute substantially to nerve cell damage (11, 26, 28, 29). Although experimental infections in animals have shown that only a small percentage (5 to 7%) of larvae actually enter the CNS, the damage they cause is extensive (25, 28). Furthermore, unlike the larvae of other helminths that more commonly cause VLM (such as T. canis), the larvae of B. procyonis exhibit very aggressive tissue migration behavior; are much more likely to cause NLM; continue to grow and molt during their migration, becoming up to 2 mm long and up to 80 ␮m in diameter; and do not readily die in tissues (11, 28). For reasons that are also not clear, B. procyonis larvae persist and migrate much longer in nervous system tissue than elsewhere in the body (28). Pathologic alterations in the CNS and other tissues caused by migrating B. procyonis larvae have been extensively investigated (26, 28, 29). Migrating larvae eventually become encapsulated in eosinophilic granulomas but remain viable in the tissues for extended periods (26, 28). Factors influencing the severity of CNS disease in humans (and other hosts) include the number of eggs ingested, the extent and location of larva migration in tissues, the severity of the inflammatory reaction caused by migrating larvae, and the amount of tissue necrosis. In any case, the resultant pathology is exacerbated by treatment delay (see “Treatment of Human Infection” below). Clinical signs of infection may develop as soon as 2 to 4 weeks after ingestion of eggs containing infective larvae and progress variably, depending on the factors mentioned above (28, 31). The infective dose of B. procyonis for humans is not precisely known, but it is known that one larva in the brain of an experimentally infected mouse can be fatal (28). Presumably, in the case of humans, the disease is most severe in young children because of their smaller size and because they are most likely to consume large numbers of infective eggs. Symptoms of baylisascariasis range from very subtle to varying degrees of mild to moderate CNS dysfunction (e.g., lethargy, irritability, agitation, somnolence, and hearing loss) to severe neurologic deficits, ataxia, developmental regression, paralysis, seizures,


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coma, blindness, and death (26–28, 31). In pediatric cases of baylisascariasis, the parents often initially notice very subtle changes in behavior or habits of their child that may or may not result in a visit to a physician (11, 44, 48). Frequently, when a child is first examined, “viral syndrome” or other similar diagnosis is made and the child is sent home on palliative care. Inevitably, the symptoms progressively worsen, prompting a return visit to the clinic (11, 44, 48). Ingestion of large numbers of B. procyonis eggs may produce rapidly fatal NLM. Another contributing factor to severity of disease is that infection with B. procyonis is often not suspected until later in the diagnostic evaluation of a patient. The delay in diagnosis contributes to the severity of the disease as the larvae continue to migrate unimpeded in body tissues. Despite treatment, progressive neurologic deterioration is the norm in human cases. The prognosis for patients with B. procyonis NLM is poor. Thirteen cases of severe human NLM or death caused by B. procyonis are known, most of which occurred in young children (8, 10, 11, 27, 28, 44, 48). Four of these cases (one of them a fatal case in a 17-year-old adolescent) have occurred in California in recent years (W. J. Murray, unpublished data). A larger number of cases of B. procyonis OLM are known (24, 26, 28, 30). From an epidemiologic perspective, reports of dogs acquiring patent B. procyonis infections warrant careful study in the future (2). Apparently, dogs either can develop VLM as a paratenic host (2, 49, 57) or, under some circumstances, may serve as hosts for the adult stage of B. procyonis (7, 16). Patent infections in dogs are troubling for three reasons: (i) this could greatly expand the range of this parasite; (ii) because of their indiscriminate defecation habits, infected dogs could further disseminate B. procyonis in the environment; and (iii) it is much more likely that children could be exposed to dog feces than raccoon feces. Why some dogs apparently can develop patent B. procyonis infections resulting in adult worms shedding viable eggs in the dog’s feces, whereas most other cases result in NLM and/or death, is not known. Presumably, because of their coprophagic tendencies, dogs may ingest raccoon feces at latrine sites or possibly they could become infected from consuming infected small animals serving as paratenic hosts for B. procyonis. The prevalence of canine B. procyonis infections is unknown. DIAGNOSIS OF INFECTION IN HUMANS B. procyonis infections in humans can be difficult to confirm. Humans are dead-end hosts for B. procyonis; therefore, ordering ova-and-parasite examinations of a patient’s stool is not useful, since the life cycle of the parasite is not completed. The antemortem diagnosis of baylisascariasis is generally achieved by excluding other known causes of VLM, NLM, or OLM. For example, the major cause of larva migrans in humans, T. canis, can generally be excluded by negative serologic results (13, 26), differing clinical symptomatology, and lack of exposure to raccoons or their feces. In addition to the appearance of CNS symptoms previously discussed, infected patients usually have peripheral blood and cerebrospinal fluid (CSF) eosinophilia, but this may not be profound (11, 44). While not pathognomonic, it is generally recognized that eosinophilic pleocytosis in the CSF in combination with peripheral blood eosinophilia in patients with progressive neurologic deficits is characteristic of baylisascariasis (25, 31, 37). Certainly, any child who presents with eosinophilic meningoencephalitis should alert the clinician to consider B. procyonis infection despite its rarity. Unfortunately, this consideration is often not made until late in the course


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of infection in most cases. Nonetheless, with increased awareness of this zoonosis, and the knowledge gained from a growing number of cases, better diagnostic clues are emerging. Neuroimaging methods such as computed tomography and magnetic resonance imaging are also helpful in supporting a diagnosis. Neuroimaging results can be normal in the early stages of infection but are especially useful in detecting subsequent pathologic changes and monitoring disease progression. Prominent features in brains of individuals infected with B. procyonis are evidence of ventricular dilatation, periventricular enhancement in contrast studies, and white matter abnormalities (10, 27, 44, 48). Serologic testing is also helpful but currently is not in routine use. Demonstration of antiBaylisascaris antibodies in patient’s serum and/or CSF by indirect fluorescent-antibody assay or by enzyme-linked immunosorbent assay can be obtained from the Department of Veterinary Pathobiology at Purdue University by contacting Kevin Kazacos (27). Additional diagnostic serology may soon be available from the Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention (W. J. Murray, unpublished data). Visual deficits or abnormal ocular clinical findings are often the first clue of a helminthic cause for a patient’s symptoms (13, 26, 44). Ophthalmologic examinations that reveal characteristic chorioretinal lesions, larval tracks, or migrating larvae can support a diagnosis of baylisascariasis (14, 24, 26, 30). If larvae are observed, they can usually be identified as B. procyonis because of their large size (up to five times larger) compared to that of the most common cause of OLM in humans, T. canis (13, 26, 30). The gold standard of diagnosis is identification of B. procyonis larvae in tissue biopsies (Fig. 6); when available, it clearly leaves no doubt as to etiology. However, obtaining permission to perform an antemortem brain biopsy is problematic at best and probably unwarranted in most cases (26, 44). Another obvious problem with biopsy of the CNS (especially fine-needle aspiration methods) is the inherent difficulty in locating the migrating larvae. From a patient history perspective, if baylisascariasis is suspected, questioning of the patient (or parents in the case of young children) often provides additional support for a diagnosis. Obviously, a history of exposure to raccoons or latrines or recollections of geophagia are helpful, but it should be kept in mind that no patient contact with raccoons or their feces may be reported. Nonetheless, conducting an investigation in the field at locations where the patient may have become exposed can be diagnostically helpful. Finding raccoon latrines is not only supportive of possible exposure to B. procyonis but can identify them for removal and thus help prevent additional cases. Because the vast majority of people cannot identify a raccoon latrine site (or raccoon feces for that matter), the assistance of public health agencies, wildlife biologists, or zoonotic disease epidemiologists familiar with the behavior of raccoons and the biology of B. procyonis is recommended. Fecal samples collected in the field should be examined by individuals skilled in the examination of fecal specimens from veterinary and wildlife sources. Raccoon fecal specimens or contaminated soil samples can be examined with modified flotation assays (23, 52). To the typical clinical laboratorian accustomed to examining human specimens, the large number of different types of eggs, larvae, and artifacts typically found in these samples may be overwhelming. Therefore, specimens should be examined by parasitologists with the requisite expertise needed to identify B. procyonis eggs because of similarities with ascarid eggs of other animals. In the absence of microscopic fecal examination


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Figure 6. Human brain biopsy sample (hematoxylin and eosin stain; magnification, ⫻140). This fine-needle aspirate was removed from the brain of an adolescent boy with severe CNS disease. A B. procyonis larva is clearly visible in cross section (at midbody level) at the edge of the tissue section. B. procyonis larvae can be easily distinguished from the larvae of Ascaris and Toxocara spp. Diameters of T. canis and Toxocara cati, which may also cause larva migrans, typically are from 17 to 21 ␮m or sometimes slightly smaller. In addition to the large size (60 to 70 ␮m in diameter) of B. procyonis larvae, conspicuous features are the prominent lateral alae on opposite sides of the cuticle and large excretory columns adjacent to the centrally located intestine. Photomicrograph courtesy of Lawrence R. Ash, UCLA School of Public Health. (Reprinted from reference 39 with permission from Elsevier Science. © 2002, Elsevier Science.)

(assuming a raccoon latrine is correctly identified), it is best to consider any sample of raccoon feces as positive for B. procyonis eggs given the prevalence and geographic distribution of the parasite until proven otherwise. TREATMENT OF HUMAN INFECTION There is no effective cure for human baylisascariasis except in cases of OLM where laser photocoagulation has been used successfully in conjunction with treatment with corticosteroids (14, 26, 27). The method of choice for patients with NLM caused by B. procyonis infection is treatment with anti-inflammatory steroids and an anthelmintic agent. However, in this patient group, progressive neurologic deterioration continues even with treatment. In any event, no treatment described to date has altered the prognosis once clinical signs of NLM develop (27, 28, 44). Without question, the biggest contributing


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factor to the poor response to B. procyonis NLM therapy is treatment delay due to failure to add this zoonosis to the list of differential diagnoses. Currently, the drug of choice for humans is albendazole, a benzimidazole anthelmintic that has good distribution into the brain and CSF (9, 11, 44, 60). Experimental B. procyonis infections in mice have demonstrated that albendazole and another anthelmintic, diethylcarbamazine, were of prophylactic value in preventing CNS disease when administered 1 to 10 days postinfection (27, 28). As a prophylactic measure, the prompt administration of albendazole is thought to have contributed to the fact that clinical symptoms of baylisascariasis did not develop in cases where children were observed ingesting raccoon feces (27). Albendazole is well tolerated by humans, and its side effects are minimal (61). In light of these facts, the current recommendation is that when geophagia near a raccoon latrine site is witnessed or in any case where B. procyonis infection is a serious consideration (e.g., a patient with eosinophilic meningoencephalitis), treatment with albendazole and steroids (e.g., methylprednisolone) should be started immediately while diagnostic tests and further investigations to document a source of exposure are carried out (11). In these situations, the patient should be immediately treated with albendazole (25 to 50 mg/kg/day for 10 days or 400 mg twice a day for 10 days) (27). Certainly, even if the diagnosis is suspected given clinical signs and patient history, the almost certain grave outcome of disease in a child with B. procyonis infection warrants treatment. CONCLUSIONS In general, with the exception of rabies, the public is not aware of zoonotic disease risks, despite an often close association with wild animals or domesticated animals. Aside from the potential threat of bites or attacks, the public does not view wildlife with any real alarm. Websites abound with information about feeding raccoons in backyards and even how to adopt them as “pets.” The promotion of such activities serves only to exacerbate the problem of nuisance wildlife. The problem of urbanized raccoons exemplifies the growing disconnect between the public’s conceptions about wildlife and the reality of the public health risks that uncontrolled wildlife can pose. No doubt another contributing factor to public apathy toward zoonoses is lack of effective education about this hazard. Wildlife education efforts should always include a discussion of zoonotic disease risks, given in a nonsensational but accurate way using terms and methods the public can understand and employ. Ascarids are the most important cause of larva migrans in humans, and of these, B. procyonis is most likely to cause serious, life-threatening disease (27, 28, 31). Without question, more public and medical practitioner awareness of this zoonosis is needed as well as the development of improved diagnostic tests. An added benefit of diagnostic improvements would be that epidemiologic surveys could be carried out in human populations. Accurate assessment of the prevalence of human exposure to B. procyonis would be of significant public health value and would begin to answer the numerous questions that remain about the epidemiology of baylisascariasis. Despite the rarity of baylisascariasis, the lack of an effective cure and the severity of the disease, especially in very young children and infants, who are most likely to become infected, cannot be overemphasized. Obviously, lack of an effective treatment for baylisascariasis in humans leaves prevention of infection of paramount importance.


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Community prevention measures include education of the public and physicians (especially pediatricians), removal of attractants (e.g., pet food and unsecured garbage), and discontinuation of practices that can induce raccoons to take up residence in an area. The number of raccoon latrines in an area is directly related to the number of raccoons living there (44). Allowing raccoons to overpopulate an area inevitably leads to widespread contamination of the environment due to natural dispersal of infective B. procyonis eggs from the action of weather and other natural forces. Raccoons should not be allowed to establish raccoon latrine sites in areas of human habitation and recreation such as parks and playgrounds. Individuals can help prevent infection by being vigilant for raccoon activity, supervising children when working or playing outdoors, admonishing young children not to indulge in pica or geophagia, and having everyone thoroughly wash their hands following outdoor activities or after handling pets. In the context as an emerging infection, perhaps baylisascariasis is best understood as a zoonosis that is being increasingly recognized and one that promises to become as prevalent as humans are willing to allow. Acknowledgments. I thank Lawrence Ash and David Bieber for their reviews of this chapter. For his invaluable and extensive contributions to the understanding of the epidemiology, pathogenesis, diagnosis, and treatment of baylisascariasis, this chapter is dedicated to Kevin R. Kazacos.

REFERENCES 1. Acha, P. N., and B. Szyfres. 1995. Zoonoses and Communicable Diseases Common to Man and Animals, 2nd ed., p. 112, 337, 428, 614, 686, 792, 813, 819, 821, 852–855. Scientific Publication No. 503. Pan American Health Organization, World Health Organization, Washington, D.C. 2. Averbeck, G. A., J. A. Vanek, B. E. Stromberg, and J. R. Laursen. 1995. Differentiation of Baylisascaris species, Toxocara canis and Toxascaris leonina infections in dogs. Compend. Cont. Ed. Pract. Vet. 17:475–511. 3. Beaver, P. C. 1956. Parasitological reviews: larva migrans. Exp. Parasitol. 5:587–621. 4. Beaver, P. C. 1969. The nature of visceral larva migrans. J. Parasitol. 55:3–12. 5. Bigler, W. J., J. H. Jenkins, P. M. Hoff, and E. C. Prather. 1975. Wildlife and environmental health: raccoons as indicators of zoonoses and pollutants in the southeastern United States. J. Am. Vet. Med. Assoc. 167:592–597. 6. Burton, M., and R. Burton. 1980. Raccoon, p. 2033–2035. In The New Funk and Wagnall’s Illustrated Wildlife Encyclopedia, vol. 18. BPC Publishing Limited, Cambridge, United Kingdom. 7. Conboy, G. 1996. Diagnostic parasitology. Can. Vet. J. 37:181–182. 8. Cunningham, C. K., K. R. Kazacos, J. A. McMillan, J. A. Lucas, J. B. McAuley, E. J. Wozniak, and L. B. Weiner. 1994. Diagnosis and management of Baylisascaris procyonis infection in an infant with nonfatal meningoencephalitis. Clin. Infect. Dis. 18:868–872. 9. de Silva, N., H. Guyatt, and D. Bundy. 1997. Antihelmintics: a comparative review of their clinical pharmacology. Drugs 53:769–788. 10. Fox, A. S., K. R. Kazacos, N. S. Gould, P. T. Heydemann, C. Thomas, and K. M. Boyer. 1985. Fatal eosinophilic meningoencephalitis and visceral larva migrans caused by the raccoon ascarid, Baylisascaris procyonis. N. Engl. J. Med. 312:1619–1623. 11. Gavin, P. J., K. R. Kazacos, T. Q. Tan, W. B. Brinkman, S. E. Byrd, A. T. Davis, M. B. Mets, and S. T. Shulman. 2002. Neural larva migrans caused by the raccoon roundworm Baylisascaris procyonis. Pediatr. Infect. Dis. J. 21:971–975. 12. Gey, A. B. 1998. Synopsis der Parasitenfauna des Waschbären (Procyon lotor) unter Berucksichtigung von befunden aus Hessen. Institute of Parasitology, Justus Liebig University, Giessen, Germany. 13. Glickman, L. T., and F. S. Shofer. 1987. Zoonotic visceral and ocular larva migrans. In J. R. August and A. S. Loar (ed.), Zoonotic Diseases. Vet. Clin. N. Am. (Small Animal Practice) 17:39–53.


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14. Goldberg, M. A., K. R. Kazacos, W. M. Boyce, E. Ai, and B. Katz. 1993. Diffuse unilateral subacute neuroretinitis: morphometric, serologic and epidemiologic support for Baylisascaris as a causative agent. Ophthalmology 100:1695–1701. 15. Goldman, E. A. 1950. Raccoons of North and Middle America. North American Fauna Series No. 60. U.S. Department of the Interior, Fish and Game Service, Washington, D.C. 16. Greve, J. H., and S. E. O’Brien. 1989. Adult Baylisascaris infections in two dogs. Companion Animal Practice 19:41–43. 17. Hoffman, C. O., and J. L. Gottschang. 1977. Numbers, distribution, and movements of a raccoon population in a suburban residential community. J. Mammal. 58:623–636. 18. Holmgren, V. C. 1990. Raccoons in Folklore, History and Today’s Backyards. Capra Press, Santa Barbara, Calif. 19. Huff, D. S., R. C. Neafie, M. J. Binder, G. A. DeLeon, L. W. Brown, and K. R. Kazacos. 1984. The first fatal Baylisascaris infection in humans: an infant with eosinophilic meningoencephalitis. Pediatr. Pathol. 2:345–352. 20. Ingles, L. G. 1965. Raccoon, p. 357–359. In Mammals of the Pacific States: California, Oregon, Washington. Stanford University Press, Stanford, Calif. 21. Jacobson, J. E., K. R. Kazacos, and F. H. Montague, Jr. 1982. Prevalence of eggs of Baylisascaris procyonis (Nematoda: Ascaroidea) in raccoon scats from an urban and rural community. J. Wildl. Dis. 18:461– 464. 22. Kazacos, K. R. 1982. Contaminative ability of Baylisascaris procyonis infected raccoons in an outbreak of cerebralspinal nematodiasis. Proc. Helminthol. Soc. Wash. 49:155–157. 23. Kazacos, K. R. 1983. Improved method for recovering ascarid and other helminth eggs from soil associated with epizootics and during survey studies. Am. J. Vet. Res. 44:896–900. 24. Kazacos, K. R. 1991. Visceral and ocular larva migrans. Semin. Vet. Med. Surg. (Small Anim.) 6:227–235. 25. Kazacos, K. R. 1996. Baylisascariasis, p. 716–717. In A. M. Rudolph, J. I. E. Hoffman, and C. D. Rudolph (ed.), Rudolph’s Pediatrics, 20th ed. Appleton & Lange, Stamford, Conn. 26. Kazacos, K. R. 1997. Visceral, ocular, and neural larva migrans, p. 1459–1473. In D. H. Connor, F. W. Chandler, H. J. Manz, D. A. Schwartz, E. E. Lack, J. B. Baird, and J. P. Utz (ed.), Pathology of Infectious Diseases, vol. II. Appleton & Lange, Stamford, Conn. 27. Kazacos, K. R. 2000. Protecting children from helminthic zoonoses. Contemp. Pediatr. 17(Suppl.):1–24. 28. Kazacos. K. R. 2001. Baylisascaris procyonis and related species. In W. M. Samuel, M. J. Pybus, and A. A. Kocan (ed.), Parasitic Diseases of Wild Animals, 2nd ed. Iowa State University Press, Ames, Iowa. 29. Kazacos, K. R., W. L. Wirtz, P. P. Burger, and C. S. Christmas. 1981. Raccoon ascarid larvae as a cause of fatal nervous system disease in subhuman primates. J. Am. Vet. Med. Assoc. 179:1089–1094. 30. Kazacos, K. R., L. A. Raymond, E. A. Kazacos, and W. A. Vestre. 1985. The raccoon ascarid: a probable cause of human ocular larva migrans. Ophthalmology 92:1735–1743. 31. Kazacos, K. R., and W. M. Boyce. 1995. Baylisascaris larva migrans. J. Am. Vet. Med. Assoc. 195:894– 903. Addendum, 1995, p. 20–30. In Zoonosis Updates from the Journal of the American Veterinary Medical Association, 2nd ed. American Veterinary Medical Association, Schaumburg, Ill. 32. Kidder, J. D., S. E. Wade, M. E. Richmond, and S. J. Schwager. 1989. Prevalence of patent Baylisascaris procyonis infection in raccoons (Procyon lotor) in Ithaca, NY. J. Parasitol. 75:870–874. 33. Kuchle, M., H. L. J. Knorr, S. Medenblik-Frysch, A. Weber, C. Bauer, and G. O. H. Naumann. 1993. Diffuse unilateral subacute neuroretinitis syndrome in a German most likely caused by the raccoon roundworm, Baylisascaris procyonis. Graefe’s Arch. Clin. Exp. Ophthalmol. 231:48–51. 34. Lotze, J.-H., and S. Anderson. 1979. Procyon lotor, p. 1–8. In Mammalian Species, no. 119. American Society of Mammalogists, Lawrence, Kans. 35. McCombie, B. 1999. Those dirty raccoons: how a once revered fur bearer became reduced to cockroach status. Field and Stream, December, p. 8. 36. Miyashita, M. 1993. Prevalence of Baylisascaris procyonis in raccoons in Japan and experimental infections of the worm in laboratory animals. J. Urban Living Health Assoc. 37:137–151. 37. Moertel, C. L., K. R. Kazacos, J. H. Butterfield, H. Kita, J. Watterson, and G. J. Gleich. 2001. Eosinophil-associated inflammation and elaboration of eosinophil-derived proteins in 2 children with raccoon roundworm (Baylisascaris procyonis) encephalitis. Pediatrics 108:1–7. [Online.] http://www. pediatrics.org/cgi/content/full/108/5/e93.


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38. Morse, E. V., D. A. Midla, and K. R. Kazacos. 1983. Raccoons (Procyon lotor) as carriers of Salmonella. J. Environ. Sci. Health A18:541–560. 39. Murray, W. J. 2002. Human infections caused by the raccoon roundworm, Baylisascaris procyonis. Clin. Microbiol. Newsl. 24:1–7. 40. Nowak, R. M., and J. L. Paradiso. 1983. Raccoons, p. 980–982. In Walker’s Mammals of the World, 4th ed. The Johns Hopkins University Press, Baltimore, Md. 41. Page, L. K., and R. K. Swihart. 1998. Raccoon latrine structure and its potential role in transmission of Baylisascaris procyonis to vertebrates. Am. Midland Naturalist 140:180–185. 42. Page, L. K., R. K. Swihart, and K. R. Kazacos. 1999. Implications of raccoon latrines in the epizootiology of baylisascariasis. J. Wildl. Dis. 35:474–480. 43. Page, L. K., R. K. Swihart, and K. R. Kazacos. 2001. Seed preferences and foraging by granivores at raccoon latrines in the transmission dynamics of the raccoon roundworm (Baylisascaris procyonis). Can. J. Zool. 79:616–622. 44. Park, S. Y., C. Glaser, W. J. Murray, K. R. Kazacos, H. A. Rowley, D. R. Fredrick, and N. Bass. 2000. Raccoon roundworm (Baylisascaris procyonis) encephalitis: case report and field investigation. Pediatrics 106:1–5. [Online.] http://www.pediatrics.org/cgi/content/full/106/4/e56. 45. Riley, S. P. D., J. Hadidian, and D. A. Manski. 1998. Population density, survival, and rabies in raccoons in an urban national park. Can. J. Zool. 76:1153–1164. 46. Roberts, L. S., and J. Janovy. 1996. Nematodes: ascardida, intestinal large roundworms, p. 419–431. In G. D. Schmidt and L. S. Roberts (ed.), Foundations of Parasitology, 5th ed. Wm. C. Brown Publishers, Dubuque, Iowa. 47. Rosatte, R. C., M. J. Powers, and C. D. MacInnes. 1991. Ecology of urban skunks, raccoons and foxes in metropolitan Toronto, p. 31–38. In L. D. Adams and D. L. Leedy (ed.), Wildlife Conservation in Metropolitan Environments. National Institute of Urban Wildlife, Columbia, Md. 48. Rowley, H. A., R. M. Uht, K. R. Kazacos, J. Sakanari, W. V. Wheaton, A. J. Barkovich, and A. W. Bollen. 2000. Radiologic-pathologic findings in raccoon roundworm (Baylisascaris procyonis) encephalitis. Am. J. Neuroradiol. 21:415–420. 49. Rudmann, D. G., K. R. Kazacos, S. T. Storandt, D. L. Harris, and E. B. Janovitz. 1996. Baylisascaris procyonis larva migrans in a puppy: a case report and update. J. Am. Anim. Hosp. Assoc. 32:73–76. 50. Schwartz, B. 1960. Evolution of knowledge concerning the roundworm Ascaris lumbricoides, p. 465–481. In Smithsonian Report for 1959. The Smithsonian Institution, Washington, D.C. 51. Sharp, D. 2000. Creature discomfort is call to battle in suburbs. USA Today, 30 August, section 4A. 52. Sloss, M. W., R. L. Kemp, and A. M. Zajac. 1994. Veterinary Clinical Parasitology, 6th ed, p. 3–8. Iowa State University Press, Ames, Iowa. 53. Sprent, J. F. A. 1955. On the invasion of the central nervous system by nematodes. I: The incidence and pathological significance of nematodes in the central nervous system. Parasitology 45:31–39. 54. Sprent, J. F. A. 1968. Notes on Ascaris and Toxascaris, with a definition of Baylisascaris gen. nov. Parasitology 58:185–198. 55. Stone, W. B. 1983. Intestinal obstruction in raccoons caused by the ascarid Baylisascaris procyonis. N. Y. Fish Game J. 30:117–118. 56. Swift, E. 2001. It’s a jungle out there. Parade, 11 February, p. 4–7. 57. Thomas, J. S. 1988. Encephalomyelitis in a dog caused by Baylisascaris infection. Vet. Pathol. 25:94–95. 58. Tiner, J. D. 1953. The migration, distribution in the brain, and growth of ascarid larvae in rodents. J. Infec. Dis. 92:105–113. 59. Vaughan, T. A. 1986. Family Procyonidae, p. 158–159. In Mammalogy, 3rd ed. Saunders College Publishing, Philadelphia, Pa. 60. Venkatesan, P. 1998. Albendazole. J. Antimicrob. Chemother. 41:145–147. 61. Webster, L. T. 1985. Drugs used in the chemotherapy of helminthiasis, p. 1012–1014. In A. G. Gilman, L. S. Goodman, T. W. Rall, and F. Murad (ed.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 7th ed. MacMillan Publishing Company, New York, N.Y.


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Chapter 13

Can Surveillance for Unexplained Deaths Be Used as a Public Health Approach for Early Recognition of New Pathogens? Marc Fischer, Rana A. Hajjeh, and Andre N. Sofair

Over the past three decades, several new infectious diseases associated with life-threatening illness have been identified in the United States, including Legionnaires’ disease, toxic shock syndrome, AIDS, and hantavirus pulmonary syndrome. Each of these diseases was first identified after an outbreak investigation of unexplained severe illnesses. However, subsequent studies found that each of these pathogens had been causing sporadic disease for years prior to their recognition. For example, Legionella pneumophila was established as the cause of Legionnaires’ disease in 1976, but subsequent evaluation identified an outbreak in 1957 and sporadic cases dating back to 1947 (34, 40). Toxic shock syndrome was first recognized in 1979, but retrospective chart reviews documented cases as early as 1960 (39). AIDS was initially described in 1981, yet subsequent investigations documented persons in the United States infected with human immunodeficiency virus (HIV) in 1968 (3, 6, 22, 25). Similarly, after the outbreak of hantavirus pulmonary syndrome in the Four Corners region in 1993, studies revealed infections due to Sin Nombre virus in the United States dating back to 1959 (19, 31, 64). These delays in the recognition of new pathogens likely resulted from limitations in both surveillance methods and diagnostic techniques. Traditional public health surveillance relies upon the reporting and monitoring of illnesses caused by a limited set of known pathogens and may not be the optimal way to detect emerging diseases. Furthermore, until recently, identification of new infectious agents relied primarily on in vitro cultivation. Therefore, even when unique clusters or patterns of illnesses were recognized, isolation of the causative agent was often complicated by the fastidious growth requirements of the new bacterium (e.g., Legionella and Bartonella spp.) or virus (e.g., HIV and Sin Nombre virus).

Marc Fischer and Rana A. Hajjeh • Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333. Andre N. Sofair • Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520. 177


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In 1992, the Institute of Medicine concluded that timely recognition of new infectious pathogens would require both advances in diagnostic techniques and modifications in the types of illnesses that should be reported to public health authorities (29). Molecular diagnostic methods had recently made it possible to identify and characterize new infectious agents in the absence of in vitro cultivation (4, 16, 28, 32, 35, 44, 45). The next step was to develop a systematic public health approach to identify illnesses that may be due to new pathogens and subject them to these novel diagnostic techniques (7, 42).

UNEXPLAINED ILLNESS SURVEILLANCE IN THE EIP SITES In 1995, a population-based project, the Surveillance for Unexplained Deaths and Critical Illnesses Due to Possibly Infectious Causes Project (UNEX), was initiated at four Emerging Infection Program (EIP) sites in California, Connecticut, Minnesota, and Oregon (26, 42). The primary objectives of this project were (i) to define the incidence and epidemiologic features of severe unexplained illness in selected populations in the United States, (ii) to apply molecular diagnostic techniques to identify potential infectious etiologies for these illnesses, (iii) to develop and test a surveillance system for early detection of previously unrecognized life-threatening infectious agents, and (iv) to create a bank of clinical specimens for future testing as new pathogens and methods are identified. The methods and results from the first 3.5 years of this project have recently been summarized and published (26).

Defining an Unexplained Death or Critical Illness For the purposes of this project, a case-patient was defined as a previously healthy resident of the surveillance area, 1 to 49 years of age, who died or was admitted to an intensive care unit owing to a possibly infectious disease in which no etiology was identified on initial testing. Exclusion criteria were extensive and included malignancy, diabetes, immunosuppressive therapy, HIV infection, and chronic cardiac, pulmonary, renal, hepatic, and rheumatologic conditions. Persons with exposures to toxic agents, ingestions of such agents, trauma, or recent hospitalization were also excluded. Hallmarks of an infectious disease included fever, leukocytosis, cerebrospinal fluid pleocytosis, and histopathologic evidence of an infection (26).

Case Finding Cases were detected through active and passive intensive care unit surveillance and death certificate review. Epidemiologic and clinical data were obtained via chart abstraction and interviews with medical practitioners, patients, and families of patients. Previously collected clinical and pathologic specimens were obtained for additional diagnostic testing. Laboratory testing decisions were made on the basis of the individual history of each case and the quality and quantity of available specimens. Over time, standard sets of syndrome-specific tests were developed and applied to all patients presenting with similar clinical features (26).


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Rates and Demographics From May 1995 to December 1998, 137 patients were identified who met the case definition for an overall annual rate of 0.5 per 100,000 persons. This rate likely represents a minimum estimate, since persons with underlying illnesses were not excluded from the denominator. Incidence rates varied by age group (Fig. 1) but were similar for males and females as well as for different racial groups. Forty-one (30%) of the case-patients died, of whom 30 (73%) had autopsies performed, reflecting a rate much higher than the national postmortem evaluation rate of ⬍11% (14). No differences were observed in the seasonal distribution of cases, and there was no clustering of cases by time or place. The most common clinical presentations were neurologic (29%), respiratory (26%), and cardiac (20%). The highest syndrome-specific case-fatality ratio was seen among cases involving cardiac syndromes (46%), and the lowest ratio was seen among cases involving neurologic syndromes (18%) (Table 1) (26). Laboratory Evaluation Of the 122 patients who had specimens available for testing, 34 (28%) had illnesses that could be attributed to a specific infectious agent (Table 2). In addition, five patients met the clinical definition for toxic shock syndrome but did not meet the study criteria for a specific etiology. All of the infectious agents identified were previously recognized bacterial and viral pathogens. However, of the 34 explained cases, 23 (68%) were explained by using serologic tests, 7 (21%) were explained by using specific-primer PCR assays, and 4 (12%) were explained by using 16S ribosomal DNA (16S rDNA) PCR. An infectious etiology was more likely to be identified in cases with paired serum specimens (14/62, 23%) than in those with single serum specimens (2/42, 5%) (P ⫽ 0.05). Explained cases were similar to unexplained cases with regard to age, sex, and race but were reported sooner after admission than unexplained cases (median, 4 days versus 7.5 days; P ⫽ 0.1) (26).

Figure 1. Incidence of cases by age group, 1995 to 1998, from UNEX. Adapted from reference 26.


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Table 1. Distribution of unexplained deaths and critical illness cases by syndrome, with proportion explaineda No. (%) of: Syndrome Neurologic Respiratory Cardiac Multisystem Hepatic Other All cases

Enrolled cases

Deaths

Explained/cases with specimens

39 (29) 36 (26) 28 (20) 18 (13) 9 (7) 7 (5)

7 (18) 11 (31) 13 (46) 4 (22) 4 (44) 2 (29)

15/37 (41) 13/33 (39) 3/22 (14) 3/15 (20) 0/8 (0) 0/7 (0)

137 (100)

41 (30)

34/122 (28)

a

Adapted from reference 26.

Lessons Learned In its first 3.5 years, active surveillance within the EIP sites defined the incidence of unexplained deaths and critical illnesses for selected populations in the United States. The overall rate of 0.5 per 100,000 remained remarkably stable from year to year and may serve as a reasonable baseline for future comparisons. On the basis of these findings, the investigators estimated that 920 cases of unexplained severe illnesses due to possibly infectious causes occur each year in the United States among previously healthy persons 1 to 49 years of age; almost 300 of these patients die. These unexplained illnesses were categorized into five primary clinical presentations that may help us to develop more efficient and effective diagnosTable 2. Infectious disease causes for explained cases, UNEX, 1995–1998, California, Oregon, Connecticut, and Minnesota (n ⫽ 34)a Etiology (n)

Tests (n)b

Mycoplasma pneumoniae (5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EIA IgM/IgG (4), PCR (1) Neisseria meningitidis (5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16S rDNA PCR (2), PCR (2), EIA IgM (1) Chlamydia pneumoniae (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MIF IgG (3) Enterovirus (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EIA IgM (3) Bartonella spp. (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR, IFA IgG (3) Adenovirus (2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EIA and IFA IgG (1), PCR (1) Legionella spp. (2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PCR (2) Streptococcus pneumoniae (2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16S rDNA PCR (2) Borrelia burgdorferi/Ehrlichia chaffeensis (1) . . . . . . . . . . . . . . . . . .EIA/IFA flagella IgG, Western blot (IgG/IgM) (1) Cytomegalovirus (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EIA and IFA IgG (1) Coxsackie B virus (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EIA IgM, viral culture (1) Epstein-Barr virus (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IFA IgG (VCA and EA) (1) Human herpesvirus 6 (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IFA and EIA (IgM and IgG) (1) Influenza A virus (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EIA and IFA IgM, EIA (IgG) (1) Influenza B virus (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EIA and IFA IgG (1) Mumps virus (1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IFA IgM, IFA and EIA IgG (1) Parainfluenza virus types 1 and 3 (1) . . . . . . . . . . . . . . . . . . . . . . . . . EIA and IFA IgG (1) a b

Adapted from reference 26. Abbreviations: EIA, enzyme immunosorbent assay; IFA, indirect immunofluorescent assay; Ig, immunoglobulin; MIF, microimmunofluorescence; VCA, viral capsid antigen; EA, early antigen.


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tic algorithms. Furthermore, despite extensive negative workups at clinical institutions, likely infectious etiologies were identified for 34 (28%) of the cases evaluated. Of the 34 cases for which a specific infectious etiology was identified, 21 (62%) were due to organisms for which either an expanded-spectrum cephalosporin or tetracycline is a first line of therapy. Although these data do not address the 72% of cases for which no etiology was identified, they provide a potentially simple empirical antibiotic regimen for severe and initially unexplained infections among immunocompetent persons and should be evaluated further. Although this project was an important first step in defining the burden of unexplained severe illness, it also identified several significant obstacles to implementing a sentinel surveillance system for the recognition of new or unusual infectious pathogens. Failure to identify an infectious cause for 72% of the cases investigated may be due to a number of reasons, including the following: (i) the illness was not due to infection; (ii) the severe clinical manifestations resulted from an immune- or toxin-mediated process, potentially days or weeks after the primary infection; (iii) empirical therapies interfered with the ability to make a specific diagnosis; (iv) adequate specimens were not obtained or tested; (v) the true pathogen was not considered; or (vi) appropriate tests for the pathogen were not available. For some patients identified through ongoing surveillance, specimens were not available from the primary site of disease, were severely limited in quantity, or were available only late in the course of the disease. In many cases, multiple serum specimens were not available, autopsies were incomplete, and tissue specimens were obtained only from unaffected organs. To move beyond these limitations, further efforts are needed to provide a simpler and more systematic approach to specimen collection and to develop, validate, and incorporate better broad-based diagnostic techniques. Applying the UNEX Approach beyond the EIP Sites Identifying these cases required the formulation of unique event-based reporting systems at each EIP site. The broad diagnostic evaluation of these cases also required the coordination of large amounts of data and specimens and a wide array of laboratory tests. As a result, the project assembled a network of investigators to assist in the evaluation of cases or clusters of unexplained disease. These links fostered collaborative efforts within the Centers for Disease Control and Prevention (CDC) and between CDC, state health departments, and academic institutions. Application of this infrastructure to selected cases occurring outside the EIP sites has resulted in the identification of specific etiologies of clinical and/or public health importance. A few examples include (i) Whipple’s encephalitis in an immigrant from Ethiopia, (ii) melioidosis in a traveler to Southeast Asia, (iii) fatal hemorrhagic pneumonitis due to herpes simplex virus type 1, (iv) hantavirus pulmonary syndrome in Nebraska, (v) fatal pneumococcal meningitis that had been attributed to Neisseria meningitidis, and (vi) meningococcemia that was initially diagnosed as toxic shock syndrome. In contrast to patients identified through ongoing surveillance at the EIP sites, all of these cases had adequate tissue specimens available for evaluation, suggesting that the diagnostic yield of UNEX could be improved if the methods were modified to allow for better specimen collection. In addition, all of these diagnoses were made by using recently developed molecular or immunohistochemical tests. These findings suggest that, under optimal conditions, surveillance for unexplained deaths can identify sporadic cases of public health significance, including atypical presentations of


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relatively common conditions. However, maintaining active surveillance, collecting adequate specimens, and performing extensive laboratory testing require substantial infrastructure and resources for relatively few diagnoses. As a result, focusing on clusters of unexplained severe infections may be a more efficient way to distinguish sentinel events that may be due to new pathogens. Surveillance for unexplained deaths has helped to identify several unusual clusters of disease, including cases of acute liver failure due to a common weight loss supplement (D. R. Stolpman, J. Petty, J. Ham, S. Orloff, and J. Schwartz, Am. Assoc. Study Liver Dis., 2002) and clostridial sepsis following femoral allograft transplantation (14). In addition, the project infrastructure proved invaluable in a number of recent outbreaks that presented as clusters of unexplained severe illness, including West Nile virus encephalitis in New York City (10), histotoxic clostridia among intravenous drug users in Scotland and Ireland (13), and adenovirus and parainfluenza virus pneumonia in a pediatric long-term-care facility (P. Kludt, K. Myers, E. Harvey, A. Demaria, B. Werner, A. Hackbarth, B. Matyas, C. Rauch, D. Fisher, J. Levin, J. Ouellette, C. Gaudet, D. Erdman, and D. Shay, Abstr. Int. Conf. Emerg. Infect. Dis. 2000, 2000). Finally, as demonstrated during the recent anthrax investigation, the ability to efficiently assess cases of unexplained illness is also an essential element in the evaluation of and response to potential acts of bioterrorism. IMPROVING THE SEARCH FOR ETIOLOGIC AGENTS Regardless of whether evaluation of unexplained deaths is being applied to active or passive surveillance, sporadic cases or clusters of disease, or naturally occurring or bioterrorism-related illnesses, recent experience has identified several principles that may improve the search for etiologic agents. First, focus and standardize the case definitions and surveillance methods to identify patients with the highest diagnostic yield for the clinical syndrome or agents of interest. Next, build a collaborative team including clinicians, pathologists, and public health practitioners to maximize the timeliness of case identification and enhance the quality of clinical specimens available for testing. Finally, systematically develop, evaluate, and incorporate better diagnostic techniques to identify causes of unexplained infections. These principles are now being employed in the next generation of projects to help identify new or emerging infectious diseases. Expanding the Role of Medical Examiners Further integration of medical examiners and coroners into case finding and evaluation is an essential component of unexplained death surveillance that could markedly improve the quality and quantity of pathologic specimens obtained from patients (27). Medical examiners investigate ⬃20% of all deaths in the United States, including deaths that occur outside of the hospital. Medical examiner jurisdictions are population based, and their investigations begin immediately after a death is reported. However, forensic pathologists are often satisfied with general pathologic diagnoses rather than organism-specific diagnoses when certifying deaths from infectious diseases. As such, specimens and tests needed to achieve an organism-specific diagnosis are frequently not collected and performed, respectively, significantly limiting the capacity to recognize emerging infectious diseases (37, 38).


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Obtaining an organism-specific diagnosis extends beyond simply performing an autopsy. Collection of autopsy specimens for microbiological evaluation is complicated by postmortem contamination of blood and tissues with gut, skin, oral, and environmental flora. Furthermore, the identification of specific infectious agents may require extensive testing beyond routine gross and histologic evaluations. By changing the timing of autopsies and improving sampling methods, the level of contamination can be reduced. Once that is accomplished, a combination of culture-based, immunohistochemical, and molecular techniques can provide an effective strategy for identifying infectious diseases (Table 3) (67). Unfortunately, many forensic pathologists remain unaware of these techniques or do not have access to the technology. Consequently, optimal specimens are sometimes not obtained or are not submitted for appropriate evaluation. An enhanced medical examiner program could strengthen surveillance for unexplained deaths by (i) improving the identification of deaths potentially due to an infectious disease, (ii) increasing the proportion of potential cases submitted for autopsy, (iii) obtaining accurate and standardized data on all suspect cases in a timely manner, (iv) collecting adequate specimens for routine and reference testing for both toxins and infectious pathogens, and (v) implementing state-of-the-art methods for autopsy-based microbiological diagnosis. To accomplish this task, the program would need to (i) increase pathologists’ awareness of the importance of organism-specific diagnoses; (ii) provide guidelines for the recognition of deaths that may be due to an infectious disease; (iii) develop a standard approach to data collection, specimen retrieval, and diagnostic testing of cases of potential infectious etiology; and (iv) provide adequate resources and training to allow forensic pathologists to perform autopsies safely and effectively on individuals with potentially transmissible infections. Since 1994, the New Mexico Office of the Medical Investigator has utilized an infectious disease death review team (IDDRT) composed of forensic and clinical pathologists, microbiologists, epidemiologists, infectious disease clinicians, and molecular diagnostic specialists to systematically review autopsied deaths (37, 38). Similar to surveillance for unexplained deaths, the objectives of the IDDRT are to (i) describe the burden of deaths due to infectious diseases and unexplained causes, (ii) perform surveillance for clusters of events and emerging infections, and (iii) improve detection and investigation of deaths Table 3. Selected examples of emerging infectious diseases that can cause severe illness and for which pathologic evaluation has made significant contributionsa Disease or syndrome

Agent(s)

AIDS Cat scratch disease Ebola hemorrhagic fever Granulomatous amebic encephalitis Hantavirus pulmonary syndrome Human ehrlichiosis

HIV Bartonella hensalae Ebola virus Balamuthia mandrillaris Sin Nombre virus Ehrlichia chaffeensis, Anaplasma phagocytophilum Legionella spp. Leptospira spp. Hendra virus, Nipah virus, Cache Valley virus, enterovirus 71

Legionellosis Leptospirosis Viral encephalitides a

Adapted from reference 66.

Selected reference(s) 5, 15, 47 1, 48, 57 23, 31, 33, 65, 66 54 24, 59, 63, 64 2, 20, 21, 55 60 9, 53, 62 11, 12, 41, 49, 50, 52, 56


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from infectious diseases by increasing the proportion of infectious disease-related deaths that receive organism-specific diagnoses. The IDDRT uses antemortem symptoms (e.g., fever and cough) to identify cases with potential pathologic syndromes (e.g., communityacquired pneumonia) that warrant an autopsy for infectious agent- or toxin-related etiologies. In turn, the pathologic syndrome helps to define a differential diagnosis, including potential bioterrorism agents (e.g., plague bacillus) and organisms of public health importance (e.g., influenza virus). During the first year of implementing this approach, the New Mexico Office of the Medical Investigator identified 150 cases that met their surveillance definition, including 24 (16%) that would not have otherwise resulted in an autopsy. Of the 150 deaths that were evaluated, 72 (48%) received an organism-specific diagnosis, including Streptococcus pneumoniae (n ⫽ 18), Haemophilus influenzae (n ⫽ 4), botulism (n ⫽ 1), and tuberculosis (n ⫽ 1) (K. B. Nolte, M. B. Nashelsky, J. S. Nine, R. A. Irvine, J. L. McLemore, P. J. McFeeley, G. R. Durka, J. D. Perrings, and R. E. Zumwalt, Natl. Syndromic Surveillance Conf., 2002). By developing standardized protocols for case ascertainment, data management, specimen procurement, and diagnostic testing, the IDDRT approach could be expanded to help identify emerging infectious diseases by increasing the medical examiners’ capacity to (i) recognize selected infectious disease syndromes, (ii) perform a core set of primary cultures and toxicology on site, and (iii) collect appropriate specimens for further broad-based evaluation. Furthermore, this approach could be exported to multiple sites based on existing infrastructure and available resources and could be applied to multiple infectious disease syndromes on the basis of public health priorities. Syndrome-Based Surveillance By design, surveillance for unexplained deaths covers a wide variety of clinical presentations and etiologies. It is precisely this wide net that provides the opportunity to detect a new or emerging infectious pathogen. However, these varied scenarios and diseases pose a significant challenge in the development and application of a standardized diagnostic approach. One potential solution is to construct a set of complementary protocols and algorithms tailored to selected infectious disease syndromes. Based on prior experience within the EIP sites, 95% of the case-patients identified as having severe, unexplained, acute community-acquired infections could be grouped into one of five primary clinical syndromes: meningitis or encephalitis, acute pulmonary syndrome, acute liver failure, myocarditis, and sepsis/multiorgan failure. Syndrome-based surveillance would apply a unique case definition, specimen requirements, and testing algorithm to each of the primary clinical presentations while maintaining a common infrastructure, protocol, methodology, data collection instrument, and database for the overall program. By focusing these companion protocols on well-defined syndromes, we hope to engage specific clinicians at each institution under surveillance to (i) identify potential cases earlier in their disease course, (ii) improve collection of essential data and specimens, and (iii) help refine diagnostic approaches to their most puzzling cases. Between 1997 and 1999, syndrome-based surveillance for acute liver failure was performed at two tertiary care referral hospitals in the United States. Of the 41 patients enrolled in this pilot study, a specific infectious etiology was identified in 26%. Drug toxicity or noninfectious etiologies accounted for another 33%, leaving 41% with uniden-


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tified etiologies. Children (age, ⬍18 years) and blacks were significantly more likely to have an unknown etiology of their liver failure. Although standard toxicologic screening and serologic evaluation for known hepatitis viruses were performed, testing for new or unusual infectious etiologies was not undertaken (W. A. Bower, E. A. Fagan, M. W. Fried, B. P. Bell, C. N. Shapiro, E. E. Mast, P. J. Coleman, and H. S. Margolis, Abstr. 37th Annu. Meet. Infect. Dis. Soc. Am., 1999). In 1998, syndrome-specific surveillance for encephalitis was initiated in California. Diagnostic testing was routinely performed for more than 13 infectious causes of encephalitis (i.e., herpesviruses, enteroviruses, arboviruses, Bartonella spp., Chlamydia spp., and Mycoplasma pneumoniae), and epidemiologic and clinical information was collected to guide further evaluation. During the first 2.5 years, 334 patients meeting the surveillance definition were enrolled. Of these, 96% had cerebrospinal fluid and 63% had acute- and convalescent-phase sera for testing. A likely infectious etiology was identified in 53 (16%) cases, including viruses (9%), bacteria (5%), parasites (1%), and prions (1%); a noninfectious cause was defined for another 33 (10%) patients (23a). Despite the dramatic differences in the epidemiology, pathophysiology, clinical manifestations, and outcomes of acute liver failure and encephalitis, the findings from these two syndrome-based projects led investigators to very similar conclusions. First, noninfectious causes accounted for a significant proportion of cases for both clinical syndromes, and these patients could not be clearly distinguished by their clinical or histopathologic features. Furthermore, even in the setting of having adequate specimens for evaluation, better diagnostic tests are an essential component in the search for new or unusual infections.

FUTURE DIRECTIONS Pathogen Identification and Discovery Two striking findings from the surveillance for unexplained deaths are that in twothirds of the cases the cause could not be determined and that all the infectious agents identified were previously recognized pathogens. A proportion of the unknown cases may have had noninfectious causes, and another subset may have been due to microbial toxins or postinfectious inflammation without the presence of the organism or nucleic acid at the time of diagnosis. However, beyond the inadequacy of clinical and pathologic specimens for testing, a likely factor contributing to the failure to identify specific infectious pathogens was the current limitation of available laboratory diagnostic techniques. Thus far in the project, diagnostic testing has relied heavily on serology, immunohistochemistry, and PCR assays for specific known pathogens. Although many of the methods employed were experimental and beyond the capacity of clinical laboratories, they remain tied to the standard clinical approach of developing and testing a differential diagnosis based on clinical presentation and epidemiologic exposures. Several approaches have previously succeeded in identifying new or emerging pathogenic agents in specimens from patients with diseases of unknown etiology, including broadly reactive PCR assays (e.g., bacillary angiomatosis, Whipple’s bacillus, Nipah virus, West Nile virus [4, 18, 32, 44, 45]), immunohistochemistry with cross-reactive antibodies (e.g., Sin Nombre virus, Nipah virus, West Nile virus [51, 61, 62]), expression libraries (e.g., hepatitis C virus [17]), and representational difference analysis (e.g., human herpesvirus 8 [16]). In order to become a


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viable method for the detection of new infectious agents, surveillance for unexplained deaths must involve not only identification of unique and sentinel cases but also acquisition of clinical and pathologic specimens and use of these specimens in novel cutting-edge diagnostic techniques. The first step in this process will be to refine and standardize broad-based 16S rDNA PCR and 18S rDNA PCR for bacterial and fungal organisms (36, 43, 46, 58). Although these approaches have great promise, they are still limited in practical application due to issues of sensitivity and environmental contamination. A similar but even more challenging process will be to develop, evaluate, or assemble broadly reactive PCR assays to cover the known families of viruses. Beyond these approaches, fully nonspecific PCR, domainspecific differential display, and host mRNA expression patterns are but a few of the promising technologies that will need to be supported and tested. For this purpose, the wellcharacterized clinical specimens obtained through surveillance for unexplained deaths may prove invaluable. However, for these nonspecific and resource-dependent methods, the targeted evaluation of cases that are clustered in time or space, or linked by a similar clinical profile, may provide a higher yield for the timely recognition of new or emerging pathogens. Alternative Approaches to Monitoring and Aberration Detection Unexplained death surveillance may serve as a sentinel surveillance system to identify new infectious agents, recognize unique presentations of known pathogens, or detect bioterrorism. However, the complexity of this active, prospective system makes it difficult to implement beyond the EIP sites. To expand surveillance, different methods may be chosen, depending on availability of resources and overall objectives. For example, if the goal is to monitor trends in unexplained deaths possibly due to infection, International Classification of Diseases, 9th revision (ICD-9), codes identified through previous active surveillance could be identified and tracked through electronic death certificates. A more ambitious and perhaps more useful alternative would be to develop and evaluate an aberration detection system for multivariate trends in demographics recorded on death certificates. If, for instance, in the early 1970s, deaths due to the burgeoning HIV epidemic were not frequent enough to register among a background of overall mortality, a system that combined the features of deaths among young adult males in Los Angeles County may have prompted some attention. Similarly, in the early 1990s, deaths among young adult Native Americans in Arizona or New Mexico may have foreshadowed a coming hantavirus pulmonary syndrome outbreak. This approach would shift the focus of surveillance for unexplained deaths from sentinel cases to sentinel clusters, which would then be pursued for their clinical, epidemiologic, and etiologic significance. Although it would take considerable time and effort for up-front development and evaluation, this system, if successful, could be applied to much larger populations with far fewer resources. CONCLUSIONS Unexplained deaths from a potentially infectious cause occur on a regular basis. Prospective surveillance is useful in the detection of clustered and sporadic cases but is costly. The surveillance and diagnostic infrastructures assembled under the UNEX have been used both domestically and internationally for the investigation of unexplained ill-


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ness and death. Although currently available serologic and molecular techniques may be used to explain a proportion of cases, this approach is limited by the quality and timing of available specimens as well as by the sensitivity and specificity of these tests. In order to move beyond these limitations, our focus needs to be on the development of more-efficient surveillance techniques and refinement of broad-based molecular and host-response diagnostic tools. Acknowledgments. We thank some of the many people whose considerable efforts and foresight contributed to this chapter, including Sherif Zaki, Wun-Ju Shieh, David Stephens, David Relman, Kurt Nolte, Carol Glaser, William Bower, Ruth Berkelman, Douglas Passaro, Jill Hacker, Stephen Ladd-Wilson, Jean Rainbow, Susan Smith, Sarah Reagan, Emmanuel Ntekop, Jordan Tappero, Christopher Woods, Simo Nikkari, Larry Anderson, Nino Khetsurani, Bradley Perkins, Jairam Lingappa, and Ashley LaMonte. REFERENCES 1. Anderson, B., K. Sims, R. Regnery, L. Robinson, M. J. Schmidt, S. Goral, C. Hager, and K. Edward. 1994. Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR. J. Clin. Microbiol. 32:942–948. 2. Bakken, J. S., J. S. Dumler, S.-M. Chen, M. R. Eckman, L. L. Van Etta, and D. H. Walker. 1994. Human granulocytic ehrlichiosis in the upper midwest United States. JAMA 272:212–218. 3. Barre-Sinoussi, F., J. C. Chermann, and F. Rey. 1983. Isolation of a T-lymphotrophic retrovirus from a patient at risk for acquired immunodeficiency syndrome (AIDS). Science 220:868–871. 4. Briese, T., X. Y. Jia, C. Huang, L. J. Grady, and W. I. Lipkin. 1999. Identification of a Kunjin/West Nilelike flavivirus in brains of patients with New York encephalitis. Lancet 354:1261–1262. 5. Brynes, R. K., W. C. Chan, T. J. Spira, E. P. Ewing, and F. W. Chandler. 1983 Value of lymph node biopsy in unexplained lymphadenopathy in homosexual men. JAMA 250:1313–1317. 6. Centers for Disease Control. 1981. Pneumocystis pneumonia—Los Angeles. Morb. Mortal. Wkly. Rep. 30:250–252. 7. Centers for Disease Control and Prevention. 1994. Addressing emerging infectious disease threats: a prevention strategy for the United States. Public Health Service, U.S. Department of Health and Human Services, Atlanta, Ga. 8. Centers for Disease Control and Prevention. 1996. Outbreak of acute febrile illness and pulmonary hemorrhage—Nicaragua, 1995. Morb. Mortal. Wkly. Rep. 44:841–843. 9. Centers for Disease Control and Prevention. 1998. Outbreak of acute febrile illness among athletes participating in triathlons—Wisconsin and Illinois, 1998. Morb. Mortal. Wkly. Rep. 47:585–588. 10. Centers for Disease Control and Prevention. 1999. Outbreak of West Nile-like viral encephalitis—New York. Morb. Mortal. Wkly. Rep. 48:845–849. 11. Centers for Disease Control and Prevention. 1999. Outbreak of hendra-like virus—Malaysia and Singapore, 1998–1999. Morb. Mortal. Wkly. Rep. 48:265–269. 12. Centers for Disease Control and Prevention. 1999. Update: outbreak of Nipah virus—Malaysia and Singapore, 1999. Morb. Mortal. Wkly. Rep. 48:335–337. 13. Centers for Disease Control and Prevention. 2000. Unexplained illness and death among injecting-drug users—Glasgow, Scotland; Dublin, Ireland; and England, April–June 2000. Morb. Mortal. Wkly. Rep. 49: 489–492. 14. Centers for Disease Control and Prevention. 2001. Public health dispatch: update: unexplained deaths following knee surgery—Minnesota, 2001. Morb. Mortal. Wkly. Rep. 50:1080. 15. Chadburn, A., C. Metrka, and J. Mouradian. 1989. Progressive lymph node histology and its prognostic value in patients with acquired immunodeficiency syndrome and AIDS-related complex. Hum. Pathol. 20:579–587. 16. Chang, Y., E. Cesarman, M. S. Pessin, F. Lee, J. Culpepper, D. M. Knowles, and P. S. Moore. 1994. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 266:1865–1869. 17. Choo, Q. L., G. Kuo, A. J. Weiner, L. R. Overby, D. W. Bradley, and M. Houghton. 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359–362.


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Fischer et al. C. A. Bolin, S. L. Bragg, B. A. Perkin, and R. A. Spiegel. 1998. Epidemic leptospirosis associated with pulmonary hemorrhage—Nicaragua, 1995. J. Infect. Dis. 178:1457–1463. Visvesvara, G. S., F. L. Schuster, and A. J. Martinez. 1993. Balamuthia mandrillaris, N. G., N. Sp., agent of amebic meningoencephalitis in humans and other animals. J. Eukaryot. Microbiol. 40:504–514. Walker, D. H., and J. S. Dumler. 1997. Human monocytic and granulocytic ehrlichioses. Discovery and diagnosis of emerging tick-borne infections and the critical role of the pathologist. Arch. Pathol. Lab. Med. 121:785–791. Wang, L. F., W. P. Michalski, M. Yu, L. I. Pritchard, G. Crameri, B. Shiell, and B. T. Eaton. 1998. A novel P/V/C gene in a new member of the Paramyxoviridae family, which causes lethal infection in humans, horses, and other animals. J. Virol. 72:1482–1490. Wear, D. J., A. M. Margileth, T. L. Hadfield, G. W. Fischer, C. J. Schlagel, and F. M. King. 1983. Cat scratch disease: a bacterial infection. Science 221:1403–1405. Wilbrink, B., I. M. van der Heijden, L. M. Schouls, J. D. van Embden, J. M. Hazes, F. C. Breedveld, P. P. Tak. 1998. Detection of bacterial DNA in joint samples from patients with undifferentiated arthritis and reactive arthritis, using polymerase chain reaction with universal 16S ribosomal RNA primers. Arthritis Rheum. 41:535–543. Williams, R. J., R. T. Bryan, J. N. Mills, R. E. Palma, I. Vera, F. De Velasquez, E. Baez, W. E. Schmidt, R. E. Figueroa, C. J. Peters, S. R. Zaki, A. S. Khan, and T. G. Ksiazek. 1997. An outbreak of hantavirus pulmonary syndrome in western Paraguay. Am. J. Trop. Med. Hyg. 57:274–282. Winn, W. C., and R. L. Myerowitz. 1981. The pathology of the Legionella pneumonias. Hum. Pathol. 12:401. Wong, K. T., W. J. Shieh, S. R. Zaki, and C. T. Tan. 2002. Nipah virus infection, an emerging paramyxoviral zoonosis. Springer Semin. Immunopathol. 24:215–228. Zaki, S. R., W.-J. Shieh, and The Epidemic Working Group at Ministry of Health in Nicaragua. 1996. Leptospirosis associated with outbreak of acute febrile illness and pulmonary haemorrhage, Nicaragua, 1995. Lancet 347:535–536. Zaki, S. R., P. W. Greer, L. M. Coffield, C. S. Goldsmith, K. B. Nolte, K. Foucar, R. M. Feddersen, R. E. Zumwalt, G. L. Miller, A. S. Khan, et al. 1995. Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease. Am. J. Pathol. 146:552–579. Zaki, S. R., A. S. Khan, R. A. Goodman, L. R. Armstrong, P. W. Greer, L. M. Coffield, T. G. Ksiazek, P. E. Rollin, C. J. Peters, and R. Khabbaz. 1996. Retrospective diagnosis of hantavirus pulmonary syndrome, 1978–1993: implications for emerging infectious diseases. Arch. Pathol. Lab. Med. 120:134–139. Zaki, S. R., and C. J. Peters. 1997. Viral hemorrhagic fevers, p. 347–364. In D. H. Connor, F. W. Chandler, D. A. Schwartz, H. J. Manz, and E. E. Lack (ed.), Diagnostic Pathology of Infectious Diseases. Appleton and Lange, Stamford, Conn. Zaki, S. R., W. J. Shieh, P. W. Greer, C. S. Goldsmith, T. Ferebee, J. Katshitski, K. Tchioko, M. A. Bwaka, R. Swanepoel, P. Calain, A. S. Khan, E. Lloyd, P. E. Rollin, T. G. Ksiazek, C. J. Peters, and the EHF Study Group. 1999. A novel immunohistochemical assay for detection of Ebola virus in skin: implications for diagnosis, spread and surveillance of Ebola hemorrhagic fever. J. Infect. Dis. 179:S36–S47. Zaki, S. R., and C. Paddock. 1999. The emerging role of pathology in infectious diseases, p. 181–200. In W. M. Scheld, W. A. Craig, and J. M. Hughes (ed.), Emerging Infections 3. ASM Press, Washington, D.C.


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Chapter 14

The International Emerging Infections Program in Thailand: an Early Report Scott F. Dowell, Supamit Chunsuttiwat, Sonja J. Olsen, Pathom Sawanpanyalert, J. Mark Simmerman, Tamara L. Fisk, and Kumnuan Ungchusak

THE INTERNATIONAL EMERGING INFECTIONS PROGRAM CONCEPT As new infectious disease threats emerge and those once believed to have been controlled are raised anew in the wake of the tumultuous events of 2001, concerns about the safety of U.S. citizens and others from new infectious disease threats have never been greater. The Centers for Disease Control and Prevention, Atlanta, Ga. (CDC), has responded to these concerns by developing an agency-wide global infectious disease strategy that includes a central commitment to establish a series of collaborations with other countries, entitled International Emerging Infections Programs (IEIPs) (6). The first such program was launched in late 2001 in collaboration with the Ministry of Public Health in Thailand; the second site, in Africa, is planned for 2003. The IEIPs are envisioned as centers of excellence that integrate disease surveillance, applied research, prevention, and control activities. Each site will represent a partnership between a ministry of health and CDC, with additional partners to include one or more local universities, medical research institutes, Field Epidemiology Training Programs, or U.S. military laboratories. Each site will maintain close ties with World Health Organization country and regional offices. The IEIP sites will strengthen national public health capacity and provide hands-on training in laboratory science, epidemiologic science, and public health administration.

Scott F. Dowell, Sonja J. Olsen, J. Mark Simmerman, and Tamara L. Fisk • International Emerging Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, and Department of Disease Control, Ministry of Public Health, Building 7, 3rd floor, Tivanon Road, Nonthaburi 11000, Thailand. Supamit Chunsuttiwat • Department of Disease Control, Ministry of Public Health, Tivanon Road, Nonthaburi 11000, Thailand. Pathom Sawanpanyalert • Department of Medical Sciences, Ministry of Public Health, Tivanon Road, Nonthaburi 11000, Thailand. Kumnuan Ungchusak • Bureau of Epidemiology, Department of Disease Control, Ministry of Public Health, Tivanon Road, Nonthaburi 11000, Thailand. 191


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The IEIP concept is modeled in part on the domestic U.S. Emerging Infections Program (EIP), whose ten sites conduct population-based surveillance, provide emergency outbreak assistance, invest in cutting edge research, and address new problems whenever they arise. Because the EIP sites combine specialized epidemiologic and laboratory expertise, they are able to go beyond the routine functions of local health departments to address important issues in infectious diseases and public health. For example, when mad cow disease was reported in the United Kingdom in 1996, the EIP surveillance sites were able to conduct active surveillance and reassure the U.S. public within a short time that the disease had not spread to the United States. Like the domestic EIP sites, the IEIP sites will perform multiple functions, including research on endemic diseases and emergency surveillance when a new threat appears. They will also provide disease surveillance data to ministries of health and finance to help assess the burden of specific diseases and evaluate the cost-effectiveness of national public health programs. Also like the EIPs, the IEIPs will incorporate preexisting institutions, work with these in an integrated fashion, and establish an international steering committee to provide guidance for core projects conducted at all of the IEIP network sites. Areas in which IEIP sites might play an especially important role are surveillance for drug-resistant forms of malaria, tuberculosis, pneumonia, and dysentery. All of the sites will be linked by electronic communications to keep health experts around the world in close contact with one another. The long-term goal of the IEIPs is to develop sustainable, in-country capacity for disease surveillance, outbreak investigation, and research on diseases of national, regional, or global importance by fostering the next generation of international public health leaders. The implementation of this goal will require extensive scientific, human, and financial resources from both private and public sources, as well as sustained efforts over many years. However, the costs will be low in relation to potential benefits in terms of human health, security, and increased global prosperity. The first IEIP was launched in October of 2001, with the opening of offices within the Ministry of Public Health complex in Nonthaburi, Thailand (Fig. 1). In its initial year of operation, IEIP-Thailand has initiated activities in each of the four priority areas described by CDC’s global strategy: surveillance, research, training and capacity development, and outbreak support (Table 1). SURVEILLANCE—THE CORE ACTIVITY The absence of high-quality and reliable data on the burden of infectious diseases worldwide impedes vaccine and other control programs. Uncertainty regarding the public health importance of specific infectious diseases in particular regions leads to indolence, despite the availability of interventions with proven effectiveness. Recognizing this need, in 1994 the United States launched the EIP, a population-based network of CDC and state health departments, working with collaborators to assess the public health impact of emerging infections and to evaluate methods for their prevention and control (11). By improving existing surveillance infrastructure, supporting ongoing epidemiologic oversight, and including reference laboratory testing, this program has allowed for accurate measurement and ongoing monitoring of the burden of disease for important pathogens across the United States. Because of their high quality and reliability, the data produced have become “gold standards” on which important public health decisions have been based,


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Figure 1. U.S. Ambassador Darryl Johnson cuts the ribbon to open the offices of IEIPThailand in December 2001.

such as the use of universal conjugate pneumococcal vaccine for infants (5), a national food safety initiative (2), and routine perinatal prophylaxis to prevent neonatal sepsis from group B streptococci (16). As in the domestic program, IEIPs will focus much of their effort initially on establishing population-based surveillance, the core objective. It is anticipated that, through the additional support offered by CDC, national ministries of health will be able to conduct high-quality surveillance and produce data compelling enough to guide national and global infectious disease control priorities. The surveillance system would then provide a broad platform from which to launch research projects. In future years, as the surveillance system becomes established and disease control priorities are clarified, efforts will shift toward implementation and evaluation of new vaccines or other control measures by using the surveillance system as the platform for such evaluations. A Unique Approach to Surveillance The surveillance approach adopted by IEIP in Thailand differs somewhat from routine surveillance in most countries. Specific geographic regions with known population denominators will be carefully defined, active surveillance for certain clearly identifiable syndromes will be conducted throughout the population, and directed laboratory confirmation for specific pathogens will be added incrementally. Although unusual in its scope and


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Dowell et al. Table 1. Selected activities of the IEIP-Thailand since its establishment in October 2001 Category

Surveillance

Activitya

Date

Official selection of Sa Kaeo as first province, pneumonia as first syndrome First pneumonia patients reported Launched pneumonia surveillance in second province Added respiratory virus and bacteria testing

August 2002 July 2003

Research

Febrile illness/leptospirosis study launched Encephalitis study launched with Bangladesh Community survey conducted

December 2001 July 2003 May–June 2003

Training and capacity development

Anthrax training course with SEARO, Thai NIH, and 64 participants from 16 countries 13 epidemiology trainees (FETP) paired with Atlanta epidemiologists for 1-month practical Two laboratory scientists to leptospirosis course in Iowa and 1-month training in Atlanta

December 2001

Caliciviruses first identified by Thai MOPH as cause of a GI outbreak Enterovirus 71 confirmed as the cause of an outbreak of hand, foot, and mouth syndrome Laboratory technology transferred to Thai NIH for identification of caliciviruses In-country and regional SARS assistance

June 2002

Outbreak support

a

March 2002

July 2003

March 2002, 2003 August 2002

July 2002 August 2002 March–June 2003

Abbreviations: SEARO, Southeast Asia Regional Office of the World Health Organization; Thai NIH, the National Institute of Health of the Ministry of Public Health in Thailand; FETP, Field Epidemiology Training Program; GI, gastrointestinal disease; MOPH, Ministry of Public Health.

expense, this approach may prove to be a practical means to conduct high-quality surveillance in selected areas of less-wealthy countries. Most countries use passive reporting mechanisms for surveillance. This type of system typically relies on the good will of physicians or public health practitioners to report diseases. As a consequence, completeness of reporting, especially for diseases with relatively mild symptoms, can be quite low. In addition, although standard, written case definitions may exist, the large number of reporting practitioners ensures various interpretations that in turn lead to misclassification. Although there may also be some capacity for laboratory testing, there is often no integration of reported cases with laboratory data. Without a wellestablished laboratory infrastructure at the local and national levels, complete laboratorybased surveillance for defined populations is not practical. For most countries with some public health infrastructure, the default system is a passive, syndromic surveillance system. This has been the situation in Thailand to date. A few wealthy countries, such as the United States, have national passive surveillance systems that are laboratory-based. Laboratory-based surveillance requires a substantial degree of laboratory capacity at the local level. In addition to routine passive surveillance systems, the United States has active, population-based laboratory surveillance in select sites. This type of surveillance is expensive and is supported in a few selected geographical areas to develop accurate estimates of the burden of various infectious diseases nationwide.


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IEIP sought a model whereby less-developed countries could collect reliable, highquality surveillance data without having uniformly strong laboratory capacity at the local level, because the costs to build and staff these laboratories would be prohibitive. The approach adopted in Thailand, using active, population-based syndromic surveillance with incremental laboratory confirmation, is a modification of the population-based laboratory surveillance system used by the U.S. EIPs that may prove to be practical for conducting high-quality surveillance in less wealthy countries. Syndrome Selection Ideally, syndromes selected for such surveillance should be easily defined, specific, and applicable in a uniform manner across the different provinces in Thailand and also across future IEIP sites in other countries. Public health officials and clinicians should recognize the burden of disease defined by these syndromes as important, even without the additional information about etiologic agents. The syndromes should capture a range of pathogens amenable to a broad assortment of future control strategies. Potential syndromes meeting these criteria include pneumonia; diarrhea; jaundice; meningitis/encephalitis; hemorrhagic fever; hand, foot, and mouth syndrome; and acute, undifferentiated febrile illness. Pneumonia was selected as the first syndrome for surveillance in Thailand because it is a leading cause of mortality worldwide, and hospitalized patients with pneumonia may be infected with any of several dozen potential pathogens. Among these are several that are amenable to a variety of current and future public health interventions. Streptococcus pneumoniae may be controllable by the newly licensed conjugate vaccine for infants, and the changing pattern of pneumococcal resistance is an important concern of clinicians worldwide. Mycobacterium tuberculosis is by itself one of the leading causes of mortality worldwide, is a leading opportunistic pathogen among human immunodeficiency virusinfected patients in Thailand, and is controllable by directly observed therapy and potentially by vaccination. Haemophilus influenzae type b causes up to 20% of cases of severe pneumonia among children and is controllable with an enhanced conjugate vaccine. Respiratory syncytial virus, influenza virus, parainfluenza viruses, and other pathogens are similarly amenable to control with existing or experimental vaccines. An additional reason for studying pneumonia is that active surveillance with aggressive laboratory testing may help identify agents of atypical pneumonia such as Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydia pneumoniae. Site Selection Provinces are selected for surveillance on the basis of several criteria, including the representativeness of the population of Thailand, accessibility from Bangkok, feasibility of capturing the majority of hospitalizations through the public health system, availability of basic laboratory facilities, existence of epidemiologic capacity, and interest and commitment from local public health authorities. Based on these criteria, IEIP and Ministry of Public Health teams evaluated the top four candidate provinces and selected Sa Kaeo, located some 300 km east of Bangkok, with a population of 439,120 (Fig. 2). In preparation for collecting surveillance data, IEIP-Thailand has worked with Sa Kaeo provincial health officials to establish a provincial working group, conducted a formal review of the existing surveillance system, proposed an


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Figure 2. Map of Thailand showing the location of Sa Kaeo (arrow), the first of a series of provinces to establish population-based surveillance with IEIP-Thailand.


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approach to data collection in consultation with a CDC pneumonia expert, and developed a written action plan. Case reporting began during the latter part of 2002. Data Collection and Reporting The surveillance system aims to capture severely ill, hospitalized patients with radiographically confirmed, community-acquired pneumonia. Briefly, surveillance officers will review hospital admission logs for patients admitted with a diagnosis that suggests pneumonia (e.g., pneumonia, lower respiratory tract infection, dyspnea, and tuberculosis). The surveillance officer will then review the medical chart for clinical and radiological findings and fill out a brief surveillance form on patients who meet the case definition. Reports will be sent to a central database via the Internet. Coordination of the system is conducted through the existing Bureau of Epidemiology surveillance infrastructure. IEIP-Thailand will enhance this system by providing routine audits, training sessions, and enhanced communication systems. Although the hospital radiologists or treating physicians will read the chest X-ray and treat the patients as per routine clinical care, the surveillance officers will make a digital image of the X-ray using either a camera or advanced medical scanners and specialized software. The digitized files will be stored on rewriteable compact disks, and data will be sent to IEIP-Thailand for evaluation by a panel of radiologists. To ensure unbiased interpretation, these readers will be blinded to each other’s readings and to all patient clinical and demographic data. Access to these medical scanners in district and provincial hospitals should serve to increase the clinical and diagnostic capacity of these facilities. The availability of the material technology may also allow training of hospital staff through teleconferences and remote consultation. At a sample of hospitals, diagnostic specimens such as serum, blood, stool, sputum, and urine will be collected to identify etiologic agents by using culture, serology, or molecular analysis. Human immunodeficiency virus serologic testing would be offered to select patients with pneumonia. The IEIP-Thailand would help ensure that diagnostic samples are obtained, help strengthen laboratory facilities for initial pathogen isolation at the hospitals, and facilitate transportation of isolates to provincial hospital laboratories, laboratories of the National Institute of Health of Thailand (Thai NIH), or other reference laboratories for specialized testing. Because of the relative ease of transporting and testing samples, viral respiratory pathogens (e.g., influenza and parainfluenza viruses, respiratory syncytial virus, respiratory adenoviruses, and human metapneumovirus) will be the first pathogens examined. The surveillance system aims to capture only severely ill, hospitalized patients with chest X-ray-confirmed pneumonia; however, illness in other populations may also be important for disease prevention and control purposes. For example, it may be important to learn about milder cases, access to care, and health-seeking behaviors. These questions will be answered by special research studies using survey methods and other methodologies. A Vision for the International Surveillance Network It is anticipated that active, population-based surveillance in centers of excellence around the globe will ultimately produce data that could be appropriately extrapolated to the entire population of the countries involved, accurately measure and monitor the burden


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of disease from infectious agents, and serve as a national and international resource to guide future public health decisions. The second IEIP site in Africa is in Kenya. The establishment of IEIPs in several developing countries has the potential to produce uniquely comparable data and form an important component of the World Health Organization’s vision of a global “network of networks.” Although current activities are all within Thailand, it is possible that in future years IEIP-Thailand may serve as a regional resource as well. Emerging diseases in neighboring countries are of great concern to Thailand. For example, in 1999 there was an outbreak of viral encephalitis among pig farmers in Malaysia that was later confirmed as an epidemic caused by Nipah virus, a newly emergent paramyxovirus (9). Authorities in Thailand, which shares a border with Malaysia in the south, are now concerned about Nipah virus as well. Border health issues are of particular interest to the Thai Ministry of Public Health, and that is one reason Sa Kaeo, which shares a border with Cambodia, was selected as the first surveillance site. Over the next few years, Thailand may be able to help share expertise in emerging infections with neighboring countries. RESEARCH The IEIP sites are intended to provide platforms for conducting a broad spectrum of research on infectious diseases of global importance. Specific research projects can further characterize new or reemerging pathogens identified in surveillance or outbreak efforts, guide future surveillance, and lead to a strengthening of research capabilities through practical training. Priority for research in IEIP sites will be placed on diseases that are identified by the host ministry of health as important public health problems in the region, those that impact socioeconomic development (for example, by having an effect on livestock), those that afford CDC and local scientists unique opportunities to learn about emerging pathogens before they spread globally, and diseases for which cost-effective control measures exist or can be developed (4, 13). The first research project of IEIP-Thailand was developed in response to a request from the Thai government to assist with an ongoing epidemic of leptospirosis. Between 1995 and 2001, the reported incidence of leptospirosis in Thailand increased by more than 100-fold, leading to widespread concern and calls for urgent control efforts (12, 15, 17). Because leptospirosis typically presents as an acute, undifferentiated febrile illness and because of the regional and global importance of other causes of febrile illness in addition to leptospirosis, the leptospirosis/febrile illness project seeks not only to prospectively define the incidence of this disease but also to evaluate other potential causes of acute undifferentiated fever, such as dengue, typhoid fever, scrub typhus and other rickettsial diseases, melioidosis, and others. As a result, clinical discriminators may be identified that can assist physicians in managing patients, not only in Thailand but also in other tropical areas where similar diseases are endemic. Furthermore, this project will evaluate field diagnostic tools for use at the hospital or national level and attempt to identify new pathogens or pathogens not previously recognized in Thailand that contribute to fever. After the first year, 743 febrile patients were enrolled at the four study hospitals and 98% had returned after 4 to 6 weeks to provide a convalescent-phase serum sample and complete the final questionnaire.


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In the future, research protocols conducted at multiple IEIP sites may provide important standardized information for disease control both in the participating nations and globally. An initial test of this concept is a project to study the causes of encephalitis by enrolling cases in both Bangladesh and Thailand. Use of an identical protocol and case definition in both countries will allow direct comparison of the relative importance of the various causes of encephalitis in these two areas. Collaborative research projects ultimately improve surveillance, outbreak response, and control of priority communicable diseases. At the same time, they provide training in research strategies and principles that can later be applied to future emerging diseases and transferred to other regions of the world. TRAINING AND CAPACITY DEVELOPMENT Capacity development through training is a priority goal of the IEIP. Training through the IEIP will include opportunities for scientists from the host nation to obtain advanced skills in CDC laboratories and also for scientists from CDC to obtain experience in the developing country setting. Training designed to develop organizational capacity facilitates interagency communication and trust, helps to define mutual objectives, and builds a longterm framework through which international partnerships succeed (3). In a five-year plan for IEIP-Thailand, which was developed in late 2001 by the U.S. CDC and senior officials from the Thai Ministry of Public Health, IEIP committed support for training opportunities for Thai scientists. These will include international fellowships as well as other short- or long-term project-specific training. In its first year of operation, IEIP-Thailand has explored several ways of developing organizational capacity and transferring technology through training in laboratory science, epidemiology, case management, and project management. Laboratory Training A few weeks after its office opened in October 2001, IEIP-Thailand responded to an urgent need to strengthen laboratory and public health capacity in Thailand and neighboring countries to identify intentional anthrax exposures. The intentional distribution of anthrax spores in the U.S. postal system prompted the mailings of hundreds of packages of suspicious white powders to many government buildings, corporate offices, and private organizations throughout Southeast Asia (7). The Thai NIH laboratories, IEIP-Thailand, the U.S. embassy, local corporations, and others received numerous requests to analyze these powders to determine their content. Subsequently, the Thai NIH, in collaboration with the Southeast Asia Regional Office of the World Health Organization, and IEIP-Thailand organized a regional training session to address this issue (J. M. Simmerman, S. Kumari, R. Bhatia, S. Dejsirilert, L. Sangsuk, P. Sawanpanyalert, T. Fisk, N. Tima, and S. F. Dowell, 10th Int. Congr. Infect. Dis., Singapore, 2002). Within 6 weeks, a 3-day training workshop was held in Bangkok for 64 participants and presenters from 16 countries. The conference addressed the diagnosis and treatment and prophylaxis of anthrax, the proper handling of suspicious packages, and the coordination of an effective public health response to intentional anthrax exposure. A wet-laboratory training course was also conducted at the Thai NIH for the identification of anthrax spores in powder samples.


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Later in its first year of operation, IEIP-Thailand coordinated a one-month laboratory training session for two Thai scientists to work with experts at CDC in Atlanta, Ga., on advanced methods to identify Leptospira, including pulsed-field gel electrophoresis, PCR testing, and microscopic agglutination techniques. A planned study of respiratory viruses in Thailand will allow a transfer of laboratory capacity to enable the Thai NIH to effectively isolate human metapneumovirus, a recently discovered pathogen thought to be a significant contributor to respiratory tract morbidity. Through its contacts at CDC in Atlanta, IEIP-Thailand will provide the protocol used to grow the virus and passage isolates in addition to practical training in PCR confirmation to detect and confirm the presence of this novel pathogen. After an outbreak of hand, foot, and mouth disease in Thailand, a Thai scientist was trained in genetic sequencing of the causative enteroviruses. Finally, IEIPThailand regularly sponsors guest lecturers in a variety of laboratory and epidemiologic topics. Recently, this has included lectures from two visiting CDC scientists, one focusing on geographic information systems and spatial statistics and the second on the molecular diagnosis of Rickettsia and Orientia species. Epidemiology Thailand has a long history of success with its Field Epidemiology Training Program (FETP), a program modeled after the U.S. Epidemic Intelligence Service. The FETP offers Thai physicians the opportunity to receive intensive training in epidemiology and handson experience in data analysis and outbreak response during a 2-year fellowship. To contribute to this valuable program and further build epidemiologic capacity, staff members from IEIP have provided classroom lectures, reviewed abstracts, and supported FETP trainees in outbreak responses. In addition, IEIP has helped to arrange for 13 FETP trainees to work with CDC staff in Atlanta to further expand their experiences in disease surveillance and outbreak investigation. Specific research projects also provide unique opportunities for capacity development through experience-based training. For example, in May and June 2003, an extensive community survey was conducted in Sa Kaeo Province in eastern Thailand. This household survey helped provide the opportunity to build capacity through training in sampling approaches, survey techniques, and the use of geographical information system technology. Technology Transfer The transfer of technology and the expertise to utilize advanced scientific tools represent additional important mechanisms to improve capacity. An important objective of IEIP-Thailand’s active, population-based infectious disease surveillance system is the establishment of an Internet-based electronic reporting system. As the environment for infectious disease surveillance systems changes, the need to have access to specific, current information about diseases increases. Electronic reporting systems can address the delays and deficiencies that often characterize passive, paper-based reporting (1). IEIP-Thailand will use experience gained from similar systems in the United States to enable the Thai Ministry of Public Health to institute the first such case reporting system in Thailand. This project will be implemented in a manner that ensures the systematic transfer of both material technology and the technical


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expertise needed to maintain and expand the system with Thai Ministry of Public Health resources in the future. OUTBREAK SUPPORT The Provincial Public Health Office of each of the 76 provinces in Thailand has an epidemiological unit comprised of three to five staff members. This unit analyzes and monitors the incidence of important diseases to detect outbreaks and initiate responses. Outbreaks are normally identified when the number of reported cases in a defined period exceeds the median number for similar periods over the previous 5 years by an arbitrary threshold. In addition, physicians often inform the Provincial Public Health Office when an abnormal number of cases are seen in their hospitals. Most outbreaks are investigated locally by district health staff along with the provincial epidemiological team. Only in the case of large-scale outbreaks, those of unknown etiology, or other complicated situations do provincial authorities request additional support from the Ministry’s Bureau of Epidemiology. The investigating team from the Bureau typically includes trainees from the FETP under close supervision of more senior staff. On average, 30 such joint investigations are performed annually. Investigating an outbreak of unknown etiology potentially caused by an emerging infectious disease requires strong laboratory support, a skilled field team, and close coordination between the two. Bringing advanced laboratory diagnostic capability to a sophisticated field investigation is one way in which IEIP promises to further strengthen the already existing Thai capacity for outbreak investigation. In the first years of IEIP operations, three examples have shown the potential for IEIP to fulfill that promise. On 25 March 2002, the Bureau of Epidemiology was called to investigate a cluster of gastroenteritis cases among foreign tourists at a resort hotel in southern Thailand. The provincial health team requested help because of their inability to identify the etiologic agent. Nearly half of the 221 guests reported illness characterized by diarrhea (77%), vomiting (74%), and abdominal pain (68%). Rectal swabs and stool specimens were collected from 20 patients, but no enteric bacteria or viruses were detected. Because the epidemiological pattern was potentially compatible with Norwalk-like virus infections, three remaining fecal specimens were sent to CDC for further testing. One of the three was positive for a calicivirus (Norwalk-like virus) by reverse transcription-PCR. Although the investigation did not fully succeed in implicating a food source, it was the first such investigation in Thailand to identify Norwalk-like viruses as the likely cause. This family of viruses is increasingly recognized as the most common cause of outbreaks of gastroenteritis in the United States (14). Determining if such viruses are equally important in Thailand is now becoming possible with the recent transfer of the necessary reagents and technology to the Department of Medical Sciences and ongoing support from the viral gastroenteritis unit in Atlanta for similar investigations in the future. In response to the devastating 1998 and 1999 outbreaks of severe encephalitis, cardiac failure, and death associated with hand, foot, and mouth syndrome in Taiwan and Malaysia (8, 10), Thailand’s Bureau of Epidemiology added this syndrome to its notifiable diseases list and the Thai NIH strengthened its laboratory capacity to identify the enteroviruses implicated as likely causative agents. In early 2002, outbreaks of hand, foot, and mouth


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syndrome were increasingly reported in Bangkok and six other provinces of Thailand. Most of the outbreaks occurred in nursery settings. To date in 2002, provincial authorities have submitted more than 100 stool and blood specimens to the Department of Medical Sciences for testing, of which 18 tested positive for enterovirus 71, one of the implicated agents in the Taiwan and Malaysia outbreaks. Because of ongoing public concern about the outbreaks and the possible relationship to the severe outbreaks of 1998 and 1999, four isolates identified as enterovirus 71 by antigenic means were sent for confirmation and further characterization in Atlanta. Three were confirmed as enterovirus 71 by genetic analysis, with sequences being most similar to those of specimens obtained from peninsular Malaysia during 1998 (B. Brown, personal communication). A plan to strengthen the Thai NIH laboratory capacity for future characterization of the viruses and a field study to attempt to identify risk factors for severe disease are among the priorities for IEIP in the coming year. The potential for IEIP to strengthen Thailand’s ability to respond to outbreaks of emerging infections was most fully realized during the recent outbreak of severe acute respiratory syndrome (SARS) in 2003. Epidemiologists from IEIP and the Bureau of Epidemiology worked together to set up mobile teams that could be deployed within hours of a SARS case report. Each team had a checklist and information packet on infection control practices. The teams’ main role was to ensure proper isolation of cases and train hospital staff in stringent infection control procedures. IEIP also served in a variety of roles for regional SARS outbreak response. In addition to rapid deployments of staff to Taiwan, Laos, Hong Kong, and Beijing, IEIP coordinated shipments of specimens from Vietnam, Thailand, and Taiwan and assisted the World Health Organization’s Thailand office to locate and distribute infection control supplies. CDC staff from Atlanta and elsewhere used IEIP as a staging area for travel arrangements, supply purchases, and communications. CONCLUSIONS The vision of the IEIPs as a network of centers of excellence in infectious disease control is beginning to assume a realistic shape with the early growth of the IEIP-Thailand and the plan for a second IEIP in Africa. The establishment in Sa Kaeo Province of active, population-based, syndromic surveillance has the potential to provide uniquely valuable data to guide national and international public health decisions, though this potential will not be fully realized for several years. Research and outbreak support activities should provide more tangible results in the short term. In its first year of activity, the IEIP-Thailand has already made modest progress toward increasing the capacity of the participating Thai institutes to identify and respond to emerging infections more effectively. A continued focus on training and technology transfer to develop capacity should enable future IEIP-Thailand collaborative projects to yield mutually beneficial, long-term results for all partner organizations. It remains to be determined whether the IEIP network will fulfill its promise to improve global capacity to control emerging infections, but the early indications are promising. REFERENCES 1. Bean, N., and S. Martin. 2001. Implementing a network for electronic surveillance reporting from public health reference laboratories: an international perspective. Emerg. Infect. Dis. 7:773–779.


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2. Binder, S., R. Khabbaz, B. Swaminathan, R. Tauxe, and M. Potter. 1998. The national food safety initiative. Emerg. Infect. Dis. 4:347–351. 3. Breman, J., and J. LeDuc. 2001. International partnerships in infectious diseases research, training, and control. Emerg. Infect. Dis. 7(Suppl.):542. 4. Broome, C. V. 1998. Effective global response to emerging infectious diseases. Emerg. Infect. Dis. 4:358–359. 5. Centers for Disease Control and Prevention. 2000. Preventing pneumococcal disease among infants and young children. Recommendations of the Advisory Committee on Immunization Practices (ACIP). Morb. Mortal. Wkly. Rep. 49:1–35. 6. Centers for Disease Control and Prevention. 2002. Protecting the Nation’s Health in an Era of Globalization: CDC’s Global Infectious Disease Strategy. U.S. Department of Health and Human Services, Washington, D.C. 7. Centers for Disease Control and Prevention. 2001. Update: investigation of bioterrorism-related anthrax, 2001. Morb. Mortal. Wkly. Rep. 50:1008–1010. 8. Chan, L. G., U. D. Parashar, M. S. Lye, F. G. Ong, S. R. Zaki, J. P. Alexander, K. K. Ho, L. L. Han, M. A. Pallansch, A. B. Suleiman, M. Jegathesan, and L. J. Anderson. 2000. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: clinical and pathological characteristics of the disease. For the Outbreak Study Group. Clin. Infect. Dis. 31:678–683. 9. Chua, K. B., K. J. Goh, K. T. Wong, A. Kamarulzaman, P. S. Tan, T. G. Ksiazek, S. R. Zaki, G. Paul, S. K. Lam, and C. T. Tan. 1999. Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 354:1257–1259. 10. Ho, M., E. R. Chen, K. H. Hsu, S. J. Twu, K. T. Chen, S. F. Tsai, J. R. Wang, and S. R. Shih. 1999. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N. Engl. J. Med. 341:929–935. 11. Hughes, J. 1998. Addressing emerging infectious diseases threats—accomplishments and future plans. Emerg. Infect. Dis. 4:360–361. 12. Kollars, T. M., Jr., D. Phulsuksombati, D. Kingnate, J. Prachumsri, N. Rachphaew, T. Monkanna, and M. Gettayakamin. 2002. Antibodies to leptospirosis in rodents from Thailand using a modified human diagnostic assay. J. Med. Assoc. Thai. 85:67–70. 13. Lam, C. L. 2000. Knowledge can flow from developing to developed countries. Br. Med. J. 321:830. 14. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607–625. 15. Panaphut, T., S. Domrongkitchaiporn, and B. Thinkamrop. 2002. Prognostic factors of death in leptospirosis: a prospective cohort study in Khon Kaen, Thailand. Int. J. Infect. Dis. 6:52–59. 16. Schrag, S. J., E. R. Zell, R. Lynfield, A. Roome, K. E. Arnold, A. S. Craig, L. H. Harrison, A. Reingold, K. Stefonek, G. Smith, M. Gamble, and A. Schuchat. 2002. A population-based comparison of strategies to prevent early-onset group B streptococcal disease in neonates. N. Engl. J. Med. 347:233–239. 17. Tangkanakul, W., P. Siriarayaporn, T. Pool, K. Ungchusak, and S. Chunsuttiwat. 2001. Environmental and travel factors related to leptospirosis in Thailand. J. Med. Assoc. Thai. 84:1674–1680.


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INDEX

Academic Alliance for AIDS Care and Prevention in Africa, 83–99 clinical care program, 88 core values, 86–87 founding members, 85 future plans, 98–99 genesis, 84–86 Infectious Disease Institute, 86–88 laboratory program, 98 mission, 86 operational research, 96–98 prevention program, 88–90 training program AIDS Treatment Information Center, 94–95 continuing education, 94–95 course examinations, 94 curriculum, training structure, and teaching methods, 91–93 goals and objectives, 90–91 health care implementer, 95 partner sites, 92 pharmacy graduate program, 96 postdoctoral fellowship in infectious disease, 95 program evaluation, 94 program management, 94 satellite training centers, 96 topical cluster program, 95–96 trainee selection, 91 trainees to date, 92 trainer selection, 91 Acquired immune deficiency syndrome, see HIV/AIDS Acute flaccid paralysis, polio surveillance, 13–23 Acute liver failure, syndrome-based surveillance, 184–185 Acute pulmonary syndrome, syndrome-based surveillance, 184 Adenovirus, 180 Aedes aegypti range, 25–27 yellow fever, 25 Africa Ebola hemorrhagic fever, 35–58 enteroaggregative E. coli diarrhea, 103

HIV/AIDS Academic Alliance for AIDS Care and Prevention in Africa, 83–99 epidemiology, 60–68, 76, 83–89 travel-related fungal infections, 133 trypanosomiasis, 143–157 African American community, HIV/AIDS, 75 African trypanosomiasis, 143–157 burden of disease, 150 clinical manifestations, 145 control measures, 143, 148–149, 154 diagnosis, 145–146 ecology, 143–145 epidemiology, 149 geographic distribution, 150 prevention, 146–148 reemergence in Africa, 148–151 stage determination, 145–146 transmission, 143–145 treatment, 146–148, 154 crisis of chemotherapy, 152–154 United States, 151–152 Age, yellow fever vaccine-associated disease and, 29–32 AggR regulon, 107–108 AIDS, see HIV/AIDS AIDS Treatment Information Center, 94–95 Albendazole, for baylisascariasis, 172 Alcohol consumption, liver disease and, 2–3, 6–7 Animal reservoir Ebola virus, 35, 38, 42, 48–51, 54 HIV, 62 Anthrax bioterrorism, 182, 199 injecting drug users, 121, 124 Antigenic variation, trypanosomes, 144 Asia enteroaggregative E. coli diarrhea, 103 HIV/AIDS epidemiology, 60–61, 63–64, 66, 69– 73, 76–77 travel-related fungal infections, 133 Australia, HIV/AIDS epidemiology, 61, 64, 66, 71 Bacillus anthracis, 121, 124 Bacillus cereus, 125 205


206

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Index

Bartonella, 180 Baylisascariasis, see also Raccoon entries diagnosis, 169–171 epidemiology, 167–169 pathogenesis, 167–169 prevention, 173 treatment, 171–172 Baylisascaris procyonis, 159–175 life cycle in raccoon, 163–167 Bioterrorism, 186 anthrax, 182, 199 Ebola virus, 50–52, 54 Black tar heroin, 111–120 culture, 117 skin popping, 112–114, 117–120 Blood products hepatitis C virus transmission, 5 HIV transmission, 66–67 Borrelia burgdorferi, 180 Building renovation, histoplasmosis and, 136 Card agglutination trypanosomiasis test (CATT), 146 Caribbean, HIV/AIDS epidemiology, 60–61, 63, 66, 73–74 Cat scratch disease, 183 CATT, see Card agglutination trypanosomiasis test Central nervous system infection, baylisascariasis, 159–175 Certification, polio eradication in the Americas, 15– 16, 22 Chlamydia pneumoniae, 180, 195 Chronic liver disease alcohol consumption and, 2–3, 6–7 disease burden, 1–4 etiology of newly diagnosed, 2–3 hepatitis C-related, 1–11 Cirrhosis, hepatitis C virus and, 1–11 Clostridial infections, injecting drug users, 121– 132, 182 risk factors, 128–129 16S rDNA testing, 126–127 subtyping and toxin testing, 126 Clostridium argentinense, 125 Clostridium botulinum, 111–113, 116–117, 119, 130 Clostridium difficile, 112 Clostridium histolyticum, 129 Clostridium innocuum, 130 Clostridium novyi, 112, 118 infections in injecting drug users, 121–132 Clostridium perfringens, 111–112, 116–119, 125– 127, 129–130 Clostridium scindens, 125, 130 Clostridium septicum, 112, 116–117, 119, 129 Clostridium sordellii, 112, 116–117, 119, 130

Clostridium sporogenes, 125, 130 Clostridium subterminale, 126–127, 130 Clostridium tertium, 112 Clostridium tetani, 111–114, 116–119, 130 Coccidioides immitis, 137–139 Coccidioides posadasii, 137 Coccidioidomycosis clinical manifestations, 137 diagnosis, 138–139 geographic distribution of pathogen, 135, 137 prevention, 139 travel-related, 133, 137–139 treatment, 139 Compatible poliomyelitis, 15 Congo, Ebola hemorrhagic fever, 35–58 Coroner, unexplained death surveillance, 182–184 Coxsackie B virus, 180 Culture, heroin, 117, 127–128 Cytomegalovirus, 180 Death, unexplained, see Unexplained illness/death Death certificates, 186 Diarrhea AIDS-associated, 104–105 enteroaggregative E. coli, 101–110 Diffuse unilateral subacute neuroretinitis (DUSN), 168 Dog, B. procyonis infection, 169 Dominican Republic, vaccine-derived polio outbreak, 13–23 DUSN, see Diffuse unilateral subacute neuroretinitis Dust exposure, coccidioidomycosis and, 137–139 EAEC, see Enteroaggregative Escherichia coli EAST1 toxin, 106–107 Ebola hemorrhagic fever (EHF), 35–58, 183 case definitions, 40, 44–45, 52 case-fatality ratio, 45–46 control measures, 38–44, 52–53 diagnostic tools, 45, 48, 52–53 history of outbreaks, 35–38 isolation wards, 45–48 media relations during outbreak, 38, 45 nosocomial infections, 40, 44–48 patient management, 38, 45–48, 52–54 response to outbreaks, 43–45 scientific publications, 40 surveillance, 38–43, 52–53 transmission, 43–44 Ebola virus bioterrorism agent, 50–52, 54 Ebola-Reston, 36–38, 49, 52 reservoir, 35, 38, 42, 48–51, 54 species, 35–36, 49 Eflornithine, for African trypanosomiasis, 147, 153


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Index Eggs, B. procyonis, 163–167 EHF, see Ebola hemorrhagic fever Ehrlichia chaffeensis, 180 EIP, see Emerging Infections Program Elderly, risks of yellow fever vaccine, 30–32 Emerging diseases, detection, 177–190 Emerging Infections Program (EIP), see also Unexplained illness/death International, see International Emerging Infections Program United States, 177–190, 192 aberration detection system, 186 case finding, 178 defining unexplained illness/death, 178 distribution of illness by syndrome, 179–180 event-based reporting system, 181–182 laboratory evaluation of illness, 179–180 pathogen identification and discovery, 185–186 rates and demographics of illness, 179–180, 186 role of medical examiners, 182–184 search for etiologic agents, 182–185 sentinel clusters, 186 specimen collection, 181, 183 syndrome-based surveillance, 184–185 Encephalitis syndrome-based surveillance, 184–185 viral, 183 England, unexplained illness/death in injecting drug users, 121–132 Enteroaggregative Escherichia coli (EAEC), 101– 110 aggregative adherence, 101–102 epidemiology, 103–104 HIV infected persons, 104–105 pathogenesis of infection inflammation, 106 mucosal adherence, 105 toxins, 105–106 virulence factors, 106–108 traveler’s diarrhea, 104 Enterovirus, 180 Enterovirus 71, Thailand, 202 Epstein-Barr virus, 180 Eradication polio in the Americas, 14–16 polio worldwide, 21–22 Escherichia coli, enteroaggregative, see Enteroaggregative Escherichia coli Europe, HIV/AIDS epidemiology, 60–64, 66, 71– 73, 76 Evolution, HIV, 62–65 Field Epidemiology Training Program, IEIP, 200 Fimbriae, enteroaggregative E. coli, 105–107

207

Fungal infections migration-related, 133–134 travel-related, 133–142 coccidioidomycosis, 133 histoplasmosis, 133–137 penicilliosis, 133, 139–140 Gabon, Ebola hemorrhagic fever, 35–58 Global Commission for the Certification of Polio Eradication, 22 Global Plan of Action for Laboratory Confinement of the Wild Poliovirus, 22 Granulomatous amebic encephalitis, 183 HAART therapy, 89 Haemophilus influenzae type b, 195 Haiti, vaccine-derived polio outbreak, 13–23 Hand, foot, and mouth disease, Thailand, 200–202 Hantavirus pulmonary syndrome, 177, 183 HCV infection, see Hepatitis C virus infection Health Information for International Travel Yellow Book, 140 Hemorrhagic fever, Ebola, 35–58 Hepatitis C virus (HCV) infection, 1–11 acute, 1, 3, 5 age at infection, 6 alcohol consumption and, 2–3, 6–7 chronic liver disease and, 1–11 community-acquired infection, 5 disease burden, 1–4 genotype differences, 8 HIV coinfection, 7 natural history, 5–7 transfusion-associated, 5 treatment, 7–8 trends in incidence and prevalence, 3–4 Heroin, 130, see also Injecting drug users black tar, 111–120 culture, 117, 127–128 laboratory evaluation, 127–128 Heterosexual transmission, HIV/AIDS, 60, 63–67, 69–76 Hispaniola, vaccine-derived polio outbreak, 13–23 Histoplasma capsulatum, 134–137 Histoplasmin skin test, 134, 136 Histoplasmosis clinical manifestations, 134 diagnosis, 136 geographic distribution of pathogen, 134–135 prevention, 136–137 travel-related, 133–137 treatment, 136 HIV evolution, 62–65 HIV-1, 62


208

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Index

HIV (continued) circulating recombinant forms, 63–64 subtype A, 63 subtype B, 63–64 subtype C, 63 subtype M, 62–63 HIV-2, 62, 65 HIV/AIDS Africa, 65–68, 83–99 in blood products, 66–67 deaths in 2001 and 2002, 60–61 emergence, 177, 183 enteroaggregative E. coli diarrhea, 104–105 global epidemiology, 59–82 hepatitis C virus coinfection, 7 heterosexual transmission, 60, 63–67, 69–76 homosexual transmission, 71, 73–75 injecting drug users, 59–60, 63–64, 69–75 molecular epidemiology, 62–65 new infections in 2002, 60 penicilliosis and, 139–140 perinatal transmission, 60, 62, 65, 75 regional epidemics Asia, 69–73 Australia and New Zealand, 71 Caribbean, 73–74 Europe, 71–73 future projections, 75–77 Latin America, 73–74 sub-Saharan Africa, 65–68 United States, 74–75 Homosexual transmission, HIV/AIDS, 71, 73–75 Human ehrlichiosis, 183 Human herpesvirus 6, 180 Human immunodeficiency virus, see HIV; HIV/AIDS ICCPE, see International Certification Commission on Polio Eradication IDDRT, see Infectious disease death review team IDU, see Injecting drug users IEIP, see International Emerging Infections Program Infectious disease death review team (IDDRT), 183–184 Infectious Disease Institute (Uganda), 86–88 Inflammation, enteroaggregative E. coli infection, 106 Influenza virus, 180 Injecting drug users (IDU) anthrax, 121, 124 clostridial infections, 121–132, 182 risk factors, 128–129 16S rDNA sequencing, 126–127 subtyping, 126 toxin testing, 126

HIV/AIDS, 59–60, 63–64, 69–75 necrotizing soft tissue infections, 111, 114–120 tetanus, 111, 113–114, 117–120 toxic shock syndrome, 124 unexplained illness/death clinical characteristics, 123 differential diagnosis, 123–124 early intervention, 124 etiology, 129 laboratory evaluation, 124–126 patient demographics, 122–123 risk factors, 128–129 Scotland, Ireland, and England, 121–132 source of outbreak, 129 surveillance, 122 wound botulism, 111–113, 117–120 Interferon alfa, for hepatitis C, 7–8 International Certification Commission on Polio Eradication (ICCPE), 15–16 International Emerging Infections Program (IEIP), 191–203 concept, 191–192 Field Epidemiology Training Program, 200 goals, 192 IEIP-Thailand, 191–203 capacity development, 194, 199–201 data collection and reporting, 197 epidemiology, 200 laboratory training, 199–200 opening, 192–193 outbreak support, 194, 201–202 research, 194, 198–199 site selection, 195–197 surveillance, 193–195 syndrome selection, 195 technology transfer, 200–201 training programs, 194, 199–200 surveillance, 192–193 vision for International Surveillance Network, 197–198 International travel African trypanosomiasis, 151–152 enteroaggregative E. coli diarrhea, 104 fungal infections, see Fungal infections, travelrelated yellow fever vaccination, 25–34 Intestinal roundworm, see Baylisascariasis Ireland, unexplained illness/death in injecting drug users, 121–132 Laboratory facilities, poliovirus storage, 22 Lactoferrin, fecal, 102 Latin America, HIV/AIDS epidemiology, 60–63, 66, 73–74 Latrine, raccoon, 162–163, 166–170, 172–173


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Index Legionella, 180, 183 Legionella pneumophila, 195 Legionnaires’ disease, 177, 183 Leptospirosis, 183 Thailand, 198, 200 Liver cancer, hepatitis C virus and, 1–11 Liver disease, chronic, see Chronic liver disease Liver transplantation, 1–2 Media relations, Ebola hemorrhagic fever outbreak, 38, 45 Medical examiner, unexplained death surveillance, 182–184 Melarsoprol, for African trypanosomiasis, 147, 152–154 Meningitis, syndrome-based surveillance, 184 Middle East, HIV/AIDS epidemiology, 60, 66 Migration-related fungal infections, 133–134 Mumps virus, 180 Mycobacterium tuberculosis, 195 Mycoplasma pneumoniae, 180, 195 Mycoses, travel-related, 133–142 Myocarditis, syndrome-based surveillance, 184 National immunization days, polio eradication, 14– 15, 18, 20 Necrotizing fasciitis, see Necrotizing soft tissue infections Necrotizing soft tissue infections, injecting drug users, 111, 114–120 Neisseria meningitidis, 180 Neural larva migrans, 168–169, 171 Neurotropic disease, yellow fever vaccine-associated, 27–32 New Zealand, HIV/AIDS epidemiology, 61, 66, 71 Nifurtimox, for African trypanosomiasis, 147, 153 Nipah virus, 198 Nonhuman primates, Ebola virus, 36, 38, 49–50, 52 North America HIV/AIDS epidemiology, 61 travel-related fungal infections, 133 Norwalk-like virus, Thailand, 201 Nosocomial disease, Ebola hemorrhagic fever, 40, 44–48 Ocular larva migrans, 168–171 Oral polio vaccine, outbreak of polio in Hispaniola, 13–23 Pacific region, HIV/AIDS epidemiology, 61, 66, 71 Paracoccidioidomycosis, 140 Parainfluenza virus, 180 Pegylated interferon, for hepatitis C, 8 Penicilliosis clinical manifestations, 139–140

209

diagnosis, 140 geographic distribution of pathogen, 139 prevention, 140 travel-related, 133, 139–140 treatment, 140 Penicillium marneffei, 139–140 Pentamidine, for African trypanosomiasis, 147–148, 153 Perinatal transmission, HIV/AIDS, 60, 62, 65, 75 Pet toxin, 106–107 Pic mucinase, 105–107 Pneumonia, surveillance in Thailand, 195, 197 Polio case definition, 15 compatible, 15 eradication in the Americas, 14–16 global eradication, 21–22 laboratory confinement of virus, 22 posteradication immunization policies, 21–22 vaccine-related, outbreak in Hispaniola, 13–23 wild poliovirus, 14, 22 Postdoctoral fellowship in infectious disease, 95 Procyon lotor, see Raccoon Raccoon behavior, 160–163 biology, 160–163 roundworm infection, 159–175, see also Baylisascariasis natural history in raccoon, 163–167 Raccoon latrine, 162–163, 166–170, 172–173 rDNA sequencing, 16S, diagnosis of infections in injecting drug users, 126–127 Reservoir, Ebola virus, 35, 38, 42, 48–51, 54 Ribavirin, for hepatitis C, 7–8 Roundworm infection, see Baylisascariasis Scotland, unexplained illness/death in injecting drug users, 121–132 Sepsis/multiorgan failure, syndrome-based surveillance, 184 Shigella enterotoxin 1, 105–106 Skin popping black tar heroin, 112–114, 117–120 heroin, 128–129 Sleeping sickness, see African trypanosomiasis South America enteroaggregative E. coli diarrhea, 103 HIV/AIDS epidemiology, 64 Spelunkers, histoplasmosis, 134 Staphylococcus aureus, 125–126, 130 Steroid therapy, for baylisascariasis, 172 Streptococci, beta-hemolytic, 125 Streptococcus intermedius, 125 Streptococcus milleri, 125


210

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Streptococcus pneumoniae, 180, 195 Sub-Saharan Africa Ebola hemorrhagic fever, 35–58 HIV/AIDS epidemiology, 59–60, 65–68, 76, 83– 99 trypanosomiasis, 143–157 Suramin, for African trypanosomiasis, 147, 153 Syndrome-based surveillance, 184–185 Technology transfer, International Emerging Infections Program, 200–201 Tetanus, injecting drug users, 111, 113–114, 117– 120 Thailand, International Emerging Infections Program, 191–203, see also International Emerging Infections Program Toxic shock syndrome, 177 injecting drug users, 124 Toxin C. novyi, 126 enteroaggregative E. coli, 105–106 Traveler’s diarrhea, enteroaggregative E. coli, 104 Travelers, see International travel Trypanosoma brucei brucei, 144 Trypanosoma brucei gambiense, 144–153 Trypanosoma brucei rhodesiense, 144–148, 151 Trypanosomes antigenic variation, 144 life cycle, 144 Trypanosomiasis, African, see African trypanosomiasis Tsetse fly, 143–144, 148, 152, 154 control measures, 148 Uganda Academic Alliance for AIDS Care and Prevention in Africa, 83–99 Ebola hemorrhagic fever, 35–58

UNEX, 177–190 Unexplained illness/death Emerging Infections Program International, 191–203 United States, 177–190 injecting drug users, 121–132 search for etiologic agents, 177–190 United States African trypanosomiasis, 151–152 Emerging Infections Program, 177–190 HIV/AIDS epidemiology, 62–63, 74–75 travel-related fungal infections, 133 Vaccine oral polio, outbreak of polio in Hispaniola, 13–23 yellow fever, 25–34 Virulence factors, enteroaggregative E. coli, 106– 107 Visceral larva migrans, 159–175 Viscerotropic disease, yellow fever vaccine-associated, 28–32 WHO Global Alert and Response Network, 43, 53 Wound botulism, injecting drug users, 111–113, 117–120 Yellow fever zones of endemicity, 25–27 epidemiology, 25–26 Yellow fever vaccine adverse events age-associated risk, 29–32 allergic reactions, 27 general, 27 neurotropic disease, 27–32 risk assessment, 30–32 viscerotropic disease, 28–32 manufacture, 27

Emerging Infections 6

Emerging Infections in Asia

Emerging Infections, Volume 5

SARS: A Case Study in Emerging Infections

Emerging Neurological Infections (Neurological Disease and Therapy)

Emerging Infections: An Atlas of Investigation and Management

Emerging issues on HPV infections: from science to practice

Musculoskeletal Infections

Musculoskeletal Infections

Biofilm Infections

Chemokines in viral infections

Immunology of Fungal Infections

Escherichia coli infections

Paleomicrobiology: Past Human Infections

Neurotropic Viral Infections

Immunology of Fungal Infections

Transplant Infections, 3rd Edition

Escherichia Coli Infections

Infections Causing Human Cancer

Neurotropic viral infections

Paleomicrobiology: Past Human Infections

Inmunology of Fungal Infections

Les infections virales émergentes

Catheter-Related Infections

Hospital-Acquired Infections

Foodborne Infections and Intoxications

Petroleum Engineering Handbook: Emerging and Peripheral Technologies. Vol. 6

Emerging Viruses

Emerging Infectious Diseases - Vol. 14, No. 6, June 2008

Emerging Infections 6

Emerging Infections in Asia

Emerging Infections, Volume 5

SARS: A Case Study in Emerging Infections

Emerging Neurological Infections (Neurological Disease and Therapy)

Emerging Infections: An Atlas of Investigation and Management

Emerging issues on HPV infections: from science to practice

Musculoskeletal Infections

Musculoskeletal Infections

Biofilm Infections

Chemokines in viral infections

Immunology of Fungal Infections

Escherichia coli infections

Paleomicrobiology: Past Human Infections

Neurotropic Viral Infections

Immunology of Fungal Infections

Transplant Infections, 3rd Edition

Escherichia Coli Infections

Infections Causing Human Cancer

Neurotropic viral infections

Paleomicrobiology: Past Human Infections

Inmunology of Fungal Infections

Les infections virales émergentes

Catheter-Related Infections

Hospital-Acquired Infections

Foodborne Infections and Intoxications

Petroleum Engineering Handbook: Emerging and Peripheral Technologies. Vol. 6

Emerging Viruses

Emerging Infectious Diseases - Vol. 14, No. 6, June 2008

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