Cumitech 30A: Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract

Selection

and Use of

Laboratory Procedures

for

Diagnosis

of Parasitic

Infections

of the

Gastrointestinal

Tract

LYNNE S. GARCIA, JAMES W. SMITH, AND THOMAS R. FRITSCHE COORDINATING

EDITOR

LYNNE S. GARCIA

Cumitech

CUMULATIVE TECHNIQUES AND PROCEDURES IN CLINICAL MICROBIOLOGY

Cumitech 1B Cumitech 2B Cumitech Cumitech Cumitech Cumitech Cumitech

3A SA 6A 7A 12A

Cumitech 13A Cumitech 16A Cumitech 18A Cumitech 19A Cumitech 21 Cumitech 23 Cumitech 24 Cumitech 25 Cumitech 26 Cumitech 27 Cumitech 28

Blood Cultures III Laboratory Diagnosis of Urinary Tract Infections Quality Control and Quality Assurance Practices in Clinical Microbiology Practical Anaerobic Bacteriology New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide Laboratory Diagnosis of Lower Respiratory Tract Infections Laboratory Laboratory Laboratory

Diagnosis of Bacterial Diarrhea Diagnosis of Ocular Infections Diagnosis of the Mycobacterioses

Laboratory Laboratory Laboratory

Diagnosis of Hepatitis Viruses Diagnosis of Chlurrrydia tvuchovvzutis Infections Diagnosis of Viral Respiratory Disease

Infections of the Skin and Subcutaneous Tissues Rapid Detection of Viruses by Immunofluorescence Current Concepts and Approaches to Antimicrobial Laboratory Diagnosis of Viral Infections Producing

Agent Susceptibility Enteritis

Testing

Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and Laboratory Animals Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals

Cumitech 29 Cumitech 30A

Laboratory Safety in Clinical Microbiology Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections Tract

Cumitech 31

Verification and Validation of Procedures in the Clinical Microbiology Laboratory Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Infections Obtained Food Animals and Wildlife Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism Laboratory Diagnosis of Mycoplasmal Infections Postmortem Microbiology

Cumitech

32

Cumitech Cumitech

33 34

Cumitech Cumitech Cumitech

35 36 37

Cumitech Cumitech

38 39

of the Gastrointestinal

Biosafety Considerations for Large-Scale Production of Microorganisms Laboratory Diagnosis of Bacterial and Fungal Infections Common to Humans, Livestock, Human Cytomegalovirus Competency

Assessment in the Clinical Microbiology

Cum/tech should be cited as follows, e g Garcia, L S , J W of Laboratory Procedures for D/agnos/s of Parasit/c lnfechons Press, WashIngton, D C

from

and Wildlife

Laboratory

Smith, and T R Fritsche. 2003 Cum/tech of the Gastro/ntest/na/ Tract Coordlnatlng

Editorial Board for ASM Cumitechs: Alice S Welssfeld, Char, Marla D Appleman, Burken, Roberta Carey, Linda Cook, Lynne Garcia. Richard M Jamlson, Karen Krlsher, Sewell, Daniel Shapiro, Susan E Sharp, James W Snyder, Allan Truant

Vi&e Susan

3OA, Seiection and Use ed , L S Garcia ASM

Baselskl, B Kay Buchanan, MItchelI L Mottice, Michael Saubolle, David

I L

Effective as of January 2000, the purpose of the Cum/tech series IS to provide consensus recommendations regarding the judicious use of clinlcal mIcrobIology and Immunology laboratories and their role In patient care Each Cumitech IS written by a team of cllnlclans, laboratonans, and other Interested stakeholders to provide a broad overview of various aspects of lnfectlous disease testing These aspects include a dIscussIon of relevant clInical conslderatlons, collection, transport, processing. and interpretive guidelines, the clinical utlllty of culture-based and non-culture-based methods and emerging technologies, and Issues surrounding coding, medical necessity, frequency limits, and reimbursement The recommendations III Curr~&x!rs do 1101 represent the official views or policies of any third-party payer CopyrIght 0 2003 ASM Press American Society for Microbiology 1752 N Street NW Washington. DC 20036-2904 All Rights

Reserved

Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract Lynne S. Garcia LSG & Associates, Santa Monica, Califohia

90402-2908

James W. Smith Department

of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120

Thomas R. Fritsche JMI Laboratories,

North Liberty, Iowa 52317

COORDINATING EDITOR: Lynne S. Garcia LSG & Associates, Santa Monica, California

Introduction Background

90402-2908

.. .. . ... .. .. .. ... . ... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. ... .. ... .. ... .. ... 2 2 .........................................................................................

Parasites Acquired Abroad and Parasite Endemicity in the United States ......................... Medical Education and Consultation Regarding Human Parasitic Infections ...................... Emerging Parasitic Infections (Coccidia and Microsporidia) ............................................. .................................. Changes in Identification Requirements for Entamoeba h&o/y&~ Factors That Influence Infection with Parasites ............................................................. Patient Symptoms ..................................................................................................... Ordering Parasite Examinations ...................................................................................

Factors Influencing

Test Performance

.....................................................

2 2 3 4 4 5 7

7

Laboratory Capabilities ............................................................................................... Communication between Clinicians and Laboratorians ................................................. Parasite Life Cycles .................................................................................................. Distribution of Parasitic Forms in Feces ......................................................................

7 10 10 12

Routine

12

Fecal Examination

Methods

.....................................................

Test Selection and Patient Preparation ....................................................................... Specimen Processing ............................................................................................... Microscopy .............................................................................................................. Direct Wet Mount .................................................................................................... Concentration Procedures ......................................................................................... Permanent Stains for Amebae, Ciliates, and Flagellates ...............................................

Organism-Specific

Detection

Methods

..................................................

12 13 16 17 18 19

21

Modified Acid-Fast, Hot Safranin, and Modified Trichrome Stains ................................. Chemofluorescent and lmmunofluorescent Agents ..................................................... Fecal lmmunoassays ................................................................................................ Gene Probes ...........................................................................................................

21 24 24 25

Other Fecal Examination

25

Methods

........................................................

Stains for Inflammatory Cells .................................................................................... Egg Count and Egg-Hatching Tests ............................................................................ Culture Techniques ..................................................................................................

25 25 25

.....................................................

26

Procedures

for Nonfecal

Specimens

Aspirates and Biopsies ............................................................................................. Sputum ................................................................................................................... Urinary Tract ............................................................................................................ Serology ..................................................................................................................

26 27 27 27

Repotting

28

........................................................................................... 1

2

Garcia et al.

CUMITECH 30A

Public Health Considerations ............................................................... Future Directions ................................................................................ References .........................................................................................

INTRODUCTION

T

his Cumitech is intended to provide guidance to the laboratory and other members of the patient care team in the appropriate selection, ordering, and application of tests for diagnosis and management of parasitic infections of the gastrointestinal (GI) tract. It reviews the reasons that examinations for parasites may be requested, discusses physician options for test selection and ordering, and discusses laboratory procedures used for examining clinical specimens, including the pros and cons of each. Detailed diagnostic procedures are not presented; readers are referred to a number of excellent books and manuals (8, 9, 12, 25, 40, 41, 54, 61) for this information. More-extensive bibliographies may be found in the articles cited at the end of this document in References. Approaches to the evaluation and examination of clinical specimens for parasites vary depending upon geographic area, patient population, laboratory resources, and preferences of laboratorians and clinicians. The goal of this Cumitech is to assist laboratorians in developing an appropriate algorithm for the evaluation of clinical specimens for intestinal parasites for their institutions. This revision of Cumitech 30 also includes extensive information regarding newer methods (e.g., immunoassays) and their relevance to the laboratory test menu. Information is provided on appropriate test ordering criteria that can be used by the physician to ensure that the most appropriate testing is performed for each patient, including routine testing and/or the newer immunoassay methods.

BACKGROUND Parasites Acquired Abroad and Parasite Endemicity in the United States With people traveling more widely than at any time in history, opportunities to contract parasitic infections continue to expand. Over 1 million U.S. citizens reside overseas, and each year over 10 million U.S. citizens travel to areas where parasitic infections are common, including the Caribbean, Latin America, Africa, Asia, or Oceania. In addition, millions of people from these areas travel to the United States each year, and several hundred thousand immigrants and refugees enter this country annually. Thus, although the parasitic infections most commonly seen in the United States are indigenous to this country, a broad range of parasitic

28 29 30

infections acquired in other countries may be brought into the United States. The pathogenic intestinal parasite most commonly seen in the United States is Giardia lam&a (Giardia intestinalis or Giardia duodenalis; this document uses G. lamblia throughout) (14, 45). Very little data are available; however, Table 1 contains data reported to the Centers for Disease Control and Prevention (CDC) from January 1992 through December 1997. The frequencies with which intestinal parasites were diagnosed in fecal specimens by state public health laboratories in 1987 are summarized in Table 2. Most of the species listed are found in the United States, though they may be encountered more frequently in other parts of the world. Note that the microsporidia and Cyclospord cayetanensis were not yet recognized as important intestinal pathogens of humans when this survey was performed and that Cryptosporidium was just beginning to be recognized as a coccidian pathogen found in both immunocompromised and immunocompetent individuals. Medical Education and Consultation Human Parasitic Infections

Regarding

Education of U.S. physicians regarding human parasitic infections is very limited. The average U.S. medical school dedicates a total of 7 h to the coverage of human parasitic diseases, usually during the first or second year of medical training. Additional education during medical residencies may be limited or nonexistent. Thus, many physicians may know little about parasitic infections and may not know what to request of the laboratory for diagnosis and management of patients with any but the most common infections. The laboratory plays a key role in the diagnosis of most parasitic infections, and clinical information provided with specimens submitted to the laboratory is particularly helpful in directing testing. However, patient clinical history is rarely submitted with the test request. One of the most important functions the laboratory performs is the distribution of relevant clinical information to physician clients regarding parasitic infections, appropriate specimen collection, utilization of specific tests, need for patient clinical history, and the interpretation of patient reports and report formats. The provision of patient clinical history must be stressed; systematic failure to provide such information must be brought to the attention of the institutional quality assurance committee. For optimal

CUMITECH Table 1.

30A National

Parasitic Giardiasis

Surveillance

System

data from

Description

1992 through

Infections

Beginning of a state-level voluntary reporting system Importance of this parasite has increased during the past few years.

43

No. of reported cases 1992 1996

Range of cases/lOO,OOO in 1997 New York State Vermont

12,793 27,778

Reported from 23 states More than double the number for 23 states; better reporting largely responsible

0.9 to 42.3; 10 states reported ~20 cases/l 00,000 3,673, or 20.3/l 00,000 (14.5% of cases nationally) Highest incidence, 42.3/l 00,000

Reflects differences in infection well as reporting efficiency Includes New York City

rates, as

Like New York, Vermont has an active surveillance program

Case information

Equal distribution by sex; highest among children aged O-5, followed by adults aged 31-40; active transmission in summer

Transmission occurs in all major geographic areas of the country; seasonal peak coincides with summer recreational water season and use of communal swimming venues; extended periods of cyst shedding and organism’s environmental resistance are factors

Estimate of infection

2.5 million cases/yr

Based on state surveillance

a From reference

3

Comments

23

2000

Tract

1997”

Data

No. of states reporting 1992

of the Gastrointestinal

16.

patient care, appropriate tests must be correctly ordered, the specimen must be collected properly, the laboratory work must be promptly and correctly performed, and the results must be appropriately interpreted by physicians. Emerging Parasitic Microsporidia)

Infections

(Coccidia

and

In recent years, several additional protozoa that may cause diarrhea in humans have been recognized (52). One of the main reasons they were previously unrecognized is that the routine parasitology methods and stains used did not allow recovery and identification of these organisms. Coccidian parasites include Cryptosporidium paruum (63) and C. cayetanensis (21); various species of microsporidia include Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis (76). C. parvum and several microsporidian species are well recognized as significant causes of severe and persistent diarrhea in AIDS patients, for whom diarrhea may be life threatening. In general, when CD4+ cell counts are >200/~1, infections are acute and resolve in approximately 2 weeks; however, when the CD4+ cell count drops below 200/~1, the infection may be chronic and persistent. C. parvum infects animals as well as humans and can contaminate inadequately treated drinking water. C. paruum has also been responsible for outbreaks traced to recreational water, such as swimming pools,

and to day care centers (17,18,44). Similar outbreaks caused by G. lamblia have been recognized for years (81). One of the largest waterborne outbreaks of cryptosporidiosis occurred in Milwaukee, Wis., and affected an estimated 400,000 persons. This outbreak led the CDC to appoint a task force to determine how to detect, prevent, and control such waterborne outbreaks (15, 50). Based on questions that arose from the 1993 Milwaukee outbreak regarding appropriate management and prognosis of patients infected with C. parvum, it is clear that cryptosporidiosis may present as an acute relapse of inflammatory bowel disease or Crohn’s disease that responds to standard therapy (53). Customary antidiarrheal antibiotics are not effective. It appears that immunosuppressive therapy does not predispose to chronic or severe illness in patients with C. parvum. Until 1995, infection with C. cayetanensis was diagnosed in the United States primarily in overseas travelers. However, in 1996,45 cases of cyclosporiasis were diagnosed in Florida residents who had no history of recent foreign travel. An epidemiological investigation failed to identify a source. In 1996, a North American outbreak resulting in more than 1,400 cases was reported from 20 states, Washington, D.C., and two Canadian provinces. Extensive investigations were undertaken to identify the vehicle of transmission. In case control studies, eating raspberries was strongly associated with cyclosporiasis (46).

4

Garcia et al.

CUMITECH

Table 2. Commonly recognized human parasites in the United States diagnosed from stool examination and prepatent periods

% Stool Organism

specimens with organisma

Prepatent (biologic incubation) period

Protozoa G. lamblia E. co/!’ B. hominis” E. hartmanni E. his tolytica lodamoeba bij tschliib D. fragilis Chilomas tix mesnilib Cryptosporidium spp. Trichomonas hominisbnd lsospora belli Balan tidium coli Unspecified protozoa

7.2 4.2 4.2 2.6 1.4 0.9 0.6 0.5 0.3 0.2 co.1 co.1 0.1

Nematodes Hookworm Trichuris trichiura A. lumbricoides Strong yloides s tercoralis En terobius vermicularis”

1.5 1.2 0.8 0.4 0.4

Cestodes Hymenolepis nana Taenia spp. Hymenolepis diminuta Diph yllobo thrium la turn Dipylidium caninum

0.4 99 (Gi) 100 (Cp) 100 (Gi) 93-98

Test

E. his tolytica TechLab Entamoeba Test

E. his tolytica/E. dispar group Tee h Lab E. his tolytica/E. dispar Test

Microplate

Entamoeba-CELISA

G. lamblia ProSpecT

EZ Microplate Assay Microplate Assay Rapid Assay Combination with Cryp tosporidium Color Vue GiardElA MeriFluor Combination with Ctyptosporidium SPP. Giardia-CELISA Giardia-Cel Giardia RIM Giardia Wampole Giardia

9

Both DFA and EIA formats available; DFA is combination reagent (Giardia-Cryptosporidium)

Contact manufacturer Requires fresh or frozen stool; combination test with Giardia and E. histolytica/E. dispar group

Can be used with fresh, frozen, or formalin-preserved stool; combination test with Giardia Can be used with fresh, frozen, or formalin-preserved stool; corn bination test with Giardia Can be used with fresh, frozen, or formalin-preserved stool

Will not differentiate E. histolytica from E. dispar; requires fresh or frozen stool. Different EIA formats; contact company for additional information

Alexon-Trend

Alexon-Trend Alexon Trend Alexon-Trend Alexon-Trend

EIA EIA EIA EIA

96-98 98-l 00 93 99.1

98 98-l 00 98 99.6

Alexon-Trend Antibodies, Inc. Meridian Diagnostics

EIA EIA DFA

100

99

Cellabs Cellabs Novocastra Remel Wampole

DFA EIA EIA EIA EIA

100 100

99 98

98-l 00 98

98-l 00 98

Both DFA and EIA formats available; DFA is combination reagent (Giardia/Cryptosporidium)

Contact manufacturer Contact manufacturer See TechLab

(Table continues)

Garcia et al.

10 Table 9.

CUMITECH

30A

Con tit-wed

Organism and kit name(sP Triage Parasite Panel

Combination with Cryp tosporidium parvum and Entamoeba histolytica/E. dispar group ColorPAC Giardial Cryp tosporidium Rapid Assay

Manufacturer and/ or distributor Biosite Diagnostics, Inc.

Type of test Cartridge device, EIA

Sensitivity (%jC

Specificity (%jC

98 (Gi) 100 (Ent)

98 (Gi) 99 (Ent)

92 Kp)

>99 (C/d

Genzyme/BectonDickinson

Cartridge device, IA

97-l 00 (Cp) 100 (Gi)

100 Kp) >99 (Gi)

ImmunoCard STATI Cryp tosporidium/ Giardia

Meridian

Cartridge device, IA

98.8 (Cp) 93.5 (Gi)

100 Kp) 100 (Gi)

TechLab Cryp to/Giardia IF Test

TechLab, Wampole

DFA

100

100

TechLab G/AM/A TEST

TechLab, Wampole

EIA

Diagnostics

Comments Requires fresh or frozen stool; combination test with Cryp tosporidium and E. his to/ ytica/ E. dispar group

Can be used with fresh, frozen, or formalin-preserved stool; combination test with Cryp tosporidium Can be used with fresh, frozen, or formalin-preserved stool; combination test with Cryp tosporidium Can be used with fresh, frozen, or formalin-preserved stool; combination test with Cryp tosporidium

98-l 00

“Adapted from reference 25. EIA, enzyme immunoassay; DFA, direct fluorescent-antibody assay; IA, immunoassay; Gi, Giardia spp.; Ent, Entamoeba spp.; Cp, Cryptosporidium spp. bA number o f t he kits are manufactured by a single manufacturer but labeled under different company names. Consequently, some of the data for sensitivity and specificity may be identifical to kits with another name and/or company. c Percentages have been rounded off to the nearest number.

minimum, a fecal concentration and a permanent stained smear of every stool specimen submitted for routine examination). Communication Laboratorians

between

Clinicians

and

The laboratorian plays a pivotal role in assessing the technical needs of the laboratory and the needs of the ordering clinician. Communication regarding the types of parasites being sought, the procedures being performed, and the parasites which may be detected must be open and clear. There is no mandated standard parasite examination. Procedures included in a standard workup vary with the type of specimen, patient population being served, parasites commonly seen, capabilities of the laboratory, cost, and preferences of the laboratory director. Some laboratories have guidelines based not only on fecal specimen consistency but also on the age of the patient. The recent discovery of previously unrecognized protozoa1 enteric pathogens (C. cayetanensis and the microsporidia) and the development of immunospecific antigen detection procedures for giardiasis and cryptosporidiosis further complicate clinician-laboratory communication (39, 51, 80). Clinical information about the patient being evaluated for enteric parasites provides important clues as to which agents are likely to be encountered and which diagnostic techniques should be employed. Patients with immunodeficiencies are especially susceptible to particular parasites and may develop severe or life-threatening infections. In most normal hosts, gi-

ardiasis is a self-limited disease that causes diarrhea lasting for 1 to 2 weeks. In patients with secretory immunoglobulin A (IgA) deficiency, giardiasis may be a chronic infection leading to a severe malabsorption syndrome. Likewise, intestinal cryptosporidiosis in AIDS patients may cause prolonged secretory diarrhea, while the disease tends to be self limiting in normal hosts. The ability of the patient to handle exposure to parasites depends on a number of factors, including age, nutritional and health status, and previous exposure. Immunity to parasitic infections, although not absolute, may be such that the infection is suppressed to a level where clinical symptoms do not develop, although parasites are sometimes detected. Examples of requests which a clinician might make, the tests the laboratory might perform, and the appropriate way to report negative results are seen in Table 10. Laboratory manuals for clients (hospitals, physician offices, and others) should explain the diagnostic test options available and their appropriate use. Categories which may appear on a requisition slip or a computer order screen include (i) routine O&P examination (concentration and permanent stained smear), (ii) GiardialCryptosporidium immunoassay, (iii) special staining for coccidia, and (iv) special staining for microsporidia. Parasite

Life Cycles

The selection of tests for detection of parasitic organisms varies with the parasite being sought and the expected diagnostic form of the parasite. A knowledge of parasite life cycles aids laboratorians in per-

CUMITECH

30A

Parasitic

Table 10.

Examples

of report

Test request and sample submitted Routine O&P examination

lmmunoassays Giardia

formats

for commonly

ordered

parasitology

Negative for G. lamblia

Giardia antigen immunoassay (EIA, FA, or immunochromatographic cartridge) Cryptosporidium antigen immunoassay (EIA, FA, or immunochromatographic cartridge) Giardia-Cryptosporidium immunoassay (EIA, FA, or immunochromatographic cartridge) E. histolytica-E. dispar immunoassay (EIA or immunochromatographic cartridge) E. histolytica immunoassay (EIA)

E. histolytica-E.

Negative for E. histolytica-E.

a Reports generated rescent antibody.

dispar group

Negative for E. histolytica

Negative for coccidia oocysts ( Cryp tosporidium, Cyclospora, and Isospora) Negative for microsporidia spores

must accurately

testsa

Concentration, permanent stained smear (direct wet smear performed on fresh, soft, or liquid stools only)

Negative for G. lamblia and C. parvum

Microsporidia

11

No helminth eggs, larvae, or protozoa seen (test will not detect coccidia oocysts or microsporidia spores; special stains required)

Giardia-Cryp tosporidium

Special stains Coccidia

Tract

Actual test(s) performed

Negative for C. parvum

E. his tolytica

of the Gastrointestinal

Report format for negatives

Cryp tosporidium

dispar group

Infections

reflect the capabilities

of the tests performed

forming their role in diagnosis. Knowledge of the prepatent and incubation periods is particularly important for the proper timing of specimen collection. The incubation period (the time from infection to appearance of symptoms) usually coincides with the appearance of diagnostic forms. When a patient with a protozoa1 GI infection presents with symptoms, diagnostic forms (trophozoites, cysts, oocysts, or spores) are usually present. In helminth infections, there is a prepatent period from the time the infection is acquired until the diagnostic forms are found in the stool; for example, in ascariasis the recently hatched larvae go through a migratory phase in the lungs before continuing on to the intestinal tract, where maturation and production of eggs occur. During the prepatent period, symptoms related to larval migration may be present, but performance of a fecal examination is not helpful in establishing a diagnosis. For helminth parasites, the length of the prepatent period varies widely. Incubation and prepatent periods for intestinal protozoa and helminths that commonly infect humans are reviewed in Table 2. The reproductive capabilities of parasites influence the manifestations that they produce in patients. Parasites capable of continued multiplication within the host are most likely to produce severe disease. Most protozoa have unlimited proliferative capacity; thus, host defenses (especially the immune system) are im-

Modified

acid-fast stains for coccidia

Modified trichrome stains for microsporidia; optical brightening agents (e.g., Calcofluor) can be used as a screening stain, but false negatives and false positives have been reported; confirm with modified trichrome stain on human fecal specimens.

EIA, enzyme

immunoassay;

FA, fluo-

portant because they may allow the host to control the infection. Most helminths do not increase their numbers within human hosts. Humans usually serve as definitive hosts, and for each egg or larval stage that is ingested or invades the skin, only one adult develops. If no additional eggs or larvae are acquired from external sources, the parasite burden does not increase and the length of infection is limited to the normal life span of the adults. Strongyloidiasis is an exception; with this infection, internal autoinfection can occur, resulting in persistence of infection for many years. If patients with such infections become immunocompromised, because of organ transplantation, corticosteroid therapy, antineoplastic chemotherapy, or other conditions, hyperinfection with large numbers of adult and larval worms may develop and can be fatal (33). Some helminths, including EL nana and Enterobius vermicularis, produce eggs that are infective immediately upon passage or within a matter of hours. Heavy infections may result, particularly in children or mentally disabled adults, probably from autoinfection. The eggs of Taenia solium are also immediately infectious for humans when passed, but in this instance the human becomes the intermediate host for the cysticerci rather than the definitive host for adult worms.

12

CUMITECH

Garcia et al.

Distribution

of Parasitic

Forms in Feces

Shedding of parasite forms varies depending upon the parasite. With infections such as ascariasis, trichuriasis, and hookworm, eggs are shed continuously. In other infections, such as taeniasis, giardiasis, dientamebiasis, and strongyloidiasis, the number of parasite forms shed can vary significantly from day to day and may not be detectable even though the patient is symptomatic. Reasons for the cyclical shedding of some protozoa are not well understood. With some helminths, including pinworms and Taenia spp., eggs are liberated from intact worms or ruptured proglottids only sporadically, resulting in uneven distribution in the fecal specimen and daily variation. Parasites that reside in the small bowel (S. stercoralis, C. parvum, and G. lamblia) or proximal large bowel (other protozoa and egg-producing helminths) usually have diagnostic forms distributed uniformly in the fecal specimen. Parasites that infect the rectum and lower colon may be distributed unevenly in or on fecal specimens. Depending upon the species, adult schistosomes release eggs in small venules in the intestine or urinary bladder. Extensive granulomatous inflammation eventually forces these eggs into the lumen of the bowel or bladder. There is a delay between the time eggs are deposited and the time they enter the bowel lumen. In chronic infections where there is much scarring, many eggs are unable to reach the lumen. Eggs of Schistosoma mansoni are more likely to be found on the surface of the stool specimen because adult worms most commonly deposit eggs in vessels of the lower large intestine. The eggs may be associated with blood or mucous flecks. Patients with E. his tolytica colitis may have focal shedding of parasites related to ulcerated areas in the colon; if the infection primarily involves the rectosigmoid portion of the colon, the parasites may be more numerous on the surface than in the center of the specimen. Again, parasites are more likely to be found in bloody flecks and in mucous strands. Careful gross examination of the stool specimen for blood and mucous strands and microscopic examination of samples from these areas may be productive. Trophozoites of protozoa1 species are more likely to be found in liquid stool specimens, whereas cysts are predominantly found in formed stools. Soft stool specimens may have both forms present. The initial portion of a formed stool is older and tends to be more dehydrated, with a relatively greater preponderance of cysts. The final portion of stool evacuated tends to be softer and is more likely to contain trophozoites. The presence of a small number of adult worms in helminth infections may lead to failure to detect the infection even after the prepatent period. With some species, such as A. lumbricoides, the number of eggs

30A

produced is quite large and eggs from one female are likely to be numerous enough to be detected by routine examination of a stool concentrate. Other species, such as T. trichiura, produce far fewer eggs that may be missed when only a few adults are present. Asymptomatic strongyloidiasis can persist for years as a result of internal autoinfection. It is important to be able to detect a low level of infection to assess cure after therapy or to ensure a patient is free of infection before institution of therapeutic immunosuppressive protocols. Because the distribution of parasitic forms in feces is variable, routine fecal examination methods periodically miss some parasites, and the use of special procedures may be necessary for diagnosis. For optimal diagnosis of pinworm infections, the cellophane tape method or similar methods that allow detection of eggs in the perianal folds are recommended. It is estimated that only 10% of pinworm infections are diagnosed from examining fecal specimens. The severity of pinworm infection is determined by the number of perianal examinations that are positive in a series of six examinations (71). In heavy infections, all of the specimens are positive, and in light infections, only one or two specimens are positive.

ROUTINE FECAL EXAMINATION METHODS Test Selection

and Patient

Preparation

The clinician should provide basic clinical information with the specimen, including the reason(s) for submitting the specimen. A history of diarrhea alerts the laboratory that intestinal protozoa are the most likely parasitic cause of the patient’s condition. If eosinophilia is present, helminths are suspected, and the physician may wish to consider whether there has been sufficient time for diagnostic stages to be detectable. If specific parasites were suspected, as with a child who is attending a day care center with an outbreak of giardiasis, this information would also be helpful and would influence the selection of methods used by the laboratory. However, this type of information usually does not accompany the test requisition. Certainly, the option always exists for laboratory personnel to call the physician to discuss the request or the patient. In most instances, there is no special preparation of the patient prior to specimen collection. Specimens should not be collected if the patient has received substances such as kaolin-containing antidiarrheal medicines, antacids, or contrast materials for GI examination. Such preparations are known to interfere with the routine examination. Purgation has been recommended by some and is known to dramatically increase yield for diagnosis of amebiasis, but it is not

CUMITECH

30A

Table 11.

Workup

Parasitic

Infections

of the Gastrointestinal

Tract

13

of fecal specimensa Specimen handling

Stool sample Fresh Formed Soft Liquid Watery Submitted

Direct wet mount (fresh specimen only) -

in fixative

Concentration

Permanent stained smea?

Modified

acid fast

+ +

+ + +c +c

+ + + +

Only Only Only Only

-

+

+

Only on request

on on on on

request request request request

Modified

trichrome

Only Only Only Only

request request request request

on on on on

Only on request

a-

not required; +, required. See Table 8 for specific test orders related to patient’s history (immunoassays, O&P examination, special stains). ‘The permanent stained smear is required for every fecal specimen submitted for an O&P examination (trichrome and iron-hematoxylin are exampies). “Although concentration is required for every fecal specimen submitted for an O&P examination, if the specimen is very liquid or watery, simple centrifugation is recommended (IO min at 500 X g).

widely used because of cost, inconvenience, and discomfort for the patient. The number of specimens to submit and the interval for collecting them vary depending upon several factors (55, 58). The suspected parasite species is an important consideration. For outpatients, it is recommended that three specimens be collected, one every 2 or 3 days, to rule out parasitic infections. Of course, if a parasite compatible with the clinical presentation were found prior to completing the series, submission of additional specimens would not be necessary. If a patient is hospitalized, it may be better to have a specimen submitted each day rather than every 3 days because of the cost of hospitalization relative to the cost of the fecal examination. It is uncommon for patients who have been hospitalized for more than 3 days to have positive specimens, and some have advocated that specimens from such patients not be examined unless specifically approved after discussion between the laboratorian and the clinician (60). On rare occasions, a second set of three specimens may be required, particularly if the patient is very ill and a parasitic agent is strongly suspected but has not been detected. Some have advocated pooling the three specimens and performing one examination (3). Although there is a slight decrease in sensitivity with this method, the major objection is the time delay for diagnosis. The routine fecal parasite examination (O&P) includes a concentration technique and a permanent stain (Table 11). For formed specimens, a concentration procedure should be performed, and a portion of the specimen should be fixed so that permanent stains can be used for definitive identification of any intestinal protozoa present. For soft or liquid specimens that are submitted fresh, a direct wet mount may allow detection of motile parasites. A concentration technique and permanent stain should also be performed. In some laboratories, these specimens are routinely stained with a modified acid-fast stain. Watery specimens are concentrated by simple centrifugation (the use of ethyl acetate is not recommended) (2.9.

Specimen

Processing

When specimens are received in the laboratory, they should be examined. If they are unsatisfactory, they should be rejected. Criteria for rejection include the presence of interfering substances, desiccation of specimens, insufficient quantity, and prolonged delays between collection and delivery to the laboratory without preservation. Interfering substances can often be recognized because they are opaque and white or tan. Rejection of any specimen should be discussed directly with the clinician or other appropriate health care provider to ensure timely submission of another specimen. Often, a patient submits a stool specimen because he or she is symptomatic; thus, the stool may be soft or liquid. When specimens are collected at home or in an off-site clinic where they may not be transported promptly, they should be placed in an appropriate fixative(s) and thoroughly mixed to prevent deterioration of any parasites present. It is important that clear verbal and written instructions be provided to patients, clinic personnel, and physicians. If the patient is unable to understand the directions, appropriate instructions can be given to relatives or other caregivers. Fresh specimens are mandatory for the recovery of motile trophozoites (amebae, flagellates, or ciliates). The protozoan trophozoite stage is normally found in cases of diarrhea, as the contents of the GI tract move through the system too rapidly for cyst formation to occur. Once the stool specimen is passed from the body, trophozoites do not encyst but may disintegrate if not examined or preserved within a short time. The time limit recommendations listed below are most relevant for the intestinal protozoa; most helminth eggs and larvae, coccidian oocysts, and microsporidian spores will survive for extended periods. However, no one can predict what organisms are present in the specimen, thus the need to use the most conservative time frames for parasite recovery. The examina-

14

Garcia et al.

tion of liquid specimens should occur within 30 min of passage, not 30 min from the time they reach the laboratory. If this general time recommendation of 30 min is not possible, then the specimen should be placed in one of the available fixatives. Soft (semiformed) specimens may have a mixture of protozoan trophozoites and cysts and should be examined within 1 h of passage; again, if this time frame is not possible, then preservatives should be used. Immediate examination of formed specimens is not as critical; in fact, if the specimen is examined any time within 24 h after passage, the protozoan cysts sho uld still be intact (25). In review, only trophozoites are usually found in liquid specimens, both protozoan trophozoites and cysts can be recovered in soft specimens, and only cysts are generally recovered from formed specimens. The time limits mentioned above are merely guidelines. However, if fresh specimens remain unpreserved for longer amounts of time before examination, many, if not all, organisms may disintegrate or become distorted. Fecal specimens should never be incubated or frozen prior to examination. Although some laboratories request fresh stool along with fixed specimens in order to perform a gross examination of the stool, this practice is no longer as popular as it was previously. If possible, the laboratory should receive information on the consistency of the stool (formed, soft, or liquid) if the specimen is received in fixative. However, this information is rarely transmitted to the receiving laboratory and has a minimal impact on the results of the stool examination. There are a number of reasons why a time lag may occur from the time of specimen passage until examination in the laboratory (e.g., laboratory workload, transit distance, or time to reach the facility). To preserve protozoan morphology and to prevent the continued development of some helminth eggs and larvae, the stool specimens can be placed in preservative either immediately after passage (by the patient using a collection kit) or once the specimen is received by the la boratory. There are several fixatives available; the more-common ones are formalin, sodium acetate-acetic acid-formalin (SAF), Schaudinn’s fluid, polyvinyl alcohol (PVA), and single-vial systems. Regardless of the fixative selected, adequate mixing of the specimen and preservative is mandatory. When selecting an appropriate fixative, keep in mind that a permanent stained smear is mandatory for a complete examination for parasites (25). It is also important to remember that disposal regulations for compounds containing mercury are becoming more strict; laboratories will have to check their applicable state and federal regulations to determine fixative options. The pros and cons of common and less-common fixatives can be seen in Table 12.

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There are two general types of fixatives: those that can be used for concentration and concentration sediment wet-mount examination and those that can be used for the preparation of permanent stained smears (e.g., trichrome or iron-hematoxylin). Formalin has been used for many years as an allpurpose fixative that is appropriate for helminth eggs and larvae and for protozoan cysts. Two concentrations are commonly used: 5 %, which is recommended for preservation of protozoan cysts, and lo%, which is recommended for helminth eggs and larvae. Although 5% is often recommended for all-purpose use, most commercial manufacturers provide 10 %, which is more likely to kill all helminth eggs. To help maintain organism morphology, the formalin can be buffered with sodium phosphate buffers, e.g., neutral formalin. Selection of specific formalin formulations is at the user’s discretion. Aqueous formalin that contains no buffers permits the examination of the specimen as a wet mount only, a technique much less accurate than a stained smear for the identification of intestinal protozoa. Protozoan cysts (not trophozoites), coccidian oocysts, helminth eggs, and larvae are well preserved for long periods of time in 10% aqueous formalin. Hot (60°C) formalin can be used for specimens containing helminth eggs, since in cold formalin some thickshelled eggs may continue to develop, become infective, and remain viable for long periods. Several grams of fecal material should be thoroughly mixed in 5 or 10 % formalin. SAF lends itself to both the concentration technique and the permanent stained smear and has the advantage of not containing mercuric chloride, as is found in Schaudinn’s fluid and PVA fixative (25). It is a liquid fixative, much like the 10% formalin previously described. The sediment is used to prepare the permanent smear, and it is frequently recommended that the stool material be placed on an albumincoated slide to improve adherence to the glass. SAF is considered to be a softer fixative than mercuric chloride. The organism morphology will not be quite as sharp after staining as with organisms originally fixed in solutions containing mercuric chloride. The pairing of SAF-fixed material with iron-hematoxylin staining provides better organism morphology than does staining SAF-fixed material with trichrome (L. S. Garcia, personal observation). Although SAF has a long shelf life and is easy to prepare, the smear preparation technique may be a bit more difficult for less-experienced laboratory personnel who are not familiar with fecal specimen techniques. Laboratories that have considered using only a single preservative have selected this option. Helminth eggs and larvae, protozoan trophozoites and cysts, coccidian oocysts,

Parasitic

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Table 12.

Stool collection:

pros and cons of fresh and preserved

Type of stool Fresh

Infections

of the Gastrointestinal

15

specimen&’

Pros 1. No requirements

Tract

Cons

for stool fixatives

I. May have excessive time lag between stool passage and fixation or processing; trophozoites may disintegrate, thus giving a false-negative result 2. O&P examination (direct wet exam, concentration, and permanent stained smear) may be negative due to lack of organism preservation and morphology integrity

2. Ability to see motile trophozoites

3. Lower cost 4. Can perform direct wet exam, concentration and permanent stained smear 5. Relevant only if time from stool passage to laboratory is acceptable; in symptomatic patients, the trophozoite form of the intestinal protozoa is present and will not encyst when outside of the body: A. liquid or watery stool (30 min) B. semi-formed (1 h) C. formed (24 h) Preserved

Organism morphology is preserved when time lag between stool passage and fixation is short; reduced lag times can be achieved by having the patients collect and fix the stool specimens at home; once the specimen is mixed with the preservative, delivery time to the laboratory is not critical L. Can perform concentration and permanent stain

1. Cost of collection vials may represent a cost increase; cost may be lower overall because of much more accurate result (improved patient outcome)

2 Disposal of vials may be a problem if the laboratory is using preservatives containing mercuric chloride

a Most products used for specimen collection are available from any major medical supply house. FA, fluorescent

and microsporidian spores are preserved by this method. Schaudinn’s fixative contains mercuric chloride and is used with fresh stool specimens or samples from the intestinal mucosal surface. Many laboratories that receive specimens from in-house patients and have no problems with delivery times often use Schaudinn’s fluid. Permanent stained smears are then prepared from the fixed material. A concentration technique using Schaudinn’s fluid-preserved material is also available but is not widely used. WA is a plastic resin that is normally incorporated into Schaudinn’s fixative (11). The PVA powder serves as an adhesive for the stool material; i.e., when the stool-WA mixture is spread onto the glass slide, it adheres because of the PVA component. Fixation is still accomplished by the Schaudinn’s fluid itself. Perhaps the greatest advantage in the use of PVA is that a permanent stained smear can be prepared. PVA-fixative solution is highly recommended as a means of preserving cysts and trophozoites for examination at a later time. The use of PVA also permits specimens to be shipped (by regular mail service) from any location in the world to a laboratory for subsequent examination. PVA is particularly useful for liquid specimens

antibody. Adapted from reference 25.

and should be used in the ratio of 3 parts of PVA to 1 part of fecal specimen. Although there has been a great deal of interest in developing preservatives without the use of mercury compounds, modified PVA substitute compounds have not provided the quality of preservation necessary for good protozoan morphology on the permanent stained smear. Copper sulfate has been tried (12) but does not provide results equal to those seen with mercuric chloride (25). Zinc sulfate has recently been proven to be a good mercury substitute and is used with trichrome stain (3 1). Although zinc substitutes are widely available, each manufacturer has a proprietary formula for the fixative. Several manufacturers now have available singlevial stool collection systems, similar to SAF or modified PVA methods. From the single vial, both the concentration and permanent stained smear can be prepared. It is also possible to perform fecal immunoassay procedures from some of these vials. Make sure to ask the manufacturer about all three capabilities (concentrate, permanent stained smear, and immunoassay procedures) and for specific information about formula components that may interfere with any of

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Garcia et al.

the three methods. Like the zinc substitutes, these formulas are proprietary. If fresh specimens are mailed, they must meet the requirements of the U.S. Postal Service. If they are placed in fixatives, specimens are not considered mfectious and do not need to be labeled as infectious agents; however, they should have sufficient absorbent material to prevent contamination if a vial leaks or breaks. Microscopy Good, clean microscopes and light sources are mandatory for the examination of specimens for parasites. Organism identification depends on morphological differences, most of which must be seen using stereoscopic (magnification of X50) or regular microscopes at low ( X loo), high dry ( x400), and oil immersion (X 1,000) magnifications. The use of a 50X or 60x oil immersion lens for scanning can be very helpful. Microscopy for detecting and identifying intestinal protozoa requires a microscope with properly adjusted illumination. For optimal illumination of wet mounts, the condenser should be set at the upper position and the light should be controlled by closing the iris diaphragm rather than by racking the condenser down. A variety of objectives may prove useful (Table 13), and proper use of these can both speed up the examination and improve its accuracy. A stereoscopic microscope is recommended for larger specimens (arthropods, tapeworm proglottids, and various artifacts). The total magnification usually varies from approximately X 10 to x45 either with a zoom capacity or with fixed objectives (0.66x, 1.3 X, and 3 X) that can be used with 5 X or 10X oculars. Depending on the density of the specimen or object being examined, you must be able to direct the light source either from under the stage or onto the top of the stage. The regular light microscope should be equipped with the following: 1. Head. A binocular head is recommended, with a diopter adjustment to compensate for focus variation in the eyes. 2. Oculars. Oculars of 10X are required; 5 X oculars can be helpful but are optional. 3. Objectives. Objectives include 10X (low-power), 40 x (high-power), and 100X (oil immersion) settings. Some laboratories are currently using 50 or 60x oil immersion lenses to screen permanent stained smears. Examination with a combination of the 50 or 60 X oil and 100 X oil immersion lenses allows screening to proceed more quickly and eliminates the problem of accidentally getting oil on the high dry objective lens when switching back and forth between the 40x (high dry) and 100~ (oil immersion) objectives.

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4. Stage. A mechanical stage for both vertical and horizontal movement is required. Graduated stages are mandatory for recording the exact location of an organism on a permanently stained smear and recommended for any facility performing diagnostic parasitology procedures. This capability is essential for consultation and teaching purposes. Condenser. A bright-field condenser equipped with an iris diaphragm is required; however, an adjustable condenser is not required with the newer microscopes. The condenser numerical aperture should be equal to or greater than the highest objective numerical aperture. Filters. Both clear blue-glass and white groundglass filters are recommended. Light source. The light source, along with an adjustable voltage regulator, is usually contained in the microscope base. This light source should be aligned according to the manufacturer’s directions. If the light source is external, the microscope must be equipped with an adjustable mirror and an adjustable condenser containing an iris diaphragm. The external light source should be a 75to 100-W bulb. The identification of protozoa and other parasites depends on several factors, one of which is size. Any laboratory doing diagnostic work in parasitology should have a calibrated ocular micrometer in the microscope available for precise measurements (25). Measurements are made using a micrometer disk that is placed in the ocular of the microscope; the disk is usually calibrated as a line divided into 50 U. Depending on the objective magnification used, the divisions in the disk represent different measurements. The ocular disk division must be compared with a known calibrated scale, usually a stage micrometer with O.land O.Ol-mm divisions. The microscope should be recalibrated at least once each year if it receives heavy use or is moved frequently to different laboratory benches. Normal or light use generally does not require yearly recalibration. However, the microscope needs to be recalibrated if an objective or an ocular lens is changed or the optical path is modified. Often the measurement of RBCs (approximately 7.5 r,tm) is used to check the calibrations of the three magnifications ( X 100, X400, and X 1,000). Latex or polystyrene beads of a standardized diameter (Sigma, J. T. Baker Diagnostics, etc.) can be used to check the calculations and measurements. Beads of lo- and 9O-pm diameter are recommended. Size is important for the differentiation of several species, for example, Entamoeba hartmanni versus E. histolytica/E. dispar, C. parvum versus C. cayetanensis, and some of the helminth eggs. Some laboratories continue to misdiagnose parasites on proficiency test-

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Parasitic

Table 13.

Use of microscope

in evaluation

of stool specimens

Wet mount

Objective 10x

objectives

Direct

of the Gastrointestinal

Examine total coverslip preparation (22 by 22 mm) Examine at least l/3 to l/2 of the coverslip preparation

50 or 60X (Oil)

Not used

Not used

100X (Oil)

Not used

Not used

Tract

17

for parasites” Permanent

Concentration

Examine total coverslip preparation (22 by 22 mm) Examine at least l/3 to l/2 of the coverslip preparation

40x (High dry)

Infections

stain

Not routinely

used

Not routinely

used

May be used for screening slide; examine at least 300 oil immersion fields (using the 100X oil immersion objective), do not report the results of the specimen without examining 300 oil immersion fields using the 100x oil immersion objective Recommended for slide examination; examine at least 300 oil immersion fields using the 100X oil immersion objective

Comments Proper light adjustment is critical for wet preparations Objective primarily used to find and identify helminth eggs and larvae; some protozoa can be identified in the wet mounts using this objective Either a 50x or 60x oil immersion objective is excellent for screening permanent stained smears; however, at least part of the smear should be examined using the 100X oil objective Used for confirmation of structures seen at lower power; used only with permanent stained slides

a The typical microscope is generally equipped with the IOX, 40x (high dry), and the 100x (oil immersion) objectives; many laboratories are now equipping their microscopes in microbiology with either an additional 50x or 60x oil immersion objective for rapid screening of permanent stained slides. A final review of the slide with the 100x oil immersion objective is recommended before reporting the specimen as negative.

ing surveys because they do not use an ocular micrometer or the micrometer has not been calculated properly. When microscopic examination is performed, it is important to consider the characteristics of a number of organisms before making a final identification. The observer should not overemphasize any one characteristic. For example, it would not be proper to identify E. histolytica/E. dispar in a specimen simply because one of the organisms had a small central karyosome when there were numerous Entamoeba coli organisms present. Staining characteristics of the cytoplasm should also differ from those of the dominant E. coli organisms. Before two species are reported, there should be distinct populations of each identified and not just slight variations. If in doubt, laboratories should not hesitate to refer specimens to experts to ensure that the identifications are correct, particularly if pathogen identification is a principal consideration and an incorrect diagnosis might lead to either unneeded therapy or lack of therapy for a serious infection. Parasites are differentiated from artifacts on the basis of size, shape, and the distinctive characteristics or structures found in parasite forms. For instance, in differentiating an A. lumbricoides egg from a plant cell, it is important that it is the right size, shape, and

color and displays the typical shell characteristics. Nematode larvae are differentiated from fibers or plant hairs by identification of typical anatomic structures, including the mouth, esophageal bulb, and genital primordium, and various distinctive features. Occasionally, it is not be possible to differentiate a rare artifact from a parasite, and it is necessary to examine additional specimens from the patient or request an outside evaluation of the specimen. Direct Wet Mount Normal mixing of fecal contents in the intestinal tract usually ensures an even distribution of organisms in stool specimens. However, depending on the level of infection, examination of the fecal material as a direct smear may or may not reveal organisms. The direct wet smear is prepared by mixing a small amount of stool (about 2 mg) with a drop of 0.85% NaCl; this mixture will provide a uniform suspension under a 22- by 22-mm coverslip. Some workers prefer a 1.5 by 3-in. (1 in. = 2.54 cm) slide for the wet preparations rather than the standard l- by 3-in. slide, which is routinely used for the permanent stained smear. A 2-mg sample of stool forms,a low cone on the end of a wooden applicator stick. If more material is used for the direct mount, the suspension is usually too thick for an accurate examination; any sample of