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
1 O-20 days IO-12 wk 21 days 4-6 wk 20-28 days

Trematodes Clonorchis/Opis thorchis spp. Schis tosoma mansoni F. hepa tica Paragonimus spp.

0.6 co.1 co.1 co.1

4 wk 5-7 wk 12-16 wk 65-90 days

7-8 days

7-21 days

7-14 days 9-15 days

7 wk 12 wk 8-12 wk 3-5 wk 5 wk

a Excerpted from reference 45; n = 216,675. ‘Considered nonpathogenic. c Status of pathogenicity controversial. d Name changed to Pentatrichomonas hominis. eAnalysis of fecal specimen not optimal for diagnosis.

These studies concluded that Guatemalan raspberries were the vehicle responsible for the 1996 Florida outbreak. Cyclospora is now accepted as a foodborne pathogen that may play a growing role in the etiology of enteric disease in the United States, as food markets become increasingly international (Table 3) (48, 74) and appropriate special diagnostic tests are more widely utilized. Diagnostic stages of C. paruum and C. cayetanensis (oocysts) and microsporidia (spores) are neither readily recovered nor easily identified by routine ova and parasite (O&P) examination methods and stains. Improved concentration methods and special staining methods or immunoassays are required to diagnose infection with these protozoa (Tables 4 and 5).

Changes in Identification Entamoeba histolytica

Requirements

30A

for

Until recently, an unexplained aspect of the epidemiology of amebiasis was the number of asymptomatic individuals who were passing cysts. Extensive work involving isoenzyme analysis and molecular techniques demonstrated conclusively that there are two genetically distinct but morphologically identical species of what was formerly known as Entamoeba histolytica (13, 23, 66). After many years of debate, agreement was reached at a recent satellite meeting of the XIII Seminar on Amebiasis held in Mexico City, Mexico, to classify the species that is associated with the disease amebiasis as E. histolytica. The other species that is more common but is not capable of causing invasive disease is now called Entamoeba dispar. Recent studies confirming the differences between the two organisms include direct sequencing of the PCR-amplified SSU rRNA gene of E. dispar and the design of primers for rapid differentiation from E. histolytica (62), differences between the phosphoglucomutases of E. histolytica and E. dispar (64), differences in the molecular biology of the hexokinase isoenzyme patterns (65), differences in acid phosphatase secretion (73), and the identification of DNA sequences unique to E. histolytica (79). E. histolytica and E. dispar are morphologically identical, except that E. histolytica trophozoites may occasionally contain ingested red blood cells (RBCs). Isoenzyme profiles, immunoassays, or DNA probes can be used to differentiate the two species. Because the laboratory diagnosis of these newly described enteric protozoa requires special techniques, discussion of recovery and identification methods follows that of the routine fecal examination methods. Factors That Influence

Infection

with

Parasites

Predisposing factors may affect patients’ susceptibility to or the severity of parasitic infections. Such factors include age, preexisting conditions such as AIDS or cancer, iatrogenic immunosuppression for transplanGeographic and environtation, and malnutrition. mental factors also influence the types of parasitic infections that may be acquired. For example, a person on a dairy farm is much more likely to acquire cryptosporidiosis than someone living in a city (47), and a child in a day care center is more likely to acquire giardiasis than a child cared for at home. Sanitary practices are particularly important among all patient groups. Most parasitic GI infections would be eliminated if there was proper disposal of human feces. Although infections for which there are animal reservoirs, such as cryptosporidiosis and giardiasis, would still be seen, the frequency would likely decrease. Handwashing after defecation and other

CUMITECH Table 3.

30A Outbreaks

Parasitic

Infections

of the Gastrointestinal

Tract

5

of C. cayetanensis

Date(s) and characteristics of outbreak

Implicated

cause(s)

Comments

26 June 1996 (48 cases); Virginia

Basil-pest0

June-July 1996 (185 cases); 25 clusters in northern Virginia, Washington, DC., and Baltimore, Md.

Food containing

fresh basil

All basil supplied by the same company

4997 (1,450 cases); United States and Canada

Fresh raspberries from Guatemala; mesclun (baby lettuce leaves, known as spring mix, field greens, or baby greens)

Majority of cases linked to ingestion of raspberries

July 1999 (62 cases); Missouri clusters)

Food containing

Basil grown in Mexico or United States; menu included chicken pasta salad and tomato-basil salad

(2

pasta salad

No raspberries

fresh basil

IO June 2000 (54 cases); Philadelphia, Pa.

Wedding filling

cake containing

2002 (34 cases); Germany

Salad side dishes (lettuce spiced with fresh green leafy herbs)

measures to prevent fecal-oral contamination are also important. Drinking water is a particularly important source of infection (IS-18), as it may be contaminated with G. lamblia cysts and C. parvum oocysts from the feces of infected humans or animals or E. histolytica Table 4. Intestinal procedure@ Organism

coccidia:

recommended

Specimen

C. parvum

Stool

Sputum Scraping (GI tract) Biopsy C. caye tanensis

Stool Biopsy

1. be/Ii

Stool

Biopsy Sarcocystis

spp.

Stool

Biopsy

diagnostic

Diagnostic

procedureQc

Modified acid-fast stains, fluorescent stains, or immunoassays Modified acid-fast stains Modified acid-fast stains or routine histology Routine histology Modified acid-fast stains and autofluorescence Routine histology Concentration sedimentation and modified acid-fast stains Routine histology Concentration sedimentation, and special stains Routine histology

a Adapted from reference 25. ‘When using i mmunoassay reagents (FA) or modified acid-fast stains for organism detection, perform these procedures using centrifuged stool (500 X g for 10 min). When using immunoassay reagents for antigen detection, centrifugation of the fecal specimen is not required. Always check procedure directions and be aware of compatibility issues between kit reagents and stool preservatives. c Fluorescent methods include the use of auramine or auramine-rhodamine. Hot safranin can also be used instead of modified acid-fast stains (25, 76).

raspberry cream

or mesclun lettuce served

Cases linked to ingestion of raspberries from Guatemala Lettuce imported from southern

Europe

cysts from humans (Tables 6 and 7). Chlorination is not totally effective in killing these organisms, particularly C. parvum oocysts; filtration or boiling of water is required. Dietary habits and food preparation customs can influence the incidence of parasitic infections in a population. Thus, people in a community who eat raw fish or raw meat may acquire infections such as clonorchiasis, taeniasis, or trichinosis, whereas their vegetarian neighbors would not be at risk for such infections. Patient

Symptoms

Except for the protozoan infections that often cause acute diarrhea, most parasitic infections of the digestive system are usually chronic and produce vague and nonspecific symptoms, such as abdominal discomfort, or are entirely asymptomatic. A variety of other infectious or noninfectious etiologies can produce similar symptoms, adding to the confusion in establishing a diagnosis. The symptoms produced with intestinal helminth infections are even less specific than those seen with protozoan infections. Helminth infections are often not suspected until an adult worm or portion thereof is passed or unless an astute clinician checks an anemic patient to determine whether he or she has a hookworm infection. Findings that should lead a physician to order a parasite exam include eosinophilia, abdominal discomfort, and diarrhea, especially following recent travel to or residence in a foreign country. Eosinophilia is common in helminth infections, particularly where the infecting organism has a tissue phase in its life cycle, such as the intestinal roundworms (Ascaris lumbricoides, hookworms, and Strongyloides sterco-

6

Garcia et al.

Table 5.

Microsporidia:

CUMITECH recommended

Technique Light microscopy Stool Modified trichrome Giemsa Optical brightening agents IF technique Other bodily fluids Modified trichrome Giemsa Optical brightening agents IF technique Routine histology Hematoxylin and eosin PAS Modified Gram stains (Brown Brenn, Brown-Hopps) Giemsa War-thin-Starry Modified trichrome IF technique

diagnostic

techniques”

Useb

Comments

++ ++ ++

Reliable and readily available; light infections difficult to identify Not recommended for routine use; hard to read Calcofluor, Fungifluor, and Unitex 2B available; sensitive but nonspecific Commercial availability limits use; products in development

++ + f+ ++

Reliable, available; light infections difficult to identify Use with urine, conjunctival swab, BAL, CSF, duodenal aspirate Calcofluor, Fungifluor, Unitex 2B available; sensitive but nonspecific Commercial availability limits use; products in development

+ + ++

Sensitivity uncertain with low parasite numbers Controversy over effectiveness Sensitive; generally recommended

+ + ++ ++

Sensitivity uncertain with low parasite numbers Not standardized; may not be necessary Reliable and sensitive Commercial availability limits use; products in development

a Adapted from reference 25. IF, lmmunofluorescence; PAS, periodic acid-Schiff stain; BAL, bronchoalveolar specimen. b- not available or recommended for routine use; f, reported use; ++, techniques in general use.

ralis), whose larvae migrate through the lungs during development. Eosinophilia may be seen before worms have matured, when diagnostic stages are not yet present in the feces (e.g., during the prepatent period). Wheezing may occur in some patients when nematodes migrate through the lungs, and fleeting infiltrates may be seen on chest radiographs. Also, during Table 6.

Risk factors

for acquisition

CSF, cerebrospinal

fluid

of cryptosporidiosisa Example(s)

syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .AIDS, other acquired or congenital immunodepression (e.g., boys with mutation of the CD154 gene or congenital X-linked immunodeficiency with hyper-IgM), immunosuppression, malnutrition

Zoonotic contact ..,..........*.......,.,..,........................,.........,.................... Occupational

lavage specimen;

hyperinfection with S. stercoralis, meningitis and polymicrobial bacteremia may occur. Diarrhea due to protozoan infections is usually caused by rapid proliferation of one of the following: E. histolytica, Dientamoeba fragilis, G. lamblia, C. parvum, C. cayetanensis, Isospora belli, or microsporidia. However, diarrhea may occasionally be mani-

Risk factor lmmunodeficiency

30A

exposure ,.....*.......................*......,..*..,............*..........*.*..

Leisure activities

such as camping, backpacking,

Veterinary, agricultural, day care centers

farm visits

nursing, medical, laboratory

Poor sanitary conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..~................... Drinking water and food, inadequate inadequate insect control

settings;

sewage or waste disposal,

Exposure to untreated water (surface or recreational). . . . . . . . . . . . . . . . . . . . . . . . Leisure activities Exposure to inadequately

Consumption

treated water supply . . . . . . . . . . . . . . . . . ..*...a.......... Inadequate treatment or breakdown in treatment process, resistance to chlorine, filtration inadequate for oocyst removal

of raw foods . . . . . . . . . . . . . . . . . ..*............*.*.*..................*.*........

Unpasteurized

milk, raw meat

Travel ..,,,....,.,..,..,,,..,..,,......,.,..........*......,...............*.............................

Travel from developed rural areas

Age (infants, young children) .,...............................,.,,..........................

Weaning, teething, infection control

Contact with case of diarrhea . . . . . . . . . . . . . . ..*.....................................*......*

Day care personnel,

a Adapted from reference

25.

to underdeveloped

countries

or urban to

pica, finger sucking, use of diapers; poor parents or household

members

CUMITECH

30A

Table 7. Factors cryptosporidiosisa

Parasitic related

potential

outbreaks

of

Factors Water related Abnormal weather conditions (heavy rainfall), increased load on water treatment facilities High turbidity of surface water, increased challenge for water treatment facilities Breakdown in integrity of groundwater aquifers (prolonged rain after drought) Suboptimal water treatment functions Problems with finished water distribution Not water related Sufficient no. of parasites (viability, infectivity, virulence) Sufficient no. of susceptible individuals for ingestion of oocysts leading to primary infection Sufficient no. of susceptible individuals for whom contact with primary case leads to secondary transmission Inadequate epidemiological and laboratory surveillance and reporting ‘Adapted

from

reference

25.

fest in patients with helminth infections with very heavy worm burdens, particularly with S. stercoralis, Trichwis trichiura, Trichinella spiralis, or Hymenolepis nana (32). The diarrhea may be either secretory or inflammatory. Secretory diarrhea is commonly caused by G. lamblia, C. parvum, or I. belli, and these patients often have watery diarrhea and evidence of malabsorption with fatty stools, particularly if they are immunocompromised. Inflammatory diarrhea is characterized by the presence of mucus and pus, and perhaps blood, in the stool. Amebiasis, balantidiasis, and severe strongyloidiasis are parasitic infections, which can cause inflammatory diarrhea characterized as dysentery, although inflammatory cells are often absent from stool in cases of amebiasis. Abdominal discomfort is often a presenting manifestation of parasitic infection. The discomfort can be mild or severe and can be constant or intermittent. Upper abdominal and epigastric pain suggests involvement of the stomach, the duodenum, the upper jejenum, or the biliary tract. Pain from these areas is referred to the upper abdominal area. Pain is commonly seen in patients with giardiasis and may lead the clinician to suspect a gastric or duodenal ulcer or gall bladder disease. Infections involving the lower small intestine usually cause pain in the middle of the abdomen, especially in the periumbilical area, and disease of the colon usually causes pain in the lower abdominal area. Inflammation in the rectal area may present as an urge to defecate, which is known as tenesmus. Ordering

Parasite

Examinations

Parasitic examinations are frequently requested to screen persons such as Peace Corps workers and missionaries returning from visits to areas where parasitic

Infections

of the

Gastrointestinal

Tract

infections are endemic, who are likely to have acquired such infections (Table 8). It is important to remember that the infection may be in the incubation or prepatent stage when the patient returns to the United States and that it may be weeks or months before diagnostic stages can be found in the feces. For example, if a patient acquired schistosomiasis shortly before returning to the United States it would be several months before eggs svould be present in the stool. If the infection was mild, these eggs may be difficult to detect. A person who has recently acquired ascariasis may present with eosinophilic pneumonia as a result of migration of A. lumbricoides larvae through the lung; however, eggs would not be detectable in the stool for a number of weeks. Another reason for requesting parasitic examination is during an outbreak situation such as in a day care center, where giardiasis, cryptosporidiosis, dientamebiasis, or enterobiasis may rapidly spread. It may be necessary to examine all of the children attending the center and perhaps family members to detect asymptomatic carriers who continue to spread the infection. Special laboratory techniques are indicated under such circumstances. Evaluation of therapy is another reason for performing parasitic examinations. Therapeutic failures occur frequently with giardiasis and certain helminth infections, particularly strongyloidiasis (33), and unless a follow-up examination is performed or the patient develops recurrent symptomatic disease, persistent infection may not be recognized. Even in this era of sophisticated laboratory technology, morphological identification of parasite life cycle stages is still required to diagnose most parasitic infections. Antigen detection methods are commercially available for several parasites (Table 9) and antigen detection or molecular biology methods have been described for others, but not all are available for routine diagnosis. Serologic tests have definite but limited lusefulness, especially with hepatic amebiasis, strongyloidiasis, trichinosis, and schistosomiasis, in which diagnostic stages of the parasite are not usually found in feces or are difficult to detect. In the years ahead, when antigen detection and molecular biological techniques are more cost effective, they will be more widely applied.

FACTORS INFLUENCING PERFORMANCE Laboratory

TEST

Capabilities

It is important that laboratories asitology provide accurate and clinicians in the management accomplish this, laboratorians recovering and identifying the

doing diagnostic partimely results to assist of their patients. To must be proficient in presumed pathogens.

8

Garcia et al.

Table 8.

Approaches

CUMITECH to stool parasitology:

Clinical history and/or setting

ordering

30A

tests Test ordereda

Follow-up

testing

Diarrhea and AIDS or other cause of immune deficiency Potential waterborne outbreak (municipal water supply)

Cryp tosporidium or GiardiaCryp tosporidium mmunoassay

If immunoassays are negative and symptoms continue, perform special tests for microsporidia (modified trichrome stain) and other coccidia (modified acid-fast stain) and O&P examination

Diarrhea (nursery school, day care center, camper, backpacker) Diarrhea and potential waterborne outbreak (resort setting) Presence in area of the United States where Giardia is most common organism found

Giardia or Giardia-Cryp tosporidium immunoassay

If immunoassays are negative and symptoms continue, perform special tests for microsporidia and other coccidia (see above) and O&P examination

Diarrhea and relevant travel history Diarrhea in a past or present resident of developing country Resident of area of the United States where parasites other than Giardia are found

O&P exam, E. histolytica-E. dispar immunoassay; immunoassay for confirmation of E. histolytica; various tests for Strongyloides may be relevant (eosinophilia)

If examination is negative and symptoms continue, perform special tests for coccidia and microsporidia

Unexplained

Consider agar pla te culture for S. s tercoralis

If test is negative and symptoms continue, perform O&P examination and special tests for microsporidia and other coccidia; consider serologic testing for helminths

Test for C. cayetanensis acid-fast stains)

If test is negative and symptoms continue, perform special procedures for microsporidia and other coccidia and O&P examination

eosinophilia

Diarrhea (suspected outbreak)

foodborne

(modified

a Depending on the particular immunoassay kit used, various single or multiple organisms may be included. Selection of a particular kit depends on many variables, including clinical relevance, cost, ease of performance, training, personnel availability, number of test orders, training of physician clients, sensitivity, specificity, equipment, and time to result. Very few laboratories will handle this type of testing exactly the same. Many options are clinically relevant and acceptable for good patient care. It is critical that the laboratory report indicate specifically which organisms could be identified with the kit; a negative report should list the organisms relevant to that particular kit. Adapted from reference 25.

Proficiency testing programs of organizations such as the College of American Pathologists (CAP) and the American Association of Bioanalysts have consistently shown that a significant number of laboratories fail to detect or incorrectly identify parasites in test specimens. It is also recognized that the survey results represent the best work of which the laboratory is capable and that routine patient specimens may be less accurately examined than proficiency testing survey specimens. Pseudo-outbreaks of infections like amebiasis have been caused by laboratory personnel who misidentified inflammatory cells as E. histolytica cysts or trophozoites (2.9. Conversely, outbreaks of amebiasis were missed because laboratory personnel did not detect amebae that were present in patient specimens. The experience of the person performing the microscopic examination is the single most important factor in the laboratory examination of fecal specimens for parasites. This individual should be well trained in diagnostic parasitology, should be adept at using the microscope, should have good visual recognition skills, and must routinely see positive specimens either from patients or from proficiency testing

or teaching materials to maintain these skills. Continuing education courses and access to appropriate texts and atlases can also assist individuals performing diagnostic parasitology. Laboratories with personnel who are unfamiliar with certain parasites or who have questions about the identification of parasites should refer these specimens to a reference laboratory for examination by more-experienced individuals. The ability of a laboratory to detect and identify parasites in fecal specimens also depends on the quality of the specimen, parasite burden, time since exposure, methods used for processing the specimen, thoroughness of the examination performed, and competence of the examiner. Common errors include failure to detect a parasite that is present, incorrect identification of a parasite, or misidentification of artifacts (such as pollen grains, spores, yeasts, plant cells, and plant hairs) as parasites (61). Laboratory examination of fecal specimens for parasites is expensive, primarily because of the technical time required. If a parasite examination by a laboratory is inexpensive, the clinician should question whether an adequate examination is being performed (i.e., as the

CUMITECH Table 9.

Parasitic

30A Summary

of commercially

Organism and kit name(sP C. parvum ProSpecT

available

Manufacturer and/ or distributor

kits for immunodetection Type of test

Infections

of the Gastrointestinal

of parasitic

Sensitivity (%F

organisms

Specificity (%Y

Tract

or antigen@ Comments Different EIA formats; contact company for additional information

Alexon-Trend

EIA EIA EIA EIA

94-l 00 93-95 76-93 99

98-l 00 98-l 00 93-l 00 99+

Diagnostics

DFA

100

99

Premier EIA Crypto-CELISA Crypto’Giardia-Cel Cryptosporidium

Meridian Diagnostics Cellabs Cellabs Novocastra

EIA EIA DFA DFA

100 89-99 100

Rim Cryptosporidium Triage Parasite Panel Combination with Giardia lamblia and Entamoeba his tolytica/E. dispar group ColorPAC Giardia/Cryp tosporidium Rapid Assay ImmunoCard STATI Cryptosporidium/Giardia

Remel Biosite Diagnostics, Inc.

EIA Cartridge device, EIA

97-l 00 98 (Gi) 100 (Ent) 92 Kp)

Genzyme/BectonDickinson

Cartridge device, IA

97-l 00 (Cp) 100 (Gi)

Meridian

Cartridge device, IA

98.8 (Cp) 93.5 (Gi)

TechLab

EIA

89-99

TechLab

EIA

93

97

Requires fresh or frozen stool; reagents will differentiate between E. histolytica and E. dispar

TechLab

EIA

93

97

E. his tolytica/E. dispar

Wampole

EIA

93

97

ProSpectT Assay

Alexon-TrendSeradyn-Remel

EIA

87

99

Will not differentiate E. histolytica from E. dispar; requires fresh or frozen stool Will not differentiate E. histolytica from E. dispar, requires fresh or frozen stool. Will not differentiate E. histolytica from E. dispar; requires fresh or frozen stool.

Cellabs

EIA

93

97

Microplate Assay Rapid Assay Color Vue Combination with Giardia lamblia MeriFluor Combination with Giardia lamblia

TechLab Cryp tosporidium

Meridian

Diagnostics

99 93-98 100 Contact manufacturer 98-l 00 98 (Gi) 99 (Ent) >99 Kp)

100 Kp) >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.

CUMITECH

30A

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

CUMITECH

30A

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

16

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 <2 mg

18

Garcia et al.

results in too thin a suspension, thus decreasing the chances of finding organisms. If present, blood or mucus should always be examined as a direct mount. For examination of the direct wet mount, the entire 22- by 22-mm coverslip should be systematically reviewed with the low-power objective (10X) and low light intensity; any suspicious objects may then be examined with the high dry objective (40x). Use of an oil immersion objective (100X) on mounts of this kind is not routinely recommended. Many workers think that the use of the oil immersion objective on this type of preparation is impractical, especially since morphological detail is more readily seen by oilimmersion examination of the permanent stained smear. This is particularly true in a busy clinical laboratory situation. The direct wet mount is used primarily to detect motile protozoan trophozoites. These organisms are very pale and transparent, two characteristics that require the use of low light intensity. Protozoan organisms in a saline preparation usually appear as refractile objects. If suspicious objects are seen on high dry power, allow at least 15 s to detect motility of slowly moving protozoa. Heat applied by placing a hot penny on the edge of a slide may enhance the motility of trophozoites. Tapping on the coverslip can also stimulate the fluid to move; objects roll over, thus providing a better view of the parasite or artifact. Helminth eggs and/or larvae, protozoan cysts, and coccidian oocysts may also be seen on the wet film, although these forms are more often detected after fecal concentration procedures. After the wet preparation has been thoroughly checked for motile amebae, a drop of iodine can be placed at the edge of the coverslip or a new wet mount can be prepared with iodine alone. A weak iodine solution is recommended; too strong a solution may obscure the organisms. Several types of iodine are available; Lugol’s and D’Antoni’s are recommended. Gram’s iodine, used in bacterial work, is not recommended for staining parasitic organisms. If preserved specimens are submitted to the laboratory, it is more clinically relevant and cost effective to delete the direct smear and begin the stool examination with the concentration procedure, particularly since motile protozoa are not viable because of the prior addition of preservative. Even if parasites are seen on a direct mount of preserved stool, they would almost certainly be seen on the concentration examination as well as on the permanent stained smear (protozoa in particular). The elimination of the direct wet mount on a fixed stool is also consistent with the CAP current checklist. A review of the direct wet preparation follows. Principle: To assess worm burden of patient, to provide quick diagnosis of heavily infected specimen,

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to check protozoan organism motility, and to diagnose organisms that may not be seen from concentration or permanent stain methods (rare, small flagellate trophozoites that may be seen as motile organisms but probably do not stain that well with the permanent stained smear). Specimen: Any fresh stool specimen that has not been refrigerated. Reagents: 0.85% NaCl; Lugol’s or D’Antoni’s iodine. Nair’s methylene blue can be used for trophozoites (not commonly used). Examination: Low-power examination ( X 100) of entire 22- by 22mm coverslip preparation (both saline and iodine); high dry power examination ( x400) of at least one-third of the coverslip area (both saline and iodine). Results and laboratory reports: Results from the direct smear examination should often be considered presumptive; however, some organisms can be definitively identified (G. lamblid and E. coli cysts, helminth eggs and larvae and I. belli oocysts). These reports should be categorized as preliminary, while the final report would be available after the results of the concentration and permanent stained smear. Procedure notes and limitations: Once iodine is added to the preparation, the organisms are ki lled and motility is lost. Specimens that arrive in the laboratory already preserved do not require a direct smear examination; proceed to the concentration and permanent stained smear. Direct smears are normally examined at low power ( X 100) and high dry power (x400); oil immersion examination ( X 1,000) is not recommended (organism morphology is not that clear). Concentration

Procedures

There are two broad categories of concentration procedures: flotation procedures, in which parasite cysts or eggs float to the surface of a solution of high specific gravity, and sedimentation procedures in which the parasitic elements sink to the bottom b y centrifu .gation. Concentration techniques vary in their effectiveness, and results may differ depending on the organisms present in the specimen. All concentrations minimize the amount of fecal debris and provide a cleaner background for the microscopic examination. Flotation Techniques Flotation techniques generally use zinc sulfate, although brine and sugar solutions have also been used. However, if the flotation method is used for routine clinical laboratory work, zinc sulfate is the solution of choice. One advantage of flotation techniques is that they provide a background free of most debris, facilitating coverslip placement and providing relatively clean microscopic fields. With unfixed specimens, flo-

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tation techniques using zinc sulfate (specific gravity, 1.18) have the disadvantages that helminth egg opercula pop, cysts and larvae rapidly collapse, and some organism forms (e.g., schistosome and other large helminth eggs) do not concentrate because the specific gravity is too low. Formalin fixation of specimens prior to flotation inhibits most popping of opercula and collapse of cysts and larvae (10). If zinc sulfate flotation is used for formalin-fixed material, the specific gravity should be increased to 1.20 rather than the 1.18 recommended for unfixed specimens. Sedimentation Techniques Sedimentation techniques vary from simple gravity sedimentation to centrifugation. Although gravity is widely used i n underdeveloped areas where centrifugation is not possible, centrifugation techniques are more rapid and provide better organism recovery. When centrifugation is performed, it is important that it be at least 500 X g for 10 min, because some stages of the recently recognized small organisms, such as oocysts of C. paruum and C. cayetanensis and spores of microsporidia, do not concentrate well with slower or shorter centrifugation. The most-common sedimentation technique uses formalin-ethyl acetate. In the past, the formalin-ether technique was widely employed, but because of the hazards of using ether, ethyl acetate is now used as a substitute. Ethyl acetate still has some flammability potential; Hemo-De (Medical Industries, Los Angeles, Calif.) and other related reagents are effective substitutes (2). In the analysis of watery specimens or those containing a lot of fat and/or mucus, centrifugation is preferred but without the ethyl acetate step. This step is a normal part of the formalin-ethyl acetate sedimentation method; however, when used with specimens containing mucus or those that are watery in consistency, specimen material may be pulled into the ring of debris for disposal at the interface between the formalin and the ethyl acetate, thus removing some of the parasites that would allow confirmation of the etiologic agent. Simple centrifugation without the use of ethyl acetate is recommended for these types of specimens: the speed and time should remain the same (500 X g for 10 min). The recovery of trophozoites by routine concentration methods often depends on the original fixative used; trophozoites are occasionally seen in concentrate sediment in specimens originally preserved in SAF fixative. However, trophozoites are generally not seen in concentrate sediment preparations. Although some workers recommend using both the flotation and sedimentation procedures, this approach is impractical for the majority of laboratories. If one technique is selected for routine use, the sedimentation procedure is recommended as the easiest to

Parasitic

Infections

of the Gastrointestinal

Tract

19

perform and the least subject to technical error. A summary of the concentration procedure follows. Principle: To concentrate the parasites present, through either sedimentation or flotation. The concentration is specifically designed to allow recovery of protozoan cysts, coccidian oocysts, microsporidian spores, and helminth eggs and larvae. The most common method is the sedimentation concentration (500 X g for 10 min). Specimen: Any stool specimen that is fresh or preserved in formalin, PVA (mercury or non-mercury based), SAF, merthiolate-iodine-formalin (MIF), or the newer single-vial system fixatives. Reagents: Either 5 or 10% formalin, ethyl acetate, zinc sulfate (specific gravity, 1.18 for fresh stool or 1.20 for preserved stool), 0.85% NaCl, or Lugol’s or D’Antoni’s iodine. Examination: Low-power examination ( X 100) of entire 22- by 22-mm coverslip preparation (iodine recommended, but optional); high dry power examination ( X400) of at least one-th ird of the coverslip area (both saline and iodine). Results and laboratory reports: Results from the concentration examination should often be considered presumptive; however, some organisms can be definitively identified (G. lamblia and E. coli cysts, helminth eggs and larvae, and I. belli oocysts). These reports should be categorized as preliminary, while the final report would be available after the results of the concentration and permanent stained smear. Procedure notes and limitations: Formalin-ethyl acetate sedimentation concentration is the most commonly used. Zinc sulfate flotation does not detect operculated or heavy eggs; by the flotation method, both the surface film and sediment need to be examined before a negative result can be reported. Smears prepared from concentrated stool are normally examined at low power ( x 100) and high dry power ( X 400); oil immersion examination ( X 1,000) is not recommended (organism morphology is not that clear). The addition of too much iodine may obscure helminth eggs (i.e., will mimic debris). Permanent Flagellates

Stains for Amebae,

Ciliates,

and

The detection and correct identification of many intestinal protozoa frequently depend on the examination of the permanent stained smear with the oil immersion lens (100X objective). These slides not only provide the microscopist with a permanent record of protozoan organisms identified but also may be used for consultations with specialists when unusual morphological characteristics are found. Considering the morphological variations that are possi-

20

Garcia et al.

ble, organisms may be found that are very difficult to identify and do not fit the pattern for any one species. Although an experienced microscopist can occasionally identify certain organisms on a wet preparation, most identifications should be considered tentative until confirmed by the permanent stained slide. The smaller protozoan organisms are frequently seen on the stained smear when they are easily missed by only the direct smear and concentration methods. For these reasons, the permanent stain is recommended for every stool sample submitted for a routine parasite examination (O&P). There are a number of staining techniques available; selection of a particular method depends on the degree of difficulty of the procedure and the amount of time necessary to complete the stain. The older classical method is the long Heidenhain iron-hematoxylin method; however, for routine diagnostic work, most laboratories select one of the shorter procedures, such as the Wheatley’s modification of Gomori’s trichrome method or one of the modified methods using ironhematoxylin. Most problems encountered in the staining of protozoan trophozoites and cysts in fecal smears occur because the specimen is too old, the smears are too dense, the smears are allowed to dry before fixation, or fixation is inadequate. There is variability in fixation in that immature cysts fix more easily than mature cysts, and E. coli cysts require a longer fixation time than do those of other species.

Trichrome Stain The single most important step in the preparation of a well-stained fecal smear is good fixation. If fixation is inad .equate, the protozoa may be distorted or shrunk, may not be stained, or may exhibit an overall pink or red- color with poor internal morphology. Slides should always be drained between solutions. Touch the end of the slide to a paper towel for 2 s to remove excess fluid before proceeding to the next step. This also maintains the integrity of the staining solutions for a longer period of time. Incomplete removal of mercuric chloride (Schaudinn’s fixative and PVA) may cause the smear to contain highly refractive crystals or granules that prevent finding or identifying any organisms present. Since the 70% ethanol-iodine solution removes the mercury complex, it should be changed at least weekly to maintain its strong tea color. A few minutes are usually sufficient to keep the slides in the iodinealcohol; too long a time in this solution may also adversely affect the staining of the organisms. When using non-mercury-based fixatives, the iodine-alcohol step (used for the removal of mercury) and the subsequent alcohol rinse can be eliminated from the procedure. The smears for staining can be prerinsed with

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70% alcohol and then placed in the trichrome stain, or they can be placed directly into the trichrome stain as the first step in the staining protocol. The prerinse step in 70% alcohol is not mandatory but may be included if the slide appears thick. Smears that are predominantly green may be due to the inadequate removal of iodine by the 70% ethanol. Lengthening the time of these steps or more frequent changing of the 70% ethanol helps. To restore weakened trichrome stain, remove the cap and allow the ethanol to evaporate (carried over on the staining rack from a previous ethanol-containing dish). After a few hours, fresh stock stain may be added to restore lost volume. Older, more-concentrated stain produces more-intense colors and requires slightly longer destaining times (an extra dip). Remember that PVA smears usually require a slightly longer staining time (8) Although the trichrome stain is used essentially as a progressive stain (that is, no destaining is necessary), best results are obtained by using the stain regressively (destaining the smears briefly in acidified alcohol). Good differentiation is obtained by destaining for a very short time (two dips only, approximately 2 to 3 s); prolonged destaining results in poor differentiation. It is essential to rinse the smears free of acid to prevent continued destaining. Since 90% alcohol continues to leach trichrome stain from the smears, it is recommended that after the acid alcohol is used, the slides are quickly rinsed in 100% alcohol and then dehydrated through two additional changes of 100% alcohol. In the final stages of dehydration, the 100% ethanol and the xylenes (or xylene substitute) should be kept as free from water as possible. Coplin jars must have tight-fitting caps to prevent both evaporation of reagents and absorption of moisture. If the xylene becomes cloudy after addition of slides from the 100% ethanol, return the slides to fresh 100% ethanol and replace the xylene with fresh stock. If the smears peel or flake off, the specimen may have been inadequately dried on the slide (in the case of PVA-fixed specimens), the smear may have been too thick, or the slide may have been greasy (e.g., from fingerprints). However, slides generally do not have to be cleaned with alcohol prior to use. If the stain, upon examination, appears unsatisfactory and it is not possible to obtain another slide to stain, the slide may be restained. Place the slide in xylene to remove the coverslip and reverse the dehydration steps, adding 50% ethanol as the last step. Destain the slide in 10% acetic acid for several hours; wash it thoroughly first in water and then in 50 and 70% ethanol. Place the slide in the trichrome stain for 8 min and complete the staining procedure.

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Parasitic

Infections

of the Gastrointestinal

Tract

21

Iron-Hematoxylin Stain The iron-hematoxylin stain is one of a number of stains used to make a permanent stained slide for detecting parasitic organisms. Iron-hematoxylin was the stain used for most of the original morphological descriptions of intestinal protozoa found in humans. With oil immersion power (magnification of X l,OOO), one can examine the diagnostic features used to identify the protozoan parasite. Although there are many modifications of iron hematoxylin techniques, two methods that are commonly used are the SpencerMonroe and Tompkins-Miller procedures. Both methods can be used with either fresh, SAF-preserved, or PVA-preserved specimens. Specimens preserved in many of the single-vial systems can also be stained by iron-hematoxylin methods. Optimal morphological detail is achieved when SAF-fixed specimens are stained with iron-hematoxylin rather than with trichrome. A procedure combining the iron-hematoxylin and modified acid-fast stains on SAF-fixed specimens, which obviates the need for performing two stains and examining two slides, has been described (J. Palmer, Letter, Clin. Microbial. Newsl. 13:39-40, 1991). The specimen usually consists of fresh stool smeared on a microscope slide that is immediately fixed in Schaudinn’s fixative, PVA-preserved stool smeared on a slide and allowed to air dry, or SAFpreserved stool smeared on an albumin-coated slide and allowed to air dry. Many of the comments related to trichrome staining are also relevant for the ironhematoxylin methods. When performing any type of permanent stain, appropriate control slides should be used according to CAP guidelines. While it is best to use slides that contain protozoa, slides containing leukocytes may be substituted if others are not available. A review of the permanent stain methods follows.

The worldwide human immunodeficiency virus epidemic was heralded by the appearance of opportunistic infections, among which were cryptosporidiosis and microsporidiosis. With the addition of C. cayetanensis, there are now multiple parasites that require specific diagnostic methods to confirm their presence in the human host.

Principle: To provide contrasting colors for both the background debris and parasites present; designed to allow examination and recognition of detailed organism morphology under oil immersion examination (100X objective for a total magnification of X 1,000). The slides can be screened using a 50 X or 60x oil immersion objective; however, the final examination should be performed using the 100 X oil immersion objective. Primarily designed to allow recovery and identification of the intestinal protozoa. Specimen: Any stool specimen that is fresh or preserved in formalin, PVA (mercury- or non-mercury-based), SAF, MIF, or the newer single-vial systern fixatives. Reagents: Trichrome, iron-hematoxylin, and their associated solutions and dehydrating solutions (alcohols and xylenes). Mounting fluid is optional.

Modified Acid-Fast Stains for Coccidia C. parvum and I. belli have been recognized as causes of severe diarrhea in immunocompromised hosts, but they can also cause diarrhea in immunocompetent hosts. Oocysts in clinical specimens may be difficult to detect without special staining. Modified acid-fast stains are recommended to demonstrate these organisms. Unlike the Ziehl-Neelsen stain, the modified acid-fast stain does not require the heating of reagents for staining. With additional reports of diarrhea1 outbreaks with Cyclospora, it is also important to remember that these organisms are acid fast and can be identified by this staining approach. Although the microsporidial spores are also acid fast, their size (1 to 2 pm) makes identification very difficult without special stains or the use of monoclonal antibody reagents. Concentrated sediment of fresh or formalin-preserved stool may be used for these stains. Other types

Examination: Oil immersion examination of at least 300 fields; additional fields may be required if suspect organisms are seen in the wet preparations from the concentrated specimen. Results and laboratory reports: The majority of the suspect protozoa and/or human cells can be confirmed with the permanent stained smear. These reports should be categorized as final and would be signed out as such (where the direct wet smear and concentration examination provide preliminary results). Procedure notes and limitations: The most commonly used stains include trichrome and iron-hematoxylin. Unfortunately, helminth eggs and larvae take up too much stain and usually cannot be identified from the permanent stained smear. Permanent stained smears are normally examined under oil immersion ( X l,OOO), and low power or high dry power is not recommended. Confirmation of the intestinal protozoa (both trophozoites and cysts) is the primary purpose of this technique. Note: coccidia and microsporidia will generally not be stained using these methods. Modified acid-fast stains (coccidia) and modified trichrome stains (microsporidia) are recommended.

ORGANISM-SPECIFIC METHODS Modified Acid-Fast, Trichrome Stains

DETECTION

Hot Safranin,

and Modified

22

Garcia et al.

of clinical specimens such as duodenal fluid, bile, and pulmonary sources (induced sputum, bronchial wash, or biopsies) may also be stained with these reagents. No method modifications are required with specimens other than stool. The oocysts of Cryptosporidium and Isospora spp. stain pink to red to deep purple. Some of the four sporozoites may be visible in the Cryptosporidium oocysts. Some of the Isospora immature oocysts (the entire oocyst) stain, while those that are mature usually appear with the two sporocysts within the oocyst wall stained a pink to purple color and a clear area between the stained sporocysts and the oocyst wall. The background stains blue. If Cyclospora oocysts are present (which is uncommon), they are approximately 10 pm in diameter, resemble C. paruum but are larger, and have no definite internal morphology; the acid-fast staining is more variable than that seen with Cryptosporidium or Zsospora spp. There is usually a range of color intensity seen in the organisms present; not every oocyst appears deep pink to purple. The most staining variation is seen with Cyclospora. If the patient has a heavy infection with microsporidia (e.g., an immunocompromised patient), small (l- to 2-pm) spores may be seen but not be recognized as anything other than bacteria or small yeast cells. A summary of modified acid-fast stains follows (25,41). Principle: To provide contrasting colors for both the background debris and parasites present; designed to allow examination and recognition of acid-fast characteristic of the organisms under high dry examination (40x lens objective for a total magnification of x400). Primarily designed to allow recovery and identification of intestinal coccidian oocysts. Internal morphology (sporozoites) will be seen in some Cryptosporidium oocysts under oil immersion (magnification of 1,000 X ). Specimen: Any stool specimen that is fresh or preserved in formalin, SAF, or the newer single-vial system fixatives. It is recommended that the specimen be centrifuged (500 X g for 10 min) and that the concentration sediment be used for staining. Reagents: Kinyoun’s acid-fast stain, modified ZiehlNeelsen stain, and their associated solutions; dehydrating solutions (alcohols and xylenes). Mounting fluid is optional. Remember that the decolorizing agents are less intense than the acid-alcohol used in routine acid-fast staining (this fact is what makes these procedures modified acid-fast procedures). Recommendation: 1% (rather than the original 3% acetic acid); this helps prevent too much color loss with Cyclospora spp. Some users also prefer to use the hot modified acid-fast stain method for more-uniform stain penetration of the Cyclospora oocysts (25). Examination: High dry examination of at least 300

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fields; additional fields may be required if suspect organisms are seen but are not clearly acid fast. R.esults and laboratory reports: The iden tification of Cryptosporidium and Isospora oocysts should be possible; Cyclospora oocysts that are twice the size of Cryptosporidium should be visible but tend to be more acid fast variable. Although microsporidia are acid fast, their small size makes their recognition very difficult. Final laboratory results depend heavily on the appearance of the quality control slides and compari son with patient specimens. Procedure notes and limitations: Both the cold and hot modified acid-fast methods are excellent for the staining of coccidial oocysts. There is some feeling that the hot method results in better stain penetration, but the differences are probably minimal. Procedure limitations are related to specimen handling (proper centrifugation speeds and time and the use of no more than two layers of wet gauze for filtration) and the complete understanding of the difficulties in recognizing microsporidial spores. There is also some controversy about whether or not the organisms lose the ability to take up acidfast stains after long-term storage in 10% formalin. The organisms are more difficult to find in specimens from patients who do not have the typical watery diarrhea (more-formed stool = more artifact material). Hot Safranin There are several hot safranin-staining methods for Cyclospora and the other coccidia. One of the rapid safranin methods uses flame heating with a counterstain of either 1% methylene or 0.1% crystal violet (11). Another option is the modified safranin technique with microwave heating (76). This method uniformly stains Cyclospora oocysts brillia .nt reddish orange and may provide more-un iform staining that that obtained with the modified acid-fast stain. This staining procedure has been reported to be fast, reliable, and easy to perform in most clinical laboratories (76) . Principle: To provide contrasting colors for both the background debris and parasites present; designed to allow examination and recognition of acid-fast characteristic of the organisms under high dry examination (40x lens objective for a total magnification of x400). Primarily designed to allow recovery and identification of intestinal coccidian oocysts. Inte rnal morphology (sporozoites) may be seen in some Cryp tosporidium oocysts under oil immersion (magnification of X 1,000). Specimen: Any stool specimen that is fresh or preserved in formalin, SAF, or the newer single-vial system fixatives. It is recommended that the speci-

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men be centrifuged (500 X g for 10 min) and that the concentration sediment be used for staining. Reagents: Safranin and optional counterstains; mounting fluid optional. Examination: High dry examination of at least 300 fields; additional fields may be required if suspect organisms are seen but do not appear typical. Results and laboratory reports: The identification of Cryptosporidium, Cyclospora, and Isospora oocysts should be possible; Cyclospora oocysts are twice the size of Cryptosporidium. All coccidian oocysts should appear brilliant reddish orange. Final laboratory results depend heavily on the appearance of the quality control slides and comparison with patient specimens. Procedure notes and limitations: The hot safranin methods provide excellent staining of coccidial oocysts (Cryptosporidium, Cyclospora, and Isospora). There is some feeling that this hot method results in more-uniform stain penetration and retention (e.g., in Cyclospora) than that seen with the room temperature-modified acid-fast staining methods. Procedure limitations are related to specimen handling (proper centrifugation speeds and time) and the use of no more than two layers of wet gauze for filtration. The selection of laboratory stains for the coccidia ultimately depends on personal preference of laboratory personnel. Modified Trichrome Stain The diagnosis of intestinal microsporidiosis (Enterocytoxoon bieneusi and Encephalitoxoon intestinalis) has depended on the use of invasive procedures and subsequent examination of biopsy specimens by histologic stains and electron microscopy methods (22, 24,70,77). However, the need for a practical method for the routine clinical laboratory has stimulated some work in the development of additional methods. Slides prepared from fresh or formalin-fixed stool specimens can be stained by a chromotrope-based technique and examined by light microscopy. This staining method is based on the fact that stain penetration of the microsporidial spore is very difficult; thus, the dye content in the chromotrope 2R is higher than that routinely used to prepare Wheatley’s modification of Gomori’s trichrome method, and the staining time is much longer (90 min). Several of these stains are available commercially from a number of suppliers. A number of variations to the modified trichrome (Weber green or Ryan blue) have been attempted, to improve the contrast between the color of the spores and the background staining (22, 70, 76). Optimal staining was achieved by modifying the composition of the trichrome solution. Both the Weber and Ryan formulations of the modified trichrome stain are available commercially from a number of suppliers.

Parasitic

Infections

of the Gastrointestinal

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23

The specimen can be fresh stool or stool preserved in 5 or 10% formalin, SAF, or some of the newer single-vial system fixatives. Actually, any specimen other than tissue thought to contain microsporidia can be stained by this method. The microsporidial spore wall stains pinkish to red; the interior of the spore is clear or perhaps shows a horizontal or diagonal stripe that represents the polar tube. The background appears green or blue, depending on the method. Other bacteria, some yeast cells, and some debris stain pink to red; the shapes and sizes of the various components help differentiate the spores from other structures. The results from this staining procedure should be reported only if the positive control sm.ears are acceptable. Future production of monoclonal antibody reagents should provide a more-specific and more-sensitive approach to the identification of the microsporidia in fecal specimens. A summary of modified trichrome stains follows. Principle: To provide contrasting colors for both the background debris and parasites present; designed to allow examination and recogn .ition of organism morphology under oil immersion (100X lens objective for a total magnification of X 1,000). Primarily designed to allow recovery and identification of microsporidial spores. Internal morphology (horizontal or diagonal stripes) may be seen in some spores under oil immersion (magnification of x 1,000). Specimen: Any stool specimen that is fresh or preserved in formalin or SAF or one of the single-vial system fixatives. Reagents: Modified trichrome stain (using high-dyecontent chromotrope 2R) and associated solutions; dehydrating solutions (alcohols and xylenes); mounting fluid optional. Examination: Oil immersion examination of at least 300 fields using the 100X oil immersion objective; additional fields may be required if suspect organisms are seen but are not clearly identified. Also, confirmation of the infection may require the examination of more than one specimen (over a period of a few weeks). Results and laboratory reports: The identification of microsporidial spores may be possible; however, in light infections their small size makes recognition very difficult. Final laboratory results depend heavily on the appearance of the quality control slides and comparison with patient specimens. Procedure notes and limitations: Because of the difficulty in getting dye to penetrate the spore wall, this staining approach can be very helpful. Procedure limitations are related to specimen handling (proper centrifugation speeds and time), use of no more than two layers of wet gauze for filtration, and a

24

Garcia et al.

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complete understanding of the difficulties in recognizing microsporidial spores due to their small size (1 to 2.5 pm). Important questions for commercial suppliers: Make sure to ask about specific fixatives and whether the fecal material can be stained with the modified trichrome stains or modified acid-fast stains. Also, ask if the fixatives prevent the use of any of the newer immunoassay methods now available for several of the intestinal amebae, flagellates, coccidia, and microsporidia. Chemofluorescent Agents

and Immunofluorescent

Another approach for the diagnosis of microsporidia involves the use of chemofluorescent agents (optical brightening agents) such as Calcofluor, Fungi-Fluor, or Uvitex 2B. These reagents are sensitive but nonspecific; objects other than microsporidial spores also fluoresce. This is a particular problem when examining stool specimens; both false-positive and falsenegative results have been seen (22, 25). When these agents are used with other bodily fluids, particularly urine, the interpretation of results is much easier than when they are used with stool. There is now general agreement that this is a simple, sensitive screening method for the detection of microsporidial spores in stool specimens (22,30,49,74). The newest approach for the identification of spores in clinical specimens uses antisera in an indirect fluorescent antibody (IFA) procedure (4, 57). Fluorescing microsporidial spores are distinguished by a darker cell wall and by internal visualization of the polar tubule as diagonal lines or cross-lines within the cell. In another study using this same antiserum, 9 of 27 patients (30%) already diagnosed with cryptosporidiosis (8 AIDS patients and 1 immunodeficient patient without AIDS) had E. bieneusi in the stool (30). Although there is some crossreactivity with bacteria, this technique offers a moresensitive approach than routine staining methods currently available for the examination of stool specimens. Commercial products are in various stages of development, and clinical testing should provide some additional, more-sensitive methods than are currently available. As clinicians begin to suspect these infections and laboratorians become more familiar with the diagnostic methods, the number of positive patients, particularly those who are immunocompromised, may increase dramatically. Current recommendations also suggest multiple diagnostic methods are necessary to diagnose microsporidiosis, particularly when examining fecal specimens (25). In general, determination of species is beyond the scope of the routine clinical laboratory, although species can be suspected on the basis of the size of the

30A

spores, as E. bieneusi is smaller (1.0 by 1.6 pm) than E. intestinalis (1.3 to 2.1 by 2.5 to 3.3 pm). Fecal lmmunoassays In addition to special staining methods for the intestinal coccidia and the microsporidia, also fecal immunoassays are commercially available for the detection of C. pawurn, G. lamblia, the E. histolytica/E. dispar group, and E. histolytica. Additional immunoassays are under development for D. fragilis and the microsporidia. Progress has been made in the development and application of molecular methods for di agnostic purposes, including the use of purified or recombinant antigens and nucleic acid probes. The detection of parasite-specific antigen is more indicati .ve of current disease. Many of the original assays were dev,eloped with polyclonal antib odies that were targeted to unpurified antigens, markedly decreasing the sensitivity and specificity of the tests. Immunoassays are generally simple to perform and al.low a large n umber of tests to be car ried out at one time, thereby reducing overall costs. A major disadvantage of antigen detection in stool specimens is that the method detects only one or two pathogens at one time. One still must perform a routine O&P examination to detect other parasitic pathogens if indicated. Currently available immunoassays have sensitivity and specificity percentages of more than 90% when used according to the ma nufacturer’s .nstructions for speci men handling and preservation (some require fresh. or frozen stools only). Some combination reagents are available for the detection and differen tiation of G. lamblia and C. paruum. Reagents currently available can be seen in Table 9 (7,26-29). As previously stated, differentiation of E. histolytica and E. dispar is not possible on morphological grounds unless ingestion of RBCs by trophozoites is recognized on the perma nent stained smear (e.g., by the trichrome or iron-hematoxylin methods) (28,59, 67). Reagents are available for the detection of the E. histolytica/E. dispar group, as well as for E. histolytica alone (1, 34-36). Results compare favorably with those obtained by established soenzyme methods or molecular biology techniques However, while formalinized specimens can be used with the available antigen detection kits for Giardia and Cryptosporidium, the reagents for E . histolytica/E. dispar or E. histolytica are limited to testing fresh and/or frozen fecal specimens. Since the laboratory is unable to determine whether the organism is E. histolytica or E. dispar on the basis of morphology (i.e., the absence of trophozoites with ingested RBCs), the report should state the presence of E. histolytica-E. dispar. An additional comment can be added to the report indicating that if further identification of the true pathogen

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E. histoEytica is required, a fresh fecal specimen can be forwarded to a reference laboratory. The development of stool immunoassays for the specific identification of G. Lz&&, C. pawurn, the E. histolytica/E. dispar group, and E. histolytica, in addition to the routine O&P examination and special stains for coccidia and microsporidia, provide very specific options for patient testing. The selection of any of these testing options depends on patient history and physical findings. Each assay should be available as a separate orderable and billable test, or they can be arranged in logical batteries addressing specific clinical situations. Specific ordering options can be seen in Table 8. Gene Probes Nucleic acid-based diagnostic tests for parasitology are primarily available in specialized research or reference centers. PCR and other nucleic acid probe tests have been reported for almost all species of parasites (78). The only nucleic acid-based probe test commercially available is for the detection of Trichomonas uaginalis. As with immunoassay techniques, cost is an issue, as separate probes are required for each parasite. As the costs of these tests decrease and the various steps necessary to perform them are automated, there will be increasing demand for commercially available reagents.

OTHER FECAL EXAMINATION Stains for Inflammatory

METHODS

Cells

Occasionally, clinicians request examination for inflammatory cells in stool (fecal leukocytes). In these cases, permanent stained smears reveal these cells (by trichrome or iron-hematoxylin staining). In some cases, Wright’s, Giemsa, or even Gram’s stain may suffice. However, successful staining of these cells very much depends on the fixative-stain combination used for the clinical specimen. If numerous inflammatory cells (e.g., neutrophils and macrophages) are present, a diagnosis of inflammatory diarrhea is suggested, whereas a lack of inflammatory cells suggests secretory diarrhea. If inflammatory cells are seen in the of routine permanent microscopic examination stained smears, these cells should be reported and quantitated on the laboratory report. Table 14 can be used for examination of permanent stained smears with the oil immersion lens (100X objective; total magnification of X 1,000). Egg Count and Egg-Hatching

Tests

Egg counts for appraisal of parasite burden are rarely indicated in certain clinical circumstances (for example, anemia in a heavy hookworm infection may be casually related, whereas anemia existing concur-

Infections

Table 14. Quantitation and a rtifacts

of the Gastrointestinal

Tract

25

of parasites,

Quantity

No. present in 10 oil immersion fields (X 1,000 magnification)

Few Moderate Many

2 3-9 10

rently with a light hookworm infection may be from other causes). Egg counts may also be helpful for determining therapeutic efficacy and quantitating residual infection or reinfection. With current therapy, the need for monitoring therapy through egg counts is no longer as relevant. However, there are several methods available that can be used if necessary. Remember that egg counts are estimates; count variations occur regardless of how carefully procedures are followed. If two or more fecal specimens are being compared, it is best to have the same individual perform the technique on both samples and do multiple counts. Egg-hatching tests are a sensitive way of detecting active intestinal or urinary tract schistosomiasis and of determining the viability of schistosome eggs after therapy to eradicate adult worms (25,40, 61). When schistosome eggs are recovered from either urine or stool, they should be carefully examined to determine viability. The presence of living miracidia within the eggs indicates an active infection that may require therapy. The viability of the miracidia can be determined in two ways: (i) the cilia of the flame cells (primitive excretory cells) may be seen on a wet smear by using high dry power and are usually actively moving and (ii) the miracidia may be released from the eggs with the use of a hatching procedure. The eggs usually hatch within several hours when placed in 10 volumes of dechlorinated or spring water (hatching begins rapidly after contact with the water). The eggs that are recovered in the urine (24-h specimen collected with no preservatives) are easily obtained from the sediment and can be exam ined under the microscope to determ ine viabi 1i tY . Culture

Techniques

Nematode Larvae Nematode infections giving rise to larval stages that hatch in soil and penetrate unbroken skin are diagnosed by using certain fecal culture methods to concentrate the larvae. S. stercoralis larvae are generally the most common larvae found in stool specimens. Depending on the fecal transit time through the intestine and the patient’s condition, rhabditiform and, rarely, filariform larvae may be present. Also, if there is delay in examination of the stool, then embryonated ova as well as rhabditiform larvae of hookworm may be present. Culture of feces for larvae is useful to (i)

26

Garcia et al.

reveal their presence when they are too scanty to be detected by concentration methods; (ii) distinguish whether the infection is due to S. stercoralis or hookworm on the basis of rhabditiform larval morphology by allowing hookworm egg hatching to occur, releasing first-stage larvae; and (iii) allow development of larvae into the filariform stage for further differentiation. The use of certain fecal culture methods (sometimes referred to as coproculture) is especially helpful for detection of light infections of hookworm, S. stercoralis, and Trichostrongylus spp. and for specific identification of parasites. The rearing of infectivestage nematode larvae also helps in the specific diagnosis of hookworm and trichostrongyle infections because the eggs of many of these species are identical and specific identifications are based on larval morphology. Additionally, such techniques are useful for obtaining a large number of infective-stage larvae for research purposes. Options include the Harada-Mori filter paper strip, the petri dish filter paper strip, the Baermann funnel, and the agar plate culture for Strongyloides (5, 37,42,43, 72). Detection of subclinical S. stercoralis infection can be difficult, as few larvae are shed at any one time. Identification of this infection is importa .nt, however, because of the risk to such patients if they become immunocompromised for any reason. Agar plate cultures are recommended for the recovery of S. stercoralis larvae and tend to be more sensitive than some of the other diagnostic methods (5, 37). Stool is placed onto agar plates, and the plates are sealed to prevent accidental infections and held for 2 days at room temperature. As the larvae crawl over the agar, they carry bacteria with them, thus creating visible tracks over the agar. The plates are examined under the microscope for confirmation of larvae, the surface of the agar is washed with 10% formalin, and final confirmation of larval identification is made via wet examination of the sediment from the formalin washings. Positive tracking on agar plates has been seen on a number of different types of agar. However, the most appropriate formula contains meat extract, peptone, salt, and agar (25). It is important to remember that more than half of S. stercoralis-infected individuals tend to have lowlevel infections. The agar plate method is a moresensitive method than the usual direct smear or fecal concentration method (42, 43). A daily search for furrows on agar plates for up to 6 consecutive days results in increased sensitivity of diagnosis of both S. stercoralis and hookworm infections. Also, a careful search for S. stercoralis should be made in all patients with comparable clinical findings before deciding on a diagnosis of idiopathic eosinophilic colitis, because consequent steroid treatment may have a fatal out-

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come by inducing parasite (20).

widespread dissemination

30A

of the

Protozoa Very few clinical laboratories offer specific culture techniques for parasites. The methods for in vitro culture are often complex, while quality control is difficult and not really feasible for the routine diagnostic labo ratory. In certain institutions, some techniques may be available, particularly where consultative services are provided (e.g. in a reference laboratory situation) and for research purposes. However, most laboratories do not offer these techniques. Clinical relevance, cost considera tion, and lack of experience in protozoa1 culture techniques preclude their widespread use. Few parasites can be routinely cultured, and the only procedures that are in general use are for E. histolytica, Naegleria fowleri, Acanthamoeba spp., T. vaginalis, Toxoplasma gondii, Trypanosoma cruzi, and the leishmanias. Culture of E. histolytica is essential to determine if the isoenzyme profile (zymodeme) indicates if the isolate is the pathogenic E. histolytica or the nonpathogenic E. dispar. Procedures for the culture of intestinal protozoa are usually available only after consultation with the laboratory and on special request. For those interested in trying these techniques, several different media are available. More-extensive options can be found in the literature (6, 19, 38, 56, 75).

PROCEDURES FOR NONFECAL SPECIMENS Aspirates

and Biopsies

Other bowel specimens may be obtained during endoscopic examination. Aspirates or scrapings from suspicious colonic lesions can be examined for E. histolytica trophozoi tes by preparation of permanent stains. Although wet mo unt examination can be useful in experienced hands, there is often a gap between the time the specimen is collected and the time it arrives in the laboratory for examination. Thus, organism motility may no longer be visible. Biopsy specimens can be examined by routine histopathology, or impression smears can be prepared, fixed, and stained with trichrome or another appropriate permanent stain and examined for amebic trophozoites. Despite repeated negative stool examinations, suspicion of schistosomiasis may elicit a biopsy of rectal mucosa. The tissue may be flattened between two glass slides and examined directly for the presence of schistosome eggs. If eggs are seen, viability can be confirmed by the presence of flame cell activity in the miracidium or movement of the miracidium itself. Examination of duodenal biopsies by histopathol-

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ogy or impression smears should be made with the understanding that a variety of organisms can be seen, including S. stercoralis, G. lamblia, C. parvum, I. belli, and microsporidia. Collection of either aspirated duodenal material or material scraped from a swallowed and retrieved string (Enterotest; Hedeco, Inc.) allows for diagnosis of occult G. lamblia and S. stercoralis infections when a routine fecal examination fails to detect these organisms. Specimens of bile may be examined to detect parasites from the biliary tree, including C. parvum, microsporidia, Clonorchis sinensis, and Fasciola hepatica. Liver aspirates submitted for examination for E. histolytica may be useful; the last portion of the aspirate is most likely to contain the organisms. Although not evaluated or approved for this indication, commercially available antigen detection tests for E. histolytica may improve the sensitivity of aspirate examination. Likewise, immunoassays and gene probe techniques may prove useful in the future. Serologic tests are very useful in diagnosing extraintestinal amebit abscesses and are often positive in patients with invasive intestinal disease. Sputum Sputum examination is usually performed only when infections with Paragonimus spp. exist or when disseminated strongyloidiasis is suspected. Occasionally, migrating larvae of A. lumbricoides or hookworms are found in such specimens, and definitive identification of the larvae may require referral to an expert. Pulmonary cryptosporidiosis or microsporidiosis can also be confirmed with the examination of sputum after special staining with modified acid-fast or modified trichrome stains, respectively. The sputum may also be examined as a direct wet mount, or it can be digested with mucolytic agents and concentrated by centrifugation. Urinary

Tract

Urine examination and bladder biopsy may be useful for diagnosing infection with Schistosoma haematobium, but urine can also contain T. vaginalis, microfilariae, or microsporidian spores. The urine sediment is usually examined when screening for these parasites. The schistosome egg-hatching test on urine sediment can detect light infections and can determine whether the eggs contain viable larvae.

In many instances, serologic methods are more practical than the routine diagnostic methods, especially when invasive procedures must be used to obtain the specimen for diagnostic workup (e.g., biopsies or aspirates). However, even with the most sophisticated technology, few serologic tests for parasitic infections

Parasitic

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27

can confirm an infection or predict the disease outcome. Interpretation of test results may also present problems, particularly when one is dealing with patients from areas where infection is endemic, who may have higher baseline titers than do patients from other areas and in whom a low titer may actually be significant. Some titers may reflect exposure rather than actual disease. Due to problems with sensitivity, specificity, and interpretation, most clinical laboratories do not offer serologic procedures for the diagnosis of parasitic infections. However, standard techniques that are the most widely used include complement fixation (CF), indirect hemagglutination (IHA), IFA, soluble antigen fluorescent antibody, bentonite flocculation, latex agglutination (LA), double diffusion, counterimmunoelectrophoresis, immunoelectrophoresis, radioimmunoassay, and intradermal tests. The CDC offers a number of serologic proced ures for diagnostic purposes, some of which are not available elsewhere. Because regulations as to submission of specimens vary from state to state, each laboratory should check with its own county or state department of public health for appropriate instructions. Serologic tests for intestinal protozoa other than E. histolytica are not widely available. Serology for E. histolytica is quite useful in diagnosing amebic liver abscess and is positive in a majority of patients with invasive intestinal disease caused by this organism (6, 69, 80). It has been suggested that patients suspected of having ulcerative colitis have an amebic serology procedure performed to rule out amebiasis. When organisms that may be E. histolytica or E. dispar are found in fecal specimens of asymptomatic patients, it has been suggested that patients who have a positive serology test for E. histolytica receive therapy for the luminal carrier state. The most used serologic tests for amebiasis are IHA, IFA, CF, enzyme-linked immunosorbent assay (ELISA), LA, counterimmunoelectrophoresis, and immunodiffusion. CF tests have not been widely used because of nonspecific and variable results (68). IHA and LA appear to detect the same antibody. Although the ELISA and IHA are some of the more-sensitive tests, antibody titers persist for years, making interpretation difficult in certain patients. The ELISA and IHA tests are useful for epidemiological surveys. IFA and immunodiffusion tests not only are sensitive tests but also have high-positive predictive values. After successful therapy, titers in many of the patients decrease, which allows physicians to monitor the therapeutic response. Although tests for the detection of IgM antibodies specific for E. histolytica have been reported, their clinical usefulness is questionable because of the lack of sensitivity. Many of the current tests use crude trophozoite extracts, whereas a few use

28

Garcia et al.

recombinant proteins. A major disadvantage for amebit serologic testing is the limited availability of commercial reagents ( 80). Serologic tests for S. stercoralis may be helpful in evaluating patients who have lived in or traveled to areas where parasite infections are endemic and who are symptomatic yet have negative parasite examinations (33). Also, individuals from areas of endemicity who are to be therapeutically immunosuppressed should be carefully evaluated to rule out occult strongyloidiasis. Although not routinely available, the ELISA and immunoblot methods have been evaluated for both the diagnosis and postchemotherapy assessment of human strongyloidiasis (25). These methods appear to be both sensitive and specific and demonstrate significant decrease in antibody levels after therapy. However, in cases where treatment was not effective, patients did not show a significant fall in antibody titers. More-detailed studies to determine appropriate intervals for serologic evaluation and criteria for successful cure are required before these tests are more widely used. Unfortunately, cross-reactions are common among serologic tests for intestinal helminths because of numerous common antigenic epitopes ( SO). Serologic tests are most accurate for patients who are not from areas of endemicity and who have not been infected with a variety of helminths. Many of the current serologic techniques use a sensitive screening method, such as enzyme immunoassay, coupled with a morespecific second assay such as the Western blot method, for specimens that are reactive in the screening test. Other methods use highly purified or recombinant antigens to increase test specificity. These approaches have greatly improved the usefulness of some serologic tests for organisms with complex antigenicity. Serologic tests for invasive amebiasis, strongyloidiasis, and schistosomiasis continue to be the most useful for the diagnosis of enteric parasite infections.

In reporting the results of a parasite examination, it is important that the report accurately reflect which parasites could have been detected by the examinations performed. It is particularly important to ensure that the results of an O&P examination (minimum of concentration and permanent stained smear) indicate that C. parvum, C. cayetanensis, or the microsporidia are normally not detected by this method. One exception for the coccidia would be the use of the ironhematoxylin permanent stain that incorporated the carbol fuchsin step; the coccidian oocysts stain pink by this method. Additional report comments can be seen in Fig. 1. Correct reporting is directly linked to correct test

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ordering, which in turn is linked to correct coding and billing (Table 15). It is critical that the physician recognize what specific tests can detect and what they cannot detect. This information should become a routine part of the educational process between clinical laboratory and clinicians. In certain situations, such as outbreaks, it may be reasonable to test for a specific parasite using a fecal immunoassay. Other patient profiles may suggest a routine O&P examination, followed by fecal immunoassays if the first test results are negative and the patient remains symptomatic.

PUBLIC HEALTH CONSIDERATIONS A number of parasites are known to be of public health concern. G. lamblia and C. parvum may be responsible for outbreaks in day care centers and waterborne outbreaks associated with drinking water supplies or recreational activities (e.g., swimming or boating). These two organisms, along with C. cayetanensis, have also been implicated in foodborne outbreaks. The presence of C. parvum in municipal water supplies is of particular concern, and the CDC has convened a task force to recommend appropriate responses (15). Outbreaks may be difficult to recognize because the affected patients are seen by numerous health care providers, with no single provider seeing enough cases to suspect an outbreak. Those laboratories that serve large and diverse patient populations are uniquely positioned to recognize outbreaks of diarrhea1 disease. When a laboratory detects a significant increase in the number of specimens positive for G. lamblia or C. parvum or notes a substantial increase in the number of stool specimens submitted to the laboratory, public health authorities should be consulted to ascertain whether an outbreak situation exists and whether preventive or control measures are indicated. Certainly other protozoa could be implicated in possible waterborne transmission, including microsporidia. Other situations that suggest the need for public health investigation include an increased frequency of reporting A. lumbricoides or other soil-transmitted helminths or the discovery of multiple patients infected with tapeworms, such as Taenia saginata or Diphyllobothrium spp. When proglottids of T. solium are identified, the referring physician should be reminded that persons in contact with the patient are at risk for acquiring cysticercosis and that a public health investigation may be needed, especially if the patient is a food handler. Although the clinical laboratory’s primary goal is to provide diagnostic services for individual patients, the greater public health must also be considered and served through consultation and education.

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1.

Infections

of the Gastrointestinal

Entamoeba histolytica/E. dispar group Note: Unless you see trophozoites containing ingested RBCs (indicates a true pathogen: E. histolytica), you cannot tell from the organism morphology whether you have actual pathogenic histolytica organisms or nonpathogenic E. dispar present. Report as seen above.

Tract

29

E.

Optional Comments: Unable to determine pathogenicity from organism morphology. A. B. Depending on patient’s clinical condition, treatment may be appropriate.

2.

If you have the kit reagents to differentiate the two organisms, comments could also be added: For differentiation of E. histolytica from E. dispar, please submit a fresh stool specimen A. for analysis. To determine the presence or absence of pathogenic E. histolytica, submit a fresh stool B. specimen. Identification of nonpathogens Comments that can be used for reporting nonpathogens include the following; however, these statements assume a complete stool exam was performed on multiple stools. You may detect nonpathogens in the first stool examination or in an unfinished examination (preliminary concentration result) but miss a pathogen (example: Dentamoeba fragilis requires the permanent stained smear for identification). Optional Comments: A. The identified organisms are considered nonpathogenic; treatment is not recommended. B. Nonpathogenic parasite(s) detected; presence of this species indicates fecal/oral contamination.

3.

Reporting BIastocystis hominis Note: Several comments are optional for reporting B. hominis. A. Clinical significance is unknown at this time. B. Status as a pathogen is controversial. You may want to add a comment: A. Presence of this species is indicative of fecal/oral agents.

4.

contamination;

consider other etiologic

Negative stool examination (O&P) Note: This comment or information must be conveyed to the clinician; many physicians think that both coccidia and microsporidia can be identified from the routine O&P examination. A. With very rare exceptions, the coccidia (C. pan/urn and C. cayetanensis) and microsporidia will not be found and/or identified with the routine O&P examination; specific examinations should be requested separately. 1. be//j is usually seen in the routine concentration sediment examination. Optional comments: A. Certain antibiotics such as metronidazole or tetracycline may interfere with the recovery of intestinal parasites, particularly protozoa. B. Antibiotics such as metronidazole or tetracycline may prevent recovery and identification of intestinal protozoa.

FIGURE 1. Laboratory test reports: optional comments. This figure is adapted from reference 25. It is important to remember that educational information for your clients is critical to the success of your test-reporting formats. The information in the figure should be shared with your clients prior to changing your report formats. Your physician group may have a preference regarding additional comments. Information updates or newsletters are appropriate for this purpose. All of these comments are optional, and wording can be changed to fit your circumstances. However, it is recommended that you select specific comments and try not to use free text to maintain uniformity. The information on O&P examination could also be conveyed to your clients via the newsletter or update formats.

FUTURE DIRECTIONS Future directions in diagnostic parasitology continue to move from traditional morphological approaches to specific diagnosis based upon antigen detection using immunologic techniques and molecular probes, especially for intestinal protozoa. Unfortunately, these methods cannot totally replace microscopic examinations until they allow accurate recognition of the important pathogenic intestinal protozoa, including G. lamblia, E. histolytica, possibly Blastocystis hominis, D. fragilis, C. parvwn, C. cayetanensis, and

the microsporidia. Due to the variable shedding of some parasites, the sensitivity of any new methods needs to be carefully evaluated. Diagnosis of intestinal helminths continues to be based on morphological detection of eggs, larvae, or adult worms. As new methods are developed, excellent candidates for improved diagnosis are S. stercoralis and Schistosoma spp. Until costs are reduced, molecular probes are unlikely to play a major role in the diagnosis of enteric parasitic infections. Simplified molecular tests con-

30 Table 15.

Garcia et al.

CUMITECH

CPT codes used for diagnostic

parasitology

Procedure

of the gastrointestinal

30A

trac@’

name

CPT code(s)

Agar plate culture (Strongyloides) Baermann concentration (plus examination) ...................................................................................... Cyclospora examination (Mod AFB) ................................................................................................... Cryptosporidium examination (Mod AFB) .......................................................................................... Cryptosporidium examination (FA) ..................................................................................................... Cryptosporidium examination (EIA) .................................................................................................... Cryptosporidium/Giardia examination (FA) ......................................................................................... Cryptosporidium/Giardia examination (EIA) Screen that detects both, but does not differentiate between the two ........................................................................................................

87081 + 87210 87015 + 87210 87207 87207 87272 87328 87272 87328 (Neg) 87328 (Pos) + 2 individual organism ElAs (87328) 82705 87272 87328 87015 + 87210 87177 87207 87015 -I- 87207 87015 + 87206 82270 82273

Fat, stool (qualitative) .......................................................................................................................... Giardia examination (FA) ..................................................................................................................... Giardia examination (EIA) .................................................................................................................... Harada-Mori concentration (plus examination) ................................................................................... Helminth egg hatching (concentration, wet mounts) ......................................................................... lsospora examination (Mod AFB) ....................................................................................................... Microsporidia, special stain (modified trichrome) (on concentration sediment) ................................ Microsporidia, fluorescent stain (Calcofluor) (on concentration sediment) ........................................ Occult blood, stool .............................................................................................................................. Occult blood, gastric ........................................................................................................................... O&P examination (direct smears, concentration and smears) (Also recommended for 87177 concentration and wet mounts) ...................................................................................................... O&P examination (permanent stain, e.g., trichrome). ....................................................................... .88313 Ova and Parasite examination (complete examination) (direct smears, concentration, permanent stain) ............................................................................................................................. 87177 -I- 88313 Parasite concentration, iodine prep (wet prep only) ........................................................................... 87015 + 87210 Parasite concentration, saline prep no. I (wet prep only) .................................................................. 87015 + 87210 Parasite examination, wet prep only (wet prep only) ......................................................................... 87210 Parasite examination, duodenum (concentration, wet mounts) ......................................................... 87177 Parasite examination, duodenum (permanent stain) .......................................................................... 88313 Parasite examination, EnteroTest (wet mounts only) ........................................................................ 87210 Parasite examination, EnteroTest (permanent stain only) .................................................................. 88313 Parasite, Calcofluor stain (direct material, no concentration) ............................................................. 87206 Parasite concentration (miscellaneous) .............................................................................................. 87015 Giemsa stain, thin blood film .............................................................................................................. 87207 Giemsa stain, thick blood film ............................................................................................................ 87015 + 87207 Parasite culture (culture, concentration, wet mount, permanent stain of culture sediment) (intestinal and blood protozoa) ........................................................................................................ 87081 + 87215 -I- 87210 + 87207 (Giemsa) or 88313 (trichrome) Parasite culture (Trichomonas) (pouch, screen) (microscope examination through plastic pouch) .............................................................................................................................................. 87081 Parasite detection (E. histo/y~&/ .. c/&par group) (EIA) ..................................................................... 87336 Parasite detection (E. histolytica) (EIA) ............................................................................................... 87337 Parasite detection (Cryptosporidium/Giardia) (EIA) Screen that detects both, but does not differentiate between the two ........................................................................................................ 87328 (Neg) 87328 (Pos) + 2 individual organism ElAs (87328) Parasite detection, EIA (Cryptosporidium only) .................................................................................. 87328 87328 Parasite detection, EIA (Giardia only) ................................................................................................. Parasite referral in (stool for O&P examination) ................................................................................. 87177 -I- 88313 Petri dish culture-Concentration (plus examination) ........................................................................... 87015 + 87210 pH, stool ............................................................................................................................................. 83986 87172 Pinworm examination ......................................................................................................................... 83986 Reducing substances, stool (pH) ........................................................................................................ Trypsin activity, stool .......................................................................................................................... 84488 Worm identification (macro) ............................................................................................................... 87169 Worm identification (wet mount) ....................................................................................................... 87210 a Mod AFB, modified acid-fast stain; FA, fluorescent antibody; EIA, enzyme immunoassay; Neg, negative; Pos, positive. bThese CPT codes are continually undergoing review and revision. It is important to update all microbiology codes yearly.

tinue to be developed for routine use by the clinical laboratory. As the health care environment continues to be faced with a shortage of personnel, financial, and diagnostic resources, optimal use of simple, accurate, and cost-effective laboratory procedures remains a primary goal for patient care.

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