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Chapter 132 : General Approaches for Detection and Identification of Parasites

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Abstract:

This chapter discusses various approaches and diagnostic methods currently in use for the diagnosis of parasitic infections. Examination of prepared wet mounts, concentrated specimens, permanent stained smears, blood films, and various culture materials can provide critical information leading to organism identification and confirmation of the suspected cause of clinical disease. The purpose of the concentration method is to separate parasites from fecal debris and to concentrate any parasites present through either sedimentation or flotation. Wet mounts prepared from concentrated stool are examined in the same manner as that used for the direct wet mount method. Although the routine ova and parasite examination consisting of the direct wet mount, the concentration, and the permanent stained smear is an excellent procedure recommended for the detection of most intestinal parasites, several other diagnostic techniques are available for the recovery and identification of specific parasitic organisms. The only human parasites for which it is reasonably possible to correlate egg production with adult worm burdens are , , and the hookworms ( and ). Several parasites may be recovered and identified from urogenital specimens. Although, the most common pathogens are probably and , other organisms such as the microsporidia are becoming much more important. Although other tests such as immunoassay diagnostic kits continue to become available commercially, the majority of medical parasitology diagnostic work depends on the knowledge and microscopy skills of the microbiologist.

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132

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Figures

Image of FIGURE 1
FIGURE 1

Processing liquid stool for O&P examination. Either PVA or Schaudinn's fixative can be used for the preparation of the permanent stained smear. Organism motility is seen when saline is used; iodine kills the organisms, so motility will no longer be visible. The use of ethyl acetate may remove the entire specimen and pull it into the layer of debris that will be discarded (liquid specimen normally contains mucus); centrifuge at 500 × for 10 min (normal centrifugation time), but do not use ethyl acetate in the procedure. In general, laboratories have switched to nonmercury substitutes; the original Schaudinn's fixative contains mercuric chloride. However, in some instances the term “Schaudinn's fixative” is still used to describe not the original fixative but a formulation that is prepared with a copper or zinc base or other proprietary compounds. When fixatives are selected, it is important to know the contents in order to comply with disposal regulations. Reprinted from reference .

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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Image of FIGURE 2
FIGURE 2

Processing preserved stool for O&P examination (two-vial collection kit). The formalin can be buffered or nonbuffered, depending on the laboratory protocol in use. PVA fixative prepared with mercuric chloride provides the best organism preservation. Alternatives are available, including zinc-based PVA, copper sulfate-based PVA, and SAF. Reprinted from reference .

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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Image of FIGURE 3
FIGURE 3

Processing preserved stool for O&P examination (one-vial collection kit). There are single-vial collection systems for which the formulas are proprietary; however, many contain zinc sulfate as one of the key ingredients. With the exception of SAF, compatibility of these fixatives with immunoassay reagents is not always possible. Other good options include Unifix, TOTAL FIX, or Z-PVA (Medical Chemical Corp.) with trichrome stain, as well as Ecofix with Ecostain (Meridian Biosciences, Cincinnati, OH). If the iron hematoxylin method containing the carbol fuchsin step is used, the coccidian oocysts will stain pink ( spp., , or ). Also, when using SAF, some recommend the use of an adhesive such as egg albumin to “glue” the fecal material onto the slide prior to staining. It is highly recommended that special stains be performed for the detection and identification of the coccidia (modified acid-fast stains) and the microsporidia (modified trichrome stains) from concentrated sediment to enhance organism recovery. Coccidian oocysts of can easily be detected in the concentration sediment wet mount; however, unless a very heavy infection is present, sp. oocysts may not be seen without special modified acid-fast stains. The small size of the microsporidian spores prevents identification without the use of special modified trichrome stains and microscopic examination with a 100× oil immersion objective. Reprinted from reference .

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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References

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1. Ash, L. R.,, and T. C. Orihel. 1991. Parasites: a Guide to Laboratory Procedures and Identification. ASCP Press, Chicago, IL.
2. Beaver, P. C.,, R. C. Jung,, and E. W. Cupp. 1984. Clinical Parasitology, 9th ed. Lea & Febiger, Philadelphia, PA.
3.Code of Federal Regulations. 1991. Occupational exposure to bloodborne pathogens. Fed. Regist. 29CFR1910.1030.
4. Garcia, L. S. 2007. Diagnostic Medical Parasitology, 5th ed. ASM Press, Washington, DC.
5. Garcia, L. S. 2009. Practical Guide to Diagnostic Parasitology, 2nd ed. ASM Press, Washington, DC.
6. Hiatt, R. A.,, E. K. Markell,, and E. Ng. 1995. How many stool examinations are necessary to detect pathogenic intestinal protozoa? Am. J. Trop. Med. Hyg. 53:3639.
7. Isenberg, H. D. (ed.). 2004. Clinical Microbiology Procedures Handbook, 2nd ed. ASM Press, Washington, DC.
8. Katanik, M. T.,, S. K. Schneider,, J. E. Rosenblatt,, G. S. Hall,, and G. W. Procop. 2001. Evaluation of ColorPAC Giardia/Cryptosporidium rapid assay and ProSpecT Giardia/ Cryptosporidium microplate assay for detection of Giardia and Cryptosporidium in fecal specimens. J. Clin. Microbiol. 39:45234525.
9. Kehl, K. S. C. 1996. Screening stools for Giardia and Cryptosporidium: are antigen tests enough? Clin. Microbiol. Newsl. 18:133135.
10. Koneman, E. W.,, S. D. Allen,, W. M. Janda,, P. C. Schreckenberger,, and W. C. Winn, Jr. 1997. Color Atlas and Textbook of Diagnostic Microbiology, 5th ed. J. B. Lippincott Co., Philadelphia, PA.
11. Markell, E.,, D. T. John,, and W. A. Krotoski. 1999. Medical Parasitology, 8th ed. The W. B. Saunders Co., Philadelphia, PA.
12. Melvin, D. M.,, and M. M. Brooke. 1982. Laboratory Procedures for the Diagnosis of Intestinal Parasites, 3rd ed. U.S. Department of Health, Education, and Welfare publication no. (CDC) 82-8282. Government Printing Office, Washington, DC.
13. Morris, A. J.,, M. L. Wilson,, and L. B. Reller. 1992. Application of rejection criteria for stool ovum and parasite examinations. J. Clin. Microbiol. 30:32133216.
14. Murray, P. R.,, E. J. Baron,, J. H. Jorgensen,, M. L. Landry,, and M. A. Pfaller (ed.). 2007. Manual of Clinical Microbiology, 9th ed. ASM Press, Washington, DC.
15.National Committee for Clinical Laboratory Standards. 2000. Laboratory Diagnosis of Blood-Borne Parasitic Diseases. Approved Guideline M15-A. National Committee for Clinical Laboratory Standards, Villanova, PA.
16.National Committee for Clinical Laboratory Standards. 1997. Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue. Approved Guideline M29-A. National Committee for Clinical Laboratory Standards, Villanova, PA.
17.National Committee for Clinical Laboratory Standards. 1997. Procedures for the Recovery and Identification of Parasites from the Intestinal Tract. Approved Guideline M28-A. National Committee for Clinical Laboratory Standards, Villanova, PA.
18. Nielsen, C. K.,, and L. A. Ward. 1999. Enhanced detection of Cryptosporidium parvum in the acid-fast stain. J. Vet. Diagn. Investig. 11:567569.
19. Ramirez, J. R.,, S. Agudelo,, C. Muskus,, J. F. Alzate,, C. Berberich,, D. Barker,, and I. D. Velez. 2000. Diagnosis of cutaneous leishmaniasis in Colombia: the sampling site within lesions influences the sensitivity of parasitologic diagnosis. J. Clin. Microbiol. 38:37683773.
20. Sharp, S. E.,, C. A. Suarez,, Y. Duran,, and R. J. Poppiti. 2001. Evaluation of the Triage Micro Parasite Panel for detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in patient stool specimens. J. Clin. Microbiol. 39:332334.

Tables

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TABLE 1

Diagnostic characteristics of organisms in wet mounts

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 2

Identification of helminth eggs

This table does not include every possible helminth that could be found as a human parasite; however, the most likely helminth infections are included.

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
Generic image for table
TABLE 3

Diagnostic characteristics of organisms in permanent stained smears

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 4

Key to identification of intestinal amebae ( permanent stained smear)

refers to the group (pathogenic) can be determined by finding red blood cells in the cytoplasm of the trophozoites. Otherwise, on the basis of morphological grounds (pathogen) and (nonpathogen) cannot be differentiated.

It is very difficult to differentiate trophozoites from those of the group or .

Although some cysts are larger than 10 μm, the majority of cysts measure 9–10 μm and the typical glycogen vacuole ensures the proper identification.

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 5

Key to identification of intestinal flagellates Fecal culture methods are especially helpful for the detection of light infections with hookworm, , and spp. and for the specific identification of parasites. Also, such techniques are useful for obtaining a large number of infective-stage larvae for research purposes. One enhanced recovery method, the agar plate culture for , and several culture techniques are described in this chapter. Since these procedures are less common, brief descriptions are included.

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 6

Commercially available immunoassays for detection of intestinal parasites

Some single-vial collection systems may use fixatives that are compatible with immunoassay testing; please consult specific manufacturer.

DFA, direct fluorescent-antibody assay.

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 7

Additional helminth recovery and identification techniques (other than O&P examination)

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 8

Recovery of parasites from other intestinal tract specimens

Immunoassay tests for the group or require fresh or frozen stool; preserved stool specimens are not acceptable for testing. Immunoassays for spp. and are approved for use on stool; immunoassay use for other intestinal tract specimens may or may not be appropriate, depending on specimen source, consistency, and volume.

Some genus-specific immunoassay reagents for the microsporidia are available commercially but are not FDA approved.

Although cysts may be present in stool, sigmoidoscopy specimens are often obtained from patients with severe diarrhea or dysentery. In such cases, the cyst forms are usually absent; trophozoites would be the most likely stage seen, particularly in the case of .

Duodenal specimens are often submitted as aspirates; in such cases the volume may be sufficient to perform concentrations. However, if small amounts of duodenal mucus and/or biopsy material are obtained, squash preparations preserved with Schaudinn's fixative are preferred. This approach may require the use of slides precoated with albumin to facilitate adhesion. Mucus obtained from the Entero-Test capsule string may be treated as a fresh specimen; the string can also be immediately placed in preservative after retrieval, and the mucus can be processed as a permanent stained smear.

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 9

Detection of urogenital parasites

Staining procedures and fluorescent antibody tests for organism detection performed on centrifuged sediment (500 × for 10 min).

Immunoassays are available commercially but are not yet FDA approved.

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 10

Specimen, possible parasite recovered, and appropriate tests (other than intestinal tract)

This table does not include every possible parasite that could be found in a particular body site. Parasite stages include trophozoites, cysts, oocysts, spores, adults, larvae, eggs, hooklets, amastigotes, and trypomastigotes. Although PCR methods have been used in the research setting for most of the organisms listed in the table, reagents are generally not commercially available.

Routine histology can be used for the detection and identification of many parasites. In some cases, it may be the only means of diagnosis.

PAS, periodic acid-Schiff stain.

Although eye specimens are much preferred, free-living amebae have been cultured from patient contact lenses and lens solutions; we would not reject these specimens. An exception would be commercial lens care solutions; these solutions would be rejected.

The examination of abscess aspirates for the presence of trophozoites is an uncommon procedure and not always reliable in diagnosing extraintestinal amebiasis; serologic tests would be preferred.

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132
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TABLE 11

Techniques for the recovery and identification of blood parasites (EDTA or heparin)

Molecular techniques are still experimental and are not always available; it is always important to verify FDA approval within the United States (contact the manufacturer).

QBC-Blood Parasite Detection Method (Becton-Dickinson Tropical Disease Diagnostics, Sparks, MD).

ParaSight F-Rapid Test for malaria (Becton-Dickinson Tropical Disease Diagnostics, Sparks, MD). This test is not licensed for diagnostic use in the United States. This test is a dipstick format.

Binax Now malaria (all four species), Binax Now ICT Filariasis () (Binax, Inc., Portland, ME). These tests are in a dipstick format.

PATH IC Falciparum malaria IC test (PATH, Seattle, WA). This test is in a dipstick format.

Filariasis Ag-CELISA (), Malaria Ag-CELISA (). Rapimal Dipstick (), Rapimal Cassette () (Cellabs, Sydney, New South Wales, Australia).

OptiMAL (differentiates between and ) (Flow, Inc., Portland, OR). This test is in a dipstick format.

TropBio (James Cook University, Townsville, Queensland, Australia).

Citation: Garcia L, Shimizu R, Paltridge G. 2011. General Approaches for Detection and Identification of Parasites, p 2071-2090. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch132

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