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Chapter 5 : Specific Test Procedures and Algorithms

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Abstract:

This section focuses on microscopic techniques and calibration of the microscope and algorithms for the ova and parasite (O&P) examination. Direct wet fecal smear, sedimentation concentration, flotation concentration methods used for the results and laboratory reports and procedure reminders and limitations are briefly discussed. Specialized stains for and are discussed using different staining methods. Modified trichrome stain for is reviewed for the clinical assay. Antigen-based fecal immunoassays have several significant advantages over other methods currently used for diagnosis of amebiasis. Different stains are analyzed for the parasites examination. Several concentration techniques such as buffy coat concentration, knott concentration and membrane filtration concentration are discussed. The section also provides a brief outline of the procedure for processing fresh stool for the O&P examination.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.1

Ocular micrometer, top scale; stage micrometer, bottom scale. Reprinted from , 5th ed.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.2

Method of scanning a direct wet film preparation with the 10× objective. (Illustration by Nobuko Kitamura.) Reprinted from , 5th ed.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.3

Fecal concentration procedures: various layers seen in tubes after centrifugation. (A) Formalin-ether (or ethyl acetate). (B) Zinc sulfate (the surface film should be within 2 to 3 mm of the tube rim). (Illustration by Sharon Belkin.)

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.4

Method used to remove surface film in the zinc sulfate flotation concentration procedure. (A) A wire loop is gently placed on (not under) the surface film. (B) The loop is then placed on a glass slide. (Illustration by Nobuko Kitamura.) Reprinted from , 5th ed.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.5

Culture methods for the recovery of larval-stage nematodes; Harada-Mori tube method and petri dish culture method. (Illustration by Nobuko Kitamura.) Reprinted from , 5th ed.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.6

Baermann apparatus. (Illustration by Nobuko Kitamura.) Reprinted from , 5th ed.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.7

Agar culture method for . (1) Agar plates are prepared; (2) agar is dried for 4 to 5 days on the bench top; (3) plates are stored in plastic bags; (4) fresh stool is submitted to the laboratory; (5) approximately 2 g of stool is placed onto an agar plate; (6) the plate is sealed with tape; (7) the culture plate is incubated at 26 to 33°C for 2 days; (8) the plate is examined microscopically for the presence of tracks (bacteria carried over the agar by migrating larvae); (9) 10% formalin is placed onto the agar through a hole made in the plastic by hot forceps; (10) material from the agar plate is centrifuged; (11) the material is examined as a wet preparation for rhabditiform or filariform larvae (high dry power; magnification, ×400). (Illustration by Sharon Belkin.) Reprinted from , 5th ed.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.8

Method of thin blood film preparation. (A) Position of spreader slide; (B) well-prepared thin film. Arrows indicate the area of the slide used to observe accurate cell morphology. (Illustration by Nobuko Kitamura.) Reprinted from , 5th ed.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Figure 5.9

Method of thick-thin combination blood film preparation. (a) Position of a drop of EDTA-blood; (b) position of the applicator stick in contact with blood and glass slide; (c) rotation of the applicator stick; and (d) completed thick-thin combination blood film prior to staining. (Illustration by Sharon Belkin.) Reprinted from , 5th ed.

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Algorithm 5.1

Procedure for processing fresh stool for the O&P examination

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Algorithm 5.2

Procedure for processing liquid specimens for the O&P examination

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Image of Algorithm 5.3
Algorithm 5.3

Procedure for processing preserved stool for the O&P examination—two-vial collection kit

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Algorithm 5.4

Procedure for processing SAF-preserved stool for the O&P examination

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Algorithm 5.5

Use of various fixatives and their recommended stains

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Image of Algorithm 5.6
Algorithm 5.6

Ordering algorithm for laboratory examination for intestinal parasites

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Image of Algorithm 5.7
Algorithm 5.7

Procedure for processing blood specimens for examination

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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References

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1. Arakaki, T.,, M. Iwanaga,, F. Kinjo,, A. Saito,, R. Asato, and, T. Ikeshiro. 1990. Efficacy of agar-plate culture in detection of Strongyloides stercoralis infection. J. Parasitol. 76: 425428.
2. Baermann, G. 1917. Eine einfache Methode zur Auffindung vor Ankylostomum (Nematoden), p. 41. In Larven in Erdproben. Meded. Geneesk Laborat. Weltever Feestbundel.
3. Baxby, D.,, N. Blundell, and, C. Hart. 1984. The development and performance of a simple, sensitive method for the detection of Cryptosporidium oocysts in feces. J. Hyg. 93: 317323.
4. Didier, E. S.,, J. M. Orenstein,, A. Aldras,, D. Bertucci,, L. B. Rogers, and, F. A. Janney. 1995. Comparison of three staining methods for detecting microsporidia in fluids. J. Clin. Microbiol. 33: 31383145.
5. Garcia, L. S. 2007. Diagnostic Medical Parasitology, 5th ed. ASM Press, Washington, DC.
6. Garcia, L. S. (coordinating ed.). 2008 Laboratory Procedures for the Diagnosis of Blood Parasites. Cumitech 46. ASM Press, Washington, DC.
7. Garcia, L. S.,, T. Brewer, and, D. A. Bruckner. 1979. A comparison of the formalin-ether concentration and trichrome-stained smear methods for the recovery and identification of intestinal protozoa. Am. J. Med. Technol. 45: 932935.
8. Garcia, L.,, D. Bruckner,, T. Brewer, and, R. Shimizu. 1983. Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens. J. Clin. Microbiol. 18: 185190.
9. Garcia, L. S.,, and R.Y. Shimizu. 1997. Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens. J. Clin. Microbiol. 35: 15261529.
10. Garcia, L. S.,, and R.Y. Shimizu. 2000. Detection of Giardia lamblia and Cryptosporidium parvum antigens in human fecal specimens using the ColorPAC combination rapid solid-phase qualitative immunochromatographic assay. J. Clin. Microbiol. 38: 12671268.
11. Hanson, K. L.,, and C.P. Cartwright. 2001. Use of an enzyme immunoassay does not eliminate the need to analyze multiple stool specimens for sensitive detection of Giardia lamblia. J. Clin. Microbiol. 39: 474477.
12. Harada, U.,, and O. Mori. 1955. A new method for culturing hookworm. Yonago Acta Med. 1: 177179.
13. Hsieh, H. C. 1962. A test-tube filter-paper method for the diagnosis of Ancylostoma duodenale, Necator americanus, and Strongyloides stercoralis. WHO Tech. Rep. Ser. 255: 2730.
14. Isenberg, H. D. (ed.). 2004. Clinical Microbiology Procedures Handbook, 2nd ed. ASM Press, Washington, DC. Parasitology Section in vol. 2 of 3 volumes.
15. Koga, K. S.,, C. Kasuya,, K. Khamboonruang,, M. Sukhavat,, M. Ieda,, N. Takatsuka,, K. Kita, and, H. Ohtomo. 1991. A modified agar plate method for detection of Strongyloides stercoralis. Am. J. Trop. Med. Hyg. 45: 518521.
16. Kokoskin, E.,, T. W. Gyorkos,, A. Camus,, L. Cedilotte,, T. Purtill, and, B. Ward. 1994. Modified technique for efficient detection of microsporidia. J. Clin. Microbiol. 32: 10741075.
17. Markell, E. K.,, and M. Voge. 1981. Medical Parasitology, 5th ed. The W. B. Saunders Co., Philadelphia, PA.
18. 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.
19. National Committee for Clinical Laboratory Standards. 2000. Use of Blood Film Examination for Parasites. Approved guideline M15-A. National Committee for Clinical Laboratory Standards, Wayne, PA.
20. National Committee for Clinical Laboratory Standards. 1997. Procedures for the Recovery and Identification of Parasites from the Intestinal Tract, 2nd ed. Approved guideline M28-A. National Committee for Clinical Laboratory Standards, Wayne, PA.
21. Neimeister, R.,, A. Logan,, B. Gerber,, J. Egleton, and, B. Kleger. 1987. Hemo-De as substitute for ethyl acetate in formalin-ethyl acetate concentration technique. J. Clin. Microbiol. 25: 425426.
22. Palmer, J. 1991. Modified iron hematoxylin/Kinyoun stain. Clin. Microbiol. Newsl. 13: 3940. (Letter to the editors.)
23. Ritchie, L. 1948. An ether sedimentation technique for routine stool examinations. Bull. U.S. Army Med. Dept. 8: 326.
24. Ryan, N. J.,, G. Sutherland,, K. Coughlan,, M. Globan,, J. Doultree,, J. Marshall,, R. W. Baird,, J. Pedersen, and, B. Dwyer. 1993. A new Trichrome-Blue stain for detection of microsporidial species in urine, stool, and nasopharyngeal specimens. J. Clin. Microbiol. 31: 32643269.
25. Spencer, F. M.,, and L.S. Monroe. 1976. The Color Atlas of Intestinal Parasites, 2nd ed. Charles C Thomas, Publisher, Springfield, IL.
26. Tompkins, V. N.,, and J.K. Miller. 1947. Staining intestinal protozoa with iron-hematoxylin-phosphotungstic acid. Am. J. Clin. Pathol. 17: 755758.
27. Visvesvara, G. S.,, H. Moura,, E. Kovacs-Nace,, S. Wallace, and, M. L. Eberhard. 1997. Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating. J. Clin. Microbiol. 35: 730733.
28. Weber, R.,, R. T. Bryan,, R. L. Owen,, C. M. Wilcox,, L. Gorelkin,, G. S. Visvesvara, and The Enteric Opportunistic Infections Working Group. 1992. Improved light-microscopical detection of microsporidia spores in stool and duodenal aspirates. N. Engl. J. Med. 326: 161166.
29. Wheatley, W. 1951. A rapid staining procedure for intestinal amoebae and flagellates. Am. J. Clin. Pathol. 21: 990991.
30. Wilson, M.,, and P.M. Schantz. 2000. Parasitic immunodiagnosis, p. 1117–1122. In G. T. Strickland (ed.): Hunter’s Tropical Medicine and Emerging Infectious Diseases, 8th ed. The W. B. Saunders Co., Philadelphia, PA.
31. Yang, J.,, and T.H. Scholten. 1977. A fixative for intestinal parasites permitting the use of concentration and permanent staining procedures. Am. J. Clin. Pathol. 67: 300304.

Tables

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Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
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Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Untitled

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Table 5.1

Body site, specimen, and recommended stain(s)

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Table 5.2

Approaches to stool parasitology: test ordering

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Table 5.3

Laboratory test reports: optional comments

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5
Generic image for table
Table 5.4

Parasitemia determined from conventional light microscopy: clinical correlation

Citation: Garcia L. 2009. Specific Test Procedures and Algorithms, p 87-217. In Practical Guide to Diagnostic Parasitology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815967.ch5

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