Symposium on Parasitic Infections
. Special Laboratory Examinations for Parasitic Infections
Lynne S. Garcia, M.S., C.L.S.(N.C.A.), M.T.(A.S.C.P.)*
Although the routine ova and parasite examination on stool is a well recognized diagnostic procedure, there are a number of other diagnostic techniques available for the recovery and identification of parasitic organisms. Most laboratories do not routinely offer all of these techniques, but many are relatively simple and inexpensive to perform. The clinician should therefore be aware of the possibilities and the clinical relevance of information obtained from using such techniques. One of the most important aspects of diagnostic parasitology work is specimen collection. Final laboratory results based on parasite recovery and identification will depend on the initial handling and fixation of the specimen. This becomes extremely important when one considers the number of compromised or immunosuppressed patients who are seen in medical practice today. These patients are often at risk for parasitic infections. However, unless the appropriate specimens are properly collected and processed, the infections may be missed (Table 1).
STOOL The traditional ova and parasite examination is the most well known diagnostic procedure for stool. This technique includes three components: the direct wet mount, the examination of material from a stool concentrate, and the permanent stained smear. This procedure is recommended for the majority of the intestinal parasites (Fig. 1). There are other techniques that may allow recovery of intestinal parasites when the ova and parasite examination is negative. These include concentration techniques, culture systems, and hatching techniques for certain helminth eggs. There are also special techniques for the recovery of intestinal coccidia, particularly Cryptosporidium (Fig. 2).7. *Manager, Microbiology Laboratory, UCLA Clinical Laboratories/Microbiology, Department of Pathology, UCLA Medical Center, Los Angeles, California
Pediatric Clinics of North America-Vol. 32, No.4, August 1985
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Table 1. Body Sites and Diagnostic Specimen Information SITE
Intestinal tract
SPECIMEN OPTIONS
Fresh stool Preserved Stool Sigmoidoscopy material Duodenal contents Anal impression smear Biopsy
Urogenital system
Vaginal discharge Urethral discharge Prostatic secretions Urine
Lung
Sputum
Liver, spleen
Tracheobronchial aspirate Brush biopsy Open lung biopsy Aspirates
CSF Cutaneous ulcers
Bone marrow Blood
Muscle
Biopsy Spinal fluid Aspirates from below surface Biopsy Aspirate Smears An ticoagulan t Biopsy
COLLECTION INFORMATION
% pint waxed container 5 or 10% Formalin, MIF, SAF, Schaudinn's, PVA Fresh, PVA or Schaudinn's smears Entero-Test or aspirates Cellulose tape (pinworm exam) Routine histology Saline swab, culturette (no charcoal), culture medium Same as above Same as above Single unpreserved specimen 24-hour unpreserved specimen True sputum/not saliva (No preservative) (10% formalin if time delay) Air dried smears Same as above Same as above Sterile, collected in 4 separate aliquots (liver/amebiasis) 1 aliquot (hydatid disease) Sterile, non sterile to histology Sterile Sterile plus air-dried smears
Sterile, nonsterile to histology Sterile Thick and Thin Films EDTA (First Choice) Heparin (Second Choice) Routine Histology or Squash Preparation
Cultures for Larval-Stage Nematodes Certain helminths produce larval stages that hatch in soil or in tissues, and these larvae can be recovered and subsequently identified using fecal culture methods that tend to concentrate the larvae. These methods are helpful for Strongyloides, hookworm, and Trichostrongylus infections. One of the better known techniques is the Baermann concentrate (Fig, 3). The technique depends on the migration of active larvae from the fecal material, through both wire and cloth gauze, and into water where they settle out. This is an excellent method for the recovery of Strongyloides larvae. 6 ,7 Several other abbreviated methods similar to the Baermann include the Harada-Mori filter paper strip culture and the filter paper/slant culture in a petri dish, both of which are helpful in the recovery of Strongyloides (Fig. 4).9. 10, 13
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STOOL (INTESTINAL PROTOZOA, HELMINTH EGGS/LARVAE)
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I
FRESH
PRESERVED (CHOICE OF PRESERVATIVE WILL DETERMINE SUBSEQUENT TECHNIQUES)
I
I
DIRECT MOUNT
I
I
NEG
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CONCENTRATE POS
I
POS
NEG
CONCENTRATE*
CONCENTRATE POS
r
POS
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52
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PERMANENT STAINED SMEAR
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PERMANENT STAINED SMEAR
Figure 1. Ova and parasite examination (stool). *Heavy eggs (unfertilized Ascaris) or operculated eggs will not Boat using the zinc-sulfate technique.
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STOOL (COCCIDIA)
FRESH
I
DIRECT MOUNT PRESERVED
I POS
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CONCENTRATE
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CONCENTRATE
CONCENTRATE 1. SHEATHER'S SUGAR 2. FORMALIN-ETHYL ACETATE POS
POS
NEG
POS
NEG
NEG
PERMANENT STAINED SMEAR* Figure 2. Examination for intestinal coccidia. *Fluorescent stain (Auramine/Auramine-Rhodamine) may be used as a screen prior to permanent stained smear (acid-fast techniques).
Egg Counts Although some of these techniques have been available for a number of years, they are rarely used. The Stoll count is perhaps the most widely used counting procedure for estimating worm burdens. 15 However, with the current therapy available for the majority of intestinal helminth infections, the need for monitoring therapy through egg counts is no longer so important. Hatching Schistosome Eggs Schistosome eggs may be recovered from either urine or stool, depending on the species involved. In either case, the eggs should be checked for viability. The presence ofliving miracidia larvae within the eggs indicates an active infection that may require therapy. Egg viability can be determined in two ways: from examination of the egg under the microscope using high-dry power (cilia on flame cells will be motile) (Fig. 5) and from hatching the eggs, releasing the miracidia larvae (Fig. 6).7 If schistosomiasis is suspected, then clinical specimens must be collected using no preservative, thus allowing egg viability to be determined.
SPECIAL LABORATORY EXAMINATIONS FOR PARASITIC INFECTIONS
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Soil or fecal material
Wire
screen------'7'7~
Water-----~===;~===::f
Rubber tubing-----;
Clamp----
Beaker------i
Figure 3. The Baermann device. (Illustration by Nobuko Kitamura.) (From Garcia, L. S.: Laboratory diagnosis of parasitic infections. In Finegold, S. M. and W. J. Martin: Diagnostic Microbiology. St. Louis: The C. V. Mosby Co. 1982.)
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SPECIAL LABORATORY EXAMINATIONS FOR PARASITIC INFECTIONS
Figure 4. Culture methods for the recovery of larval stage nematodes: Harada-Mori tube method and Petri dish culture method. (Illustration by Nobuko Kitamura.) (From Garcia, L. S., and Ash, L. R: Diagnostic Parasitology: Clinical Laboratory Manual. 2nd ed. St. Louis, The C. V. Mosby Co., 1979.)
Search for Tapeworm Scolex Occasionally, there may be a request to find the scolex of a tapeworm to confirm successful therapy.7 In the event the scolex is not recovered and is still attached to the mucosa, then it is possible for the tapeworm to regenerate proglottids. Considering the therapy that is presently available, the need for this technique is no longer as important, since elimination of the total tapeworm is usually accomplished.
Flame cell
Figure 5. Schistosoma mansoni egg shOwing flame cells. (Illustration by Nobuko Kitamura; modified from Faust, E. C., et al.: Craig and Faust's Clinical Parasitology. 8th ed. Philadelphia Lea and Febiger, 1970. From Garcia, L. S. and Ash, L. R: Diagnostic Parasitology: Clinical Laboratory Manual. 2nd ed. St. Louis, C. V. Mosby Co., 1979.)
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Figure 6. Side-arm hatching flask used to recover miracidia from viable schistosome eggs. (Illustration by Nobuko Kitamura) (From Garcia, L. S., and Ash, L. R.: Diagnostic Parasitology: Clinical Laboratory Manual. 2nd ed. St. Louis, The C. V. Mosby Co., 1979.)
OTHER SPECIMENS FROM THE INTESTINAL TRACT Sigmoidoscopy Specimens Specimens obtained at sigmoidoscopy can be useful in the diagnosis of amebiasis. This procedure, however, does not replace the recommended three ova and parasite examinations. Specimens from the mucosal surface must be handled quickly for fixation, smear preparation, and delivery to the laboratory. The trophozoite stage of Entamoeba histolytica tends to disintegrate outside of the body, so the specimen should be immediately examined or should be preserved for examination at a later time. Material should be scraped or aspirated from the mucosal surface and should not be obtained with a cotton swab. The material should be processed immediately without additional transit time to a laboratory. Smears are prepared for permanent staining and either or both Schaudinn's fixative and polyvinyl alcohol fixative (PVA) can be used during smear preparation. Although the material obtained can be examined as a fresh, wet mount, the ability to specifically diagnose E. histolytica from such a preparation can be very difficult. It is recommended that any wet mount identification be considered tentative and be confirmed on a permanent stained smear.7 The presence of a tray containing appropriate fixatives in the procedure room can be helpful for proper specimen preparation. Since there are human cells that can be confused with organisms, it is also mandatory that trained personnel evaluate the smears. Usually, several smears from different areas of the mucosa are examined before a negative result is reported. 6, 7 Duodenal Contents In suspected infections with Giardia lamblia or Strongyloides stercoralis, a series of stool examinations may not reveal the organisms. Duodenal contents can be obtained several ways and may allow recovery of the organisms. Duodenal drainage may be submitted in a tube containing no preservative and should be examined as a wet preparation for motile organisms, again mandating rapid delivery to the laboratory. It is particularly important to examine any mucus that may be present in the sample.
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Figure 7. ENTERO-TEST capsule for sampling duodenal contents. (Illustration by Nobuko Kitamura.) (From Garcia, L. S., and Ash, L. R.: Diagnostic Parasitology: Clinical Laboratory Manual. 2nd ed. St. Louis, The C. V. Mosby Co., 1979.)
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Another method for sampling duodenal contents that eliminates the need for intestinal intubation is the Entero-Test capsule (Fig. 7).1 This device consists of a length of nylon yarn that has a weight on the end of the string and is coiled inside a gelatin capsule. The line protrudes through one end of the capsule; this end of the string can be taped to the patient's cheek. The gelatin capsule dissolves in the stomach and the weighted string is carried by peristalsis into the duodenum. The weight is released and passes out in the stool when the line is retrieved after approximately four hours. The mucus is removed from the string and examined as several wet preparations. If organisms are present, the remainder of the material can be preserved and prepared as a permanent stained smear. Duodenal biopsy specimens could also be obtained for histologic processing. Organisms that might be present would include Isospora, Cryptosporidium, Giardia lamblia and Strongyloides stercoralis. Examination for Pinworm Eggs of the pinworm Enterobius vermicularis are rarely recovered in a routine fecal examination. The female worm deposits eggs on the perianal skin; diagnosis of this infection is usually based on egg recovery using the cellullose tape technique. Sticky tape is applied to the perianal skin, and the eggs and sometimes adult female worms adhere to the tape (Fig. 8).2.8 Because the female worm migration is sporadic, it has been recommended that from four to six consecutive tapes be obtained and examined before a pinworm infection is ruled out. Specimens should be obtained several
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Figure 8. Collection of Enterobius vermicularis eggs using cellulose tape method. (From Hunter, Schwartzwelder, and Clyde: Tropical Medicine. 5th ed. Philadelphia, W. B. Saunders Co., 1976.)
hours after the individual has retired or the first thing in the morning before going to the bathroom or bathing. After collection, the tape is pressed onto a glass slide, which is then submitted to the laboratory. In some cases, the patient may be treated ba~ed on past history of infection and recurrence of symptoms.
UROGENITAL SPECIMENS
Trichomonas vaginalis recovery and identification are usually based on the examination of wet preparations of vaginal and urethral discharges and prostatic secretions. The specimen can be obtained using sterile, cottontipped applicators or a sterile, nonlubricated dry speculum. If there will be a time-delay prior to examination, stained smears can be prepared (trichrome or Giemsa). Frequently, false-positive and false-negative results are reported on the basis of stained smears. Therefore, observation of
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motile organisms, either from direct wet preparations or from culture media, may be the preferred method for identification of the organism. 14 Often T. vaginalis can be recovered in urine sediment. The eggs of Schistosoma haematobium can be recovered in urine; however, a series of several specimens may be necessary for egg recovery and identification. Recommendations include several specimens passed around noon and a 24-hour specimen, all of which should be collected with no preservatives. Several specimens must be examined in order to recover schistosome eggs. SPUTUM, ASPIRATES, AND BIOPSY SPECIMENS Sputum Several parasites can be recovered from sputum, including the migrating larvae of Ascaris lumbricoides, Strongyloides stercoralis, and hookworm; E. histolytic and Echinococcus granulosus from lung involvement; and Paragonimus westermani eggs. Sputum is usually examined as a saline or iodine wet mount under low and high dry power of the microscope-a technique that is simple but does not allow any' concentration of the specimen. A sputum specimen can be concentrated using the acid-ether or formalin-ether technique. Also, some of the specimen can be preserved for permanent staining at a later time. The examination of permanent stains can be particularly important when differentiating E. histolytica from Entamoeba gingivalis, which is a commensal organism found in the mouth. If examination of the specimen must be delayed, particularly for echinococcosis or paragonimiasis, then it should be preserved in 10 per cent formalin. Aspirates The recovery of parasites from aspirated material can be extremely helpful in diagnosing certain infections, including amebiasis where there is liver involvement. Although this technique is infrequently used in this country, if performed correctly it can be diagnostic. The aspirated liver abscess material should be examined immediately and should be collected in at least two aliquots, the last portion being the most likely to contain organisms. 7, 12 Another organism that may be recovered by the examination of material aspirated from the lung or from. brush biopsy material is Pneumocystis carinii. Although tracheobronchial aspirates are also used, the specimen of choice is often listed as the open lung biopsy. The sputum specimen is generally considered unacceptable for organism recovery. Cutaneous ulcers, lymph nodes, spleen, liver, bone marrow, and spinal fluid are often aspirated to obtain material for organism recovery and identification. Specific infections that may be diagnosed from these types of specimens include Mrican trypanosomiasis, leishmaniasis, Chagas' disease, and primary amebic meningoencephalitis. Obviously, specimens of this type must be immediately transported to the laboratory, particularly since one may be looking for motile organisms and/or may wish to inoculate culture media.
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Biopsy Specimens In certain cases a biopsy may be helpful in confirming a suspected parasitic infection. Specimens that are not going to be handled by routine histologic techniques should immediately be submitted to the laboratory. Although skin snips are rarely requested, they can be the specimen of choice for the diagnosis of certain human filarial infections such as Onchocerca volvulus and Dipetalonema streptocerca. For best results, the skin snip specimen should be thick enough to include the outer part of the dermal papillae. Specimens can be obtained by cutting a small slice from a skinfold held between thumb and forefinger, or a slice may be taken from a small "cone" of skin pulled up by a needle. 7 Lymph node biopsies can be very helpful in the diagnosis of trypanosomiasis, leishmaniasis, Chagas' disease, and toxoplasmosis. Impression smears of this material should be prepared and tissue submitted for routine histology. Artificial culture medium can also be used for organism recovery and immediate transport of the specimen to the laboratory is mandatory. Although the diagnosis of trichinosis is often based on the patient history, and possibly serologic results, confirmation can be obtained by the recovery and identification of Trichinella spiralis larvae in muscle. Larvae are most often recovered from the diaphragm, masseter muscle, or tongue at necropsy. The gastrocnemius muscle can be biopsied, and squash preparations examined as well as routine histologic preparations. Occasionally, human infection with larval cestodes may present diagnostic problems with the final diagnosis being dependent on surgical removal and examination of the organism. This diagnosis is often based on the recognition of calcareous corpuscles that occur in tapeworm tissue; species identification may not be possible without expert consultation. Rectal, colon, and bladder tissue may be submitted for the diagnosis of schistosomiasis. Fresh tissue, kept moist with saline, should be submitted to the laboratory. It is important to check for egg viability; thus fresh tissue and rapid transit time are necessary. Additional samples can also be submitted for routine histologic processing. The examination of tissue and bloody fluids for protozoa can be the most difficult diagnostic request. Some additional information can be seen in Table 2. BLOOD For accurate identification of blood parasites, Giemsa stain is recommended, although organisms can be seen with other blood stains such as Wright's stain. It is also important to realize that certain blood parasites, especially malaria, may not be diagnosed on the basis of a single specimen, and additional specimens and/or procedures may be required to recover and identifY the organisms. Blood can be submitted for the diagnosis of malaria, babesiosis, trypanosomiasis, leishmaniasis, and filariasis. Depending on the circumstances and the suspect organism, finger-stick blood or blood collected with anticoagulants can be acceptable (Fig. 9).
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Table 2. Examination of Tissue/Body Fluids (Protozoa) SUSPECT CAUSATIVE AGENT
DISEASE
APPROPRIATE TEST
1. Wet exam of CSF (not in counting chamber) 2. Stained prep. of CSF sediment Routine histology
POSITIVE RESULTS
Trophozoites present and identified
N aegleria fowleri
Primary amebic meningoencephalitis (PAM)
Entameba histolytica
Amebiasis
Giardia lamblia
Giardiasis
1. Duodenal aspirate 2. Duodenal biopsy 3. Entero-Test Capsule
Leishmania sp. (cutaneous lesions)
Cutaneous leishmaniasis
Leishmania sp. (mucocutaneous lesions)
Mucocutameous leishmaniasis
Leishmania sp. (visceral)
Viseral leishmaniasis (kala-azar)
Pneumocystis carinii
Interstitial plasma cell pneumonia
Toxoplasma gondii
Toxoplasmosis
Cryptosporidium
Cryptosporidiosis
1. Material from Amastigotes recovered in under bed of ulcer macrophages of skin, or (a) smear animals, other stages (b) culture recovered in culture (c) animal inoculation 2. Punch biopsy (a) routine histology (b) squash prep (c) culture (d) animal inoculation Amastigotes recovered in As above macrophages of skin and/or mucous membranes, or animals; other stages recovered in culture Amastigotes recovered in 1. Buffy Coat (a) stain cells of reticuloendothelial (b) culture system (c) animal inoculation 2. Bone marrow as above 3. Liver/spleen biopsy (a) routine histology (b) as above 1. Open lung biopsy Trophozoites or cysts present 2. Lung needle and identified aspirate 3. Bronchial brush 4. ITA (Silver methenamine stain recommended) 1. Lymph node biopsy Identification of tachyzoites (a) routine histology PLUS appropriate serologic (b) tissue culture test results isolation (c) animal inoculation 1. Duodenal scraping Identification of organisms in 2. Duodenal biopsy microvillus border (a) stain (b) routine histology 3. Punch biopsy (a) routine histology (b) squash prep (c) culture (d) animal inoculation
Trophozoites present and identified Trophozoites and/or cysts present and identified
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BLOOD (MALARIA) FINGER STICK (CAPILLARY BLOOD) EDTA
THICK FILM (200-300 FIELDS AT 1000 X)
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·------'1--
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REDRAW EVERY 6 HRS (COVER 36 HR) Figure 9. Examination for blood parasites (malaria).
Blood collected for the diagnosis of trypanosomiasis can be processed by the- examination of buffY coat films, a direct examination of the white cell layer in a centrifuged capillary tube, the triple centrifugation technique, or culture in artificial medium. 7 Although these tests, with the possible exception of culture, are relatively easy to perform, they are generally not available in most laboratories (Fig. 10). If visceral leishmaniasis is suspected, buffY coat films could be prepared from peripheral blood prior to examination of bone marrow. Although both spleen and liver biopsies are available, they are not without risk and are not routinely performed. Specimens from bone marrow or tissues could be examined as stained smears or touch preparations, both of which should be stained with Giemsa stain. These specimens can also be cultured, although this is not routinely available in most laboratories.
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BUFFY COAT CAPILLARY TUBE DIRECT EXAM TRIPLE CENTRIFUGATION CULTURE
Figure 10. Examination for blood parasites (trypanosomiasis).
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L _ _ _ _ _ _.,--_ _ _ _
1. 2. 3. 4.
(SPECIATE)
I
REVIEW HISTORY FOR POSSIBLE PERIODICITY BUFFY COAT KNOTT CONCENTRATE MEMBRANE FILTRATION
Figure 11. Examination for blood parasites (filariasiS).
If filariasis is suspected, a careful history should be taken to determine if there is a possibility of periodicity involved. This will certainly impact on the recommended time frame for blood drawing. After the specimen is obtained at the proper time, then thick films and buffy coat films can be examined. A blood concentration technique called the Knott concentration can also be used to recover microfilariae from a larger volume of blood.ll A somewhat different approach can also be used where dilute whole blood can be expelled through a membrane filter with the microfilariae being trapped on the membrane 3-5 (Fig. 11).
REFERENCES 1. Beal, C. B., Viens, P., Grant, R. G. L., et al.: A new technique for sampling duodenal contents. Demonstration of upper small-bowel pathogens. Am. J. Trop. Med. Hyg., 19:349-352, 1970. 2. Brooke, M. M., and Melvin, D. M.: Morphology of diagnostic stages of intestinal parasites of man. U.S. Department of Health, Education and Welfare Publication No. (HSM) 72-8116, Atlanta, 1969. 3. Dennis, D. T., and Kean, B. H.: Isolation of microfilariae: Report of a new method, J. Parasitol., 57:1146-1147, 1971. 4. Desowitz, R. S., and Hitchcock, J. C.: Hyperendemic Bancroftian filariasis in the Kingdom of Tonga: The application of the membrane filter concentration technique to an agestratified blood survey. Am. J. Trop. Med. Hyg., 23:877-879, 1974. 5. Desowitz, R. S., Southgate, B. A., and Mataika, J. U.: Studies on filariasis in the Pacific. 3. Comparative efficacy of the stained blood-film, counting-chamber, and membranefiltration techniques for the diagnosis of Wuchereria bancrofti microfilaraemia in untreated patients in areas of low endemicity. Southeast Asiao J. Trop. Med. Public Health, 4:329-335, 1973. 6. Finegold, S. M. and Martin, W. J.: Diagnostic Microbiology. St. Louis, The C. V. Mosby Co., 1982. 7. Garcia, L. S., and Ash, L. R.: Diagnostic Parasitology: Clinical Laboratory Manual. 2nd ed., St. Louis, The C. V. Mosby Co., 1969.
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7a. Garcia, L. S., Bruckner, D. A., Brewer, T. C., et al.: Techniques for the recovery and identification of C ryptosporidium oocysts from stool specimens. J. Clin. Microbiol., 18:185-190, 1983. 8. Graham, C. F.: A device for the diagnosis of Enterobius infection. Am. J. Trop. Med., 21:159-161, 1941. 9. Harada, Y., and Mori, 0.: A new method for culturing hookworm. Yonago Acta Med., 1:177-179, 1955. 10. Hsieh, H. C.: A test-tube filter-paper method for the diagnosis of Ancylosto1TUl duodenale, Necator americanus and Strongyloides stercoralis. WHO Tech. Rep. Ser., 255:27-30, 1963. 11. Knott, J.: A method for making microfilarial surveys on day blood. Trans. R. Soc. Trop. Med. Hyg., 33:191-196, 1939. 12. Lennette, E. H., Spaulding, E. H., and Truant, J. P.: Manual of Clinical Microbiology. 2nd ed. Washington, D.C., American Society of Microbiology, 1974. 13. Little, M. D.: Comparative morphology of six species of Strongyloides (Nematoda) and redefinition of the genus. J. Parasitol., 52:69-84, 1966. 14. Peri, G.: Errors in the diagnosis of Trichmonas vaginalis infection. Obstet. Gynecol., 39:7-9, 1972. 15. Stoll, N. R., and Hausheer, W. C.: Concerning two options in dilution egg counting; small drop and displacement. Am. J. Hyg., 6(Suppl.):134-145, 1926. Microbiology Laboratory Clinical Laboratory UCLA Medical Center Los Angeles, California 90024