Nonnutrient agar plate seeded with bacteria (see chapter 8): add drops to center of seeded agar plate, and incubate at 35 to 37°C (isolation of Acanthamoeba or Naegleria spp.).
6. Examine cultures (wear gloves at all times).
A. NNN agar for promastigotes of Leishmania spp. (see chapter 8): using a Pasteur pipette, remove a drop of fluid from interface of agar slant and culture tube, place on glass slide, cover with coverslip, and examine microscopically (×400) under low light for motile promastigotes (Fig. 6.17).
Figure 6.17 Leishmania culture sediment. Note the promastigotes from the culture sediment. The typical “rosette” formation in the right image is frequently seen. doi:10.1128/9781555819002.ch6.f17
B. TYSGM-9 medium for amebae (see chapter 8): using a sterile Pasteur pipette, remove 1 or 2 drops of material from interphase of agar and overlay, place on glass slide, cover with coverslip, and examine microscopically (×100) under low light for trophozoite motility.
C. Nonnutrient agar plate with lawn of Escherichia coli or Enterobacter sp. for detection of free-living amebae (see chapter 8): examine microscopically at low power (×100) for changes in bacterial lawn, particularly patches and tracks, indicating that the protozoan trophozoites have ingested bacteria as they move over the agar.
D. Mouse passage for detection of Toxoplasma gondii:
a. Wear canvas gloves to handle mice; sacrifice the animal(s).
b. Pin mouse to board, spray with 70% ethyl alcohol, and open peritoneal cavity with sterile scissors.
c. Using a sterile Pasteur pipette, remove fluid from the peritoneal cavity, and place in small tube or prepare smears.
d. Using one of the blood stains, prepare stained smears of peritoneal exudate, and examine microscopically at a magnification of ×1,000 for T. gondii tachyzoites.
e. Place mice and all contaminated disposable materials in bag to be autoclaved and destroyed. Place nondisposable materials (scissors) in bag to be autoclaved prior to washing.
7. Correlate all examination results (wet mount, stains, and cultures) to determine presence of organisms.
Results
The majority of the protozoa are found on the permanent stained smears (impression smears, touch or squash preparations, or teased preparations). When culture is used, permanent stained smears of the culture medium or sediment may also reveal some of the protozoa. Although infrequently used, material from animals (at autopsy) can be examined as both wet and permanent stained preparations for confirmation of protozoa. Filarial infections may be confirmed by the recovery and identification of microfilariae in skin scrapings and/or biopsy specimens.
If identifications are certain, report the organisms detected; if a presumptive identification is made, confirmation by another laboratory is suggested.
Onchocerca volvulus and Mansonella streptocerca
The use of skin snips is the method of choice for the diagnosis of human filarial infections with O. volvulus and M. streptocerca. Microfilariae of both species occur chiefly in the skin, although O. volvulus microfilariae are on rare occasions found in the blood and occasionally in the urine. For best results, the skin snip specimens should be thick enough to include the outer part of the dermal papillae. Snips may be taken in various ways. A small slice may be cut (using a razor blade) from a skin fold held between thumb and forefinger, or a slice may be taken from a small “cone” of skin pulled up by a needle. The skin snip should be so thin that significant bleeding does not occur, just a slight oozing of fluid. Corneal-scleral punches (either Holth or Walser type) have been found to be successful in taking skin snips of uniform size and depth and an average weight of 0.8 mg (range, 0.4 to 1.2 mg); this procedure is easy to perform and is painless. It has been demonstrated that in African onchocerciasis, it is preferable to take skin snips from the buttock region (above the iliac crest); in Central American onchocerciasis, the preferred skin snip sites are from the shoulders (over the scapula).
Skin snips are placed immediately in a drop of normal saline or distilled water and covered so that they will not dry; teasing the specimen with dissecting needles is not necessary but may facilitate release of the microfilariae. Microfilariae tend to emerge more rapidly in saline; however, in either fluid, the microfilariae usually emerge within 30 min to 1 h and can be examined under low-intensity light with the 10× objective of the microscope. To see definitive morphologic details of the microfilariae, allow the snip preparation to dry, fix it in absolute methyl alcohol, and stain it with Giemsa or one of the other blood stains (Fig. 6.18). For the differential diagnosis of O. volvulus and M. streptocerca, see chapter 23.
Figure 6.18 Microfilariae (Giemsa stain). (Upper) In this image, the microfilariae from a skin snip saline preparation are visible after staining. (Lower) Microfilariae from blood; note that the characteristic nuclei are more easily seen in this preparation. doi:10.1128/9781555819002.ch6.f18
Cutaneous Amebiasis and Cutaneous Leishmaniasis
Skin biopsy specimens for the diagnosis of cutaneous amebiasis and cutaneous leishmaniasis should be processed for tissue sectioning and subsequently stained by the hematoxylin-eosin technique.
Itch Mites (Sarcoptes scabiei)
Several genera infect the skin of mammals, with S. scabiei being found in humans. S. scabiei is microscopic and lives in cutaneous burrows, where the fertilized female deposits eggs (Fig. 6.16). Scabies is transmitted by close contact with infested individuals, including touching, shaking hands, sexual contact, or day care centers with children or the elderly. The usual skin sites that are susceptible to infection are the interdigital spaces, backs of the hands, elbows, axillae, groin, breasts, umbilicus, penis, shoulder blades, small of the back, and buttocks. The most dramatic symptom is intense itching. Scratching commonly causes weeping, bleeding, and sometimes secondary infection. Specific and detailed diagnostic methods for the recovery of mites, eggs, or scybala (fecal pellets) can be found in the chapter on arthropods (chapter 35).
Trypanosomiasis, Leishmaniasis, Chagas’ Disease, and Toxoplasmosis
Material obtained from lymph nodes should be processed for tissue sectioning and as impression smears that should be processed as thin blood films and stained with Giemsa or other blood stains. Appropriate culture media can also be inoculated, again making sure that the specimen has been collected under sterile conditions.
