pressed between two slides, and a smear is formed when the slides are pulled apart (one across the other).
3. The smears are allowed to air dry and then processed like a thin blood film (fixed in absolute methanol and stained with Giemsa stain or one of the other blood stains).
4. Appropriate culture media should be inoculated with any remaining material (see chapter 8).
5. If microsporidia are suspected, modified trichrome stains (Ryan blue, Weber green) can be used; calcofluor white and immunoassay methods (currently under development) are also excellent options (36–43).
Primary amebic meningoencephalitis is rare, but the examination of spinal fluid may reveal the amebae, usually Naegleria fowleri (Fig. 6.10). Granulomatous amebic encephalitis is caused by Acanthamoeba spp., Balamuthia mandrillaris, or Sappinia diploidea. Uncentrifuged sedimented spinal fluid should be placed under a coverslip on a slide and observed for motile amebae; smears can also be stained with Wright’s, Giemsa, or one of the other blood stains (including the rapid stains). Spinal fluid, exudate, or tissue fragments can be examined by light microscopy or phase-contrast microscopy. Care must be taken not to confuse leukocytes with actual organisms and vice versa. The appearance of the spinal fluid may vary from cloudy to purulent (with or without red blood cells), with a cell count from a few hundred to over 20,000 white blood cells (primarily neutrophils) per ml. Failure to find bacteria in this type of spinal fluid should alert one to the possibility of primary amebic meningoencephalitis. These organisms can be isolated from tissues or soil with special media (see chapter 8).
Figure 6.10 Naegleria fowleri. Note the large karyosome and lobate pseudopodia. Spinal fluid should be examined on a slide, not in a counting chamber, where protozoan trophozoites could mimic white blood cells. (Armed Forces Institute of Pathology photograph.) doi:10.1128/9781555819002.ch6.f10 (Armed Forces Institute of Pathology photograph.)
Note When spinal fluid is placed in a counting chamber, any organisms that settle to the bottom of the chamber will tend to round up and look very much like white blood cells. For this reason, it is better to examine the spinal fluid on a slide directly under a coverslip, not in a counting chamber.
A rapid method for the diagnosis of Acanthamoeba or microsporidial keratitis involves the use of calcofluor white, which is a chemofluorescent dye with an affinity for the polysaccharide polymers of amebic cysts and microsporidial spores (Fig. 6.11 and 6.12). The following method has proven to be very successful for examination of corneal scrapings or biopsy material (42).
Figure 6.11 Acanthamoeba cyst. Note the fluorescence of the cyst wall. Trophozoites do not fluoresce when calcofluor white is used. doi:10.1128/9781555819002.ch6.f11
Figure 6.12 Microsporidial spores. (Upper left) Spores in a corneal scraping specimen; (upper right) spores in a fecal specimen (both images stained with silver stain). (Lower left) Microsporidial spores in a urine sediment; (lower right) spores from nasopharyngeal aspirate (both images stained using calcofluor white). doi:10.1128/9781555819002.ch6.f12
1. Place corneal scrapings on slides, and air dry them.
2. Fix the slides in absolute methyl alcohol for 3 to 5 min.
3. Add several drops of solution (0.1% calcofluor white and 0.1% Evans blue dissolved in distilled water); leave on for 5 min.
4. Turn the slide on its side, and allow excess stain to run off onto paper towels.
5. Add a coverslip, and examine under the fluorescence microscope for pale blue chemofluorescence of the amebic cysts (will not stain trophozoite). The microsporidial spores usually stain brighter; however, fluorescence can vary between 1+ and 3+.
Note Select UV irradiation with an exciter filter which transmits the 365-nm group of intense mercury spectral emission lines (Zeiss UGI or G365). View through a barrier filter which removes UV while emitting visible blue light and longer wavelengths (Zeiss no. 41 or LP420).
Leishmaniasis
Material containing intracellular Leishmania organisms must be aspirated from below the ulcer bed through the uninvolved skin, not from the surface of the ulcer (Fig. 6.13). It is very important that the surface of the ulcer be thoroughly cleaned before specimens are taken; any contamination of the material with bacteria or fungi may prevent recovery of organisms from culture. Cutaneous ulcers can be seen in Fig. 6.14.
Figure 6.13 Proper way to aspirate material from below the ulcer bed (Leishmania spp.); sterile saline (0.5 to 1.0 ml) can be injected under the ulcer prior to aspiration. (Illustration by Sharon Belkin.) doi:10.1128/9781555819002.ch6.f13
Figure 6.14 Leishmania cutaneous lesions. doi:10.1128/9781555819002.ch6.f14
Biopsy specimens are recommended for the diagnosis of tissue parasites. The following procedures may be used for this purpose in addition to standard histologic preparations: impression smears and teased and squash preparations of biopsy tissue from the skin, muscle, corneas, intestines, liver, lungs, and brain. Tissue to be examined by permanent sections or electron microscopy should be fixed as specified by the laboratories that will process the tissue. In certain cases, examination of a biopsy specimen may be the only means of confirming a suspected parasitic infection. Specimens that are going to be examined as fresh material rather than as tissue sections should be kept moist in saline and submitted to the laboratory immediately.
Importance of Biopsy Specimens
Success in detection of parasites in tissue depends in part on specimen collection and on the presence of sufficient material to perform the recommended diagnostic procedures. Biopsy specimens are usually quite small and may not be representative of the diseased tissue. Examination of multiple tissue samples often improves diagnostic results. To optimize the yield from any tissue specimen, examine all areas and use as many procedures as possible. Tissues are obtained by invasive procedures, many of which are very expensive and lengthy; consequently, these specimens deserve the most comprehensive procedures possible. It is also important to remember that if the tissue specimen is not sufficient for all diagnostic procedures requested, the procedures should be prioritized after consultation with the physician.
Submission of Specimens
