by open or brush biopsy. P. jirovecii can also be seen in stained smears of tracheobronchial aspirates, although examination of lung tissue is more likely to reveal the organisms. Sputum specimens are generally considered unacceptable for the recovery of P. jirovecii; however, in patients with severe, progressive disease, such specimens may be acceptable. To avoid the possibility of false-negative results, acceptance of sputum specimens should be carefully monitored and reviewed. Also, even for patients with fulminant disease, multiple specimens may have to be submitted to recover and identify the organisms. With a number of the stains that are available, the cyst walls stain but the cyst contents do not (Fig. 6.4); however, only the methenamine silver stain is discussed here (15).
Amebiasis
Examination of aspirates from lung or liver abscesses may reveal trophozoites of E. histolytica (Fig. 6.6); demonstration of the organisms is often very difficult. In many cases, serologic confirmation is recommended (35). Liver aspirate material should be taken from the margin of the abscess rather than the necrotic center (Fig. 6.7). The organisms are often trapped in the viscous pus or debris and may not exhibit typical motility. The Amoebiasis Research Unit, Durban, South Africa, has recommended using proteolytic enzymes to free the organisms from the aspirate material.
Figure 6.6 Entamoeba histolytica containing ingested red blood cells within the cytoplasm; morphologically this organism is E. histolytica, and this would be the species designation if organisms were isolated from liver abscess material (trichrome stain). doi:10.1128/9781555819002.ch6.f6
Figure 6.7 (Upper) Liver abscess caused by Entamoeba histolytica. Amebae would be found at the advancing margin of the lesion; the last portion of the aspirated material might reveal the organisms. (Illustration by Sharon Belkin.) (Lower) Amebic abscess; note the “flask”-shaped ulcer. doi:10.1128/9781555819002.ch6.f7
The digestion technique is performed as follows.
1. A minimum of two separate portions of exudate should be removed (more than two are recommended). The first portion of the aspirate, usually yellowish white, rarely contains organisms. The last portion of the aspirated abscess material is reddish and is more likely to contain amebae. The best material to examine is that obtained from the actual wall of the abscess.
2. Add 10 U of streptodornase per ml of thick pus; incubate this mixture for 30 min at 37°C, and shake repeatedly.
3. Centrifuge the mixture at 500 × g for 5 min. The sediment may be examined microscopically as wet mounts or used to inoculate culture media. Some of the aspirate can be mixed directly with a fecal fixative on a slide, allowed to air dry, stained, and examined as a permanent stained smear.
Hydatid Disease
Aspiration of cyst material for the diagnosis of hydatid disease is a dangerous procedure and is normally performed only when open surgical techniques are used for cyst removal. Aspirated fluid usually contains hydatid sand (intact and degenerating protoscolices, hooklets, and calcareous corpuscles) (Fig. 6.8). Some older cysts contain material that resembles curded cottage cheese, and the hooklets may be very difficult to see. Some of this material can be diluted with saline or 10% KOH; usually, protoscolices or daughter cysts will have disintegrated. However, the diagnosis can be made by seeing the hooklets, which can be stained using the Ryan blue modified trichrome stain (see chapter 3) (Fig. 6.9).
Figure 6.8 Hydatid disease (Echinococcus granulosus). (Upper left) Hydatid cyst with protoscolices budding off from the germinal layer. (Upper right) Immature protoscolices, with the dark area being the hooklets. (Lower left) Higher magnification of the protoscolices taken from the hydatid cyst fluid. (Lower right) Hooklets from disintegrating protoscolices. Reprinted from reference 3. doi:10.1128/9781555819002.ch6.f8
Figure 6.9 Hydatid disease (Echinococcus granulosus). (Upper) Hydatid protoscolex revealed by Ryan modified trichrome stain. (Lower) Hydatid hooklets revealed by Ryan blue modified trichrome stain (reprinted from reference 30). doi:10.1128/9781555819002.ch6.f9
Examination of hydatid cyst material is carried out as follows.
1. If the cyst material is fluid, centrifuge at 500 × g for 3 min.
2. Carefully remove some of the sediment and prepare a wet mount.
3. Examine the material under low (100×) and high dry (430×) power (remember to use low light intensity, since some of the material may be very transparent).
4. If the cyst material is more viscous or solid, the material can be mixed with saline or 10% KOH and then centrifuged at 500× g for 3 min.
5. Some of the very viscous material can be placed on a glass slide (undiluted). Another glass slide can be placed on top of the first (material will now lie between the two slides). Rub the glass slides back and forth over each other and listen for a grating sound (like grains of sand being scratched). If this occurs, you may be hearing evidence of the presence of calcified hooklets. Place the material from the smears (may be diluted with saline) under the microscope to try to confirm the presence of hooklets (which may be extremely difficult to see with only low power).
6. A dried smear of the cyst aspirate can be stained using the Ryan blue modified trichrome stain (see chapter 3). The hooklets will stain reddish-purple in color; remember they are quite small, measuring 20 to 40 µm long.
Note Remember, the absence of protoscolices or hooklets does not rule out the possibility of hydatid disease, since some cysts are sterile and contain no protoscolices and/or daughter cysts. Review of the cyst wall from pathology (tissue sections) should be able to confirm the diagnosis.
Lymph Nodes, Spleen, Liver, Bone Marrow, Spinal Fluid, Eyes, and Nasopharynx
African Trypanosomiasis, Leishmaniasis, Chagas’ Disease, Primary Amebic Meningoencephalitis, Granulomatous Amebic Encephalitis, Amebic Keratitis, and Microsporidial Keratitis
Material from lymph nodes, spleen, liver, bone marrow, spinal fluid, eye specimens, or nasopharynx may be examined for the presence of parasites and should be processed as follows.
1. A portion of the fluid material can be examined under low (×100) and high dry (×400) power as a wet mount (diluted with saline) for the presence of motile organisms. Spinal fluid should not be diluted before examination.
2. Impression smears from tissues should be prepared and stained with Giemsa or another blood stain. The material
