antibodies specific for human strain P. jirovecii are now available. The commercial systems vary; some are indirect stains, and some are direct stains. Reports with all systems have been variable. Many laboratories are now using these immunospecific stains.
9. Select an organism stain and a cyst wall stain or immunospecific stain; use of a pair of stains will help avoid both false-negative and false-positive reporting.
10. In addition to organism detection, cytocentrifuge preparations of sputum can be used to determine cell populations for further patient evaluation.
The examination of aspirated material for the diagnosis of parasitic infections may be extremely valuable, particularly when routine testing methods have failed to demonstrate the organisms. These types of specimen should be transported to the laboratory immediately after collection. Aspirates include liquid specimens collected from a variety of sites where organisms might be found. Aspirates most commonly processed in the parasitology laboratory include fine-needle aspirates and duodenal aspirates. Fluid specimens collected by bronchoscopy include bronchoalveolar lavage fluid and bronchial washings.
Procedural details for sigmoidoscopic aspirates and scrapings for the recovery of E. histolytica are presented in chapter 5. Techniques for preparation of duodenal aspirate material are also presented in that chapter.
Specimens Obtained from Aspiration Procedures
Fine-needle aspirates are often collected by cytopathology staff, who process the specimens, or they may be collected and sent to the laboratory directly for slide preparation and/or culture. Suggested stains are Giemsa and methenamine silver for P. jirovecii, Giemsa for Toxoplasma gondii (25), trichrome for amebae, modified acid-fast stains for Cryptosporidium (2), and modified trichrome stains for the microsporidia (see chapter 3).
Aspirates of cysts and abscesses to be evaluated for amebae may require concentration by centrifugation, digestion, microscopic examination for motile organisms in direct preparations, and cultures and microscopic evaluation of stained preparations.
Duodenal aspirates to be evaluated for S. stercoralis, Giardia lamblia (G. duodenalis, G. intestinalis). Cryptosporidium spp., Cyclospora cayetanensis, or the microsporidia require concentration by centrifugation prior to microscopic examination for motile organisms and permanent stains (26–29).
Cyst aspirates to be evaluated for hydatid “sand” (daughter cysts, small protoscolices, hooklets) can be examined as wet direct mounts after centrifugation. Several stains can be used to visualize the hooklets; one of the best is the Ryan blue modified trichrome stain for the microsporidia (see chapter 3). The stained smears can be examined using routine microscopy with transmitted light (30).
Bone marrow aspirates to be evaluated for Leishmania amastigotes, Trypanosoma cruzi amastigotes, or Plasmodium spp. require staining with Giemsa or another blood stain (31, 32).
Fluid specimens collected by bronchoscopy may be lavages or washings, with bronchoalveolar lavages preferred. Specimens usually are concentrated by centrifugation prior to microscopic examination of stained preparations. Organisms included here which may be detected are P. jirovecii, Toxoplasma gondii, and Cryptosporidium spp.
Pneumocystosis
Although formerly classified with the sporozoa, P. jirovecii has been reclassified with the fungi. It is an important cause of pulmonary infections, particularly in patients who are immunosuppressed as a result of therapy or from congenital or acquired immunologic deficiencies (33, 34). Clinically, the infection may involve both lungs diffusely, may be localized, or may be disseminated. For immunosuppressed patients, in whom the disease may progress very quickly, correct specimen collection and rapid diagnostic techniques are very important. The organisms can be demonstrated in stained impression smears of lung material obtained 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