Enterocytozoon bieneusi or Encephalitozoon intestinalis; a note containing this information can be attached to the report.
Notes on Staining Procedures
1. Use at least two stains for detection and identification of P. jirovecii. With traditional histochemical stains, a trophozoite stain such as Giemsa and a cyst wall stain such as methenamine-silver nitrate are recommended.
2. There are many cyst wall stains in addition to the one described, and there are other modifications of the silver stain (12, 23).
3. Stain effectiveness varies. Other counterstains may be used; a counterstain with Giemsa is useful if referral slides will be submitted for examination.
4. When selecting a cyst wall stain, consider stain quality, reagent stability, and potential testing frequency (including STAT requests). Toluidine blue O stains vary in dye lots and in the stability of sulfation reagents (24). In the modified procedure, the sulfation reagent made of glacial acetic acid and concentrated sulfuric acid presents disposal problems.
5. The rapid silver stain described is a modification of the procedure described by Brinn (11). Heating the chromic acid may cause nonspecific staining, making the background too dark. Also, buildup of silver on the stain container may interfere with staining. Bleach should be added to the stain jar after staining, and jars should be scrubbed periodically with a brush. Additional tips for getting good stain results include the following.
A. Before use, inspect glassware to ensure that it does not have residual silver deposits.
B. Use fresh reagents for each run (5% chromic acid, 1% sodium bisulfite, methenamine-silver nitrate working solution, and 2% sodium thiosulfate).
C. Distilled deionized water must be used throughout the procedure, including sliderinses (microwave procedure).
D. Methenamine-silver nitrate working solution must be clear; if it becomes opaque at any time, the reduction step may take longer or may not occur at all. Check the distilled deionized water source, and prepare fresh methenamine-silver nitrate working solution.
6. If mounted slides appear opaque or cloudy, the dehydration and clearing with xylene (or xylene substitute) were not adequate. Soak slides in xylene to remove the coverslips. Using fresh ethanol and xylene, repeat the dehydration steps.
7. Degenerating polymorphonuclear leukocytes may resemble P. jirovecii.
8. Monoclonal 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
