can also be seen in the background. doi:10.1128/9781555819002.ch6.f5
Other methods such as fluorescent-antibody and immunoperoxidase techniques have been used for P. jirovecii (13–17). The development of tissue culture procedures for P. jirovecii may lead to additional diagnostic procedures for this infection. Immunoassay reagents are also available (14–18) and are being widely used in many diagnostic laboratories.
When noninduced sputum and the direct fluorescent-antibody technique were used, the sensitivity was 55%; this is within the range reported in the literature for the diagnosis of Pneumocystis pneumonia from induced sputum (19). Although detection of P. jirovecii has increased with the use of PCR, particularly in sputum specimens, some workers recommend that sputum samples of HIV-infected patients be tested by both PCR and immunofluorescence. The results of PCR in this patient group could be misleading without careful clinical evaluation (20). However, the level of sensitivity seen using PCR and other molecular tests indicates that these procedures should be considered for patients in whom Pneumocystis pneumonia is suggestive clinically and the specimen is negative by the immunofluorescence test (21, 22).
Quality Control Measures
Quality control measures for all stains should include the following, and control slides must be incorporated into all stain procedures.
1. Examine a control slide for each stain prior to examination of specimen stains. The stain intensity of controls will be a guide to the stain appearance of organisms in specimens.
2. Examine stained specimen smears by systematically moving from field to field until the majority of the smear has been covered (total area for silver stain).
3. In Giemsa stains, Pneumocystis trophozoite clumps of various sizes may be detected. In large clumps, it may be difficult to differentiate individual organisms. Look at organisms at the edges of clumps, and look for small, more dispersed clumps.
4. In silver- or other cyst wall-stained smears, look for the various cyst forms, including those that show dark centers, cup-shaped cysts, and cysts with foldlike lines (they look like “punched-in” ping-pong balls). If dark-staining organisms appear more oval, look carefully for budding forms, which indicate that the organisms are yeasts. It is important to review thinner areas of the smear for microsporidial spores.
A. P. jirovecii cysts: 70% should have delicately stained walls, usually brown or gray. They appear somewhat transparent, with structures described as “parentheses” staining black; these curved structures are usually thick (much thicker than the cyst wall) rather than thin like a line drawing.
B. Other fungi and actinomycetes: gray to black. Microsporidial spores stain dark gray to black; some of the spores may contain the polar tubule (horizontal or diagonal stripe), but they are more difficult to see in the silver stain than in the modified trichrome stains.
C. Glycogen, mucin, and red blood cells: rose taupe to gray.
D. Background: pale green (from the light green counterstain).
5. Detection of P. jirovecii in specimen smears from one type of stain always suggests a careful examination of the other stain, hopefully to confirm the identification.
6. Retain all positive stained specimen slides and control slides for reference.
Reporting Smear Results
1. Report P. jirovecii as follows (high dry power; total magnification, ×400).
A. “No Pneumocystis jirovecii seen.”
B. “Pneumocystis jirovecii seen” (no quantitation should be included).
2. Report other fungi that may be present as follows (low power; total magnification, ×100).
A. “No mycotic elements seen.”
B. “Budding yeasts” or “Budding yeasts and pseudohyphae resembling Candida species” are quantitated as few, moderate, or many.
C. Hyphae are reported with no quantitation:
a. “Septate hyphae seen.”
b. “Nonseptate hyphae seen.”
3. Report actinomycetes (oil immersion; total magnification, ×1,000) as follows.
A. “No filamentous branching bacteria seen.”
B. “Filamentous branching bacteria seen” (no quantitation).
4. Report microsporidia (oil immersion; total magnification, ×1,000) as follows.
A. “No microsporidial spores seen.”
B. “Microsporidial spores seen; identification to genus not possible.” However, if the specimen is stool or other gastrointestinal tract specimens, the organisms are probably 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.