to detect eggs, larvae, oocysts, or amebic trophozoites.
6. If results are inconclusive, prepare smears for permanent staining.
A. Place 1 drop of sediment in the center of each of three 1- by 3-in. glass slides, add 2 to 3 drops of fecal fixative to one of the slides, and mix/spread the material with the tip of the pipette.
B. Allow all three smears to air dry a minimum of 30 min (they can be incubated for drying at 37°C).
C. Trichrome stain the slide fixed in fecal fixative.
D. Fix the other two air-dried smears in methanol; stain one with Giemsa and the other with a modified acid-fast stain. The modified trichrome stain may have to be used if infection with microsporidia is suspected (6, 7).
E. Put immersion oil on stained smears; examine the Giemsa-stained smear with the 10× objective and the trichrome-stained smear with the 50× oil objective if available; otherwise, use the 100× oil objective. Read at least 300 oil immersion fields (total magnification, ×1,000) before reporting the specimen as negative.
F. The methenamine silver stain can be used; however, expectorated sputum is generally not recommended as an acceptable specimen for the recovery and identification of P. jirovecii.
7. Report any organisms found as follows.
A. Give genus and species, if necessary, after confirmation with permanent stain.
B. “No parasites found” in expectorated sputum is considered a normal/negative report; therefore, call the physician if any organisms are found.
Note Care should be taken not to confuse E. gingivalis, which may be found in the mouth and saliva, with E. histolytica, which could result in an incorrect suspicion of pulmonary abscess. E. gingivalis usually contains ingested polymorphonuclear leukocytes, while E. histolytica may contain ingested red blood cells but not polymorphonuclear leukocytes (Fig. 6.2). T. tenax would also be found in saliva from the mouth and thus would be an incidental finding and normally not an indication of pulmonary problems (Fig. 6.3).
Figure 6.2 (Upper) Entamoeba gingivalis containing ingested polymorphonuclear leukocytes (PMNs) within large vacuoles. (Lower) Entamoeba histolytica containing ingested red blood cells (RBCs). Note: the nuclear characteristics are very similar; however, the vacuole sizes are quite different (large vacuoles contain ingested PMNs, small vacuoles contain ingested RBCs). doi:10.1128/9781555819002.ch6.f2
Figure 6.3 (Left) Trichomonas tenax from the mouth (stained with Giemsa stain). (Right) Trichomonas vaginalis from a genital specimen (stained with Giemsa stain). Note the large nucleus in T. tenax and the fact that this flagellate is somewhat smaller than T. vaginalis. doi:10.1128/9781555819002.ch6.f3
Concentrated stained preparations of induced sputum specimens are commonly used to detect P. jirovecii and differentiate trophozoite and cyst forms from other possible causes of pneumonia, particularly in AIDS patients (8). Organisms must be differentiated from other fungi such as Candida spp. and Histoplasma capsulatum. Although induced sputum specimens have been used successfully in the diagnosis of P. jirovecii in some institutions, they have not been useful in others. This difference may be due in part to careful adherence to specimen rejection criteria. If the clinical evaluation of a patient suggests P. jirovecii pneumonia and the induced sputum specimen is negative, a bronchoalveolar lavage fluid specimen should be evaluated with the stains presented in this protocol or newer molecular methods (9).
Collection and Examination of the Specimen
Induced sputum specimens are collected by pulmonary or respiratory therapy staff after patients have used appropriate cleansing procedures to reduce oral contamination. Nebulizing procedures are generally determined by the respiratory therapy staff collecting the specimens. The induction protocol is critical for the success of the procedure, and it is mandatory for well-trained individuals to be involved in the recovery of organisms. Patients with Pneumocystis pneumonia usually have dry, nonproductive coughs. Organisms are rarely detected in expectorated sputum, which is not accepted by many laboratories as a clinically relevant specimen. In cooperation with the pulmonary staff, the laboratory processing the specimens must establish a protocol for this diagnostic procedure. Induced sputum specimens are most useful for detection of P. jirovecii in HIV-infected individuals, because others have fewer, less readily detected organisms. Stains that can be used for organism detection include the rapid methenamine silver stain, the rapid Giemsa stain (Diff-Quik or Giemsa Plus), the Giemsa stain, and immune-specific staining (10).
Preparation of the Specimen Prior to Staining
1. Wear gloves when handling specimens.
2. Specimens which contain mucus should be treated with a mucolytic agent by adding the agent in a 1:1 ratio with the specimen, usually 2 or 3 ml, and incubating the specimen at room temperature for 15 min. Large-volume specimens usually are watery. These specimens (up to 20 to 25 ml) should be concentrated by centrifugation prior to the addition of a mucolytic agent.
3. Centrifuge the specimen at 500 × g for 5 min in capped centrifuge tubes and closed carriers.
4. Decant supernatant fluids into a disinfectant solution (1:10 dilution of bleach).
5. If the sediment contains a significant amount of blood, treat a portion (one-half to one-third) with a red-cell lytic agent such as saponin or Lyse in one-half to one-third of the volume of the sediment, leave at room temperature for 5 min, and recentrifuge.
6. Decant supernatant fluid from treated specimens.
7. Use Pasteur pipettes to resuspend remaining sediment.
8. Using 1- by 3-in. glass slides, place drops of sediment in the center of each slide. For specimens treated to lyse red cells, prepare two smears, one from material before lytic treatment and one from the treated specimen.
9. Spread the drops with the pipette so that they are thin and even.
10. Air dry slides, and dip them in methanol prior to Giemsa or silver staining. Fix slides for immune-specific staining as specified in the package insert directions.
Reagents Used in Staining Procedures
1. Mucolytic agent: Sputalysin Stat-Pack dithiothreitol solution (Behring Diagnostics, Inc.). Red cell lytic agent: saponin (Aldrich, Milwaukee, WI; ICN Biochemicals, Costa Mesa, CA), Lyse (Curtin Matheson Scientific, Inc.), or Hematall LA-Hgb reagent (Fisher Scientific).
2. Rapid Giemsa stain: Diff-Quik (Baxter Scientific Products) or Wright’s Dip Stat (Medical Chemical). Follow the manufacturer’s instructions or use Giemsa stain.
3. Giemsa stain: azure B alcoholic stock (Harleco,
