jirovecii (previously P. carinii) has been reclassified with the fungi, this organism is no longer included in this book or discussed in terms of organism, disease, pathogenesis, and diagnosis. However, other organisms such as the microsporidia can be stained using silver stains, so these methods and references have been kept in this chapter and will periodically refer to P. jirovecii.
Although it is not one of the more common specimens, expectorated sputum may be submitted for examination for parasites. Organisms in sputum that may be detected and may cause pneumonia, pneumonitis, or Loeffler’s syndrome include the migrating larval stages of Ascaris lumbricoides, Strongyloides stercoralis, and hookworm; the eggs of Paragonimus spp.; Echinococcus granulosus hooklets; and P. jirovecii (now classified with the fungi), Entamoeba histolytica, Entamoeba gingivalis, Trichomonas tenax, Cryptosporidium spp., and possibly the microsporidia (now classified with the fungi) (1). In a Paragonimus infection, the sputum may be viscous and tinged with brownish flecks, which are clusters of eggs (“iron filings”), and may be streaked with blood (Fig. 6.1).
Figure 6.1 Paragonimus spp. eggs. (Left) Operculated eggs in a sputum specimen. (Right) Eggs photographed at a higher magnification. doi:10.1128/9781555819002.ch6.f1
Collection and Examination of the Specimen
Sputum is usually examined as a wet mount (saline or iodine), using low and high dry power (×100 and ×400). The specimen is not concentrated before preparation of the wet mount. If the sputum is thick, an equal amount of 3% sodium hydroxide (or undiluted chlorine bleach) can be added; the specimen is thoroughly mixed and then centrifuged. NaOH should not be used if Entamoeba spp. or T. tenax is being sought; the protozoa will be destroyed. After centrifugation, the supernatant fluid is discarded and the sediment can be examined as a wet mount with saline or iodine. If examination has to be delayed for any reason, the sputum should be fixed in 5 or 10% formalin to preserve helminth eggs or larvae or in one of the fecal fixatives to be stained later for protozoa. Usually, sputum is not a recommended specimen for the diagnosis of P. jirovecii; however, if such a specimen is accepted by the laboratory, other stains such as silver methenamine, Giemsa, or immunoassay reagents are used. If Cryptosporidium is suspected (rare), acid-fast or immunoassay techniques normally used for stool specimens can be used (see chapters 3 and 15) (2–5). Trichrome stains of material may aid in differentiating E. histolytica from E. gingivalis, and Giemsa stain may better define larvae and juvenile worms.
A sputum specimen should be collected properly so the laboratory receives a “deep sputum” sample for examination rather than a specimen that is primarily saliva from the mouth rather than from the lower respiratory passages. If the sputum is not induced, the patient can be instructed as follows.
1. Expectorated sputum specimens are collected after the patient is instructed in the appropriate measures to ensure high-quality specimens, including prior mouth washing with hydrogen peroxide and exclusion of saliva from specimens. Try to obtain the specimen early in the morning, when the chances of obtaining a deep sputum specimen are increased.
2. Transport the specimen to the laboratory in clean, closed containers as quickly as possible. Select any blood-tinged, viscous areas for sampling.
3. If the specimen is uniformly mucoid:
A. Wear gloves when handling specimens.
B. Remove a 1.0-ml portion to a 15-ml conical tube.
C. Add 1.0 ml of mucolytic agent such as Sputalysin (Sputalysin Stat-Pack dithiothreitol solution [Behring Diagnostics, Inc.]) which has been prepared as specified by the manufacturer. This reagent can be stored unopened at room temperature until the expiration date. Date and store working solution at 4°C (include the expiration date). Discard working solution after 48 h. Prepare working solution by removing 1.0 ml from the 10-ml bottle and diluting with 9.0 ml of sterile water.
D. Incubate at room temperature for 15 min.
E. Add 2.0 ml of phosphate buffer (pH 6.8; M/15 [0.067 M]).
F. Centrifuge the material at 500 × g for 5 min.
G. Decant supernatant fluid, and use sediment to prepare wet mounts and smears.
H. Quality control measures should include the following.
a. Ensure that the saline and mucolytic agent are free of contamination (particulate matter) as determined by a clear appearance. If cloudy, discard and make new working solution.
b. Control the trichrome stain for each new set of reagents with a specimen containing blood to ensure that white cells stain with purple nuclei and blue-green cytoplasm. If cells do not stain appropriately, change reagents.
c. With each new lot of Giemsa stain or new buffer, check the stain with a specimen containing blood to ensure that red cells stain grayish, white cell nuclei stain red-purple, and cytoplasm stains bluish. If cells do not stain appropriately, check the stock stain and buffer to find cause.
d. Yeast-containing material may be used as a positive control for silver staining.
e. Both P. jirovecii-positive and P. jirovecii-negative and yeast-positive material should be used for immune-specific staining (check with suppliers).
f. Stains cannot be evaluated if controls do not stain appropriately. A stain is not within acceptable results when:
(i) P. jirovecii does not stain or stains black without delineation of “parentheses.”
(ii) Other fungi and actinomycetes do not stain.
g. Store stock stain solutions in area away from light; discard any reagent (prior to the expiration date) that is cloudy or appears contaminated.
h. Evaluate the fluorescence microscope for correct light wavelength, using commercially available quality control slides.
i. Record all quality control results.
Procedure for Examination of Sputum
1. Wear gloves when performing these procedures.
2. For expectorated sputum (no addition of mucolytic agent):
A. Using a Pasteur pipette, place 1 or 2 drops (50 µl) on one side of a 2- by 3-in. (1 in. = 2.54 cm) glass slide, and cover with a 22- by 22-mm (no. 1) coverslip.
B. Place a second drop on the slide, add 1 drop of saline, and cover with a coverslip.
3. Expectorated sputum (treated with a mucolytic agent) can be resuspended in 100 µl of saline; place 1 drop on a 2- by 3-in. slide, and cover with a coverslip.
4. Save the untreated specimen and remaining treated specimen for permanent smear preparation if stains are required.
5. Examine the entire 22- by 22-mm coverslip wet preparation under low light with the
