Laboratory diagnosis of Pneumocystis jiroveci
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p769
2025-12-04
4
SPECIMEN COLLECTION AND TRANSPORT
Respiratory specimens from the deep portions of the lung, such as bronchoalveolar lavage fluid (BALF), are best for detection of P. jiroveci. A sputum specimen sub mitted for direct examination should be an induced sputum obtained by a trained respiratory therapist; otherwise, the rate of false-negative results may be unacceptably high.
SPECIMEN PROCESSING
See the following section for specific details for specimen processing required for different test methods.
DIRECT DETECTION METHODS
Stains
The diagnosis of P. jiroveci pneumonia currently is based on the clinical presentation, radiographic studies, and direct and/or pathologic examination of bronchoalveolar lavage fluid or biopsy material. The flexible-walled trophozoites are the predominant form of the organism, but these are difficult to visualize. They are somewhat discernible in Giemsa-stained material, but their pleomorphic appearance makes this form of the organism difficult to identify. A firm-walled cystic form also exists, although the cysts are outnumbered by the trophozoites 10 to 1. Cysts are more easily recognized than trophozoites and may be definitively identified using a variety of stains, such as calcofluor white, methenamine silver, and immunofluorescent staining (Figure 1). The cysts are spherical to concave, uniform in size (4 to 7 μm in diameter), do not bud, and contain distinctive intracystic bodies.
Fig1. Cystic forms of Pneumocystis jiroveci (arrows) stain well with methenamine silver and hematoxylin and eosin stain (×500).
A comparison of the four most common staining methods used for P. jiroveci (i.e., Giemsa, immunofluorescent, calcofluor white, and methenamine silver) has demonstrated that immunofluorescent staining (Meri fluor Pneumocystis; Meridian Bioscience, Cincinnati, Ohio), calcofluor white staining (Fungifluor; Poly sciences, Warington, Pennsylvania), and methenamine silver staining (GMS and DiffQuick; Baxter Scientific, McGraw Park, Illinois) likely represent the best balance between sensitivity and specificity and have the best overall positive and negative predictive values. The immunofluorescent method showed greater sensitivity than the other three but a smaller negative predictive value. Therefore, if this method is used as a screening tool for pneumocystis, a confirmatory method should be performed because of the high number of false-positive results.
Antigen-Protein
Commercial kits use monoclonal antibodies directed against P. jiroveci to stain both the cysts and the trophozoites. These tests are a highly sensitive microscopic method of detection, but they are expensive. Also, because they use nonspecific staining, which may be thought to represent the pleomorphic trophozoite forms, the specificity of this assay may be limited.
Nucleic Acid Amplification
A variety of nucleic acid amplification assays for P. jiroveci have been developed, including, most recently, real-time polymerase chain reaction (PCR) methods. These methods are making their way from the research setting into the clinical molecular microbiology laboratory, but commercial kits are not yet available.
Cultivation
P. jiroveci is very difficult to cultivate outside the lung; therefore, routine culture methods are not performed.
Approach to Identification
See Direct Detection Methods.
Serodiagnosis
Serology is not useful for the diagnosis of pneumocystosis.
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