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Preferred test for detection of Pneumocystis
Pneumocystis pneumonia is an important cause of opportunistic infection in immunocompromised patients, particularly those with HIV. The causative agent, Pneumocystis jiroveci, cannot be cultured in vitro and, therefore, laboratory detection has historically relied upon microscopic identification directly from patient specimens using fluorescent stains or antibodies. Unfortunately, stains often lack sensitivity and require expertise on the part of the reader in order to differentiate Pneumocystis jiroveci from staining artifacts and other fungi. This real-time PCR assay provides sensitive (21% more sensitive than direct detection using fluorescent calcofluor white stain), specific, and objective detection of Pneumocystis from bronchoalveolar lavage fluid and other specimens.
A positive result indicates the presence of Pneumocystis DNA.
A negative result indicates the absence of detectable Pneumocystis DNA.
Test results should be used as an aid in diagnosis and should not be considered diagnostic in themselves. The literature indicates that Pneumocystis can cause asymptomatic colonization of healthy and immunocompromised individuals. Therefore, test results should be correlated with patient symptoms and clinical presentation.
A negative result does not rule out the presence of Pneumocystis or active disease because the organism may be present at undetectable levels.
A total of 221 bronchoalveolar lavage (BAL) fluid samples were evaluated for the presence of Pneumocystis DNA by the LightCycler and compared to fluorescent microscopy using calcofluor white staining. Of the 221, 24 were positive and 190 were negative by both detection methods. The remaining 7 were positive by PCR and negative by microscopy. The 7 specimens that were positive using LightCycler PCR alone were tested using another PCR assay targeting a second Pneumocystis gene. All 7 specimens were positive using the second target suggesting that they were true positives that were undetected using the microscopic method. The sensitivity, specificity, positive and negative predictive values of this real-time PCR assay is 100%, 96%, 77%, and 100%, respectively. The analytical sensitivity of the method is 5.6 copies/mcL of positive plasmid control or approximately 28 copies/reaction. The analytical sensitivity in spiked, pooled BAL specimens was found to be 56 targets/mcL using the positive control plasmid. PCR inhibition was tested by spiking 50 extracted negative respiratory specimens (including 10 BAL fluid specimens) with 100 copies of target/mcL using a positive control plasmid. No PCR inhibition was detected. The specificity of the PCR assay was determined by evaluating DNA extracted from pure cultures of a variety of bacteria and fungi. Extracted human DNA was analyzed as well. None of the microbial or human DNA was amplified by the Pneumocystis LightCycler assay indicating that the assay is specific for Pneumocystis species.
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