Development and Validation of a Diagnostic Real-time PCR Assay for the Rapid and Accurate Detection of Pneumocystis jirovecii from Bronchoalveolar Lavage Fluid Samples
Pneumocystis jirovecii causes severe interstitial pneumonia called Pneumocystis pneumonia (PcP) among immunocompromised patients. Laboratory diagnosis of PcP currently relies on direct microscopic examination of respiratory specimens. The purpose of this study was to develop and validate an in-house PCR Assay for the rapid, reliable, and accurate detection of Pneumocystis jirovecii from bronchoalveolar lavage fluid (BALF) samples.
This study analyzed 92 BALF samples sent to Calgary Laboratory Services (CLS) between May 2011 and September 2012 for detection of P.jirovecii. DNA from the BALF samples was subjected to an in-house real-time PCR assay targeting the mitochondrial large subunit ribosomal RNA gene. Results were compared to the existing gold standard immunofluorescence assay (IFA) at CLS. Discrepant positive PCR samples were sent to St. Paul's hospital (SPH, Vancouver) for repeat PCR testing through their in-house PCR assay. Test characteristics were re-calculated using a modified gold standard that defined a positive test as one that was positive through both PCR assays in an immunocompromised patient.
54% of the samples were derived from immunosuppressed patients. The immunosuppressed samples included those with HIV (18%), malignancies (28%), solid-organ transplants (46%), hematopoietic stem cell transplant (4%), and others (4%). 13 of the 92 samples tested positive with the IFA at CLS. The in-house PCR assay identified an additional 7 positive samples, besides confirming the positivity of the previous 13 samples. Using the IFA as the available gold standard, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR assay was 100%, 91%, 65%, and 100% respectively. The 7 additional positive samples were subsequently confirmed as true positives through an independent PCR assay at SPH. Using the modified gold standard, the sensitivity, specificity, PPV, and NPV of PCR assay were all 100%. In comparison, the IFA had a sensitivity, specificity, PPV and NPV of 65%, 100%, 100%, and 91%, respectively
The in-house real-time PCR assay aids in the rapid and accurate diagnosis of PcP from BALF samples, with a considerable increase in sensitivity as compared to the currently existing IFA at CLS.
D. Church, None
See more of: Poster Abstract Session