Real-time polymerase chain reaction (PCR) for Pneumocystis jirovecii detection in lower respiratory tract samples

To date, the gold standard for Pneumocystis jirovecii pneumonia (PjP) diagnosis is direct microscopic examination (ME). However, several studies have shown that the DNA copy number of the P. jirovecii, measured by quantitative real-time PCR (qPCR), is significantly higher in patients with PjP than in colonized individuals. Nevertheless, this approach is not standardized yet and requires further investigation. In this study, we aimed to compare the results obtained using the Pneumocystis ELITe MGB Kit (ELITech Group S.p.a., Italy) with those from ME, evaluating specificity, sensitivity, and the ability to distinguish infection from colonization through the identification of a cut-off value. A total of 163 bronchoalveolar lavage (BAL) or sputum samples, collected and tested with direct ME for P. jirovecii in our Unit over the past 10 years, were also analyzed using a qPCR assay. The results were concordant between the two methods in all 63 ME-positive samples. Among 100 ME-negative samples, 21 (21%) tested positive by qPCR. A receiver operating characteristic curve analysis was performed to assess the qPCR assay's prognostic performance in identifying patients already diagnosed with PjP by ME on BAL or sputum samples. A cut-off value of 150 DNA copies/10⁵ cells yielded a sensitivity of 100% (95% confidence interval [CI]: 94%-100%) and a specificity of 99% (95% CI: 95%-100%) in the diagnosis of the disease. qPCR may be used alongside careful clinical evaluation to ensure accurate diagnosis and appropriate treatment.IMPORTANCERecently, molecular approaches have been proposed for the detection of Pneumocystis jirovecii. Thanks to their high sensitivity, these methods can detect small amounts of fungal DNA. However, it is necessary to establish a cut-off value to distinguish between colonization and infection. Since P. jirovecii is closely related to epithelial cells in the lung environment, normalizing the fungal DNA load on the host cellular DNA content could represent an important step toward standardizing the procedure.