A New PCR-Based Assay for Testing Bronchoalveolar Lavage Fluid Samples from Patients with Suspected <i>Pneumocystis jirovecii</i> Pneumonia

• Main Authors: Flora Marzia Liotti, Brunella Posteraro, Giulia De Angelis, Riccardo Torelli, Elena De Carolis, Domenico Speziale, Giulia Menchinelli, Teresa Spanu, Maurizio Sanguinetti
• Format: Article
• Language: English
• Published: MDPI AG 2021-08-01
• Series: Journal of Fungi
• Subjects: BAL fluid; dihydrofolate reductase gene; <i>Pneumocystis jirovecii</i> pneumonia; qPCR assay
• Online Access: https://www.mdpi.com/2309-608X/7/9/681

To support the clinical laboratory diagnosis of <i>Pneumocystis jirovecii</i> (<i>PJ</i>) pneumonia (PCP), an invasive fungal infection mainly occurring in HIV-negative patients, in-house or commercial <i>PJ</i>-specific real-time quantitative PCR (qPCR) assays are todays’ reliable options. The performance of these assays depends on the type of <i>PJ</i> gene (multi-copy mitochondrial versus single-copy nuclear) targeted by the assay. We described the development of a <i>PJ</i>-PCR assay targeting the dihydrofolate reductase (DHFR)-encoding gene. After delineating its analytical performance, the <i>PJ</i>-PCR assay was used to test bronchoalveolar lavage (BAL) fluid samples from 200 patients (only seven were HIV positive) with suspected PCP. Of 211 BAL fluid samples, 18 (8.5%) were positive and 193 (91.5%) were negative by <i>PJ</i>-PCR. Of 18 <i>PJ</i>-PCR-positive samples, 11 (61.1%) tested positive and seven (38.9%) tested negative with the immunofluorescence assay (IFA). All (100%) of the 193 <i>PJ</i>-PCR-negative samples were IFA negative. Based on IFA/PCR results, patients were, respectively, classified as having (<i>n</i> = 18) and not having (<i>n</i> = 182) proven (<i>PJ</i>-PCR+/IFA+) or probable (<i>PJ</i>-PCR+/IFA−) PCP. For 182 patients without PCP, alternative infectious or non-infectious etiologies were identified. Our <i>PJ</i>-PCR assay was at least equivalent to IFA, fostering studies aimed at defining a qPCR-based standard for PCP diagnosis in the future.