Abstract
To support the clinical laboratory diagnosis of Pneumocystis jirovecii (PJ) pneumonia (PCP), an invasive fungal infection mainly occurring in HIV-negative patients, in-house or commercial PJ-specific real-time quantitative PCR (qPCR) assays are todays’ reliable options. The performance of these assays depends on the type of PJ gene (multi-copy mitochondrial versus single-copy nuclear) targeted by the assay. We described the development of a PJ-PCR assay targeting the dihydrofolate reductase (DHFR)-encoding gene. After delineating its analytical performance, the PJ-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 PJ-PCR. Of 18 PJ-PCR-positive samples, 11 (61.1%) tested positive and seven (38.9%) tested negative with the immunofluorescence assay (IFA). All (100%) of the 193 PJ-PCR-negative samples were IFA negative. Based on IFA/PCR results, patients were, respectively, classified as having (n = 18) and not having (n = 182) proven (PJ-PCR+/IFA+) or probable (PJ-PCR+/IFA−) PCP. For 182 patients without PCP, alternative infectious or non-infectious etiologies were identified. Our PJ-PCR assay was at least equivalent to IFA, fostering studies aimed at defining a qPCR-based standard for PCP diagnosis in the future.
Highlights
Fungal infections, mainly Pneumocystis jirovecii (PJ; pneumocystosis), Aspergillus, or Mucorales, account for about 5% of all severe respiratory infections that occur in immunocompromised patients [1]
Molecular-based methods for detecting pneumonia agents in multiple or single assays have been developed to overcome the limits of conventional identification methods, which are familiar in medical mycology [2] especially with PJ organisms [3]
We report on the detection of PJ dihydrofolate reductase (DHFR) DNA in bronchoalveolar lavage (BAL) fluid samples, prospectively collected from patients hospitalized at our institution, using a new in-house quantitative PCR (qPCR) assay
Summary
Flora Marzia Liotti 1,2,† , Brunella Posteraro 1,3,† , Giulia De Angelis 1,2 , Riccardo Torelli 2 , Elena De Carolis 2 , Domenico Speziale 2 , Giulia Menchinelli 1,2 , Teresa Spanu 1,2 and Maurizio Sanguinetti 1,2, *. Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario A. Dipartimento di Scienze Mediche e Chirurgiche, Fondazione Policlinico Universitario A. The first two authors contributed to this work
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