A New PCR-Based Assay for Testing Bronchoalveolar Lavage Fluid Samples from Patients with Suspected Pneumocystis
Flora Marzia Liotti1,2, Brunella Posteraro1,3, Giulia De Angelis1,2
1Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Roma, Italy.
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.
Insights
A new Pneumocystis jirovecii (PJ) PCR assay targeting the DHFR gene shows diagnostic performance equivalent to immunofluorescence assays for Pneumocystis pneumonia (PCP). This development supports future qPCR-based diagnostic standards for PCP.
Area of Science:
- Medical diagnostics
- Clinical microbiology
- Mycology
Background:
- Pneumocystis jirovecii pneumonia (PCP) is an invasive fungal infection primarily affecting HIV-negative individuals.
- Accurate laboratory diagnosis of PCP is crucial for timely treatment.
- Current reliable methods include in-house or commercial Pneumocystis jirovecii (PJ)-specific real-time quantitative PCR (qPCR) assays.
Purpose of the Study:
- To develop and evaluate a novel PJ-PCR assay targeting the dihydrofolate reductase (DHFR)-encoding gene.
- To assess the clinical performance of this new assay in diagnosing PCP.
- To compare the assay's performance against the immunofluorescence assay (IFA).
Main Methods:
- Development of a PJ-PCR assay targeting the DHFR gene.
- Analytical performance assessment of the developed PJ-PCR assay.
- Testing of bronchoalveolar lavage (BAL) fluid samples from 200 patients with suspected PCP using the PJ-PCR assay and IFA.
Main Results:
- The PJ-PCR assay detected PJ in 18 out of 211 BAL fluid samples (8.5%).
- Of the 18 PJ-PCR positive samples, 11 were also IFA positive (proven PCP), and 7 were IFA negative (probable PCP).
- All 193 PJ-PCR negative samples were IFA negative, indicating high specificity.
Conclusions:
- The developed PJ-PCR assay demonstrates diagnostic performance equivalent to IFA for PCP.
- This assay provides a reliable option for detecting Pneumocystis jirovecii.
- The findings encourage further research towards establishing a qPCR-based standard for PCP diagnosis.
