Standardization and Clinical Evaluation of a Real-Time PCR Assay for the Diagnosis of Pneumocystis jirovecii Pneumonia

Background Pneumocystis jirovecii pneumonia (PjP) is a life-threatening infection in immunocompromised individuals. Its diagnosis remains challenging, especially in microscopy-negative cases. This study aimed to standardize and validate a qPCR assay targeting the mtLSU rRNA gene for detecting P. jirovecii DNA in respiratory samples from patients in Argentina. Materials and Methods The assay was optimized using plasmid dilutions containing the target gene. Analytical specificity was evaluated against 60 fungal, 21 mycobacterial, and 16 bacterial species. Clinical validation included 101 respiratory samples from symptomatic patients and 37 from healthy individuals. An internal positive control (IPC) was included in all reactions to detect inhibitors. Results The qPCR assay showed a detection limit of 8.8 copies/µL and no cross-reactivity. Among microscopy-confirmed cases, 95.5% were qPCR-positive. Notably, 14.9% of microscopy-negative but clinically compatible cases tested positive. ROC analysis yielded an AUC of 0.96, with an optimal Ct cutoff ≤ 36, providing 90.7% sensitivity and 95.8% specificity. No healthy controls tested positive. A “grey zone” (Ct 36–37.8) was observed, requiring clinical correlation. Conclusions This qPCR assay is highly sensitive and specific, offering a valuable diagnostic tool for PjP. Its performance supports implementation in routine diagnostics, especially when microscopy is inconclusive. However, interpretation in the grey zone requires complementary clinical or biomarker data.