Evaluation of the ELITe InGenius PCR assay compared with immunofluorescence for Pneumocystis jirovecii detection in respiratory samples

Pneumocystis jirovecii (PJ) causes pneumonia primarily in immunocompromised individuals. Although direct immunofluorescence (IF) remains widely used in routine diagnostics, PCR assays are increasingly being adopted because they offer higher sensitivity and reduced observer dependency. This study evaluates the ELITe InGenius PJ PCR assay compared with IF for PJ detection in respiratory specimens. Respiratory samples submitted for IF-based PJ testing at the University Hospital Zurich over a 19-month period were retrospectively analyzed using the ELITe InGenius PCR assay. Diagnostic accuracy was assessed using IF as the reference method, and performance was evaluated by receiver operating characteristic (ROC) analysis. A total of 222 samples from 213 patients were included (70 sputum, 152 bronchoalveolar lavage). PCR and IF results were concordant in 175 (78.8%) specimens, including 18 positives and 157 negatives. 47 (21.2%) samples were discrepant: six IF-positive/PCR-negative and 41 IF-negative/PCR-positive. IF reexamination was possible for three IF-positive/PCR-negative and eight IF-negative/PCR-high-positive samples (> 100’000 copies/ml (cp/ml) or Ct < 29), resulting in revised IF findings in 3/3 and 2/8 cases, respectively. Three IF-negative/PCR-positive cases were clinically confirmed as true infections. ROC analysis identified an optimal PCR threshold of 6’233 cp/ml, yielding 70.8% sensitivity and 88.3% specificity, improving to 82.6% and 89.5% after reexamination. These findings support the higher sensitivity of PCR relative to IF for direct PJ detection. Clinical confirmation of IF-negative/PCR-positive cases and known limitations of IF underscore the diagnostic value of the ELITe InGenius assay. Its performance supports its use in routine diagnostics to improve early detection and guide timely treatment.