Combined multiplex polymerase chain reaction-based targeted next−generation sequencing and serum 1, 3-β-D-glucan for differential diagnosis of Pneumocystis pneumonia and Pneumocystis colonization

Background and Objective Pneumocystis jirovecii pneumonia (PjP) remains an important cause of morbimortality worldwide, and differentiating Pneumocystis jirovecii ( P. jirovecii ) infection from P. jirovecii colonization (PjC) is crucial for guiding treatment strategies. Multiplex polymerase chain reaction-based targeted next-generation sequencing (mp-tNGS) is a promising tool for identifying lower respiratory tract infections, with a detectable pathogen spectrum that covers more than 95% of clinical infectious cases. This study evaluated mp-tNGS for P. jirovecii identification in bronchoalveolar lavage fluid (BALF) samples combined with serum 1,3-β-D-glucan (BDG) level detection to differentiate PjP and PjC. Methods A total of 73 patients were enrolled and the final diagnosis was used as a reference criterion, and patients were divided into the PjP group and PjC group. The clinical data and detection performance of mp-tNGS/serum BDG were analyzed. Results The median fungal reads (normalized sequence counts) detected by mp-tNGS were 1522.00 (interquartile range [IQR], 581.5, 4898.0) in the PjP group versus 117.00 (IQR, 79.00, 257.00) in the PjC group ( p < 0.0001). Correspondingly, BDG levels were 122.5 (88.75,239.3) pg/ml in PjP patients compared to 59.00 (51.0,79.0) pg/ml in PjC patients ( p < 0.0001). Area under the receiver operator characteristic curve (AUROC) for discriminating PjP from colonization was 0.935 (95% CI: 0.88–0.99) for BALF mp-tNGS and 0.822 (95% CI: 0.72–0.93) for serum BDG. The optimal diagnostic thresholds were determined to be 355 reads for mp-tNGS (sensitivity: 89.1%; specificity: 85.2%) and 84.5 pg/ml for BDG (sensitivity: 85.2%; specificity: 80.4%). Conclusion BALF mp-tNGS and serum BDG serve as valuable adjunct diagnostic tools, providing reliable differentiation between P. jirovecii colonization and active infection.