Multiplex digital PCR for rapid quantitative detection and dynamic monitoring of respiratory pathogens in bronchoalveolar lavage fluid.

Lower respiratory tract infections (LRTIs) remain a leading cause of mortality worldwide. Rapid and comprehensive etiological diagnosis is critical yet challenging: existing approaches, from culture to multiplex PCR and mNGS, carry distinct limitations in one or more of turnaround time, sensitivity, cost, or quantitative resolution. We developed a 33-plex digital PCR (dPCR) assay for absolute quantification of bacteria, viruses, fungi, and atypical pathogens in bronchoalveolar lavage fluid (BALF). Systematic evaluation of five liquefaction protocols identified dithiothreitol (DTT) as the optimal universal pretreatment, ensuring balanced RNA and DNA recovery. The assay demonstrated reliable quantification from 10 to 105 copies per test for all 33 targets (R2 > 0.99), with 27 targets showing linearity across full range and 6 targets exhibiting linearity from 10 to 104 copies per test (105 as upper detection limit). The limit of detection was 5 copies per test for all targets. In 105 BALF samples, dPCR achieved a significantly higher positive rate than culture (96.19% vs. 83.81%, p < 0.05), particularly for polymicrobial infections (85.71% vs. 40.00%). Concordance with mNGS for primary pathogens was 86.96% (20/23) for 14 primary pathogens across 23 positive samples. The complete workflow from sample pre-treatment to results required only 2.5 hours. Serial BALF dPCR quantification of P. aeruginosa and K. pneumoniae in one severe LRTI case tracked pathogen loads in parallel with clinical course and antibiotic responses, preliminarily supporting the technical feasibility of dynamic monitoring. This study demonstrates the feasibility of a rapid 33-plex dPCR assay for comprehensive respiratory pathogen profiling in BALF, with potential for dynamic monitoring in critically ill patients.