Pathogen detection profile and mixed-pathogen patterns in bronchoalveolar lavage fluid from respiratory and critical care patients using targeted next-generation sequencing

Targeted next-generation sequencing (tNGS) is increasingly used for pathogen detection in lower respiratory tract infections. However, real-world data on bronchoalveolar lavage fluid (BALF)-dominant tNGS findings in respiratory and critical care settings remain limited. This single-center retrospective real-world study included 580 tNGS records from the respiratory and critical care department between November 2024 and April 2026. Reported pathogens and resistance- or virulence-related genetic signals were extracted and standardized. Detection frequencies, pathogen-category distributions, mixed-pathogen detection patterns, co-detection pairs, age-stratified differences, and monthly variation were analyzed. Among 580 records, BALF accounted for 551 specimens (95.0%). At least one pathogen or related signal was detected in 543 records (93.6%). Among pathogen-positive records, 125 (23.0%) showed single-pathogen detection, whereas 418 (77.0%) showed detection of two or more pathogens. The most frequently detected pathogens were Epstein–Barr virus (EBV) (38.1%), Haemophilus influenzae (20.2%), Candida albicans (18.4%), Klebsiella pneumoniae (15.3%), and Pseudomonas aeruginosa (15.2%). Bacteria were detected in 388 records (66.9%), viruses in 366 (63.1%), fungi in 186 (32.1%), and resistance- or virulence-related genetic signals in 117 (20.2%). Common co-detection pairs included EBV with C. albicans , EBV with H. influenzae , and C. albicans with K. pneumoniae . Frequently observed pairs were not always significantly associated after FDR correction, indicating that absolute pair frequency and chance-adjusted association provide different report-level information. Significant age-stratified differences after false discovery rate correction were observed across multiple pathogen categories and individual pathogens, including bacteria, viruses, fungi, atypical or special bacteria, EBV, C. albicans , K. pneumoniae , CMV, A. baumannii , E. coli , and HSV-1 (all adjusted P < 0.05). Monthly variation was significant for overall detection, mixed-pathogen detection, bacteria, viruses, fungi, EBV, H. influenzae , and C. albicans (all adjusted P < 0.05). BALF-dominant tNGS records showed a complex and frequently mixed pathogen detection profile. However, incomplete patient-level clinical data and the absence of paired conventional microbiology limited differentiation between infection, colonization, and incidental detection and restricted the generalizability of these findings. Co-detection should therefore be interpreted together with clinical and microbiological evidence.