Exploring lung microbiota and clinical application of BALF-mNGS in patients with pulmonary mycobacterial diseases: a multicenter retrospective study

Background Pulmonary mycobacterial diseases (PMDs) remain a leading cause of infectious disease-related mortality worldwide, with the majority of cases attributed to the Mycobacterium tuberculosis complex (MTBC). However, non-tuberculous mycobacteria (NTM) can also cause PMDs, and the incidence of non-tuberculous mycobacterial pulmonary disease (NTM-PD) has been increasing in recent years. Objectives This study aimed to explore the lung microbiota and assess the clinical application of bronchoalveolar lavage fluid metagenomic next-generation sequencing (BALF-mNGS) in patients with PMDs caused by MTBC or NTM. Methods This multicenter, retrospective study included patients with suspected PMDs between July 2021 to June 2025. mNGS and conventional diagnostic methods (CDTs), including GeneXpert, BALF culture, acid-fast bacillus (AFB) staining, and T-SPOT, were performed. Based on the microbiological diagnosis, patients were classified into TB and NTM-PD groups. We further analyzed the clinical impact of different MTBC/NTM abundance levels. The relative abundance of MTBC/NTM was represented by reads ten per million (RTPM). Patient clinical characteristics, length of hospital stay (LOHS), laboratory results, and treatment effectiveness were collected from the electronic medical record system. Results Compared with the TB group, patients with NTM-PD exhibited a higher prevalence of immunosuppression (34.96% vs. 53.85%, P = 0.013), particularly prolonged corticosteroid or immunosuppressant therapy (8.94% vs. 21.54%, P = 0.016). In the TB group, higher MTBC abundance was associated with increased positivity of CDTs and alterations in pulmonary microbiota, including enrichment of Candida albicans and other opportunistic pathogens. In the NTM-PD group, although CDTs positivity did not significantly differ between high- and low-abundance subgroups (21.21% vs. 20.00%, P = 0.906), higher NTM abundance was linked to distinct microbial community patterns and a markedly higher ineffective treatment rate (66.67% vs. 39.39%, P = 0.043). Notably, in both TB and NTM-PD groups, elevated MTBC or NTM abundance was associated with longer hospital stays and lower treatment effectiveness, indicating that pathogen abundance is significantly associated with clinical outcomes in pulmonary mycobacterial diseases. Conclusion BALF-mNGS not only provides superior pathogen detection in patients with PMDs but also shows that lower MTBC/NTM abundance is associated with better clinical prognosis, including shorter hospital stay and better treatment effectiveness, highlighting its potential role as a prognostic indicator.