Nanopore-targeted sequencing: A new and effective technique for the diagnosis of non-tuberculous mycobacteria pulmonary disease.

Non-tuberculous mycobacteria pulmonary disease (NTM-PD) has become a significant public health issue threatening human health in recent years. Traditional mycobacterial culture is complex, time-consuming, and unable to identify NTM at the species level. Nanopore-targeted sequencing (NTS) is a novel molecular detection technology that integrates the high sensitivity of targeted multiplex PCR with the high specificity of third-generation long-read sequencing. NTS has significant advantages in diagnosing, precise differentiation of mixed infection pathogens and antibiotic resistance genes in infectious diseases. The present study compared the diagnostic performance of NTS with fluorescence-stained smears and BACTEC 960 culture in NTM-PD. In respiratory specimens from suspected NTM-PD individuals, NTS exhibited a sensitivity of 88.2 %, significantly higher than culture (74.0 %) and smears (37.8 %) (both P < 0.05). The AUC for NTS was 0.893, surpassing that of culture (0.869) and smears (0.605). The sensitivity varied among respiratory specimens: bronchoalveolar lavage fluid (BALF) 90.1 %, sputum 78.6 %, and lung puncture fluid 50.0 %. The NTS sensitivity and specificity for predicting resistance to amikacin and clarithromycin were 42.9 %, 35.5 % and 98.8 %, 98.3 %, respectively. Compared to culture and smears, NTS demonstrated higher sensitivity, with slightly lower specificity than culture. However, its predictive value for drug-resistant mycobacteria is limited. BALF specimens exhibit maximal sensitivity among different respiratory samples. Taken together, NTS is a novel, rapid, and efficient molecular diagnostic method for NTM-PD.