Targeted next-generation sequencing for comprehensive diagnosis and drug resistance detection in pulmonary and extrapulmonary tuberculosis: a single-center retrospective study

, drug resistance mutations, and co-infecting pathogens. This study evaluates the effectiveness of tNGS for TB detection across various pulmonary and extrapulmonary TB samples. We retrospectively enrolled 159 patients with suspected TB at Kunming Third People's Hospital. Specimens included 126 pulmonary and 33 extrapulmonary samples. All samples underwent tNGS, and results were compared against conventional microbiological tests and NAATs. tNGS demonstrated high diagnostic sensitivity for both pulmonary TB and extrapulmonary TB, achieving 83.9% sensitivity in bronchoalveolar lavage fluid, 89.5% in sputum, and 100% in extrapulmonary samples. tNGS showed an 83.3% agreement with Xpert in detecting rifampicin resistance. Additionally, tNGS also identified 22 drug resistance mutations, which are critical for predicting multidrug-resistant TB and pre-extensively drug-resistant TB. Additionally, tNGS effectively detected co-infecting respiratory pathogens, enhancing the understanding of complex TB cases. tNGS offers a highly sensitive and comprehensive approach for detecting TB and drug resistance, outperforming traditional methods in both pulmonary and extrapulmonary samples. It effectively identifies co-infections, providing a holistic view that enhances patient management, particularly in cases involving multidrug-resistant strains. These findings underscore tNGS's potential to improve TB diagnostics and patient management through faster and more precise detection methods.IMPORTANCETuberculosis (TB) continues to be a leading cause of morbidity and mortality worldwide, particularly among marginalized populations. Timely and accurate diagnosis of TB, particularly extrapulmonary TB, remains challenging in low-resource settings, primarily due to non-specific clinical presentations that hinder early suspicion, along with limitations in both diagnosis and drug resistance detection. Additionally, targeted next-generation sequencing can identify co-infections with other clinically relevant pathogens, providing a more comprehensive understanding of each patient's infectious profile. By leveraging advanced sequencing technologies, our findings highlight a powerful diagnostic approach that can improve TB diagnosis, support appropriate treatment strategies, and may increasingly benefit patients in underserved settings as sequencing platforms become more accessible and costs continue to decrease.