Diagnostic performance of tNGS mycobacterium tuberculosis sequence reads in differentiating bacteriological-positive from bacteriological-negative pulmonary tuberculosis

Targeted next-generation sequencing (tNGS) has demonstrated substantial value in pulmonary tuberculosis (PTB) diagnosis, particularly in cases that are refractory to detection by other methods. However, whether this parameter can be utilized to precisely differentiate bacteriological-positive from bacteriological-negative PTB has not been systematically investigated. This study evaluated the diagnostic performance of tNGS Mycobacterium tuberculosis (MTB) sequence reads in distinguishing bacteriological-positive from bacteriological-negative PTB. This retrospective study initially evaluated 482 patients with suspected PTB from January 2023 to May 2025, of whom 369 were finally included in the analysis after applying exclusion criteria. Clinical data, laboratory parameters, and tNGS reports were analyzed. The diagnostic efficacy of MTB sequence reads was assessed using receiver operating characteristic (ROC) curve, decision curve analysis (DCA), precision-recall curve, and calibration curve analysis. Concordance with conventional diagnostic tests was evaluated using Cohen’s kappa coefficient. High tNGS MTB sequence reads are closely associated with enhanced CRP, low BMI, weight loss, and AFB and rapid culture-positive. Patients with positive rapid culture or AFB exhibited substantially higher tNGS MTB sequence reads. tNGS MTB sequence reads were significantly and independently predictive of bacteriological-positive status. ROC analysis demonstrated an area under the curve of 0.825 for distinguishing bacteriological-positive from negative PTB, with an optimal cutoff of 10.163, sensitivity of 68.2%, and specificity of 85.7%. DCA and calibration curves confirmed excellent clinical utility. tNGS sequence reads showed the highest agreement with the AFB and rapid culture-positive detection (Kappa = 0.559). tNGS MTB sequence reads show promise as a potential indicator for identifying highly infectious bacteriological-positive PTB, with superior diagnostic performance compared to existing immunological assays, supporting their potentially clinical application in PTB infection control and management. Not applicable.