Diagnostic performance of metagenomic next-generation sequencing in Mycobacterium and non-Mycobacterium infection patients: a retrospective study at a large comprehensive medical center

Background Metagenomic next-generation sequencing (mNGS) has become a potentially valuable complementary test, if not an essential one, in some challenging clinical cases. This study comprehensively compared mNGS with other methods, including TB-IgG, TB-IGRA, TB-DNA, Xpert, culture, and AFS for the diagnosis of and differentiation between TB infection from NTM infection using multiple sample types, including BALF, CSF, blood, pus, tissue, hydrothorax, and ascites. Methods From 2021 to 2024, the clinical data, including imaging, laboratory results, and medical records, of 425 patients with suspected TB infection were retrospectively collected and classified as PTB [306 (72%)], EPTB [67 (15.76%)], and non-TB [52 (12.24%)], according to the final clinical diagnosis. Results Among the three groups, significant differences were observed in age, sex, cough, smoking history, hsCRP, IFN-γ, and hypertension ( P < 0.05). According to the final clinical diagnosis, the sensitivity and specificity of mNGS in all samples were 90.1% (95% CI: 0.866–0.927) and 75.0% (95% CI: 0.618–0.848), respectively. The sensitivity of mNGS was higher than that of TB-IGRA (79.1%), Xpert (72.4%), culture (52.3%), TB-DNA (45.6%), TB-IgG (36.4%), and AFS (10.7%). For distinguishing TB from non-TB, the sensitivity of mNGS was approximately 37.8% higher than that of standard test culture (90.1% vs 36.4%; P < 0.001). Further analysis also showed that MTB read counts from mNGS were highly correlated with MTB burden measured by other laboratory tests ( P < 0.05). Analysis of the ROC curve of mNGS reads showed that the AUC for MTB diagnosis was 0.9073, with a sensitivity of 73.19% and specificity of 96.15% when the recommended cutoff value of 4 reads (Youden’s index, 0.6934) was applied. Conclusions For patients with suspected TB or NTM infection, mNGS may serve as a helpful adjunctive tool, particularly when routine diagnostic tests are unrevealing or indeterminate. It has the potential to assist in distinguishing MTB from NTM and to provide additional evidence to guide appropriate treatment choices, though its results should always be interpreted alongside clinical findings and conventional microbiological testing.