Mycobacterium tuberculosis identification and antibiotic resistance gene profiling using nanopore sequencing: A new approach for tailoring more effective treatments for patients

Rapid diagnosis of TB infections and effective drug treatment is central to reducing disease morbidity and mortality. Here we evaluate the performance of TBseek, a multiplex targeted PCR nanopore sequencing-based test designed for TB identification and antibiotic resistance genotyping. From the analysis of 185 clinical samples, TBseek test demonstrated significantly superior diagnostic performance compared to Xpert PCR test, fluorescent microscopy and culture, achieving a sensitivity of 89.47%, specificity of 96.97%, and an AUC of 0.93. Retrospective analysis of 136 TBseek-positive specimens showed rapid detection: 78.68% met the ≥ 10 MTB-specific reads threshold within 1 h, 91.18% by 3 h, and 100% by 8 h. In 43 MTB-positive samples, full-length gene primers consistently yielded a higher proportion of MTB sequences compared to hotspot-targeted primers, demonstrating strong specificity. Compared to phenotypic drug sensitivity testing results for 76 TB positive culture samples, TBseek had high detection sensitivities for resistance to rifampicin (100.00%), isoniazid (83.33%), ethambutol (100.00%), streptomycin (95.24%) and levofloxacin (90%). Based on these findings, TBseek delivered high performance for detection of TB and antibiotic resistance. With further validation, TBseek has clinical potential as a first-tier screen to evaluate suspected TB cases and for guiding and monitoring personalized treatments.