Accuracy of nanopore-based targeted next-generation sequencing assay for detection of Mycobacterium tuberculosis and drug resistance from non-sputum specimens: a multicenter prospective study in China.

Tuberculosis (TB) remains the leading cause of mortality caused by a single infectious agent. Targeted next-generation sequencing (tNGS) has become a promising molecular method for TB diagnosis, but its accuracy and application for non-sputum specimens still remain unexplored. In this multicenter prospective lab-developed assay, 701 participants from five designated TB hospitals from different provinces in China were recruited. Non-sputum specimens were collected for tNGS, Xpert Mycobacterium tuberculosis (MTB)/RIF (Xpert), and culture tests. The diagnostic accuracy of tNGS for TB patients was evaluated compared with the microbiological reference standard (MRS). Phenotypic drug susceptibility tests (DSTs) were conducted using culture-positive isolates and employed to assess the accuracy of tNGS for detecting drug resistance. We found that the tNGS assay exhibited high diagnostic accuracy with sensitivity and specificity of 93.4% (95% CI, 91.5%-95.2%) and 93.2% (95% CI, 91.3%-95.0%) when utilizing MRS as the gold standard. The diagnostic performance of tNGS was robust regardless of specimen types and clinical symptoms. tNGS also showed a detection potential for other coinfecting respiratory pathogens. More than 90% of tNGS-positive individuals gained drug susceptibility results, which were mainly dependent on bacterial loads. Compared with phenotypical DSTs, tNGS had high sensitivities and specificities for the detection of rifampicin, isoniazid, streptomycin, ethambutol, and levofloxacin resistance. Our findings illustrated that tNGS assay is a rapid and highly sensitive test for TB diagnosis and simultaneous detection of drug resistance in non-sputum specimens. Its accuracy gain compared with conventional methods is most remarkable in TB patients with low bacterial loads.IMPORTANCETuberculosis (TB) diagnosis still remains challenging, especially in non-sputum patients. Nanopore-based targeted next-generation sequencing (tNGS) is a promising technology for the detection of TB cases and drug resistance, which has capacities to provide a panel of drug resistance profiles. The purpose of this study is to explore the diagnostic performance of tNGS for non-sputum specimens from five designated TB hospitals from different regions in China. Compared with the microbiological reference standard (MRS), tNGS exhibited high sensitivity and specificity, which are associated with bacterial loads in samples. Meanwhile, tNGS has capacities to detect coinfecting respiratory pathogens and produce drug-resistant profiles. Therefore, our findings suggested that tNGS is an alternative method for the diagnosis of non-sputum TB patients.CLINICAL TRIALSThis study is registered with Chinese Clinical Trial Registry as ChiCTR2400088518.