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

Overview

This multicenter prospective study evaluated the diagnostic accuracy and clinical utility of nanopore-based targeted next-generation sequencing (tNGS) for detection of Mycobacterium tuberculosis and antimicrobial drug resistance using non-sputum specimens across five designated TB hospitals in China. The study enrolled 701 participants and compared tNGS performance against microbiological reference standards and conventional phenotypic drug susceptibility testing.

Methods and approach

Non-sputum specimens were collected from 701 participants across five TB hospitals in different Chinese provinces. Specimens underwent concurrent testing via tNGS, Xpert MTB/RIF assay, and culture. Diagnostic accuracy of tNGS was evaluated against microbiological reference standards. Phenotypic drug susceptibility testing was performed on culture-positive isolates to assess the accuracy of tNGS for detecting resistance to rifampicin, isoniazid, streptomycin, ethambutol, and levofloxacin.

Key Findings

The tNGS assay demonstrated sensitivity of 93.4% (95% CI 91.5%-95.2%) and specificity of 93.2% (95% CI 91.3%-95.0%) using microbiological reference standards as the gold standard. Diagnostic performance remained robust across different specimen types and clinical symptomatology. More than 90% of tNGS-positive individuals yielded drug susceptibility results, with performance dependent on bacterial load. The assay showed high sensitivity and specificity for detection of resistance to all five tested antimicrobial agents. tNGS additionally demonstrated capacity to detect coinfecting respiratory pathogens. Accuracy gains compared with conventional methods were most pronounced in TB patients with low bacterial loads.

Implications

The findings establish tNGS as a rapid, highly sensitive diagnostic method for tuberculosis and simultaneous drug resistance detection in non-sputum specimens, addressing a significant gap in TB diagnostic capabilities for non-sputum-producing patients. The assay's performance is primarily dependent on bacterial load, suggesting potential clinical utility particularly in scenarios where conventional methods demonstrate reduced sensitivity. The multiplex detection capability for both TB and coinfecting respiratory pathogens represents a diagnostic advantage over single-target conventional approaches, though implementation considerations regarding bacterial load thresholds and specimen quality remain relevant for clinical translation.