A comprehensive evaluation of a novel targeted-sequencing workflow forspecies identification and anti-tuberculosis drug-resistance detection.

BACKGROUND: Although several World Health Organization-endorsed targeted next-generation sequencing (tNGS) assays exist for tuberculosis (TB) drug-resistance detection, their target selection and diagnostic accuracy vary widely. In this study, we developed a novel tNGS workflow (the TB Pro assay) and evaluated its performance in identifyingspecies and predicting drug resistance.

METHODS: The TB Pro assay was validated for identifying 10complex (MTBC) and 39 nontuberculous mycobacterial (NTM) species, as well as predicting resistance to 4 first-line and 13 second-line anti-TB drugs. The limit of detection (LOD) was determined using 11 reference strains spiked in sputum. The prediction of resistance to anti-tuberculous drugs/drug classes was compared with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS) using 435 clinical isolates.

RESULTS: The assay demonstrated high sensitivity with a calculated LOD of 3.0 CFU/ml for MTB and 1.4-16.2 CFU/ml for most NTMs, except forwith 117.9 CFU/ml. Using pDST as the reference standard, the sensitivity of the TB Pro assay for the detection of resistance ranged from 74.3% (ethambutol) to 94.4% (rifampicin), with specificity values >98% for all drugs. Compared with WGS, the sensitivity of the TB Pro assay was over 98.0% for all drugs except pyrazinamide (66.7%), and the specificity values were all nearly 100.0%. Directly on sputum, the TB Pro assay showed 100% agreement with smear- and culture-positive sputum specimens.

CONCLUSIONS: The TB Pro assay represents a sensitive and specific solution for simultaneous mycobacterial identification and comprehensive drug-resistance profiling, performing robustly on both cultured isolates and direct clinical specimens.