Summary

Drug-resistant tuberculosis (DR-TB) remains one of the most challenging infectious diseases globally, and treatment is constrained by the lack of rapid diagnostics that comprehensively predict resistance to across drugs classes. While NGS has transformed surveillance, its culture-free (cf) application has been limited by analytical sensitivity and incomplete drug coverage.

This study provides the first head-to-head evaluation of two novel assays for cf MTBC sequencing: a high-resolution tNGS panel and an enrichment-based cfWGS assay, enabling full resistance profiling and genotyping directly from sputum. In our study, the new version of the WHO-approved tNGS assay achieves analytical sensitivity comparable to the most sensitive rapid TB assays, accurately predicting resistance across standard and newly introduced DR-TB drugs. On the other side, cfWGS enables robust genome-wide analysis, detection of emergent drug resistance, lineage assignment, and transmission inference without culture.

These findings position cfNGS as a realistic alternative to culture-based WGS, shortening turnaround time and overcoming MTBC culture limitations, with immediate clinical and research relevance.