Targeted Next-Generation Sequencing in Drug-Resistant Tuberculosis: WHO Guidance and Practical Implementation Priorities

Targeted next-generation sequencing (tNGS) closes the gap between point-of-care rapid tests and phenotypic drug susceptibility testing (pDST) in drug-resistant tuberculosis (DR-TB). The 2025 World Health Organization (WHO) consolidated guidelines and the operational handbook place tNGS after initial automated nucleic acid amplification tests (aNAATs) for the delivery of catalogue-linked molecular drug susceptibility testing (DST) for a broad drug panel, reserving whole-genome sequencing (WGS) and/or pDST for discordance resolution, confirmation, and surveillance. This review summarizes (i) the core tNGS principles and panel design; (ii) platform-specific workflows for Illumina and Nanopore, including direct-from-sample implementations and typical turnaround times; (iii) catalogue-based interpretation against the 2023 WHO <i>Mycobacterium tuberculosis</i> mutation catalogue, with emphasis on bedaquiline/clofazimine (BDQ/CFZ) resistance and management of uncertain variants; (iv) pooled accuracy and sources of genotype-phenotype discordance; and (v) practical requirements for bioinformatics, quality assurance/external quality assessment (QA/EQA), and standardized reporting. We summarize operational and economic considerations (throughput, batching, and network design) to clarify where tNGS adds value compared with alternative strategies and to outline priority research needs, including (i) performance standards for culture-free tNGS, (ii) robust heteroresistance detection, (iii) standardized variant curation, and (iv) data-sharing frameworks to refine genotype-phenotype links. When embedded within validated QA/EQA frameworks and catalogue-linked reporting systems, tNGS can shorten the time to effective therapy by rapidly informing fluoroquinolone (FQ) susceptibility and providing early, tiered resistance signals for newer agents (e.g., BDQ), with indeterminate findings prompting reflex pDST/WGS.