Towards culture-free sequencing of Mycobacterium tuberculosis: evaluating new targeted and whole-genome approaches for genotyping and drug resistance profiling

BACKGROUND: The effectiveness of current drug-resistant tuberculosis (DR-TB) regimens is limited by the absence of rapid diagnostics that comprehensively predict resistance to included drugs. Next-generation sequencing (NGS), through culture-free targeted sequencing (tNGS) and culture-based whole-genome sequencing (cWGS) of the Mycobacterium tuberculosis complex (MTBC), offers a powerful framework for precision diagnosis, surveillance, and trial applications. We evaluated two novel assays enabling high-resolution tNGS and enrichment-based direct WGS (dWGS) on respiratory samples, focusing on analytical sensitivity, DR and genotyping concordance. METHODS: tNGS Deeplex Myc-TB XL tNGS (Genoscreen, beta-testing) and dWGS QIAseq xHYB MTB (Qiagen) were evaluated on 96 MTBC-positive decontaminated sputum samples from a vaccine trial, spanning a wide range of bacillary loads. DNA was extracted using a host-depletion protocol and quantified by MTBC-specific real-time PCR. Libraries were sequenced on Illumina platforms and analysed using assay-specific pipelines. Associations between genome copy (gc) number and sequencing coverage were assessed. DR concordance was benchmarked against cWGS and the WHO mutation catalogue across first-, second-line, newer, and repurposed drugs. WGS-based phylogenetic trees were constructed using Ridom SeqSphere + . RESULTS: Bacillary loads ranged from < 10 to > 1,000 MTBC gc/µL. tNGS generated interpretable resistance profiles in 96.6% of specimens, achieving a limit of detection (LoD) of ~ 10 gc, with 100% positive agreement for all evaluated drugs and 100% negative agreement except for isoniazid/ethionamide (≥ 97%). dWGS yielded data suitable for DR analysis in 75% of samples, with a LoD of ~ 100gc. No false negatives were observed for most drugs; one fluoroquinolone-resistant case was missed due to low-frequency variant thresholds, and resistance to delamanid and clofazimine was misclassified in one case each owing to interpretation rules. Negative agreement was 100% except for rifampicin (≥ 97%). Lineage assignment was concordant with cWGS for both approaches, and dWGS supported transmission analysis in 65% of samples with adequate genome coverage, confirming the cluster detected by cWGS. CONCLUSIONS: In this predominantly drug-susceptible TB cohort, tNGS showed high overall agreement with cWGS for DR profiling with a LoD within the range of low-complexity automated assays used for initial diagnosis. dWGS, while requiring higher DNA input, enables robust culture-free genome-wide analysis, including transmission inference and exploration of candidate DR loci. Bacillary load-guided integration of both approaches may optimize DR-TB clinical management and genomic surveillance. Larger studies are needed to validate clinical and epidemiological predictive performance. TRIAL REGISTRATION: This trial was registered on April 19, 2018, on the ClinicalTrials.gov database under the title: Study to Evaluate H56:IC31 in Preventing Rate of TB Recurrence, with the identifier NCT03512249.