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

backgroundThe 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.

methodstNGS 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 + .

resultsBacillary 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.

conclusionsIn 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 registrationThis 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.