Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS (tNGS) offers a streamlined approach by focusing on specific genes associated with drug resistance, bypassing the need for traditional culture-based methods with turnaround times ranging from weeks to months. The World Health Organization (WHO) has recommended tNGS as a valuable strategy for improving tuberculosis (TB) diagnosis to guide treatment and improve treatment outcomes, particularly in resource-limited settings. Among the WHO-recommended tNGS assays, we have selected a method that provides rapid and comprehensive drug susceptibility testing, lineage determination, and strain typing. While standardized DNA extraction methods are available, they can be time-consuming and labor-intensive. To address this challenge, we optimized a simplified, matrix-based DNA extraction protocol in combination with magnetic bead purification. This method offers a rapid and efficient approach for extracting DNA directly from decontaminated sputum sediments, enabling rapid downstream tNGS analysis. By streamlining the DNA extraction process from sputum sediment, this protocol could facilitate wider adoption of tNGS in routine clinical settings, ultimately contributing to improved patient outcomes and lending to global TB control efforts.

Whole-genome sequencing (WGS) holds promise for accurate and comprehensive diagnosis of drug resistance in Mycobacterium tuberculosis and identification of transmission events. ‘Early positive cultures’ (EPC) are increasingly used when WGS is implemented to guide clinical care to reduce the turnaround time. We performed a systematic literature review to compare methods used for EPC-based WGS and performed an individual sample data meta-analysis to identify variables associated with bioinformatic quality measures. Of 423 studies identified, 15 met eligibility criteria. We analysed 1,065 FASTQ files from 11 studies using Illumina sequencing; 96.1% passed all quality control thresholds. Median genome coverage was 65× (IQR, 63–82), with a pooled mapping percentage of 91.2%. The meta-analysis showed that the number of sequencing cycles was significantly associated with improved sequencing quality, while other laboratory variables had no consistent effect. Based on these findings, we suggest replacing the term EPC with ‘clinical primary culture’ and propose a standardized workflow and reporting checklist for WGS on primary Mycobacteria Growth Indicator Tube (MGIT) cultures.

Background: Whole genome sequencing (WGS) can guide effective rifampicin-resistant tuberculosis (RR-TB) treatment but the cost is a perceived barrier.Methods: We performed a health-economic evaluation of culture-based WGS combined with automated recommendation for an all-oral 6-month individualized regimen. Provider, patient and household costs for RR-TB management were analyzed for 203 participants randomized to the WGS intervention or standard of care (SOC) in the South African SMARTT trial (Sept 2021-Feb 2023). Cost-effectiveness was assessed by incremental cost-effectiveness ratio (ICER); monetary value by Incremental Net Monetary Benefit (INMB) and Return on Investment (ROI).Findings: WGS-guided treatment was dominant, reducing the per patient mean total cost (-$370) and increasing the per patient health benefit (-0.05 DALYs), resulting in an INMB of $618. Society gained $3.61 for every dollar invested. Among people living with HIV, WGS-guided treatment resulted in greater cost reduction (-$582 vs -$22), greater DALY reduction (-0.08 vs -0.01) and higher INMB ($969 vs $51). The benefit was larger for women than men (cost savings $694 vs $191, DALY reduction -0.06 vs -0.03, and INMBs $997 vs $319). People with expanded resistance experienced the greatest health benefit (0.46 DALYs averted), but the intervention was not cost-effective in this population (INMB $-1231) due to higher drug ($1690 vs $1260) and inpatient costs ($4,244 vs $1,201).Interpretation: WGS-guided RR-TB treatment was cost-effective and society gained $3.6 benefit for every dollar invested. Future research should evaluate the cost-effectiveness of WGS-guided treatment in contexts where an all-oral 6-month regimen is the SOC.

Nontuberculous mycobacteria (NTM) pose significant diagnostic challenges in high tuberculosis (TB)-burden settings, particularly when routine molecular assays suggest multidrug-resistant TB (MDR-TB). Current diagnostic algorithms in high TB-burden settings are primarily designed to detect members of the Mycobacterium tuberculosiscomplex (MTBC) and may inadequately distinguish NTM when discordant laboratory findings are encountered. The value of this case lies not in demonstrating that whole-genome sequencing (WGS) should guide real-time treatment, but in illustrating how diagnostic uncertainty can arise when routine MTBC-focused tests fail to identify NTM and how genomic surveillance can contribute to species-level characterization in such settings. We report a complex case which was initially diagnosed as rifampicin- and isoniazid-resistant MTBC and subsequently managed according to programmatic MDR-TB guidelines. Persistent acid-fast bacilli positivity in the presence of negative MTBC antigen testing during follow-up raised suspicion of NTM involvement. Whole-genome sequencing (WGS), performed retrospectively on a follow-up isolate, identified an NTM belonging to the Mycobacterium avium complex (MAC) which was phylogenetically closest to genomes provisionally designated Mycobacterium europaeum_A and distinct from Mycobacterium europaeum sensu stricto, a member of the Mycobacterium simiae complex. The identification of a MAC-associated NTM provided an explanation for the observed diagnostic discordance but could not determine whether the organism represented colonization, sequential infection, or concurrent infection. Although WGS did not inform clinical management, it provided high-resolution species identification and highlighted important limitations of MTBC-focused diagnostic workflows when discordant microbiological findings are encountered. This case underscores the need for the targeted investigation of NTM in patients with acid-fast bacilli-positive cultures and negative MTBC antigen testing and supports the use of genomic surveillance to improve species-level identification and understanding of NTM epidemiology in high TB-burden settings such as Limpopo Province, South Africa.

Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS (tNGS) offers a streamlined approach by focusing on specific genes associated with drug resistance, bypassing the need for traditional culture-based methods with turnaround times ranging from weeks to months. The World Health Organization (WHO) has recommended tNGS as a valuable strategy for improving tuberculosis (TB) diagnosis to guide treatment and improve treatment outcomes, particularly in resource-limited settings. Among the WHO-recommended tNGS assays, we have selected a method that provides rapid and comprehensive drug susceptibility testing, lineage determination, and strain typing. While standardized DNA extraction methods are available, they can be time-consuming and labor-intensive. To address this challenge, we optimized a simplified, matrix-based DNA extraction protocol in combination with magnetic bead purification. This method offers a rapid and efficient approach for extracting DNA directly from decontaminated sputum sediments, enabling rapid downstream tNGS analysis. By streamlining the DNA extraction process from sputum sediment, this protocol could facilitate wider adoption of tNGS in routine clinical settings, ultimately contributing to improved patient outcomes and lending to global TB control efforts.

Background: Comprehensive knowledge of resistance to the novel all-oral regimens for treatment of rifampicin-resistant tuberculosis (RR-TB) is important, but challenging variant detection by next-generation sequencing (NGS) in combination with variant interpretation software can diagnose resistance early. We aimed to generate a consensus on strategic issues for the implementation of NGS for routine RR-TB care. Methods: We employed a stakeholder consensus process informed by Delphi principles. First, stakeholders participated in an online survey to provide feedback on statements using a 3-point Likert scale and free text. Responses were then summarised and follow-up discussion fora with stakeholders were held to revise statements until a consensus was reached. Findings: Of the 116 invited stakeholders, 58 participated, representing RR-TB research (n=28), clinical care (n=11), diagnosis (n=5), RR-TB survivors and advocacy communities (n=5) from 14 countries in five of the six WHO regions. A consensus was rapidly reached for most statements with limited editorial changes needed. The debate mostly focused on statements covering performing sequencing at centralised facilities, acting on variants without statistical association with resistance in the WHO catalogue of mutations, strengthening WHO-endorsed regimens and the need for informed consent for the use of NGS for care and surveillance. Interpretation: The consensus-building process grounded in real-world experience from a diverse group of stakeholders generated 32 consensus statements covering five strategic domains. The blueprint generated is not a definite prescriptive framework but a document that can support policymakers when developing country-specific policies for NGS-guided precision medicine and RR-TB care. Sources of funding: This work was supported by the Global Health EDCTP3 Joint Undertaking and the Bill & Melinda Gates Foundation under the GenPath Africa project (101103171), the Research Foundation Flanders (FWO) Odysseus (G0F8316N) and TBM grants (T001018N).