RATIONALE:Transmission is the primary driver of tuberculosis (TB) and drug-resistant TB (DR-TB) in high-burden countries; however, where and between whom spread occurs is poorly understood. OBJECTIVES:We conducted universal whole genome sequencing (WGS) to evaluate the role of casual contact in Mycobacterium tuberculosis transmission. METHODS:We recruited persons diagnosed with second-line DR-TB (eg, extensively drug-resistant [XDR], pre-XDR-TB) from June 2018 to December 2022 in metropolitan Durban, South Africa. We collected named contacts and GPS coordinates of homes, clinics, and community locations visited regularly before diagnosis. Among participants genotypically clustered by whole genome sequencing (≤12 single-nucleotide polymorphisms), we quantified the proportion attributable to close versus casual contact. Close contact was defined as person-to-person links or overlapping hospitalizations. Casual contact links were based on geographic proximity of homes and community locations, or shared outpatient clinics. MEASUREMENTS AND MAIN RESULTS:We enrolled 305 of 383 (80%) persons diagnosed with second-line DR-TB. TB isolates were sequenced for 251 (83%) participants; 141 (56%) were genotypically linked, forming 25 clusters (range, 2-49 persons/cluster). Among clustered participants, 69 (49%) were epidemiologically linked by casual contact and 13 (9%) through close contact. Multivariable analysis identified living within 1 km (OR, 17.9), visiting proximate community locations (OR, 1.88), shared outpatient clinic (OR, 1.72), and person-to-person links (OR, 5.38) as significant risk factors associated with genotypic clustering. CONCLUSIONS:Casual contact in community locations accounted for half of transmission among genotypic clusters in a high-burden setting. Efforts to curb TB will require a greater emphasis on community-based measures to identify cases from casual contact or undetected intermediate cases, in addition to the current mainstay of contact tracing.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is spread through the air. Although extensive data has shown that TB case notifications decreased during the COVID-19 pandemic, there is little known about the extent to which these reductions were due to a decrease in transmission, rather than delays in healthcare seeking and diagnosis. We used data from CONTEXT, a population-based cross-sectional study which enrolled newly diagnosed cases of extensively drug-resistant (XDR) or pre-extensively drug resistant (pre-XDR) TB in KwaZulu-Natal, South Africa from 2019 to 2023 and recorded information on their contacts. We found that close contacts declined by 36

Background:One major challenge in the implementation of household contact investigation (HCI) for tuberculosis (TB) in high burden settings is finding contact persons in the home for screening. Conducting HCI during evenings, weekends, or holidays, particularly in settings with high levels of poverty, may improve effectiveness and implementation. Methods:We conducted a pragmatic, individually randomized controlled trial of HCI for TB at two sites in South Africa, comparing the effectiveness of two novel strategies for timing (during evenings and weekends in an urban area and during three annual holiday periods in a rural area) to weekday working hours. The primary outcome was the number of secondary cases identified and started on TB treatment per index participant, comparing novel versus standard timing at each site. Clinicaltrials.gov registration: NCT04520113. Findings:From September 2020 to August 2023, we randomized 1335 index participants with TB in Limpopo to receive standard HCI and 666 to receive holiday = based HCI, and 1616 to receive standard HCI and 805 to receive evening/weekend HCI in Soshanguve. In Limpopo, standard HCI and holidy-based HCI and resulted in 0.6 and 0.7 secondary TB diagnoses started on treatment per 100 index participants, respectively (difference: 0.1 [95% CI: -0.7, 0.8, p = 0.84]). In Soshanguve, evening/weekend-based HCI and standard HCI generated 0.4 and 0.6 diagnoses started on TB treatment per 100 index participants, respectively (difference 0.3 [95% CI: -0.8, 0.4, p = 0.54]). Interpretation:HCI conducted either during evenings/weekends or during holiday did not increase effectiveness compared to HCI conducted during weekday working hours. Funding:Funding was provided by the United States National Institute of Allergy and Infectious Diseases (Grant # 5R01AI147681).

RATIONALE:Bedaquiline is a critical component of new drug-resistant tuberculosis (TB) treatment regimens. Emergence of bedaquiline-resistant Mycobacterium tuberculosis (Mtb) threatens to undermine already limited treatment options. Understanding the evolution and transmission of bedaquiline-resistant strains is a high priority for TB control. OBJECTIVES:We evaluated the prevalence, strain genetic diversity, and transmission of bedaquiline resistance among people with drug-resistant TB. METHODS:We conducted a prospective study of all patients diagnosed with TB resistant to a fluoroquinolone or a second-line injectable drug in KwaZulu-Natal province, South Africa, between 2018 and 2022. We utilized whole genome sequencing (WGS), SNP-based clustering, and Bayesian reconstruction of transmission trees to examine the genomic characteristics and transmission networks of bedaquiline-resistant Mtb strains. MEASUREMENTS AND MAIN RESULTS:We enrolled 843 of 1070 eligible individuals and obtained WGS for 632 participants. Of the sequenced isolates, 14% (n = 89) were genotypically bedaquiline-resistant, 67% (n = 60) of which were in clusters of recently transmitted infections. Resistance was present across all Mtb lineages and across strains with different resistance profiles to other TB drugs. We observed no differences in lineage distribution, bedaquiline resistance-associated mutations, or drug resistance profiles between clustered and nonclustered isolates. Transmission inference and geographic reconstruction of transmission networks indicate considerable unsampled cases within transmission networks. CONCLUSIONS:Bedaquiline resistance was common in this cohort of individuals with highly drug-resistant TB. Most bedaquiline-resistant infections were attributable to recent transmission, involving Mtb strains with diverse genetic backgrounds and a variety of causative mutations. The diversity and transmissibility of bedaquiline-resistant Mtb signal a growing public health threat.

BACKGROUND:The efficacy and safety of the M72/AS01E tuberculosis (TB) candidate vaccine were evaluated in the phase 2b randomized, placebo-controlled trial NCT01755598. In the trial, participants with Mycobacterium tuberculosis sensitization by a positive interferon-gamma release assay received either M72/AS01E or placebo (randomized 1:1) and were followed for 3 years. In this descriptive study, we genetically characterized M. tuberculosis isolates from participants who developed pulmonary TB during the trial to assess variability among isolates and evaluate the possibility of preferential progression from infection to active TB by certain strains in M72/AS01E and placebo recipients. METHODS:M. tuberculosis isolates were recovered from sputum samples and underwent DNA extraction and sequencing to assess lineage, antibiotic resistance profile, genetic variation, and genomic clusters. RESULTS:One hundred isolates (37 M72/AS01E, 63 placebo) were genetically characterized from 50 participants who developed microbiologically confirmed active pulmonary TB during the trial (20 M72/AS01E, 30 placebo). Diverse M. tuberculosis lineages were identified in both M72/AS01E and placebo recipients. No multidrug-resistant strains were detected. Genetic sequences corresponding to the antigenic components of M72/AS01E, showed low variation in pepA and high variation in PPE18. However, no apparent preferential distribution of variants was observed in M72/AS01E or placebo recipients. Cluster analyses identified 10 genomic clusters with isolates from >1 participant, illustrating complex transmission dynamics and potential of co-infection by multiple strains within individuals. CONCLUSION:This descriptive analysis showed no apparent preferential distribution of local M. tuberculosis strains in M72/AS01E or placebo recipients. These results support the continued development of this candidate vaccine.

In vitro experiments with Mycobacterium tuberculosis showed that clofazimine exposure selected for delamanid and pretomanid resistance and mutations in fbiA, fbiC, or fbiD-after the acquisition of Rv0678 mutations where this could be determined. Whether this is also possible in vivo and in an Rv0678 wild-type background has to be studied further. Based on the available evidence, however, we propose that nitroimidazole resistance should not be considered an exclusion criterion for the use of clofazimine.

Tuberculosis remains a global health problem. Making it easier and quicker to identify which antibiotics an infection is likely to be susceptible to will be a key part of the solution. Whilst whole-genome sequencing offers many advantages, the processing of the genetic reads to produce the relevant public health and clinical information is, surprisingly, often the responsibility of the end user, which inhibits uptake. Here, we characterize how well a freely available tool we have developed, gnomonicus, predicts the antibiotic resistance profile of a sample (given its variant call file) using our implementation of the second edition of the World Health Organization (WHO) catalogue of resistance-associated variants (WHOv2). To facilitate this, we have constructed a diverse test set of 2,663 publicly available Mycobacterium tuberculosis samples, which have both genetic and drug susceptibility testing (DST) data. We have chosen to apply the catalogue such that our tool will return a result of (i) Fail if there are insufficient reads at a genetic locus associated with resistance, (ii) Unknown if a genetic variant in a resistance gene not listed in the catalogue is encountered and (iii) Resistant if three or more short-reads support the presence of a resistance-associated variant. The last step increases the sensitivity for all 15 antibiotics but only reaches significance in a few in our test set. Comparing our results with those of TB-Profiler, an existing tool, highlights the different design choices and demonstrates that the performance of both tools on our diverse test set is comparable. By only considering high-confidence DST results, we show that gnomonicus, in combination with our translation of WHOv2, achieves sensitivities and specificities in excess of 95% for both isoniazid and rifampicin.

Bedaquiline (BDQ) has only been approved for use for just over a decade and is a key drug for treating multidrug-resistant tuberculosis; however, rising levels of resistance threaten to reduce its effectiveness. Catalogues of mutations associated with resistance to BDQ are key to detecting resistance genetically for either diagnosis or surveillance. At present, building catalogues requires considerable expert knowledge, often requires the use of complex grading rules and is an irreproducible process. We developed an automated method, catomatic, that associates genetic variants with resistance (or susceptibility) using a two-tailed binomial test with a stated background rate and applied it to a dataset of 11,867 Mycobacterium tuberculosis samples with whole-genome and BDQ susceptibility testing data. Using this framework, we investigated how to best classify variants and the phenotypic significance of minor alleles. The genes mmpS5 and mmpL5 are not directly associated with BDQ resistance, and our catalogue of Rv0678 , atpE and pepQ variants attains a cross-validated sensitivity and specificity of 79.4±1.8% and 98.5±0.3%, respectively, for 94±0.4% of samples. Identifying samples with subpopulations containing Rv0678 variants improves sensitivity, and detection thresholds in bioinformatic pipelines should therefore be lowered. By using a more permissive and deterministic algorithm trained on a sufficient number of resistant samples, we have reproducibly constructed a catalogue of BDQ resistance-associated variants that is comprehensive and accurate.

Background Economic and behavioural factors lead to poor outcomes in patients with tuberculosis. We investigated the effects of a package of interventions consisting of pre-test and post-test tuberculosis counselling with conditional cash transfers on patient outcomes in adults undergoing investigation for pulmonary tuberculosis. Methods This pragmatic, open-label, individual randomised controlled trial was done in nine clinics in Johannesburg, South Africa. Participants (aged >= 18 years) undergoing investigation for tuberculosis were randomly assigned (1:1) to the intervention group or control group (standard of care) via permuted block randomisation, stratified by clinic; group assignment was concealed using opaque envelopes. The intervention group received pre-test and post-test tuberculosis counselling, and for participants diagnosed with rifampicin-susceptible tuberculosis, a digital payment (R150; approximately US$10) at treatment initiation and each monthly treatment visit. Payments were contingent on timely attendance: 14 days from initial sputum sample collection and within 7 days on either side of their scheduled monthly appointment. The primary endpoint was successful patient outcome (patients who were cured or completed treatment) or unsuccessful patient outcome (pretreatment loss-to-follow-up, on-treatment loss-to-follow-up, development of rifampicin-resistant tuberculosis while on treatment, treatment failure [ie, smear or culture positive at 5 months or later after commencing treatment], or death). The primary outcome was analysed in the modified intention-to-treat population, defined as all randomly assigned participants with rifampicin-susceptible tuberculosis confirmed before the commencement of tuberculosis treatment. Weighted outcome prevalence, relative risks (RRs), and risk differences were calculated using a multivariable Poisson model with robust standard errors. This trial is registered with the Pan African Clinical Trials Registry (PACTR202410708311054) and is completed. Findings Between Oct 25, 2018, and Dec 9, 2019, 4110 participants were enrolled and randomly assigned, 2059 to the intervention group and 2051 to the control group. 381 (93%) participants had microbiologically confirmed rifampicinsusceptible pulmonary tuberculosis (195 [95%] of 2059 in the intervention group vs 186 [91%] of 2051 in the control group; median age 37 years [IQR 30 to 45], 257 [675%] male, 124 [325%] female). At study closure, primary outcome data were available for 128 (656%) of 195 participants in the intervention group and 139 (747%) of 186 participants in the control group. 105 (820%) of 128 participants in the intervention group and 93 (669%) of 139 participants in the control group had a successful patient outcome; 23 (180%) of 128 participants in the intervention group and 46 (331%) of 139 participants in the control group had an unsuccessful patient outcome. The weighted regression analysis showed a substantial reduction in the risk of unsuccessful patient outcomes in the intervention group compared with the control group (weighted prevalence 159% vs 286%; RR in weighted population 052, 95% CI 033 to 082; risk difference in weighted population-141 percentage points, 95% CI-233 to-48). Pretreatment loss to follow-up was lower in the intervention group than in the control group (unweighted population: five [39%] of 128 participants vs 22 [158%] of 139 participants; risk difference in weighted population-96 percentage points, 95% CI-149 to-42). Interpretation The package of interventions consisting of pre-test and post-test tuberculosis counselling with conditional cash transfers significantly reduced the risk of unsuccessful tuberculosis patient outcomes, bringing one of the 90-90-90 targets within reach (ie, achieving 90% tuberculosis treatment success). Furthermore, reduction in pretreatment loss to follow-up is expected to reduce transmission and lower incidence of the disease over time. Funding South African Medical Research Council, UK Medical Research Council, and Newton Fund. Crown Copyright (c) 2025 Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license.

BACKGROUND:Drug-resistant tuberculosis remains a major obstacle in ending the global tuberculosis epidemic. Deployment of molecular tools for comprehensive drug resistance profiling is imperative for successful detection and characterisation of tuberculosis drug resistance. We aimed to assess the diagnostic accuracy of a new class of molecular diagnostics for drug-resistant tuberculosis. METHODS:We conducted a prospective, cross-sectional, multicentre clinical evaluation of the performance of two targeted next-generation sequencing (tNGS) assays for drug-resistant tuberculosis at reference laboratories in three countries (Georgia, India, and South Africa) to assess diagnostic accuracy and index test failure rates. Eligible participants were aged 18 years or older, with molecularly confirmed pulmonary tuberculosis, and at risk for rifampicin-resistant tuberculosis. Sensitivity and specificity for both tNGS index tests (GenoScreen Deeplex Myc-TB and Oxford Nanopore Technologies [ONT] Tuberculosis Drug Resistance Test) were calculated for rifampicin, isoniazid, fluoroquinolones (moxifloxacin, levofloxacin), second line-injectables (amikacin, kanamycin, capreomycin), pyrazinamide, bedaquiline, linezolid, clofazimine, ethambutol, and streptomycin against a composite reference standard of phenotypic drug susceptibility testing and whole-genome sequencing. FINDINGS:Between April 1, 2021, and June 30, 2022, 832 individuals were invited to participate in the study, of whom 720 were included in the final analysis (212, 376, and 132 participants in Georgia, India, and South Africa, respectively). Of 720 clinical sediment samples evaluated, 658 (91%) and 684 (95%) produced complete or partial results on the GenoScreen and ONT tNGS workflows, respectively, with 593 (96%) and 603 (98%) of 616 smear-positive samples producing tNGS sequence data. Both workflows had sensitivities and specificities of more than 95% for rifampicin and isoniazid, and high accuracy for fluoroquinolones (sensitivity approximately ≥94%) and second line-injectables (sensitivity 80%) compared with the composite reference standard. Importantly, these assays also detected mutations associated with resistance to critical new and repurposed drugs (bedaquiline, linezolid) not currently detectable by any other WHO-recommended rapid diagnostics on the market. We note that the current format of assays have low sensitivity (≤50%) for linezolid and more work on mutations associated with drug resistance is needed. INTERPRETATION:This multicentre evaluation demonstrates that culture-free tNGS can provide accurate sequencing results for detection and characterisation of drug resistance from Mycobacterium tuberculosis clinical sediment samples for timely, comprehensive profiling of drug-resistant tuberculosis. FUNDING:Unitaid.

Background Catalogues of genetic variants associated with resistance underpin whole-genome sequencing (WGS)-based predictions of drug susceptibility in Mycobacterium tuberculosis , and are essential for molecular diagnostics and surveillance. The current gold standard catalogues released by the WHO represent substantial progress in using standardised data and methods to associate phenotypes to genotypes, but they remain opaque. The underlying data are not fully released and the catalogues are difficult to interpret. Open and reproducible methods would help address these problems, extending the important work already done. Methods We have developed an automated method, catomatic, that uses a binomial test to associate informative isolates with resistance or susceptibility, and built a catalogue (catomatic-1) from the same 39,358 samples used to construct the first WHO catalogue (WHOv1). We performed sensitivity analysis to optimise statistical and bioinformatic parameters for each drug, and benchmarked catomatic-1 against WHOv1 using an independent set of 14,380 isolates. Findings By using simpler statistics, catomatic-1 algorithmically classified 1,329 variants, ranging from 5 for linezolid to 440 for pyrazinamide. WHOv1 included generalisable rules added by a panel of experts, which increase the predictive coverage of WHOv1, but at the cost of reproducibility. Despite excluding rules, catomatic-1 achieves comparable performance for all drugs, with sensitivities for first-line agents above 88% on the independent test set. The automated process allowed us to efficiently explore the parameter space; for instance, detecting resistant variants with low read support improved the sensitivity for all drugs. Interpretation Accurate resistance catalogues can be built automatically using transparent and reproducible statistical methods. As more data are collected, catalogue content and performance will evolve, highlighting the need for proper version control, machine/human readability, and open access. This approach provides a foundation for real-time surveillance, diagnostics, and flexible application to diverse use cases in drug-resistant tuberculosis. ### Competing Interest Statement ZI, DWC & PWF work as consultants for the Ellison Institute of Technology, Oxford Ltd. NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance, NIHR207397 NIHR Oxford Biomedical Research Centre EPSRC Sustainable Approaches to Biomedical Science: Responsible & Reproducible Research CDT ORACLE Corporation

BACKGROUND:Rapid and accurate detection of drug-resistant tuberculosis (DR-TB) is crucial for ensuring effective treatment, halting transmission and preventing the amplification of resistance. Comparative evaluations of molecular diagnostic assays in high-burden settings are essential for informing clinical decision-making for DR-TB treatment. METHODS:The Seq&Treat clinical study previously evaluated the performance of two targeted next-generation sequencing (tNGS) workflows, GenoScreen Deeplex Myc-TB and Oxford Nanopore Technologies Tuberculosis Drug Resistance Test, on direct sediment samples from persons at risk for DR-TB. Hain Line Probe Assay (LPAs-MTBDRplus and MTBDRsl) were run as a comparator test using an aliquot of the same sediment samples. Diagnostic performance of the LPAs and previously established tNGS performance were compared, including sensitivity and specificity, for rifampicin, isoniazid, fluoroquinolones (moxifloxacin, levofloxacin), and amikacin, using a composite reference standard of phenotypic drug susceptibility testing and whole-genome sequencing. FINDINGS:Among 720 clinical samples tested, MTBDRplus LPA sensitivity for rifampicin and isoniazid was 92.3% (95% CI 88.9-94.8) and 91.9% (88.4-94.4), each significantly lower than ≥95% achieved by both tNGS workflows (p < 0.01). For fluoroquinolones (moxifloxacin and levofloxacin), the MTBDRsl LPA and ONT had similar sensitivities (94.3% and 92.7%, and 94.8% and 93.9%, respectively), while GenoScreen outperformed both (97.3% and 96.6%). GenoScreen also demonstrated the highest sensitivity for amikacin resistance (94.6%) compared to LPAs (88.7%) and ONT (88.3%). Complete assay failure rates were low for LPAs (4.9%) and ONT (5.0%) and moderately higher for GenoScreen (8.6%), with differences in single-target failures across all assays. INTERPRETATION:LPAs demonstrated lower sensitivity and more limited drug resistance detection compared to tNGS workflows, underscoring the advantages of tNGS for improving DR-TB diagnostic algorithms. These findings provide critical evidence to guide updates in DR-TB diagnostic programs. FUNDING:Support for the Seq&Treat project was provided through funding from Unitaid (2019-32-FIND MDR).

BACKGROUND:Whole genome sequence data can generate insights about Mycobacterium tuberculosis (Mtb) transmission. We used whole genome sequencing and linked epidemiology data from a recent randomized trial to characterize Mtb relatedness across 3 geographically distinct South African sites. METHODS:We sequenced culture isolates from participants with culture-positive tuberculosis in the Kharituwe study, which evaluated household contact investigation strategies in 1 urban and 2 rural sites. We adapted a previous bioinformatic pipeline to clean, extract, and filter Mtb reads; perform reference alignment; calculate single-nucleotide polymorphism (SNP) distances between isolates; and group isolates into clusters linked by recent transmission based on 3 SNP-based cutoffs. Sequence data were linked to individual data on demographics and risk factors. We analyzed clustering across and within study sites and used log-binomial regression to assess characteristics associated with clustering. RESULTS:At a cutoff of 12 SNPs, 213 of 714 sequenced isolates passing quality control filters were clustered. While only 3 of 45 pairs included participants from different sites, the majority of clusters with ≥4 participants included representation from at least 2 sites. Expanding to a 20-SNP cutoff revealed a large cluster containing 10% of isolates, with urban/rural representation mirroring that of all the isolates (61% urban, 39% rural). Participants from the urban site, TB household contacts, and participants reporting a history of incarceration were more likely to be in a cluster. CONCLUSIONS:Observed clustering and strain diversity across sites indicate the presence of multiple ongoing and geographically dispersed outbreaks in this setting.

ABSTRACT Rapid and comprehensive drug susceptibility testing (DST) is essential for diagnosing and treating drug-resistant tuberculosis effectively, and next-generation sequencing can be an effective genotypic DST method. We implemented and evaluated the performance of a nanopore targeted sequencing assay, called the Tuberculosis Drug Resistance Test (TBDR, Oxford Nanopore Diagnostics, Ltd., United Kingdom), which predicts drug resistance to 16 TB drugs, at a South African reference laboratory and a district diagnostic laboratory in Zambia. We compared the sequencing success rates between unprocessed and decontaminated sputum samples and determined the diagnostic accuracy against local DST (Xpert MTB/RIF Ultra, Xpert MTB/XDR, and BD BACTEC MGIT phenotypic DST). We prospectively sequenced 236 samples and have 148 samples with sequencing results from unprocessed and decontaminated sputum. We obtained successful sequencing results from 66.4% (94/148) unprocessed sputum samples and 75% (111/148) decontaminated samples. Sequencing success rates at the two sites differed, with 50.7% (36/71) successful sequencing results from unprocessed sputum in Zambia and 75.3% (58/77) in South Africa. Samples with “low” bacterial load, measured by Xpert MTB/RIF Ultra, tended to produce fewer successful sequencing results. TBDR sequencing predicted resistances in 48 samples, detecting resistance for rifampicin ( n = 41) and isoniazid ( n = 20), as well as 10 second-line drugs ( n = 15). Sensitivity was variable compared to phenotypic DST, ranging from 33 (ethionamide) to 94% (rifampicin), while specificity remained above 90% for all drugs, except clofazimine. The TBDR assay can provide rapid, comprehensive genotypic DST. Technical and operational challenges need to be addressed for its broader implementation in high tuberculosis-burden settings. IMPORTANCE This study illustrates the use of the Tuberculosis Drug Resistance Test (TBDR, Oxford Nanopore Diagnostics, Ltd., United Kingdom) as a rapid drug susceptibility testing (DST) approach for diagnosing drug-resistant TB in the high TB-burden countries of South Africa and Zambia. The TBDR assay predicts resistance to 16 TB drugs, including first- and second-line treatments. By implementing the TBDR assay in a national reference laboratory in South Africa and a district diagnostic laboratory in Zambia, we demonstrate how this technology can provide faster diagnostic results (days) compared to traditional phenotypic DST methods (~2 months), with adequate sensitivity. Missed resistances compared to phenotypic DST indicate that technical improvements are needed. Successful sequencing from unprocessed and decontaminated sputum samples at different sites suggests feasibility in diverse settings, though operational challenges remain. Implementing this rapid, comprehensive DST approach could enhance drug-resistant tuberculosis diagnosis and treatment, ultimately improving patient outcomes and helping to combat tuberculosis in high-burden regions.

Background: High levels of multidrug and rifampicin resistant tuberculosis (MDR/RR-TB) are a global concern, although they have declined over the last decade. TB patients are at increased risk of acquired rifampicin resistance if they have HIV coinfection, especially at low CD4 counts, but this dynamic has not previously been modelled. Methods: We extended a previously-developed model that simulates HIV and TB in South African adults, to include the acquisition and transmission of rifampicin resistance. In line with systematic reviews, the risk of acquiring RR with TB treatment is modelled as being negatively associated with patients' CD4 counts. We allow for temporal changes in drug susceptibility testing, both before treatment initiation and at treatment failure, as well as other changes in TB prevention and treatment. The model is calibrated to data from national TB drug-resistance surveys, and recorded numbers of MDR/RR-TB laboratory diagnoses and patients initiating second-line TB treatment, using a Bayesian approach. Results: The model estimates that the proportion of South African TB patients with rifampicin resistance at diagnosis increased from 2.0% (95% CI: 1.7-2.3%) in 1986 to 5.9% (5.2-6.9%) in 2013, in line with survey data. In the absence of HIV, the prevalence of MDR/RR-TB would have increased to 4.1% (2.7-5.1%) in 2013, suggesting a third of rifampicin resistance in 2013 was attributable to HIV. In the absence of antiretroviral treatment (ART), the prevalence of rifampicin resistance would have been higher (6.5% [5.6-7.6%] in 2013, rising to 6.9% [5.7-8.2%] in 2019). ART reduced the prevalence of rifampicin resistance in 2019 by 17%. Conclusions: In countries with high HIV prevalence, HIV may be a major driver of rifampicin resistance in people with TB. ART programmes have the potential to reduce the emergence of resistance substantially. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported, in part, by the Bill & Melinda Gates Foundation (INV-019496 and INV-063625). The conclusions and opinions expressed in this work are those of the authors alone and shall not be attributed to the Foundation. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Human Research Ethics Committee of the University of Cape Town gave approval for this work (ref. 740/2025). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. Most of the data used in the model calibration are from publicly available sources cited in the paper.

ABSTRACT Rapid molecular diagnostics have significantly improved access to tuberculosis (TB) drug resistance detection and reduced turnaround times. However, these tools remain limited in their capacity to generate comprehensive resistance profiles, particularly for newer and repurposed anti-TB drugs. Targeted next-generation sequencing (tNGS) offers a promising alternative, enabling a broader resolution of Mycobacterium tuberculosis genomes and faster reporting compared to phenotypic drug susceptibility testing (DST). We conducted a prospective, cross-sectional, multicenter evaluation of the DeepChek 13-Plex KB Drug Susceptibility Testing (ABL Diagnostics S.A., France) tNGS assay for direct detection of TB drug resistance from clinical sputum samples. The study was performed at three reference laboratories in India, South Africa, and Georgia between April 2021 and June 2022. Adults (≥18 years) with confirmed pulmonary TB were enrolled. Sensitivity and specificity were assessed for key anti-TB drugs against a composite reference standard combining phenotypic DST and whole-genome sequencing. Of 832 participants enrolled, 694 (83.4%) were included in the final analysis. Sequencing was successful in 75.6% of samples. Failure rates were higher in samples with low or very low Xpert MTB/RIF categories. The ABL tNGS workflow showed ≥95% sensitivity for rifampicin, isoniazid, and levofloxacin; 92%–93% for pyrazinamide and moxifloxacin; 88% for ethambutol; and 72%–82% for bedaquiline and clofazimine. Specificity was ≥95% for all drugs. The ABL tNGS workflow enables comprehensive resistance profiling, including for new and repurposed TB drugs. However, higher bacillary loads are required to provide a valid test compared to current class-based tNGS assays recommended by the WHO, thus requiring further improvements. IMPORTANCE Drug-resistant tuberculosis (TB) threatens progress in global TB control, yet current molecular tests detect resistance to only a few drugs. Targeted next-generation sequencing (tNGS) can read many resistance-related genes at once, offering faster and broader results than conventional culture-based testing. We evaluated a commercial tNGS workflow (DeepChek 13-Plex KB, ABL Diagnostics) for direct detection of drug resistance in sputum samples from adults with pulmonary TB in India, South Africa, and Georgia. Among 832 participants, sequencing produced valid results for most samples with moderate or high bacterial loads. The assay accurately identified resistance to key drugs—including rifampicin, isoniazid, fluoroquinolones, and newer medicines such as bedaquiline and clofazimine—while maintaining high specificity. These findings show that tNGS can deliver comprehensive resistance profiles, supporting tailored treatment for people with drug-resistant TB. Further refinement in sample preparation may expand its use to specimens with lower bacterial counts. CLINICAL TRIALS This study is registered with ClinicalTrials.gov as NCT04239326 .

[This corrects the article DOI: 10.1371/journal.pgph.0001788.].

OBJECTIVES/GOALS: DR-TB care in South Africa includes decentralized treatment with shorter, all-oral regimens. Treatment guidelines direct regular clinical and laboratory evaluation to assess patient improvement. We therefore measured sputum collection frequency and follow-up time to assess fidelity to these guidelines in Gauteng Province, South Africa. METHODS/STUDY POPULATION: We included Rifampicin-resistant (RR) sputum specimens from the South African National Health Laboratory Service, which provides pathology services to 80% of the population, submitted between August 2022-September 2023. Patient data were obtained from a DR-TB registry and additional sputum specimen data were collected from follow-up laboratory worksheets. Follow-up spanned from first sputum collection date (baseline) to patient outcome date (e.g., completion, lost) or study closure date (if still on treatment). Monthly sputum submission rate was measured for those with ≥1 additional sputum submitted. We compared patient data by treatment site: at the specialized hospital vs. any other site, using Wilcoxon ranksum and χ2 tests. RESULTS/ANTICIPATED RESULTS: Baseline RR-TB specimens were available for 142 patients, of whom 28 (20%) had specimens submitted from the specialized hospital. Patients at the specialized hospital were older (median age 41 vs. 35.5 years, p=0.03), had higher baseline fluoroquinolone resistance (10% vs. 1%, p=0.01), and longer follow-up (median 5.2 vs. 3.5 months, p=0.01) compared to patients elsewhere. Further, 43 (30%) patients had ≥1 additional sputum submitted during follow-up. Among these, monthly sputum collection rates did not differ by site (0.3 vs. 0.3 sputum per month, p=0.89). We anticipate that increased sputum frequency will be associated with successful TB treatment outcomes based on preliminary findings. DISCUSSION/SIGNIFICANCE: These findings highlight ongoing challenges with routine laboratory follow-up according to DR-TB guidelines across treatment sites in South Africa. Future research is needed to determine reasons for low sputum collection rates, such as low patient adherence, variation in practice of healthcare workers, loss to follow-up, and clinical challenges.

ABSTRACTPrevious work reported unprecedented differences in the intrinsic in vitro susceptibility of the Mycobacterium tuberculosis complex (MTBC) to pretomanid (Pa) using the Mycobacteria Growth Indicator Tube (MGIT) system. We tested 125 phylogenetically diverse strains from all known MTBC lineages (1–9) without known Pa resistance mutations and four strains with known resistance mutations as controls. This confirmed that MTBC, unlike most bacteria-antimicrobial combinations, displayed substantial differences in the intrinsic susceptibility relative to the technical variation of Pa MIC testing. This was also the case for the Middlebrook 7H11 (7H11) medium, demonstrating that these differences were not specific to MGIT. Notably, lineage 1 was confirmed to have intrinsically elevated MICs compared with lineages 2, 3, 4, and 7 (L2–4/7), underlining the urgent need for WHO to publish its decision of whether lineage 1 should be deemed treatable by BPaL(M), the now preferred all-oral regimen for treating rifampin-resistant tuberculosis. Lineages 5 and 6, which are most frequent in West Africa, responded differently to Pa, with lineage 5 being more similar to L2–4/7 and lineage 6 being more susceptible. More data are needed to determine whether 7H11 MICs are systematically lower than those in MGIT.IMPORTANCEThis study confirmed that the Mycobacterium tuberculosis complex lineage 1, responsible for 28% of global tuberculosis cases, is less susceptible to pretomanid (Pa). It also refined the understanding of the intrinsic susceptibilities of lineages 5 and 6, most frequent in West Africa, and lineages 8 and 9. Regulators must review whether these in vitro differences affect the clinical efficacy of the WHO-recommended BPaL(M) regimen and set breakpoints for antimicrobial susceptibility testing accordingly. Notably, regulators should provide detailed justifications for their decisions to facilitate public scrutiny.

BACKGROUND:Emerging resistance to bedaquiline (BDQ) threatens to undermine advances in the treatment of drug-resistant tuberculosis (DRTB). Characterizing serial Mycobacterium tuberculosis (Mtb) isolates collected during BDQ-based treatment can provide insights into the etiologies of BDQ resistance in this important group of DRTB patients. METHODS:We measured mycobacteria growth indicator tube (MGIT)-based BDQ minimum inhibitory concentrations (MICs) of Mtb isolates collected from 195 individuals with no prior BDQ exposure who were receiving BDQ-based treatment for DRTB. We conducted whole-genome sequencing on serial Mtb isolates from all participants who had any isolate with a BDQ MIC >1 collected before or after starting treatment (95 total Mtb isolates from 24 participants). RESULTS:Sixteen of 24 participants had BDQ-resistant TB (MGIT MIC ≥4 µg/mL) and 8 had BDQ-intermediate infections (MGIT MIC = 2 µg/mL). Participants with pre-existing resistance outnumbered those with resistance acquired during treatment, and 8 of 24 participants had polyclonal infections. BDQ resistance was observed across multiple Mtb strain types and involved a diverse catalog of mmpR5 (Rv0678) mutations, but no mutations in atpE or pepQ. Nine pairs of participants shared genetically similar isolates separated by <5 single nucleotide polymorphisms, concerning for potential transmitted BDQ resistance. CONCLUSIONS:BDQ-resistant TB can arise via multiple, overlapping processes, including transmission of strains with pre-existing resistance. Capturing the within-host diversity of these infections could potentially improve clinical diagnosis, population-level surveillance, and molecular diagnostic test development.