The review, authored by Patrick K. Moonan of the U.S. Centers for Disease Control and Prevention’s Division of Global HIV and Tuberculosis and Timothy H. Holtz of the George Washington University Milken Institute School of Public Health, draws on a structured PubMed search that identified 12,351 tuberculosis-related records published between January 1 and December 31, 2025. Rather than a formal systematic review, the authors describe their work as a narrative synthesis guided by scientific rigor, novelty, translational relevance, and policy impact, prioritizing randomized trials, systematic reviews, high-impact mechanistic studies, and major policy updates. Their central conclusion is both encouraging and cautionary: the science accelerated dramatically in 2025, but health-system limitations and inequitable access continue to constrain real-world impact, and without deliberate strategies to bridge the gap between discovery and delivery, breakthroughs may fail to generate population-level change.

Nowhere was the progress more visible than in diagnostics. Artificial intelligence–assisted chest radiography emerged as one of the year’s most consequential tools, improving diagnostic sensitivity and reader consistency while reducing time to triage in high-volume screening programs across India, Eastern Europe, and Southeast Asia. A systematic review of 152 studies found that AI-based methods, particularly deep learning applied to radiographic data, achieved consistently high diagnostic performance, with pooled sensitivity estimates commonly exceeding 85 percent depending on study design and reference standard. The WHO subsequently recognized computer-aided detection tools as critical for lowering the proportion of undetected disease. In Nigeria, researchers found that ultraportable chest radiography paired with AI interpretation was a cost-effective approach for active case finding, improving diagnostic yield while reducing reliance on scarce expert readers.

Equally transformative were advances in noninvasive specimen collection. Tongue-swab polymerase-chain-reaction tests demonstrated diagnostic accuracy comparable to sputum-based GeneXpert MTB/RIF assays when evaluated against the same reference standards. The multi-country SMART4TB study, which enrolled 1,168 participants with presumptive tuberculosis in the Philippines, South Africa, Nigeria, and Zambia, found that tongue-swab testing achieved remarkably high specificity of 99.6 percent and moderate sensitivity of 66.0 percent against a culture-based reference standard, with low rates of non-actionable results at most sites. These characteristics make the approach particularly valuable for individuals unable to produce sputum, including children, older adults, and people living with HIV. The authors caution, however, that moderate sensitivity in adults means these tests are best deployed within diagnostic algorithms rather than as standalone tools for treatment initiation, with confirmatory testing remaining essential for clinical decision-making.

Stool-based molecular testing also matured significantly. Optimization studies of the Simple One-Step stool method for GeneXpert Ultra showed that real-world variation in stool quantity, transport time, and ambient temperature did not meaningfully affect error rates or positivity, reinforcing suitability for decentralized, child-friendly testing. Programmatic evaluations demonstrated high feasibility and acceptability among health workers and increased bacteriologic confirmation of pediatric tuberculosis when stool testing complemented respiratory sampling. Beyond diagnosis, a multi-country study found that stool-based quantification of Mycobacterium tuberculosis correlated closely with sputum culture, provided results more rapidly, and identified individuals at elevated risk for drug resistance and persistent two-month culture positivity, though no single biomarker reliably predicted symptom resolution or mortality. The WHO responded by issuing guidance early in 2025 emphasizing concurrent testing using multiple specimen types in children, including parallel collection of stool and respiratory samples, with urine lipoarabinomannan testing recommended for children living with HIV to improve microbiologic confirmation and reduce diagnostic delay.

On the therapeutic front, 2025 cemented the shift toward shorter, all-oral regimens. For drug-susceptible tuberculosis, randomized trials established the effectiveness of a four-month regimen combining rifapentine, moxifloxacin, isoniazid, and pyrazinamide, with Study 31 demonstrating noninferiority to the traditional six-month standard. A 2025 subgroup analysis among participants with diabetes reported favorable efficacy and safety, with the rifapentine-moxifloxacin arm showing the lowest proportion of unfavorable outcomes. For rifampin-resistant and multidrug-resistant tuberculosis, a large multinational Phase 3 trial evaluated five all-oral regimens lasting six to nine months, finding that four were noninferior to existing standards and achieved favorable outcomes of approximately 80 percent with improved tolerability compared with injectable-containing regimens. These regimens deliberately combine mechanistically orthogonal agents, including ATP synthase inhibition through bedaquiline, protein-synthesis inhibition through linezolid, and DNA gyrase inhibition through fluoroquinolones, to maximize sterilization across bacterial subpopulations. Updated clinical practice guidelines from the American Thoracic Society, CDC, European Respiratory Society, and Infectious Diseases Society of America formally endorsed these shortened approaches, signaling broad consensus that will shape national policy adoption throughout the decade.

The mechanistic foundation underlying these therapeutic advances is increasingly sophisticated. Antimycobacterial drugs act on three principal domains of M. tuberculosis physiology: cell-wall biosynthesis, energy metabolism and oxidative phosphorylation, and protein or nucleic acid synthesis. Bedaquiline and next-generation diarylquinolines collapse intracellular ATP stores by binding the c-subunit of ATP synthase, while cytochrome bc1 inhibitors such as telacebec disrupt the electron transport chain. Benzothiazinones including BTZ-043 target DprE1, an essential epimerase in arabinogalactan synthesis, and oxazolidinones block peptide bond formation at the 50S ribosomal subunit. Emerging compounds reported in 2025 include sorfequiline (TBAJ-876), which in the multi-country Phase 2 NC-009 trial demonstrated greater early bactericidal activity in combination with pretomanid and linezolid than standard first-line therapy, and TBAJ-587, now in Phase 1 evaluation with preclinical data showing higher potency and improved safety margins relative to bedaquiline. A Phase 2a trial of telacebec demonstrated dose-dependent reductions in bacterial load among patients with drug-susceptible pulmonary tuberculosis, and a Phase 1b-2a trial of BTZ-043 in South Africa reported dose-proportional bactericidal activity with favorable tolerability.

Prevention also advanced meaningfully. Two randomized controlled trials evaluated fluoroquinolone-based tuberculosis preventive treatment for household contacts of patients with multidrug-resistant tuberculosis. The VQUIN trial in Vietnam randomized 2,041 household contacts to receive six months of daily levofloxacin or placebo, finding tuberculosis incidence of 0.6 percent in the levofloxacin group compared with 1.1 percent in the placebo group at 30 months. The TB-CHAMP trial in South Africa randomized 922 children exposed to multidrug-resistant tuberculosis to levofloxacin or placebo. Although neither trial reached statistical significance individually due to lower-than-expected incidence, a preplanned individual participant data meta-analysis demonstrated a 60 percent relative reduction in tuberculosis incidence among adult and pediatric contacts receiving levofloxacin, though musculoskeletal adverse events were more frequent among participants aged 10 years and older. Ongoing trials including PHOENIx, evaluating six months of delamanid, and BREACH-TB, evaluating six months of bedaquiline, are expected to further inform preventive strategies for contacts exposed to fluoroquinolone-resistant disease.

Vaccine development progressed on multiple fronts. MTBVAC, a live-attenuated M. tuberculosis vaccine, demonstrated a favorable safety profile and elicited significantly stronger Th1-restricted CD4-positive T-cell responses than BCG in a Phase 1b-2a trial, supporting advancement to Phase 3 efficacy evaluation. The M72/AS01E-4 vaccine completed enrollment of approximately 20,000 participants in its Phase 3 trial in 2025, a major milestone toward generating definitive evidence on protection against tuberculosis. A Phase 2 trial in virally suppressed adolescents and adults living with HIV showed durable antigen-specific T-cell and antibody responses, addressing a critical evidence gap. Not all results were positive: H56:IC31 failed to reduce recurrence among adults who recently completed treatment despite strong immunogenicity, and a Phase 2b evaluation of BCG revaccination did not reduce sustained interferon-gamma release assay conversion in adolescents, underscoring continued uncertainty regarding immunologic correlates of protection. Meanwhile, advances in immunometabolism revealed that tuberculosis infection induces strong upregulation of indoleamine 2,3-dioxygenase-1, shifting tryptophan metabolism toward kynurenine and generating an immunosuppressive granuloma microenvironment, with plasma kynurenine-to-tryptophan ratios emerging as a potential biomarker for infection activity and treatment response.

Perhaps most conceptually significant, 2025 saw a fundamental challenge to how tuberculosis is classified and described. Analyses demonstrated that commonly used symptom-based dichotomies, such as asymptomatic versus symptomatic or subclinical versus clinical tuberculosis, are inadequate classifiers of disease state lacking both biological and operational validity. Nearly half of individuals with bacteriologically confirmed tuberculosis in prevalence surveys do not report symptoms at screening, and a sizeable proportion of reported symptoms in both tuberculosis and non-tuberculosis populations likely arise from unrelated causes. The authors of the review argue that symptoms lie on a continuum rather than a categorical boundary and should not serve as the foundation for disease definitions, epidemiologic estimates, or treatment algorithms, calling instead for objective, reproducible indicators including bacteriologic burden, radiographic extent, and functional impairment. This shift parallels progress in characterizing post-tuberculosis lung disease, for which a 2025 Lancet Infectious Diseases Personal View proposed the field’s first research case definition anchored in lung function, respiratory symptoms, and imaging abnormalities. Despite this remarkable scientific momentum, the WHO Global TB Report documented persistent gaps in case detection, suboptimal treatment success for drug-resistant tuberculosis, and chronic underfinancing, with global funding still far below the estimated US$22 billion needed annually. As the late Dr. Paul Farmer consistently argued, tuberculosis is not only a biomedical problem but a political and moral one, and scientific advances will translate into population-level impact only if matched by sustained commitments to equity, strong health systems, and the social conditions required to deliver care.

Subject of Research: 2025 advances in tuberculosis diagnostics, treatment, prevention, and elimination strategy

Article Title: Research overview—2025: integrating scientific progress to accelerate global tuberculosis elimination

Article References: Moonan, P. K., & Holtz, T. H. (2026). Research overview—2025: integrating scientific progress to accelerate global tuberculosis elimination. Frontiers in Tuberculosis, 4, Article 1805962. https://doi.org/10.3389/ftubr.2026.1805962

Image Credits: AI Generated

DOI: 10.3389/ftubr.2026.1805962

Keywords: tuberculosis, diagnostics, drug-resistant TB, vaccines, AI chest radiography, tongue swab testing, stool-based testing, bedaquiline, host-directed therapy, post-tuberculosis lung disease, WHO End TB Strategy, global health

TB-Profiler was developed to profile M. tuberculosis directly from next-generation sequencing data. Rather than waiting days or weeks for laboratory cultures to grow, the tool scans raw sequencing reads for known resistance-conferring mutations and assigns the isolate to one of the major phylogenetic lineages of the tuberculosis bacillus. This approach, known as genotypic antimicrobial susceptibility prediction, has become increasingly important as sequencing costs fall and national tuberculosis programmes adopt whole-genome sequencing for routine diagnostics. The original version of the tool covered 16 anti-TB drugs; the updated release adds delamanid and pretomanid, two newer agents that are central to modern regimens for multidrug-resistant and extensively drug-resistant disease.

The technical heart of the upgrade lies in its expanded and refined mutation libraries. TB-Profiler does not attempt to predict resistance from first principles; instead, it relies on carefully curated catalogues of mutations that have been experimentally or epidemiologically linked to reduced drug susceptibility. The updated version incorporates interpretation rules endorsed by the World Health Organization, ensuring that the tool’s output aligns with internationally recognised standards for reporting resistance to drugs such as bedaquiline and clofazimine, cycloserine and terizidone, and para-aminosalicylic acid. The developers have also added curated loss-of-function mutations, which are changes that disable a gene entirely and are increasingly recognised as important drivers of resistance to newer drug classes.

Why does this matter clinically? Regimens for drug-resistant tuberculosis have changed dramatically in recent years, shifting towards shorter, all-oral combinations built around bedaquiline, pretomanid and linezolid. Choosing the right regimen requires knowing exactly which drugs the infecting strain can tolerate, and phenotypic antimicrobial susceptibility testing, the traditional gold standard, can take weeks because M. tuberculosis grows so slowly. Genotypic prediction from sequencing data can deliver a resistance profile in a fraction of that time, and tools like TB-Profiler make that analysis accessible to laboratories that lack deep bioinformatics expertise, through both a command-line version and a web interface.

The second major pillar of the update is population context. The team has integrated a continuously expanding global genomic database of more than 170,000 isolates, representing all major lineages of the M. tuberculosis complex and spanning 136 countries. This resource allows the tool to place any newly sequenced genome within its global population framework, comparing allele frequencies and identifying how unusual or common a given mutation is across the worldwide diversity of the pathogen. That context is technically important because not every mutation observed in a resistant isolate actually causes resistance; some are simply neutral variants that happen to be common in certain lineages. A global reference panel helps distinguish the two.

Built on top of this database is a new analytical capability for calculating and visualising genomic relatedness between isolates. In tuberculosis surveillance, genomic relatedness is the key to identifying transmission. When two isolates from different patients are nearly identical across their genomes, it strongly suggests that one infected the other, or that both were infected by a common source. By computing distances between genomes and presenting the results in accessible visualisations, the updated TB-Profiler allows public health teams to spot potential transmission chains and outbreaks directly within the same workflow they already use for resistance prediction, rather than juggling separate tools.

To demonstrate the new functionality, the researchers applied it to isolates from Uganda, where they were able to identify potential transmission events among the sampled genomes. The demonstration illustrates the practical value of combining resistance profiling and relatedness analysis in a single platform. In high-burden settings, distinguishing between reactivation of a latent infection picked up years ago and recent active transmission changes the public health response entirely: recent transmission calls for intensified contact tracing and community screening, whereas reactivation points towards the management of latent infection in vulnerable populations.

Accessibility has also been broadened in a way that could prove decisive for global uptake. The updated tool introduces multilingual reporting capabilities, meaning that the clinical reports generated from sequencing data can be delivered in the languages spoken by the health workers who use them. This may sound like a modest feature, but in tuberculosis control it addresses a real barrier: the countries carrying the greatest burden of drug-resistant disease are often those where English is not the primary working language of laboratory and clinical staff. By lowering the language barrier, the developers hope to widen implementation of genomic surveillance in precisely the regions where it is needed most.

The work is a product of an international collaboration spanning the London School of Hygiene and Tropical Medicine, Thailand’s Ministry of Public Health Medical Genetics Center, the South African National Bioinformatics Institute at the University of the Western Cape, Nottingham Trent University and the University of Cambridge. The informatics development was supported by a UKRI EPSRC award in artificial intelligence innovation to accelerate health research, with additional support from the UKRI Medical Research Council. The tool and its associated databases are available in both stand-alone and web-based versions at the team’s hosted platform, and the underlying article is published open access under a Creative Commons licence.

The developers are explicit that this release is a stepping stone rather than a destination. Future versions will leverage the ever-expanding sequencing database to deploy artificial intelligence approaches aimed at refining lineage assignment, improving drug-resistance prediction and sharpening transmission classification, including the identification of resistance-associated mutations that have not yet been characterised. As the global database grows with every sequenced isolate, the statistical power available to such machine-learning methods grows with it, promising a feedback loop in which each new genome makes the tool smarter for the next patient. For a disease that kills well over a million people each year, turning sequencing data into faster treatment decisions and earlier outbreak detection is a goal worth the engineering effort, and the updated TB-Profiler brings that goal measurably closer.

Subject of Research: Genomic prediction of antimicrobial resistance and transmission in Mycobacterium tuberculosis using the updated TB-Profiler tool

Article Title: Updated TB-Profiler: enhanced genotypic antimicrobial resistance prediction and relatedness analysis powered by a database of over 171,000 Mycobacterium tuberculosis genomes

Article References: Phelan, J. E., Sawaengdee, W., Thorpe, J., Thawong, N., Piboonsiri, P., Billows, N., Wang, L., van Heusden, P., Meehan, C., Köser, C. U., Mahasirimongkol, S., Campino, S., & Clark, T. G. (2026). Updated TB-Profiler: enhanced genotypic antimicrobial resistance prediction and relatedness analysis powered by a database of over 171,000 Mycobacterium tuberculosis genomes. Genome Medicine. https://doi.org/10.1186/s13073-026-01767-y

Image Credits: AI Generated

DOI: 10.1186/s13073-026-01767-y

Keywords: tuberculosis, TB-Profiler, drug resistance, whole-genome sequencing, Mycobacterium tuberculosis, antimicrobial susceptibility prediction, genomic surveillance, transmission analysis, WHO mutation catalogue, bedaquiline, pretomanid, public health genomics

The study, led by Dmytro Butov of Kharkiv National Medical University together with Stefan Niemann and Viola Dreyer of the Research Center Borstel in Germany, enrolled 4,828 patients and successfully sequenced 4,162 isolates after quality-control exclusions. Participants were divided into two groups: a drug-resistant tuberculosis cohort of 3,112 patients with rifampicin-resistant, multidrug-resistant, pre-extensively drug-resistant, or extensively drug-resistant disease, and a comparator cohort of 1,050 patients with non-MDR or drug-susceptible tuberculosis. Within the drug-resistant group, 62.3 percent of isolates were classified as multidrug-resistant, nearly a third as pre-XDR, and 2.5 percent as extensively drug-resistant.

The population structure of the circulating bacteria tells a striking story. Fully 70.6 percent of all sequenced isolates belonged to Lineage 2, the so-called Beijing lineage, which predominates across Eastern Europe and is associated with enhanced transmissibility and a greater propensity to acquire resistance mutations. Among drug-resistant cases, Lineage 2 accounted for 83.7 percent of isolates, rising to 89.7 percent among extensively drug-resistant infections. By contrast, Lineage 4 Euro-American strains, which are more genetically diverse, dominated the comparator cohort, particularly among patients with drug-susceptible disease in western Ukraine.

Even more striking is how little diversity exists within the drug-resistant Lineage 2 population. More than 82 percent of MDR, pre-XDR, and XDR isolates belonged to just four previously described Beijing clades: the Europe/Russia W148 outbreak clone, which alone made up nearly 40 percent of the resistant cohort, the Ukraine outbreak clone, the Central Asia outbreak clone, and the broader Central Asia clade. Geospatial analysis showed Lineage 2 dominance in almost every participating region, with the highest proportions in the central, southern, northern, and eastern parts of the country, while Lineage 4 remained comparatively more common in the west.

Genomic clustering analysis, which identifies isolates separated by five or fewer allelic differences at 2,891 core-genome loci, revealed that nearly half of all isolates belonged to clusters, a signature of recent or ongoing transmission. Clustering was far more frequent in the drug-resistant cohort, affecting 55 percent of isolates, than in the comparator group at 28.9 percent. The three largest clusters, all exclusively Lineage 2, contained 671 drug-resistant isolates, over a fifth of that cohort. Cluster 1, assigned to the Ukraine outbreak clone, comprised 307 isolates, more than half of them pre-XDR or XDR. Cluster 2, the W148 clone, included 296 isolates, mostly MDR, and Cluster 3 contained 76 Central Asia outbreak isolates.

The resistance mutations embedded in these clusters indicate that resistance was largely fixed in common ancestors and propagated through clonal expansion rather than acquired independently. Canonical mutations such as KatG S315T and RpoB S450L, which confer resistance to isoniazid and rifampicin at low fitness cost, dominated all three major clusters. Fluoroquinolone resistance, present in roughly a third of drug-resistant isolates, was driven mainly by GyrA D94G and A90V substitutions, while bedaquiline and clofazimine resistance was linked primarily to loss-of-function variants in Rv0678, notably the 192_ins_g and 141_ins_c frameshift insertions.

Resistance to the newer Group A agents central to modern all-oral therapy remained uncommon, offering a measure of reassurance. WGS-inferred bedaquiline resistance was detected in only 90 isolates overall, about 2.8 percent of the drug-resistant cohort, and linezolid resistance in just 33 isolates, or 1.1 percent. Yet the researchers identified 11 isolates carrying variants associated with combined resistance to fluoroquinolones, bedaquiline, and linezolid, a combination that would compromise both BPaL and BPaLM regimens. Notably, seven bedaquiline-resistant XDR isolates carried atpE variants, six of them in Lineage 2 strains scattered across different clades, suggesting independent acquisition. Comparative analysis showed the Ukrainian atpE-mutant isolates were separated by at least 28 SNPs from publicly available Russian genomes, ruling out a single shared clone and pointing to a possible regional propensity for this resistance mechanism that the authors say warrants urgent investigation.

Temporal analysis across four consecutive 12-month intervals revealed significant declines in resistance to moxifloxacin and levofloxacin, each falling from around 41 percent in 2019–2020 to roughly 28 percent in 2022–2023, with pyrazinamide resistance also declining modestly. The fluoroquinolone trends held up in sensitivity analyses restricted to the 12 consistently sampled regions and within the pooled major genomic clusters. In parallel, recorded treatment success rose from 59.6 percent to 73.4 percent over the study period. The authors, however, caution strongly against causal interpretation: the trends coincided with changes in regional sampling after the February 2022 invasion, population displacement, diagnostic disruptions, evolving treatment policies, and the pandemic, and individual-level regimen data were unavailable.

The study also highlights the social epidemiology of the epidemic. Patients with drug-resistant disease were younger and more often male, and more frequently reported recurrent tuberculosis, previous treatment failure, homelessness, drug use, incarceration, HIV infection, and viral hepatitis. Lineage 2 infection was independently associated with rifampicin-resistant or MDR status, fluoroquinolone resistance, and genomic clustering in multivariable models adjusted for demographic, clinical, social, temporal, and regional factors.

The authors conclude that sustained genomic surveillance of drug-resistant tuberculosis in Ukraine and neighbouring countries is essential, particularly for dominant Lineage 2 clones and for resistance to fluoroquinolones, bedaquiline, and linezolid. They call for systematic fluoroquinolone testing before deployment of shorter all-oral regimens, targeted phenotypic confirmation of bedaquiline and linezolid susceptibility, integration of whole-genome sequencing into routine care, and cross-border data sharing to track tuberculosis among displaced, migrant, and refugee populations. As Ukraine continues to rebuild its health system amid ongoing conflict, the genomic record preserved in this cohort provides both a warning about the entrenched clones fuelling the epidemic and a template for surveillance elsewhere in Eastern Europe.

Subject of Research: Genomic surveillance of drug-resistant Mycobacterium tuberculosis in Ukraine from 2019 to 2023

Article Title: Drug-resistance profiles, population structure, genomic clustering, and temporal trends in drug resistance among Mycobacterium tuberculosis complex isolates in Ukraine, 2019–2023: a multicentre cohort study

Article References: Butov, D., Butova, T., Miasoiedov, V., Feshchenko, Y., Nakonechna, O., Kuzhko, M., Rosenthal, A., Grinev, A., Hoppes, D., Kilmnick, J., Levandovska, D., Hryhorieva, A., Vekshyn, V., Abramova, L., Niemann, S., & Dreyer, V. (2026). Drug-resistance profiles, population structure, genomic clustering, and temporal trends in drug resistance among Mycobacterium tuberculosis complex isolates in Ukraine, 2019–2023: a multicentre cohort study. The Lancet Regional Health – Europe, 70, Article 101865. https://doi.org/10.1016/j.lanepe.2026.101865

Image Credits: AI Generated

DOI: 10.1016/j.lanepe.2026.101865

Keywords: tuberculosis, drug resistance, whole-genome sequencing, Lineage 2 Beijing, MDR-TB, Ukraine, fluoroquinolone, bedaquiline, linezolid, genomic clustering, outbreak clones, public health surveillance

The assay under scrutiny is Deeplex Myc-TB, a targeted next-generation sequencing test developed to detect mutations in the tuberculosis genome that are associated with resistance to a broad range of first- and second-line anti-TB drugs. Unlike conventional rapid molecular tests, which typically interrogate only a handful of genetic regions, targeted sequencing approaches can survey dozens of genes and intergenic regions in a single run. This breadth matters enormously in clinical practice, because treatment decisions for multidrug-resistant and extensively drug-resistant tuberculosis depend on knowing, quickly and reliably, which drugs are likely to fail and which are likely to work.

To evaluate the assay, the researchers turned to an unusual but powerful resource: isolates distributed through the World Health Organization global Tuberculosis Drug-Susceptibility Testing Proficiency Testing Program, organized and administered by the TB Supranational Reference Laboratory in Antwerp, Belgium. A total of 41 isolates were included in the study. These samples are particularly valuable for verification work because they have been exhaustively characterized by reference laboratories using multiple independent methods. In this study, phenotypic drug susceptibility testing performed on Löwenstein-Jensen medium and in mycobacteria growth indicator tube culture, together with whole-genome sequencing, served as the reference standards against which the Deeplex assay was judged.

The analytical workflow was straightforward in concept. Remnant DNA extracted from the proficiency testing isolates was subjected to targeted next-generation sequencing using the Deeplex Myc-TB assay. The resulting sequence data were interpreted using the manufacturer’s Deeplex web application, and, in parallel, with TBProfiler, an open-source tool for predicting drug resistance from mycobacterial sequence data, applying a composite interpretation rule. Concordance, sensitivity, and specificity were then calculated for each drug class, providing a detailed picture of where the assay excels and where it falls short.

The headline result is striking: the Deeplex Myc-TB assay achieved 100 percent specificity across all drug targets examined. In diagnostic terms, this means that whenever the assay reported a resistance mutation, the isolate genuinely carried a mutation associated with resistance. False positives, which can lead clinicians to withhold effective drugs unnecessarily, were entirely absent from the dataset. For several drugs, the assay’s sensitivity was equally impressive. It correctly identified resistance in every tested isolate for rifampicin, detecting 17 of 17 resistant strains; for pyrazinamide, 7 of 7; for fluoroquinolones, 13 of 13; and for linezolid, 6 of 6. For isoniazid, sensitivity was 94.1 percent, with 16 of 17 resistant isolates correctly flagged.

The single isoniazid miss is instructive rather than alarming. The one false-susceptible result traced back to a specific insertion mutation in the katG gene, designated katG c.45_46insA, which fell outside the region targeted by the assay. This illustrates a fundamental property of targeted sequencing: its performance is bounded by the mutations it is designed to detect. When a resistance-conferring variant lies outside the panel’s target regions, even a technically flawless assay will report susceptibility. The finding underscores why laboratories adopting such assays must understand the local epidemiology of resistance mutations in the strains they encounter.

Performance was more mixed for the newer and repurposed drugs that have become central to modern regimens for drug-resistant tuberculosis. For clofazimine, the assay detected resistance in 4 of 5 phenotypically resistant isolates, a sensitivity of 80 percent. For bedaquiline, sensitivity dropped to 44.4 percent, with only 4 of 9 resistant strains correctly identified. In five cases, isolates that were resistant by phenotypic testing carried no resistance variants detected by the assay. The authors traced much of this gap to the biology of resistance to these drugs. Mutations in the mmpR5 gene, also known as Rv0678, a major regulator of efflux pump expression, were largely detected by the assay, but resistance mediated through the atpE gene was not identified, because that gene is not adequately covered by the assay’s target panel.

Overall concordance between the Deeplex assay and the reference standards ranged from 87.5 percent for bedaquiline to a perfect 100 percent for rifampicin, pyrazinamide, fluoroquinolones, and linezolid. Notably, the assay consistently detected several recurrent mutations associated with resistance to the different drugs, reinforcing the idea that for the common, well-established resistance mechanisms, targeted sequencing performs at or near the level of whole-genome sequencing while being faster and cheaper to deploy in routine laboratory settings. For a disease where every week of delayed or inappropriate treatment increases the risk of transmission, disease progression, and death, that speed advantage carries real clinical weight.

The implications of this verification study extend well beyond one laboratory in Uganda. Proficiency testing panels offer a rare opportunity to benchmark a diagnostic assay against isolates whose resistance profiles are known with high confidence, free from the uncertainties that complicate evaluations using routine clinical samples. By demonstrating near-perfect specificity and strong sensitivity for the cornerstone drugs of tuberculosis therapy, the study provides laboratories considering adoption of targeted next-generation sequencing with concrete, independently grounded performance data. At the same time, the documented gaps for bedaquiline and clofazimine serve as a caution against treating any single assay as a complete solution. The authors emphasize that understanding local resistance mutation patterns is essential when implementing sequencing-based resistance detection, because an assay that misses the mutations circulating in a given region will systematically underreport resistance there.

The broader context makes these findings timely. Drug-resistant tuberculosis kills thousands of people each year, and the introduction of bedaquiline, linezolid, and other newer agents has transformed outcomes for patients with resistant disease, but only when those drugs are deployed appropriately. Molecular diagnostics that can rapidly and accurately profile resistance across the full range of current and repurposed drugs are a critical link in that chain. This study shows that targeted sequencing platforms have matured to the point where they can match reference standards for the most important drugs, while also mapping precisely where the remaining blind spots lie. For laboratory directors, national tuberculosis programs, and diagnostic developers alike, the message is twofold: the technology is ready for prime time in the core of TB drug resistance testing, but careful attention to target coverage, and to the mutation landscape of the populations being served, remains indispensable.

Subject of Research: Analytical performance verification of a targeted next-generation sequencing assay for detecting drug-resistant Mycobacterium tuberculosis

Article Title: Analytical performance verification of the Deeplex Myc-TB assay using well-characterized Mycobacterium tuberculosis proficiency testing isolates

Article References: Kabahita, J. M., Lunkuse, J. M., Batte, D. N., Nakato, H. S., Namutebi, J., Kabugo, J., Adam, I., Kanyerezi, S., Makoha, C., Nsubuga, M. L., Oundo, H. R., Wenka, G. T., Sseruyange, J., Murungi, M., Kasule, G. W., Lutaaya, P., Kyokushaba, J., Byabajungu, H., Ademun, P., … Joloba, M. L. (2026). Analytical performance verification of the Deeplex Myc-TB assay using well-characterized Mycobacterium tuberculosis proficiency testing isolates. BMC Genomics. https://doi.org/10.1186/s12864-026-13336-z

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13336-z

Keywords: tuberculosis, drug resistance, Deeplex Myc-TB, targeted next-generation sequencing, whole-genome sequencing, diagnostics, bedaquiline, clofazimine, isoniazid, rifampicin, proficiency testing, Uganda