Tuberculosis

Background: With the emergence of drug-resistant strains and an unprecedented threat to control initiatives, tuberculosis remains to be a major public health risk in Ethiopia. Resistance to rifampicin (RR) is an important indicator, since RR is an acceptable surrogate for multidrug-resistant TB (MDR-TB). Over 95% of RR is based on mutations in an 81base pair segment of the rpoB gene, detected using rapid molecular assays. Despite this, detailed molecular epidemiological information is scarce. This study characterized the specific rpoB gene mutation patterns among patients in Addis Ababa, Ethiopia. Methods: A cross-sectional study was conducted in 753 Mycobacterium tuberculosis complex (MTBC) clinical samples, corroborated as positive for MTBC from 2020 to 2024; respective probe mutation patterns were generated by the Xpert MTB/RIF platform. Demographic and clinical variables were also assessed for detecting the potential risk factors. Results: The overall RR-TB rate was 2.3% (17/753). Molecular analysis showed a distinct pattern of mutation, with codon 526 mutations being the most frequent, occurring in 54.3% of the resistance mechanisms. This was followed by those at codons 531 (21.7%) and 533 (15.2%). Most significant was the fact that 100% of RR-TB was observed among treatment-naive patients, providing unequivocal evidence that primary transmission is the exclusive cause of resistance in this population. Moreover, there were no statistically significant correlations between RR-TB and demographic factors, including sex, age, or HIV co-infection. Conclusion: The study demonstrates a steady, low-grade epidemic of RR-TB in Addis Ababa, dominated by a virulent bacterial strain with a distinctive mutation at codon 526. These observations highlight the imperative necessity for a strategic shift from a reactive, clinically-oriented model to proactive public health measures. To effectively break the chains of transmission, we recommend the universal application of drug susceptibility testing, enhanced and socially-directed contact tracing, and integrating molecular surveillance into the TB control program.

The lack of a reliable chronic murine model limits drugs evaluation against Mycobacterium abscessus. Models show discrepancies, especially regarding host factors (mouse strain, sex and age). Using beads-model, we compared BALB/cJRJ and C57BL/6NCrl across sexes and ages. BALB/cJRJ showed more sustained infection and lower variability, with no significant sex- or age-related differences. Considering these results and the higher prevalence of NTM pulmonary infections in female patients, 5-6 weeks-old female BALB/cJRJ are appropriate for M. abscessus beads-model.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), has regained its position as the worlds leading killer among infectious diseases. Despite extensive research progress across epidemiology, diagnosis, drug development, treatment regimens, vaccines, drug resistance, virulence factors, and immune mechanisms, MTB-related knowledge remains fragmented across thousands of publications, limiting its effective use. To address this gap, we present MTB-KB, a literature-curated knowledgebase that systematically integrates high-impact findings from eight major sections of TB research. The current release contains 75,170 associations from 1,246 publications, covering 18,439 entities standardized using authoritative databases and WHO-endorsed classifications. A central feature is the interactive knowledge graph, which links cross-section associations to reveal and infer MTB-host interactions, treatment strategies, and vaccine development opportunities. MTB-KB also provides a user-friendly interface with browsing, advanced search, and statistical visualization. Overall, by consolidating dispersed MTB knowledge into a structured and accessible platform, MTB-KB provides a valuable resource for researchers, clinicians, and policymakers, supporting both basic and clinical TB research, enabling evidence-based TB prevention, diagnosis, and treatment, and contributing to global elimination efforts. MTB-KB is accessible at https://ngdc.cncb.ac.cn/mtbkb/.

Rationale: Sputum-based testing using Xpert MTB/RIF Ultra (Xpert) is the most common molecular testing method for diagnosing tuberculosis (TB). Objectives: To evaluate whether sputum quality influences Xpert positivity and diagnostic accuracy. Methods: We screened consecutive people for presumptive TB in India, the Philippines, Vietnam, Nigeria, South Africa, Uganda, and Zambia as part of the R2D2 TB Network and ADAPT studies. Participants provided 2-3 sputum samples for Xpert and culture reference testing. The quality of the first sputum sample was graded following standardized procedures by trained research staff and used for Xpert testing. We performed logistic regression to evaluate whether sputum grade was independently associated with Xpert positivity, and calculated sensitivity and specificity of Xpert against a culture-based microbiological reference standard (MRS). Measurements and Main Results: Among 1,855 participants, 798 (43%) were female, 348 (19%) were living with HIV (PLHIV), and 1795 (97%) had a cough of [≥]2 weeks. Overall, 313 (17%) had a positive Xpert result. Most sputum samples were salivary (83%). Xpert positivity was lowest among salivary samples (16.1%) and highest among purulent samples (31.2%). After adjusting for demographic and clinical variables, there was no significant association between any sputum grade and Xpert positivity. Xpert sensitivity (salivary: 89%, mucoid: 91%, mucopurulent: 87%, purulent: 100%) and specificity (>98%) were high across sputum grades. Conclusions: Sputum quality was not independently associated with Xpert positivity and Xpert sensitivity was high across all sputum grades. These findings support molecular testing of all sputum samples for TB diagnosis regardless of macroscopic appearance.

Background: Host genetics alone explains limited susceptibility to tuberculosis (TB), particularly in people with HIV (PWH). Protein quantitative trait loci (pQTLs), genetic variants that regulate plasma protein levels, may bridge genetic and immunological mechanisms underlying TB progression. Methods: We conducted cis-pQTL mapping in 60 PWH who progressed to active TB and 194 matched controls from the Swiss HIV Cohort Study. Plasma proteomes were quantified via high-resolution mass spectrometry (dia-PASEF), and genotype-protein associations were analyzed separately in TB and control groups. Results: TB progressors harbored 26 cis-pQTLs linked to 12 proteins uniquely enriched in immune pathways (antigen presentation, complement activation, phagocytosis, and T-cell regulation). Controls showed 107 cis-pQTLs linked to 14 targets. Gene Ontology enrichment revealed 46 immune biological processes in TB versus only 1 in controls, with HLA-C, C4B, and CHIT1 as key TB-specific proteins. Conclusions: Integrating proteomics with genomics suggests differential regulation of immune proteins associated with TB progression in PWH. hese genetically anchored protein candidates support follow-up studies and future biomarker evaluation for TB risk prediction.

Background: In Rwanda, genomic surveillance identified a dominant multidrug-resistant tuberculosis (MDR-TB) strain, the R3clone, responsible for approximately 70% of rifampicin-resistant TB cases. Its presence beyond Rwanda remains unexplored. Methods: Unique genetic signatures of the R3clone were defined using whole-genome sequencing of MDR-TB isolates from Rwanda. We developed a targeted qPCR assay detecting a clone-specific single-nucleotide polymorphism. With these tools, we screened isolates from neighbouring countries and public genomic repositories. Results: We identified 375 R3clone isolates, including 264 from historical Rwandan collections (1991-2021), 49 from recent Rwandan diagnostic routine (2021-2024), 25 from historical Burundi isolates (2002-2013), and 37 among public repositories from several countries. The R3clone-specific qPCR showed 100% specificity in distinguishing the R3clone from other MTBC (sub-)lineages. Transmission analysis revealed cross-border transmission of the R3clone within the Great Lakes Region. Conclusion: This study comprehensively assesses cross-border transmission of a dominant MDR-TB strain, highlighting the need for coordinated international surveillance.

Despite multiple treatment strategies and extensive research on resistance mechanisms, tuberculosis (TB) remains a major global health threat, largely because of the rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. Among various mechanisms complicating the situation, active antibiotic export via efflux pumps is particularly significant, yet largely unexplored. Mycobacterium sp. encodes numerous transporters, many of which are overexpressed in clinical isolates or under drug stress. Here, we examined the possible role of Rv0783c, a putative transporter that is reportedly overexpressed in drug-stressed conditions. Rv0783c conferred resistance to multiple structurally diverse antibiotics, fluoroquinolones and anti-TB drugs in the heterologous hosts, namely, Escherichia coli and Mycobacterium smegmatis. Reduced drug accumulation and active efflux of ethidium bromide (EtBr) confirmed its transport activity, which in turn gets nullified upon using the proton-motive force blocker, CCCP. On the other hand, its expression enhanced biofilm formation, linking antibiotic resistance to persistence-associated phenotype. Furthermore, site-directed mutagenesis confirmed the presence of crucial interacting residues with antibiotics that were identified by in silico analysis. Overall, we demonstrate the role of Rv0783c in the extrusion of first and second-line anti-TB drugs and enhancing biofilm formation.

Aminoglycoside drugs, amikacin, streptomycin, and amikacin liposome inhalation suspension are crucial for treating refractory Mycobacterium avium-intracellulare complex pulmonary disease. In Mycobacterium tuberculosis, cross-resistance occurs between amikacin and kanamycin, but not between amikacin and streptomycin in genetic drug susceptibility testing. However, the occurrence of cross-resistance among aminoglycosides remains unclear in M. avium-intracellulare complex. We aimed to evaluate cross-resistance among aminoglycosides to determine whether streptomycin or kanamycin remains effective after the development of amikacin resistance. This single-center retrospective study included 20 patients with amikacin-resistant M. avium-intracellulare complex harboring rrs mutations. Paired analyses of streptomycin and kanamycin minimum inhibitory concentration values before and after amikacin resistance development were performed. In addition, streptomycin- and kanamycin-resistant strains were generated in vitro and resistance-associated mutations were identified using whole-genome sequencing. No significant increase was observed in streptomycin minimum inhibitory concentration values following amikacin resistance. In contrast, kanamycin values uniformly increased to >256 g/mL after the acquisition of amikacin resistance. Furthermore, amikacin- and kanamycin-resistant isolates shared mutations at position 1408 in the rrs gene, whereas streptomycin-resistant isolates exhibited mutations at position 20 in the rrs gene. These results suggest that amikacin and kanamycin exhibit cross-resistance in M. avium-intracellulare complex, whereas amikacin and streptomycin may not. Two cases in our cohort in which streptomycin treatment was effective after the acquisition of amikacin resistance further support these findings. In conclusion, streptomycin may be a potential therapeutic alternative for amikacin-resistant M. avium-intracellulare complex pulmonary disease. Future studies correlating streptomycin minimum inhibitory concentration values with clinical outcomes are required.

BackgroundART effectively suppresses HIV replication and restores CD4+ T cells; however, long-lived HIV latent reservoirs enable viral persistence. Tuberculosis (TB) co-infection further impacts HIV latency and enhances viral replication. Given the high prevalence of latent TB infection (LTBI) in TB-endemic settings, understanding its impact on HIV biology is critical. Our study aims to investigate the influence of TB co-infection on HIV reservoir dynamics, viral diversity, and drug resistance mutations in ART-naive individuals. MethodologySamples from 90 ART-naive HIV-1C individuals, stratified based on IGRA and TB diagnosis, were used in this study. Plasma and PBMCs were isolated for viral RNA and DNA extraction respectively. Total proviral DNA was quantified using gag PCR. Full-length env and pol genes were amplified, purified and sequenced using ONT and Illumina platforms. Pol sequences were subjected to Drug Resistance Mutation (DRM) analysis via Stanford HIVdb with a minimum threshold mutation frequency of [≥]10%. Full length env sequences were used for phylogenetic analysis by aligning with Indian Subtype C reference sequence and phylogenetic tree was generated using ggplot2. ResultProviral load analysis showed no significant differences across HIV+LTBI-, HIV+LTBI+, and HIV+TB+ groups, although a trend toward higher levels was observed in HIV+TB+ individuals. Correlation analysis revealed distinct immune associations, with HIV+LTBI+ individuals showing positive correlations with activation and PD-1 expression. Longitudinal analysis of proviral loads demonstrated a modest decline in proviral load post-ART but remained persistent for up to 18-20 months following initiation of ART accompanied by low level ongoing viral replication. DRM analysis revealed a 33% prevalence in ART-naive individuals, with higher occurrence in HIV+LTBI+ group. Of the identified DRMs, 38% (5/13) and 71% (5/7) in sequences obtained from PBMC and plasma respectively were attributed to polymorphic mutations associated with Integrase strand transfer inhibitors (INSTIs). DRMs within plasma and PBMC derived viruses showed high concordance. Phylogenetic analysis of env sequences indicated overlapping viral populations between the 3 groups, with greater diversity in PBMCs compared to plasma. ConclusionThe study highlights that HIV reservoir dynamics, drug resistance, and viral diversity are significantly influenced by TB co-infection. While proviral loads were comparable, LTBI-associated immune activation and granuloma niches may have driven viral diversification and DRM emergence. High concordance between compartments and presence of transmitted resistance underscore the need for baseline screening, multi-compartment analysis, and sustained surveillance.

Background: Although the relaxation of COVID-19 containment measures in China has altered the transmission dynamics of respiratory pathogens, regional data on post-pandemic epidemiological characteristics remain limited.Objective: This study aimed to investigate the pathogen spectrum and epidemiological characteristics of acute respiratory infections (ARIs) in Quzhou City from 2023 to 2024, providing a scientific basis for local prevention and control strategies.Methods: A total of 2,800 respiratory specimens were collected from November 2023 to July 2024, comprising 1,960 influenza-like illness (ILI) cases from outpatient/emergency departments and 840 severe acute respiratory infection (SARI) cases from inpatient departments. All samples were tested for 13 common respiratory pathogens using multiplex fluorescence quantitative PCR. Etiological and epidemiological analyses were performed based on detection results and case information. Results: The overall ARI positivity rate was 59.28% (1,660/2,800), with a male-to-female ratio of 1.07:1 (1,447/1,353). The three most prevalent pathogens were influenza virus (Flu, 23.21%, 650/2,800), Streptococcus pneumoniae (SP, 13.14%, 368/2,800), and adenovirus (ADV, 8.39%, 235/2,800). Single pathogen infections accounted for 73.55% (1,221/1,660) of positive cases, while co-infections with two or more pathogens accounted for 26.45% (439/1,660), yielding an overall co-infection rate of 15.68% (439/2,800). No significant gender difference was observed in detection rates. However, significant differences were found across case types, temporal periods, age groups, and geographic regions (P < 0.01). Children aged [&le;]5 years exhibited the highest positivity rate (78.00%, 378/525), while adults aged [&ge;]65 years showed the lowest (34.53%, 144/417). Among surveillance regions, Kaihua County had the highest positivity rate (72.47%), and Changshan County the lowest (40.55%). Conclusions: Multiple respiratory pathogens and co-infections are prevalent in Quzhou City, with distinct age-specific and seasonal patterns. These findings underscore the need for continuous multi-pathogen surveillance and integrated prevention strategies for influenza and other respiratory infectious diseases in the post-pandemic era.

BACKGROUNDRapid, interoperable whole-genome tools for Mycobacterium tuberculosis complex (MTBC) surveillance remain limited for harmonised lineage assignment across recognised lineages and simultaneous resistance-associated variant detection. AIMTo develop and validate Pathotypr, an alignment-free tool for harmonised MTBC lineage assignment and resistance genotyping from assemblies and raw reads. METHODSWe reconstructed an MTBC phylogeny from 26,813 genomes using 609,003 polymorphic sites, derived an updated lineage marker backbone, and implemented a k-mer/Random Forest framework with marker-based lineage and WHO catalogue-based resistance calling. Performance was evaluated on 498 RefSeq assemblies, 88,071 UShER-TB typed sequencing samples, 162 clinical read sets for closest-reference matching, and 7,148 CRyPTIC isolates with phenotypic drug susceptibility data. RESULTSPathotypr supported all 14 currently recognised MTBC lineages (L1-L10, A1-A4). On 498 complete genomes, marker-based and alignment-free lineage calls were 100% concordant, and prediction accuracy remained 100% on 254 independent assemblies. In 88,071 non-ambiguous UShER-TB samples, root-lineage concordance with TB-Profiler was 100%, while Pathotypr additionally identified lineage 10, A1 and A2. Resistance predictions showed 85.0% genotype-phenotype concordance overall, with high performance for rifampicin (95.8% sensitivity, 95.0% specificity) and isoniazid (93.0%, 97.9%). Runtime was about 1 second per sample, enabling analysis of 88,071 samples in approximately 24 hours on four threads. In the MDR-enriched CRyPTIC collection, Pathotypr supported reconstruction of 135 probable introduction events into Germany, Italy and Ukraine; 33.7% of introduction-associated isolates carried MDR/pre-XDR genotypes. CONCLUSIONPathotypr enables rapid, harmonised MTBC lineage assignment and high-confidence resistance screening, supporting near real-time and cross-border tuberculosis surveillance.

WHO recommends bedaquiline-pretomanid-linezolid- (BPaL) and BPaL-moxifloxacin (BPaLM) for treatment of rifampicin-resistant tuberculosis, informed by the TB-PRACTECAL results. However, clinical explanatory data of these drugs exposure and Mycobacterium tuberculosis clearance rates and toxicity relationships remain understudied. We therefore investigated the relationship between the patients exposure to anti-TB drugs in TB-PRACTECAL trial investigational regimens and their treatment outcomes. PRACTECAL-PKPD was a prospective pharmacokinetics and pharmacodynamics study nested in TB-PRACTECAL. Patients with rifampicin-resistant pulmonary tuberculosis were enrolled from Belarus and South Africa. The first objective was to develop drug exposure metrics for bedaquiline, pretomanid, linezolid, moxifloxacin and clofazimine. The efficacy objectives were to establish an exposure-response model for each drug and regimen to both bactericidal activity and long-term treatment outcomes. The safety objective was to investigate the exposure-toxicity relationship of each drug. Antimicrobial exposure did not correlate with the speed of sputum bacterial clearance, however there was a 20% increased bacillary killing rate with BPaLM compared to the standard of care arm whilst BPaL and BPaL-clofazimine (BPaLC) displayed a 15% decreased bacillary killing rate compared to the standard of care arm. Linezolid plasma exposure was higher amongst patients with anaemia or neutropenia compared to those without. No other exposure-toxicity relationships were identified for all other drugs. Absence of correlation between drug exposure and bacillary clearance suggest that the dosages used achieve saturation of bacillary killing, while remaining safe.

Digital pathology, coupled with advanced image recognition algorithms, represents a transformative frontier in histopathological diagnosis. This sub-Saharan African laboratorys exploratory study investigates the application of a Convolutional Neural Network (CNN) model, specifically leveraging the VGG16 architecture with transfer learning, for automated analysis and classification of selected gastrointestinal (GIT) and liver tissue samples, incorporating both routine and specialized staining protocols. The study utilized a dataset comprising 114 samples (18 liver, 96 GIT images) derived from archival formalin-fixed paraffin-embedded tissue blocks at University College Hospital, Ibadan, Nigeria. Specialized staining techniques included Alcian Yellow for GIT mucin visualization and Massons Trichrome for liver fibrosis assessment, alongside conventional H&E staining. Model performance was evaluated using statistical methodologies including Wilson Score confidence intervals (CI), Bayesian probability assessment, and effect size analysis. Results reveal a striking dichotomy in model performance. The GIT tissue model achieved perfect classification accuracy (100% test accuracy) with exceptional statistical significance (Z=10.0, p<0.0001), Wilson CI [96.29%, 99.99%], Cohens h=1.571, and Bayesian probability >99.99%. Conversely, the liver tissue model demonstrated diagnostic failure (42.86% test accuracy), with Z=-1.428, p=0.9236, Wilson CI [33.59%, 52.65%], Cohens h=-0.144, and Bayesian probability of 7.64%. This performance divergence correlates with training data availability, as the liver dataset fell far below empirically established thresholds (>100-200 samples) for reliable classification. The liver models failure reveals limitations in transfer learning with insufficient data. These findings underscore critical implications for AI-enhanced digital pathology, demonstrating potential deployment of the GIT model as a promising one that supports tissue-specific model development.

BackgroundDelayed tuberculosis (TB) treatment remains a major challenge to TB control and is associated with increased mortality, drug resistance, and onward transmission. Food insecurity may contribute to delayed TB treatment through economic, physical, and psychosocial pathways. Depression and anxiety are also associated with delayed TB treatment and may mediate the relationship between food insecurity and delayed TB treatment. This study examined the association between food insecurity and delayed TB treatment initiation and assessed the mediation roles of depression and anxiety for this relationship among people newly diagnosed with TB. MethodsWe recruited 180 participants newly diagnosed with TB in Gaborone, Botswana. Food insecurity, depression, and anxiety were measured using the Household Food Insecurity Access Scale, PHQ-9, and Zung Self-Rating Anxiety Scale, respectively. Delayed TB treatment was defined as > 2 months since first TB symptoms. Logistic regression was used to examine the association between food insecurity and delayed TB treatment. Causal mediation analysis was conducted to assess the mediating roles of depression and anxiety. ResultsAmong the 180 participants, 45 (25%) experienced delayed TB treatment initiation. Participants with delayed TB treatment had slightly higher median scores for food insecurity (2 vs. 1, p = 0.11), depression (9 vs. 6, p = 0.001), and anxiety (37 vs. 34, p = 0.05). There was insufficient evidence of an overall association between food insecurity and delayed TB treatment initiation (OR = 1.04, 95% CI 0.98-1.11, p = 0.20). Mediation analysis found insufficient evidence of total and direct effects through depression and anxiety. However, there was evidence of significant indirect effect through depression (OR = 1.04, 95% CI 1.01-1.08, p < 0.001) and a borderline indirect effect through anxiety (OR = 1.02, 95% CI 1.00-1.04, p = 0.05). ConclusionMediation analysis revealed associations between food insecurity and delayed TB treatment initiation mediated by depression and anxiety which were not evident in total effects analysis. These findings highlight the importance of considering both socioeconomic and psychological factors in addressing delayed TB treatment. Further studies are needed to confirm these pathways.

BackgroundOnly 54% of people with TB had an initial molecular diagnostic test in 2024, due to barriers including high test costs. Pooled testing is implemented in Cameroon as a strategy to increase testing efficiency on the Xpert MTB/RIF Ultra (Ultra) assay and extend molecular testing to more people when test reagents are limited, as recently recommended by the World Health Organization. At GeneXpert sites, laboratory personnel decide whether to test individually or in pools and pool size based on locally available information, including smear microscopy results, lab positivity rates, daily testing volume, availability of Ultra cartridges and GeneXpert modules, and patient characteristics. MethodsWe conducted a retrospective evaluation of Ultra testing at GeneXpert laboratories that implemented both individual and pooled testing in pools of 2 to 8. Ultra test results and duration were extracted from GeneXpert instruments. Testing efficiency, instrument time to result, and assay cost were analyzed overall and by pool size. ResultsFrom October 2023 to March 2025, 71,328 sputum specimens were tested at 16 GeneXpert laboratories. For 59,164 specimens tested in pools, including 1,999 (3.4%) with TB detected, 20,838 Ultra cartridges were used, or 0.35 cartridges per result, enabling an additional 38,326 people to have molecular test results compared to if specimens were tested individually. The average time to result varied from 45 minutes to 10 minutes for specimens tested in pools of 2 or 8, respectively, as compared to 66 minutes for individual testing. The calculated assay cost per result was $2.81 for specimens tested in pools (from $5.29 to $1.19 for specimens in pools of 2 or 8, respectively) as compared to $7.97 for individual testing. DiscussionImplementation of pooled testing enabled many more people to have a molecular test result for TB, with significant time and cost savings compared to individual testing. What is already known on this topicPooled testing is a strategy used to increase testing efficiency by combining specimens from multiple individuals prior to testing; if the pool tests negative, a negative result is reported for each specimen with no further testing, and if the pool tests positive, then each specimen from the pool is re-tested individually and the individual result is reported. The World Health Organization has recently recommended the use of pooled testing to increase access to molecular diagnostic testing for tuberculosis when resources are constrained. What this study addsThis is the first report of large-scale programmatic implementation of pooled testing for the detection of TB. Pooled testing was performed by laboratory personnel who decided whether to test individually or in pools of size 2 to 8, based on the information available to them. Implementation of pooled testing enabled many more people to be tested with existing resources, with significant reductions in time to result and assay cost per specimen tested. How this study may affect research, practice or policyThis demonstration of the successful scale up of pooled testing for TB should contribute to uptake of pooled testing by national TB programs and laboratories to increase access to molecular testing for TB when resources are constrained.

BackgroundShort-read genetic sequencing technologies (mainly Illumina) have been extensively used for around a decade for Mycobacterium tuberculosis complex (MTBC) outbreak analysis and genomic drug susceptibility testing (gDST) with the result that Illumina has become the de facto gold standard. Long-read sequencing, as exemplified by Oxford Nanopore Technologies (ONT), offer the prospect of faster, simpler, and portable sequencing. In this work, we carry out the largest to date comparison of how well Illumina and ONT technologies sequence MTBC samples, making use of R10.4 ONT flowcells, updated basecalling models and deep-learning variant calling. MethodsA total of 508 samples were sequenced using both short and long-read platforms. All samples originated from South Africa or Vietnam and were over-selected for drug resistance and also included several local outbreaks and a range of lineages. The South African and Vietnamese samples had already been Illumina sequenced. Samples with [&ge;]50 read depth by Illumina were selected for sequencing by ONT using one of the GridION or PromethION platforms. Bioinformatics processing was done using a modified online cloud platform which included reference-based variant calling, catalogue-based gDST and identified related samples via SNP counting to inform outbreak detection. The lineages and gDST predictions obtained by short-and long-sequencing were compared for all samples as were all putative clusters identified via SNP counting. For convenience Illumina was used as the reference method. FindingsOf the 508 samples, 425 (83.7%) had sufficient read depths to permit comparison between the two sequencing technologies. The assigned lineages were identical for 407/425 (95.8%) samples and all discordances were due to mixed lineages being identified by one technology. Evidence of non-tuberculous mycobacterium (NTM) subpopulations were found in nine samples. Using Illumina as the reference method, the very major error (VME) rate of ONT for predicting resistance to all 15 drugs is 1.0% (0.6-1.5%) whilst the major error (ME) rate is 1.7% (1.3-2.2%) with an unclassified rate of 6.9% (6.3-7.5%). This is below the thresholds specified by the CLSI. Considering each of the 15 drugs individually they had VME and ME point estimates below [&le;]3% in 29/30 cases; and most 25/30 below [&le;]1.5%. Filtering out all samples containing mixtures left 382 isolates. By appropriate masking of the reference genome we were able to obtain a mean SNP distance between the two platforms of 0.13 (median of zero) for the same sample and for 376/382 samples (98.4%, CI:96.6-99.4%) the difference was [&le;]1 SNPs. The high concordance in SNP identification ensured that few differences in the 43 putative clusters among 172 isolates were observed. InterpretationThe differences between the two sequencing platforms for the key clinical outputs is so small that it is now within the tolerances set by regulatory agencies. Provided the sequencing is of sufficient quality, we have therefore reached a threshold whereby sequencing data from long-and short-read platforms can be aggregated. This will enable large scale analyses by national and international public health agencies whilst allowing the MTBC community to take advantage of the portability and speed of long-read sequencing. FundingThe NIHR Health Protection Research Unit: Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford (NIHR200915), a partnership between the UK Health Security Agency (UKHSA) and the University of Oxford, the National Institute for Health and Care Research Biomedical Research Centre: Oxford (BRC) and the Ellison Institute of Technology, Oxford Ltd. The CRyPTIC project was funded by Wellcome [214560/Z/18/Z], a Wellcome Trust/Newton Fund-MRC Collaborative Award (200205/Z/15/Z); and the Bill & Melinda Gates Foundation Trust (OPP1133541). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe conducted a PubMed Central full text search for "tuberculosis" AND ("drug resistance prediction" OR "drug susceptibility prediction") AND ("genome" OR "genomic" OR "geno-typic") AND ("ont" OR "oxford nanopore") between 2022 and 2026 (conducted 1 April 2026). This returned 62 papers; of which, six used both Illumina and ONT sequencing. One of these, published in 2023, directly compared the performance of the two platforms on 151 M. tuberculosis isolates oversampled for resistance. The investigation yielded comparative results for the earlier generation ONT flow cell (R9{middle dot}4{middle dot}1) and base-caller (guppy version 5{middle dot}0{middle dot}16). Another, published in 2026, investigated a targeted next-generation sequencing panel of 20 amplicons using ONT sequencing on R10.4.1 flow cells with guppy 6{middle dot}4{middle dot}6. They compared the results on 71 isolates against phenotypic data and Illumina whole genome sequencing (for 53 isolates) but had low rates of resistance, with all drugs but isoniazid being limited to under five resistant isolates. Two other small studies (10 and 13 samples, respectively) conducted feasibility studies comparing ONT with Illumina, also using earlier generation flow cells and base-calling technology from ONT. Two further studies compared Illumina with ONT for direct sputum sequencing and did not investigate the comparative performance of the two platforms for variant call accuracy, resistance prediction, and outbreak detection. Illumina sequencing technology is widely used for genomic sequence analysis in research, and clinical and public health contexts. Consequently, it has become the de facto reference standard for generating whole genome sequence data. Whilst previous studies established the promise and limitations of long-read (ONT) sequencing as an alternative to short-read sequencing (mainly Illumina), the enhanced performance arising from newer flowcells (e.g. R10.4.1), V14 chemistry, and the latest basecallers (dorado v4.3.0/5.0.0) has not been analysed. Neither has any ONT analysis incorporating the new deep-learning variant callers been evaluated in a large-scale comparative study. Thus, it is currently unclear whether data generated by either platform can be used safely in aggregated analyses for research and clinical or public health service. Added value of this studyWe compared how well short-(Illumina) and long-read (ONT) sequencing platforms identify the genetic variants in M. tuberculosis, predict antituberculous drug resistance and recog-nise outbreaks. The long-reads were generated using the latest generation ONT R10.4.1 flows cells, V14 chemistry, super high accuracy basecalling (dorado v4.3.0/5.0.0) and a bioinformatics analysis pipeline built using the Clair3 deep-learning based variant caller. A total of 508 clinical samples were sequenced using both technologies, substantially more than previous studies. The sampling frame was much larger than previously investigations and included a large proportion of isolates with resistance to first-line and second-line antibiotics as well as bedaquiline. Thus, providing greater statistical power for resistance prediction than before. In particular, the inclusion of bedaquiline resistance provided evidence useful for predicting resistance to this newly deployed drug for treating multi-drug resistant (MDR) TB. We find that the differences between technologies are small meaning that either technology can be used alone safely, and services using both technologies can confidently aggregate the data for analysis. Implications of all the available evidenceThis will be a benefit to local, regional and international organisations, particularly public health agencies, which often have a mix of the two main sequencing technologies for characterising TB whole genome sequences. It also opens up the sequence based diagnostic market to greater competition, particularly if the observed performance can be replicated for other pathogen species.

Mycoplasma bovis was first detected in cattle in New Zealand in 2017, prompting an eradication programme that incorporated extensive surveillance and a test-and-cull policy. Genome sequence data and phylodynamic models were used to inform decision making throughout the eradication programme. Isolates from 697 cattle on 126 farms were collected and sequenced between July 2017 and December 2023. Phylodynamic models were used to estimate the time of most recent common ancestor, the effective reproduction number (Reff) and effective population size, and long-range and local between-farm transmission dynamics. The analysis revealed the dramatic impact of movement restrictions and culling up to early 2020, with a sharp reduction in the Reff to less than 1 in 2018/9 and the extinction of two of three major lineages in 2020. This was followed by three-years of residual infection in farms in the South Island, associated with persistent infection of a large feedlot farm and nearby farms. The comprehensive dataset of genomic and epidemiological data provided a unique opportunity to study the dynamics of a country-wide outbreak of a single-host pathogen from first detection to potential eradication, underlining the utility of integrated genomic surveillance during an outbreak response. Author summaryThe economically important cattle pathogen, Mycoplasma bovis, was first detected in New Zealand in 2017. This led to a large-scale, successful control programme aimed at eradication of the pathogen. The decision to undertake an eradication programme was informed by initial analyses of whole genome sequences from isolates collected as part of the surveillance programme. The analysis showed that the bacteria had entered New Zealand relatively recently and was unlikely to be widespread. Over the subsequent years, genome sequencing and modelling of transmission dynamics informed important policy decisions made by the New Zealand Government and the cattle industry, and helped to monitor progress of the eradication programme. The impact of the detection, movement control and culling programme was profound, with sharp reductions in transmission between 2018 and 2020. This was followed by a long tail of localised infection in the South Island, involving transmission from a large feedlot farm. Provisional eradication was achieved after depopulation of this feedlot. This analysis highlights the role of genomic surveillance and modelling to inform decision making during an infectious disease outbreak.

BackgroundXpert(R) MTB/RIF Ultra (Xpert Ultra) is a WHO recommended molecular test for rapid tuberculosis (TB) detection, but cartridge cost limits large-scale use of Xpert Ultra in high TB burden, resource-constrained countries. This study assessed the diagnostic performance of Xpert Ultra on pooled sputum for detection of pulmonary TB (PTB)in Bangladesh. MethodsBetween July 2024 and February 2025, adults with presumptive PTB were prospectively enrolled from primary and secondary healthcare facilities through facility-based case finding, and from urban slum communities through community-based case finding. Participants provided two sputum samples, one for individual and pooled Xpert Ultra testing, and another for culture. Pooled and individual Xpert Ultra results were compared to the microbiological reference standard of culture. Cost analysis was performed by comparing cartridge usage between individual and pooled Xpert Ultra testing. ResultsA total of 3043 individuals were tested individually and as part of 771 pooled samples. Compared with culture, the overall sensitivities of pooled and individual Xpert Ultra were 85.8% (95% CI: 79.8-90.6) and 89.2% (83.7-93.4), respectively, while specificities were 98.9% (98.4-99.2) and 98.1% (97.5-98.6). Pooled Xpert Ultra detected 100% of the high, medium, and low burden categories identified by individual testing, but showed lower detection for very low (81.8%) and trace (31.4%). Compared with individual testing, pooled testing reduced cartridge use and cost by 55.8%. ConclusionsPooled sputum testing with Xpert Ultra demonstrated high diagnostic performance similar to individual testing, while substantially reducing the cartridge costs. This approach offers a scalable molecular TB testing in resource-limited, high-burden countries such as Bangladesh.

Background: Tuberculosis recurrence accounts for a substantial proportion of incident tuberculosis in many settings. Distinguishing between its mechanisms can inform public health interventions for prevention. Methods: We conducted a retrospective study of individuals with multiple culture-confirmed TB episodes and available sequential isolates from 2012 to 2023 in Dourados and Campo Grande, Mato Grosso do Sul state, Brazil. Patients were classified as having recurrent TB after treatment completion or retreatment following non-curative outcomes. Whole-genome sequencing was used to assess pairwise genetic distances between isolates, classifying relapse or persistent infection ([&le;]12 single-nucleotide polymorphisms [SNPs]) versus reinfection or retreatment with reinfection (>12 SNPs). Results: Among 9,293 individuals with TB, 772 recurrent or retreatment episodes were identified. Paired isolates from 82 individuals were available for comparisons. Among individuals who completed treatment, reinfection accounted for 74.1% (40/54) of recurrent episodes, while 25.9% (14/54) were classified as relapse. Among individuals with non-curative outcomes, persistent infection (53.6%, 15/28) and retreatment with reinfection (46.4%, 13/28) occurred at similar frequencies. Persistent infection and relapse occurred earlier after the initial episode, whereas reinfection and retreatment with reinfection predominated after two years. Incarceration history was strongly associated with reinfection after treatment completion (92.5%, p=0.012) and after non-curative outcomes (76.9%, p=0.016). Conclusions: In this high-burden setting, reinfection drives TB recurrence among individuals who complete treatment, particularly at longer intervals after initial disease, reflecting sustained exposure risk. Relapse and persistent infection remain clinically important, especially following non-curative outcomes. These findings underscore the need for integrated strategies combining adherence support to prevent treatment-related recurrence with interventions to reduce transmission, particularly in high-risk settings.

BackgroundUnited Kingdom Standards for Microbiology Investigations limits the pre-analytical delay of blood cultures to a maximum of four-hours between collection and incubation. Compliance with this delay standard is a measure of the ability of a microbiology service to support the management of sepsis which is a life-threatening complication of infection. A positive blood culture confirms the infection and an early result is critical to the effective management of the condition. Delayed results lead to the prolongation of empiric broad spectrum antimicrobial therapy which is considered a causal factor in the emergence of antimicrobial resistance. This retrospective observational study documents compliance with the standard by microbiology services in England in 2022/23. The impact of laboratory centralisation on the ability of microbiology services to comply with this standard is examined. MethodsFreedom of Information requests were submitted to 116 National Health Service Trusts/administrative units in England requesting retrospective audit data showing compliance with the recommended pre-analytical delay standard. Data relating to service configuration and cost were also requested. ResultsResponses were received from 89 Trusts (76.7%) managing 146 hospitals. Overall, the rate of compliance was low, with only four hospitals (2.7%) showing full compliance and 31.5% showing >80% compliance. ConclusionsPoor rates of compliance with the PAD standard are a concern as prompt attention to blood cultures improves patient outcomes from sepsis and supports antimicrobial stewardship. Laboratory centralisation has resulted in withdrawal of staff and facilities from some hospitals with insufficient investment in others, leading to a demonstrable inability of many hospitals to comply with this standard. Compliance will require investment in microbiology services. The financial implications of the improvements proposed should be evaluated in the context of overall health care and community benefits.