Tuberculosis

Background: Tuberculosis (TB) remains the leading infectious disease cause of mortality worldwide. A subset of individuals exposed to Mycobacterium tuberculosis (Mtb) remain TST/IGRA-negative despite sustained exposure, termed "TB resisters". The biological mechanisms underlying this resistance remain poorly understood. We applied untargeted high-resolution metabolomics to determine whether systemic metabolic profiles differ between TB resisters and matched Mtb-infected close contacts. Methods: We compared untargeted high-resolution plasma metabolomics using liquid chromatography mass spectrometry between 269 TB resisters and 269 matched Mtb-infected close contacts, enrolled across Brazil, India, and South Africa. TB resisters were defined as highly exposed close contacts who slept in the same room or spent at least 5 hours indoors per day with an infectious pulmonary TB index patient, but remained TST/IGRA-negative. Mtb-infected close contacts were those who were TST/IGRA positive. Metabolome-wide association studies (MWAS) were conducted using paired t-tests. Results: There were 1,787 features with nominal differences between TB resisters and matched Mtb-infected contacts (p < 0.05). Pathway enrichment identified fructose and mannose metabolism and bile acid biosynthesis in the overall cohort, while lipid-related pathways were enriched in Brazil. Among metabolites with confirmed chemical identities, glyceric acid concentrations were higher in TB resisters by 0.53 uM, whereas hydroxyproline was lower by 0.90 uM, butyrobetaine was lower by 0.06 uM, and homogentisate was lower by 0.0036 uM at false discovery rate of 0.2. Conclusions: Plasma metabolomic profiles differed between TB resisters and matched Mtb-infected close contacts. These findings indicate that systemic metabolic differences are associated with resistance to Mtb infection.

As drug-resistant tuberculosis (DR-TB) cases rise, resistance detection in a timely manner is essential to lead effective treatment and limit transmission. Targeted next-generation sequencing (tNGS) offers quick results with multiple important drugs covered, but assessments regarding its performance for DR-TB diagnostic use compared to whole genome sequencing (WGS) as the most comprehensive genomic-based tool are still limited. This cross-sectional study compared resistance profiles generated by Deeplex Myc-TB tNGS assay with WGS for 116 prospectively-collected rifampicin resistant TB samples from West Java, Indonesia. All 116 samples were subject to paired analysis, the clinical samples were split to be directly processed for tNGS and to be cultivated for culture-based WGS. Both WGS and tNGS were carried out using Illumina MiSeq platform. High concordance of tNGS and WGS were observed across thirteen anti-TB drugs evaluated, particularly for drugs included in the BPaLM regimen. Isoniazid had the lowest concordance of 86.73%. Of 116 samples, 31.03% (n = 36) had discrepant resistance calling from the two methods for one or more drugs, which came from 73 discordant variants identification. The most common source of discrepancy was when tNGS detected a resistance-conferring mutation while WGS did not (54.8%). tNGS could detect mixed infection better than WGS, but WGS was superior in identifying detailed major Mycobacterium tuberculosis lineage of the sample. tNGS showed a good level concordance with WGS in detecting resistance-conferring mutations in rifampicin-resistant TB samples, with a more rapid turnaround time. Continuous update to tNGS panel and mutation catalogue is needed to keep the tool clinically relevant. ImportanceDrug-resistant tuberculosis (DR-TB) continues to pose worldwide threat, and newer diagnostic tools to generate quick, comprehensive resistance profile are crucial to provide timely appropriate treatment. Targeted next-generation sequencing (tNGS) is a promising new alternative, but more evidence on its performance is needed to support programmatic adoption. By analysing DR-TB samples with both tNGS and whole genome sequencing (WGS) and evaluating their results agreement, this study shows that tNGS works just as well as WGS in detecting TB drug resistance-conferring mutations, confirming its potential for routine diagnostic use. This study also observed that while WGS is superior in identifying Mycobacterium tuberculosis lineage with high resolution, it did not detect mixed infection better than tNGS. Notably, this study demonstrated that tNGS is clinically relevant for DR-TB detection in a high burden setting, providing evidence for programmatic consideration in Indonesia and other settings with similar demographics and TB situation.

Objectives: The near point-of-care (nPOC) Pluslife MiniDock MTB (MiniDock) assay does not report semiquantitative values. We evaluated whether categorized MiniDock time-to-positivity (TTP) serves as a quantitative proxy for Mycobacterium tuberculosis (Mtb) bacterial load. Methods: Presumptive tuberculosis (TB) patients enrolled across 27 health facilities in Indonesia were tested with sputum GeneXpert MTB/RIF Ultra (Xpert), MiniDock sputum swabs, and tongue swabs. Positive results were categorized using a median split at 13 minutes ([&le;]13, 13-25, and 25 minute). MiniDock TTP categories were evaluated against Xpert semiquantitative grades and BACTEC MGIT 960 liquid culture TTP (days). Results: Of 2974 presumptive TB participants tested with sputum Xpert, 426 (14.3%) were sputum Xpert-positive. MiniDock detected Mtb in 248/299 (83.0%) sputum and 263/382 (68.8%) tongue swab. Among 248 Minidock sputum-positive results, 99 (39.9%) turned positive [&le;]13 minutes, 107 (43.1%) between 13 and 25 minutes, and 42 (16.9%) at 25 minutes. Minidock TTP categories correlated with sputum and tongue swab semiquantitative results as well as with culture time to positivity (p<0.001). Conclusions: MiniDock TTP categories ([&le;]13, 13-25, 25 minutes) provide meaningful stratification which correlates with both Xpert semiquantitative and culture TTP. Time to Positivity from nPOC could thus serve as a proxy for bacterial burden and infectiousness, strongly increasing its utility for clinical care, public health and research.

Riboflavin biosynthesis is required for in vitro survival of the human pathogen Mycobacterium tuberculosis (Mtb). However, despite the lack of a known transporter, growth can be rescued by exogenous riboflavin. The riboflavin biosynthesis pathway is also predicted essential in vivo, but whether riboflavin levels available in the host can support survival has not been directly tested. Here we constructed a set of inducible CRISPR interference (CRISPRi) knockdown and targeted gene deletion strains for known riboflavin biosynthesis genes (ribA2, ribG, ribH, ribC) as tools to characterize riboflavin requirements, uptake, and metabolite changes and to assess in vivo essentiality. We found that riboflavin, but not flavin adenine dinucleotide or flavin mononucleotide, rescued auxotrophy for all strains tested. Further, riboflavin uptake did not show strong evidence of being dependent on active or facilitated transport, supporting the mechanism of passive diffusion. Targeted metabolite profiling after removal of riboflavin from growth medium confirmed reduced riboflavin levels. While other riboflavin intermediates were not detected, significant accumulation of aromatic amino acids (Phe, Tyr) was observed across all assayed strains, as well as alteration in a vitamin B9 metabolite. Selecting the ribC knockout as a representative strain, we found that riboflavin depletion had a bacteriostatic effect as late as 3 weeks after removal. Unexpectedly, {Delta}ribC lacked infectivity in an aerosol mouse infection, suggesting that the potential to scavenge riboflavin from the host is not sufficient to survive in vivo. Overall, our results show that altered metabolism upon loss of riboflavin biosynthesis leads to compromised Mtb infectivity. IMPORTANCETuberculosis remains one of the worlds most deadly infectious diseases, underscoring the need to explore new drug targets. Riboflavin (vitamin B2) biosynthesis has emerged as a promising target because Mycobacterium tuberculosis (Mtb) depends on this pathway for survival. The riboflavin pathway also produces metabolites that modulate host mucosal-associated invariant T (MAIT) cell activity, towards understanding potential strategies for host-directed therapies. Here we found that disrupting riboflavin biosynthesis led to not only compromised survival, but also widespread changes to metabolism and loss of the ability to establish infection in an animal model. These findings improve our understanding of how Mtb adapts to metabolic stress, with implications for developing drugs that target riboflavin biosynthesis and for alterations in host immunity to be explored in future studies.

Interferon-gamma release assays detect Mycobacterium tuberculosis infection but cannot distinguish infection from active tuberculosis (TB). We evaluated the diagnostic information provided by QuantiFERON-TB Gold Plus (QFT-Plus) among adults investigated for active TB in Brazil and South Africa. Adults with presumptive TB were prospectively enrolled by convenience sampling. QFT-Plus was the index test, and diagnostic classification required microbiological confirmation of TB or an established alternative diagnosis. QFT-Plus results did not inform diagnostic classification. Accuracy was assessed among participants with determinate results, primarily using likelihood ratios. Of 723 participants tested, 48 (6.6%) had indeterminate results, all due to low mitogen responses. Among 675 participants with determinate results, 147 (21.8%) had confirmed TB. Sensitivity was 68.0% (95% CI, 60.1-75.0), specificity was 59.3% (95% CI, 55.0-63.4), and the negative likelihood ratio was 0.54 (95% CI, 0.42-0.69). At the observed prevalence, a negative result left a 13.1% probability of active TB. Negative likelihood ratio confidence intervals included 1 in both South African HIV strata. Specificity was higher among participants with HIV in both countries. Adding TB2 to TB1 alone identified 15 additional TB cases and generated 18 additional positive results among participants without active TB. HIV and active TB were independently associated with indeterminate results in exploratory regression. A negative QFT-Plus result provided insufficient information to exclude active TB. Higher negative predictive values among participants with HIV reflected lower disease prevalence and higher specificity rather than greater sensitivity. Negative or indeterminate results should not terminate diagnostic evaluation.

The COVID-19 pandemic substantially disrupted tuberculosis (TB) prevention, diagnosis, and treatment services worldwide. Despite increased TB notifications in Nepal during the post-pandemic recovery period, the geographic distribution and temporal clustering of notifications remain poorly understood. This study examined the spatial and temporal patterns of TB notifications across Nepal during the COVID-19 recovery period. We conducted a nationwide retrospective ecological analysis of district-level TB notifications across all 77 districts of Nepal from FY 2019/20 to FY 2023/24. Space-time, purely spatial, and purely temporal scan statistics were performed using a discrete Poisson model in SaTScan. Statistical significance was assessed using Monte Carlo simulation with 999 replications. A total of 172,155 TB cases were notified during the study period. TB notification rates increased by 51%, from 92 to 139 per 100,000 population between FY 2019/20 and FY 2023/24. Four statistically significant high-notification space-time clusters were identified (all p < 0.001). The primary cluster included Kathmandu, Lalitpur, Bhaktapur, Makwanpur, Bara, and Parsa districts during FY 2021/22-FY 2023/24 (RR = 1.60). Additional clusters were identified in western Nepal, the eastern Terai, and Rupandehi district. Purely temporal analysis showed a nationwide increase in notifications during FY 2022/23-FY 2023/24 (RR = 1.26, p = 0.001). TB notifications increased substantially during Nepals post-pandemic recovery period, with marked geographic and temporal heterogeneity. Integrating spatial and temporal surveillance may help identify areas requiring geographically targeted TB control strategies.

L-Serine is an important metabolic and immunomodulatory biomolecule with promising role in managing infections, and autoimmune diseases. L-Serine provides the energy requirements and triggers the toll-like receptor signalling collaterally. However, the role of L-Serine in host antimicrobial response against Mycobacterium tuberculosis (Mtb) remains unexplored. In this study, we investigated whether this metabolite could modulate the antibiotics efficacy against Mtb. Although L-Serine exhibits limited intrinsic anti-mycobacterial activity, but L-Serine demonstrates a synergistic effect when combined with rifampicin and moxifloxacin against both drug-sensitive and multidrug-resistant Mtb. Moreover, L-Serine particularly in combination with palmitic acid showed the enhanced intracellular bacterial clearance in a dose- and time-dependent manner in murine and human macrophages. This synergistic effect was accompanied by increased nitric oxide production and modulation of the host immune response. We identified elevated levels of pro-inflammatory cytokines and reduced IL-10 expression. Furthermore, the metabolic supplementation demonstrated enhanced antimicrobial activity in isolated primary CD14+ monocytes from TB patients. Similarly, the metabolic supplementation of L-Serine in combination with isoniazid and rifampicin significantly reduced bacterial burdens in the lungs and spleen, while improving tissue architecture in murine infection model. Our observations suggest that L-Serine contributes to the observed therapeutic effects. Collectively, this study concludes that L-Serine acts as a promising host-directed therapeutic adjunct, which enhances antimicrobial immunity and potentiating antibiotic efficacy, providing a potential strategy for improving tuberculosis treatment outcomes.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells primarily through the angiotensin-converting enzyme 2 (ACE2) receptor. Genetic variation in the ACE2 gene may influence host inflammatory responses and clinical outcomes during COVID-19. Tuberculosis (TB) on the other hand increases the level of ACE2 expression through interferon stimulation. The objective of this study was to investigate the association between specific single-nucleotide polymorphism (SNP) in the ACE2 gene and their impact on susceptibility to and severity of SARS-CoV-2 and Mycobacterium tuberculosis Co-infection. Methods: This retrospective cross-sectional study included 120 symptomatic individuals recruited between September 2020 and December 2023 from four hospitals in Yaounde, Cameroon. Participants were classified into four groups: COVID-19, TB, TB/COVID-19 co-infection, and infection-negative controls. Diagnoses were confirmed using RT-PCR and standard microbiological methods. Serum biochemical markers and cytokines were quantified, and genotyping of ACE2 polymorphisms was performed using TaqMan assays. Results: Statistical analysis showed a significant association between ACE2 expression and inflammatory or biochemical markers, particularly for rs147311723. Several ACE2 polymorphisms (rs6632677, rs147311723, rs4646140, rs2285666, and rs4646142) were significantly associated with increased pro-inflammatory cytokine levels. Selected biochemical parameters demonstrated suggestive associations with ACE2 variants, including creatinine (rs147311723, p = 0.0522), AST (rs2285666, p = 0.0536), and ALT (rs4646142, p = 0.0582), indicating potential renal and hepatic involvement. Principal component analysis identified distinct immunometabolic profiles across patient groups, with co-infected individuals exhibiting heightened inflammatory and metabolic perturbations. Conclusions: These findings suggest that ACE2 genetic variability is associated with modulation of inflammatory responses and may contribute to organ-specific dysfunction in COVID-19 and TB across different populations. This highlights the potential role of host genetics in shaping disease severity and underscore the need for larger studies to validate these associations in diverse populations. Keywords: ACE2 polymorphisms; COVID-19; tuberculosis; co-infection; cytokines; biomarkers; genetic susceptibility;

Mycobacterium tuberculosis has a flavin-intensive metabolism, with more than 180 proteins that require flavin or deazaflavin cofactors. M. tuberculosis requires riboflavin biosynthesis for growth in standard culture conditions in vitro, which can be overcome by providing exogenous riboflavin. It is unknown whether M. tuberculosis also requires riboflavin synthesis to grow or persist in infected host cells or tissues. Here we used a M. tuberculosis strain that conditionally expresses ribA2, which encodes a bifunctional enzyme that catalyzes the first steps in both branches of riboflavin biosynthesis, to determine whether M. tuberculosis requires riboflavin biosynthesis during infection. Transcriptional repression of ribA2 caused death of M. tuberculosis in vitro without riboflavin supplementation. RNAseq revealed that riboflavin starved M. tuberculosis induce a transcriptional program indicating decreased electron transport chain function and broad changes to central metabolism. Transcriptional repression of ribA2 prevented M. tuberculosis growth in cultured macrophages and caused clearance of M. tuberculosis from infected tissues at all stages of infection in both C57BL/6 mice that form cellular lung lesions and C3HeB/FeJ mice that develop necrotic lung granulomas. Our results demonstrate that M. tuberculosis requires RibA2 for viability at all stages of infection and support further characterization of the riboflavin biosynthesis pathway as a potential target for development of new anti-tubercular drugs.

Macrophages, alongside mammary epithelial cells, are major contributors to the innate immune response triggered by bacterial invasion of the mammary gland. To investigate breed-specific immune mechanisms, we stimulated monocyte-derived macrophages from Holstein and Normande cattle with Escherichia coli and Streptococcus uberis, two major mastitis pathogens. We monitored bacterial survival using [methode] and profiled gene expression via RNA-seq. E. coli exhibited significantly higher survival 6 hours post-infection compared to S. uberis (p < 0.05). Moreover, E. coli induced a robust transcriptional response, with 4403 differentially expressed genes (FDR < 0.05, |log2FC| > 1), versus only 813 for S. uberis. Qualitative differences were also observed: genes such as CCL2 and CSF1 were uniquely induced by S. uberis. Additionally, macrophages from Holstein and Normande cattle showed distinct expression profiles: 185, 45, and 174 genes were differentially expressed at steady state or after E. coli or S. uberis stimulation, respectively. These genes were enriched in immune-related pathways. Overall, our results demonstrate a stronger response to E. coli, although S. uberis specifically induces a subset of genes. The observed breed-specific differences may reflect underlying immune mechanisms that could influence disease susceptibility, warranting further functional validation.

Background. Urine-based testing offers a promising non-sputum approach for diagnosing paediatric tuberculosis. However, the currently available lipoarabinomannan (LAM) assay shows limited sensitivity in children and is primarily indicated for those living with HIV. Co-detection of LAM with Mycobacterium tuberculosis (Mtb) proteins in urine could provide complementary pathogen-derived biomarkers that improve diagnostic performance. Methods. We developed an ultrasensitive multiplex electrochemiluminescence (ECL) immunoassay to measure Ag85B, CFP-10, ESAT-6, MPT32, and MPT64 in urine. We determined the analytical limits of detection and evaluated the diagnostic performance of individual proteins and LAM using urine samples from children with Confirmed, Unconfirmed, and Unlikely pulmonary tuberculosis enrolled across five high-burden countries (The Gambia, India, Peru, South Africa, and Uganda). Performance was assessed overall, by HIV and nutritional status, and across biomarker combinations. Findings. Urine samples from 630 children were analysed (median age was 4 years [IQR 2-8]; 44% female, 15% living with HIV, 19% underweight, 24% with Confirmed tuberculosis). The ECL assay achieved femtomolar limits of detection (1.5 to 4.0 fM). The sensitivity and specificity of individual Mtb proteins were 12-33% and 98-100%, respectively. Ag85B had the highest sensitivity (33%, 95% CI 26-41) for Confirmed tuberculosis and was similar to LAM. A four-antigen signature (Ag85B, MPT64, MPT32, LAM) was 50% sensitive (95% CI 42-58) and 94% specific (95% CI 90-96), and was significantly more sensitive than LAM alone, in particular among those without HIV. An additional sixteen (10%) of children with Unconfirmed TB had at least one Mtb protein or LAM detected. Interpretation. Multiple Mtb proteins are detectable in paediatric urine with high specificity, and multi-antigen signatures can augment sensitivity versus LAM alone. These findings demonstrate the potential of multi-antigen urine detection for childhood TB and define analytical targets for the development of future point-of-care diagnostics. Funding. National Institutes of Health.

Brain tuberculosis remains associated with high mortality, and many survivors exhibit cognitive impairments. Progress in understanding the disease is hindered by the lack of human models. In this study, human neural organoids were infected, revealing that a subpopulation of neural progenitor cells (NPCs) is directly infected by apoptotic cell receptors expressed by NPCs, mediating bacterial uptake. Phagocytosed bacteria were localized in late endosomes, lysosomes, and the cytoplasm. Cytoplasmic bacteria frequently formed cords, indicating limited control of bacterial expansion. Immunostaining demonstrated that infected NPCs produce a type I interferon (IFN) response, corroborated by increased expression of type I IFN and IFN-regulated genes detected by RNA sequencing. Pathways related to innate immune response, cell death, and proliferation were also activated following Mycobacterium tuberculosis (Mtb) uptake by NPCs. The addition of color-coded microglia and monocytes to 3D neural organoids and NPCs revealed cross-infection of NPCs and other phagocytes by Mtb, suggesting a mechanism by which NPCs may access the bacteria. Infection of NPCs resulted in increased cell death, inhibition of neural differentiation, and reduced proliferation, effects that were partially mitigated by anti-IFN treatment. Differentiated neurons were not infected. These findings indicate that brain organoids and NPC-based in vitro platforms provide a novel approach for studying brain tuberculosis. Decreased NPC function may contribute to brain tuberculosis-induced cognitive disease.

Tuberculosis (TB) is a life-threatening disease with heterogenous presentation. Approximately one-quarter of the global population is infected with Mycobacterium tuberculosis (Mtb), yet a much smaller fraction develops active TB disease. Host genetics, immune system function, and environmental factors have all been implicated in susceptibility to Mtb, yet no one factor fully explains TB heterogeneity. Strikingly, many of these same factors are linked to gut microbiome composition, which is intimately linked to systemic development of the immune system. Antibiotic treated mouse models suggest that increased gut microbiota diversity is protective against Mtb infection. In contrast, Helicobacter hepaticus colonization is correlated with exacerbated Mtb burden. However, antibiotics can have both microbial and nonmicrobial targets and studies to date have not deconvoluted these effects. Focused testing of specific microbiome members has been impossible without a gnotobiotic model for Mtb. Here, we develop the first gnotobiotic mouse model for Mtb infection and test how microbial diversity in the gut microbiome impacts host susceptibility to Mtb. Surprisingly, after intranasal challenge with Mtb, germ-free wild type mice had no difference in lung burden when compared to mice born with either a defined gut microbiome community (OMM-12) or a diverse, conventional microbiota. H. hepaticus gut colonization of OMM-12 and conventional mice also did not impact Mtb burden in the lungs in this controlled setting. H. hepaticus colonization of the gut did perturb lung immune responses associated with TB infection control. CD4+ T cells were decreased, CD8+ T cells were increased, and IL-6 production was decreased. While the gut microbiome may yet play a role in immune compromised mouse models or human disease, comparing drastically different gut microbiotas in gnotobiotically controlled C57BL/6 mice did not yield any evidence of alteration in Mtb lung burden. The hosts unique immune response to Mtb may in part make the pathogen resistant to immune disruption caused by gut microbiome changes.

Linezolid is a key component of the bedaquiline, pretomanid, linezolid (BPaL) regimen for rifampin-resistant tuberculosis, but dose- and duration-dependent mitochondrial toxicity frequently limits its use. TBD09 (formerly MK-7762) is a novel oxazolidinone developed to maintain antitubercular potency comparable to linezolid while reducing related adverse events. We evaluated the contribution of TBD09 to the bactericidal and sterilizing activities of BPa-based regimens in a murine tuberculosis model. BALB/c mice were aerosol-infected with Mycobacterium tuberculosis H37Rv and treated starting 2 weeks post-infection. In Experiment 1, dose-ranging bactericidal activity of TBD09 (50, 100, 200 mg/kg) was assessed alone and in combination with BPa, with comparison to linezolid and sutezolid. In Experiment 2, the sterilizing activity of TBD09 (200 mg/kg) in combination with BPa and BPa plus moxifloxacin (M) was assessed by evaluating relapse after treatment durations of 4-22 weeks. TBD09 monotherapy was bacteriostatic, similar to linezolid over the first 4 weeks of treatment. The addition of TBD09 to BPa significantly enhanced bactericidal activity comparable to the addition of linezolid. In relapse assessments, BPa+TBD09 resulted in significantly fewer relapses compared to BPa alone after 14 weeks of treatment, and BPaM+TBD09 resulted in significantly fewer relapses compared to BPaM after 10 weeks. Model-based analysis estimated the treatment duration to 95% cure for BPa+TBD09 at 2.7 weeks, compared to 3.2 weeks for BPaL, although the time to 50% cure was similar. These results demonstrate that TBD09 provides bactericidal and sterilizing activity similar to linezolid in combination with BPa(M), supporting further clinical development of TBD09 for tuberculosis.

In primary healthcare centers lacking advanced imaging, community-based active tuberculosis (TB) case finding often relies on basic symptom screening. This approach often misses cases and leads to the inefficient allocation of rapid molecular testing (RMT). We aimed to develop and internally validate a simple clinical triage scorecard to improve TB detection and guide RMT use in resource-constrained settings. We conducted a retrospective cross-sectional study of 15,137 adults ([&ge;]18 years) evaluated within the Zero TB Yogyakarta program (2020-2025). Participants with complete clinical assessments and confirmatory GeneXpert results were included. Using multivariable logistic regression, we identified independent clinical predictors, which were subsequently transformed into an integer-based point scorecard. Model performance was evaluated via discrimination and calibration, utilizing bootstrap resampling (1,000 iterations) for internal validation. Among the 15,137 participants, 251 (1.7%) were GeneXpert-positive. The final multivariable model identified eight independent predictors: age, male sex, body mass index, prolonged cough, hemoptysis, unexplained weight loss, TB contact history, and diabetes mellitus. The model demonstrated strong predictive accuracy, with an optimism-adjusted AUROC of 0.836 and good calibration. When translated to the integer scorecard and compared directly to standard national symptom screening, the scorecard performed (AUROC 0.81 vs. 0.73; p<0.001). At a high sensitivity cut off score of [&ge;] 0, the tool achieved 93.63% sensitivity and 41.33% specificity. This point-of-care clinical scorecard provides higher diagnostic accuracy than standard symptom screening algorithms. By offering flexible operational thresholds, it empowers local health programs to dynamically balance the urgency of case detection with available diagnostic capacity, optimizing GeneXpert allocation where advanced radiological imaging is unavailable.

A substantial fraction of the Mycobacterium tuberculosis proteome remains functionally uncharacterised. Rv1025, a 155-residue protein carrying the domain of unknown function DUF501 (Pfam PF04417), is essential by transposon mutagenesis and vulnerable by CRISPR interference, an attractive but neglected drug target, yet has never been functionally described. The family (4,370 proteins, no Gene Ontology term, no solved structure) is uncharacterised across all organisms and essential in three Actinobacterial genera. A Foldseek search of the AlphaFold model against complete structural databases finds no significant homolog, indicating a novel fold. The operon eno-divIC-Rv1025-ppx2 is conserved across the Actinobacteria phylum, yet AlphaFold-Multimer finds no direct complex between Rv1025 and its neighbour DivIC. Instead, conservation across 8,700 homologous sequences reveals a near-invariant Cys113-His115-Glu59 cluster forming a pocket. Holo AlphaFold3 predictions with Zn, Fe and Mn confidently place a divalent metal on this triad at 2.25-2.47 A; mutating the triad relocates the metal, and an independent backbone-geometry predictor recovers the same site, confirming specificity. The triad is universal across the family: present in all 1,472 near-complete bacterial sequences of the Pfam alignment, with no non-conservative substitution among the 2,228 sequences examined, a defining feature of bacterial DUF501 rather than a mycobacterial peculiarity. We propose that DUF501 is a metal-binding protein and candidate metalloenzyme, the first functional hypothesis for this family, whose conserved, essential metal pocket is a promising drug target. As the predictions build on a conservation-defined site within a fully computational study, they are supportive rather than proof of metal occupancy and warrant experimental validation.

BackgroundMultiple Sclerosis (MS) is a chronic autoimmune disorder characterized by inflammation and demyelination in central nervous system (CNS). Although increasing evidence suggests that gut microbial dysbiosis contributes to MS pathogenesis through the microbiota-gut-brain axis, reproducible microbial signatures associated with disease progression across independent clinical cohorts remain incompletely characterized. ObjectiveThis study aimed to identify conserved gut microbial alterations associated with Multiple Sclerosis by integrating publicly available human gut microbiome datasets and characterizing disease-associated microbial signatures linked to immune dysregulation. DesignHuman gut metagenomic 16S rRNA sequencing data from MS patients and healthy controls obtained from publicly available repositories (NCBI, Bioproject). Raw sequencing reads were processed using a standardized microbiome analysis workflow, including quality control, denoising, taxonomic assignment, phylogenetic reconstruction, diversity analyses, and differential abundance testing. Microbial community structure was evaluated using alpha- and beta-diversity analyses, while statistically significant differences between study groups were assessed using PERMANOVA, Kruskal-Wallis, and ANCOM to identify disease-associated bacterial taxa. ResultsIntegration of independent cohorts revealed consistent alterations in the gut microbial composition of MS patients compared with healthy controls. Significant reductions in microbial diversity and distinct microbial community structures were observed in MS. Differential abundance analysis demonstrated enrichment of the pro-inflammatory family Streptococcaceae, whereas beneficial short-chain fatty acid-producing taxa, particularly Lachnospiraceae, were significantly depleted in MS patients. These conserved microbial alterations indicate disruption of immune-regulatory bacterial communities and support the involvement of gut microbial dysbiosis in MS-associated neuroinflammation. ConclusionThis study identifies a reproducible gut microbial dysbiosis signature associated with Multiple Sclerosis, characterized by expansion of pro-inflammatory bacterial taxa and depletion of beneficial SCFA-producing microorganisms. These findings strengthen the evidence supporting the microbiota-gut-brain axis in MS pathogenesis and highlight microbial community signatures that may contribute to future biomarker development and microbiome- based therapeutic strategies.

Background Community-based tuberculosis screening can identify asymptomatic tuberculosis, but microbiological confirmation has been sputum-dependent. We evaluated the diagnostic performance, yield, and operational characteristics of centralized qPCR testing of community-collected tongue swabs (TS). Methods We conducted a cross-sectional study among household contacts (HHCs; [&ge;]18 years) of individuals receiving treatment for pulmonary TB in South Africa. During household visits, health workers collected TS specimens and, where possible, sputum, which was tested in participants' homes using Xpert MTB/RIF Ultra. TSs were transported non-refrigerated in molecular transport medium to a distant central laboratory for manual qPCR assay. The primary outcome was diagnostic performance of TS qPCR compared with sputum Xpert Ultra using paired results. Secondary outcomes were diagnostic yield by TS and operational indicators of the centralized testing workflow. Findings 909 HHCs were enrolled; median age 39 years (IQR 28-55), 532 (58.5%) were asymptomatic, 617 (67.9%) were sputum scarce. TS were collected from 901/909 (99.1%) and sputum from 292/909 (32.1%) participants. Among 271 paired TS-sputum results, TS qPCR sensitivity was 60.9% (95% CI 38.5-80.3), specificity 95.2% (91.7-97.5), and overall agreement 92.3% (88.5-95.0). Yield increased from 1.8% (95% CI 1.0-2.8) with symptom-restricted sputum testing to 3.2% (2.1-4.5) with symptom-agnostic sputum testing and 5.9% (4.5-7.7) when TS testing was implemented among sputum-scarce individuals, identifying 25 additional HHCs with positive molecular test results (86.2% increase). Operational indicators demonstrated successful implementation, with 2.3% specimen loss and valid molecular results for 93.8% who provided a TS. Interpretation Despite lower per-test sensitivity than sputum testing, TS may provide a scalable strategy for expanding microbiological screening beyond sputum-dependent pathways, thereby substantially increasing tuberculosis detection among HHCs. Funding U.S. NIH; Australian Department of Foreign Affairs and Trade; UK Foreign, Commonwealth and Development Office

The pathogenicity of Mycobacterium tuberculosis relies on its ability to withstand hostile host environments. Coenzyme F420 is critical for redox metabolism in Mycobacterium tuberculosis an organism distinguished by its abundant use of this low-redox-potential cofactor and a repertoire of F420-dependent enzymes. While F420-dependent mechanisms are known to confer protection against antimicrobials and oxidative stress, the specific enzymes responsible remain largely unidentified. In this study, using the F420-dependent detoxification of malachite green as a phenotypic marker, we performed a genome-wide screen in Mycobacterium tuberculosis using transposon mutagenesis and whole genome sequencing. The results of transposon mutagenesis and whole genome sequencing revealed that, among the 28 genes encoding F420-dependent enzymes, only mutations in fgd (essential for F420H2 re-generation) and Rv2061c, a gene encoding a putative F420-dependent oxidoreductase, were highly susceptible to malachite green. Subsequent experiments confirmed that Rv2061c and its homolog MT2120 are required for detoxifying malachite green and crystal violet. Furthermore, the Rv2061c mutant exhibited increased susceptibility to isoniazid and pretomanid; as well as menadione-induced oxidative stress. We also identified mutations in the transcriptional repressor Rv0678 as a key determinant of resistance to malachite green and crystal violet. This study establishes Rv2061c as a pivotal F420-dependent oxidoreductase that protects Mycobacterium tuberculosis from malachite green and other antimicrobial compounds as well as oxidative stress and further supports Rv0678 as a master regulator of multidrug resistance. These findings expand our knowledge of Mycobacterium tuberculosis defense mechanisms and highlight F420-dependent pathways as a potential target for new drug development.

Tuberculosis (TB) disease consistently causes the highest number of annual deaths from infectious disease and is responsible for over 100 million disability-adjusted-life-years annually. Despite the dramatic impact on human health, TB treatments are intensive and moderately effective, while Bacille Calmette-Guerin (BCG), the primary TB vaccine strategy and the oldest vaccine in circulation, needs updating. One gap in knowledge is that the vaccine primed lung immune environment has not been systematically profiled following TB challenge, making it challenging to interpret how various vaccine strategies provide protection. Here we profiled the male and female mouse lung immune environment using Mass Cytometry by Time of Flight (CyTOF) and bacterial burden (colony forming units, CFU), to systematically identify immune cells contributing to Mycobacterium tuberculosis (Mtb) infection or vaccine-induced protection. We investigated the mouse immune system after various infection outcomes including primary Mtb infection, Mtb reinfection, Mtb challenge after BCG vaccination and Mtb challenge after vaccination with a live attenuated Mtb vaccine. Across all conditions we identified Thy1.2 expression on NK cells as a new candidate for a sex-specific correlate of protection. Our results further replicate and support known immune responses for vaccine-induced protection, such as T-cell activation and infiltration. These findings highlight the pan-immune response to vaccine-induced protection, showcase the importance of sex in rodent models of TB, and identify novel host immune candidates to protect the lung against TB disease.