Tuberculosis

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.

Tuberculosis (TB) diagnosis remains challenging, particularly for extrapulmonary TB (EPTB), where invasive sampling, low bacillary burden, and suboptimal sensitivity of nucleic acid-based tests in peripheral specimens hinder timely detection. Here, we report an immunology-driven strategy for biomarker discovery and development of a peptide-based serological assay targeting Mycobacterium tuberculosis zinc metalloprotease-1 (Zmp1). Leveraging fundamental principles of adaptive immunity that antigenic regions containing overlapping B-cell and CD4 T-helper cell epitopes would preferentially generate high antibody titers through linked recognition and cognate T-cell help, we used an immunoinformatics pipeline to identify two nested immunodominant peptide regions within Zmp1 (Mtb-Zp-NT and Mtb-Zp-CT) enriched for overlapping B- and T-cell epitopes. The diagnostic potential of these peptides was evaluated through ELISA-based serological assays. A blinded pilot study (N=137) demonstrated a clear discrimination between active TB and TB-recovered individuals. The assay was subsequently validated in an expanded cohort (N=875) by screening 6,086 individuals, which identified 457 TB-positive cases. The cohort included pulmonary TB (PTB), EPTB, TB-recovered individuals, household contacts, non-specific infections, and healthy controls. Receiver operating characteristic analyses, supported by DeLong and bootstrap comparisons, revealed superior diagnostic performance of the peptide-based assays relative to full-length Zmp1. Mtb-Zp-CT exhibited the highest accuracy (AUC=0.93; specificity >90%), while Mtb-Zp-NT also demonstrated strong discriminatory power (AUC{approx}0.89). These findings establish that the immunologically optimized Zmp1 peptides are highly promising serological biomarkers for TB and EPTB. More broadly, they demonstrate how mechanistically informed epitope selection can accelerate translation of pathogen-specific immune signatures into sensitive, minimally invasive, and potentially point-of-care diagnostic platforms for resource-limited settings.

The phenotypic plasticity, slow replication, and complex, hydrophobic cell envelope of Mycobacterium tuberculosis contribute to its successful survival as a pathogen and its drug tolerance. Consequently, the global threat of multidrug-resistant Tuberculosis (MDR-TB), coupled with lengthy and highly toxic treatment regimens, necessitates the development of innovative treatment solutions. Mycobacteriophages are natural viruses of mycobacteria that typically encode two endolysins, which cooperatively facilitate host cell lysis at the end of the lytic life cycle: LysA, a peptidoglycan hydrolase, and LysB, a lipolytic enzyme, targeting the mycolylarabinogalactan-peptidoglycan complex. Their precise and efficient lytic activity, along with their low propensity to induce resistance, make them, particularly LysBs, promising candidates for new treatment solutions. In this study, we report MTB-LysB1, a novel LysB enzyme from an F1 sub-cluster mycobacteriophage isolated from our laboratory collection. While studying its structural features by comparing the modelled structure with representative mycobacteriophage LysB homologues, we found that the /{beta}-hydrolase fold and key motifs are conserved. Also, we identified putative membrane-interaction motifs that may play a role in LysB1s cell permeation. Significantly, we found MTB-LysB1 to be active against both drug-susceptible and multidrug-resistant (MDR) M. tuberculosis strains at nanomolar concentrations, comparable to the well-characterised D29 LysB reference enzyme. Beyond its standalone activity, MTB-LysB1 exhibits an additive effect when combined with the TB drugs rifampicin and moxifloxacin, and co-administration reduces the drugs minimum inhibitory concentrations (MICs), which holds clinical significance. By structurally damaging the mycobacterial cell wall, the enzyme appears to act as a permeability enhancer for the chemotherapeutic drugs, thereby improving antibiotic efficacy. Collectively, our findings position the enzyme not only as a novel antimycobacterial agent but also provide a structural framework for its rational engineering as a promising next-generation adjunct to TB drug regimens. HighlightsO_LIA novel F1 sub-cluster phage-derived LysB is discovered and characterised using integrated computational, biochemical and microbiological methods. C_LIO_LIAlphaFold2 modelling, molecular dynamics simulations and comparative structural analyses revealed an /{beta}-hydrolase fold with conserved catalytic and membrane-interaction features. C_LIO_LIThe enzyme exhibited high esterase activity, thermal stability and potent lytic activity against Mycobacterium tuberculosis. C_LIO_LIAn additive effect with TB drugs rifampicin and moxifloxacin highlights MTB-LysB1s potential as an adjunct therapeutic. C_LI

Males incur greater risk of tuberculosis (TB) than females, but the contribution of sex-associated immune differences remains unclear. We addressed this using sex-stratified transcriptomic analyses across four independent studies spanning active pulmonary TB, subclinical TB and latent infection, in peripheral blood, bronchoalveolar lavage (BAL) and by using the tuberculin skin test (TST) as a standardised in vivo antigenic challenge. In blood of active TB patients, expression of TNF- and type I interferon-regulated signatures, genome-wide gene expression, and performance of leading host-response biomarkers of TB were comparable between sexes. Similarly, blood transcriptomic biomarkers showed no meaningful sex-related differences for predicting asymptomatic or incident TB. In the TST of people with latent infection, bulk and single-cell RNA sequencing identified only limited differences, largely restricted to sex chromosome-linked transcripts, with no consistent evidence of dimorphism in immune-regulated pathways. Single-cell RNA sequencing of BAL samples identified reduced abundance of B cells in male TB patients, with gene expression differences again largely restricted to sex chromosome-linked transcripts. These findings suggest that canonical immune responses associated with TB are broadly similar between the sexes, and that increased TB risk among males more likely reflects differential exposure rather than intrinsic immunological susceptibility.

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.

Purpose: Central nervous system tuberculosis (CNS TB) presenting with vasculitis or arachnoiditis causes significant morbidity and mortality. The purpose of this study was to characterise the clinical spectrum and outcomes of patients diagnosed with TB arachnoiditis and TB-associated CNS vasculitis Methods A retrospective study was conducted between October 2020 and September 2023, screening patients admitted with suspected CNS TB to a tertiary care hospital. Patients diagnosed with proliferative arachnoiditis, and TB-associated CNS vasculitis were recruited. Their clinical details, follow-up records, and outcomes were assessed. Results Among 318 patients admitted with suspected CNS TB, 87 patients had complications, with follow-up data available for 69 patients. Vasculitis, spinal arachnoiditis (SA), and optochiasmatic arachnoiditis (OCA) was diagnosed in 66 (76%), 41 (47%), and 26 (30%) patients respectively. Median duration of follow-up was 490 days. Median mRS at discharge was 4. 18 (69%) OCA patients and 14 (35%) SA patients received pulse methylprednisolone. Intrathecal hyaluronidase was administered in 14 patients and thalidomide was given to 9 patients. 30 (46%) patients with vasculitis were treated with aspirin. 69 patients completed follow-up, 49% died. Among the remaining, 88.6% had improvement with treatment with a median mRS of 2 (1-3). Among patients with OCA, 3(23.1%) showed complete improvement with a median improvement of 3 points on Likert scale. In the SA patients, 19 (55.9%, 34) patients were alive on follow-up, with a median mRS of 1. Aspirin use was not associated with better mRS or survival in patients with vasculitis. A multivariable Cox proportional model showed age at diagnosis to be the only predictor of mortality (HR 1.04, 95% CI (1.01- 1.08), p =0.012). Conclusions TB arachnoiditis and CNS vasculitis are severe complications of CNS TB, and management remains a challenge. The poor therapeutic response to intrathecal hyaluronidase, thalidomide, and aspirin highlights need for further larger prospective trials and search for alternative agents.

Background The World Health Organization (WHO) recommends 4-month treatment for children with non-severe pulmonary tuberculosis, outlining eligibility criteria for settings with and without chest X-ray (CXR). We evaluated the diagnostic accuracy of the WHO eligibility criteria in settings without CXR (WHO-criteria) and developed clinical scores to support disease classification. Methods Using data from an individual participant dataset (IPD; Decide TB) of children with confirmed/unconfirmed tuberculosis from four diagnostic studies (RaPaed-TB, Umoya, TB-Speed HIV, TB-Speed Decentralisation), we assessed the diagnostic accuracy of the WHO-criteria (with/without bacteriological testing) using expert CXR interpretation as a reference. We developed two multivariable logistic regression models with (Score 1) and without (Score 2) bacteriological testing, converted coefficients into integer scores with a threshold of >10 corresponding to a sensitivity [&ge;]70%. Results Of 2,383 children in the Decide TB-IPD, 633 (26.6%) met the eligibility criteria for a 4-month regimen, of whom 116 (18.3%) had radiologically severe disease. With and without bacteriological testing, the WHO-criteria had sensitivities of 30.1% (95%CI: 20.3%-40.2%) and 21.7% (95%CI: 10.4%-34.5%), and specificities of 83.4% (95%CI: 80.2%-86.4%) and 81.9% (95%CI: 78.8%-84.9%), respectively. Score 1 and Score 2 had sensitivities of 41.1% (95%CI: 32.4%-49.5%) and 30.9% (95%CI: 22.6%-40.4%), and specificities of 77.3% (95%CI: 73.6%-80.8%) and 83.0% (95%CI: 79.5%-86.3%) respectively. Using WHO-criteria, 91/116 (78.4%) and 105/116 (90.5%) of children were at risk of undertreatment, compared to 68/116 (58.6%) and 80/116 (68.9%) when using developed scores. Conclusions Developed scores demonstrated better sensitivity than WHO-criteria, however, performance remains suboptimal. Implementing shorter antituberculosis regimens without CXR remains challenging in children.

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.

Background: Access to accurate tuberculosis (TB) diagnostics remains limited, particularly in high-burden settings. The Pluslife MTB assay is among the first molecular tests specifically designed for swab-based detection of Mycobacterium tuberculosis complex (MTBC) and offers near point-of-care use. Methods: We conducted a prospective diagnostic accuracy study in South Africa to evaluate the performance of the Pluslife assay on tongue swabs (TSs) and sputum swabs (SSs) among symptomatic and asymptomatic adults. Results were compared against liquid culture as the reference standard and Xpert MTB/RIF Ultra (Xpert Ultra) as a comparator. Operational characteristics and ease-of-use were assessed through structured observation and Likert-scale scoring by testing personnel. Results: Of 256 participants enrolled, 92 [36%] were people with HIV (PHIV) and 217 were included in the final analysis. Culture confirmed TB in 41/217 (19%). The Pluslife assay demonstrated sensitivity of 85% (95% CI: 70.8-94.4) on SSs, comparable to Xpert Ultra on sputum (83%, 95% CI: 67.9-92.8), and detected one additional case missed by Xpert Ultra. Sensitivity on TSs was lower (63%, 95% CI: 46.9-77.9), particularly among PHIV. Specificity exceeded 97% across specimen types. Concordance on TSs between Pluslife and Xpert Ultra increased with higher bacterial loads. Operational evaluation showed short hands-on time and high ease-of-use scores, though limitations were noted for patient identifier recording and troubleshooting on the Pluslife MiniDock device. Conclusions: The Pluslife MTB assay on SSs shows comparable performance to existing rapid diagnostics and favorable usability. Tongue swabs remain feasible but less reliable, supporting sputum as the preferred first specimen for TB diagnosis.

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.

Immune biomarkers of tuberculosis (TB) disease severity present a challenging area of research that remains poorly understood. New technologies are able to perform larger, unbiased studies that can unravel the complex host-pathogen dynamics occurring during a Mycobacterium tuberculosis infection, the causative agent of TB. In this study, we designed a high dimensional approach combining viable bacterial burden (CFU, colony forming units) with time-of-flight mass cytometry (CyTOF) analysis to profile differences in cell-type abundance and cell-type specific protein expression during states of low, intermediate and high TB disease burden. Broadly, we segregated cell-type specific immune responses into those driven by bacterial burden and/or the mycobacterial infection strain. Interrogating these immune signatures allowed us to identify ATP-catabolizing protein CD39 as a correlate of disease severity. Treatment of mice with a small molecule inhibitor of CD39 promoted effector T cell functions and CD4 T cell expansion during Mtb infection. Collectively, our data defines the differential lung immune environment between various mycobacterial disease severity states and uncovers a potential immune biomarker of infection and therapeutic immunomodulating target to aid in the treatment of TB.

Tongue swabs (TSs) are a non invasive specimen type for the detection of Mycobacterium tuberculosis complex (MTBC) and can expand access to testing for individuals unable to produce sputum. This study evaluated the diagnostic performance and user acceptability of self collected and health worker (HW) collected tongue swabs using the Xpert MTB/RIF Ultra (Ultra) assay and assessed participant perspectives on self collection. In this prospective, cross sectional study, symptomatic and asymptomatic adults under investigation for TB were enrolled from a high HIV prevalence setting. Each participant provided both a self collected and a HW collected TS, which were tested using Ultra. Ultra TS results were compared to liquid culture as the reference standard and sputum Ultra as a comparator. Participant perspectives on self collection were captured via questionnaires. Sensitivity on Ultra for both self and HW collected TSs was 68% (95% CI:51.9 to 81.9), compared to liquid culture. This sensitivity was significantly higher than that of sputum smear microscopy (46%, 95% CI: 30.7 to 62.6; McNemar's p = 0.003). Tongue swab sensitivity was lower than sputum Ultra (80.5%; p<0.001) and decreased with low bacillary loads. Importantly, TSs enabled MTBC detection in six participants unable to produce sputum. Most participants (>90%) found self collection instructions easy to follow, reporting high confidence and comfort, and trust in results from self collected TSs. This study demonstrates that self collected TSs perform comparably to those collected by health workers for TB detection using Ultra and are both feasible and acceptable in a high TB/HIV burden setting. To maximize impact, clear training instructions and robust linkage to care remain critical priorities.

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.

Neurocysticercosis (NCC), the infection of the central nervous system by Taenia solium larvae, is a leading cause of acquired epilepsy in endemic regions. While viable cysticerci can persist asymptomatically for extended periods, their spontaneous or drug-induced degradation triggers marked perilesional inflammation and severe neurological symptoms. Despite well-documented histopathological characterisation of these lesion states, the host transcriptional programmes associated with viable parasite persistence and early post-treatment lesion disruption remain poorly understood. To address this gap, we performed the first bulk RNA sequencing of pericystic brain tissue using a physiologically relevant porcine model of NCC. Comparing uninfected controls (n = 3), infected untreated pigs with intact viable cysts (n = 6), and antiparasitic-treated pigs with disrupted cysts (n = 3), we identified distinct transcriptional signatures associated with each disease state. Viable infection was associated with broad transcriptional changes (461 upregulated and 175 downregulated genes), characterised by local immune activation alongside suppression of blood-brain barrier (BBB) remodelling, vascular, and neuronal signalling molecular signatures. The post-treatment state with confirmed BBB disruption was associated with a smaller but directionally distinct response (160 upregulated and 57 downregulated genes), marked by inflammatory signalling and increased expression of genes associated with endothelial activation, vascular regulation, and BBB-associated remodelling. Together, these findings suggest that, while immune engagement is a feature shared across both lesion states, the BBB-associated transcriptional axis shifts substantially following treatment. These results provide an exploratory transcriptomic framework for understanding parasite persistence, treatment-induced neuroinflammation, and neurovascular remodelling in NCC, and highlight candidate pathways and genes for future mechanistic investigation.

Recognition of infected macrophages by CD4+ T cells is essential to immune protection against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). However, not all infected macrophage subsets successfully elicit T cell activation. We recently discovered that M2-like macrophages fail to efficiently activate memory CD4+ T cells when infected with Mtb, yet successfully elicit T cell activation when loaded with peptides, {gamma}-irradiated bacteria, or Mtb whole cell lysate. Since the mechanisms underlying CD4+ T cell evasion by infected M2 but not M1-like macrophages remain underexplored, we sought to determine the genes and pathways unique to Mtb infection of M2-like cells, including alveolar macrophages. RNA sequencing of human macrophages infected with virulent Mtb identified enrichment of IL-10 and type I interferon (IFN) signaling genes, including IL10RA and HERC5, respectively, in infected M2-like monocyte-derived and alveolar macrophages. However, genes involved in MHC-II trafficking, such as AP1M2, were higher in infected M1-like macrophages. In complementary experiments using fluorescence microscopy and flow cytometry, we observed impaired trafficking of newly synthesized MHC-II to the plasma membrane of Mtb-infected M2-like macrophages despite high total surface MHC-II levels. Neutralization of IL-10 or knockdown of HERC5 restored MHC-II trafficking to the cell surface among infected M2-like macrophages and significantly enhanced activation of memory CD4+ T cells in an MHC-II-dependent manner. These findings identify coordinated IL-10 and type I IFN signaling as key mechanisms that restrict MHC-II trafficking to the plasma membrane in Mtb-infected M2-like macrophages, thereby limiting antigen presentation and CD4+ T cell activation. We propose that host-directed therapies targeting these pathways in infected alveolar macrophages will facilitate T cell recognition for the prevention or treatment of active TB. Author SummaryRecognition of infected macrophages by CD4+ T cells is essential to immune protection against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). However, not all infected macrophage subsets successfully elicit T cell activation. We recently discovered that M2-like macrophages fail to efficiently activate memory CD4+ T cells when infected with Mtb, yet they successfully elicit T cell activation when treated with peptides, {gamma}-irradiated bacteria, or Mtb whole cell lysate. In this study, we identified genes and pathways uniquely upregulated in Mtb-infected M2-like macrophages that are linked to inefficient CD4+ T cell activation, including IL-10 signaling and type I interferon (IFN) pathways. These pathways were linked to reduced MHC-II trafficking to the plasma membrane in Mtb-infected M2-like macrophages. Neutralization of IL-10 or knockdown of HERC5 restored MHC-II trafficking and augmented memory CD4+ T cell activation. Our study demonstrates that IL-10 signaling and type I IFN pathways play detrimental roles in macrophages during Mtb infection, impairing MHC-II trafficking and CD4+ T cell activation. Since lung-resident alveolar macrophages express a dominant M2-like phenotype, these findings suggest that targeting IL-10 and type I IFN signaling may offer a strategy to enhance CD4+ T cell-mediated immunity and improve TB outcomes.

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.

Naturally occurring variation in the bovine mannose receptor C-type 1 gene (MRC1) may shape macrophage responses to Mycobacterium (M.) bovis, a key driver of bovine tuberculosis (bTB). We identified four coding region SNPs in MRC1 across Bos taurus (Holstein Friesian, Brown Swiss) and Bos indicus (Boran, Sahiwal) cattle breeds, including a non-synonymous variant, rs380943118 (c.2963G>A; Ser988Asn) in C-type lectin-like domain (CTLD) 6, most prevalent in Sahiwal cattle. Structural modelling suggested that the S988N substitution, which is spatially separated from the monosaccharide binding site of CTLD4, might indirectly affect glycan binding, perhaps through a conformational change in the receptor. Monocyte-derived macrophages upregulated MR expression during differentiation, with heterozygous (G/A) animals showing higher MR expression and increased uptake of GFP-M. bovis BCG, although differences were not statistically significant. Anti-CD206 blockade did not inhibit BCG internalization, either indicating that this specific antibody did not bind to a CTLD involved in ligand binding or that MR is not the sole entry receptor. These results highlight naturally occurring MRC1 polymorphisms that may influence MR structure and macrophage function, providing a foundation for future studies to assess their role in bTB susceptibility.

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.

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.