Tuberculosis (TB) continues to pose a significant public health challenge. An effective immune response against Mycobacterium tuberculosis (M.tb) is widely dependent on T cell memory responses, particularly in Latent tuberculosis infection (LTBI) and active tuberculosis disease (ATB). We quantified and phenotypically characterized peripheral blood mononuclear cells (PBMCs) from Southern Thai cohort after stimulation with established M.tb specific antigen: PPD and novel antigens: IrtA, PE9 and PPE68. We compared immune responses in 60 individuals (ATB n = 20, LTBI n = 20 and healthy controls (HC) n = 20) using flow cytometry and Enzyme linked Immunospot (ELISpot) assays. PPD induced the strongest IFN-γ responses across all groups. PPE68 demonstrated the highest immunogenicity among the novel antigens, inducing stronger IFN-γ responses. PE9 uniquely distinguished disease states by showing significantly reduced CD4+ TNF-α production in ATB and LTBI. PD-1 expression was markedly elevated on PPD-stimulated CD4+ T cells in ATB and LTBI, reflecting antigen load, T cell activation and/or exhaustion, whereas PE9 induced higher PD-1 expression in HC. These findings highlight the distinct immune signatures elicited by classical and novel M.tb antigens, which support further evaluation for future diagnostic or biomarker applications.
The diagnosis of pleural tuberculosis (Pl-TB) remains challenging. Histopathological analysis and pathogen detection in pleural biopsies are informative but limited. We investigated differentially expressed proteins and metabolites in pleural effusions from patients with Pl-TB, malignancies, and other pathologies. A proteomic analysis of pooled pleural effusions identified 45 proteins exclusively detected or upregulated in Pl-TB samples, many linked to infectious processes. Conversely, 18 proteins were uniquely found or upregulated in malignant pleural effusions, mainly associated with detoxification and hemostasis. To validate these findings, we employed targeted proteomics in individual samples. Eight proteins were validated: S100-A9, histone H4, insulin-like growth factor-binding protein 2, fibrinogen beta chain, ficolin-3, immunoglobulin heavy constant alpha 1, sulfhydryl oxidase 1, and histidine-rich glycoprotein. Additionally, NMR-based metabolomics identified 13 metabolites with differential abundance between Pl-TB and non-TB samples. Notably, N-acetyl-glycoprotein and the branched-chain amino acids, alanine and lysine differed between groups. Proteomic and metabolomic analyses revealed distinct molecular profiles between Pl-TB and non-TB patients, despite intra-group variability. To address this, we applied classification models. Histone H4 and alanine consistently emerged as discriminative features. Overall, this study provides novel insights into the molecular landscape of Pl-TB. The combined quantification of proteins and metabolites may improve differential diagnosis, although should be further validated in larger, independent cohorts before clinical application.
Understanding host-pathogen interactions between Mycobacterium bovis and Mycobacterium tuberculosis is critical for elucidating pathogen-specific infection dynamics. This pilot study was designed to compare the progression of infection in mice following inoculation with M. bovis or M. tuberculosis via intratracheal or oral gavage routes. We utilized the C3H/HeJ strain- a susceptible mouse model that exhibits severe pulmonary inflammation and poor bacterial control, a valuable tool for studying the spectrum of tuberculosis infection. Direct pulmonary inoculation of M. bovis was associated with earlier mortality, stronger inflammation, severe pathology, and earlier dissemination than pulmonary M. tuberculosis, whereas oral-gastrointestinal exposure to M. bovis produced a distinct systemic IL-1β-associated response without overt signs of disease. This work establishes the feasibility of using these infection modalities to guide investigations into pathogen-specific host-pathogen interactions.
Tuberculosis (TB) and diabetes mellitus (DM) represent a growing dual global health burden, with chronic hyperglycaemia recognized as a major modifier of host immunity against Mycobacterium tuberculosis (Mtb). Macrophages, central to pathogen recognition, phagocytosis, antigen presentation, and intracellular killing, may be particularly vulnerable to diabetic metabolic dysregulation. This study evaluated phenotypic and functional macrophage alterations in individuals with pulmonary TB, type 2 DM, TB-DM comorbidity, and healthy controls. Surface receptor expression was analysed by multicolour flow cytometry, while phagocytosis and intracellular bacterial clearance were assessed using FITC-labelled Mtb assays and colony-forming unit enumeration. Hyperglycaemia was associated with reduced CD11b, MARCO, and TLR2 expression alongside upregulation of the mannose receptor CD206, which correlated positively with HbA1c levels, indicating a shift toward a permissive M2-like phenotype. Phagocytic uptake of Mtb was significantly impaired and inversely correlated with HbA1c. Antigen-presenting capacity was selectively compromised, with reduced CD80 and CD86 expression in DM and TB-DM groups, while HLA-DR remained unchanged. Intracellular Mtb killing was markedly diminished in diabetic macrophages. These findings demonstrate that chronic hyperglycaemia profoundly disrupts macrophage innate immunity, contributing to increased TB susceptibility and poor infection control in diabetic populations.
Tuberculous meningitis (TBM) is the most severe form of tuberculosis and is associated with high mortality and long-term neurological disability. Detection of drug resistance remains challenging because cerebrospinal fluid (CSF) specimens are typically paucibacillary and often available only in limited volumes. This retrospective laboratory based study characterized drug-resistance patterns and diagnostic attrition across the laboratory testing cascade using CSF samples from patients with presumed TBM referred between January 2023 and December 2025 in a high tuberculosis-burden setting. Samples underwent Mycobacterium tuberculosis cartridge-based nucleic acid amplification testing (CBNAAT), automated liquid culture, line probe assays and phenotypic drug susceptibility testing according to national guidelines. Of 8926 CSF samples tested by CBNAAT, 812 (9.0%) were positive for M. tuberculosis. Rifampicin resistance was detected in 17.0% (138/812; 95% CI: 14.6-19.7) of positive cases. Only 837 samples had sufficient volume for culture, of which 58 (6.9%; 95% CI: 5.4-8.9) were culture-positive. Among these isolates, 29 (50.0%; 95% CI: 37.5-62.5) were multidrug resistant (MDR-TB). Six MDR isolates were also fluoroquinolone resistant, including one extensively drug-resistant (XDR-TB) isolate. These findings demonstrate a high burden of drug resistance, substantial diagnostic attrition, and the need to expand access to comprehensive drug-susceptibility testing in TBM.
This study investigated immune cell distributions, cell-specific immune markers, and selected biomarker targets in pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) using multiparametric flow cytometry (MFC). Whole blood was collected from 45 individuals, including healthy controls (HC), EPTB, and PTB patients (n = 15/group). Peripheral blood leukocytes were analysed by MFC to characterize CD4+ and CD8+ T cells, natural killer (NK), invariant NKT (iNKT) and NKT cells, classical (CM), intermediate (IM) and non-classical monocytes (NCM), and activated monocytes (AM). Expression of GBP1, CALCOCO2, IFIT3, SNX10, ARG1, PD-1, and PD-L1 was assessed across these immune subsets. Increased frequencies of NK, NKT, and monocytes were observed in PTB and EPTB compared with HC, while CD4+, CD8+, iNKT, and AM were reduced. Monocyte-to-lymphocyte ratios were incrementally elevated in EPTB and PTB compared with HC. Despite variability of expression within groups, median biomarker fold-change expression changes were found between HC, EPTB and PTB groups; (i) (>2.0FC) for ARG1 in CD4, CD8, CM and AM, for CALCOCO2 in AM, GBP1 in CD8 and NCM, PD-1 in CD4, CD8, NK, IM and AM, PD-L1 in CD4, CD8, iNKT and NKT, NK, IM and AM and SNX10 in CD4, CD8, NCM, IM and AM (ii) (<2.0FC) in TB vs HC for CALCOCO2 in iNKT and NKT, IFIT3 in NCM, PD-1 in NK and NCM, PD-L1 in NCM, IM and AM and SNX10 in AM. Statistical significance was achieved for ARG1 (P = 0.017) in CD4 cells. Our findings highlight distinct immune cell and biomarker signatures in PTB and EPTB.
A diverse set of transcriptional regulators (TRs) enables Mycobacterium tuberculosis (M. tuberculosis) to reprogram its gene expression profiles to adapt and counteract the challenges imposed by the host during infection. FadR subfamily TRs regulate different physiological aspects of bacteria, including stress adaptation and virulence. The genome of M. tuberculosis encodes five homologs of the FadR subfamily of TRs. Here, we have comprehensively characterised the three FadR subfamily TRs, namely Rv0043c, Rv0165c, and Rv3060c, to assess their role in M. tuberculosis stress adaptation and pathogenesis. We demonstrate that the individual deletion of these TRs does not affect the growth of M. tuberculosis in vitro under various host-mimicking stress conditions or during infection of macrophages, mice, or guinea pigs. RNA-seq analysis revealed that, compared to the wild type strain, the relative transcript levels of the majority of differentially expressed genes were increased in the ΔRv0043c, ΔRv0165c and ΔRv3060c strains of M. tuberculosis, suggesting that these proteins function as transcriptional repressors. Taken together, these results suggest that functional redundancy exists between the FadR subfamily of TRs.
Despite significant efforts to improve diagnostics, tuberculosis (TB) diagnosis still relies on clinical and laboratory methods limited by cost, expertise, and time. Early diagnosis is essential for effective management of pulmonary TB (PTB), pleural TB (PLTB), and latent TB infection (LTBI), highlighting the urgent need for rapid, accurate, and accessible TB diagnostics based on low-invasive sampling. In this study, a quantitative rapid test, previously evaluated in European and African cohorts, was applied to a Brazilian cohort to measure six host-derived proteins in serum from individuals with PTB, PLTB, other pleural diseases (OPLD), LTBI, and controls. Serum levels of CRP, ferritin, and SAA1/A2 were significantly elevated in PTB and PLTB compared to controls. SAA1/A2 and IP-10 levels were higher in PLTB compared to LTBI. Furthermore, IP-10 serum levels showed diagnostic potential, as they were elevated in PTB compared with both LTBI and controls, and significantly increased in PLTB compared to OPLD. In pleural effusion, IP-10, IL-6, and S100A12 were significantly higher in PLTB than in OPLD, while ferritin levels were higher in OPLD. Our study shows that quantitative detection of host-derived biomarkers identified for active PTB in European and African populations can support clinical decision-making for TB spectrum in a Brazilian setting.
Background Tuberculous meningitis (TBM), the most severe form of tuberculosis, may involve the gut-brain axis. This study investigated the link between TBM, brain white matter integrity measured by diffusion tensor imaging (DTI), and the gut microbiome. Methods 22 TBM patients and 31 healthy controls underwent MRI and provided fecal samples. Gut microbiota (16S rRNA sequencing), fecal metabolites (metabolomics), and DTI metrics (FA, MD, RD, AD) were analyzed. Tract-based spatial statistics and correlation analyses were employed. Results TBM patients showed lower gut microbiota α-diversity. The abundance of the Escherichia genus correlated negatively with FA and positively with MD in specific white matter tracts. Metabolomics revealed elevated acetic acid in TBM patients, which correlated with both Escherichia abundance and DTI metrics. KEGG analysis indicated altered arginine and proline metabolism pathways. Conclusion TBM is associated with differences in gut microbiota composition. Higher relative abundance of Escherichia is linked to white matter microstructural damage, potentially mediated by specific bacterial metabolites.
Inflammatory microenvironments regulate immune cell differentiation, activation, and programming. Although traditionally considered terminally differentiated effectors, neutrophils can acquire distinct functional states in response to environmental cues. To examine context-dependent neutrophil programming, cells from healthy donors were cultured in vitro under cytokine regimens mimicking opposing inflammatory environments (GM-CSF/IFN-γ or IL-4/IL-13/TGF-β), alone or combined with PMA and Mycobacterium tuberculosis, to assess functional and transcriptional responses. GM-CSF/IFN-γ-conditioned neutrophils exhibited increased cell size, altered nuclear morphology, enhanced MHC class II and CD86 expression, and elevated IL-8 and reactive oxygen species production. This profile was associated with increased TNF-α, IL-10, TLR2, and TLR4 gene expression and reduced global DNA methylation. In contrast, IL-4/IL-13/TGF-β-conditioned neutrophils resembled non-conditioned controls regarding surface markers and cytokine production but showed reduced ROS generation and increased DNMT3A expression. Upon in vitro infection with M. tuberculosis, GM-CSF/IFN-γ-conditioned neutrophils produced higher IL-8 and IL-1β levels and formed more neutrophil extracellular traps. Additionally, plasma from patients with severe tuberculosis modulated TLR4, CCR7, and IP-10 expression in healthy neutrophils, indicating systemic inflammatory influences. These findings demonstrate that inflammatory conditioning induces coordinated transcriptional and epigenetic remodeling, shaping neutrophil antimicrobial and immunoregulatory potential in infectious contexts.
BACKGROUND:The Bacille Calmette-Guérin (BCG) vaccine against tuberculosis is the most widespread vaccine in the world. Discovered by French investigators Albert Calmette and Camille Guérin at the Pasteur Institute, it remains the only effective vaccine against tuberculosis infection. This report describes the recognition and identification of a previously unknown French handwritten laboratory notebook prepared by Drs. Calmette and Camille Guérin recording their experiments performed during the development of the BCG vaccine. METHODS:The notebook was examined, translated into English, photographed and the experiments analyzed. RESULTS:The manuscript laboratory notebook consists of 69 leaves written in 2 hands, one of which corresponds to that of Albert Calmette. It contains details of experiments that were performed during the development of the BCG vaccine at the Pasteur Institute by Drs. Calmette and Guérin. These include experimental inoculations of rabbits and guinea pigs; descriptions of the pathology of skin lesions, inflammatory reactions, and organ pathology; and survival outcome. The experiments describe varying inoculative dosages of the bacteria, and different routes of administration including intraperitoneal and subcutaneous injections, and administration of bacilli in the ear. In those cases where the animal had died following inoculation of tubercle bacilli, necropsy was performed and the organs examined and the pathology findings described. Details of culture experiments and vaccine passage are listed. CONCLUSIONS:This previously unknown notebook is a highly organized and detailed record of investigations using tuberculosis in animal experiments and microbiological culture to produce a safe and effective vaccine, first used in humans in 1921.
OBJECTIVE:The aim of the study was to determine the associations between vitamin D (VD) and cytokine levels in active pulmonary tuberculosis (PTB) patients with chronic pulmonary aspergillosis (CPA). METHODS:One hundred seventy-two active PTB patients, twenty-eight PTB patients with CPA (PTB-CPA), and sixty healthy subjects were included in the study. The concentrations of serum 25-hydroxyvitamin D, interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF-α) were measured by ELISA. RESULTS:A significantly lower VD level was detected in PTB-CPA patients (14.96 ± 10.89 ng/mL) than in both PTB patients (20.72 ± 8.07 ng/mL, p < 0.01) and controls (23.90 ± 8.16 ng/mL, p < 0.001). VD deficiency was significantly more prevalent in PTB-CPA patients than in PTB patients (OR = 2.86, 95% CI: 1.22-6.69; p = 0.01) or controls (OR = 5.33, 95% CI: 2.02-14.11; p = 0.0005). In the PTB group, VD levels exhibited a significant inverse correlation with only IL-6 (r = -0.376, p = 0.0001), whereas in PTB-CPA patients, stronger inverse correlations were observed with IL-6 (r = -0.691, p = 0.0005) and IL-8 (r = -0.538, p = 0.01). In cavitary PTB patients, a significant inverse link was found between VD and IL-1β and IL-6 levels (all p ≤ 0.05). CONCLUSION:Our data revealed a high prevalence of VD deficiency in all groups, predominantly in PTB-CPA patients. The inverse correlations between VD levels and IL-6 and IL-8 levels in PTB-CPA patients as well as between VD and IL-1β and IL-6 levels in cavitary PTB patients, provide further evidence for the role of VD as a regulator of inflammatory pathways in the context of coinfection.
Pulmonary and skin infections caused by Mycobacterium abscessus, a rapidly growing non-tuberculous mycobacterium (NTM), are becoming increasingly prevalent worldwide. Managing these infections is particularly challenging due to the bacterium's inherent resistance to many antibiotics, often resulting in extended treatment periods or therapeutic failure. With few effective drugs available, identifying new therapeutic targets is critical. A promising approach involves targeting the pathogen's energy metabolism, specifically the electron transport chain (ETC), which plays a vital role in its survival and virulence. This review explores the essential components of the ETC in Mycobacterium abscessus as potential drug targets to support and expedite the development of novel treatments.
A significant proportion of individuals heavily exposed to infectious tuberculosis patients do not acquire Mycobacterium tuberculosis (Mtb) infection, as detected by an interferon gamma release assay (IGRA). Trained immunity may contribute to this host resistance to Mtb infection, also termed early clearance. From a prospective tuberculosis household study in Indonesia we selected 80 heavily exposed IGRA-negative household contacts, of whom 40 converted their baseline-negative IGRA to positive after three months (IGRA converters) and 40 remained IGRA-negative (early clearers). From all individuals we measured circulating β-D-glucan and used their serum for induction of trained immunity in vitro, using peripheral blood mononuclear cells from healthy unexposed Dutch donors, that were stimulated with unrelated stimuli six days after exposure to serum from household contacts. β-D-glucan concentrations and positivity did not correlate with early clearance, nor with serum-induced in-vitro trained immunity as measured by heterologous cytokine responses. These findings suggest that early clearance is unlikely to be maintained by circulating β-D-glucan or other serum factors present at exposure to Mtb, and may instead depend on cell-intrinsic or local host processes not captured by serum-based assays.
Background Type 2 diabetes mellitus elevates the risk of developing tuberculosis, posing a substantial challenge to TB treatment and control. However, the underlying molecular mechanisms remain poorly understood. Methods Non-targeted metabolomics analysis of serum samples collected from 20 patients with concurrent DM and TB (DM-TB) and 20 patients with TB only (TB) was performed using UPLC-MS/MS. Logistic regressions were applied to evaluate the associations between the metabolites and lung pathology. Results Pathway analysis revealed that TCA cycle, steroidogenesis, ketone body metabolism were the key pathways enriched in the differential metabolites distinguishing DM-TB from TB patients. Multivariable logistic regression analysis suggested, high levels of C18 Sphingosine (OR = 2.207, 95% CI = 1.049-4.645), L-Saccharopine (OR =4.407, 95% CI = 1.045-18.582) and arachidonic acid (AA) (OR = 2.845, 95% CI = 1.272-6.366) were associated with an increased risk of extensive lung lesions. In addition, C18 Sphingosine (OR = 3.596, 95% CI = 1.239-10.440) and AA levels (OR = 3.306, 95% CI = 1.200-9.112) were positively associated with the risk of prevalent multiple cavities. Conclusions Our results suggests that lipid metabolism disorders are significantly associated with severe lung lesions in TB-DM patients, suggest a potential link to exacerbated inflammatory and immunomodulatory responses.
Rv2660c is present in both Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis) and has been reported as a protective antigen. In this study, Rv2660c was linked with another M. tuberculosis specific antigen Rv2645, which is present in M. tuberculosis but not in M. bovis, to construct a fusion protein Rv2645-Rv2660c (LT25 in short). LT25 failed to confer protection following M. bovis challenge. Structural predictions via AlphaFold 3 revealed that LT25 retained the native conformation of Rv2645, whereas Rv2660c underwent significant conformational alterations. LT25 elicited strong Rv2645 and Rv2660c-specific cellular immune responses and high levels of Rv2645-specific antibodies, but lower levels of Rv2660c-specific antibodies than Rv2660c did. Further investigation showed that the Rv2660c-induced polyclonal antibody possessed stronger phagocytosis-promoting activity than LT25-induced antibodies did. These findings suggest that the structural integrity of Rv2660c is essential for its protective function.
