Tuberculosis

Humans are highly genetically diverse, and most are resistant to Mycobacterium tuberculosis. However, lung tissue from genetically resistant humans is not readily available to identify potential mechanisms of resistance. To address this, we model M. tuberculosis infection in Diversity Outbred mice. Like humans, Diversity Outbred mice also exhibit genetically determined susceptibility to M. tuberculosis infection: Progressors who succumb within 60 days of a low dose aerosol infection due to acute necrotizing granulomas, and Controllers who maintain asymptomatic infection for at least 60 days, and then develop chronic pulmonary TB with occasional necrosis and cavitation, over months to greater than 1 year. Here, we identified specific regions of granuloma-associated lymphoid tissue (GrALT) and B-cell gene expression pathways as key features of asymptomatic lung infection using cytokine, antibody, granuloma image, and gene expression datasets. Cytokines and anti-M. tuberculosis cell wall antibodies discriminated acute vs chronic pulmonary TB but not asymptomatic lung infection. To find unique features of asymptomatic lung infection, we trained a weakly supervised, deep-learning neural network on lung histology images. The neural network accurately produced an interpretable imaging biomarker: perivascular and bronchiolar lymphocytic cuffs, a type of GrALT. We expected CD4 T cell genes would be highly expressed in asymptomatic lung infection. However, the significantly different, highly expressed genes in lungs of asymptomatically infected Diversity Outbred mice corresponded to B-cell activation, proliferation, and antigen-receptor signaling, including Fcrl1, Cd79, Pax5, Cr2, and Ms4a1. Overall, our results suggest that genetically controlled B-cell responses are important for establishing asymptomatic M. tuberculosis lung infection.

BackgroundBovine tuberculosis (bTB) is a chronic infectious disease primarily caused by Mycobacterium bovis, which inflicts significant economic losses on the global livestock industry worldwide and can also cause tuberculosis (TB) disease in other mammalian species, including humans. Alveolar macrophages are the host cells targeted by the pathogen during the early stages of infection. While they play a crucial role in controlling infection, the exact nature of the host-pathogen interaction and the genetic and epigenetic factors that modulate infection outcome remain poorly understood. ResultsHere, we used transcriptomics (RNA-seq) and chromatin configuration analyses (ChIP-seq and ATAC-seq) to examine the effects of intracellular mycobacterial infection on the bovine alveolar macrophage (bAM) transcriptome and epigenome. The primary focus was M. bovis infection, but we also conducted parallel comparative analyses using M. tuberculosis (the primary cause of human TB--hTB), M. bovis BCG (the vaccine strain), and gamma-irradiated (killed) M. bovis. Integration of RNA-seq, ChIP-seq, and ATAC-seq data revealed coordinated remodelling of chromatin accessibility and histone modification landscapes underpinning transcriptional activation of key immune and metabolic pathways in response to infection. The identification of candidate genes, including ERBB4, LRCH1, MRTFA, and RNPC3, through integrative analysis with a genome-wide association study (GWAS) for M. bovis infection susceptibility underscores the functional relevance of these regulatory networks. ConclusionsOur results demonstrate that M. bovis drives extensive reprogramming of the bAM epigenome, distinct from the responses elicited by other members of the M. tuberculosis complex (MTBC). The results of this multi-omics comparison provide new insights into the function of pivotal response genes and support the hypothesis that pathogen-driven epigenetic reprogramming of the bovine host macrophage is key to M. bovis survival. It also identifies molecular targets that may inform genome-enabled breeding strategies to enhance bTB disease resilience in cattle.

Mycobacterium tuberculosis (Mtb) infection activates type I interferons (IFNs) which are crucial mediators of tuberculosis (TB) pathogenesis. Despite assumptions that IFN and IFN{beta} are secreted by macrophages, direct protein quantification in primary human monocyte-derived macrophages is surprisingly lacking. Here, we demonstrate measurable IFN and IFN{beta} secretion by MDMs infected with both virulent (H37Rv) and attenuated (H37Ra) Mtb strains as early as 48 h post-infection, with levels persisting at 120 h. These findings challenge existing assumptions about type I IFN kinetics and highlight the importance of timing in experimental designs and provides a foundation for exploring their role in host-pathogen interactions.

ObjectivesTo better understand the pathogenesis of pericardial tuberculosis (PCTB), we sought to characterize the systemic inflammatory profile in HIV-1-infected participants with latent TB infection (LTBI), pulmonary TB (PTB) and PCTB. MethodsUsing Luminex, we measured 39 analytes in pericardial fluid (PCF) and paired plasma from 18 PCTB participants, and plasma from 16 LTBI and 20 PTB. Follow-up plasma samples were also obtained from PTB and PCTB participants. HLA-DR expression on Mtb-specific CD4 T cells was measured in baseline samples using flow cytometry. ResultsAssessment of the overall systemic inflammatory profile by principal component analysis showed that the inflammatory profile of active TB participants was distinct from the LTBI group, while PTB patients could not be distinguished from those with PCTB. In the LTBI group, 12 analytes showed a positive association with plasma HIV-1 viral load, and most of these associations were lost in the diseased groups. When comparing the inflammatory profile between PCF and paired blood, we found that the concentrations of most analytes (24/39) were elevated at site of disease. However, the inflammatory profile in PCF partially mirrored inflammatory events in the blood. After TB treatment completion, the overall plasma inflammatory profile reverted to those observed in the LTBI group. Lastly, HLA-DR expression showed the best performance for TB diagnosis compared to previously described biosignatures built from soluble markers. ConclusionOur results describe the inflammatory profile associated with PTB and PCTB and emphasize the potential role of HLA-DR as a promising biomarker for TB diagnosis.

BackgroundThe immune response to Mycobacterium tuberculosis (M. tuberculosis) is central to the pathogenesis of tuberculosis (TB), yet the immune dynamics induced by drug-resistant strains remain underexplored. Understanding the hosts immune response to both drug-sensitive and drug-resistant M. tuberculosis isolates is crucial for elucidating the mechanisms of pathogenesis and resistance. This study aims to assess the cellular immune responses, including PBMC proliferation, cytokine secretion (IL-4 and IL-17a), and reactive oxygen species (ROS) production in response to live drug-sensitive and drug-resistant M. tuberculosis clinical isolates. MethodsPeripheral blood mononuclear cells (PBMCs) from PPD-negative and PPD-positive healthy volunteers were stimulated with live M. tuberculosis isolates, including MDR, SI-resistant, and sensitive strains. The immune responses were assessed by evaluating cell proliferation, secretion of IL-4 and IL-17a cytokines, and ROS production over a 9-day period. ResultsPBMCs from PPD-positive individuals exhibited a higher proliferative response compared to PPD-negative individuals, indicating more robust immune memory. IL-4 secretion was low but varied among samples, with higher levels observed in response to MDR isolates, suggesting a potential role in immunopathology. IL-17a levels increased over time, particularly in PPD-positive individuals, and MDR strains elicited a stronger response than sensitive isolates. ROS production was significantly elevated in response to resistant strains, reflecting the hosts oxidative defense mechanisms. ConclusionThis study demonstrates distinct immune responses to drug-resistant M. tuberculosis isolates, with variations in cell proliferation, cytokine secretion, and ROS production. These findings provide insights into the immune dynamics during infection with resistant strains and underscore the importance of genotype-environment interactions in TB pathogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/626310v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1bb9ad6org.highwire.dtl.DTLVardef@1b268e7org.highwire.dtl.DTLVardef@52800org.highwire.dtl.DTLVardef@85234b_HPS_FORMAT_FIGEXP M_FIG C_FIG

We successfully employed a single cell RNA sequencing (scRNA-seq) approach to describe the cells and the communication networks characterizing granulomatous lymph nodes of TB patients. When mapping cells from individual patient samples, clustered based on their transcriptome similarities, we uniformly identify several cell types that known to characterize human and non-human primate granulomas. Whether high or low Mtb burden, we find the T cell cluster to be one of the most abundant. Many cells expressing T cell markers are clearly quantifiable within this CD3 expressing cluster. Other cell clusters that are uniformly detected, but that vary dramatically in abundance amongst the individual patient samples, are the B cell, plasma cell and macrophage/dendrocyte and NK cell clusters. When we combine all our scRNA-seq data from our current 23 patients (in order to add power to cell cluster identification in patient samples with fewer cells), we distinguish T, macrophage, dendrocyte and plasma cell subclusters, each with distinct signaling activities. The sizes of these subclusters also varies dramatically amongst the individual patients. In comparing FNA composition we noted trends in which T cell populations and macrophage/dendrocyte populations were negatively correlated with NK cell populations. In addition, we also discovered that the scRNA-seq pipeline, designed for quantification of human cell mRNA, also detects Mtb RNA transcripts and associates them with their host cells transcriptome, thus identifying individual infected cells. We hypothesize that the number of detected bacterial transcript reads provides a measure of Mtb burden, as does the number of Mtb-infected cells. The number of infected cells also varies dramatically in abundance amongst the patient samples. CellChat analysis identified predominating signaling pathways amongst the cells comprising the various granulomas, including many interactions between stromal or endothelial cells and the other component cells, such as Collagen, FN1 and Laminin. In addition, other more selective communications pathways, including MIF, MHC-1, MHC-2, APP, CD 22, CD45, and others, are identified as originating or being received by individual immune cell components. Author SummaryThe research conducted describes the cellular composition and communication networks within granulomatous lymph nodes of tuberculosis (TB) patients, employing a single-cell RNA sequencing (scRNA-seq) approach. By analyzing individual patient samples and clustering cells based on their transcriptome similarities, the study reveals several consistent cell types described to be present in both human and non-human primate granulomas. Notably, T cell clusters emerge as abundant in most samples. Additionally, variations in the abundance of B cells, plasma cells, macrophages/dendrocytes, and NK cells among patient samples are observed. Pooling scRNA-seq data from 23 patients enabled the identification of T, macrophage, dendrocyte, and plasma cell subclusters, each displaying distinct signaling activities. Moreover, the study uncovers a surprising capability of the scRNA-seq pipeline to detect Mtb RNA transcripts within host cells, providing insights into individual infected cells and Mtb burden. CellChat analysis unveils predominant signaling pathways within granulomas, highlighting interactions between stromal/endothelial cells and other immune cell components. Moreover, selective communication pathways involving molecules such as Collagen, FN1, Laminin, CD99, MIF, MHC-1, APP and CD45 are identified, shedding light on the intricate interplay within granulomatous lymph nodes during TB infection.

Dysregulated iron metabolism is reported in tuberculosis patients; therefore, it represents an opportunity for developing host-directed therapeutics. This study monitored the antimycobacterial properties of an iron chelator, i.e., Deferoxamine (DFO/D), and its impact on the transcript and metabolite levels of Mycobacterium tuberculosis (Mtb) in vitro. For in vivo validation, a group of mice received ferric carboxymaltose to create an iron overload condition, and controls were aerosol-infected with Mtb H37Rv. Mtb-infected mice received isoniazid (INH/H) and rifampicin (RIF/R) in combination with or without DFO before tissue-specific CFU assay, liver metabolite screening and iron quantification using mass spectrometry. DFO showed antimycobacterial properties comparable to INH in vitro. DFO treatment deregulated (log2DFO/control>{+/-}1.0) Mtb transcript (n=137) levels, the majority of which encode for iron-containing proteins and proteins involved in stress response. DFO treatment up-regulated Rv3622c (PE32), Rv2353c (PPE39) and Rv3022A (PE29) genes and conditional knocking down of ABC transporter like irtA by anhydrotetracycline (Atc) inducible CRISPR interference (CRISPRi) approach compromised Mtb growth showing their potential involvement in iron metabolism. Global Mtb metabolite analysis using GC-MS identified a set of 5 deregulated metabolites indicating a perturbed pentose phosphate pathway and inositol phosphate metabolism in the host upon DFO treatment. Iron-overloaded mice exhibited significantly higher tissue mycobacterial burden at two weeks post-infection, and the efficacy of INH and RIF were compromised, corroborating with previous reports. Iron chelation by DFO or combined with/adjunct to RIF and INH significantly reduced the lung/tissue mycobacterial burden at four weeks post-treatment, specifically in the first ([~]0.5 log) and second weeks ([~]0.5 log) of treatment. The intracellular pro-inflammatory cytokine levels in the lung CD4+ T cells of INH and RIF-treated groups with or without DFO were similar, suggesting DFO has a direct role in Mtb survival and metabolism rather than improved infection, and the efficacy of INH and RIF were compromised, corroborating with previous reports. DFO adjunct to RIF and INH treatment significantly altered liver arginine biosynthesis, which directly neutralizes ammonia and is immune-supportive. Conventionally, DFO is used for treating acute iron toxicity that is common in thalassemic patients, and this study demonstrates DFO has potential as adjunct therapeutics for tuberculosis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/543389v3_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@5066fdorg.highwire.dtl.DTLVardef@99a292org.highwire.dtl.DTLVardef@80426dorg.highwire.dtl.DTLVardef@892436_HPS_FORMAT_FIGEXP M_FIG C_FIG

Tuberculosis (TB) represents a global public health threat and is the leading cause of morbidity and mortality worldwide. Effective control of TB is complicated with the emergence of multidrug resistance. Yet, there is still a fundamental gap in understanding the complex and dynamic interactions between different Mycobacterium tuberculosis strains and the host. In this project, we investigated the host immune response to different M. tuberculosis strains, including avirulent or virulent and rifampin-resistant or isoniazid-resistant strains in THP-1 cells. We identified major differences in the gene response profiles in response to infection with these strains. The expression of IDO1 and IL-1{beta} in the infected cells was stronger in all virulent M. tuberculosis strains. The most striking result was the overexpression of many interferon-stimulated genes (ISGs) in cells infected with the isoniazid-resistant strain, compared to the rifampin-drug resistant strain and the drug-sensitive strain. A transcription regulation analysis of the differentially expressed genes in infected THP-1 cells implicated two major transcription factors, NF-{kappa}B and STAT1. The differentially expressed ISGs in response to the isoniazid-resistant M. tuberculosis strain were associated with STAT1 signaling, while the expression of many cytokines, such IL-1{beta}, was associated with NF-{kappa}B signaling. Our data suggest that the isoniazid-resistant M. tuberculosis strain preferentially activates STAT1 in response to cGAS-STING activation and induces a host immune response signature that is characteristic of isoniazid resistance. This study has a potential to provide important new insights into TB pathogenesis and to characterize host gene signatures specifically involved in isoniazid-resistant TB.

Pulmonary infections caused by the group of nontuberculosis mycobacteria (NTM), Mycobacterium avium complex (MAC), are increasing worldwide and a growing public health concern. Pulmonary granulomas are the hallmark of MAC lung infection, yet reliable correlates of granuloma progression and susceptibility in immunocompetent hosts are poorly defined. The development of mouse models that recapitulate the diversity of granulomas seen in MAC pulmonary disease in humans is crucial to study mechanisms of susceptibility in humans and for preclinical evaluation of therapeutics. Unlike widely used inbred mouse strains, mice that carry the mutant allele at the genetic locus sst1 develop human-like pulmonary tuberculosis featuring well-organized caseating granulomas. These mice became instrumental in pre-clinical testing of novel interventions. In this study we tested whether the B6.Sst1S that carries the sst1 mutant allele on standard B6 background develop more advanced pulmonary infection with NTM M. avium spp. hominissuis (M.av). To assess pulmonary disease progression, we utilized traditional semi-quantitative histomorphological evaluation and fluorescent multiplex immunohistochemistry (fmIHC) in combination with whole slide imaging and digital image analysis. After infection with the laboratory M.av strain 101, the B6.Sst1S pulmonary lesions progressed 12 - 20 weeks post infection, although we did not observe the formation of necrotic granulomas during this interval. Using fmIHC, we determined that the disease progression was associated with a steadily increasing proportion of mycobacteria infected Arg1+ and double positive iNOS+/Arg1+ macrophages. The B6.Sst1S granulomas had a greater proportion of Arg1+ and double positive iNOS+/Arg1+ macrophages, and decreased T cell density, as compared to wild type B6 mice. Thus, the genetic composition of the B6.Sst1S mice renders them more susceptible to pulmonary M.av infection. In combination with more virulent clinical isolates of M.av these mice could provide an improved mouse model that recapitulates more severe pulmonary disease in humans. The Arg1 macrophage expression in this model combined with automated fmIHC could serve as a sensitive biomarker for the unbiased assessment of medical countermeasures against NTM infection.

BackgroundTuberculosis (TB) is the leading cause of infectious disease mortality worldwide. Numerous blood-based gene expression signatures have been proposed in the literature as alternative tools for diagnosing TB infection. Ongoing efforts are actively focused on developing additional signatures in other TB-related contexts. However, the generalizability of these signatures to different patient contexts is not well-characterized. There is a pressing need for a well-curated database of TB gene expression studies for the systematic assessment of existing and newly developed TB gene signatures. ResultsWe built the curatedTBData, a manually-curated database of 49 TB transcriptomic studies. This data resource is freely available through GitHub and as an R Bioconductor package that allows users to validate new and existing biomarkers without the challenges of harmonizing heterogeneous studies. We also demonstrate the use of this data resource with cross-study comparisons for 72 TB gene signatures. For the comparison of subjects with active TB from healthy controls, 19 gene signatures had weighted mean AUC of 0.90 or greater, with the highest result of 0.94. In active TB disease versus latent TB infection, 7 gene signatures had weighted mean AUC of 0.90 or greater, with a maximum of 0.93. We also explore ensembling methods for averaging predictions from multiple gene signatures to significantly improve diagnostic ability beyond any single signature. ConclusionsThe curatedTBData data package offers a comprehensive resource of curated gene expression and clinically annotated data. It could be used to identify robust new TB gene signatures, to perform comparative analysis of existing TB gene signatures, and to develop alternative gene set scoring or ensembling methods, among other things. This resource will also facilitate the development of new signatures that are generalizable across cohorts or more applicable to specific subsets of patients (e.g. with rare comorbid conditions, etc.). We demonstrated that these blood-based gene signatures could distinguish patients with distinct TB outcomes; moreover, the combination of multiple gene signatures could improve the overall predictive accuracy in differentiating these subtypes, which point out an important aspect for the translation of genomics to clinical implementation.

If we are to break new grounds in TB research, we need to have a complete understanding of what is occurring at the site of infection in humans. Postmortem studies give us an opportunity to compare TB-involved and -uninvolved tissues, in both diseased and non-diseased individuals. We examined the feasibility of carrying out a postmortem study in Mulago and Kiruddu National Referral Hospitals in Uganda, to determine whether we could use immune cells collected postmortem for immunological studies. We report that we can consent the Next-of-Kin, perform postmortem procedures and process tissues within 8 hours of death, and that immune cells remain viable and functional up to 14 hours after death. We found subtle differences in T cell subsets within TB groups. We found a depletion of the CD4 CD69+CD103+ T cell subset in the lungs and BAL, which was associated with HIV, and that the CD8 CD69+CD103- T cell subset was depleted in BAL only, and was associated with TB. Our data show overall changes Tissue Resident Memory T cells within, and between, TB-infected and TB-uninfected human lungs. SummaryO_LICoroner led postmortem studies are possible in Uganda, samples processed within 8 hours from death C_LIO_LICells from samples collected postmortem are viable and functional C_LIO_LIHIV associated depletion of CD4 CD69+/CD103+ T cell subset in lungs and BAL C_LIO_LICD8 CD69+/CD103- depletion in BAL associated with TB C_LI

BackgroundZoonotic tuberculosis (zTB) is the transmission of Mycobacterium tuberculosis complex (MTBC) subspecies from animals to humans. zTB is generally quantified by determining the proportion of human isolates that are Mycobacterium bovis. Although India has the worlds largest number of human TB cases and the largest cattle population, where bovine TB is endemic, the burden of zTB is unknown. MethodsTo obtain estimates of zTB in India, a PCR-based approach was applied to sub-speciate positive MGIT(R) cultures from 940 patients (548 pulmonary, 392 extrapulmonary disease) at a large referral hospital in India. Twenty-five isolates of interest were subject to whole genome sequencing (WGS) and compared with 715 publicly available MTBC sequences from South Asia. FindingsA conclusive identification was obtained for 939 samples; wildtype M. bovis was not identified (95% CI: 0 - 0.4%). There were 912 M. tuberculosis sensu stricto (97.0%, 95% CI: 95.7 - 98.0), 7 M. orygis (95% CI: 0.3 - 1.5%); 5 M. bovis BCG, and 15 non-tuberculous mycobacteria. WGS analysis of 715 MTBC sequences again identified no M. bovis (95% CI: 0 - 0.4%). Human and cattle MTBC isolates were interspersed within the M. orgyis and M. tuberculosis sensu stricto lineages. InterpretationM. bovis prevalence in humans is an inadequate proxy of zTB in India. The recovery of M. orygis from humans, together with the finding of M. tuberculosis in cattle, underscores the need for One Health investigations to assess the burden of zTB in countries with endemic bovine TB. FundingBill & Melinda Gates Foundation, Canadian Institutes for Health Research

Tuberculosis remains a major health threat, infecting nearly a third of the worlds population. Of those infected, 5-10% progress from latent infection to active tuberculosis (TB) disease and biomarkers to identify which individuals will progress are needed to allow targeted prophylactic treatment. Several risk biomarkers have been developed to predict progression but have not been tested head-to-head on the same platform. Here, we used the NanoString platform and compared the performance of 15 published gene signatures in predicting progression at baseline in a household contact cohort. Expression of gene signatures was profiled in RNA extracted from whole blood and scored using GSVA and PLAGE. We found that specificity is enhanced by combining signatures and report that the performance of a combined signature that includes a newly derived parsimonious signature through machine learning and a published signature met WHO TPP levels for a triage test. The combined signature had a 90.9% sensitivity and 88% specificity with a PPV of 0.24 and NPV of 1. This combined signature has potential clinical utility in identifying high-risk individuals for targeted prophylaxis to prevent TB morbidity and mortality.

IntroductionPredictive biomarkers for symptomatic tuberculosis (TB) progression would transform targeted prevention efforts. Although interferon-gamma release assays (IGRAs), including QuantiFERON(R) TB-Gold Plus (QFT-Plus), have been studied for this purpose, systematic evaluation of the QFT-Plus TB1 and TB2 Interferon-Gamma (IFN{gamma}) concentrations remains limited, particularly in high-burden TB settings. MethodsBaseline TB1 and TB2 IFN{gamma} concentrations from 5,259 participants in TB-endemic regions were analyzed in relation to subsequent TB outcomes over a median of 525 days follow-up. Participants were categorized as controls (no TB), suspected TB (no microbiological confirmation), or laboratory-confirmed TB, including a subset meeting a stringent case definition ([&ge;]2 positive microbiologic tests). Associations between baseline IFN{gamma} concentrations and progression to symptomatic TB were assessed. ResultsIn the full cohort (IGRA+/-participants), baseline TB2 IFN{gamma} concentrations were significantly higher compared to controls among participants who developed suspected TB (p=0.01), laboratory-confirmed TB (p=0.01), or met the stringent case definition (p<0.0001). In IGRA+ participants, baseline TB2 concentrations were significantly higher than controls in suspected (p=0.01) and laboratory-confirmed (p=0.02) groups. Associations with baseline TB1 IFN{gamma} concentrations and TB progression were observed for participants meeting the stringent case definition within the full cohort (p=0.009). Among stringent definition cases, TB2 concentrations achieved an area under the Receiver Operating Characteristic curve of 0.84, with a sensitivity of 80% and specificity of 78%. ConclusionsQuantitative IFN{gamma} concentrations from QFT-Plus, particularly TB2, were associated with progression to symptomatic TB, met or exceeded WHO-recommended sensitivity and specificity thresholds for predictive biomarkers, and may support biomarker-based stratification in TB clinical research. SummaryIn an exploratory analysis from a global tuberculosis (TB) epidemiology study, higher QuantiFERON-TB Gold Plus IFN{gamma} concentrations were associated with progression to symptomatic TB and met WHO predictive biomarker thresholds, supporting their potential value for risk stratification in TB research.

BackgroundTuberculous meningitis (TBM) is the most sinister form of extrapulmonary tuberculosis (EPTB), associated with high mortality due to delayed diagnosis and limited sensitivity of conventional and molecular tests. Current study evaluated the diagnostic utility of Lipoarabinomannan antigen (LAM) detection in CSF and urine and explored host inflammatory biomarkers for diagnosis and prognosis of TBM. MethodsThis prospective observational study enrolled 80 patients with presumptive TBM at a tertiary care centre. CSF samples were subjected to AFB microscopy, liquid culture(MGIT-960), GeneXpert MTB/RIF (GX), and LAM lateral flow assay. Urine LAM was performed at baseline. Serum and CSF levels of IL-1{beta}, IL-6, TNF-, IFN-{gamma}, IL-17A, and IP-10 were measured at baseline and after 1 month treatment. ResultsAmong 80 participants, 23 (28.7%) had definite TBM and 46 (57.5%) had probable TBM. CSF LAM sensitivity and specificity against microbiological reference standards was 43.5% and 80.7%, while urine LAM sensitivity (60.9%) and specificity 82.5% was higher. Against composite reference standards, both CSF and urine LAM showed reduced sensitivity but achieved 100% specificity. Serum IL-1{beta} showed the best diagnostic performance (AUC 0.943; sensitivity 88.9%, specificity 90.9%). Elevated serum and CSF IP-10 levels were associated with poor outcomes, whereas declining IL-6 and TNF- levels correlated with treatment response. ConclusionLAM detection in CSF and urine may serve as a highly specific, rapid rule-in test for TBM. Host inflammatory biomarkers, especially IL-1{beta} and IP-10, show additional diagnostic and prognostic value. Combining LAM testing with cytokine biomarkers may improve early diagnosis and efficient clinical management of TBM.

Mouse models have been key to studies of tuberculosis pathogenesis and drug efficacy, but many, such as those employing BALB/c mice, fail to reproduce the full range of heterogenous microniches observed in well-structured human lesions, which feature hypoxic caseous cores of necrotic debris surrounded by infected foamy macrophages. The granuloma presents a variety of environments differing in levels of oxygen, ions, nutrients, and intra versus extracellular residence, which determine the physiological state of the infecting bacillus and its susceptibility to immune or drug control. Recently, alternative mouse strains such as C3HeB/FeJ have allowed the study of infection and treatment in the context of these varied environments but exhibit substantial inconsistency in development of human-like lesions, both within and between individual mice. Building on the observation that inducible nitric oxide synthase (Nos2)-deficient mice consistently develop hypoxic necrotic lesions, we have established two simplified models with infection by the aerosol route. The first uses the slightly attenuated M. tuberculosis R1Rv strain, which produces a progressive infection that is contained at a high stable burden by an adaptive immune response. In the second model, vaccination with the attenuated {Delta}RD1, pantothenate auxotroph mc2 6230 protects from an otherwise lethal infection with virulent M. tuberculosis Erdman. This model reflects most contemporary tuberculosis infections, which take place in the context of a pre-existing immune response from vaccination. Both variations uniformly develop well-structured hypoxic necrotic lesions harboring drug tolerant bacteria. These refined models will be useful in studies of M. tuberculosis infection and treatment.

Host innate immune cells, including alveolar macrophages, have been identified as key players in the early eradication of Mycobacterium tuberculosis and in the maintenance of an anti-mycobacterial immune memory, which is believed to be induced through epigenetic changes. The aim of the study was to elucidate whether exposure to M. tuberculosis induced a different DNA methylation pattern of alveolar macrophages and pulmonary T lymphocytes. Alveolar macrophages and T lymphocytes were isolated from induced sputum obtained from individuals living in Lima, which is an area high endemic for tuberculosis. To determine the latent tuberculosis infection status of the subjects, an interferon-{gamma} release assay was performed. We evaluated the DNA methylomes of the alveolar macrophages and T lymphocytes using the Illumina Infinium Human Methylation 450K Bead Chip array, revealing a distinct DNA methylation pattern in alveolar macrophages allowing the discrimination of asymptomatic individuals with latent tuberculosis infection from non-infected individuals. Pathway analysis revealed that cell signalling of inflammation and chemokines in alveolar macrophages play a role in latent tuberculosis infection. In conclusion, we demonstrated that DNA methylation in alveolar macrophages can be used to determine the tuberculosis infection status of individuals in a high endemic setting.

Mycobacterium tuberculosis (Mtb) remains a global human health threat and a significant cause of human morbidity and mortality. We document here the capture of Mtb transcripts in libraries designed to amplify eukaryotic mRNA. These reads are often considered spurious or nuisance and are rarely investigated. Because of early literature suggesting the possible presence of polyadenylated transcripts in Mtb RNA, we included the H37Rv Mtb reference genome when assembling scRNA seq libraries from fine needle aspirate samples from patients presenting at the TB clinic, Port Moresby General Hospital, Papua New Guinea. We used 10X Genomics single-cell RNA sequencing transcriptomics pipeline, which initiates mRNA amplification with poly-T primers on [~]30-micron beads designed to capture, in this case, human mRNA associated with individual cells in the clinical samples. Utilizing the 10X Genomics Cell Ranger tool to align sequencing reads, we consistently detected bacterial small and large ribosomal subunit RNA sequences (rrs and rrl, respectively) and other bacterial gene transcripts in the cell culture and patient samples. We interpret Mtb reads associated with the host cells unique molecular identifier (UMI) and transcriptome to indicate infection of that individual host cell. The Mtb transcripts detected showed frequent sequence variation from the reference genome, with greater than 90% of the rrs or rrl reads from many clinical samples having at least 1 sequence difference compared to the H37Rv reference genome. The data presented includes only bacterial sequences from patients with TB infections that were confirmed by the hospital pathology lab using acid-fast microscopy and/or GeneXpert analysis. The repeated, non-random nature of the sequence variations detected in Mtb rrs and rrl transcripts from multiple patients, suggests that, even though this appears to be a stochastic process, there is possibly some selective pressure that limits the types and locations of sequence variation allowed. The variation does not appear to be entirely artefactual, and it is hypothesized that it could represent an additional mechanism of adaptation to enhance bacterial fitness against host defenses or chemotherapy.

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with {Delta}vapC12 induced a proinflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with {Delta}vapC12 succumbed early to the disease. Finally, we reported that the above phenotype of {Delta}vapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggest that the inability of {Delta}vapC12 to resolve neutrophil-mediated inflammation reduced bacterial killing by altering the T-cell response. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the hosts innate immune response in ways that favor the long-term survival of the pathogen inside the host.

BackgroundClinical manifestations of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) show lineage-specific differences contributed by genetic polymorphism such as phylo-single nucleotide variations (PhyloSNPs) and insertion or deletions (INDELs). Intragenomic rearrangement events, such as gene duplications and deletions, may cause gene copy number differences in Mtb, contributing to lineage-specific phenotypic variations, if any, which need better understanding. ResultsThe relative gene copy number differences in high-quality publicly available whole genome sequencing datasets of 9,482 clinical Mtb isolates were determined by repurposing and modifying an RNA-seq data analysis pipeline. The pipeline included various steps, viz., alignment of reads, sorting by coordinate, GC bias correction, and variant stabilising transformation. The strategy showed maximum separation of lineage-specific clusters in two principal components, capturing [~]54% variability. Unsupervised hierarchical clustering of the top 100 genes and pairwise comparisons between Mtb lineages revealed an overlapping subset of genes (n=42) having significantly perturbed copy numbers (Benjamin Hochberg adjusted P-value < 0.05 and log2(drug-resistant/sensitive) > {+/-} 1). These 42 genes formed multiple tandem gene clusters and are known to be involved in virulence, pathogenicity and defence response to invading phages. A separate comparison showed a significantly high copy number of phage genes and a recently reported druggable target Rv1525 in pre- and extensively drug-resistant (Pre-XDR, XDR) compared to drug-sensitive clinical Mtb isolates. ConclusionThe identified gene sets in Mtb clinical isolates may be useful targets for lineage-specific therapeutics and diagnostics development.