Anatomic and Cellular Niches for Mycobacterium tuberculosis in Latent Tuberculosis Infection.

Latent tuberculosis has been recognized for over a century, but discovery of new niches, where Mycobacterium tuberculosis resides, continues. We evaluated literature on M.tuberculosis locations during latency, highlighting that mesenchymal and hematopoietic stem cells harbor organisms in sensitized asymptomatic individuals.

Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses.

New vaccine platforms are needed to address the time gap between pathogen emergence and vaccine licensure. RNA-based vaccines are an attractive candidate for this role: they are safe, are produced cell free, and can be rapidly generated in response to pathogen emergence. Two RNA vaccine platforms are available: synthetic mRNA molecules encoding only the antigen of interest and self-amplifying RNA (sa-RNA). sa-RNA is virally derived and encodes both the antigen of interest and proteins enabling RNA vaccine replication. Both platforms have been shown to induce an immune response, but it is not clear which approach is optimal. In the current studies, we compared synthetic mRNA and sa-RNA expressing influenza virus hemagglutinin. Both platforms were protective, but equivalent levels of protection were achieved using 1.25 µg sa-RNA compared to 80 µg mRNA (64-fold less material). Having determined that sa-RNA was more effective than mRNA, we tested hemagglutinin from three strains of influenza H1N1, H3N2 (X31), and B (Massachusetts) as sa-RNA vaccines, and all protected against challenge infection. When sa-RNA was combined in a trivalent formulation, it protected against sequential H1N1 and H3N2 challenges. From this we conclude that sa-RNA is a promising platform for vaccines against viral diseases.

Mycobacterium tuberculosis-Infected Hematopoietic Stem and Progenitor Cells Unable to Express Inducible Nitric Oxide Synthase Propagate Tuberculosis in Mice.

Persistence of Mycobacterium tuberculosis within human bone marrow stem cells has been identified as a potential bacterial niche during latent tuberculosis. Using a murine model of tuberculosis, we show here that bone marrow stem and progenitor cells containing M. tuberculosis propagated tuberculosis when transferred to naive mice, given that both transferred cells and recipient mice were unable to express inducible nitric oxide synthase, which mediates killing of intracellular bacteria via nitric oxide. Our findings suggest that bone marrow stem and progenitor cells containing M. tuberculosis propagate hallmarks of disease if nitric oxide-mediated killing of bacteria is defective.

Seasonally Varying Secondary Organic Aerosol Formation From In-Situ Oxidation of Near-Highway Air.

The extent to which motor vehicles contribute to ambient secondary organic aerosol (SOA) remains uncertain. Here, we present in situ measurements of SOA formation at a near-highway site with substantial tree-cover 10 m from Interstate 40 near Durham, North Carolina. In July 2015 (summer) and February 2016 (winter), we exposed ambient air to a range of oxidant (O3 and OH) concentrations in an oxidation flow reactor (OFR), resulting in hours to weeks of equivalent atmospheric aging. We observed substantial seasonal variation in SOA formation upon OFR aging; diurnally varying OA enhancements of ∼3-8 µg m-3 were observed in summer and significantly lower enhancements (∼0.5-1 µg m-3) in winter. Measurements in both seasons showed consistent changes in bulk OA properties (chemical composition; volatility) with OFR aging. Mild increases in traffic-related SOA precursors during summer partly explains the seasonal variation. However, biogenic emissions, with sharp temperature dependence, appear to dominate summer OFR-SOA. Our analysis indicates that SOA observed in the OFR is similar (within a factor of 2) to that predicted to form from traffic and biogenic precursors using literature yields, especially in winter. This study highlights the utility of the OFR for studying the prevalence of SOA precursors in complex real-world settings.

Spatial-Temporal Analysis of PM2.5 and NO2 Concentrations Collected Using Low-Cost Sensors in Peñuelas, Puerto Rico.

The U.S. Environmental Protection Agency (EPA) is involved in the discovery, evaluation, and application of low-cost air quality (AQ) sensors to support citizen scientists by directly engaging with them in the pursuit of community-based interests. The emergence of low-cost (<$2500) sensors have allowed a wide range of stakeholders to better understand local AQ conditions. Here we present results from the deployment of the EPA developed Citizen Science Air Monitor (CSAM) used to conduct approximately five months (October 2016⁻February 2017) of intensive AQ monitoring in an area of Puerto Rico (Tallaboa-Encarnación, Peñuelas) with little historical data on pollutant spatial variability. The CSAMs were constructed by combining low-cost particulate matter size fraction 2.5 micron (PM2.5) and nitrogen dioxide (NO2) sensors and distributed across eight locations with four collocated weather stations to measure local meteorological parameters. During this deployment 1 h average concentrations of PM2.5 and NO2 ranged between 0.3 to 33.6 µg/m³ and 1.3 to 50.6 ppb, respectively. Peak concentrations were observed for both PM2.5 and NO2 when conditions were dominated by coastal-originated winds. These results advanced the community's understanding of pollutant concentrations and trends while improving our understanding of the limitations and necessary procedures to properly interpret measurements produced by low-cost sensors.

Primary and Photochemically Aged Aerosol Emissions from Biomass Cookstoves: Chemical and Physical Characterization.

Secondary organic aerosol (SOA) formation during photo-oxidation of primary emissions from cookstoves used in developing countries may make important contributions to their climate and air quality impacts. We present results from laboratory experiments with a field portable oxidation flow reactor (F-OFR) to study the evolution of emissions over hours to weeks of equivalent atmospheric aging. Lab tests, using dry red oak, measured fresh and aged emissions from a 3 stone fire (TSF), a "rocket" natural draft stove (NDS), and a forced draft gasifier stove (FDGS), in order of increasing modified combustion efficiency (MCE) and decreasing particulate matter emission factors (EF). SOA production was observed for all stoves/tests; organic aerosol (OA) enhancement factor ranged from 1.2 to 3.1, decreasing with increased MCE. In primary emissions, OA mass spectral fragments associated with oxygenated species (primary biomass burning markers) increased (decreased) with MCE; fresh OA from FDGS combustion was especially oxygenated. OA oxygenation increased with further oxidation for all stove emissions, even where minimal enhancement was observed. More efficient stoves emit particles with greater net direct specific warming than TSFs, with the difference increasing with aging. Our results show that the properties and evolution of cookstove emissions are a strong function of combustion efficiency and atmospheric aging.

Macrophage arginase-1 controls bacterial growth and pathology in hypoxic tuberculosis granulomas.

Lung granulomas develop upon Mycobacterium tuberculosis (Mtb) infection as a hallmark of human tuberculosis (TB). They are structured aggregates consisting mainly of Mtb-infected and -uninfected macrophages and Mtb-specific T cells. The production of NO by granuloma macrophages expressing nitric oxide synthase-2 (NOS2) via l-arginine and oxygen is a key protective mechanism against mycobacteria. Despite this protection, TB granulomas are often hypoxic, and bacterial killing via NOS2 in these conditions is likely suboptimal. Arginase-1 (Arg1) also metabolizes l-arginine but does not require oxygen as a substrate and has been shown to regulate NOS2 via substrate competition. However, in other infectious diseases in which granulomas occur, such as leishmaniasis and schistosomiasis, Arg1 plays additional roles such as T-cell regulation and tissue repair that are independent of NOS2 suppression. To address whether Arg1 could perform similar functions in hypoxic regions of TB granulomas, we used a TB murine granuloma model in which NOS2 is absent. Abrogation of Arg1 expression in macrophages in this setting resulted in exacerbated lung granuloma pathology and bacterial burden. Arg1 expression in hypoxic granuloma regions correlated with decreased T-cell proliferation, suggesting that Arg1 regulation of T-cell immunity is involved in disease control. Our data argue that Arg1 plays a central role in the control of TB when NOS2 is rendered ineffective by hypoxia.

Neonatal Fc Receptor Regulation of Lung Immunoglobulin and CD103+ Dendritic Cells Confers Transient Susceptibility to Tuberculosis.

The neonatal Fc receptor (FcRn) extends the systemic half-life of IgG antibodies by chaperoning bound Fc away from lysosomal degradation inside stromal and hematopoietic cells. FcRn also transports IgG across mucosal barriers into the lumen, and yet little is known about how FcRn modulates immunity in the lung during homeostasis or infection. We infected wild-type (WT) and FcRn-deficient (fcgrt(-/-)) mice with Pseudomonas aeruginosa or Mycobacterium tuberculosis to investigate whether recycling and transport of IgG via FcRn influences innate and adaptive immunity in the lung in response to bacterial infection. We found that FcRn expression maintains homeostatic IgG levels in lung and leads to preferential secretion of low-affinity IgG ligands into the lumen. Fcgrt(-/-) animals exhibited no evidence of developmental impairment of innate immunity in the lung and were able to efficiently recruit neutrophils in a model of acute bacterial pneumonia. Although local humoral immunity in lung increased independently of the presence of FcRn during tuberculosis, there was nonetheless a strong impact of FcRn deficiency on local adaptive immunity. We show that the quantity and quality of IgG in airways, as well as the abundance of dendritic cells in the lung, are maintained by FcRn. FcRn ablation transiently enhanced local T cell immunity and neutrophil recruitment during tuberculosis, leading to a lower bacterial burden in lung. This novel understanding of tissue-specific modulation of mucosal IgG isotypes in the lung by FcRn sheds light on the role of mucosal IgG in immune responses in the lung during homeostasis and bacterial disease.

Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics.

General interest in the biological functions of IFN type I in Mycobacterium tuberculosis (Mtb) infection increased after the recent identification of a distinct IFN gene expression signature in tuberculosis (TB) patients. Here, we demonstrate that TB-susceptible mice lacking the receptor for IFN I (IFNAR1) were protected from death upon aerogenic infection with Mtb. Using this experimental model to mimic primary progressive pulmonary TB, we dissected the immune processes affected by IFN I. IFNAR1 signaling did not affect T-cell responses, but markedly altered migration of inflammatory monocytes and neutrophils to the lung. This process was orchestrated by IFNAR1 expressed on both immune and tissue-resident radioresistant cells. IFNAR1-driven TB susceptibility was initiated by augmented Mtb replication and in situ death events, along with CXCL5/CXCL1-driven accumulation of neutrophils in alveoli, followed by the discrete compartmentalization of Mtb in lung phagocytes. Early depletion of neutrophils rescued TB-susceptible mice to levels observed in mice lacking IFNAR1. We conclude that IFN I alters early innate events at the site of Mtb invasion leading to fatal immunopathology. These data furnish a mechanistic explanation for the detrimental role of IFN I in pulmonary TB and form a basis for understanding the complex roles of IFN I in chronic inflammation.

For better or for worse: the immune response against Mycobacterium tuberculosis balances pathology and protection.

Tuberculosis (TB) is a complex disease, and the success of the bacterium as an intracellular pathogen is the outcome of its close and longstanding coevolution with the mammalian host. The dialogue between Mycobacterium tuberculosis and the host is becoming understandable at the molecular, cellular, and tissue level. This has led to the elucidation of the (i) interaction between pattern recognition receptors and pathogen-associated molecular patterns, (ii) cross-talk between immune cells, and (iii) mechanisms underlying granuloma development. Disease as an eventual but not a necessary consequence of infection results from a sensitive balance between protective immunity and destructive pathology. Early events, governed largely by conserved mechanisms of host recognition, impact not only on type and course of adaptive immunity but also on lung parenchymal function. New interpretations of how these responses shape the lung environment and direct granuloma development emphasize that the disease results from pathologic consequences of non-resolving inflammation. We review recent advances in TB research within the context of this ambitious view of TB.

Toward a molecular microbial blood test for tuberculosis infection.

The World Health Organization's aim to end the global tuberculosis (TB) epidemic by 2050 cannot be achieved without taking measures to identify people with asymptomatic Mycobacterium tuberculosis (Mtb) infection and offer them an intervention to reduce the risk of disease progression, such as preventive antimicrobial therapy. Implementation of this strategy is limited by the fact that existing tests for Mtb infection, which use immunosensitization to Mtb-specific antigens as a proxy for infection, have low positive predictive value for progression to TB. A blood test that detects Mtb deoxyribonucleic acid (DNA) could allow preventive therapy to be targeted at individuals with microbiological evidence of persistent infection. In this review, we summarize recent advances in the development of molecular microbial blood tests for Mtb infection and discuss potential explanations for discordance between their results and those of immunodiagnostic tests in adults with recent exposure to an infectious index case. We also present a roadmap for further development of molecular microbial blood tests for Mtb infection, and highlight the potential for research in this area to provide novel insights into the biology of Mtb infection and yield new tools to support efforts to control the global TB epidemic.

Combining machine learning with high-content imaging to infer ciprofloxacin susceptibility in isolates of Salmonella Typhimurium.

Antimicrobial resistance (AMR) is a growing public health crisis that requires innovative solutions. Current susceptibility testing approaches limit our ability to rapidly distinguish between antimicrobial-susceptible and -resistant organisms. Salmonella Typhimurium (S. Typhimurium) is an enteric pathogen responsible for severe gastrointestinal illness and invasive disease. Despite widespread resistance, ciprofloxacin remains a common treatment for Salmonella infections, particularly in lower-resource settings, where the drug is given empirically. Here, we exploit high-content imaging to generate deep phenotyping of S. Typhimurium isolates longitudinally exposed to increasing concentrations of ciprofloxacin. We apply machine learning algorithms to the imaging data and demonstrate that individual isolates display distinct growth and morphological characteristics that cluster by time point and susceptibility to ciprofloxacin, which occur independently of ciprofloxacin exposure. Using a further set of S. Typhimurium clinical isolates, we find that machine learning classifiers can accurately predict ciprofloxacin susceptibility without exposure to it or any prior knowledge of resistance phenotype. These results demonstrate the principle of using high-content imaging with machine learning algorithms to predict drug susceptibility of clinical bacterial isolates. This technique may be an important tool in understanding the morphological impact of antimicrobials on the bacterial cell to identify drugs with new modes of action.

Activation of the NLRP3 inflammasome by Mycobacterium tuberculosis is uncoupled from susceptibility to active tuberculosis.

As a hallmark of tuberculosis (TB), Mycobacterium tuberculosis (MTB) induces granulomatous lung lesions and systemic inflammatory responses during active disease. Molecular regulation of inflammation is associated with inflammasome assembly. We determined the extent to which MTB triggers inflammasome activation and how this impacts on the severity of TB in a mouse model. MTB stimulated release of mature IL-1ß in macrophages while attenuated M. bovis BCG failed to do so. Tubercle bacilli specifically activated the NLRP3 inflammasome and this propensity was strictly controlled by the virulence-associated RD1 locus of MTB. However, Nlrp3-deficient mice controlled pulmonary TB, a feature correlated with NLRP3-independent production of IL-1ß in infected lungs. Our studies demonstrate that MTB activates the NLRP3 inflammasome in macrophages in an ESX-1-dependent manner. However, during TB, MTB promotes NLRP3- and caspase-1-independent IL-1ß release in myeloid cells recruited to lung parenchyma and thus overcomes NLRP3 deficiency in vivo in experimental models.

Intranasal immunization with outer membrane vesicles (OMV) protects against airway colonization and systemic infection with Acinetobacter baumannii.

OBJECTIVES: The multidrug-resistant bacteria Acinetobacter baumannii is a major cause of hospital-associated infection; a vaccine could significantly reduce this burden. The aim was to develop a clinically relevant model of A. baumannii respiratory tract infection and to test the impact of different immunization routes on protective immunity provided by an outer membrane vesicle (OMV) vaccine. METHODS: BALB/c mice were intranasally challenged with isolates of oxa23-positive global clone GC2 A. baumannii from the lungs of patients with ventilator-associated pneumonia. Mice were immunized with OMVs by the intramuscular, subcutaneous or intranasal routes; protection was determined by measuring local and systemic bacterial load. RESULTS: Infection with A. baumannii clinical isolates led to a more disseminated infection than the prototype A. baumannii strain ATCC17978; with bacteria detectable in upper and lower airways and the spleen. Intramuscular immunization induced an antibody response but did not protect against bacterial infection. However, intranasal immunization significantly reduced airway colonization and prevented systemic bacterial dissemination. CONCLUSIONS: Use of clinically relevant isolates of A. baumannii provides stringent model for vaccine development. Intranasal immunization with OMVs was an effective route for providing protection, demonstrating that local immunity is important in preventing A. baumannii infection.

Determinants of QuantiFERON Plus-diagnosed tuberculosis infection in adult Ugandan TB contacts: A cross-sectional study.

BACKGROUND: The tuberculin skin test is commonly used to diagnose latent tuberculosis infection (LTBI) in resource-limited settings, but its specificity is limited by factors including cross-reactivity with BCG vaccine and environmental mycobacteria. Interferon-gamma release assays (IGRA) overcome this problem by detecting M. tuberculosis complex-specific responses, but studies to determine risk factors for IGRA-positivity in high TB burden settings are lacking. METHODS: We conducted a cross-sectional study to determine factors associated with a positive IGRA by employing the QuantiFERON-TB® Gold-plus (QFT Plus) assay in a cohort of asymptomatic adult TB contacts in Kampala, Uganda. Multivariate logistic regression analysis with forward stepwise logit function was employed to identify independent correlates of QFT Plus-positivity. RESULTS: Of the 202 participants enrolled, 129/202 (64%) were female, 173/202 (86%) had a BCG scar, and 67/202 (33%) were HIV-infected. Overall, 105/192 (54%, 95% CI 0.48-0.62) participants had a positive QFT Plus result. Increased risk of QFT-Plus positivity was independently associated with casual employment/unemployment vs. non-casual employment (adjusted odds ratio (aOR) 2.18, 95% CI 1.01-4.72), a family vs. non-family relation to the index patient (aOR 2.87, 95% CI 1.33-6.18), living in the same vs. a different house as the index (aOR 3.05, 95% CI 1.28-7.29), a higher body mass index (BMI) (aOR per additional kg/m2 1.09, 95% CI 1.00-1.18) and tobacco smoking vs. not (aOR 2.94, 95% CI 1.00-8.60). HIV infection was not associated with QFT-Plus positivity (aOR 0.91, 95% CI 0.42-1.96). CONCLUSION: Interferon Gamma Release Assay positivity in this study population was lower than previously estimated. Tobacco smoking and BMI were determinants of IGRA positivity that were previously unappreciated.

Clinical and laboratory factors associated with neonatal sepsis mortality at a major Vietnamese children's hospital.

Sepsis is a major cause of neonatal mortality and children born in low- and middle-income countries (LMICs) are at greater risk of severe neonatal infections than those in higher-income countries. Despite this disparity, there are limited contemporaneous data linking the clinical features of neonatal sepsis with outcome in LMICs. Here, we aimed to identify factors associated with mortality from neonatal sepsis in Vietnam. We conducted a prospective, observational study to describe the clinical features, laboratory characteristics, and mortality rate of neonatal sepsis at a major children's hospital in Ho Chi Minh City. All in-patient neonates clinically diagnosed with probable or culture-confirmed sepsis meeting inclusion criteria from January 2017 to June 2018 were enrolled. We performed univariable analysis and logistic regression to identify factors independently associated with mortality. 524 neonates were recruited. Most cases were defined as late-onset neonatal sepsis and were hospital-acquired (91.4% and 73.3%, respectively). The median (IQR) duration of hospital stay was 23 (13-41) days, 344/524 (65.6%) had a positive blood culture (of which 393 non-contaminant organisms were isolated), and 69/524 (13.2%) patients died. Coagulase-negative staphylococci (232/405; 57.3%), Klebsiella spp. (28/405; 6.9%), and Escherichia coli (27/405; 6.7%) were the most isolated organisms. Sclerema (OR = 11.4), leukopenia <4,000/mm3 (OR = 7.8), thrombocytopenia <100,000/mm3 (OR = 3.7), base excess < -20 mEq/L (OR = 3.6), serum lactate >4 mmol/L (OR = 3.4), extremely low birth weight (OR = 3.2), and hyperglycaemia >180 mg/dL (OR = 2.6) were all significantly (p<0.05) associated with mortality. The identified risk factors can be adopted as prognostic factors for the diagnosis and treatment of neonatal sepsis and enable early risk stratification and interventions appropriate to reduce neonatal sepsis in LMIC settings.

Secondary lymphoid organs are dispensable for the development of T-cell-mediated immunity during tuberculosis.

Tuberculosis causes 2 million deaths per year, yet in most cases the immune response successfully contains the infection and prevents disease outbreak. Induced lymphoid structures associated with pulmonary granuloma are observed during tuberculosis in both humans and mice and could orchestrate host defense. To investigate whether granuloma perform lymphoid functions, mice lacking secondary lymphoid organs (SLO) were infected with Mycobacterium tuberculosis (MTB). As in WT mice, granuloma developed, exponential growth of MTB was controlled, and antigen-specific T-cell responses including memory T cells were generated in the absence of SLO. Moreover, adoptively transferred T cells were primed locally in lungs in a granuloma-dependent manner. T-cell activation was delayed in the absence of SLO, but resulted in a normal development program including protective subsets and functional recall responses that protected mice against secondary MTB infection. Our data demonstrate that protective immune responses can be generated independently of SLO during MTB infection and implicate local pulmonary T-cell priming as a mechanism contributing to host defense.

Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study.

BACKGROUND: Haematopoietic stem cells expressing the CD34 surface marker have been posited as a niche for Mycobacterium tuberculosis complex bacilli during latent tuberculosis infection. Our aim was to determine whether M tuberculosis complex DNA is detectable in CD34-positive peripheral blood mononuclear cells (PBMCs) isolated from asymptomatic adults living in a setting with a high tuberculosis burden. METHODS: We did a cross-sectional study in Ethiopia between Nov 22, 2017, and Jan 10, 2019. Digital PCR (dPCR) was used to determine whether M tuberculosis complex DNA was detectable in PBMCs isolated from 100 mL blood taken from asymptomatic adults with HIV infection or a history of recent household or occupational exposure to an index case of human or bovine tuberculosis. Participants were recruited from HIV clinics, tuberculosis clinics, and cattle farms in and around Addis Ababa. A nested prospective study was done in a subset of HIV-infected individuals to evaluate whether administration of isoniazid preventive therapy was effective in clearing M tuberculosis complex DNA from PBMCs. Follow-up was done between July 20, 2018, and Feb 13, 2019. QuantiFERON-TB Gold assays were also done on all baseline and follow-up samples. FINDINGS: Valid dPCR data (ie, droplet counts >10 000 per well) were available for paired CD34-positive and CD34-negative PBMC fractions from 197 (70%) of 284 participants who contributed data to cross-sectional analyses. M tuberculosis complex DNA was detected in PBMCs of 156 of 197 participants with valid dPCR data (79%, 95% CI 74-85). It was more commonly present in CD34-positive than in CD34-negative fractions (154 [73%] of 197 vs 46 [23%] of 197; p<0·0001). Prevalence of dPCR-detected M tuberculosis complex DNA did not differ between QuantiFERON-negative and QuantiFERON-positive participants (77 [78%] of 99 vs 79 [81%] of 98; p=0·73), but it was higher in HIV-infected than in HIV-uninfected participants (67 [89%] of 75 vs 89 [73%] of 122, p=0·0065). By contrast, the proportion of QuantiFERON-positive participants was lower in HIV-infected than in HIV-uninfected participants (25 [33%] of 75 vs 73 [60%] of 122; p<0·0001). Administration of isoniazid preventive therapy reduced the prevalence of dPCR-detected M tuberculosis complex DNA from 41 (95%) of 43 HIV-infected individuals at baseline to 23 (53%) of 43 after treatment (p<0·0001), but it did not affect the prevalence of QuantiFERON positivity (17 [40%] of 43 at baseline vs 13 [30%] of 43 after treatment; p=0·13). INTERPRETATION: We report a novel molecular microbiological biomarker of latent tuberculosis infection with properties that are distinct from those of a commercial interferon-γ release assay. Our findings implicate the bone marrow as a niche for M tuberculosis in latently infected individuals. Detection of M tuberculosis complex DNA in PBMCs has potential applications in the diagnosis of latent tuberculosis infection, in monitoring response to preventive therapy, and as an outcome measure in clinical trials of interventions to prevent or treat latent tuberculosis infection. FUNDING: UK Medical Research Council.

Combination of host susceptibility and Mycobacterium tuberculosis virulence define gene expression profile in the host.

Progression and outcome of tuberculosis is governed by extensive crosstalk between pathogen and host. Analyses of global changes in gene expression during immune response to infection with Mycobacterium tuberculosis (M.tb) can help identify molecular markers of disease state and progression. Global distribution of M.tb strains with different degrees of virulence and drug resistance, especially for the immunocompromised host, make closer analyses of host responses more pressing than ever. Here, we describe global transcriptional responses of inducible nitric oxide synthase-deficient (iNOS(-/-)) and WT mice infected with two related M.tb strains of markedly different virulence, namely the M.tb laboratory strains H37Rv and H37Ra. Both hosts exhibited highly similar resistance to infection with H37Ra. In contrast, iNOS(-/-) mice rapidly succumbed to H37Rv, whereas WT mice developed chronic course of disease. By differential analyses, virulence-specific changes in global host gene expression were analyzed to identify molecular markers characteristic for chronic versus acute infection. We identified several markers unique for different stages of disease progression and not previously associated with virulence-specific host responses in tuberculosis.

Detection of Mycobacterium tuberculosis DNA in CD34 + peripheral blood mononuclear cells of Ugandan adults with latent infection: a cross-sectional and nested prospective study.

Background: Tuberculin skin test and interferon gamma release assay (IGRA) show limitations in diagnosing latent tuberculosis infection (LTBI) and poorly predict progression to active tuberculosis. This study will explore detection of Mycobacterium tuberculosis ( M.tb) DNA in CD34 + peripheral blood mononuclear cells (PBMCs) as a biomarker for LTBI and monitoring chemoprophylaxis response. Methods: In a cross-sectional study, 120 household contacts (60 HIV positive and 60 HIV negative) will be recruited. Also, 10 patients with sputum positive pulmonary tuberculosis and 10 visitors from low incidence countries with no history of TB treatment will be recruited as positive and negative controls, respectively. Participants will donate 100 ml (50 ml for TB patients) of blood to isolate PBMCs using density gradient centrifugation. Isolated PBMCs will be separated into CD34 + and CD34 - enriched cellular fractions. DNA from each fraction will be purified, quantified and subjected to droplet digital PCR targeting IS6110 (a M.tb Complex multi-copy gene) and rpoB, a single copy gene. Also, 4 ml of blood will be drawn for IGRA. In a nested prospective study, 60 HIV positive participants will be given 300 mg of Isoniazid Preventive Therapy (IPT) daily for six months, after which they will donate a second 100 ml blood sample that will be processed as described above. Data from the cross-sectional study will be analysed to determine the proportion of individuals in whom M.tb DNA is detectable in CD34 + and CD34 - fractions and number of M.tb genomes present. Data from the prospective study will be analysed to compare the proportion of individuals with detectable M.tb DNA in CD34 + and CD34 - fractions, and median M.tb genome copy number, post vs pre-IPT. Discussion: This study will determine whether detection of M.tb DNA in CD34 + PBMCs holds promise as a biomarker for LTBI and monitoring chemoprophylaxis response.