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Mycobacterium tuberculosis (Mtb) exposure leads to a range of outcomes including clearance, latent TB infection (LTBI), and pulmonary tuberculosis (TB). Some heavily exposed individuals resist tuberculin skin test (TST) and interferon-gamma (IFNγ) release assay (IGRA) conversion (RSTR), which suggests that they employ IFNγ-independent mechanisms of Mtb control. Here, we compare monocyte epigenetic profiles of RSTR and LTBI from a Ugandan household contact cohort. Chromatin accessibility did not differ between uninfected RSTR and LTBI monocytes. By contrast, methylation significantly differed at 174 CpG sites and across 63 genomic regions. Consistent with previous transcriptional findings in this cohort, differential methylation was enriched in lipid- and cholesterol-associated pathways including the genes APOC3, KCNQ1, and PLA2G3. In addition, methylation was enriched in Hippo signaling, which is associated with cholesterol homeostasis and includes CIT and SHANK2. Lipid export and Hippo signaling pathways were also associated with gene expression in response to Mtb in RSTR as well as IFN stimulation in monocyte-derived macrophages (MDMs) from an independent healthy donor cohort. Moreover, serum-derived high-density lipoprotein from RSTR had elevated ABCA1-mediated cholesterol efflux capacity (CEC) compared to LTBI. Our findings suggest that resistance to TST/IGRA conversion is linked to regulation of lipid accumulation in monocytes, which could facilitate early Mtb clearance among RSTR subjects through IFNγ-independent mechanisms.IMPORTANCETuberculosis (TB) remains an enduring global health challenge with millions of deaths and new cases each year. Despite recent advances in TB treatment, we lack an effective vaccine or a durable cure. While heavy exposure to Mycobacterium tuberculosis often results in latent TB latent infection (LTBI), subpopulations exist that are either resistant to infection or contain Mtb with interferon-gamma (IFNγ)-independent mechanisms not indicative of LTBI. These resisters provide an opportunity to investigate the mechanisms of TB disease and discover novel therapeutic targets. Here, we compare monocyte epigenetic profiles of RSTR and LTBI from a Ugandan household contact cohort. We identify methylation signatures in host lipid and cholesterol pathways with potential relevance to early TB clearance before the sustained IFN responses indicative of LTBI. This adds to a growing body of literature linking TB disease outcomes to host lipids., Competing Interests: The authors declare no conflict of interest.

Female and latent genital tuberculosis (FGTB and LGTB) in young women may lead to infertility by damaging ovarian reserve function, but the regulatory mechanisms remain unclear. In this study, we investigated the effects of FGTB and LGTB on ovarian reserve function and potential regulatory mechanisms by untargeted metabolomics of follicular fluid, aiming to provide insights for the clinical management and treatment approaches for afflicted women. We recruited 19 patients with FGTB, 16 patients with LGTB, and 16 healthy women as a control group. Clinical data analysis revealed that both the FGTB and LGTB groups had significantly lower ovarian reserve marker levels compared to the control group, including lower anti-Müllerian hormone levels (FGTB: 0.82 [0.6, 1.1] μg/L; LGTB: 1.57 [1.3, 1.8] μg/L vs. control: 3.29 [2.9, 3.5] μg/L), reduced antral follicular counts (FGTB: 6 [5.5, 9.5]; LGTB: 10.5 [7, 12.3] vs. control: 17 [14.5, 18]), and fewer retrieved oocytes (FGTB: 3 [2, 5]; LGTB: 8 [4, 8.3] vs. control: 14.5 [11.5, 15.3]). Conversely, these groups exhibited higher ovarian response marker levels, such as longer gonadotropin treatment days (FGTB: 12 [10.5, 12.5]; LGTB: 11 [10.8, 11.3] vs. control: 10 [8.8, 10]) and increased gonadotropin dosage requirements (FGTB: 3300 [3075, 3637.5] U; LGTB: 3037.5 [2700, 3225] U vs. control: 2531.25 [2337.5, 2943.8] U). All comparisons were statistically significant at P < 0.05. The results suggested that FGTB and LGTB have adverse effects on ovarian reserve and response. Untargeted metabolomic analysis identified 92 and 80 differential metabolites in the control vs. FGTB and control vs. LGTB groups, respectively. Pathway enrichment analysis revealed significant alterations in metabolic pathways in the FGTB and LGTB groups compared to the control group (P < 0.05), with specific changes noted in galactose metabolism, biotin metabolism, steroid hormone biosynthesis, and nicotinate and nicotinamide metabolism in the FGTB group, and caffeine metabolism, primary bile acid biosynthesis, steroid hormone biosynthesis, and glycerophospholipid metabolism in the LGTB group. The analysis of metabolic levels has revealed the potential mechanisms by which FGTB and LGTB affect ovarian reserve function, namely through alterations in metabolic pathways. The study emphasizes the importance of comprehending the metabolic alterations associated with FGTB and LGTB, which is of considerable relevance for the clinical management and therapeutic approaches in afflicted women., (© 2024. The Author(s).)

During tuberculosis (TB) infection, B-lymphocytes migrate to the lungs and form B-cell follicles (BCFs) in the vicinity of TB granulomata. B-cell-lacking mice display enhanced susceptibility to TB infection, and early B-cell depletion in infected non-human primates alters T-lymphocyte cytokine responses and increases bacterial burdens in the lungs. However, the role of B cells during late TB stages remained unaddressed. Here, we demonstrate that B cells and BCFs persist up to weeks 25-45 post-challenge in the lungs of TB-resistant C57BL/6 (B6) mice. In hyper-susceptible I/St mice, B-cell content markedly drops between weeks 12-16 post-infection, paralleled by diffuse lung tissue inflammation and elevated gene expression levels for pro-inflammatory cytokines IL-1, IL-11, IL-17a, and TNF-α. To check whether B-cells/BCFs control TB infection at advanced stages, we specifically depleted B-cells from B6 mice by administrating anti-CD20 mAbs at week 16 post-infection. This resulted in more rapid cachexia, a shortened lifespan of the infected animals, an increase in (i) lung-infiltrating CD8 + T cells, (ii) IL-6 production by F4/80 + macrophages, (iii) expression levels of genes for neutrophil-attracting factors CXCL1 and IL-17, and tissue-damaging factors MMP8, MMP9, and S100A8. Taken together, our results suggest that lung B cells and BCFs are moderately protective against chronic TB infection.

Studies using the nonhuman primate model of Mycobacterium tuberculosis/simian immunodeficiency virus coinfection have revealed protective CD4+ T cell-independent immune responses that suppress latent tuberculosis infection (LTBI) reactivation. In particular, chronic immune activation rather than the mere depletion of CD4+ T cells correlates with reactivation due to SIV coinfection. Here, we administered combinatorial antiretroviral therapy (cART) 2 weeks after SIV coinfection to study whether restoration of CD4+ T cell immunity occurred more broadly, and whether this prevented reactivation of LTBI compared to cART initiated 4 weeks after SIV. Earlier initiation of cART enhanced survival, led to better control of viral replication, and reduced immune activation in the periphery and lung vasculature, thereby reducing the rate of SIV-induced reactivation. We observed robust CD8+ T effector memory responses and significantly reduced macrophage turnover in the lung tissue. However, skewed CD4+ T effector memory responses persisted and new TB lesions formed after SIV coinfection. Thus, reactivation of LTBI is governed by very early events of SIV infection. Timing of cART is critical in mitigating chronic immune activation. The potential novelty of these findings mainly relates to the development of a robust animal model of human M. tuberculosis/HIV coinfection that allows the testing of underlying mechanisms.

The PD-1/PD-L1 pathway is critical in T cell biology; however, the role of the PD-1/PD-L1 pathway in clinical characteristics and treatment outcomes in pulmonary tuberculosis (PTB) patients is unclear. We prospectively enrolled PTB, latent TB infection (LTBI), and non-TB, non-LTBI subjects. The expression of PD-1/PD-L1 on peripheral blood mononuclear cells (PBMCs) was measured and correlated with clinical characteristics and treatment outcomes in PTB patients. Immunohistochemistry and immunofluorescence were used to visualize PD-1/PD-L1-expressing cells in lung tissues from PTB patients and from murine with heat-killed MTB (HK-MTB) treatment. A total of 76 PTB, 40 LTBI, and 28 non-TB, non-LTBI subjects were enrolled. The expression of PD-1 on CD4+ T cells and PD-L1 on CD14+ monocytes was significantly higher in PTB cases than non-TB subjects. PTB patients with sputum smear/culture unconversion displayed higher PD-L1 expression on monocytes. PD-L1-expressing macrophages were identified in lung tissue from PTB patients, and co-localized with macrophages in murine lung tissues. Mycobacterium tuberculosis (MTB) whole cell lysate/EsxA stimulation of human and mouse macrophages demonstrated increased PD-L1 expression. In conclusion, increased expression of PD-L1 on monocytes in PTB patients correlated with higher bacterial burden and worse treatment outcomes. The findings suggest the involvement of the PD-1/PD-L1 pathway in MTB-related immune responses.

Latent tuberculosis infection (LTBI) represents a major challenge to curing TB disease. Current guidelines for LTBI management include only three older drugs and their combinations-isoniazid and rifamycins (rifampicin and rifapentine). These available control strategies have little impact on latent TB elimination, and new specific therapeutics are urgently needed. In the present mini-review, we highlight some of the alternatives that may potentially be included in LTBI treatment recommendations and a list of early-stage prospective small molecules that act on drug targets specific for Mycobacterium tuberculosis latency.

Accurate detection and risk stratification of latent tuberculosis infection (LTBI) remains a major clinical and public health problem. We hypothesize that multiparameter strategies that probe immune responses to Mycobacterium tuberculosis can provide new diagnostic insights into not only the status of LTBI infection, but also the risk of reactivation. After the initial proof-of-concept study, we developed a 13-plex immunoassay panel to profile cytokine release from peripheral blood mononuclear cells stimulated separately with Mtb-relevant and non-specific antigens to identify putative biomarker signatures. We sequentially enrolled 65 subjects with various risk of TB exposure, including 32 subjects with diagnosis of LTBI. Random Forest feature selection and statistical data reduction methods were applied to determine cytokine levels across different normalized stimulation conditions. Receiver Operator Characteristic (ROC) analysis for full and reduced feature sets revealed differences in biomarkers signatures for LTBI status and reactivation risk designations. The reduced set for increased risk included IP-10, IL-2, IFN-γ, TNF-α, IL-15, IL-17, CCL3, and CCL8 under varying normalized stimulation conditions. ROC curves determined predictive accuracies of > 80% for both LTBI diagnosis and increased risk designations. Our study findings suggest that a multiparameter diagnostic approach to detect normalized cytokine biomarker signatures might improve risk stratification in LTBI., (© 2021. The Author(s).)

Tuberculosis (TB) remains a prominent health concern worldwide. Besides extensive research and vaccinations available, attempts to control the pandemic are cumbersome due to the complex physiology of Mycobacterium tuberculosis (Mtb). Alongside the emergence of drug-resistant TB, latent TB has worsened the condition. The tubercle bacilli are unusually behaved and successful with its strategies to modulate genes to evade host immune system and persist within macrophages. Under latent/unfavorable conditions, Mtb conceals itself from immune system and modulates its genes. Among many intracellular modulated genes, important are those involved in cell entry, fatty acid degradation, mycolic acid synthesis, phagosome acidification inhibition, inhibition of phagosome-lysosome complex and chaperon protein modulation. Though the study on these genes date back to early times of TB, an insight on their inter-relation within and to newly evolved genes are still required. This review focuses on the findings and discussions on these genes, possible mechanism, credibility as target for novel drugs and repurposed drugs and their interaction that enables Mtb in survival, pathogenesis, resistance and latency., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

After extensive exposure to Mycobacterium tuberculosis (Mtb), most individuals acquire latent Mtb infection (LTBI) defined by a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA). To identify mechanisms of resistance to Mtb infection, we compared transcriptional profiles from highly exposed contacts who resist TST/IGRA conversion (resisters, RSTRs) and controls with LTBI using RNAseq. Gene sets related to carbon metabolism and free fatty acid (FFA) transcriptional responses enriched across 2 independent cohorts suggesting RSTR and LTBI monocytes have distinct activation states. We compared intracellular Mtb replication in macrophages treated with FFAs and found that palmitic acid (PA), but not oleic acid (OA), enhanced Mtb intracellular growth. This PA activity correlated with its inhibition of proinflammatory cytokines in Mtb-infected cells. Mtb growth restriction in PA-treated macrophages was restored by activation of AMP kinase (AMPK), a central host metabolic regulator known to be inhibited by PA. Finally, we genotyped AMPK variants and found 7 SNPs in PRKAG2, which encodes the AMPK-γ subunit, that strongly associated with RSTR status. Taken together, RSTR and LTBI phenotypes are distinguished by FFA transcriptional programs and by genetic variation in a central metabolic regulator, which suggests immunometabolic pathways regulate TST/IGRA conversion.

Mycobacterium tuberculosis (MTB) is one of the most threatening pathogens for its latent infection in macrophages. The intracellular MTB isolated itself from drugs and could spread via macrophages. Therefore, a mannose-modified macrophage-targeting solid lipid nanoparticle, MAN-IC-SLN, loading the pH-sensitive prodrug of isoniazid (INH), was designed to treat the latent tuberculosis infection. The surface of SLNs was modified by a synthesized 6-octadecylimino-hexane-1,2,3,4,5-pentanol (MAN-SA) to target macrophages, and the modified SLNs showed a higher cell uptake in macrophages (97.2%) than unmodified SLNs (42.4%). The prodrug, isonicotinic acid octylidene-hydrazide (INH-CHO), was synthesized to achieve the pH-sensitive release of INH in macrophages. The INH-CHO-loaded SLNs exhibited a pH-sensitive release profile and accomplished a higher accumulated release in pH 5.5 media (82.63 ± 2.12%) compared with the release in pH 7.4 media (58.83 ± 3.84%). Mycobacterium smegmatis was used as a substitute for MTB, and the MAN-IC-SLNs showed a fourfold increase of intracellular antibiotic efficacy and enhanced macrophage uptake because of the pH-sensitive degradation of INH-CHO and MAN-SA in SLNs, respectively. For the in vivo antibiotic efficacy test, the SLNs group displayed an 83% decrease of the colony-forming unit while the free INH group only showed a 60% decrease. The study demonstrates that macrophage targeting and pH-sensitive SLNs can be used as a promising platform for the latent tuberculosis infection. Graphical Abstract Table of contents: Macrophage-targeting and pH-sensitive solid lipid nanoparticles (SLN) were administrated to the lung via nebulization. Macrophage targeting was achieved by appropriate particle size and surface mannose modification with synthesized MAN-SA. After being swallowed by macrophages, the prodrug, Isonicotinic acid octylidene-hydrazide (INH-CHO), quickly released isoniazid, which was triggered by the intracellular acid environment. The SLNs exhibited higher intracellular antibiotic efficacy due to their macrophage-targeting and pH-sensitive properties.

Although 23% of world population is infected with Mycobacterium tuberculosis ( M. tb ), only 5-10% manifest the disease. Individuals surely exposed to M. tb that remain asymptomatic are considered potential latent TB (LTB) cases. Such asymptomatic M. tb .-exposed individuals represent a reservoir for active TB cases. Although accurate discrimination and early treatment of patients with active TB and asymptomatic M. tb .-exposed individuals are necessary to control TB, identifying those individuals at risk of developing active TB still remains a tremendous clinical challenge. This study aimed to characterize the differences in the serum metabolic profile specifically associated to active TB infected individuals or to asymptomatic M. tb .-exposed population. Interestingly, significant changes in a specific set of metabolites were shared when comparing either asymptomatic house-hold contacts of active TB patients (HHC-TB) or active TB patients (A-TB) to clinically healthy controls (HC). Furthermore, this analysis revealed statistically significant lower serum levels of aminoacids such as alanine, lysine, glutamate and glutamine, and citrate and choline in patients with A-TB, when compared to HHC-TB. The predictive ability of these metabolic changes was also evaluated. Although further validation in independent cohorts and comparison with other pulmonary infectious diseases will be necessary to assess the clinical potential, this analysis enabled the discrimination between HHC-TB and A-TB patients with an AUC value of 0.904 (confidence interval 0.81-1.00, p -value < 0.0001). Overall, the strategy described in this work could provide a sensitive, specific, and minimally invasive method that could eventually be translated into a clinical tool for TB control.

Background: Latent Tuberculosis Infection (LTBI) remains a major driver of the TB epidemic, and individuals with Human Immuno-deficiency Virus (HIV) are particularly at a heightened risk of developing LTBI. However, LTBI screening among HIV-infected individuals in resource limited setting is largely based on a negative symptom screen, which has low specificity., Methods: In a cross sectional diagnostic study, 115 HIV infected participants with a negative symptom screen will be consented and enrolled. They will be requested to donate 5 ml of blood for complete blood count (CBC) and interferon gamma release assay (IGRA) testing. In a nested prospective study, the 115 participants will be initiated on Tuberculosis Preventive Therapy and the CBC testing repeated after 3 months. In the analysis of study finding, the monocyte to lymphocyte ratio (MLR) will be derived from the dividend of the absolute monocyte and lymphocyte counts. The optimal MLR positivity cut-off for elevated or normal MLR will be the highest value of Youden's index, J (sensitivity + specificity-1). The MLR will be cross tabulated with the IGRA status to determine the sensitivity, specificity, negative and positive predictive values of the MLR. The area under the receiver operating characteristic (ROC) curve will be determined to give the overall diagnostic accuracy of MLR. The baseline and 3 month CBC will be used to determine the change in MLR, and a random effect logistic regression will be used to determine factors associated with the change in the MLR., Discussion: If positive results are realized from this study, the MLR could become an inexpensive alternative biomarker with potential to improve the specificity of the negative symptom screen in identifying individuals that should be targeted for TB preventive therapy., Competing Interests: The authors have declared that no competing interests exist.

Background: Tuberculosis (TB) is an infectious disease and its mortality rate ranks first. Latent tuberculosis infection (LTBI) means that a patient is infected with Mycobacterium tuberculosis, but has no relative clinical symptoms. It has been estimated that approximately 10% of patients with LTBI would develop into active tuberculosis. Therefore, it was urgent to search for more efficient biomarkers to discriminate LTBI from healthy population., Methods: The Luminex assay was employed to detect the quantity of cytokines secreted by mononuclear cells from peripheral blood stimulated with the ESAT6 protein among TB, LTBI and healthy controls. The cytokine profile was analyzed by principal components analysis and the receiver operating characteristic curve analysis., Results: The principal components analysis indicated that LTBI and TB were clearly separated from healthy controls, and that LTBI was also successfully differentiated from healthy controls. The cytokine profiling method to distinguish LTBI from healthy controls has a sensitivity and specificity of 100%. Nine potential biomarkers, including IL-23, IL-21, HGF, Bngf, IL-27, IL-31, IL-1β, IL-22 and IL-18, were identified, and these cytokines were considered as a potential cytokine complex for more effectively discriminating LTBI from healthy controls., Conclusion: IL-23, IL-21, HGF, Bngf, IL-27, IL-31, IL-1β, IL-22 and IL-18 were demonstrated to be the potential cytokine complex for the assessment between LTBI and healthy controls., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: With a marginally effective vaccine and no significant breakthroughs in new treatments, a sensitive and specific method to distinguish active tuberculosis from latent tuberculosis infection (LTBI) would allow for early diagnosis and limit the spread of the pathogen. The analysis of multiple cytokine profiles provides the possibility to differentiate the two diseases., Design: Systematic review and meta-analysis., Data Sources: PubMed, Cochrane Library, Clinical Key and EMBASE databases were searched on 31 December 2019., Eligibility Criteria: We included case-control studies, cohort studies and randomised controlled trials considering IFN-γ, TNF-α, IP-10, IL-2, IL-10, IL-12 and VEGF as biomarkers to distinguish active tuberculosis and LTBI., Data Extraction and Synthesis: Two students independently extracted data and assessed the risk of bias. Diagnostic OR, sensitivity, specificity, positive and negative likelihood ratios and area under the curve (AUC) together with 95% CI were used to estimate the diagnostic value., Results: Of 1315 records identified, 14 studies were considered eligible. IL-2 had the highest sensitivity (0.84, 95% CI: 0.72 to 0.92), while VEGF had the highest specificity (0.87, 95% CI: 0.73 to 0.94). The highest AUC was observed for VEGF (0.85, 95% CI: 0.81 to 0.88), followed by IFN-γ (0.84, 95% CI: 0.80 to 0.87) and IL-2 (0.84, 95% CI: 0.81 to 0.87)., Conclusion: Cytokines, such as IL-2, IFN-γ and VEGF, can be utilised as promising biomarkers to distinguish active tuberculosis from LTBI., Prospero Registration Number: CRD42020170725., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In our work, we aim to identify new candidate host biomarkers to discriminate between active TB patients (n = 28), latent infection (LTBI; n = 27) and uninfected (NoTBI; n = 42) individuals. For that, active TB patients and their contacts were recruited that donated serum and saliva samples. A multiplex assay was performed to study the concentration of different cytokines, chemokines and growth factors. Proteins with significant differences between groups were selected and logistic regression and the area under the ROC curve (AUC) was used to assess the diagnostic accuracy. The best marker combinations that discriminate active TB from NoTBI contacts were [IP-10 + IL-7] in serum and [Fractalkine + IP-10 + IL-1α + VEGF] in saliva. Best discrimination between active TB and LTBI was achieved using [IP-10 + BCA-1] in serum (AUC = 0.83) and IP-10 in saliva (p = 0.0007; AUC = 0.78). The levels of TNFα (p = 0.003; AUC = 0.73) in serum and the combination of [Fractalkine+IL-12p40] (AUC = 0.83) in saliva, were able to differentiate between NoTBI and LTBI contacts. In conclusion, different individual and combined protein markers could help to discriminate between active TB and both uninfected and latently-infected contacts. The most promising ones include [IP-10 + IL-7], [IP-10 + BCA-1] and TNFα in serum and [Fractalkine + IP-10 + IL-1α + VEGF], IP-10 and [Fractalkine+IL-12p40] in saliva., Competing Interests: A González-Fernández is co-promotor of the company NanoImmunoTech. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Interferon-γ release assays cannot differentiate latent from active tuberculosis (TB), nor identify the recently infected with increased risk of active disease. The objective of this study was to identify biomarkers of recent infection following exposure to tuberculosis, to increase the positive predictive value for incipient TB. Contacts to patients with pulmonary TB were tested repeatedly with interferon-γ release assays and flow-cytometry. Proliferative CD4 + T cell responses to purified protein derivative (PPD) and 11 M. tuberculosis antigens were analysed. The individual probability of recent and remote infection was estimated using clinical data in a novel mathematical model and compared with CD4 + responses in a prediction model. The most specific prediction of recent infection was high CD4 + proliferative responses to CFP-10 and PPD and a low CD4 + response to ESAT-6. CD4 + proliferative responses to Rec85a, Rec85b and Rv1284 were also observed in recent infection, but did not reach significance in the prediction model. CONCLUSIONS: High CD4 + proliferative responses to CFP-10 and PPD and a low response to ESAT-6 may be used as biomarkers to improve positive predictive values for recent LTBI and thus, increased risk of incipient TB. Rec85a, Rec85b and Rv1284 are also of interest to study further in this context., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

There is limited understanding of the role of host metabolism in the pathophysiology of human tuberculosis (TB). Using high-resolution metabolomics with an unbiased approach to metabolic pathway analysis, we discovered that the tryptophan pathway is highly regulated throughout the spectrum of TB infection and disease. This regulation is characterized by increased catabolism of tryptophan to kynurenine, which was evident not only in active TB disease but also in latent TB infection (LTBI). Further, we found that tryptophan catabolism is reversed with effective treatment of both active TB disease and LTBI in a manner commensurate with bacterial clearance. Persons with active TB and LTBI also exhibited increased expression of indoleamine 2,3-dioxygenase-1 (IDO-1), suggesting IDO-1 mediates observed increases in tryptophan catabolism. Together, these data indicate IDO-1-mediated tryptophan catabolism is highly preserved in the human response to Mycobacterium tuberculosis and could be a target for biomarker development as well as host-directed therapies.

Background: Latent tuberculosis infection (LTBI) is the major source of active TB and is an obstacle to the strategy of World Health Organization to end TB by 2035. In Ethiopia, there are hundreds of prisons and they are conducive settings for the transmission of TB and could serve as the sources of infection to the general public. However, there is little data on the epidemiology of TB in prisons in Ethiopia. The objective of the present study was to estimate the prevalence of LTBI and evaluate associated risk factors in prisons in East Wollega Zone in western Ethiopia., Methods: A cross-sectional design and systematic sampling technique were used to select 352 prisoners from a total of 2620 prisoners during the two months (May and June, 2019). The selected inmates were consented for their willingness to participate in the study. Thereafter, they were interviewed and 2ml of blood sample was collected from each prisoner and screened for LTBI using interferon-gamma release assay (IGRA). The data were analyzed using SPSS version 25 and logistic regression was used to model the likelihood of LTBI occurrence and to identify risk factors associated with LTBI., Results: The prevalence of LTBI was 51.2% (95% CI: 46.45-57%) and higher prevalence was recorded in males (53%) than in females (43.5%) although the difference was not significant. Prisoners whose age ≥45 years (AOR = 2.48, 95%CI, 1.04-5.9), who chewed khat (AOR = 2.27; 95% CI, 1.27-4.19), who were prisoned over a year (AOR = 1.81, 95%CI, 1.04-3.18) and who were in overcrowded pens (AOR = 1.91, 95% CI, 1.002-3.65) were at higher risk of LTBI., Conclusions: The prevalence of LTBI in prisoners in West Wollega Zone of western Ethiopia was high and could serve as sources of infection to the public. Hence optimum handling of prisoners, and regular follow up and treatment of TB cases in prisons were recommended to minimize the burden of TB in the Zone., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium tuberculosis ( M. tuberculosis ) produces a diverse range of antigenic proteins in its dormant phase. The cytokine profiles of CD4 + T cell responses, especially subsets other than Th1 type (non-Th1 type), against these latency-associated M. tuberculosis antigens such as α-crystallin (Acr), heparin-binding hemagglutinin (HBHA), and mycobacterial DNA-binding protein 1 (MDP-1) remain elusive in relation to the clinical stage of M. tuberculosis infection. In the present study, peripheral blood mononuclear cells (PBMCs) collected from different stages of M. tuberculosis -infected cases and control PBMCs were stimulated with these antigens and ESAT-6/CFP-10. Cytokine profiles of CD4 + T cells were evaluated by intracellular cytokine staining using multicolor flow cytometry. Our results demonstrate that Th1 cytokine responses were predominant after TB onset independent of the type of antigen stimulation. On the contrary, non-Th1 cytokine responses were preferentially induced by latency-associated M. tuberculosis antigens, specifically IL-10 response against Acr in latent M. tuberculosis infection. From these results, we surmise a shift in the CD4 + T cell response from mixed non-Th1 to Th1 dominant type during TB progression., (Copyright © 2019 Yamashita, Oe, Kawakami, Osada-Oka, Ozeki, Terahara, Yasuda, Edwards, Tanaka, Tsunetsugu-Yokota, Matsumoto and Ariyoshi.)

Background/aim: The present study aimed to prospectively examine the usefulness of interferon-gamma (IFN-γ) release (IGR) as a biomarker in non-small-cell lung cancer patients receiving immune checkpoint inhibitor treatment (ICI-Tx)., Patients and Methods: IGR was measured using enzyme-linked immunosorbent assay at four time points: within 14 days before ICI-Tx (T1), and 8±3 (T2), 22±7 (T3), and 43±7 (T4) days after ICI-Tx., Results: Twenty-nine patients were divided into three groups based on IFN-γ levels in the IGR-positive control: Group-1 (n=8) with <10 IU/ml at T1, Group-2 (n=12) with a decrease in IFN-γ levels to <10 IU/ml at T3 and/or T4, and Group-3 (n=9) without changes in IFN-γ levels. Early progression and ICI-induced interstitial pneumonitis were frequently observed in Group-1 and Group-2, respectively. Group-3 exhibited more treatment cycles than the other groups. All three groups showed clear differences in clinical outcomes., Conclusion: IFN-γ levels could be a biomarker for ICI-Tx., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: The difference of macrophage-specific interleukin-1 beta (IL-1b) response between latent tuberculosis infection (LTBI) and active tuberculosis (TB) remains less studied., Method: We performed this prospective study and recruited active TB patients, contacts with LTBI, and uninfected contacts. The gene and protein expression of human monocyte-derived macrophage (hMDM) after ex vivo stimulation by early secretory antigenic target-6KD (ESAT-6) and tuberculin purified protein derivatives (PPD) was studied by real-time PCR and flow cytometry. The effect of caspase-1 inhibitor was also studied., Result: The IL-1b gene expression after 6 hr ESAT-6 1  μ g/ml stimulation was different among active TB patients ( n = 12), LTBI cases ( n = 12), and uninfected contacts ( n = 23) (log fold change: 0.98 ± 1.26 vs. 2.20 ± 0.96 vs. 2.20 ± 0.96, P = 0.013). The IL-1b gene expression at 24 hours was higher than that at 6 hours in LTBI cases ( n = 4) and uninfected contacts ( n = 6). After 24 hr ESAT-6 1  μ g/ml stimulation, the percentage of IL-1b-expressed hMDM was borderline lower in the active TB patients ( n = 9) than in the LTBI cases ( n = 10) (14.0 ± 11.2% vs. 31.6 ± 22.5%, P = 0.065). Compared with ESAT-6 1  μ g/ml stimulation but without the addition of caspase-1 inhibitor (CasI) (55.6 ± 16.3%), the percentage of IL-1b-positive hMDMs decreased after addition of CasI (50  μ g/ml CasI: 49.8 ± 18.2%, P = 0.078; 100  μ g/ml CasI: 46.6 ± 20.8%, P = 0.030; 150  μ g/ml CasI: 33.7 ± 15.5%, P = 0.016)., Conclusions: This study revealed that macrophage-specific IL-1b response differed among different stages of Mycobacterium tuberculosis infection. The role of IL-1b and inflammasome in the process of LTBI progressing to active TB warrants further investigation., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Meng-Rui Lee et al.)

Background: HIV-infected individuals with latent TB infection are at increased risk of developing active TB. HAART greatly reduces the incidence rate of TB in HIV-infected patients and reconstitutes Mycobacterium tuberculosis (M. tuberculosis)-specific immune response in the first 12 months of therapy. The durability of the anti-mycobacterial immune restoration after a year of HAART however remains less investigated., Method: A cross-sectional study was conducted to evaluate M. tuberculosis-specific functional immune responses in HIV/latent TB co-infected patients who were on HAART for at least 1.5 up to 9 years as compared to HAART-naïve patients. Three-hundred sixteen HIV-infected patients without active TB were screened by tuberculin skin testing for M. tuberculosis infection and peripheral blood mononuclear cells (PBMCs) were isolated from 61 HIV/latent TB co-infected patients (30 HAART-naïve and 31 HAART-treated). IFN-γ and IL-2 ELISPOT as well as CFSE cell proliferation assays were performed after stimulation with M. tuberculosis antigens PPD and ESAT-6., Result: The median frequency of PPD and ESAT-6 specific IFN-γ secreting cells was significantly higher in the HAART-treated patients as compared to HAART-naïve patients, p = 0.0021 and p = 0.0081 respectively. However, there was no significant difference in the median frequency of IL-2 secreting cells responding to PPD (p = 0.5981) and ESAT-6 (p = 0.3943) antigens between HAART-naïve and-treated groups. Both IFN-γ and IL-2 responses were independent of CD4 + T cell count regardless of the HAART status. Notably, the frequency of PPD and ESAT-6 specific IL-2 secreting cells was positively associated with CD4 + T cell proliferation while inversely correlated with duration of HAART, raising the possibility that M. tuberculosis-specific IL-2 response that promote the antigen-specific CD4 + T cell proliferation diminish with time on antiretroviral therapy in HIV/latent TB co-infected patients., Conclusion: This study shows an increased M. tuberculosis-specific IFN-γ, but not IL-2, response in HIV/latent TB co-infected patients with long-term HAART, consistent with only partial immune restoration. Future studies should, therefore, be done to prospectively define the rate and extent to which functional immune responses to M. tuberculosis are restored after long-term HAART.

Protective immunity against Mycobacterium tuberculosis is poorly understood. The role of interleukin (IL)-4, the archetypal T-helper type 2 (Th2) cytokine, in the immunopathogenesis of human tuberculosis remains unclear.Blood and/or bronchoalveolar lavage fluid (BAL) were obtained from participants with pulmonary tuberculosis (TB) (n=23) and presumed latent TB infection (LTBI) (n=22). Messenger RNA expression levels of interferon (IFN)-γ, IL-4 and its splice variant IL-4δ2 were determined by real-time PCR. The effect of human recombinant (hr)IL-4 on mycobacterial survival/containment (CFU·mL -1 ) was evaluated in M. tuberculosis- infected macrophages co-cultured with mycobacterial antigen-primed effector T-cells. Regulatory T-cell (Treg) and Th1 cytokine levels were evaluated using flow cytometry.In blood, but not BAL, IL-4 mRNA levels (p=0.02) and the IL-4/IFN-γ ratio (p=0.01) was higher in TB versus LTBI. hrIL-4 reduced mycobacterial containment in infected macrophages (p<0.008) in a dose-dependent manner and was associated with an increase in Tregs (p<0.001), but decreased CD4 + Th1 cytokine levels (CD4 + IFN-γ + p<0.001; CD4 + TNFα + p=0.01). Blocking IL-4 significantly neutralised mycobacterial containment (p=0.03), CD4 + IFNγ + levels (p=0.03) and Treg expression (p=0.03).IL-4 can subvert mycobacterial containment in human macrophages, probably via perturbations in Treg and Th1-linked pathways. These data may have implications for the design of effective TB vaccines and host-directed therapies., Competing Interests: Conflict of interest: A. Pooran has nothing to disclose. Conflict of interest: M. Davids has nothing to disclose. Conflict of interest: A. Nel has nothing to disclose. Conflict of interest: A. Shoko has nothing to disclose. Conflict of interest: J. Blackburn has nothing to disclose. Conflict of interest: K. Dheda has nothing to disclose., (Copyright ©ERS 2019.)

Tuberculosis (TB) is the ninth leading cause of death worldwide, ranking above human immunodeficiency virus. Latency is the major obstacle in the eradication of this disease. How the physiology of the pathogen changes in transition to the latent stage needs to be understood. The latent bacteria extracted from animal hosts exist in a nonculturable (NC) phase, whereas bacteria extracted from most in vitro models are culture-positive. In the present study, we observed that nitrite, up to a concentration of 5 mM, shows the growth of Mycobacterium tuberculosis (MTB) in liquid media, but this effect starts reversing at higher concentrations. At a concentration of 10 mM, nitrite induces rapid nonculturability of MTB at the aerobic stage. This noncultivable dormancy was confirmed by analyzing the characteristics of NC bacteria. Further differential gene expression analyses clearly supported the formation of a dormancy phenotype. This study will be helpful for the use of this bacillus as a dormancy model in future studies on TB latency.

Background: Rewiring cellular metabolism is important for activation of immune cells during host defense against Mycobacterium tuberculosis. Glutamine has been implicated as an immunomodulatory nutrient, but its role in the response to M. tuberculosis is unknown., Methods: We assessed expression of glutamine pathway genes in M. tuberculosis-infected macrophages and blood transcriptomic profiles of individuals with latent M. tuberculosis infection or tuberculosis. Subsequently, we studied the effect of blocking glutaminolysis on M. tuberculosis-induced cytokines. Finally, we examined whether polymorphisms in genes involved in the glutamine pathway influence M. tuberculosis-induced cytokines in a cohort of 500 individuals., Results: Glutamine pathway genes were differentially expressed in infected macrophages and patients with tuberculosis. Human peripheral blood mononuclear cells stimulated with M. tuberculosis displayed decreased cytokine (ie, interleukin 1β, interferon γ, and interleukin 17) responses when medium was devoid of glutamine. Specific inhibitors of the glutamine pathway led to decreased cytokine responses, especially T-cell cytokines (ie, interferon γ, interleukin 17, and interleukin 22). Finally, genetic polymorphisms in glutamine metabolism genes (including GLS2, SLC1A5, and SLC7A5) influenced ex vivo cytokine responses to M. tuberculosis, especially for T-cell cytokines., Conclusions: Cellular glutamine metabolism is implicated in effective host responses against M. tuberculosis. Targeting immunometabolism may represent new strategies for tuberculosis prevention and/or treatment., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

HIV infection markedly increases the likelihood of latent tuberculosis infection progressing to active TB. Information on expression of TLR-2, myeloid differentiation factor (MyD88), IL-1R- associated kinase-4 (IRAK4) and nuclear factor kappa B (NF-kB) in HIV+LTBI+ and HIV+ patients with active TB disease is limited. We found significantly higher percentages of CD14+TLR2+ cells in PBMCs of HIV+LTBI+ patients compared to HIV-LTBI+ individuals. γ-irradiated Mtb was unable to induce MyD88, IRAK4 expression and IL-1β, MCP-1, IP-10 production in HIV+LTBI+ patients. Pleural fluids from HIV+TB+ patients had low IL-1β, MCP-1, IP-10 and high IL-10, TNF-α production. γ-irradiated Mtb stimulated CD14+ cells from HIV+TB+ patients had low IL-1β, MCP-1, IP-10 production and MyD88, IRAK4 and similar NF-kB expression compared to those from of HIV-TB+ patients. Our results suggest defective MyD88, IRAK4 but not NF-kB inhibit IL-1β, MCP-1 and IP-10 production by CD14+ cells of HIV+ individuals with LTBI and active TB disease in peripheral blood and at the site of disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: A simple blood test for detecting active tuberculosis (TB) could be key to this epidemic containment, given that a large proportion of patients are unable to produce sputum for testing. Currently available interferon-γ release assays (IGRAs) are inadequate to diagnose active TB, with reported pooled sensitivity and specificity both under 81%., Objective: To explore whether cytokines/chemokines other than interferon-γ in response to long-term cell stimulation could improve the ability to distinguish between different TB infection status., Methods: We prospectively enrolled subjects with newly diagnosed pulmonary TB and their household contacts in Santiago. All contacts were tested with IGRA. Peripheral blood mononuclear cells were obtained and antigen-specific stimulated for 72 h before collecting their culture supernatants., Results: Subjects with active TB displayed markedly low cytokines/chemokines secretion upon PBMC stimulation, with lower GM-CSF being the best differentiator from IGRA(+) contacts, with 71% (95% CI 53-85) sensitivity, 86% (95% CI 65-97) specificity and AUC = 0.79 (p = 0.0003). On the other hand, when compared to the uninfected IGRA(-) contacts, higher level of IL-2 secretion was the best indicator of active TB, with 73.5% (95% CI 56-87) sensitivity, 85% (95% CI 66-96) specificity and AUC = 0.79 (p = 0.0001). No single cytokine/chemokine released upon stimulation could accurately differentiate between active TB and all TB contacts grouped together., Conclusion: GM-CSF and IL-2 provided the best yield to differentiate active TB from latent TB and from TB uninfected, respectively, with higher specificities than that reported for IGRAs. However, none of both resulted sensitive enough to be used as a stand-alone biomarker for active TB., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

With the ageing population globally, tuberculosis (TB) in older people becomes a major clinical and public health challenge. In many Asian countries, especially those located in the eastern and southeastern parts of the continent, geriatric TB is a significant problem. TB in the older patients is more difficult to diagnose in the early course of disease, and has poorer treatment outcomes, largely as increased failure and death. More drug-induced adverse reactions are also experienced by this population during TB therapy. Oxidative stress and mitochondrial dysfunction are now well recognized to be associated with the ageing process, and it is likely that the cellular and molecular perturbations interact inextricably with the immunological dysfunction biophysiologically inherent to ageing. These underlying mechanistic bases putatively contribute to the development of TB in the geriatric population and worsen the disease outcomes, especially when the TB is compounded by co-morbid conditions such as smoking and diabetes mellitus. Unravelling these mechanisms further would yield knowledge that might potentially help to prevent reactivated TB in older people, and also to better manage the established disease with drug regimens and other new therapeutic strategies. In addition, addressing the social elements associated with geriatric TB is also imperative in the relief of individual patient suffering and improvement of overall disease control., (© 2018 Asian Pacific Society of Respirology.)

Granulomas are the histopathologic hallmark of tuberculosis (TB), both in latency and active disease. Diagnostic and therapeutic strategies that specifically target granulomas have not been developed. Our objective is to develop a probe for imaging relevant immune cell populations infiltrating the granuloma. We report the binding specificity of Cyanine 3 (Cy3)-labeled cFLFLFK-PEG 12 to human leukocytes and cellular constituents within a human in vitro granuloma model. We also report use of the probe in in vivo studies using a mouse model of lung granulomatous inflammation. We found that the probe preferentially binds human neutrophils and macrophages in human granuloma structures. Inhibition studies showed that peptide binding to human neutrophils is mediated by the receptor formyl peptide receptor 1 (FPR1). Imaging the distribution of intravenously administered cFLFLFK-PEG 12 -Cy3 in the mouse model revealed probe accumulation within granulomatous inflammatory responses in the lung. Further characterization revealed that the probe preferentially associated with neutrophils and cells of the monocyte/macrophage lineage. As there is no current clinical diagnostic imaging tool that specifically targets granulomas, the use of this probe in the context of latent and active TB may provide a unique advantage over current clinical imaging probes. We anticipate that utilizing a FPR1-targeted radiopharmaceutical analog of cFLFLFK in preclinical imaging studies may greatly contribute to our understanding of granuloma influx patterns and the biological roles and consequences of FPR1-expressing cells in contributing to disease pathogenesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Accurate diagnosis and subsequent treatment of latent tuberculosis infection (LTBI) is essential for TB elimination. However, the absence of a gold standard test for diagnosing LTBI makes assessment of the true prevalence of LTBI and the accuracy of diagnostic tests challenging. Bayesian latent class models can be used to make inferences about disease prevalence and the sensitivity and specificity of diagnostic tests using data on the concordance between tests. We performed the largest meta-analysis to date aiming to evaluate the performance of tuberculin skin test (TST) and interferon-gamma release assays (IGRAs) for LTBI diagnosis in various patient populations using Bayesian latent class modelling., Methods: Systematic search of PubMeb, Embase and African Index Medicus was conducted without date and language restrictions on September 11, 2017 to identify studies that compared the performance of TST and IGRAs for LTBI diagnosis. Two IGRA methods were considered: QuantiFERON-TB Gold In Tube (QFT-GIT) and T-SPOT.TB. Studies were included if they reported 2x2 agreement data between TST and QFT-GIT or T-SPOT.TB. A Bayesian latent class model was developed to estimate the sensitivity and specificity of TST and IGRAs in various populations, including immune-competent adults, immune-compromised adults and children. A TST cut-off value of 10 mm was used for immune-competent subjects and 5 mm for immune-compromised individuals., Findings: A total of 157 studies were included in the analysis. In immune-competent adults, the sensitivity of TST and QFT-GIT were estimated to be 84% (95% credible interval [CrI] 82-85%) and 52% (50-53%), respectively. The specificity of QFT-GIT was 97% (96-97%) in non-BCG-vaccinated and 93% (92-94%) in BCG-vaccinated immune-competent adults. The estimated figures for TST were 100% (99-100%) and 79% (76-82%), respectively. T-SPOT.TB has comparable specificity (97% for both tests) and better sensitivity (68% versus 52%) than QFT-GIT in immune-competent adults. In immune-compromised adults, both TST and QFT-GIT display low sensitivity but high specificity. QFT-GIT and TST are equally specific (98% for both tests) in non-BCG-vaccinated children; however, QFT-GIT is more specific than TST (98% versus 82%) in BCG-vaccinated group. TST is more sensitive than QFT-GIT (82% versus 73%) in children., Conclusions: This study is the first to assess the utility of TST and IGRAs for LTBI diagnosis in different population groups using all available data with Bayesian latent class modelling. Our results challenge the current beliefs about the performance of LTBI screening tests, and have important implications for LTBI screening policy and practice. We estimated that the performance of IGRAs is not as reliable as previously measured in the general population. However, IGRAs are not or minimally affected by BCG and should be the preferred tests in this setting. Adoption of IGRAs in settings where BCG is widely administered will allow for a more accurate identification and treatment of LTBI.

Protein kinase G (PknG), a thioredoxin-fold-containing eukaryotic-like serine/threonine protein kinase, is a virulence factor in Mycobacterium tuberculosis , required for inhibition of phagolysosomal fusion. Here, we unraveled novel functional facets of PknG during latency-like conditions. We found that PknG mediates persistence under stressful conditions like hypoxia and abets drug tolerance. PknG mutant displayed minimal growth in nutrient-limited conditions, suggesting its role in modulating cellular metabolism. Intracellular metabolic profiling revealed that PknG is necessary for efficient metabolic adaptation during hypoxia. Notably, the PknG mutant exhibited a reductive shift in mycothiol redox potential and compromised stress response. Exposure to antibiotics and hypoxic environment resulted in higher oxidative shift in mycothiol redox potential of PknG mutant compared with the wild type. Persistence during latency-like conditions required kinase activity and thioredoxin motifs of PknG and is mediated through phosphorylation of a central metabolic regulator GarA. Finally, using a guinea pig model of infection, we assessed the in vivo role of PknG in manifestation of disease pathology and established a role for PknG in the formation of stable granuloma, hallmark structures of latent tuberculosis. Taken together, PknG-mediated GarA phosphorylation is important for maintenance of both mycobacterial physiology and redox poise, an axis that is dispensable for survival under normoxic conditions but is critical for non-replicating persistence of mycobacteria. In conclusion, we propose that PknG probably acts as a modulator of latency-associated signals., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The diagnosis of a latent tuberculosis infection (LTBI) is based on detection of immunity against Mycobacterium tuberculosis (Mtb). The tuberculin skin test (TST), the Quantiferon (QFT) and a prolonged lymphocyte stimulation test using either ESAT-6/CFP-10 (LST-EC) or PPD (LST-PPD) were evaluated in a cohort of 495 individuals, suspected to have LTBI, in a low endemic country. While the TST and LST-PPD were both positive in the majority (75%) of individuals, only one third responded in the LST-EC and in the QFT. The choice for LTBI treatment was significantly associated with ESAT6/CFP10 recognition, however the LST-EC detected considerably more individuals (21%) with immunity against Mtb, who might also be at risk for development of active TB, although none of them did during follow up. Follow-up for 2 years showed 7% conversions and 32% reversions for the QFT. The LST-EC showed higher conversion rates (∼45%), although the percentage of individuals positive in the LST-EC did not change significantly within the follow-up period. LTBI treatment did not alter immune recognition of Mtb antigens. In conclusion, the sensitivity of tests for detection of cellular immunity to Mtb specific antigens depends on test methodology and may vary considerably over time in a low endemic region., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In patients with diabetes mellitus, TB treatment outcomes are poorer. Most parameters, when measured, reflect the slower bacteriological conversion from positivity to negativity and higher risks of disease relapse and mortality, as well as a greater propensity to develop drug-resistant TB. Aside from the well-known immunological dysfunction inherent to patients with diabetes mellitus, oxidative stress is likely a major underlying mechanism adversely impacting their TB treatment outcomes. Mycobacterium tuberculosis persisters, formed as a result of the core dormancy response to stress, possibly play a central role in this hypothesis. This hypothetical model also underscores the paramount importance of programmatic management of TB and diabetes mellitus, in collaboration, to improve the outcomes of patients with both diseases. The validity of these ideas could be further ascertained by laboratory and clinical research., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Upregulation of CD137 on recently activated CD8 + T cells has been used to identify rare viral and tumour antigen-specific T cells from the peripheral blood. We aimed to evaluate the accuracy of CD137 for identifying Mycobacterium tuberculosis (Mtb)-reactive CD4 + T cells in the peripheral blood of infected individuals by flow cytometry and to investigate the characteristics of these CD137 + CD4 + T cells. We initially enrolled 31 active tuberculosis (TB) patients, 31 individuals with latent TB infection (LTBI) and 25 healthy donors. The intracellular CD137 and interferon-γ (IFN-γ) production by CD4 + T cells was simultaneously detected under unstimulated and CFP10-stimulated (culture filtrate protein 10, a Mtb-specific antigen) conditions. In unstimulated CD4 + T cells, we found that the CD137 expression in the TB group was significantly higher than that in the LTBI group. Stimulation with CFP10 largely increased the CD4 + T cell CD137 expression in both the TB and LTBI groups. After CFP10 stimulation, the frequency of CD137 + CD4 + T cells was higher than that of IFN-γ + CD4 + T cells in both the TB and LTBI groups. Most of the CFP10-activated IFN-γ-secreting cells were CD137-positive, but only a small fraction of the CD137-positive cells expressed IFN-γ. An additional 20 patients with TB were enrolled to characterize the CD45RO + CCR7 + , CD45RO + CCR7 - and CD45RO - subsets in the CD137 + CD4 + T cell populations. The Mtb-specific CD137 + CD4 + T cells were mainly identified as having an effector memory phenotype. In conclusion, CD137 is a useful marker that can be used for identifying Mtb-reactive CD4 + T cells by flow cytometry., (© 2017 The Foundation for the Scandinavian Journal of Immunology.)

QuantiFERON-TB gold in-tube has been used for screening latent tuberculosis infection in newly employed health care workers in Japan. There have been a few studies concerning quality control. We retrospectively analysed QuantiFERON-TB gold in-tube results in a hospital in Japan. Interferon-γ values in three blood collection tubes for QuantiFERON-TB gold in-tube were analysed in association with the positivity rate. The data set consisted of health care workers aged 20-29 years during the 7 years between 2010 and 2016. The yearly QuantiFERON-TB gold in-tube positivity rate was 0.9%, 16.4%, 3.0%, 39.3%, 2.8%, 0.9% and 1.5%, and was extremely high in 2011 and 2013. The interferon-γ values in the tuberculosis antigen tube were elevated in these two years, as indicated by higher median and wider interquartile range. The interferon-γ value in the negative control tube was also higher in 2011. The higher interferon-γ values in collection tubes (tuberculosis antigen tube and/or negative control tube) resulted in higher QuantiFERON-TB gold in-tube positivity rate. The distribution of interferon-γ in tuberculosis antigen tube and negative control tube, as evaluated by median and interquartile range, proved to be an effective index for the quality control of QuantiFERON-TB gold in-tube., (Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Helminth infections are known to modulate cytokine responses in latent tuberculosis (LTB). However, very few studies have examined whether this modulation is reversible upon anthelmintic therapy. We measured the systemic and mycobacterial (TB) antigen-stimulated levels of type 1, type 2, type 17, and regulatory cytokines in individuals with LTB and with or without coexistent Strongyloides stercoralis infection before and after anthelmintic therapy. Our data reveal that individuals with LTB and coexistent S. stercoralis infection have significantly lower levels of systemic and TB antigen-stimulated type 1 (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and type 17 (IL-17A and/or IL-17F) cytokines and significantly higher levels of systemic but not TB antigen-stimulated type 2 (IL-4 and IL-5) and regulatory (transforming growth factor beta [TGF-β]) cytokines. Anthelmintic therapy resulted in significantly increased systemic levels of type 1 and/or type 17 cytokines and in significantly decreased systemic levels of type 2 and regulatory (IL-10 and TGF-β) cytokines. In addition, anthelmintic therapy resulted in significantly increased TB antigen-stimulated levels of type 1 cytokines only. Our data therefore confirm that the modulation of systemic and TB antigen-stimulated cytokine responses in S. stercoralis -LTB coinfection is reversible (for the most part) by anthelmintic treatment., (Copyright © 2017 American Society for Microbiology.)

It is known that cholesterol plays a key role for Mycobacterium tuberculosis (Mtb) adaptation and survival within the host, thus contributing to the establishment of dormancy. It has been extensively demonstrated that fatty acids are the main energy source of Mtb during infection and dormancy, and it has been proposed that these molecules are implicated in reactivation of bacilli from a dormant state. We used in vitro models to analyze Mtb gene expression during dormancy and reactivation when fatty acids and cholesterol are the unique carbon source in the media. Our results suggest that cholesterol might function as a signal to trigger Mtb expression of some genes required for stress protection earlier than the one induced by fatty acids alone, indicating that cholesterol is very favorable for its development. This process is so conducive that cholesterol-adapted bacilli can reactivate their growth after NRP2 dormancy state even 10 min post ventilation. Thus, we hypothesize that cholesterol is not only involved in Mtb dormancy but that it also plays a critical role for favorable and almost immediate reactivation from an in vitro long-lasting dormant state induced by hypoxia., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tuberculosis control relies on the identification and preventive treatment of people who are latently infected with Mycobacterium tuberculosis (Mtb). PE/PPE proteins have been reported to elicit CD4 and/or CD8 responses either in the form of whole recombinant proteins or as individual peptides. Very few of the PE and PPE proteins have been previously tested for responses in patients with TB and healthy donors. This is the first study to evaluate the Interferon Gamma Release Assay (IGRA) after stimulation with PE35 and PPE68. The antigen-specific levels of IFN-γ following stimulation with QuantiFERON-TB gold in-tube (QFT-G-IT) antigens, and PE35 and PPE68 recombinant proteins were evaluated in 79 children and 102 adults, respectively. Using QFT-G-IT kit, latent tuberculosis infection (LTBI) was detected in 26 children (33%) and 41 adults (40%); IGRA following stimulation with PE35 and PPE68 recombinant proteins, was positive, respectively, in 36 (46%) and 32 (40.5%) children, respectively. In addition, 53 adults (52%) had positive results following stimulation with these two proteins. The sensitivity and specificity of IGRA following stimulation with recombinant PE35 in children were 76% and 80%, and following stimulation with recombinant PPE68 in this group, it was 73% and 75%, respectively. Meanwhile, there is no gold standard test for LTBI. Our designed tests using PE35 and PPE68 PE/PPE proteins, two PE/PPE proteins not present in BCG vaccins, which elicit CD4 and/or CD8 responses, might be helpful for rapid diagnosis of TB and improve the detection of LTBI. However, further validation studies to determine the advantage of IGRAs using these proteins, alone or combined, are highly recommended.

It has been shown that inactivation of the VapC toxin from the VapВС toxin-antitoxin system prevents mycobacterial cells from transitioning to an ovoid state that meets the criteria of dormancy. The results indicate a potential target for medicines that prevent the development of latent tuberculosis infection and provide a basis to obtain bacterial cells for the testing of compounds that are active towards dormant forms of mycobacteria.

This report demonstrates that using interferon gamma release assays to screen for latent tuberculosis infection in female commercial sex workers in an outreach sexual health clinic is feasible and acceptable. Routine interferon gamma release assay use successfully identified high numbers of latent tuberculosis infection. Innovative approaches to treatment and follow up were required to improve treatment adherence in this group. Direct observation of therapy within the sexual health clinic was also feasible. Successful follow up was dependent on the support of outreach workers, interpreters and tuberculosis nurses., (© The Author(s) 2015.)

Objectives: To characterize at high resolution DNA methylation changes of cytokines which occur in the genome of macrophages in association with Mycobacterium tuberculosis (MTB) infection, Methods: We studied the methylation profiles of THP-1 derived macrophage cells infected with clinical MTB strains [Beijing/W & non-Beijing/W lineage, sensitive (INH(S), RIF(S)) & resistant (INH(R), RIF(R)) strains] and of host macrophages from MTB infected cohorts (active & latent patients) with the human methylation CpG islands microarrays., Results: Methylated modification on the promoter sequences of cytokines and their receptors were found to be associated with MTB infection in a strain- and host-dependent manner. Our epigenetic analyses revealed that infection with Beijing/W MTB strains enhanced IL6R, IL4R and IL17R hyper-methylations in infected macrophages. Validation of IL6R methylated sequence confirmed that MTB infection induced DNA methylation of CpG67 region in the IL6R promoter. In addition, studies on the human macrophage methylation profiles from the patient cohorts indicated that the methylation rate of IL17 family members and related genes were significantly altered in patients with active MTB infections., Conclusions: Our study offered novel insights into the epigenetic changes in the interaction of host macrophages in MTB infections and warrant further explorations into these changes in modulating the immune response in active and latent MTB infections., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The QuantiFERON®-TB Gold In-Tube test (QFT), an interferon-γ release assay, is used to diagnose Mycobacterium tuberculosis, but its inaccuracy in distinguishing active tuberculosis from latent infection is a major concern. There is thus a need for an easy and accurate tool for achieving that goal in daily clinical settings. This study aimed to identify candidate cytokines for specifically differentiating active tuberculosis from latent infection. Our study population consisted of 31 active TB (tuberculosis) patients, 29 LTBI (latent tuberculosis infection) patients and 10 healthy control subjects. We assayed for 27 cytokines in QFT supernatants of both specific antigen-stimulated blood samples (TBAg) and negative-control samples (Nil). We analyzed their specificities and sensitivities by creating receiver operating characteristic (ROC) curves and measuring the area under those curves (AUCs). In TBAg-Nil supernatants, IL-10, IFN-γ, MCP-1 and IL-1RA showed high AUCs of 0.8120, 0.7842, 0.7419 and 0.7375, respectively. Compared with each cytokine alone, combined assay for these top four cytokines showed positive rates in diagnosing active TB, and GDA analysis revealed that MCP-1 and IL-5 are potent in distinguishing active TB from LTBI, with Wilk's lambda = 0.718 (p < 0.001). Furthermore, utilizing the unique characteristic of IL-2 that its TBAg-Nil supernatant levels are higher in LTBI compared to active TB, the difference between IFN-γ and IL-2 showed a large AUC of 0.8910. In summary, besides IFN-γ, IL-2, IL-5, IL-10, IL-1RA and MCP-1 in QFT supernatants may be useful for distinguishing active TB from LTBI. Those cytokines may also help us understand the difference in pathogenesis between active TB and LTBI.

In cancer and chronic infectious diseases, immune checkpoint-blockade of inhibitory receptors can enhance T-cell immunity. In tuberculosis (TB), a chronic infectious disease, prolonged antigen exposure can potentially drive terminal T-cell differentiation towards functional 'exhaustion': in human TB T-cells express PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T-lymphocyte-associated protein-4). However, in murine TB not PD-1 but rather killer cell lectin-like receptor subfamily-G1 (KLRG1) was a superior indicator of terminal T-cell differentiation. We therefore compared expression of KLRG1, PD-1 and CTLA-4 on T-cells in different stages of human TB, and also analysed their induction following BCG-vaccination. KLRG1, PD-1 and CTLA-4-expression were highest on in vitro BCG-stimulated CD4(+) T-cells following recent TB-treatment; KLRG1 and PD-1-expression on CD4(+) T-cells in active--but not latent--TB were only slightly increased compared to healthy donors. BCG-vaccination induced KLRG1-expression on BCG-stimulated CD8(+) but not CD4(+) T-cells, while neither PD-1 nor CTLA-4-expression increased. KLRG1-expressing CD8(+) T-cells exhibited markedly decreased proliferation, whereas PD-1(+) T-cells proliferated after in vitro BCG-stimulation. Thus, we demonstrate the presence of increased KLRG1-expressing T-cells in TB-treated individuals, and present KLRG1 as a marker of decreased human T-cell proliferation following BCG-vaccination. These results expand our understanding of cell-mediated immune control of mycobacterial infections., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Current Tuberculosis treatment is long and expensive, faces the increasing burden of MDR/XDR strains and lack of effective treatment against latent form, resulting in an urgent need of new anti-TB drugs. Key to TB biology is its capacity to fight the host's RNOS mediated attack. RNOS are known to display a concentration dependent mycobactericidal activity, which leads to the following hypothesis "if we know which proteins are targeted by RNOS and kill TB, we we might be able to inhibit them with drugs resulting in a synergistic bactericidal effect". Based on this idea, we performed an Mtb metabolic network whole proteome analysis of potential RNOS sensitive and relevant targets which includes target druggability and essentiality criteria. Our results, available at http://tuberq.proteinq.com.ar yield new potential TB targets, like I3PS, while also providing and updated view of previous proposals becoming an important tool for researchers looking for new ways of killing TB., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Type 2 diabetes mellitus (DM) is a risk factor for tuberculosis among individuals with latent Mycobacterium tuberculosis infection. To explore the influence of DM on CD8(+) T-cell responses during latent M. tuberculosis infection, we estimated the cytokine and cytotoxic marker expression pattern in individuals with latent M. tuberculosis infection with DM and those with latent M. tuberculosis infection without DM. Among individuals with latent M. tuberculosis infection, those with DM had diminished frequencies of CD8(+) T-helper type 1 (Th1), Th2, and Th17 cells following stimulation by M. tuberculosis antigen and enhanced frequencies of CD8(+) T cells expressing cytotoxic markers, compared with those without DM. Thus, our results suggest that coincident DM modulates CD8(+) T-cell function during latent M. tuberculosis infection., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background: In the absence of a gold standard for the diagnosis of latent tuberculosis (TB) infection (LTBI), the current tests available for the diagnosis of LTBI are limited by their inability to differentiate between LTBI and active TB disease. We investigated IP-10 as a potential biomarker for LTBI among household contacts exposed to sputum positive active TB cases., Methods: Active TB cases and contacts were recruited into a cohort with six months' follow-up. Contacts were tested for LTBI using QuantiFERON®-TB Gold In-Tube (QFN) assay and the tuberculin skin test (TST). Baseline supernatants from the QFN assay of 237 contacts and 102 active TB cases were analysed for Mycobacterium tuberculosis (MTB) specific and mitogen specific IP-10 responses., Results: Contacts with LTBI (QFN+TST+) had the highest MTB specific IP-10 responses at baseline, compared to uninfected contacts (QFN-TST-) p<0.0001; and active cases, p = 0.01. Using a cut-off of 8,239 pg/ml, MTB specific IP-10 was able to diagnose LTBI with a sensitivity of 87.1% (95% CI, 76.2-94.3) and specificity of 90.9% (95% CI, 81.3-96.6). MTB specific to mitogen specific IP-10 ratio was higher in HIV negative active TB cases, compared to HIV negative latently infected contacts, p = 0.0004. Concentrations of MTB specific IP-10 were higher in contacts with TST conversion (negative at baseline, positive at 6-months) than in those that were persistently TST negative, p = 0.001., Conclusion: IP-10 performed well in differentiating contacts with either latent or active TB from those who were uninfected but was not able to differentiate LTBI from active disease except when MTB specific to mitogen specific ratios were used in HIV negative adults. In addition, IP-10 had the potential to diagnose 'recent TB infection' in persons classified as having LTBI using the TST. Such individuals with strong IP-10 responses would likely benefit from chemoprophylaxis.

Interferon-gamma Release Assays (IGRAs) significantly increases the possibility for early diagnosis of tuberculosis, but IGRAs alone cannot discriminate active TB from LTBI. Therefore, fast and reliable discrimination of active tuberculosis, especially bacteriology negative tuberculosis, from LTBI is a great necessity. Here we established an assay based on flow cytometric multiparameter assay assessing expression of CD161 along with CD3, CD4, and CD8, whereby a set of indices formulated by the percentages of CD3(+)CD161(+), CD3(+)CD4(+)CD161(+) and CD3(+)CD8(+)CD161(+) T cells multiplied with lymphocyte/monocyte ratio were established. Application of the CD3(+)CD8(+)CD161(+) index to compare a cohort of active tuberculosis with a cohort of LTBI or health control yielded 0.7662 (95% confidence interval [CI] 0.6559-0.8552) or 0.7922 (95% CI 0.6846-0.8763) for sensitivity and 0.9048 (95% CI 0.8209-0.9580) or 0.8939 (95% CI 0.8392-0.9349) for specificity when the TB cohort was AFB(+); the corresponding results were 0.7481 (95% CI 0.6648-0.8198) or 0.7557 (95% CI 0.6730-0.8265) for sensitivity and 0.8571 (95% CI 0.7637-0.9239) or 0.8603 (95% CI 0.8008-0.9075) for specificity when the TB cohort was AFB(-). Our results reveal that in combination with IGRAs, CD161-based indices provide a novel, fast diagnostic solution addressing the limitation of current tuberculosis diagnostics.

Latent TB infection (LTBI) is one of the major contributing factors for the high incidence of TB in India that in turn significantly contributes to the pool of active TB. Hence, identification and treatment of LTBI is of utmost importance. Currently, no specific diagnostic test is available for LTBI. Earlier, in our immunoproteomic analysis, we identified Rv2204c and Rv0753c protein-containing fractions induced significantly higher interferon-gamma (IFN-γ) response in LTBI than in active TB. In this study, we evaluated cytokine and chemokine response against M. tuberculosis antigens for improving LTBI identification. Two M. tb proteins Rv2204c and Rv0753c were cloned, over expressed in E. coli and purified by affinity chromatography. Antigen-specific immune response was evaluated in 39 pulmonary TB patients (PTB) and 35 healthy house-hold contacts (HHC). After whole blood culture for 6 days, the secretion of cytokines and chemokines were quantified in culture supernatants using Enzyme Linked Immune Sorbent Assay (ELISA). Antigen specific cytokines such as interferon gamma (IFN-γ), interleukin-6 (IL-6), IL-8, IL-12p40 and chemokines like monocyte chemotactic proteins MCP-1, MCP-2 were significantly higher in HHC than PTB. In contrast to other cytokines, tumor necrosis factor-alpha (TNF)-α response was significantly increased in PTB compared with HHC. Both Rv2204c and Rv0753c antigen specific IFN-γ response showed 86% positivity in HHC; whereas in PTB, these antigens showed 18% and 21% positivity respectively. Rv2204c antigen-specific IFN-γ/TNF-α response displayed maximum positivity of 91% in HHC and minimum positivity of 10% (4/39) in PTB. Rv2204c and Rv0753c specific IFN-γ and IFN-γ/TNF-α responses showed the most promising accuracy in identifying LTBI., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: The ability to monitor the response to therapy for tuberculosis (TB) and confirm adequate treatment would be a major advance. The utility of interferon gamma assays (IGRA) for this purpose remains uncertain., Methods: A systematic search of all studies investigating commercial IGRA to monitor anti-tuberculous treatment was done. Studies were included if they included an IGRA before the start of, and at least once during, treatment for active or latent TB., Results: We identified 30 studies, of which 24 used QuantiFERON-TB (QFT), three used T-SPOT.TB and three used both QFT and T-SPOT.TB. Most studies were done in low TB incidence countries. No uniform pattern was seen in IGRA conversion and reversion rates at the end of treatment for active or latent TB. In most studies, the majority of IGRA results remained positive at the end of treatment. In many studies, the quantitative levels of IFN-γ decreased during treatment, particularly in active TB. There was significant heterogeneity in the included studies., Conclusion: While quantitative IGRA responses generally fall during treatment for TB, the large degree of variation in results between participants in each study means that IGRAs are unlikely to be useful for monitoring anti-tuberculous treatment in clinical practice for any individual patient., (Copyright © 2015 Elsevier Ltd. All rights reserved.)