TCA metabolism regulates DNA hypermethylation in LPS and Mycobacterium tuberculosis-induced immune tolerance.

Severe and chronic infections, including pneumonia, sepsis, and tuberculosis (TB), induce long-lasting epigenetic changes that are associated with an increase in all-cause postinfectious morbidity and mortality. Oncology studies identified metabolic drivers of the epigenetic landscape, with the tricarboxylic acid (TCA) cycle acting as a central hub. It is unknown if the TCA cycle also regulates epigenetics, specifically DNA methylation, after infection-induced immune tolerance. The following studies demonstrate that lipopolysaccharide and Mycobacterium tuberculosis induce changes in DNA methylation that are mediated by the TCA cycle. Infection-induced DNA hypermethylation is mitigated by inhibitors of cellular metabolism (rapamycin, everolimus, metformin) and the TCA cycle (isocitrate dehydrogenase inhibitors). Conversely, exogenous supplementation with TCA metabolites (succinate and itaconate) induces DNA hypermethylation and immune tolerance. Finally, TB patients who received everolimus have less DNA hypermethylation demonstrating proof of concept that metabolic manipulation can mitigate epigenetic scars.

Circulating Biomarkers, Fraction of Exhaled Nitric Oxide, and Lung Function in Patients With Human Immunodeficiency Virus and Tuberculosis.

BACKGROUND: Identification of proinflammatory factors responding to Mycobacterium tuberculosis is important to reduce long-term sequelae of pulmonary tuberculosis (TB). METHODS: We examined the association between plasma biomarkers, the fraction of exhaled nitric oxide (FeNO), and lung function among a prospective cohort of 105 adults newly diagnosed with TB/human immunodeficiency virus (HIV) in South Africa. Participants were followed for 48 weeks from antiretroviral therapy (ART) initiation with serial assessments of plasma biomarkers, FeNO, lung function, and respiratory symptoms. Linear regression and generalized estimating equations were used to examine the associations at baseline and over the course of TB treatment, respectively. RESULTS: At baseline, higher FeNO levels were associated with preserved lung function, whereas greater respiratory symptoms and higher interleukin (IL)-6 plasma levels were associated with worse lung function. After ART and TB treatment initiation, improvements in lung function were associated with increases in FeNO (rate ratio [RR] = 86 mL, 95% confidence interval [CI] = 34-139) and decreases in IL-6 (RR = -118 mL, 95% CI = -193 to -43) and vascular endothelial growth factor ([VEGF] RR = -178 mL, 95% CI = -314 to -43). CONCLUSIONS: Circulating IL-6, VEGF, and FeNO are associated with lung function in adults being treated for TB/HIV. These biomarkers may help identify individuals at higher risk for post-TB lung disease and elucidate targetable pathways to modify the risk of chronic lung impairment among TB survivors.

Quantitative Systems Pharmacology Modeling Framework of Autophagy in Tuberculosis: Application to Adjunctive Metformin Host-Directed Therapy.

Quantitative systems pharmacology (QSP) modeling of the host immune response against Mycobacterium tuberculosis can inform the rational design of host-directed therapies (HDTs). We aimed to develop a QSP framework to evaluate the effects of metformin-associated autophagy induction in combination with antibiotics. A QSP framework for autophagy was developed by extending a model for host immune response to include adenosine monophosphate-activated protein kinase (AMPK)-mTOR-autophagy signaling. This model was combined with pharmacokinetic-pharmacodynamic models for metformin and antibiotics against M. tuberculosis. We compared the model predictions to mice infection experiments and derived predictions for the pathogen- and host-associated dynamics in humans treated with metformin in combination with antibiotics. The model adequately captured the observed bacterial load dynamics in mice M. tuberculosis infection models treated with metformin. Simulations for adjunctive metformin therapy in newly diagnosed patients suggested a limited yet dose-dependent effect of metformin on reduction of the intracellular bacterial load when the overall bacterial load is low, late during antibiotic treatment. We present the first QSP framework for HDTs against M. tuberculosis, linking cellular-level autophagy effects to disease progression and adjunctive HDT treatment response. This framework may be extended to guide the design of HDTs against M. tuberculosis.

Pulmonary restriction predicts long-term pulmonary impairment in people with HIV and tuberculosis.

BACKGROUND: While tuberculosis is considered a risk factor for chronic obstructive pulmonary disease, a restrictive pattern of pulmonary impairment may actually be more common among tuberculosis survivors. We aimed to determine the nature of pulmonary impairment before and after treatment among people with HIV and tuberculosis and identify risk factors for long-term impairment. METHODS: In this prospective cohort study conducted in South Africa, we enrolled adults newly diagnosed with HIV and tuberculosis who were initiating antiretroviral therapy and tuberculosis treatment. We measured lung function and symptoms at baseline, 6, and 12 months. We compared participants with and without pulmonary impairment and constructed logistic regression models to identify characteristics associated with pulmonary impairment. RESULTS: Among 134 participants with a median CD4 count of 110 cells/µl, 112 (83%) completed baseline spirometry at which time 32 (29%) had restriction, 13 (12%) had obstruction, and 9 (7%) had a mixed pattern. Lung function was dynamic over time and 30 (33%) participants had impaired lung function at 12 months. Baseline restriction was associated with greater symptoms and with long-term pulmonary impairment (adjusted odds ratio 5.44, 95% confidence interval 1.16-25.45), while baseline obstruction was not (adjusted odds ratio 1.95, 95% confidence interval 0.28-13.78). CONCLUSIONS: In this cohort of people with HIV and tuberculosis, restriction was the most common, symptomatic, and persistent pattern of pulmonary impairment. These data can help to raise awareness among clinicians about the heterogeneity of post-tuberculosis pulmonary impairment, and highlight the need for further research into mediators of lung injury in this vulnerable population.

A Common NLRC4 Gene Variant Associates With Inflammation and Pulmonary Function in Human Immunodeficiency Virus and Tuberculosis.

BACKGROUND: Inflammasomes mediate inflammation in adults living with both human immunodeficiency virus (HIV) and tuberculosis (TB), but the relevance of inflammasome gene polymorphisms in TB-associated pulmonary damage is unknown. We hypothesized that functional single-nucleotide polymorphisms (SNPs) in inflammasome pathway genes modify systemic and pulmonary inflammation, contributing to respiratory impairment in adults living with HIV/pulmonary TB. METHODS: This was a prospective cohort study set in South Africa following individuals living with HIV/TB up to 48 weeks post-antiretroviral therapy (ART) initiation. Ten functional SNPs in 5 inflammasome pathway genes were related to circulating inflammatory biomarkers and lung function assessed by spirometry pre- and post-ART initiation. Analyses used 2-sided t tests, Wilcoxon rank sum tests, Spearman correlation coefficients, linear regression, and generalized estimating equation models. RESULTS: Among 102 patients with baseline samples, the minor allele (T) in NLRC4 rs385076 was independently associated with lower levels of interleukin (IL)-18 and IL-6 before and up to 12 weeks post-ART initiation (Benjamini-Hochberg corrected P values < .02). Patients with the CT/TT genotypes also had improved lung function vs CC patients up to 48 weeks post-ART initiation (forced vital capacity, 206 mL higher; 95% confidence interval [CI], 67-345 mL; P = .004 and forced expiratory volume in 1 second, 143 mL higher; 95% CI, 11-274 mL; P = .034). CONCLUSIONS: A common SNP in the NLRC4 inflammasome may modify TB-associated inflammation in clinically relevant ways. This SNP may identify high-risk groups for lung damage in TB. Inhibition of NLRC4 activity may be an important approach for TB host-directed therapy.

Lung Injury on Antiretroviral Therapy in Adults With Human Immunodeficiency Virus/Tuberculosis.

BACKGROUND: Immune restoration on antiretroviral therapy (ART) can drive inflammation in people living with human immunodeficiency virus (HIV) who have pulmonary tuberculosis (TB), but its effects on the lungs have not been assessed. We evaluated associations between pulmonary inflammation, recovery of pathogen-specific CD4 T-cell function, and lung injury prior to and after ART initiation in adults with HIV and pulmonary TB. METHODS: This was a prospective cohort study in South Africa, following adults with HIV and pulmonary TB prior to and up to 48 weeks after ART initiation. Pulmonary-specific inflammation was defined as total glycolytic activity (TGA) on [18]F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) at baseline and 4 weeks after ART initiation. Spirometry, respiratory symptom tests, and flow cytometry were performed at the same times to assess lung involvement and the frequency of mycobacteria-specific CD4 T-cells. In addition, we evaluated lung function longitudinally up to 48 weeks after ART initiation. RESULTS: Greater lung TGA on FDG PET-CT was associated with worse lung function and respiratory symptoms prior to ART initiation, and nearly half of subjects experienced worsening lung inflammation and lung function at Week 4 of ART. Worsening Week 4 lung inflammation and pulmonary function were both associated with greater increases in pathogen-specific functional CD4 T-cell responses on ART, and early decreases in lung function were independently associated with persistently lower lung function months after TB treatment completion. CONCLUSIONS: Increases in pulmonary inflammation and decreases in lung function are common on ART, relate to greater ART-mediated CD4 T-cell restoration, and are associated with the persistent impairment of lung function in individuals with HIV/TB.

Early Biomarkers and Regulatory Innovation in Multidrug-Resistant Tuberculosis.

Biomarkers play an essential role in accelerating drug development. Sputum culture conversion using solid medium is the best-characterized tuberculosis biomarker, having been examined at the patient and trial levels in studies with thousands of subjects, and having recently been validated using data from 3 unsuccessful phase 3 trials. We presently are poised at the threshold of regulatory innovation for antibacterials to treat drug-resistant infections, in which Special Medical Use authorization restricted to patients with limited options could be based on the results of small clinical trials. Patients worldwide would be well served by licensing of new regimens for multidrug-resistant tuberculosis based on biomarker evidence commensurate with the urgency of the current global crisis.

Population pharmacokinetic/pharmacodynamic analysis of the bactericidal activities of sutezolid (PNU-100480) and its major metabolite against intracellular Mycobacterium tuberculosis in ex vivo whole-blood cultures of patients with pulmonary tuberculosis.

Sutezolid (PNU-100480 [U-480]) is an oxazolidinone antimicrobial being developed for the treatment of tuberculosis. An active sulfoxide metabolite (PNU-101603 [U-603]), which reaches concentrations in plasma several times those of the parent, has been reported to drive the killing of extracellular Mycobacterium tuberculosis by sutezolid in hollow-fiber culture. However, the relative contributions of the parent and metabolite against intracellular M. tuberculosis in vivo are not fully understood. The relationships between the plasma concentrations of U-480 and U-603 and intracellular whole-blood bactericidal activity (WBA) in ex vivo cultures were examined using a direct competitive population pharmacokinetic (PK)/pharmacodynamic 4-parameter sigmoid model. The data set included 690 PK determinations and 345 WBA determinations from 50 tuberculosis patients enrolled in a phase 2a sutezolid trial. The model parameters were solved iteratively. The median U-603/U-480 concentration ratio was 7.1 (range, 1 to 28). The apparent 50% inhibitory concentration of U-603 for intracellular M. tuberculosis was 17-fold greater than that of U-480 (90% confidence interval [CI], 9.9- to 53-fold). Model parameters were used to simulate in vivo activity after oral dosing with sutezolid at 600 mg twice a day (BID) and 1,200 mg once a day (QD). Divided dosing resulted in greater cumulative activity (-0.269 log10 per day; 90% CI, -0.237 to -0.293 log10 per day) than single daily dosing (-0.186 log10 per day; 90% CI, -0.160 to -0.208 log10 per day). U-480 accounted for 84% and 78% of the activity for BID and QD dosing, respectively, despite the higher concentrations of U-603. Killing of intracellular M. tuberculosis by orally administered sutezolid is mainly due to the activity of the parent compound. Taken together with the findings of other studies in the hollow-fiber model, these findings suggest that sutezolid and its metabolite act on different mycobacterial subpopulations.

Repurposing N-acetylcysteine for management of non-acetaminophen induced acute liver failure: an evidence scan from a global health perspective.

Background: The World Health Organization (WHO)'s Essential Medicines List (EML) plays an important role in advocating for access to key treatments for conditions affecting people in all geographic settings. We applied our established drug repurposing methods to one EML agent, N-acetylcysteine (NAC), to identify additional uses of relevance to the global health community beyond its existing EML indication (acetaminophen toxicity). Methods: We undertook a phenome-wide association study (PheWAS) of a variant in the glutathione synthetase (GSS) gene in approximately 35,000 patients to explore novel indications for use of NAC, which targets glutathione. We then evaluated the evidence regarding biologic plausibility, efficacy, and safety of NAC use in the new phenotype candidates. Results: PheWAS of GSS variant R418Q revealed increased risk of several phenotypes related to non-acetaminophen induced acute liver failure (ALF), indicating that NAC may represent a therapeutic option for treating this condition. Evidence review identified practice guidelines, systematic reviews, clinical trials, retrospective cohorts and case series, and case reports. This evidence suggesting benefit of NAC use in this subset of ALF patients. The safety profile of NAC in this literature was also concordant with existing evidence on safety of this agent in acetaminophen-induced ALF. Conclusions: This body of literature indicates efficacy and safety of NAC in non-acetaminophen induced ALF. Given the presence of NAC on the EML, this medication is likely to be available across a range of resource settings; promulgating its use in this novel subset of ALF can provide healthcare professionals and patients with a valuable and safe complement to supportive care for this disease.

Adjunctive N-Acetylcysteine and Lung Function in Pulmonary Tuberculosis.

BACKGROUND: Tuberculosis remains a global health concern, and half of cured patients have permanent lung injury. N-acetylcysteine (NAC) has shown beneficial antimicrobial, antioxidant, and immunomodulatory effects in preclinical tuberculosis models. We examined its effects on tuberculosis treatment outcomes. METHODS: This prospective, randomized, controlled trial nested within the TB SEQUEL cohort study enrolled 140 adults with moderate or far-advanced tuberculosis. Participants were randomly assigned 1:1 to standard therapy with or without 1200 mg of oral NAC twice daily for days 1 to 112. Clinical evaluations, sputum culture, and spirometry were performed at specified intervals through day 168, after which participants returned to the TB SEQUEL cohort. The primary outcome was culture conversion. Secondary outcomes included whole-blood glutathione levels and lung function. RESULTS: Participants were predominantly young, male, and human immunodeficiency virus 1-negative and had heavy sputum Mycobacterium tuberculosis (MTB) infection burdens. NAC increased glutathione levels (NAC × day interaction, 8.48; 95% confidence interval [CI], 1.93 to 15.02) but did not increase stable culture conversion (hazard ratio, 0.84; 95% CI, 0.59 to 1.20; P=0.33). NAC treatment was associated with improved recovery of lung function (NAC × month, 0.49 [95% CI, 0.02 to 0.95] and 0.42 [95% CI, -0.06 to 0.91] for forced vital capacity and forced expiratory volume in the first second, respectively, as percentages of predicted values). The effects of NAC on lung function were greatest in participants with severe baseline lung impairment and appeared to persist beyond the period of NAC administration. Rates of serious or grade 3 to 4 nonserious adverse events did not differ between the groups. CONCLUSIONS: Despite increasing whole-blood glutathione levels, NAC did not affect eradication of MTB infection in adults with pulmonary tuberculosis that was moderate to far advanced. Secondary outcomes of lung function showed changes that merit further investigation. (Funded by TB SEQUEL grant 01KA1613 of the German Ministry for Education and Research, the Health Africa Project, and the German Center for Infection Research; ClinicalTrials.gov number, NCT03702738.).

Sustainable tuberculosis drug development.

Six new antituberculosis compounds in 4 classes are presently in clinical trials. Although these show substantial promise for drug-resistant (DR) tuberculosis, the presently planned studies of these compounds will not inform their optimal use, as each will be tested singly vs placebo with existing drugs, rather than in new regimens. Each successive regulatory approval will increase the size, cost, and complexity of trials for those that follow, causing delays during which suboptimal use will occur and resistance will emerge. This paper proposes the development of a novel regimen with the potential for use in both drug-sensitive (DS) and DR tuberculosis. Adaptive licensing for DR tuberculosis based on 2-month sputum culture would shorten time to initial approval by several years. A global outcomes registry would confirm safety and effectiveness in both DS and DR tuberculosis, making possible the second transformation of tuberculosis treatment. We should do our utmost to see it succeed.

Host-directed immunotherapy of viral and bacterial infections: past, present and future.

The advent of COVID-19 and the persistent threat of infectious diseases such as tuberculosis, malaria, influenza and HIV/AIDS remind us of the marked impact that infections continue to have on public health. Some of the most effective protective measures are vaccines but these have been difficult to develop for some of these infectious diseases even after decades of research. The development of drugs and immunotherapies acting directly against the pathogen can be equally challenging, and such pathogen-directed therapeutics have the potential disadvantage of selecting for resistance. An alternative approach is provided by host-directed therapies, which interfere with host cellular processes required for pathogen survival or replication, or target the host immune response to infection (immunotherapies) to either augment immunity or ameliorate immunopathology. Here, we provide a historical perspective of host-directed immunotherapeutic interventions for viral and bacterial infections and then focus on SARS-CoV-2 and Mycobacterium tuberculosis, two major human pathogens of the current era, to indicate the key lessons learned and discuss candidate immunotherapeutic approaches, with a focus on drugs currently in clinical trials.

IFN-γ independent markers of Mycobacterium tuberculosis exposure among male South African gold miners.

BACKGROUND: The prevalence of tuberculosis among men who work in the gold mines of South Africa is among the highest in the world, but a fraction of miners demonstrate consistently negative results upon tuberculin skin test (TST) and IFN-γ release assay (IGRA). We hypothesized that these "resisters" (RSTRs) may display unconventional immune signatures of exposure to M. tuberculosis (M.tb). METHODS: In a cohort of RSTRs and matched controls with latent TB infection (LTBI), we profiled the functional breadth of M.tb antigen-specific T cell and antibody responses using multi-parameter flow cytometry and systems serology, respectively. FINDINGS: RSTRs and LTBI controls both exhibited IFN-γ independent T-cell and IgG antibody responses to M.tb-specific antigens ESAT-6 and CFP-10. Antigen-specific antibody Fc galactosylation and sialylation were higher among RSTRs. In a combined T-cell and antibody analysis, M.tb lysate-stimulated TNF secretion by T cells correlated positively with levels of purified protein derivative-specific IgG. A multivariate model of the combined data was able to differentiate RSTR and LTBI subjects. INTERPRETATION: IFN-γ independent immune signatures of exposure to M.tb, which are not detected by approved clinical diagnostics, are readily detectable in an occupational cohort uniquely characterized by intense and long-term infection pressure. Further, TNF may mediate a coordinated response between M.tb-specific T-cells and B-cells. FUNDING: This work was supported by the US National Institutes of Health (R01-AI124348 to Boom, Stein, and Hawn; R01-AI125189 and R01-AI146072 to Seshadri; and 75N93019C00071 to Fortune, Alter, Seshadri, and Boom), the Doris Duke Charitable Foundation (Davies), the Bill & Melinda Gates Foundation (OPP1151836 and OPP1109001 to Hawn; and OPP1151840 to Alter), Mass Life Science Foundation (Fortune), and Good Ventures Fund (Fortune).

Sterilizing activities of novel combinations lacking first- and second-line drugs in a murine model of tuberculosis.

Novel oral regimens composed of new drugs with potent activity against Mycobacterium tuberculosis and no cross-resistance with existing agents are needed to shorten and simplify treatment for both drug-susceptible and drug-resistant tuberculosis. As part of a continuing effort to evaluate novel drug combinations for treatment-shortening potential in a murine model, we performed two long-term, relapse-based experiments. In the first experiment, several 3- and 4-drug combinations containing new agents currently in phase 2/3 trials (TMC207 [bedaquiline], PA-824 and PNU-100480 [sutezolid], and/or clofazimine) proved superior to the first-line regimen of rifampin, pyrazinamide, and isoniazid. TMC207 plus PNU-100480 was the most effective drug pair. In the second experiment, in which 3- and 4-drug combinations composed of TMC207 and pyrazinamide plus rifapentine, clofazimine, PNU-100480, or both rifapentine and clofazimine were evaluated, the rank order of drugs improving the sterilizing activity of TMC207 and pyrazinamide was as follows: rifapentine plus clofazimine ≥ clofazimine ≥ rifapentine > PNU-100480. The results revealed potential new building blocks for universally active short-course regimens for drug-resistant tuberculosis. The inclusion of pyrazinamide against susceptible isolates may shorten the duration of treatment further.

SQ109 and PNU-100480 interact to kill Mycobacterium tuberculosis in vitro.

OBJECTIVES: To investigate in vitro interaction between two compounds, SQ109 and PNU-100480, currently in development for the treatment of Mycobacterium tuberculosis (MTB). METHODS: The two-drug interactions between SQ109 and PNU-100480 and its major metabolite PNU-101603 were assessed by chequerboard titration, and the rate of killing and intracellular activity were determined in both J774A.1 mouse macrophages and whole blood culture. RESULTS: In chequerboard titration, interactions between SQ109 and either oxazolidinone were additive. In time-kill studies, SQ109 killed MTB faster than PNU compounds, and its rate of killing was further enhanced by both oxazolidinones. The order of efficacy of single compounds against intracellular MTB was SQ109 > PNU-100480 > PNU-101603. At sub-MIC, combinations of SQ109 + PNU compounds showed improved intracellular activity over individual drugs; at ≥MIC, the order of efficacy was SQ109 > SQ109 + PNU-100480 > SQ109 + PNU-101603. In whole blood culture, the combined bactericidal activities of SQ109 and PNU-100480 and its major metabolite against intracellular M. tuberculosis did not differ significantly from the sum of the compounds tested individually. CONCLUSIONS: SQ109 and PNU combinations were additive and improved the rate of MTB killing over individual drugs. These data suggest that the drugs may work together cooperatively to eliminate MTB in vivo.

Impact of N-Acetyl Cysteine (NAC) on Tuberculosis (TB) Patients-A Systematic Review.

Sustained TB infection overproduces reactive oxygen species (ROS) as a host defense mechanism. Research shows ROS is destructive to lung tissue. Glutathione (GSH) neutralizes ROS, although it is consumed. NAC is a precursor of GSH synthesis, and administering an appropriate dose of NAC to patients with respiratory conditions may enhance lung recovery and replenish GSH. The present review searched for articles reporting on the effects of NAC in TB treatment from 1960 to 31 May 2022. The PICO search strategy was used in Google Scholar, PubMed, SciFinder, and Wiley online library databases. The COVIDENCE tool was used to delete inappropriate content. We eventually discovered five clinical trials, one case report, seven reviews, in vitro research, and four experimental animal studies from the twenty-four accepted articles. The use of NAC resulted in increased GSH levels, decreased treatment time, and was safe with minimal adverse events. However, the evidence is currently insufficient to estimate the overall effects of NAC, thus the study warrants more NAC clinical trials to demonstrate its effects in TB treatment.

Lung and blood early biomarkers for host-directed tuberculosis therapies: Secondary outcome measures from a randomized controlled trial.

BACKGROUND: Current tuberculosis treatments leave most patients with bronchiectasis and fibrosis, permanent conditions that impair lung function and increase all-cause post-TB mortality. Host-directed therapies (HDTs) may reduce lung inflammation and hasten eradication of infection. Biomarkers can accelerate tuberculosis regimen development, but no studies have yet examined early biomarkers of TB-HDTs. METHODS: Biomarkers of inflammation and microbicidal activity were evaluated as a part of a recent phase-2 randomized controlled trial of four HDTs in 200 patients with pulmonary tuberculosis and baseline predictors of poor outcome, including CC-11050 (PDE4i), everolimus (mTORi), auranofin (oral gold salt), and ergocalciferol (vitamin D). Two of the 4 arms (CC-11050 and everolimus) showed superior recovery of lung function at day 180 compared to control; none showed accelerated eradication of MTB infection. Patients underwent 18F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) on entry and day 56. PET signals were analyzed according to total, maximal, and peak glycolytic activity; CT was analyzed according to total modified Hounsfield units to assess radiodensity. Mycobactericidal activity in ex vivo whole blood culture was measured on days 42, 84, and 140. C-reactive protein (CRP) was measured at multiple time points. RESULTS: All PET/CT parameters showed highly significant reductions from baseline to day 56; however, only maximal or peak glycolytic activity showed further experimental reduction compared to controls, and only in everolimus recipients. CRP dropped precipitously during early treatment, but did so equally in all arms; over the entire period of treatment, the rate of decline of CRP tended to be greater in CC-11050 recipients than in controls but this fell short of statistical significance. Whole blood mycobactericidal activity in ex-vivo culture was enhanced by auranofin compared to controls, but not by other HDTs. CONCLUSIONS: None of these early biomarkers correctly predicted HDT effects on inflammation or infection across all four experimental arms. Instead, they each appear to show highly specific responses related to HDT mechanisms of action.

Monocyte Transcriptional Responses to Mycobacterium tuberculosis Associate with Resistance to Tuberculin Skin Test and Interferon Gamma Release Assay Conversion.

Heavy exposure to Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) and among the top infectious killers worldwide, results in infection that is cleared, contained, or progresses to disease. Some heavily exposed tuberculosis contacts show no evidence of infection using the tuberculin skin test (TST) and interferon gamma release assay (IGRA); yet the mechanisms underlying this "resister" (RSTR) phenotype are unclear. To identify transcriptional responses that distinguish RSTR monocytes, we performed transcriptome sequencing (RNA-seq) on monocytes isolated from heavily exposed household contacts in Uganda and gold miners in South Africa after ex vivo M. tuberculosis infection. Gene set enrichment analysis (GSEA) revealed several gene pathways that were consistently enriched in response to M. tuberculosis among RSTR subjects compared to controls with positive TST/IGRA testing (latent TB infection [LTBI]) across Uganda and South Africa. The most significantly enriched gene set in which expression was increased in RSTR relative to LTBI M. tuberculosis-infected monocytes was the tumor necrosis factor alpha (TNF-α) signaling pathway whose core enrichment (leading edge) substantially overlapped across RSTR populations. These leading-edge genes included candidate resistance genes (ABCA1 and DUSP2) with significantly increased expression among Uganda RSTRs (false-discovery rate [FDR], <0.1). The distinct monocyte transcriptional response to M. tuberculosis among RSTR subjects, including increased expression of the TNF signaling pathway, highlights genes and inflammatory pathways that may mediate resistance to TST/IGRA conversion and provides therapeutic targets to enhance host restriction of M. tuberculosis intracellular infection. IMPORTANCE After heavy M. tuberculosis exposure, the events that determine why some individuals resist TST/IGRA conversion are poorly defined. Enrichment of the TNF signaling gene set among RSTR monocytes from multiple distinct cohorts suggests an important role for the monocyte TNF response in determining this alternative immune outcome. These TNF responses to M. tuberculosis among RSTRs may contribute to antimicrobial programs that result in early clearance or the priming of alternative (gamma interferon-independent) cellular responses.

Biomarker-assisted dose selection for safety and efficacy in early development of PNU-100480 for tuberculosis.

Tuberculosis is a serious global health threat for which new treatments are urgently needed. This study examined the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple ascending doses of the oxazolidinone PNU-100480 in healthy volunteers, using biomarkers for safety and efficacy. Subjects were randomly assigned to PNU-100480 or placebo (4:1) at schedules of 100, 300, or 600 mg twice daily or 1,200 mg daily for 14 days or a schedule of 600 mg twice daily for 28 days to which pyrazinamide was added on days 27 and 28. A sixth cohort was given linezolid at 300 mg daily for 4 days. Signs, symptoms, and routine safety tests were monitored. Bactericidal activity against Mycobacterium tuberculosis was measured in ex vivo whole-blood culture. Plasma drug and metabolite concentrations were compared to the levels required for inhibition of M. tuberculosis growth and 50% inhibition of mitochondrial protein synthesis. All doses were safe and well tolerated. There were no hematologic or other safety signals during 28 days of dosing at 600 mg twice daily. Plasma concentrations of PNU-100480 and metabolites at this dose remained below those required for 50% inhibition of mitochondrial protein synthesis. Cumulative whole-blood bactericidal activity of PNU-100480 at this dose (-0.316 ± 0.04 log) was superior to the activities of all other doses tested (P < 0.001) and was significantly augmented by pyrazinamide (-0.420 ± 0.06 log) (P = 0.002). In conclusion, PNU-100480 was safe and well tolerated at all tested doses. Further studies in patients with tuberculosis are warranted. Biomarkers can accelerate early development of new tuberculosis treatments.

Biomarkers and diagnostics for tuberculosis: progress, needs, and translation into practice.

Human infection with Mycobacterium tuberculosis can progress to active disease, be contained as latent infection, or be eradicated by the host response. Tuberculosis diagnostics classify a patient into one of these categories. These are not fixed distinct states, but rather are continua along which patients can move, and are affected by HIV infection, immunosuppressive therapies, antituberculosis treatments, and other poorly understood factors. Tuberculosis biomarkers-host or pathogen-specific-provide prognostic information, either for individual patients or study cohorts, about these outcomes. Tuberculosis case detection remains difficult, partly because of inaccurate diagnostic methods. Investments have yielded some progress in development of new diagnostics, although the existing pipeline is limited for tests for sputum-smear-negative cases, childhood tuberculosis, and accurate prediction of reactivation of latent tuberculosis. Despite new, sensitive, automated molecular platforms for detection of tuberculosis and drug resistance, a simple, inexpensive point-of-care test is still not available. The effect of any new tests will depend on the method and extent of their introduction, the strength of the laboratories, and the degree to which access to appropriate therapy follows access to diagnosis. Translation of scientific progress in biomarkers and diagnostics into clinical and public health programmes is possible-with political commitment, increased funding, and engagement of all stakeholders.