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OBJECTIVE: This study sought to evaluate the utility of the Global Health Security (GHS) index in predicting the launch of COVID-19 vaccine rollout by Organization for Economic Cooperation and Development (OECD) member countries. METHODS: Country-level data on the preparedness to respond to infectious disease threats through vaccination rollout were collected using the GHS index. OECD member countries were rank-ordered based on the percentage of their populations fully vaccinated against COVID-19. Rank-ordering was conducted from the lowest to the highest, with each country assigned a score ranging from 1 to 33. Spearman's rank correlation between the GHS index and the percentage of the population that is fully vaccinated was also performed. RESULTS: Israel, ranked 34th in the world on the GHS index for pandemic preparedness, had the highest percentage of the population that was fully vaccinated against COVID-19 within 2 months of the global vaccine rollout. The Spearman rank correlation coefficient between GHS index and the percentage of population fully vaccinated was -0.1378, with a p-value of 0.43. CONCLUSION: The findings suggest an absence of correlation between the GHS index rating and the COVID-19 vaccine rollout of OECD countries, indicating that the preparedness of OECD countries for infectious disease threats may not be accurately reflected by the GHS index.

Background Although the incidence of tuberculosis is higher in men than in women, the relationship of sex with tuberculosis treatment outcomes has not been adequately studied. Methods We performed a retrospective cohort study and a systematic review and meta-analysis of observational studies during the last 10 years to assess sex differences in clinical and microbiological outcomes in tuberculosis. Results In our cohort of 2894 Taiwanese patients with drug-susceptible pulmonary tuberculosis (1975 male and 919 female), male patients had higher adjusted hazards of 9-month mortality due to all causes (hazard ratio, 1.43 [95% confidence interval (CI), 1.03–1.98]) and infections (1.70 [1.09–2.64]) and higher adjusted odds of 2-month sputum culture positivity (odds ratio [OR], 1.56 [95% CI, 1.05–2.33]) compared with female patients. Smear positivity at 2 months did not differ significantly (OR, 1.27 [95% CI, .71–2.27]) between the sexes. Among 7896 articles retrieved, 398 were included in our systematic review describing a total of 3 957 216 patients. The odds of all-cause mortality were higher in men than in women in the pooled unadjusted (OR, 1.26 [95% CI, 1.19–1.34]) and adjusted (1.31 [1.18–1.45]) analyses. Men had higher pooled odds of sputum culture (OR, 1.44 [95% CI, 1.14–1.81]) and sputum smear (1.58 [1.41–1.77]) positivity, both at the end of the intensive phase and on completion of treatment. Conclusions Our retrospective cohort showed that male patients with tuberculosis have higher 9-month all-cause and infection-related mortality, with higher 2-month sputum culture positivity after adjustment for confounding factors. In our meta-analysis, male patients showed higher all-cause and tuberculosis-related mortality and higher sputum culture and smear positivity rates during and after tuberculosis treatment.

Pediatric tuberculosis (TB) remains a major global health problem. Improved pediatric diagnostics using readily available biosources are urgently needed. We used liquid chromatography-mass spectrometry to analyze plasma metabolite profiles of Indian children with active TB (n = 16) and age- and sex-matched, Mycobacterium tuberculosis-exposed but uninfected household contacts (n = 32). Metabolomic data were integrated with whole blood transcriptomic data for each participant at diagnosis and throughout treatment for drug-susceptible TB. A decision tree algorithm identified 3 metabolites that correctly identified TB status at distinct times during treatment. N-acetylneuraminate achieved an area under the receiver operating characteristic curve (AUC) of 0.66 at diagnosis. Quinolinate achieved an AUC of 0.77 after 1 month of treatment, and pyridoxate achieved an AUC of 0.87 after successful treatment completion. A set of 4 metabolites (gamma-glutamylalanine, gamma-glutamylglycine, glutamine, and pyridoxate) identified treatment response with an AUC of 0.86. Pathway enrichment analyses of these metabolites and corresponding transcriptional data correlated N-acetylneuraminate with immunoregulatory interactions between lymphoid and non-lymphoid cells, and correlated pyridoxate with p53-regulated metabolic genes and mitochondrial translation. Our findings shed new light on metabolic dysregulation in children with TB and pave the way for new diagnostic and treatment response markers in pediatric TB.

Background Hypertension induces systemic inflammation, but its impact on the outcome of infectious diseases like tuberculosis (TB) is unknown. Calcium channel blockers (CCB) improve TB treatment outcomes in preclinical models, but their effect in patients with TB remain unclear. Methods This retrospective cohort study, including all patients > 18 years receiving treatment for culture-confirmed, drug-sensitive TB from 2000 to 2016 at the National Taiwan University Hospital, assessed the association of hypertension and CCB use with all-cause and infection-related mortality during the first 9 months of TB treatment, as well as sputum smear microscopy and sputum culture positivity at 2 and 6 months. Results Of the 2894 patients, 1052 (36.4%) had hypertension. A multivariable analysis revealed that hypertension was associated with increased mortality due to all causes (hazard ratio [HR], 1.57; 95% confidence interval [CI], 1.23–1.99) and infections (HR, 1.87; 95% CI, 1.34–2.6), but there were no statistical differences in microbiological outcomes when stratified based on hypertensive group. Dihydropyridine-CCB (DHP-CCB) use was associated only with reduced all-cause mortality (HR, 0.67; 95% CI, .45–.98) by univariable Cox regression. There were no associations between DHP-CCB use and infection-related mortality (HR, 0.78; 95% CI, .46–1.34) or microbiological outcomes in univariable or multivariable regression analyses. Conclusions Patients with hypertension have increased all-cause mortality and infection-related mortality during the 9 months following TB treatment initiation. DHP-CCB use may lower all-cause mortality in TB patients with hypertension. The presence of hypertension or the use of CCB did not result in a significant change in microbiological outcomes.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

The main advantage of animal models of infectious diseases over in vitro studies is the gain in the understanding of the complex dynamics between the immune system and the pathogen. While small animal models have practical advantages over large animal models, it is crucial to be aware of their limitations. Although the small animal model at least needs to be susceptible to the pathogen under study to obtain meaningful data, key elements of pathogenesis should also be reflected when compared to humans. Well-designed small animal models for HIV, hepatitis viruses and tuberculosis require, additionally, a thorough understanding of the similarities and differences in the immune responses between humans and small animals and should incorporate that knowledge into the goals of the study. To discuss these considerations, the NIAID hosted a workshop on ‘Small Animal Models for HIV, Hepatitis B, and Tuberculosis’ on May 30, 2019. Highlights of the workshop are outlined below.

The chemokine MIP-3α (CCL20) binds to CCR6 on immature dendritic cells. DNA vaccines fusing MIP-3α to melanoma-associated antigens have shown improved efficacy and immunogenicity in the B16F10 mouse melanoma model. Here, we report that the combination of type-I interferon therapy (IFNα) with 5-Aza-2′-deoxycitidine (5Aza) profoundly enhanced the therapeutic efficacy of a MIP-3α-Gp100-Trp2 DNA vaccine. Beginning on day 5 post-transplantation of B16F10 melanoma, vaccine was administered intramuscularly (i.m.) by electroporation. CpG adjuvant was given 2 days later. 5Aza was given intraperitoneally at 1 mg/kg and IFNα therapy either intratumorally or i.m. as noted. Tumor sizes, tumor growth, and mouse survival were assessed. Tumor lysate gene expression levels and tumor-infiltrating lymphocytes (TILs) were assessed by qRT-PCR and flow cytometry, respectively. Adding IFNα and 5Aza treatments to mice vaccinated with MIP-3α-Gp100-Trp2 leads to reduced tumor burden and increased median survival (39% over vaccine and 95% over controls). Tumor lysate expression of CCL19 and CCR7 were upregulated ten and fivefold over vaccine, respectively. Vaccine-specific and overall CD8+ TILs were increased over vaccine (sevenfold and fourfold, respectively), as well as the proportion of TILs that were CD8+ (twofold). Efficient targeting of antigen to immature dendritic cells with a chemokine-fusion vaccine offers an alternative to classic and dendritic cell vaccines. Combining this approach with IFNα and 5Aza treatment significantly improved vaccine efficacy. This improved efficacy correlated with changes in chemokine gene expression and CD8+ TIL infiltration and was dependent on the presence of all therapeutic components.

Mycobacterium avium complex (MAC) is one of the most prevalent causes of nontuberculous mycobacteria pulmonary infection in the United States, yet it remains understudied. Current MAC treatment requires more than a year of intermittent to daily combination antibiotic therapy, depending on disease severity. In order to shorten and simplify curative regimens, it is important to identify the innate bacterial factors contributing to reduced antibiotic susceptibility, namely antibiotic tolerance genes. In this study, we performed a genome-wide transposon screen to elucidate M. avium genes that play a role in the bacterium’s tolerance to first- and second-line antibiotics. We identified a total of 193 unique M. avium mutants with significantly altered susceptibility to at least one of the four clinically used antibiotics we tested, including two mutants (in DFS55_00905 and DFS55_12730) with panhypersusceptibility. The products of the antibiotic tolerance genes we have identified may represent novel targets for future drug development studies aimed at shortening the duration of therapy for MAC infections.ImportanceThe prolonged treatment required to eradicate Mycobacterium avium complex (MAC) infection is likely due to the presence of subpopulations of antibiotic-tolerant bacteria with reduced susceptibility to currently available drugs. However, little is known about the genes and pathways responsible for antibiotic tolerance in MAC. In this study, we performed a forward genetic screen to identify M. avium antibiotic tolerance genes, whose products may represent attractive targets for the development of novel adjunctive drugs capable of shortening curative treatment for MAC infections.

The stringent response is well conserved across bacterial species and is a key pathway involved both in bacterial survival and virulence and in the induction of antibiotic tolerance in Mycobacteria. It is mediated by the alarmone (p)ppGpp and the regulatory molecule inorganic polyphosphate in response to stress conditions such as nutrient starvation. Efforts to pharmacologically target various components of the stringent response have shown promise in modulating mycobacterial virulence and antibiotic tolerance. In this review, we summarize the current understanding of the stringent response and its role in virulence and tolerance in Mycobacteria, including evidence that targeting this pathway could have therapeutic benefit.

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 105TCID50of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFNβ and TNFα, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population.ImportanceMen experience more severe outcomes from COVID-19 than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human clinical isolate of SARS-CoV-2. After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in females than male hamsters. Greater lung pathology during the acute phase combined with reduced antiviral antibody responses during the recovery phase of infection in males than females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection.One Sentence SummaryFollowing SARS-CoV-2 infection, male hamsters experience worse clinical disease and have lower antiviral antibody responses than females.

Background Tuberculosis (TB) treatment is lengthy and complicated and patients often develop chronic lung disease. Recent attention has focused on host-directed therapies aimed at optimizing immune responses to Mycobacterium tuberculosis (Mtb), as adjunctive treatment given with antitubercular drugs. In addition to their cholesterol-lowering properties, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have broad anti-inflammatory and immunomodulatory activities. Methods In the current study, we screened 8 commercially available statins for cytotoxic effect, anti-TB activity, synergy with first-line drugs in macrophages, pharmacokinetics and adjunctive bactericidal activity, and, in 2 different mouse models, as adjunctive therapy to first-line TB drugs. Results Pravastatin showed the least toxicity in THP-1 and Vero cells. At nontoxic doses, atorvastatin and mevastatin were unable to inhibit Mtb growth in THP-1 cells. Simvastatin, fluvastatin, and pravastatin showed the most favorable therapeutic index and enhanced the antitubercular activity of the first-line drugs isoniazid, rifampin, and pyrazinamide in THP-1 cells. Pravastatin modulated phagosomal maturation characteristics in macrophages, phenocopying macrophage activation, and exhibited potent adjunctive activity in the standard mouse model of TB chemotherapy and in a mouse model of human-like necrotic TB lung granulomas. Conclusions These data provide compelling evidence for clinical evaluation of pravastatin as adjunctive, host-directed therapy for TB.

To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

Background: Lipids play a central role in the pathogenesis of tuberculosis (TB). The effect of serum lipid levels on TB treatment (ATT) outcomes and their association with serum inflammatory markers have not yet been characterized.Methods: Our retrospective cohort study on drug-susceptible TB patients, at the National Taiwan University Hospital, assessed the association of baseline serum lipid levels such as low-density lipoprotein (LDL), high-density lipoprotein (HDL), total cholesterol (TC) and triglycerides (TG) with all-cause and infection-related mortality during first 9 months of ATT and baseline inflammatory markers namely C-reactive protein (CRP), total leukocyte count (WBC), and neutrophil-lymphocyte ratio (NL ratio).Results: Among 514 patients, 129 (26.6%) died due to any-cause and 72 (14.0%) died of infection. Multivariable Cox-regression showed a lower adjusted hazard ratio (aHR) of all-cause mortality in the 3rd tertiles of HDL (aHR 0.17, 95% CI 0.07–0.44) and TC (aHR 0.30, 95% CI 0.14-0.65), and lower infection-related mortality in the 3rd tertile of HDL (aHR 0.30, 95% CI 0.14–0.65) and TC (aHR 0.30, 95% CI 0.14–0.65) compared to the 1st tertile. The 3rd tertiles of LDL and TG showed no association in multivariable analysis. Similarly, 3rd tertiles of HDL and TC had lower levels of baseline inflammatory markers such as CRP, WBC, and NL ratio using linear regression analysis. Body mass index (BMI) did not show evidence of confounding or effect modification.Conclusions: Higher baseline serum cholesterol levels were associated with lower hazards of all-cause and infection-related mortality and lower levels of inflammatory markers in TB patients. BMI did not modify or confound this association.

To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

Tuberculosis (TB) accounts for disproportionate morbidity and mortality among persons living with HIV (PLWH). Conventional methods of TB diagnosis, including smear microscopy and Xpert MTB/RIF, have lower sensitivity in PLWH. Novel high-throughput approaches, such as miRNAomics and metabolomics, may advance our ability to recognize subclinical and difficult-to-diagnose TB, especially in very advanced HIV. We conducted a case-control study leveraging REMEMBER, a multi-country, open-label randomized controlled trial comparing 4-drug empiric standard TB treatment with isoniazid preventive therapy in PLWH initiating antiretroviral therapy (ART) with CD4 cell counts

Objective: Tuberculosis (TB) and atherosclerotic cardiovascular disease (ASCVD) have a close epidemiological and pathogenetic overlap. Thus, it becomes essential to understand the relationship between ASCVD and TB outcomes.Methods:From our retrospective cohort on drug-susceptible TB patients at the National Taiwan University Hospital, we assessed the association of pre-existing ASCVD (coronary artery disease (CAD) and atherothrombotic stroke (ATS)) with 9-month all-cause and infection-related mortality and the extent of mediation by systemic inflammatory markers. We determined the effect of pre-existing ASCVD on 2-month sputum microbiological status. Among ASCVD patients, we assessed the association of statin use on mortality.Results:Nine-month all-cause mortality was higher in CAD patients with prior acute myocardial infarction (CAD+AMI+) (adjusted HR 2.01, 95%CI 1.38-3.00) and ATS patients (aHR 2.79, 95%CI 1.92-4.07) and similarly, for infection-related mortality was higher in CAD+AMI+ (aHR 1.95, 95%CI 1.17-3.24) and ATS (aHR 2.04, 95%CI 1.19-3.46) after adjusting for confounding factors. Pre-existing CAD (AMI- or AMI+) or ATS did not change sputum culture conversion or sputum smear AFB positivity at 2 months. The CAD+AMI+ group had significantly higher levels of CRP at TB diagnosis in the multivariable linear regression analysis (Adjusted B(SE) 1.24(0.62)). CRP mediated 66% (p=0.048) and 25% (p=0.033) of the association all-cause mortality with CAD+AMI- and CAD+AMI+, respectively. ConclusionsIn summary, patients with ASCVD have higher hazards of 9-month all-cause and infection-related mortality, with elevated serum inflammation mediating one to three-quarters of this association when adjusted for confounders. Statin use was associated with lower all-cause mortality among patients with ASCVD.

COVID-19 is a novel disease caused by SARS-CoV-2. During the global vaccination rollout, it is vital to thoroughly understand the modes of transmission of the virus in order to prevent further spread of variants and ultimately to end the pandemic. The current literature suggests that SARS-CoV-2 is transmitted among the human population primarily through respiratory droplets and, to a lesser extent, via aerosols. Transmission appears to be affected by temperature, humidity, precipitation, air currents, pH, and radiation in the ambient environment. Finally, the use of masks or facial coverings, social distancing, and hand washing are effective public health strategies in reducing the risk of exposure and transmission. Additional research is needed to further characterize the relative benefits of specific nonpharmaceutical interventions.

RationaleAlthough the incidence of tuberculosis (TB) is higher in males compared to females, the relationship of sex with TB treatment outcomes has not been adequately studied.Objectives and MethodsWe performed a retrospective cohort study and a systematic review and meta-analysis of observational studies during the last 10 years to assess the sex differences in clinical and microbiological outcomes in tuberculosis.Measurements and Main ResultsIn our cohort of 2,894 patients with drug-susceptible pulmonary TB (1,975 males and 919 females), males had higher adjusted hazards of mortality due to all causes (HR 1·43,95%CI 1.03-1.98) and infections (HR 1.70, 95%CI 1.09-2.64) at 9 months and higher adjusted odds ratio for sputum culture (OR 1.56,95%CI 1.05-2.33) and similar odds ratio for smear positivity (OR 1.27, 0.71-2.27) at 2 months compared to females. Among 7896 articles retrieved, 398 articles were included in our systematic review with a total of 3,957,216 patients. The odds of all-cause mortality was higher in males compared to females in the pooled unadjusted (OR 1.26, 95%CI 1.19-1.34) and adjusted (OR 1.31, 95%CI 1.18-1.45) analyses. Relative to females, males had higher pooled odds of sputum culture (OR 1.44,95% CI 1.14-1.81) and sputum smear (OR 1.58,95%CI 1.41-1.77) positivity at the end of the intensive phase, and upon completion of treatment.ConclusionsDuring TB treatment, males have higher all-cause-, infection- and TB-related mortality, as well as higher rates of sputum smear and culture positivity, both after the intensive phase and at the completion of TB treatment, after adjusting for confounding factors.

Mycobacterium avium complex (MAC) is an increasingly important cause of morbidity and mortality, and is responsible for pulmonary infection in patients with underlying lung disease and disseminated disease in patients with AIDS. MAC has evolved various virulence strategies to subvert immune responses and persist in the infected host. Current treatment for MAC is challenging, requiring a combination of multiple antibiotics given over a long time period (for at least 12 months after negative sputum culture conversion). Moreover, even after eradication of infection, many patients are left with residual lung dysfunction. In order to address similar challenges facing the management of patients with tuberculosis, recent attention has focused on the development of novel adjunctive, host-directed therapies (HDTs), with the goal of accelerating the clearance of mycobacteria by immune defenses and reducing or reversing mycobacterial-induced lung damage. In this review, we will summarize the evidence supporting specific adjunctive, HDTs for MAC, with a focus on the repurposing of existing immune-modulatory agents targeting a variety of different cellular pathways. We also highlight areas meriting further investigation.

Background: Given the relatively modest benefit of currently available treatments in improving COVID-19 clinical outcomes, there is an urgent need for novel therapeutic strategies, especially in reversing associated lung inflammation. Attention has focused on repurposing existing agents with immune modulatory properties to accelerate their incorporation into clinical practice. Recent evidence has demonstrated that the cholesterol-lowering agents, statins, are associated with reduced mortality in patients with influenza, sepsis, community-acquired pneumonia, and chronic obstructive pulmonary diseases (COPD). We sought to investigate the relationship between statin use and COVID-19 disease severity in hospitalized patients. Methods: A retrospective analysis of COVID-19 patients admitted to the Johns Hopkins Medical Institutions between March 1, 2020 and June 30, 2020 was performed. The outcomes of interest were mortality and severe COVID-19 infection, as defined by prolonged hospital stay (≥ 7 days) and/ or invasive mechanical ventilation. Logistic regression, Cox proportional hazards regression and propensity score matching were used to obtain both univariable and multivariable associations between covariates and outcomes in addition to the average treatment effect of statin use. Results: Of the 4,447 patients who met our inclusion criteria, 594 (13.4%) patients were exposed to statins on admission, of which 340 (57.2%) were male. The mean age was higher in statin users compared to non-users [64.9 ± 13.4 vs. 45.5 ± 16.6 years, p

Statins are a class of drugs that inhibit HMG-CoA reductase, the rate-limiting step in cholesterol biosynthesis, and are commonly used for primary and secondary prevention of cardiovascular disease. In addition to lowering cholesterol, statins also have broad immunomodulatory properties, and their use has been associated with improved clinical outcomes in patients with community-acquired pneumonia and sepsis. Multiple retrospective cohort studies have shown that statin use is associated with reduced tuberculosis (TB) incidence. Although at clinically recommended drug exposures they lack direct activity against Mycobacterium tuberculosis (Mtb), statins enhance the ability of Mtb-infected macrophages to clear intracellular infection by promoting autophagy and phagosome maturation. Recent data have revealed that these biological effects are dependent on inhibition of cholesterol biosynthesis and are mediated through the AMPK-mTORC1-TFEB-autophagy axis. Animal model studies have supported a role for statin adjunctive therapy against TB. Thus, the addition of simvastatin to the first-line antitubercular regimen reduced the duration of curative treatment by one month in the standard mouse model of chronic tuberculosis (TB), and pravastatin similarly showed potent, dose-dependent adjunctive activity in this model, as well as in a novel mouse model with human-like necrotic TB lung granulomas. An ongoing randomized clinical trial, Statin Adjunctive Therapy for TB (StAT-TB) is evaluating the safety, pharmacokinetics and adjunctive activity of pravastatin in patients with drug-susceptible, pulmonary TB.

Mycobacterium avium complex (MAC) species are the most commonly isolated nontuberculous mycobacteria to cause pulmonary infections worldwide. The lengthy and complicated therapy required to cure lung disease due to MAC is at least in part due to the phenomenon of antibiotic tolerance. In this review, we will define antibiotic tolerance and contrast it with persistence and antibiotic resistance. We will discuss physiologically relevant stress conditions that induce altered metabolism and antibiotic tolerance in mycobacteria. Next, we will review general molecular mechanisms underlying bacterial antibiotic tolerance, particularly those described for MAC and related mycobacteria, including Mycobacterium tuberculosis, with a focus on genes containing significant sequence homology in MAC. An improved understanding of antibiotic tolerance mechanisms can lay the foundation for novel approaches to target antibiotic-tolerant mycobacteria, with the goal of shortening the duration of curative treatment and improving survival in patients with MAC.

BackgroundHypertension induces systemic inflammation, but its impact on the outcome of infectious diseases like tuberculosis (TB) is unknown. Calcium channel blockers (CCB) improve TB treatment outcomes in pre-clinical models, but their effect in patients with TB remain unclear.MethodsThis retrospective cohort study, including all patients > 18 years receiving treatment for culture-confirmed, drug-sensitive TB from 2000 to 2016 at the National Taiwan University Hospital, assessed the association of hypertension and CCB use with all-cause and infection-related mortality during the first 9 months of TB treatment, as well as sputum-smear microscopy and sputum-culture positivity at 2 and 6 months.Results1052 of the 2894 patients (36.4%) had hypertension. Multivariable analysis revealed that hypertension was associated with increased mortality due to all causes (HR 1.57, 95% confidence interval[CI], 1.23-1.99) and infections (HR 1.87, 95%CI, 1.34-2.6), but there was no statistical difference in microbiological outcomes when stratified based on hypertensive group. Dihydropyridine-CCB (DHP-CCB) use was associated with reduced all-cause mortality (HR 0.67, 95%CI: 0.45-0.98) only by univariate Cox regression. There was no association between DHP-CCB use and infection-related mortality (HR 0.78, 95%CI: 0.46-1.34) or microbiological outcomes in univariate or multivariate regression analyses.ConclusionsPatients with hypertension have increased all-cause mortality and infection-related mortality during the 9 months following TB treatment initiation. DHP-CCB use may lower all-cause mortality in TB patients with hypertension. The presence of hypertension or the use of CCB did not result in a significant change in microbiological outcomes.

BackgroundUnderstanding the factors associated with disease severity and mortality in Coronavirus disease (COVID-19) is imperative to effectively triage patients. We performed a systematic review to determine the demographic, clinical, laboratory and radiological factors associated with severity and mortality in COVID-19.MethodsWe searched PubMed, Embase and WHO database for English language articles from inception until May 8, 2020. We included Observational studies with direct comparison of clinical characteristics between a) patients who died and those who survived or b) patients with severe disease and those without severe disease. Data extraction and quality assessment were performed by two authors independently.ResultsAmong 15680 articles from the literature search, 109 articles were included in the analysis. The risk of mortality was higher in patients with increasing age, male gender (RR 1.45, 95%CI 1.23–1.71), dyspnea (RR 2.55, 95%CI 1.88–2.46), diabetes (RR 1.59, 95%CI 1.41–1.78), hypertension (RR 1.90, 95%CI 1.69–2.15). Congestive heart failure (OR 4.76, 95%CI 1.34–16.97), hilar lymphadenopathy (OR 8.34, 95%CI 2.57–27.08), bilateral lung involvement (OR 4.86, 95%CI 3.19–7.39) and reticular pattern (OR 5.54, 95%CI 1.24–24.67) were associated with severe disease. Clinically relevant cut-offs for leukocytosis(>10.0 ×109/L), lymphopenia(< 1.1 ×109/L), elevated C-reactive protein(>100mg/L), LDH(>250U/L) and D-dimer(>1mg/L) had higher odds of severe disease and greater risk of mortality.ConclusionKnowledge of the factors associated of disease severity and mortality identified in our study may assist in clinical decision-making and critical-care resource allocation for patients with COVID-19.Primary Funding SourceNone.

The lengthy and complicated current regimen required to treat drug-susceptible tuberculosis (TB) reflects the ability of Mycobacterium tuberculosis (Mtb) to persist in host tissues. The stringent response pathway, governed by the dual (p)ppGpp synthetase/hydrolase, RelMtb, is a major mechanism underlying Mtb persistence and antibiotic tolerance. In the current study, we addressed the hypothesis that RelMtb is a “persistence antigen” presented during TB chemotherapy and that enhanced immunity to RelMtb can enhance the tuberculocidal activity of the first-line anti-TB drug, isoniazid, which has reduced efficacy against Mtb persisters. C57BL/6 mice and Hartley guinea pigs were aerosol-infected with Mycobacterium tuberculosis (Mtb) and, 4 weeks later, received either human-equivalent daily doses of isoniazid alone, or isoniazid in combination with a DNA vaccine targeting relMtb. After isoniazid treatment, there was a significant reduction in dominant antigen ESAT6-specific CD4+ or TB10.4-specific CD8+ T cells in the lungs and spleens of mice. However, the total number of RelMtb-specific CD4+ T cells remained stable in mouse lungs and spleens, as did the number of RelMtb-specific CD8+T cells. Therapeutic vaccination with relMtb DNA vaccine enhanced the activity of isoniazid in Mtb-infected C57BL/6 mice and guinea pigs. When treatment with isoniazid was discontinued, mice immunized with the relMtb DNA vaccine showed a lower mean lung bacterial burden at relapse compared to the control group. Our work shows that antitubercular treatment shapes the antigenic environment, and that therapeutic vaccination targeting the Mtb stringent response may represent a novel approach to enhance immunity against Mtb persisters, with the ultimate goal of shortening curative TB treatment.

Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. We investigated the mechanism of anti-tubercular action of simvastatin in Mycobacterium tuberculosis-infected human monocytic cells. We found that the anti-tubercular activity of statins was phenocopied by cholesterol-branch but not prenylation-branch inhibitors. Moreover, statin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Overall, our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, AMPK-mTORC1-TFEB axis, and intracellular infection control, and uncover new anti-tubercular therapy targets.

A long duration of treatment and emerging drug resistance pose significant challenges for global tuberculosis (TB) eradication efforts. Therefore, there is an urgent need to develop novel strategies to shorten TB treatment regimens and to treat drug-resistant TB. Using an albumin-fusion strategy, we created a novel albumin-fused granulocyte-macrophage colony-stimulating factor (albGM-CSF) molecule that harnesses albumin’s long half-life and targeting abilities to enhance the biostability of GM-CSF and direct it to the lymph nodes, where the effects of GM-CSF can increase dendritic cell populations crucial for eliciting a potent immune response. In this study, we demonstrate that albGM-CSF serves as a novel immunotherapy for chronic Mycobacterium tuberculosis (Mtb) infections by enhancing GM-CSF biostability in serum. Specifically, albumin is very safe, stable, and has a long half-life, thereby enhancing the biostability of GM-CSF. In the lungs and draining lymph nodes, albGM-CSF is able to increase the numbers of dendritic cells, which are crucial for the activation of naive T cells and for eliciting potent immune responses. Subcutaneous administration of albGM-CSF alone reduced the mean lung bacillary burden in mice with chronic tuberculosis infection. While GM-CSF administration was associated with IL-1β release from Mtb-infected dendritic cells and macrophages, higher IL-1β levels were observed in albGM-CSF-treated mice with chronic tuberculosis infection than in mice receiving GM-CSF. Albumin fusion with GM-CSF represents a promising strategy for the control of chronic lung tuberculosis infections and serves as a novel therapeutic vaccination platform for other infectious diseases and malignancies.

The ongoing COVID-19 pandemic has devastated many countries with ripple effects felt in various sectors of the global economy. In November 2019, the Global Health Security (GHS) Index was released as the first detailed assessment and benchmarking of 195 countries to prevent, detect, and respond to infectious disease threats. This paper presents the first comparison of Organization for Economic Cooperation and Development OECD countries’ performance during the pandemic, with the pre-COVID-19 pandemic preparedness as determined by the GHS Index. Using a rank-based analysis, four indices were compared between select countries, including total cases, total deaths, recovery rate, and total tests performed, all standardized for comparison. Our findings suggest a discrepancy between the GHS index rating and the actual performance of countries during this pandemic, with an overestimation of the preparedness of some countries scoring highly on the GHS index and underestimation of the preparedness of other countries with relatively lower scores on the GHS index.

Background Understanding the factors associated with disease severity and mortality in Coronavirus disease (COVID-19) is imperative to effectively triage patients. We performed a systematic review to determine the demographic, clinical, laboratory and radiological factors associated with severity and mortality in COVID-19. Methods We searched PubMed, Embase and WHO database for English language articles from inception until May 8, 2020. We included Observational studies with direct comparison of clinical characteristics between a) patients who died and those who survived or b) patients with severe disease and those without severe disease. Data extraction and quality assessment were performed by two authors independently. Results Among 15680 articles from the literature search, 109 articles were included in the analysis. The risk of mortality was higher in patients with increasing age, male gender (RR 1.45, 95%CI 1.23–1.71), dyspnea (RR 2.55, 95%CI 1.88–2.46), diabetes (RR 1.59, 95%CI 1.41–1.78), hypertension (RR 1.90, 95%CI 1.69–2.15). Congestive heart failure (OR 4.76, 95%CI 1.34–16.97), hilar lymphadenopathy (OR 8.34, 95%CI 2.57–27.08), bilateral lung involvement (OR 4.86, 95%CI 3.19–7.39) and reticular pattern (OR 5.54, 95%CI 1.24–24.67) were associated with severe disease. Clinically relevant cut-offs for leukocytosis(>10.0 x109/L), lymphopenia(< 1.1 x109/L), elevated C-reactive protein(>100mg/L), LDH(>250U/L) and D-dimer(>1mg/L) had higher odds of severe disease and greater risk of mortality. Conclusion Knowledge of the factors associated of disease severity and mortality identified in our study may assist in clinical decision-making and critical-care resource allocation for patients with COVID-19.

Background: Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase the efficacy of anti-tubercular antibiotics in animal models. A previous report proposed that the anti-tubercular activity of statins was associated with induction of autophagy and resided in the ability of these drugs to inhibit protein prenylation. Methods: We used an in vitro human macrophage infection model to inhibit each branch of the mevalonate pathway and evaluate treatment effects on mycobacterial burden. The role of the AMP-activated protein kinase (AMPK) - mechanistic target of rapamycin complex 1 (mTORC1) - transcription factor EB (TFEB)-autophagy axis in simvastatin anti-tubercular activity was determined by western blot analyses, transcriptomics, use of pathway-specific compounds, AMP:ATP assay, and addition of exogenous cholesterol to pharmacological treatments. Findings: We found that the anti-tubercular activity of statins was phenocopied by inhibitors of cholesterol biosynthesis rather than by prenylation inhibitors. Simvastatin treatment blocked mTORC1 activation, activated AMPK through increased intracellular AMP:ATP ratios, and favored TFEB nuclear translocation. These mechanisms all induce autophagy, which is anti-mycobacterial. The simvastatin effects on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Interpretation: Our data demonstrate that the anti-tubercular activity of simvastatin resides in inhibiting cholesterol biosynthesis. Our work also reveals novel links between cholesterol homeostasis, AMPK-mTORC1-TFEB axis, and intracellular infection control, and uncovers new anti-tubercular therapy targets. Funding Statement: NIH grants to PK (AI122309) and MLG (HL149450, AI104615, UL1TR003017); New Jersey Health Foundation grant to MLG (PC10-15). Declaration of Interests: The authors declare no competing interests.

BackgroundThere is an urgent need for novel therapeutic strategies for reversing COVID-19-related lung inflammation. Recent evidence has demonstrated that the cholesterol-lowering agents, statins, are associated with reduced mortality in patients with various respiratory infections. We sought to investigate the relationship between statin use and COVID-19 disease severity in hospitalized patients.MethodsA retrospective analysis of COVID-19 patients admitted to the Johns Hopkins Medical Institutions between March 1, 2020 and June 30, 2020 was performed. The outcomes of interest were mortality and severe COVID-19 infection, as defined by prolonged hospital stay (≥ 7 days) and/ or invasive mechanical ventilation. Logistic regression, Cox proportional hazards regression and propensity score matching were used to obtain both univariable and multivariable associations between covariates and outcomes in addition to the average treatment effect of statin use.ResultsOf the 4,447 patients who met our inclusion criteria, 594 (13.4%) patients were exposed to statins on admission, of which 340 (57.2%) were male. The mean age was higher in statin users compared to non-users [64.9 ± 13.4 vs. 45.5 ± 16.6 years, p ConclusionStatin use was not associated with altered mortality, but with an 18% increased risk of severe COVID-19 infection.

The lengthy and complicated current regimen required to treat drug-susceptible tuberculosis (TB) reflects the ability of Mycobacterium tuberculosis (Mtb) to persist in host tissues. The stringent response pathway, governed by the dual (p)ppGpp synthetase/hydrolase, RelMtb, is a major mechanism underlying Mtb persistence and antibiotic tolerance. In the current study, we addressed the hypothesis that RelMtb is a “persistence antigen” presented during TB chemotherapy and that enhanced T-cell immunity to RelMtb can enhance the tuberculocidal activity of the first-line anti-TB drug, isoniazid, which has reduced efficacy against Mtb persisters, C57BL/6 mice and Hartley guinea pigs were aerosol-infected with Mycobacterium tuberculosis (Mtb) and, 4 weeks later, received either human-equivalent daily doses of isoniazid alone, or isoniazid in combination with a DNA vaccine targeting relMtb. After isoniazid treatment, the total number of Mtb antigen-specific CD4+ T cells remained stable in mouse lungs and spleens, as did the number of RelMtb-specific T cells, although there was a significant reduction in dominant antigen ESAT6-specific CD4+ or TB10.4-specific CD8+ T cells in the lungs and spleens of mice, Therapeutic vaccination enhanced the activity of isoniazid in Mtb-infected C57BL/6 mice and guinea pigs. When treatment with isoniazid was discontinued, mice immunized with the relMtb DNA vaccine showed a lower mean lung bacterial burden at relapse compared to the control group. Our work shows that antitubercular treatment shapes antigen presentation and antigen-specific T-cell responses, and that therapeutic vaccination targeting the Mtb stringent response may represent a novel approach to enhance immunity against Mtb persisters, with the ultimate goal of shortening curative TB treatment.

The stringent response enables Mycobacterium tuberculosis (Mtb) to shut down its replication and metabolism under various stresses. Here we show that Mtb lacking the stringent response enzyme RelMtb was unable to slow its replication rate during nutrient starvation. Metabolomics analysis revealed that the nutrient-starved relMtb-deficient strain had increased metabolism similar to that of exponentially growing wild-type bacteria in nutrient-rich broth, consistent with an inability to enter quiescence. Deficiency of relMtb increased the susceptibility of mutant bacteria to killing by isoniazid during nutrient starvation and in the lungs of chronically infected mice. We screened a pharmaceutical library of over 2 million compounds for inhibitors of RelMtb and showed that the lead compound X9 was able to directly kill nutrient-starved M. tuberculosis and enhanced the killing activity of isoniazid. Inhibition of RelMtb is a promising approach to target M. tuberculosis persisters, with the potential to shorten the duration of TB treatment.

Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis , while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1 -deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2 , together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE , did not show protective activity against aerosol challenge with M. tuberculosis , but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo .

The turnaround times for conventional methods used to detect Mycobacterium tuberculosis in sputum samples and to obtain drug susceptibility information are long in many developing countries, including Panama, leading to delays in appropriate treatment initiation and continued transmission in the community. We evaluated the performance of a molecular line probe assay, the Genotype MTBDR plus version 2.0 assay, in detecting M. tuberculosis complex directly in respiratory specimens from smear-positive tuberculosis cases from four different regions in Panama, as well as the most frequent mutations in genes conferring resistance to isoniazid ( katG and inhA ) and rifampin ( rpoB ). Our results were confirmed with the nitrate reductase assay and genomic sequencing. M. tuberculosis complex was detected by the Genotype MTBDR plus 2.0 assay with 100% sensitivity and specificity. The sensitivity and specificity for rifampin resistance were 100% and 100%, respectively, and those for isoniazid resistance were 90.7% and 100%. Isoniazid monoresistance was detected in 5.2% of new cases. Genotype MTBDR plus 2.0 is highly accurate in detecting M. tuberculosis complex in respiratory specimens and is able to discriminate isoniazid-monoresistant cases from multidrug-resistant cases within 2 days.

Novel drugs are required to shorten the duration of treatment for tuberculosis (TB) and to combat the emergence of drug resistance. One approach has been to identify and target Mycobacterium tuberculosis (Mtb) virulence factors, which promote the establishment of TB infection and pathogenesis. Mtb produces a number of virulence factors, including two protein tyrosine phosphatases (PTPs), mPTPA and mPTPB, to evade the antimicrobial functions of host macrophages. To assess the therapeutic potential of targeting the virulent Mtb PTPs, we developed highly potent and selective inhibitors of mPTPA (L335-M34) and mPTPB (L01-Z08) with drug-like properties. We tested the bactericidal activity of L335-M34 and L01-Z08 alone or together in combination with the standard antitubercular regimen of isoniazid-rifampicin-pyrazinamide (HRZ) in the guinea pig model of chronic TB infection, which faithfully recapitulates some of the key histological features of human TB lesions. Following a single dose of L335-M34 50mg/kg and L01-Z08 20 mg/kg, plasma levels were maintained at levels 10-fold greater than the biochemical IC50 for 12–24 hours. Although neither PTP inhibitor alone significantly enhanced the antibacterial activity of HRZ, dual inhibition of mPTPA and mPTPB in combination with HRZ showed modest synergy, even after 2 weeks of treatment. After 6 weeks of treatment, the degree of lung inflammation correlated with the bactericidal activity of each drug regimen. This study highlights the potential utility of targeting Mtb virulence factors, and specifically the Mtb PTPs, as a strategy for enhancing the activity of standard anti-TB treatment.

Multidrug-resistant tuberculosis (TB), defined as TB resistant to the two first-line drugs, isoniazid and rifampin, is a serious challenge to global TB eradication efforts. Although mutations in rpoA or rpoC have been proposed to compensate for this fitness cost due to rpoB mutation in rifampicin-resistant Mycobacterium tuberculosis mutants, whether the compensatory effect exists and the underlying mechanisms of compensation remain unclear. Here, we used RNA sequencing to investigate the global transcriptional profiles of 6 rifampin-resistant clinical isolates with either single mutation in rpoB or dual mutations in rpoB/rpoC, as well as 3 rifampin-susceptible clinical isolates, trying to prove the potential compensatory effect of rpoC by transcriptomic alteration. In rifampin-free conditions, rpoC mutation was associated with M. tuberculosis upregulation of ribosomal protein-coding genes, dysregulation of growth-related essential genes and balancing the expression of arginine and glutamate synthesis-associated genes. Upon rifampin exposure of M. tuberculosis isolates, rpoC mutations were associated with the upregulation of the oxidative phosphorylation machinery, which was inhibited in the rpoB single mutants, as well as stabilization of the expression of rifampin-regulated essential genes and balancing the expression of genes involved in metabolism of sulfur-containing amino acids. Taken together, our data suggest that rpoC mutation may compensate for the fitness defect of rifampicin-resistant M. tuberculosis by altering gene expression in response to rifampin exposure.

New therapeutics to augment current approaches and shorten treatment duration are of critical importance for combating tuberculosis (TB), especially those with novel mechanisms of action to counter the emergence of drug-resistant TB. Host-directed therapy (HDT) offers a novel strategy with mechanisms that include activating immune defense mechanisms or ameliorating tissue damage. These and related concepts will be discussed along with issues that emerged from the workshop organized by the Stop TB Working Group on New Drugs, held at the Gordon Research Conference for Tuberculosis Drug Development in Lucca, Italy in June 2017, titled "Strategic Discussion on Repurposing Drugs & Host Directed Therapies for TB." In this review, we will highlight recent data regarding drugs, pathways, and concepts that are important for successful development of HDTs for TB.

The development of antibiotic tolerance is believed to be a major factor in the lengthy duration of current tuberculosis therapies. In the current study, we have modeled antibiotic tolerance in vitro by exposing Mycobacterium tuberculosis to two distinct stress conditions: progressive hypoxia and nutrient starvation [phosphate-buffered saline (PBS)]. We then studied the bacterial transcriptional response using RNA-seq and employed a bioinformatics approach to identify important transcriptional regulators, which was facilitated by a novel Regulon Enrichment Test (RET). A total of 17 transcription factor (TF) regulons were enriched in the hypoxia gene set and 16 regulons were enriched in the nutrient starvation, with 12 regulons enriched in both conditions. Using the same approach to analyze previously published gene expression datasets, we found that three M. tuberculosis regulons (Rv0023, SigH, and Crp) were commonly induced in both stress conditions and were also among the regulons enriched in our data. These regulators are worthy of further study to determine their potential role in the development and maintenance of antibiotic tolerance in M. tuberculosis following stress exposure.

Background: Mycobacterium tuberculosis (Mtb) rpoB mutations are associated with global metabolic remodeling. However, the net effects of rpoB mutations on Mtb physiology, metabolism and function are not completely understood. Based on previous work, we hypothesized that changes in the expression of cell wall molecules in Mtb mutant RpoB 526D lead to changes in cell wall permeability and to altered resistance to environmental stresses and drugs. Methods: The phenotypes of a fully drug-susceptible clinical strain of Mtb and its paired rifampin-monoresistant, RpoB H526D mutant progeny strain were compared. Results: The rpoB mutant showed altered colony morphology, bacillary length and cell wall thickness, which were associated with increased cell wall permeability and susceptibility to the cell wall detergent sodium dodecyl sulfate (SDS) after exposure to nutrient starvation. Relative to the isogenic rifampin-susceptible strain, the RpoB H526D mutant showed altered bacterial cellular metabolic activity and an eightfold increase in susceptibility to the cell-wall acting drug vancomycin. Conclusion: Our data suggest that RpoB mutation H526D is associated with altered cell wall physiology and resistance to cell wall-related stress. These findings are expected to contribute to an improved understanding of the pathogenesis of drug-resistant M. tuberculosis infections.

Background: The long duration of treatment and emerging drug resistance pose significant challenges for global tuberculosis eradication efforts. Therefore, novel strategies to shorten TB treatment regimens are urgently needed. Methods: Using an albumin fusion strategy, we created an albumin-fused granulocyte-macrophage colony stimulating factor (named albGM-CSF) novel molecule that harnesses albumin's long-half life and targeting abilities to enhance the biostability of GM-CSF and direct it to the lymph nodes where the effects of GM-CSF can increase dendritic cell populations crucial to eliciting a potent immune response. Findings: In our study, we found that subcutaneous administration of albGM-CSF reduces the mean lung bacillary burden in mice chronically-infected with tuberculosis. This effect was dependent on both innate immunity and CD4 T cells. GM-CSF induced IL-1β release from Mtb-infected dendritic cells and macrophages, and higher IL-1β levels were observed in albGM-CSF-treated mice with chronic TB infection. Interpretation: Albumin fusion with GM-CSF represents a promising tissue-targeting strategy for controlling chronic lung TB infections and serves as a novel in situ therapeutic vaccination platform for other infectious diseases and malignancies. Funding Statement: This research was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R21AI22922. Declarations of Interest: The authors declare no conflict of interest. Ethics Approval Statement: All procedures were performed according to the Johns Hopkins Institutional Animal Care and Use Committee and in accordance with recommendations for proper use and care of laboratory animals.

The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our understanding of the biological role of miRNAs involved in the disease. Datasets deposited in publically available databases from microarray studies on infectious diseases and malignancies were retrieved, screened, and subjected to further analysis. Effect sizes were combined using the inverse-variance model and between-study heterogeneity was evaluated by the random effects model. 35 miRNAs were differentially expressed (12 up-regulated, 23 down-regulated; p

Tuberculosis (TB) is a communicable disease of major global importance and causes metabolic disorder of the patients. In a previous study, we found that the plasma metabolite profile of TB patients differs from that of healthy control subjects based on nuclear magnetic resonance (NMR) spectroscopy. In order to evaluate the TB specificity of the metabolite profile, a total of 110 patients, including 40 with diabetes, 40 with malignancy, and 30 with community-acquired pneumonia (CAP), assessed by NMR spectroscopy, and compared to those of patients with TB. Based on the orthogonal partial least-squares discriminant analysis (OPLS-DA), the metabolic profiles of these diseases were significant different, as compared to the healthy controls and TB patients, respectively. The score plots of the OPLS-DA model demonstrated that TB was easily distinguishable from diabetes, CAP and malignancy. Plasma levels of ketone bodies, lactate, and pyruvate were increased in TB patient compared to healthy control, but lower than CAP and malignancy. We conclude that the metabolic profiles were TB-specific and reflected MTB infection. Our results strongly support the NMR spectroscopy-based metabolomics could contribute to an improved understanding of disease mechanisms and may offer clues to new TB clinic diagnosis and therapies.

Mycobacterium tuberculosis (Mtb) remains a global health challenge due to the limited efficacy of the Mtb vaccine in current use, Bacillus Calmette–Guérin (BCG). To date, there is no available vaccine for immunocompromised individuals. Thus, there is an urgent need to develop a new vaccine candidate which can induce mucosal immunity in hosts with different immune statuses. DnaK (HSP70) has been shown to induce protective immunity against Mtb infection when administered by DNA vaccine; however, the protection is inferior to that induced by the BCG vaccine. In our study, we vaccinated C57BL/6J mice with DnaK protein alone. Subcutaneous or intranasal vaccination with DnaK generated IFNγ-secreting CD4+ T cells in the spleen, but only intranasal vaccination generated IL-17-releasing CD4+ T cells in the lungs, even when circulating CD4+ T cells were diminished. Furthermore, intranasal vaccination with DnaK generated tissue resident CD4+ T cells in the lungs. Vaccination with DnaK alone resulted in protective immunity comparable to BCG vaccination against tuberculosis in mice. Our results demonstrate that intranasal vaccination with DnaK can generate mucosal immunity in immunocompromised or immunocompetent mice and DnaK vaccination can generate protection against Mtb similar to BCG, underscoring its potential utility as an Mtb vaccine candidate in humans.