A Perspective on the Success and Failure of BCG

TB continues to be one of the major public health threats. BCG is the only available vaccine against TB and confers significant protection against the childhood disease. However, the protective efficacy of BCG against adult pulmonary TB, which represents a larger burden of disease, is highly variable. It has been suggested that prior exposure to environmental mycobacteria (EMb) mitigates the anti-TB efficacy of BCG by blocking its duplication or masking its immunogenicity. However, its effectiveness against childhood TB and failure of repeated administration to provide additional benefit against pulmonary TB, suggest of some other mechanisms for the variable efficacy of BCG against the pulmonary disease. Importantly, TB is a heterogeneous disease occurring in different forms and having distinct mechanisms of pathogenesis. While inability of the immune system to contain the bacilli is responsible for TB pathogenesis in infants, an aggravated immune response to Mtb has been blamed for the development of adult pulmonary TB. Available data suggest that EMb play a key role in heightening the immune response against Mtb. In this article, differential efficacy of BCG against childhood and adult TB is explained by taking into account the heterogeneity of TB, mechanisms of TB pathogenesis, and the effect of EMb on anti-Mtb immunity. It is believed that a refined understanding of the success and failure of BCG will help in the development of effective anti-TB vaccines.

Disseminated Mycobacterium simiae Infection in a Patient with Complete IL-12p40 Deficiency

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare group of genetic disorders characterized by infections with weakly virulent environmental mycobacteria (EM) or Mycobacterium bovis bacillus Calmette-Guérin (BCG). Herein, we described the case of a 4.5-yearold boy with protein-losing enteropathy, lymphoproliferation, and candidiasis, who was found to have disseminated Mycobacterium simiae infection. A homozygous mutation in the IL12B gene, c.527_528delCT (p.S176Cfs*12) was identified, responsible for the complete IL-12p40 deficiency. He was resistant to anti-mycobacterial treatment and finally died due to sepsis-related complications.

Clinical and Molecular Findings in Mendelian Susceptibility to Mycobacterial Diseases: Experience From India

Mendelian Susceptibility to Mycobacterial diseases (MSMD) are a group of innate immune defects with more than 17 genes and 32 clinical phenotypes identified. Defects in the IFN-γ mediated immunity lead to an increased susceptibility to intracellular pathogens like mycobacteria including attenuated Mycobacterium bovis-Bacillus Calmette-Guérin (BCG) vaccine strains and non-tuberculous environmental mycobacteria (NTM), Salmonella, fungi, parasites like Leishmania and some viruses, in otherwise healthy individuals. Mutations in the IL12RB1 gene are the commonest genetic defects identified. This retrospective study reports the clinical, immunological, and molecular characteristics of a cohort of 55 MSMD patients from 10 centers across India. Mycobacterial infection was confirmed by GeneXpert, Histopathology, and acid fast bacilli staining. Immunological workup included lymphocyte subset analysis, Nitro blue tetrazolium (NBT) test, immunoglobulin levels, and flow-cytometric evaluation of the IFN-γ mediated immunity. Genetic analysis was done by next generation sequencing (NGS). Disseminated BCG-osis was the commonest presenting manifestation (82%) with a median age of presentation of 6 months due to the practice of BCG vaccination at birth. This was followed by infection with Salmonella and non-typhi Salmonella (13%), Cytomegalovirus (CMV) (11%), Candida (7%), NTM (4%), and Histoplasma (2%). Thirty-six percent of patients in cohort were infected by more than one organism. This study is the largest cohort of MSMD patients reported from India to the best of our knowledge and we highlight the importance of work up for IL-12/IL-23/ISG15/IFN-γ circuit in all patients with BCG-osis and suspected MSMD irrespective of age.

Defining an intermediate category of tuberculin skin test: A mixture model analysis of two high-risk populations from Kampala, Uganda

One principle of tuberculosis control is to prevent the development of tuberculosis disease by treating individuals with latent tuberculosis infection. The diagnosis of latent infection using the tuberculin skin test is not straightforward because of concerns about immunologic cross reactivity with the Bacille Calmette-Guerin (BCG) vaccine and environmental mycobacteria. To parse the effects of BCG vaccine and environmental mycobacteria on the tuberculin skin test, we estimated the frequency distribution of skin test results in two divisions of Kampala, Uganda, ten years apart. We then used mixture models to estimate parameters for underlying distributions and defined clinically meaningful criteria for latent infection, including an indeterminate category. Using percentiles of two underlying normal distributions, we defined two skin test readings to demarcate three ranges. Values of 10 mm or greater contained 90% of individuals with latent infection; values less than 7.2 mm contained 80% of individuals without infection. Contacts with values between 7.2 and 10 mm fell into an indeterminate zone where it was not possible to assign infection. We conclude that systematic tuberculin skin test surveys within populations at risk, combined with mixture model analysis, may be a reproducible, evidence-based approach to define meaningful criteria for latent tuberculosis infection.

Liver bacterial dysbiosis occurs in SIV-infected macaques and persists during antiretroviral therapy

Background: Liver disease remains a significant contributor to morbidity and mortality in HIV-infected individuals, even during successful treatment with combination antiretroviral therapy (cART). In non-human primates, SIV infection is associated with gut microbiome dysbiosis as well as bacterial translocation into the colonic lamina propria and liver via the portal vein. Here the liver microbiome was evaluated in rhesus macaques to discern the influence of SIV infection alone (SIV+) and during cART administration (SIV+cART) on liver bacterial dysbiosis and neutrophil infiltration.Results: Dysbiosis in liver bacterial composition was observed, encompassing changes in a number of genera, during SIV infection in the absence and presence of cART. The most striking finding was an increase in the level of Mycobacterium, which while barely detectable in the uninfected macaques, was the most abundant genus observed in the livers of a majority SIV+ and SIV+cART macaques. Multi-gene sequencing analyses identified a species of environmental mycobacteria similar to the opportunistic pathogen M. smegmatis. The effect of M. smegmatis on host gene expression in primary hepatocytes was evaluated in vitro utilizing PILAM, a glycolipid cell wall component found in atypical Mycobacteria. PILAM induced an upregulation of inflammatory responses, including an increase in the chemokines associated with neutrophil chemotaxis (CXCL1, CXCL5, and CXCL6). Assessment of the macaque livers by microscopy determined that neutrophil levels were reduced in SIV+cART macaques, suggesting that the SIV infection and/or cART treatment influence the liver-associated neutrophil response. Conclusions: A number of liver bacteria genera were altered following SIV infection even in the context of cART, possibly as a consequence of reduced neutrophil recruitment. Mycobacteria became a major component of the SIV infected macaque liver microbiome, raising the possibility that bacteria of this genus might contribute to liver disease in HIV infected patients.

MmpL3 Inhibition: A New Approach to Treat Nontuberculous Mycobacterial Infections

Outside of Mycobacterium tuberculosis and Mycobacterium leprae, nontuberculous mycobacteria (NTM) are environmental mycobacteria (>190 species) and are classified as slow- or rapid-growing mycobacteria. Infections caused by NTM show an increased incidence in immunocompromised patients and patients with underlying structural lung disease. The true global prevalence of NTM infections remains unknown because many countries do not require mandatory reporting of the infection. This is coupled with a challenging diagnosis and identification of the species. Current therapies for treatment of NTM infections require multidrug regimens for a minimum of 18 months and are associated with serious adverse reactions, infection relapse, and high reinfection rates, necessitating discovery of novel antimycobacterial agents. Robust drug discovery processes have discovered inhibitors targeting mycobacterial membrane protein large 3 (MmpL3), a protein responsible for translocating mycolic acids from the inner membrane to periplasm in the biosynthesis of the mycobacterial cell membrane. This review focuses on promising new chemical scaffolds that inhibit MmpL3 function and represent interesting and promising putative drug candidates for the treatment of NTM infections. Additionally, agents (FS-1, SMARt-420, C10) that promote reversion of drug resistance are also reviewed.

BCG VACCINES MAY NOT REDUCE COVID-19 MORTALITY RATES

ABSTRACTThe reason for the observed country-wise variability in incidence and severity of the COVID-19 outcome remains unknown. Few recent studies have suggested a positive protective correlation of the BCG vaccination policy of the countries with the observed COVID-19 severity. The current study was undertaken to reassess the existing data as of 4th April 2020. The incidence rates (cases per million population), Case Fatality Rates (CFR) and inherently more robust Infection Fatality Rates (IFR) were calculated across countries accounting for about 99% COVID-19 deaths. The initial scrutiny suggested a weaker association with BCG vaccination policy or BCG coverage, so positivity to the Tuberculin Sensitivity Test (TST)/ Interferon Gamma Release Assay (IGRA) as a measure of the potential protective effect of the resident populations’ exposure to Mycobacterium spp. whether from BCG vaccination or as a result of exposure to environmental mycobacteria was analyzed. The incidence rates (the number of cases per million population) decreased with an increase in % LTBI (TST/IGRA positivity) for the analyzed countries with R2 =0.6343, suggesting an exponentially negative covariation. However, the covariation of CFR estimates that ranged from 0.29% to 12.25 % (average 5.39%) among countries, was tenuous. Interim estimates of IFR (i-IFR), a more dependable measure for such studies, for the best and worst-case scenarios, i.e., i-IFR-l and i-IFR-h, predict on an average 20.57% to 30.15 % COVID-19 fatality rates globally, but individual country estimates display huge variation. Among countries accounting for 92.14% deaths (11 countries; top 20% countries included in current study) the estimate for lowest IFRs (i-IFR-l=4.16 (China) & i-IFR-h=4.61 (China)) and highest IFRs (i-IFR-l=96.39% (UK); & i-IFR-h=96.54% (UK)) displayed huge difference (average for the group: CFR=6.8±3.6%; i-IFR-l=34.97±30.55%; & i-IFR-h=44.20±29.08%). Currently, the worst affected countries Italy (CFR=12.25%; i-IFR-l=42.63%; i-IFR-h=48.69%) and Spain (CFR=9.39%; i- IFR-l=26.85%; i-IFR-h=36.60%) would seemingly cope with COVID-19 better than UK, Netherlands and USA while the countries Germany (CFR=1.40%; i-IFR-l=4.93%; i-IFR-h=17.49%) and Switzerland (CFR=3.01%; i-IFR-l=10.87%; i-IFR-h=16.23%) along with China could fare the best. The rest of the 80% countries (accounting for 6.74% deaths), seemed to have reduced mortality (CFR=2.45±2.01; i-IFR-l= 30.62±28.24%; i-IFR-h=40.99±30.47%) with associated high % LTBI (17.28±8.87) than top 20% countries. The inherent issues in the data set (e.g., heterogeneity, non- random sampling, different criteria of sampling and reporting, access to health care, genetic composition, underlying co-morbidities, etc) need to be taken into account for making informed decisions.

A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN–FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.