South Africa

Drug-resistant (DR) tuberculosis (TB) threatens to derail the progress made in global control of TB and HIV. Bedaquiline (Bdq) and pretomanid (Pa) are medications from the first novel TB drug classes created since 1968. Combined with a repurposed medication, linezolid (Lzd), these new drugs have provided substantial improvements in survival and cure rates. The emergence of widespread Bdq, Pa, or Lzd resistance could undermine these drugs’ potential. In the proposed study, we will examine the emergence of Bdq, Pa, and Lzd resistance in South Africa as treatment with these new drugs is expanded to all drug-resistant TB cased. In Aim 1, we will prospectively characterize changes in resistance-conferring mutations for Bdq, Pa, and Lzd using whole genome sequencing (WGS) on isolates from patients in three provinces. In Aim 2, we will examine minimum inhibitory concentrations (MIC) to assess for meaningful shifts in the level of resistance to Bdq, Pa, and Lzd over the course of the study. In Aim 3, we will use WGS, prior treatment exposure, and geospatial analysis to identify increased transmissibility and geographic spread of Bdq, Pa, and Lzd resistance. The proposed study will provide essential information that can inform the development of new rapid diagnostic assays for these critical new TB drugs.

Goals to “eliminate” TB by 2035 are unlikely to be achieved in this century with the currently available control strategies. Development of new therapeutic and prevention tools, such as TB vaccine, is needed, but such efforts are hampered by insufficient understanding of the mechanisms of protection against Mycobacterium tuberculosis (Mtb) infection. In the proposed study, we will enroll a cohort of 4,000 household contacts who have been recently exposed to active TB disease. We will identify contacts who remain uninfected, despite a well-characterized, high degree of exposure to a TB index case, and compare them with household contacts who become infected with Mtb. The study will take place in the high TB incidence settings of India and South Africa. In Aim 1, we will characterize a phenotype for resistance to Mtb infection using responses to both tuberculin skin test (TST) and interferon-gamma release assays (IGRA) in a cohort recently exposed to a culture-confirmed active TB index case. By integrating these TST and IGRA results with rigorous characterization of contacts’ exposure to active TB index cases, we will be able to identify individuals who have resisted Mtb infection despite a high degree of exposure. In Aim 2, we will conduct a genome-wide association study (GWAS) to identify common and rare genetic variants associated with resistance to Mtb infection. We will also investigate the candidate SNPs in previously reported TB-related genetic loci. In Aim 3, we will leverage the emerging field of metabolomics to identify metabolic profiles that distinguish individuals resistant to Mtb infection. Identification of metabolic clusters associated with resistance will reveal cellular pathways involved in resisting or clearing Mtb infection and will also enhance the GWAS findings by providing a functional output of the downstream effects of any genetic polymorphisms. This unbiased and integrated approach will provide an unprecedented opportunity to identify genes and pathways involved in resistance to Mtb infection and understand the multi-layered molecular mechanisms underlying TB infection.

Although efforts to control TB have primarily focused on treating active Tb cases, greater emphasis on preventing new infections will be needed to achieve the End TB goal of fewer than 10 TB cases per 100,000 population by 2035. However, the development of biomedical interventions to prevent TB infection, such as a TB vaccine, has been hampered by a lack of understanding of host factors that mediate susceptibility and resistance to Mycobacterium tuberculosis (Mtb) infection following exposure. A genetic basis for resistance to Mtb infection has long been postulated. Gene expression can also be influenced by epigenetic modifications, such as DNA methylation, which can mediate inherited, behavioral, and co-morbid effects (e.g., smoking, aging, diabetes), and can influence immunological function. Thus, epigenetics may play a pivotal role in a variety of diseases, particularly infectious diseases. In the proposed R21, wewill conduct a hypothesis-generating, epigenome-wide study of >850,000 DNA methylation sites to identify epigenetic predictors of resistance among household contacts who remain uninfected, despite well-characterized exposure to active TB. The proposed R21 will utilize clinical and exposure data and DNA specimens from participants enrolled in the parent R01 study, TB GWAS, making this epigenetic study highly feasible. In Aim 1, we will conduct agnostic searches for epigenomic associations, in addition to targeted analyses of candidate genes. In Aim 2, we will combine GWAS data from the parent R01 and DNA methylation sites identified in Aim 1 to elucidate the causal relationships between epigenetic and resistance using Mendelian Randomization. The findings of this R21 would provide preliminary data to facilitate a future, definitive investigation of epigenetics and resistance to Mtb infection.

Linezolid is recommended for treating drug-resistant TB. Adverse events are a concern to prescribers but have not been systematically studied at the standard dose, and the relationship between linezolid exposure and clinical toxicity is not completely elucidated. We conducted an observational cohort study to describe the incidence and determinants of linezolid toxicity, among patients with rifampicin-resistant TB in South Africa. Linezolid exposures were estimated from a population pharmacokinetic model. Mixed-effects modelling was used to analyze toxicity outcomes. We concluded that permanent discontinuation of linezolid was common, but linezolid-containing therapy was associated with average improvement in toxicity measures. HIV co-infection was not independently associated with linezolid toxicity. Linezolid trough concentration of 2.5 mg/L should be evaluated as a target for therapeutic drug monitoring.

Following exposure to Mycobacterium tuberculosis (Mtb), a coordinated host response comprising both pro-and anti-inflammatory cytokines is critical for pathogen control. Although tuberculosis (TB) remains the leading cause of death among people with human immunodeficiency virus (HIV), the impact of HIV infection on Mtb-specific immune responses remains unclear. In this cross-sectional study of TB-exposed household contacts with and without HIV, we collected remaining supernatant from interferon-gamme release assay (IGRA) testing and measured Mtb-specific pro-inflammatory, anti-inflammatory, and regulatory cytokine responses with a multiplex assay of 11 analytes. This was a cross-sectional sub-study of participants enrolled in a prospective observational cohort study of household contacts of people with pulmonary TB disease in Botshabelo, South Africa. In this retrospective cohort study of TB household contacts, we measured a panel of pro-inflammatory, anti-inflammatory, and regulatory cytokines in IGRA supernatants in order to compare the immune response to TB exposure. Overall, household contacts without HIV had higher cytokine responses than household contacts with HIV, both in response to the mitogen and in response to the TB1 and TB2.