Bayesian reconstruction of Mycobacterium tuberculosis transmission networks in a high incidence area over two decades in Malawi reveals associated risk factors and genomic variants.

Understanding host and pathogen factors that influence tuberculosis (TB) transmission can inform strategies to eliminate the spread of Mycobacterium tuberculosis (Mtb). Determining transmission links between cases of TB is complicated by a long and variable latency period and undiagnosed cases, although methods are improving through the application of probabilistic modelling and whole-genome sequence analysis. Using a large dataset of 1857 whole-genome sequences and comprehensive metadata from Karonga District, Malawi, over 19 years, we reconstructed Mtb transmission networks using a two-step Bayesian approach that identified likely infector and recipient cases, whilst robustly allowing for incomplete case sampling. We investigated demographic and pathogen genomic variation associated with transmission and clustering in our networks. We found that whilst there was a significant decrease in the proportion of infectors over time, we found higher transmissibility and large transmission clusters for lineage 2 (Beijing) strains. By performing evolutionary convergence testing (phyC) and genome-wide association analysis (GWAS) on transmitting versus non-transmitting cases, we identified six loci, PPE54, accD2, PE_PGRS62, rplI, Rv3751 and Rv2077c, that were associated with transmission. This study provides a framework for reconstructing large-scale Mtb transmission networks. We have highlighted potential host and pathogen characteristics that were linked to increased transmission in a high-burden setting and identified genomic variants that, with validation, could inform further studies into transmissibility and TB eradication

Reliability of reporting of HIV status and antiretroviral therapy usage during verbal autopsies: a large prospective study in rural Malawi.

OBJECTIVE: Verbal autopsies (VAs) are interviews with a relative or friend of the deceased; VAs are a technique used in surveillance sites in many countries with incomplete death certification. The goal of this study was to assess the accuracy and validity of data on HIV status and antiretroviral therapy (ART) usage reported in VAs and their influence on physician attribution of cause of death. DESIGN: This was a prospective cohort study. METHODS: The Karonga Health and Demographic Surveillance Site monitors demographic events in a population in a rural area of northern Malawi; a VA is attempted on all deaths reported. VAs are reviewed by clinicians, who, with additional HIV test information collected pre-mortem, assign a cause of death. We linked HIV/ART information reported by respondents during adult VAs to database information on HIV testing and ART use and analysed agreement using chi-square and kappa statistics. We used multivariable logistic regression to analyse factors associated with agreement. RESULTS: From 2003 to 2014, out of a total of 1,952 VAs, 80% of respondents reported the HIV status of the deceased. In 2013-2014, this figure was 99%. Of those with an HIV status known to the study, there was 89% agreement on HIV status between the VA and pre-mortem data, higher for HIV-negative people (92%) than HIV-positive people (83%). There was 84% agreement on whether the deceased had started ART, and 72% of ART initiation dates matched within 1 year. CONCLUSIONS: In this population, HIV/ART information was often disclosed during a VA and matched well with other data sources. Reported HIV/ART status appears to be a reliable source of information to help classification of cause of death

Whole Genome Sequencing Shows a Low Proportion of Tuberculosis Disease Is Attributable to Known Close Contacts in Rural Malawi.

BACKGROUND: The proportion of tuberculosis attributable to transmission from close contacts is not well known. Comparison of the genome of strains from index patients and prior contacts allows transmission to be confirmed or excluded. METHODS: In Karonga District, Malawi, all tuberculosis patients are asked about prior contact with others with tuberculosis. All available strains from culture-positive patients were sequenced. Up to 10 single nucleotide polymorphisms between index patients and their prior contacts were allowed for confirmation, and ≥ 100 for exclusion. The population attributable fraction was estimated from the proportion of confirmed transmissions and the proportion of patients with contacts. RESULTS: From 1997-2010 there were 1907 new culture-confirmed tuberculosis patients, of whom 32% reported at least one family contact and an additional 11% had at least one other contact; 60% of contacts had smear-positive disease. Among case-contact pairs with sequences available, transmission was confirmed from 38% (62/163) smear-positive prior contacts and 0/17 smear-negative prior contacts. Confirmed transmission was more common in those related to the prior contact (42.4%, 56/132) than in non-relatives (19.4%, 6/31, p = 0.02), and in those with more intense contact, to younger index cases, and in more recent years. The proportion of tuberculosis attributable to known contacts was estimated to be 9.4% overall. CONCLUSIONS: In this population known contacts only explained a small proportion of tuberculosis cases. Even those with a prior family contact with smear positive tuberculosis were more likely to have acquired their infection elsewhere

Contribution of remote M. tuberculosis infection to tuberculosis disease: A 30-year population study

Background: The importance of remote infection with M.tuberculosis as a cause of tuberculosis disease (TB) is unclear, with limited evidence of impact on TB rates beyond 10 years. Our objective was to assess rates of tuberculosis over 30 years by M.tuberculosis infection status at baseline in Karonga District, Northern Malawi. Materials and methods: Population-based surveys of tuberculin skin testing (TST) from the 1980s were linked with follow-up and TB surveillance in Karonga district. We compared rates of microbiologically-confirmed TB by baseline TST induration <5mm (no evidence of M.tuberculosis infection) and those with baseline TST >17mm (evidence of M.tuberculosis infection), using hazard ratios by time since baseline and attributable risk percent. The attributable risk percent was calculated to estimate the proportion of TB in those infected that can be attributed to that prior infection. We analysed whole genome sequences of M.tuberculosis strains to identify recent transmission. Results: Over 412,959 person-years, 208 incident TB episodes were recorded. Compared to the small induration group, rates of TB were much higher in the first two years in the large induration group, and remained higher to 20 years: age, sex and area-adjusted hazard ratios (HR) 2–9 years post-TST 4.27 (95%CI 2.56–7.11); 10–19 years after TST 2.15 (1.10–4.21); ≥20 years post-TST 1.88 (0.76–4.65). The attributable risk percent of remote infection was 76.6% (60.9–85.9) 2–9 years post-TST, and 53.5% (9.1–76.2) 10–19 years post-TST. Individuals with large TST indurations had higher rates of unique-strain TB (HR adjusted for age, sex and area = HR 6.56 (95% CI 1.96–22.99)), suggesting disease following remote infection, but not of linked-strain TB (recent transmission). Conclusions: M.tuberculosis infection can increase the risk of TB far beyond 10 years, accounting for a substantial proportion of TB occurring among those remotely infected

Identifying mixed Mycobacterium tuberculosis infections from whole genome sequence data.

BACKGROUND: Mixed, polyclonal Mycobacterium tuberculosis infection occurs in natural populations. Developing an effective method for detecting such cases is important in measuring the success of treatment and reconstruction of transmission between patients. Using whole genome sequence (WGS) data, we assess two methods for detecting mixed infection: (i) a combination of the number of heterozygous sites and the proportion of heterozygous sites to total SNPs, and (ii) Bayesian model-based clustering of allele frequencies from sequencing reads at heterozygous sites. RESULTS: In silico and in vitro artificially mixed and known pure M. tuberculosis samples were analysed to determine the specificity and sensitivity of each method. We found that both approaches were effective in distinguishing between pure strains and mixed infection where there was relatively high (> 10%) proportion of a minor strain in the mixture. A large dataset of clinical isolates (n = 1963) from the Karonga Prevention Study in Northern Malawi was tested to examine correlations with patient characteristics and outcomes with mixed infection. The frequency of mixed infection in the population was found to be around 10%, with an association with year of diagnosis, but no association with age, sex, HIV status or previous tuberculosis. CONCLUSIONS: Mixed Mycobacterium tuberculosis infection was identified in silico using whole genome sequence data. The methods presented here can be applied to population-wide analyses of tuberculosis to estimate the frequency of mixed infection, and to identify individual cases of mixed infections. These cases are important when considering the evolution and transmission of the disease, and in patient treatment

Risk of Mycobacterium tuberculosis transmission in an antiretroviral therapy clinic.

OBJECTIVE: The risk of transmission of Mycobacterium tuberculosis in antiretroviral therapy (ART) clinics is recognized, particularly, when HIV and tuberculosis services are unified, but the degree of potential exposure to patients with infectious tuberculosis has not been measured. We aimed to quantify this clinic exposure. METHODS: Over 1 year, we recorded all visits to a clinic in northern Malawi that offers HIV testing and counselling, HIV care, ART, and TB diagnostic and treatment services. We included patients and guardians, noting timing and reason for the visit, using a palm vein reader to assist recognition of individuals and record times automatically. Screening for tuberculosis was enhanced, including induced sputum if necessary. RESULTS: Information was collected on 5011 individuals and 19 426 visits. During the period, 90 individuals with bacteriologically confirmed pulmonary tuberculosis attended the clinic when they were likely to have been infectious (taken as 6 weeks before diagnosis to 2 weeks after the start of treatment), including 76 who attended before tuberculosis was diagnosed or suspected. We estimated that 19% of visits had at least 1 h of potential exposure to patients with infectious tuberculosis, half to patients attending prediagnosis. CONCLUSION: There was considerable risk of exposure, including of immunosuppressed patients, to patients with infectious tuberculosis, especially as repeated visits are made. Much of this exposure could not be avoided by separation of patients with known tuberculosis. Good ventilation and avoidance of crowding is essential to minimize transmission of M. tuberculosis in this type of setting

BCG re-vaccination in Malawi: 30-year follow-up of a large, randomised, double-blind, placebo-controlled trial.

BACKGROUND: A large, double-blind, randomised, placebo-controlled trial of repeat BCG found 49% efficacy against leprosy but no protection against tuberculosis after 6-9 years' follow-up in 1995. We report here additional follow-up, which resulted in greatly increased tuberculosis case numbers, and allowed subgroup analysis. METHODS: Nearly 47 000 individuals of all ages living in northern Malawi with a BCG vaccine scar were randomly assigned (1:1) between 1986 and 1989 to receive a second BCG or placebo. The investigators and project staff remained masked to all interventions. Enhanced passive surveillance ensured ascertainment of tuberculosis and leprosy to the end of 2018. Tuberculosis case definitions included rigorous microbiological or histological confirmation. Prespecified subgroup analyses were by tuberculosis type, age at vaccination, time since vaccination, previous tuberculin reactivity, HIV status and Mycobacterium tuberculosis lineage. The original trial is registered with ISRCTN registry, ISRCTN11311670. FINDINGS: In follow-up until Dec 31, 2018, 824 participants had developed tuberculosis, including 786 with pulmonary disease, of whom 383 (63%) of 607 with known HIV status were HIV positive. There was no effect of a second BCG overall (odds ratio [OR] 0·92; 95% CI 0·80-1·05), or for pulmonary (0·93; 0·81-1·07), or lymph node tuberculosis (0·60; 0·31-1·17). The OR was lower for those with known HIV-negative tuberculosis (0·77; 0·59-1·00), for those vaccinated as children (aged <5 years, 0·74; 0·41-1·35; aged 5-14 years, 0·77; 0·60-0·99), and for cases arising at least 20 years after vaccination (0·79; 0·63-1·01). There were no differences by tuberculin status at vaccination, or lineage. There was no evidence of protection against leprosy beyond 10 years after vaccination (although there have been only nine diagnostically certain cases since 1995). INTERPRETATION: There was no evidence that repeat BCG vaccination provides appreciable protection against overall tuberculosis in this rural African population with a high prevalence of HIV. Subgroup effects should not be overinterpreted given the multiple analyses done. However, the evidence for modest protection against HIV-negative tuberculosis, and for a delayed benefit in those vaccinated as children, is consistent with other observations in the literature. FUNDING: LEPRA, Wellcome Trust, Bill & Melinda Gates Foundation

Incidence of tuberculosis infection among adolescents in rural KwaZulu-Natal, South Africa, 2017-2018

The datasets provided will be used for analyses in estimating prevalence of M. tuberculosis infection among adolescents aged 10-19 year

Identification of immunological biomarkers which may differentiate latent tuberculosis from exposure to environmental nontuberculous mycobacteria in children.

A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic