To improve understanding of the factors influencing tuberculosis transmission and the role of pathogen variation, we sequenced all available specimens from patients diagnosed over 15 years in a whole district in Malawi. Mycobacterium tuberculosis lineages were assigned and transmission networks constructed, allowing ≤10 single nucleotide polymorphisms (SNPs) difference. We defined disease as due to recent infection if the network-determined source was within 5 years, and assessed transmissibility from forward transmissions resulting in disease. High-quality sequences were available for 1687 disease episodes (72% of all culture-positive episodes): 66% of patients linked to at least one other patient. The between-patient mutation rate was 0.26 SNPs/year (95% CI 0.21–0.31). We showed striking differences by lineage in the proportion of disease due to recent transmission and in transmissibility (highest for lineage-2 and lowest for lineage-1) that were not confounded by immigration, HIV status or drug resistance. Transmissions resulting in disease decreased markedly over time.DOI: http://dx.doi.org/10.7554/eLife.05166.001

Background. Recurrent tuberculosis is a major health burden and may be due to relapse with the original strain or reinfection with a new strain.Methods. In a population-based study in northern Malawi, patients with tuberculosis diagnosed from 1996 to 2010 were actively followed after the end of treatment. Whole-genome sequencing with approximately 100-fold coverage was performed on all available cultures. Results of IS6110 restriction fragment-length polymorphism analyses were available for cultures performed up to 2008.Results. Based on our data, a difference of ≤10 single-nucleotide polymorphisms (SNPs) was used to define relapse, and a difference of >100 SNPs was used to define reinfection. There was no evidence of mixed infections among those classified as reinfections. Of 1471 patients, 139 had laboratory-confirmed recurrences: 55 had relapse, and 20 had reinfection; for 64 type of recurrence was unclassified. Almost all relapses occurred in the first 2 years. Human immunodeficiency virus infection was associated with reinfection but not relapse. Relapses were associated with isoniazid resistance, treatment before 2007, and lineage-3 strains. We identified several gene variants associated with relapse. Lineage-2 (Beijing) was overrepresented and lineage-1 underrepresented among the reinfecting strains (P = .004).Conclusions. While some of the factors determining recurrence depend on the patient and their treatment, differences in the Mycobacterium tuberculosis genome appear to have a role in both relapse and reinfection.

BackgroundMixed, 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.ResultsIn 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.ConclusionsMixed 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.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4988-z) contains supplementary material, which is available to authorized users.

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.

Background: Sub-Saharan Africa faces region-specific risk factors for chronic kidney disease (CKD), such as nephrotoxic herbal medicines, antiretroviral therapy and infections, in addition to hypertension and diabetes. However, large epidemiological studies from this area are scarce. Methods: In a cross-sectional survey of non-communicable diseases, we conducted a prevalence sub-study of CKD in two Malawian populations. Study participants (N=5264) of 18 years of age and above were recruited and data on demographics and CKD risk factors were collected. Glomerular filtration rate was estimated (eGFR) using the CKD-EPI equation. Results: The prevalence of eGFR<60ml/min/1.73m 2 was 1.4% (95% CI 1.1 – 1.7%) and eGFR<90ml/min/1.73m 2 was 20.6% (95% CI 19.5 – 21.7%). The rural area had higher age-standardized prevalence of both eGFR<60ml/min/1.73m 2, at 1.8% (95% CI 1.4 – 2.3) and eGFR <90 ml/min/1.73m², at 21.1% (95% CI 19.9 – 22.3), than urban location, which had a prevalence of 1.5%, (95% CI 1.0 – 2.2) and 19.4% (95% CI 18.0 – 20.8), respectively, with overlapping confidence intervals. The prevalence of CKD was lower in females than in males in both rural and urban areas. Older age (p < 0.001), a higher level of education (p = 0.03) and hypertension (p < 0.001) were associated with a higher adjusted odds ratio (aOR) of low eGFR. Diabetes was associated with a reduced aOR of eGFR<90ml/min/1.73m 2 of 0.69 (95% CI 0.49–0.96; p=0.03). Of participants with eGFR<60ml/min/1.73m 2, 14 (19.4%) had no history of hypertension, diabetes or HIV, while 36 (50%) had a single risk factor of being hypertensive. Conclusion s: Impaired renal function is prevalent, but lower than expected, in rural and urban Malawi. Further research is needed to increase understanding of CKD incidence, survival and validation of eGFR calculations in this population.