M. tuberculosis microvariation is common and is associated with transmission: analysis of three years prospective universal sequencing in England

Background: The prevalence, association with disease status, and public health impact of infection with mixtures of M. tuberculosis strains is unclear, in part due to limitations of existing methods for detecting mixed infections. Methods: We developed an algorithm to identify mixtures of M. tuberculosis strains using next generation sequencing data, assessing performance using simulated sequences. We identified mixed M. tuberculosis strains when there was at least one mixed nucleotide position, and where both the mixture's components were present in similar isolates from other individuals. We determined risk factors for mixed infection among isolations of M. tuberculosis in England using logistic regression. We used survival analyses to assess the association between mixed infection and putative transmission. Findings 6,560 isolations of TB were successfully sequenced in England 2016-2018. Of 3,691 (56%) specimens for which similar sequences had been isolated from at least two other individuals, 341 (9.2%) were mixed. Infection with lineages other than Lineage 4 were associated with mixed infection. Among the 1,823 individuals with pulmonary infection with Lineage 4 M. tuberculosis, mixed infection was associated with significantly increased risk of subsequent isolation of closely related organisms from a different individual (HR 1.43, 95% CI 1.05,1.94), indicative of transmission. Interpretation Mixtures of transmissible strains occur in at least 5% of tuberculosis infections in England; where present in pulmonary disease, such mixtures are associated with an increased risk of tuberculosis transmission. Funding Statement: This study was supported by the National Institute for Health Research (NIHR) Health Protection Research Unit (NIHR HPRU) in Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford in partnership with Public Health England [HPRU-2012-10041], and by the NIHR Biomedical Research Centre, Oxford. VN received funding from the European Centre for Disease Control under Grants 2014/001 and 2018/001. TEAP, DCW and ASW are NIHR Senior Investigators. Declaration of Interests: The authors state: "No conflicts of interest are declared." Ethics Approval Statement: The authors declared: "The Health and Social Care Act 2012 and related statutory instruments provide a legal basis for the activity. This work is part of service development carried out under this framework, and as such explicit ethical approval is unnecessary."