Background: Tuberculosis (TB) is a major public health problem globally, and particularly in Brazil, which remains one of the 22 TB high-burden countries defined by the World Health Organization. Pulmonary TB is primarily caused by Mycobacterium tuberculosis (Mtb), but other nontuberculous mycobacteria (NTMs) are increasingly being isolated from such patients. The global TB epidemic is partially driven by co-infection with HIV and worsening due to the emergence of multidrug-resistant and extensively drug-resistant Mtb strains. One of the striking facts about TB is that patients can be re-infected following successful treatment. Hence, prior TB infection does not protect against reinfection. Similarly, and against common believes, TB patients can be infected with multiple Mtb genotypes at the same time. Indeed, studies have been reporting mixed infection in up to 60% of patients depending on TB incidence and study setting, but mathematical models predict that in most cases these figures are underestimates, because of the inherent limitations of the methods used to detect mixed infections. Moreover, little is known on the co-occurrence of Mtb and NTMs in TB patients. Recent advances in sample preparation and DNA sequencing offer an opportunity to explore mixed-strain infections in TB. Because mixed infections in TB have been highly neglected, little empirical data exist with respect to their impact on treatment outcome. Another neglected field in TB research is hetero-resistance, which refers to the co-occurrence of drug-susceptible and drug-resistant Mtb strains in the same individual. Hetero-resistance can emerge de novo during the development of drug resistance in a patient failing anti-TB drug therapy, or through initial co-infection or sequential superinfection of different Mtb strains with varying drug susceptibility profiles. The relative importance of these different phenomena for the evolution of drug-resistant Mtb and the outcome of patient treatment has not been determined. A few studies have reported that HIV co-infected TB patients were more likely to carry multiple Mtb genotypes, but it is unknown if and how this relates to the comparably poor treatment outcome of TB/HIV co-infected patients. More generally, little is known on the impact of HIV co-infection on the evolution of drug-susceptible and drug-resistant Mtb. To address these knowledge gaps, we will study TB patients in Rio de Janeiro and apply state-of-the art genotyping and whole genome sequencing (WGS) of Mtb isolates directly from sputum samples. Goal, Hypotheses and Objectives: The overall goal of this project is to study the biology and epidemiology of mixed infections with different strains of Mtb and/or NTMs and hetero-resistance in patients who start anti-TB treatment and the association with treatment outcome. We hypothesise that i) mixed infections (as defined above) and hetero-resistance occur more often in TB patients suffering from treatment failure, ii) mixed infection and hetero-resistance occur more often in patients co-infected with HIV, and iii) mixed infection and hetero-resistance occur more often in patients with previous TB treatment and/or drug-resistant TB. We will test these hypotheses by addressing the following Objectives: 1. To study the nature and frequency of mixed infections and hetero-resistance in TB patients from Rio de Janeiro; 2. To analyse the association of mixed infections and hetero-resistance with epidemiological and laboratory variables; 3. To link mixed infections and/or hetero-resistance to treatment failure in TB patients; 4. To investigate the nature and frequency of mixed infections and hetero-resistance as a function of HIV co-infection and anti-TB drug resistance. Study Setting, General Approach, and Methods: This project is embedded in a larger study funded by the National Institutes of Health and the Departamento Ciência Tecnologia – Secretaria de Ciência Tecnologia do Ministério da Saúde, on which the Brazilian Principal Investigator of this proposal (Prof. Kritski) is a Co-Principal Investigator. This larger study focuses on the immunology of progression from latent to active TB. The proposed project will take advantage of the available infrastructure and resources to recruit TB patients at four TB clinics in Rio de Janeiro during a period of 18 months. A total of 780 culture-positive TB patients will be recruited and their sputum samples subjected to standard genotyping to detect signals of mixed infection by different Mtb and/or NTM strains. Based on our preliminary data, we anticipate that a minimum of 10-20% of patients will harbour more than one mycobacterial strain, depending on HIV-status and drug resistance profile. All patient samples showing evidence of mixed infection will be subjected to WGS performed directly on sputum to avoid any potential bias introduced during the culturing step. As a back-up, WGS will be performed on multiple colonies obtained from bacterial cultures. Because TB patients with HIV co-infection and/or drug resistance will be comparably few, these will all be subjected to WGS analysis, irrespective of whether they show evidence of mixed infection based on standard Mtb genotyping. Moreover, to study hetero-resistance in detail and differentiate intra-host evolution from mixed or superinfection, we will clone individual PCR products obtained from sputum samples and directly sequence. This will allow us to reconstruct the specific haplotypes in mixed bacterial populations, which currently cannot easily be inferred from Illumine sequencing data alone. We will combine these Mtb genetic data to detailed clinical and epidemiological data obtained through our ongoing cohort study and look for associations between mixed strain infections and patient variables, including culture conversion at 2 months and treatment outcome at 6 months post treatment initiation. We will use a nested case-control study to compare TB patients with and without mixed infections and identify risk factors and patient characteristics associated with mixed infections. Moreover, we will use the WGS data to study the intra-host evolution of Mtb as a function of HIV co-infection and anti-TB drug resistance. Significance: This project will generate for the first time detailed data on the nature and frequency of mixed-strain infections in TB patients from Brazil as well as its impact on patient treatment outcome. Moreover, we will define risk factors and other patient variables associated with mixed infections. Because we will be able to combine the data generated in this project with data from the larger immunological study, this project will also shed new light on the impact of mixed-strain infections on the immune response to TB infection, and inform the development of new TB vaccines and biomarkers. Finally, our project will explore the diversity of Mtb in individual patients during treatment and generate new knowledge on the evolution of Mtb in the context of HIV co-infection and drug-resistant TB, as well as contribute to a better understanding of the role of hetero-resistance in the emergence of anti-TB drug resistance and its impact on treatment outcome.