The overall goal of this proposal is to fundamentally advance molecular diagnostics for pre-XDR and XDR TB. Targeted next-generation deep sequencing of all known genes and gene regions associated with Mycobacterium tuberculosis drug resistance will be performed on stored culture isolates from all patients diagnosed with pre-XDR and XDR TB and a random sample of patients diagnosed with MDR TB through the Philippines Programmatic Management of Drug-resistant Tuberculosis (PMDT) Program. Results will be used to assess the characterize the frequency of hetero-resistance for different anti-TB drugs, determine the ability of current molecular assays to identify resistance in the presence of mixed bacillary populations, and assess the impact of hetero-resistance on treatment outcomes. In addition, whole genome next-generation sequencing will be performed in cases (FQ- and SLI-resistant isolates without a known mutation) and controls (FQ- and SLI-susceptible isolates) to identify novel genetic markers of FQ and SLI resistance. Prediction models will be developed to quantify improvements in diagnostic accuracy for FQ and SLI resistance when adding the novel genetic markers and experiments will be performed to identify which novel mutations directly cause FQ or SLI resistance. The analyses will result in a comprehensive description of reasons for discordance between phenotypic and genotypic tests for resistance to FQ and SLI, evidence on the impact of hetero-resistance on treatment outcomes, and a list of novel mutations that improve prediction of and direclty cause FQ and SLI resistance.