PROJECT SUMMARY / ABSTRACT Rifampin-resistant tuberculosis (RR-TB) remains a global public health crisis. Molecular TB assays such as Xpert have led to dramatic increases in RR-TB case detection and ongoing expansion of the global estimated need for newer treatments. Tremendous financial and scientific resources are directed toward the investigation of new and repurposed drugs, but efforts to optimize and scale-up shorter course, all-oral RR-TB regimens are hindered significantly by the scarcity of availability and access to phenotypic or molecular susceptibility testing for these agents. Longstanding critical barriers of routine phenotypic drug susceptibility testing include prolonged turnaround time and infrastructure requirements that preclude efficient scale-up, contributing significantly to the DR-TB diagnostic gap. Patients are often committed to months of ineffective treatments, leading to acquisition of further drug resistance through selective drug pressure and worse clinical outcomes. Our goal in proposing this work is to improve patient outcomes through strategic and evidence-based use of genomics tools in high burden settings. We will leverage collaborations with international non-profit organizations, a South African MRC- funded cohort, and commercial partners to translate our established targeted deep sequencing assay onto a cost-efficient, handheld nanopore-based sequencing platform (Aim 1, near-clinic solution); and prospectively sequence patient samples early in the course of their treatment in two diverse geographic regions with differing RR-TB and HIV epidemics (Aim 2, centralized solution). These efforts will translate modern-day pathogen genomics into population benefits and contribute to extending the effective lifespan of hard fought new and repurposed anti-TB drugs.