Project Summary In this grant application, we propose to develop a rapid phenotypic drug susceptibility test for tuberculosis (TB) based on our rapid sputum diagnostic test for TB. The emergence of drug resistance in tuberculosis (TB), already the most frequent cause of infectious death in humans worldwide, constitutes a significant concern in global health. The World Health Organization (WHO) set as a critical milestone in their End TB Strategy, a goal of a 75% reduction in tuberculosis deaths over the ten years from 2015 to 2025. However, the emergence of TB strains with drug resistance threatens global progress toward this milestone and hinders all tuberculosis eradication programs. Factors contributing to drug resistance include the treatment of patients with a drug that does not affect resistant organisms, poor management of the supply and quality of drugs, and higher transmission of TB in public places. Moreover, patients face catastrophic costs while undergoing treatment for drug resistance and often go untreated as a result, further exacerbating the problem. The key to the reduction and containment of these antibiotic-resistant strains is the early detection of all forms of drug resistance in TB. To solve this problem, we are developing a rapid phenotypic drug susceptibility test based on our novel REFtb sputum-based TB diagnostic system that can dramatically transform how the drug susceptibility diagnosis is currently made in low-resource public settings. The broad objective of the proposed research in this Phase 1 application is to develop the rapid phenotypic drug susceptibility test to the front-line tuberculosis drugs. In this Phase 1 application, we plan to carry out development of an optimized rapid phenotypic drug susceptibility test and test antibiotic-resistant BCG spiked sputum samples. For the Phase 2, we plan to continue development to extend these findings to drug resistant TB in preliminary clinical trials. Thus, the long-term goal of our research is to develop a drug susceptibility test TB diagnostic that is stable in storage at ambient temperature and can be shipped worldwide without reliance on a cold-chain. The central hypothesis of this research is that our sputum- based REFtb diagnostic can be adapted to include drug susceptibility testing. Without the right drug susceptibility tests available near where patients seek care, the majority of patients with MDR-TB will go undetected, second- line treatments will not be started, and the WHO goal of TB elimination will not be met. Successful completion of the above tasks in Phase I will begin the development of a rapid drug susceptibility test that can be used to reach more of these patients in low-resource settings so they can be referred for additional testing and treatment, thus significantly contributing to the goal of TB elimination.