Summary/Abstract: 30 lines Drug-resistant tuberculosis (TB) threatens our goal of ending the global TB epidemic by 2035. Early diagnosis of drug-resistant TB for timely initiation of appropriate treatment remains a challenge. Phenotypic drug susceptibility testing (DST) requires Mycobacterium tuberculosis (MTB) culture and takes 8-12 weeks. Molecular-based methods are rapid but require previously identified and validated resistance-conferring mutations. Unfortunately, mutations are not completely known for key drugs included in newer all-oral regimens. Our long-term goal is to develop a non-culture-based method for phenotypic DST that provides results in 48-72 hours from sputum collection and that can be used directly on sputum. Our innovative approach is based on MPT64, a protein secreted only during active growth that is highly specific for MTB. We demonstrated that MPT64 secretion in MTB cultures decreases significantly within 48–72 hours of anti-TB drug exposure for drug-susceptible but not drug-resistant MTB isolates. We have previously measured MPT64 directly in sputum samples with a limit of detection comparable with Xpert MTB/RIF Ultra (Cepheid, Sunnyvale, CA, USA). In this study we will validate culture MPT64 DST chemiluminescence-enzyme immunoassay (CLEA) for isoniazid (INH), rifampin (RIF), fluoroquinolones (FQs), bedaquiline (BDQ), linezolid (LZD), and delamanid (DLM). Our proposed approach will provide results within 72 hours of the time the culture is identified as MTB, compared to the 2-3 weeks required to obtain results from conventional DST (Aim 1). We will use the principles of culture MPT64 DST to develop a sputum MPT64 DST CLEIA, which will provide results in 48-72 hours from the time of sputum collection. We will identify protocols for sputum liquefaction, antibiotic exposure times, and CLEIA that result in the lowest limit of detection (LOD) for resistant MTB cells (Aim 2). We will prospectively evaluate sputum MPT64 DST in 860 patients with TB in the Philippines and in the country of Georgia (Aim 3). We hypothesize that both culture MPT64 DST and sputum MPT64 DST will meet or exceed minimum sensitivity (>95%) and specificity (>98%), which are the WHO high priority target product profiles for rapid DST. The innovative MPT64 DST assays will transform TB management by enabling early initiation of effective treatment based on rapid drug susceptibility data, thus improving patient outcomes and curtailing the spread of drug resistant TB that is critical if TB is to be eliminated by 2035.