Project Summary Leprosy is caused by Mycobacterium leprae, a slow-growing bacterium that cannot be cultured in laboratory media. The disease is curable, and disabilities can be prevented with the early administration of multi-drug therapy (MDT). The implementation of MDT successfully decreased the number of new cases reported from 10 million in 1981 to 250,000 in 2005 but plateaued since then with 200,000 new cases in 2019, suggesting that disease transmission is still active. One important factor to consider in the sustained transmission of leprosy is treatment efficacy. In clinical practice, treatment effectiveness is subjectively assessed based on clinical examination of skin lesions at the end of the 6 to 12-month-long treatment, a long period of time during which patients may develop permanent disabilities. Relapse is common in endemic settings but might only manifest clinically 10 to 15 years after completion of therapy which represents a considerable obstacle to follow-up studies and renders the clinical testing of new drug regimens extremely challenging. There is thus a clear need for easy methods that rapidly and accurately assesses treatment efficacy directly from clinical samples during chemotherapy rather than at the end of treatment. Screening and optimization of anti-leprosy drugs and drug combination regimens with the potential to shorten treatment duration is similarly complicated by the fact that M. leprae cannot be cultured in vitro. The current gold standard method to measure bacterial viability in clinical and pre-clinical samples involves the inoculation of bacteria prepared from these samples into the footpads of immunocompromised mice followed by the microscopic assessment of bacterial replication in the footpads 6 to 12 months post-infection. Besides being time-consuming, this method is expensive, technically challenging, and requires maintaining a high number of mice for several months in BSL2 or BSL3 animal facilities. In this project, we propose to evaluate a number of molecular methods, including one recently developed in-house, to rapidly monitor response to chemotherapeutic treatments using both human samples (Aim 1) and an animal model of M. leprae infection (Aim 2). Success in this project could be transformative in monitoring treatment response in leprosy patients by enabling clinicians to assess in “real-time” treatment success or failure. The molecular read-outs developed are further expected to provide much-needed methods to rapidly evaluate treatment efficacy both in animal models and in humans that may be used in the screening and optimization of new drugs and drug combination regimens.