Project Summary Tuberculosis (TB) is a major global health burden and led to the death of 1.6 million people in 2017. A major driver of this high mortality rate is delay in timely diagnosis. Conventional TB diagnostic tests such as smear microscopy or culture used to confirm TB disease rely on sputum, but many TB patient have culture-negative disease or cannot produce sputum (e.g., children or people living with HIV), which hampers case detection and treatment initiation. Therefore, there is an urgent need for diagnostic tools using other sample types to confirm TB disease. Fragments of cell-free Mycobacterium tuberculosis (Mtb) DNA can pass the kidneys and be detected in urine as transrenal (tr) DNA, offering promise as a diagnostic tool. However, test sensitivity has been variable and factors influencing presence of bacterial trDNA are unknown. As part of a large cohort study in Lima, Peru, we have collected urine samples from TB patients and extracted DNA from these samples. Our preliminary data suggest that levels of Mtb trDNA increase shortly after initiation of treatment due to increased bacterial killing leading to release of nucleic acids in the bloodstream. In this study, we will (1) conduct short-fragment real-time PCR to detect changes in Mtb trDNA over time in extracts from 200 TB cases, of whom we have three serial samples, collected pre-, four, and seven days post treatment start; and (2) associate levels of Mtb trDNA in urine with host factors (such as smear status or comorbidities) and urine factors. This work will provide important information to develop strategies for trDNA-based tools to confirm TB disease and monitor TB treatment response.