In response to RFA AI-19-036, investigators from Rutgers University, Makerere University, the Foundation for Innovative New Diagnostics (FIND), McGill University, Frontier Sciences Boston, the US Centers for Disease Control and Prevention, the Kenyan Medical Research Institute and Oxford University propose to study Novel and Optimized Diagnostics (NOD) for Pediatric TB in a prospective study of well-characterized cohorts of children <5 years of age in Uganda and by accessing the biorepository from a pediatric diagnostic study in Kenya. The study populations are enriched for human immunodeficiency virus (HIV)-infected and HIV-exposed, uninfected (HEU) children. The major advances in the diagnosis of adult tuberculosis (TB) have not extended to pediatric TB. There are several reasons for this. Most children in high burden countries with symptoms compatible with TB have limited access to clinical and laboratory facilities necessary for the diagnosis. Even at referral centers where diagnosis is feasible, because of the paucibacillary nature of pediatric TB only a small proportion of children that have a compatible clinical presentation can be bacteriologically confirmed. The current bacteriologic reference standard for TB diagnosis requires difficult to obtain respiratory samples and has limited sensitivity. The impact of HIV infection of and HIV-exposure on diagnostic accuracy is uncertain. Under- and misdiagnosis of pediatric TB contributes to the high case-fatality rates that are highest in children <5 years of age and in those with HIV infection. The novel approaches for TB diagnosis in children < 5 year of age that we propose address the current needs: a) Point-of-care (POC) tests that avoid collections of respiratory specimens; b) increasing the proportion of bacteriologically-confirmed cases with more sensitive diagnostics; and, c) stratification of children with unconfirmed TB by their likelihood of having TB. We hypothesize that novel diagnostics that are currently available or further optimized and new diagnostics based on whole genome sequencing (WGS) of cell free DNA can address each of these needs. We will accomplish these goals in 3 specific aims: 1. Evaluate and develop novel assays that diagnose TB by detecting Mycobacterium tuberculosis (MTB) bacterial products in non-sputum body fluids comparing highly characterized children with microbiologically confirmed TB versus children suspected but deemed unlikely to have TB (unlikely TB). 2. Evaluate and develop novel assays that diagnose TB by detecting host biomarkers in non-sputum body fluids comparing children with confirmed TB versus unlikely TB. 3. Identify combinations of assays that applied together could be used to diagnose TB among children suspected of having TB but that are culture negative (unconfirmed TB). We will propose a new reference standard for the diagnosis of TB that might contain host and bacteria-based assays. Once validated in large observational studies and clinical trials, it could allow targeting of TB treatment in children currently classified as unconfirmed.