PROJECT SUMMARY/ABSTRACT Children under 5 years of age account for ~50% of the 1.2 million new cases of pediatric TB each year, but are least likely to be diagnosed, and are at highest risk of death without prompt treatment. Young children are more likely to present with disseminated or extrapulmonary TB and paucibacillary disease, often missed by respiratory sampling and our currently available diagnostics. Non-sputum diagnostic tools for TB detection and treatment response in young children, using easily obtained specimens are urgently needed. Our team has successfully developed an ultra-sensitive CRISPR-based approach (CRISPR-TB) that detects M. tuberculosis (Mtb) cell free DNA (cfDNA). In pilot evaluation with repository blood samples, CRISPR-TB demonstrated high sensitivity (94%) and specificity (95%) among adults and children with TB and their asymptomatic household contacts in Eswatini. Among hospitalized Kenyan children with HIV (median age 2 years), CRISPR-TB detected 100% with microbiologically confirmed TB, and an additional 85% with clinically diagnosed TB (i.e. missed by respiratory-based diagnostics). We now propose to expand CRISPR-TB evaluation in a large prospective cohort of 400 children with suspected TB (majority <5 years) to evaluate CRISPR-TB diagnostic performance (Aim 1) including longitudinal evaluation for treatment response (Aim 2), and extend evaluation to urine samples (Aim 3). Additional cohorts of adults with confirmed TB and their asymptomatic household controls, and recently BCG-immunized asymptomatic infants will be evaluated. Exploratory aims will pilot a POC platform, assess utility of CRISPR-TB to identify early incipient or subclinical disease in participants with missed diagnosis at baseline who develop incident TB, and estimate “true” prevalence of pediatric TB using Bayesian latent class analysis given imperfect reference tests. We hypothesize CRISPR-TB will have similar/improved diagnostic performance to Xpert on respiratory samples among children with confirmed TB without the need for sputum, and identify additional children missed by respiratory samples, will provide a useful surrogate marker of treatment response with decline in quantitative levels during successful treatment, and be adaptable to urine samples.