Project Summary Tuberculosis (TB) is the leading infectious disease killer globally, causing 1.5 million deaths annually, and because most TB diagnoses currently rely on sputum-based tests that are complex or have low sensitivity, TB is massively underdiagnosed leading to poor outcomes worldwide. Assays that detect Mycobacterium tuberculosis (Mtb) antigens in blood could revolutionize TB diagnostics, because blood is easy to collect and can detect TB not limited to the lungs; yet blood-base assays remain elusive due to ultra-low antigen concentrations and cross-reacting elements present in blood. The objective of this proposal is to develop and evaluate a highly innovative, blood-based point-of-care (POC) assay for rapid diagnosis of active TB disease (ATB) based on detection of exosome-bound Mtb antigens in blood. We will exploit Alternating Current Electrophoresis (ACE) on a POC platform to isolate and concentrate exosome-bound Mtb antigens in unprocessed patient blood samples to diagnose active TB with accuracy that meets or exceeds World Health Organization’s Target Product Profile (TPP) performance criteria for a TB triage assay (sensitivity >95%, specificity >80%). This groundbreaking assay, originally developed for cancer diagnoses, uses ACE technology to capture small (~0.1 to 1 micron) biomolecules such as exosomes from blood to concentrate Mtb Ag while excluding potential inhibitors allowing them to be imaged using fluorescent antibody probes. This study aims to: 1) evaluate the proof-of-concept Mtb Ag microchip assay on the ExoVerita Flex platform against 200 archived serum samples from patients with culture-confirmed ATB, LTBI, and culture-negative control patients to quantify assay performance drivers and identify opportunities for optimizing assay performance, potentially with additional Mtb antigens; 2) transfer the optimized Mtb Ag microchip assay from the ExoVerita Flex platform to the OmniVerita-M POC reader using the same 200 archived serum samples from UCSD’s biobank and characterizing the assay’s POC performance using prospectively collected whole blood (n=100); and 3) determine the clinical sensitivity and specificity of the optimized assay on the POC reader in a field- based study with prospectively collected blood samples from patients at risk for TB in settings with high (Pakistan), medium (Mexico) and low (U.S.) TB burden (n=1,500). We will evaluate and optimize the Mtb Ag assay using serum samples from a well-characterized Biobank and whole blood from patients with suspected ATB (n=100) at the University of California, San Diego, and then assess its performance compared to standard Mtb bacteriological confirmatory methods among patients with suspected ATB in field settings with a high (Pakistan), medium (Mexico) and low (U.S.) TB burden (n=1,500). The proposed work is highly innovative in that isolation of exosome-bound Mtb antigens in whole blood is a first-of-its-kind approach for TB diagnosis, which may also be able ...