PROJECT ABSTRACT Tuberculosis (TB) is a leading cause of mortality in children worldwide. Difficulty in obtaining sputum and a low sputum bacillary load are major barriers to diagnosis, and necessitate the development of a non-sputum, biomarker-based assay for childhood intrathoracic TB disease. Host biomarker discovery for childhood TB requires a greater focus on downstream proteins and their post-translational modifications (PTMs) that are more likely to be specific to a disease phenotype and can be more easily translated into a point-of-care test. With the support of this K23 award, Dr. Devan Jaganath will identify a host proteomic biosignature in urine that can achieve the goal accuracy for a triage and/or diagnostic test for pulmonary TB in children. To complete this objective, he will leverage an ongoing prospective cohort of symptomatic children being evaluated for intrathoracic TB in Kampala, Uganda, with the extensive proteomic facilities available at the University of California, San Francisco (UCSF). In Aim 1, he will perform targeted mass spectrometry on urine samples from children with confirmed vs. unlikely TB, and examine the abundance and ubiquitylation of 10 host proteins that have prior evidence of specific interactions with M. tuberculosis (Mtb) proteins as candidate biomarkers. In Aim 2, he will use shotgun mass spectrometry on the urine samples to identify all host proteins and their PTMs that can differentiate TB status as novel biomarkers, and perform pathway analysis to determine the subset with functional relevance to Mtb pathogenesis. In Aim 3, he will apply machine learning analyses to identify the smallest combination of biomarkers that can achieve the target accuracy thresholds for a triage and/or diagnostic test for intrathoracic TB disease. He will then evaluate the performance of promising biosignatures in an independent, prospectively enrolled test set. Through this approach, Dr. Jaganath seeks to optimize biomarker discovery for childhood TB diagnosis by coupling prospective clinical cohorts with a targeted and untargeted high-throughput approach to comprehensively examine non-sputum samples for host biomarkers for children. Dr. Jaganath's career goal is to be a physician scientist who translates non-sputum biomarkers into clinical tools that can improve the care of children with TB. To support his path to independence, the proposed work will be paired with a dedicated, multidisciplinary mentorship team and training in international pediatric TB biomarker studies, bioinformatics for proteomic analysis, and machine learning. UCSF is an outstanding environment that is committed to junior investigators with extensive resources for research and career development, and Mulago National Referral Hospital in Uganda is a leader in pediatric TB research, and has the established infrastructure for ongoing enrollment and sample collection. The findings will support an NIH R01 application to validate the biomarkers and biosignatures in large, diverse cohorts in comparison to existing non-sputum diagnostics. Thus, the K23 award will provide Dr. Jaganath with the critical mentorship, training, resources and experience to become an independent investigator who can make important contributions to the field of childhood TB.

PROJECT NARRATIVE More than half of the estimated tuberculosis (TB) disease cases in children worldwide have not been reported to the health system, and 96% of pediatric TB deaths are in children not started on treatment. The proposed project seeks to address the key barriers in diagnosing pulmonary TB in children through discovery of a host proteomic biosignature in urine. The findings will be relevant to the public health efforts to reduce the burden of TB in children and will support the development of a non-invasive, non-sputum based assay for childhood TB disease at the point-of-care.