ABSTRACT Our current bedside tests for urinary tract infections (UTI) are not sufficiently accurate. The best available test for UTI, the leukocyte esterase (LE) test, has a sensitivity of only 79%, meaning that 21% of children with a true UTI are missed, placing them at increased risk for permanent renal scarring. The 87% specificity of the LE test means that 13% of those without UTI will be incorrectly diagnosed as having UTI and receive antimicrobial treatment. Accordingly, development of more sensitive and specific tests for the diagnosis of UTI, which is our first aim, represents an important contribution. We will collect urine specimens from 3,334 consecutive febrile children aged 1 month to 3 years with presumed UTI and, in a sample of cases and controls from this larger population, we will perform transcriptomics (mRNA) and multiplex inflammatory protein analysis to identify urinary markers that distinguish children with UTI from febrile children without UTI. Promising markers will be validated in a sample selected from 3,334 children from a second site. A second limitation in the current clinical approach to UTI stems from the lack of reliable markers to distinguish children with bladder infection from children with kidney infection (pyelonephritis). Because accurate markers for pyelonephritis have not been identified, all febrile children with UTI are generally assumed to have pyelonephritis (i.e., treated with antibiotics for 10 days and undergo renal ultrasonography) even though approximately only 40% have pyelonephritis. To identify accurate markers for pyelonephritis, which is our second aim, we will enroll 100 febrile children with UTI and perform transcriptomics and multiplex inflammatory protein analysis on their urine samples. Pyelonephritis will be confirmed using Tc99m-dimercaptosuccinic acid (DMSA) scan. We will validate promising markers in an independent sample of 100 children from a second site. Diagnosis of UTI is especially challenging in children with infections caused by bacteria other than Escherichia coli. Accordingly, our third aim will be to develop tests for the diagnosis of UTI in these children. Features that will differentiate our study include (1) enrollment of consecutive febrile subjects (which will minimize bias);; (2) use of catheterization to collect all urine samples (which will minimize misclassification);; (3) timely processing of the urine samples, (4) use of a novel method of assembling high-quality RNA libraries from small amounts of urine, and (5) selection of different sites for training and validation. Results from the proposed project are expected to (1) identify easy to measure urinary proteins that can be used to accurately diagnose UTI in children with and without E. coli infection, (2) develop RNA signatures for UTI and pyelonephritis which can be used as a gold standard in future studies, and (3) establish a large specimen bank for use in future studies.