PROJECT SUMMARY Nanopath proposes to develop a point-of-care molecular diagnostic that can identify UTI-causing pathogens and key genetic markers of antibiotic resistance directly from a patient urine sample in 15 minutes, without the need for nucleic acid amplification. Despite the severity and prevalence of UTIs, the current diagnostic standard-of-care relies on culture-based methods, which take an average several days to return species-level and antimicrobial resistance information. This time- intensive diagnostic workflow leaves many patients in pain and/or at-risk of severe infection before they are prescribed targeted antibiotic therapy. Critically, empiric treatment of UTIs is one of the largest contributors to the growing problem of antibiotic resistance. Nanopath’s product is a single- use assay cartridge coupled to a low-cost readout instrument. The assay cartridge integrates sample preparation with our proprietary molecular nanosensor. Our multiplexed molecular nanosensor is functionalized with an array of probes complementary to 1) species-specific genetic sequences and 2) genetic markers of antimicrobial resistance. In our preliminary work, we designed a 5-target proof-of-concept panel encompassing three bacterial species and two antimicrobial resistance genes that are commonly observed in UTIs. Our preliminary data demonstrate highly selective and robust target detection in a variety of sample matrices, including healthy patient urine. Our Phase II effort will focus on four Specific Aims. First, we will expand our target panel to encompass >95% of UTI-causing organisms and key markers of antimicrobial resistance. This will involve rational design and bench-top validation of candidate PNA probes to inform a finalized panel and probe set. Second, we will optimize the design of the assay cartridge to integrate sample handling functions, lower limit-of-detection, and enable high throughput sample processing. This aim will also support low-volume manufacturing of the assay cartridge in preparation for verification studies. Third, we will perform a series of comprehensive analytical verification studies on the assay. Finally, we will characterize clinical performance of Nanopath’s UTI assay in pilot clinical study (n>200) in collaboration with Brigham & Women’s Hospital. Successful commercialization of this technology will enable point-of-care characterization of UTIs in <15 minutes, enabling the immediate targeting of antibiotic therapy and alleviating ongoing contributions to antibiotic resistance. The applications of the proposed platform extend beyond UTIs and are translatable to other clinical scenarios that currently employ lengthy culture-based steps and molecular testing workflows, such as respiratory infections, bloodstream infections, and prosthetic joint infections.