PROJECT SUMMARY There is a large unmet need for fast and effective point-of-care (POC) molecular diagnostic (MDx) testing for congenital and adult acquired syphilis. Current syphilis detection methods utilize dark-field microscopy and/or serum antibody tests that are performed in a centralized lab, require a trained operator, have inadequate sensitivity and specificity without confirmatory reflex testing, or have long turnaround times on the order of days. These challenges prevent clinicians from obtaining immediate and definitive test results to provide patients with the correct treatment before patients leave the clinic. The goal of our proposal is to develop a POC PCR-based syphilis assay as part of a multiplexed genital ulcer disease (GUD) assay panel on GMR biosensors and to determine the best sample type or pooling of sample types, for molecular detection of congenital, secondary, and latent syphilis. GMR biosensors are magnetic-based field sensors that provide several advantages over conventional fluorescent and optical-based detection methods. They offer lower background noise and a higher signal-to-noise ratio due to sample matrix insensitivity. Additionally, GMR biosensors do not need complex and expensive components for signal readout and processing or light compensation that are required for more traditional methods. Therefore, our MagChipRTM platform can be made truly point-of-care, portable, affordable, and accessible by reducing footprint and cost while maintaining a simple user workflow for untrained operators. This platform has previously demonstrated successful multiplexed detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) in a single test. Given the advantages of our platform, and our knowledge and skill sets in developing GMR-based molecular assays, we aim to achieve the goal of our proposal with the following Specific Aims: 1) Develop POC multiplexed PCR/GMR-based molecular assay for Treponema pallidum and other pathogens causing genital ulcer disease, and 2) Clinical validation of GUD assay in patients with primary syphilitic lesions and discovery of novel biospecimens and/or pooled specimens for various syphilis disease stages. Ultimately, this assay panel will be run on our fully automated, raw sample-to-result MagChipRTM platform to provide clinicians with diagnostic tests results in 20 minutes and enable them to give patients immediate and definitive treatment before their patients leave the clinic. This assay would be one of the first commercially available POC NAAT for syphilis, and in combination with our other STI assay panels, we hope to offer clinicians and patients with a solution for comprehensive STI testing.