ABSTRACT [30 lines] The continued growth of syphilis cases in the United States is associated with significant maternal-fetal morbidity and mortality, health disparities for racial/ethnic minorities, and men who have sex with men (MSM). Rising case counts have led to a shortage of penicillin, the treatment of choice for the etiologic agent of syphilis, Treponema pallidum subsp. pallidum, and the only approved therapy for pregnant patients and neonates. Penicillin shortages have led to increased reliance on doxycycline for treatment and prophylaxis, but patient compliance is suboptimal, particularly for doxycycline treatment which requires twice daily dosing for > 14 days. These conditions - high case counts, increased use of doxycycline, and potential incomplete treatment – are ripe for selection of doxycycline resistant T. pallidum strains. A previous second line treatment, single dose azithromycin, is no longer recommended due to high rates of resistance (> 79%) observed among MSM. However, susceptible strains may circulate among non-MSM populations where resistance rates have historically been much lower (11%) suggesting there are still patients who could benefit from azithromycin therapy. Unfortunately, there are no clinical assays that detect T. pallidum resistance to either drug. Such assays would serve the dual purposes of public health surveillance and informing therapeutic antibiotic selection, particularly for patients unlikely/unable to comply with multiday doxycycline treatment. Resistance to azithromycin is mediated by single nucleotide polymorphisms (SNPs) in the 23S rRNA gene and doxycycline resistance is predicted to emerge as SNPs in the 16S rRNA gene. Resistance-conferring SNPs are amenable to molecular detection by allele-specific nucleic acid amplification assays. We have previously developed and validated in our clinical laboratory a sensitive and specific Reverse-Transcription Loop-mediated isothermal Amplification (RT-LAMP) molecular test for T. pallidum and are currently validating the assay in a multiplexed, point-of-care (POC) format. In this project, we propose to build upon our existing assays by developing complementary approaches to detect antibiotic resistance in T. pallidum. The deliverables will be a POC allele-specific LAMP for drug resistance (AS- LAMP) multiplexed with organism detection and an internal amplification control, and a parallel next- generation sequencing (NGS) assay for high-complexity clinical laboratories. In Aim 1, we propose to develop and validate both a POC AS-LAMP and an amplicon-NGS assay for detection of SNPs mediating azithromycin resistance. In Aim 2, we propose to develop and validate an amplicon-NGS assay for detection of doxycycline resistance and to develop a panel of AS-LAMP primers that can be deployed following detection of emerging doxycycline resistance genotypes. After primer and protocol optimization, we will validate the assays following established procedures for deploying clinical...