ABSTRACT Background: The resurgence of syphilis in the USA and around the globe has hastened the need for widely available syphilis testing. Early and rapid detection of syphilis is crucial for prompt treatment, avoiding serious clinical complications, and preventing the spread of infection. Self administered rapid tests enhance access to testing and promote timely treatment for positive cases. The present report describes the evaluation of diagnostic performance of First to Know Syphilis Test, the first over the counter (OTC) treponemal rapid test. Methods: A prospective multi-site clinical study with 1270 subjects was conducted, where the subjects self-collected capillary blood from a finger stick and executed self-testing without the assistance of any site personnel. The test results were compared with a composite reference standard to evaluate the diagnostic performance of the First To Know Syphilis Test. Additional analysis was performed in a panel of 125 pre-characterized clinically staged syphilis patient samples. Results: The First to Know test exhibited an overall sensitivity of 93.4% (95% CI, 87.0% to 96.8%) and specificity of 99.5% (95% CI, 98.9% - 99.8%) and accuracy of 99%. The overall agreement was 98.9%, with Cohens Kappa (ⱪ) value 0.93 (95% CI, 0.90 to 0.97) showing excellent agreement between the reference standard and First to Know test results. In addition, the test was validated in a panel of 125 clinically staged syphilis patient samples and the results showed 100% agreement in detecting anti-treponemal antibodies in all the samples tested. Conclusions: The present study findings showed an excellent diagnostic performance of the First to Know Syphilis Test demonstrating its utility in the screening and diagnosis of syphilis. The availability of this OTC test and its excellent performance at low prevalence, will have a profound impact on syphilis detection and prevention strategies and could reduce co morbidities such as HIV and other STI transmission. Keywords: Syphilis, Treponema pallidum, Diagnosis, Rapid syphilis test, Self-test, Over-the Counter Syphilis Test

Competing Interest Statement All authors are employees of Now Diagnostics. AGJ is a scientific consultant of Now Diagnostics. LN is marketing consultant and RP is project management consultant for Now Diagnostics. All other authors have disclosed that they have no conflicts of interest

This study was funded internally by NOW Diagnostics.

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Ethics Statement This study was conducted in accordance with the ethical standards of the Helsinki Declaration. IRB approval for the multi-site study was obtained from Advarra IRB #Pro00032035. 13 The initial approval was on Dec 9, 2019, and latest approval was on July 25, 2023. Informed consent was obtained from all study subjects, and all the participant data were de-identified in the database.

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