An integrated multiplexed diagnostic assay targeting fusion lipoproteins and cardiolipin for detection of treponemal and nontreponemal antibodies in syphilis patients

Abstract Syphilis cases in the US are increasing at an alarming rate and the need for a rapid diagnostic test that can quickly inform treatment and management decisions is desperately needed. An accurate diagnosis of syphilis relies on recognizing a constellation of symptoms, reviewing medical and sexual history, and performing multiple laboratory tests. The mainstay of routine laboratory diagnosis of active syphilis infection remains serological detection of treponemal and nontreponemal antibodies. Detection of treponemal-specific antibodies is evidence of infection, however, it may indicate past, successfully treated infections since these antibodies persist for decades. Non-treponemal serological response to cardiolipins is suggestive of potential syphilis infection but is not sufficiently specific to support treatment decisions. Further, responses to non-treponemal antigens are generally titered in the laboratory to allow clinicians to assess increases or decreases in titer that support clinical decision making regarding the stage of the disease. There are only two FDA-cleared syphilis rapid tests that would support immediate decision making. However, both are of limited clinical utility because they detect only treponemal-specific antibodies. Currently, there is no point-of-care device available in the US that can test for active syphilis infection, which requires the addition of non-treponemal antibody testing, preferably with a semi- quantitative result. This is necessary to determine appropriate treatment with high sensitivity and low false positive rate. Innovations in this area have yet to be translated into clinical practice. Combining treponemal and nontreponemal assays within the same platform will provide a more definitive, stand-alone diagnosis of syphilis. The proposed rapid assay will fill this gap by utilizing ultra-sensitive electrochemical detection of resistance potential changes that results from the direct interaction of nontreponemal and treponemal antibodies against lipoidal material and T. pallidum antigens respectively. By using 2D nanomaterials (e.g., graphene), which have been successfully used in the development of assays for other pathogens, this electrochemical test can detect subtle interactions between the sensing platform and the targeted analytes. We proposed to use modified cardiolipin for the detection of nontreponemal antibodies and antigens from T. pallidum for the detection of treponemal antibodies. If the target antigen-antibody complex is present, graphene's carrier mobility will change, resulting in a change in electrochemical signal. The proposed electrochemical platform demonstrated ultra- sensitivity of 1 fg/mL for treponemal antibodies, and opened avenues for fast, accurate detection of nontreponemal sera. This project aims to develop a sensing platform that allows simultaneous detection and semi-quantification of treponemal and nontreponemal antibodies that can identify and differentiate active from past cases of syphilis within 10 minutes.