A-262 Analytical and Clinical Validation of a Multiplex RT-PCR based Mpox Assay

Abstract Background The 2022 non-endemic spread of Mpox in the western hemisphere was a unique public health challenge in the backdrop of the COID19 pandemic. To address the public health emergency where Mpox transmission was unlike previous instances, HealthtrackRX was one of the first independent diagnostics labs to develop a multiplexed RT-PCR based test for Mpox. In the present study analytical and clinical validation results for the Mpox RT-PCR test are presented. Methods The multiplexed pan-orthopox/pan-Mpox RT-PCR test was validated using inactivated Mpox (Clade I and II) viral samples establishing the limit of detection (LoD). Clinical validation of the test was performed against the FDA approved test at CDC and a commercially available pan-Orthpox assay. Sensitivity and specificity performance against interfering substance, pathogens and other poxviruses was also established. All statistical analysis were performed using R version 3.6.0. Results The multiplexed pan-Orthopox/pan-Mpox RT-PCR test successfully detected both Clade I and II Mpox and the LoD was established at 102 RNA copies/µl.The in-house developed Mpox test displayed 100% concordance against the FDA approved CDC pan-orthopox test and the pan-orthopox RT-PCR test from Thermo Fisher. The test showed no interference from a number of sexually transmitted pathogens and could successfully discern Mpox from several other poxviruses. With the exception of ethanol, no substances (from the FDA recommended list) interfered with the test performance. A total of 1712 patient samples were collected (July 25th, 2022, to February 1st, 2023) and tested for the presence of Mpox. Conclusion We have demonstrated the development, validation, and commercialization of a multiplexed RT-PCR based pan-orthopox/pan-Mpox diagnostic test for Mpox. Our data shows the feasibility of rapidly developing and deploying a PCR based test against an emerging infectious disease.