Evaluation of a Zoonotic Orthopoxvirus PCR Assay for the Detection of Monkeypox Virus Infection

An epidemic caused by an outbreak of mpox in May 2022 rapidly spread internationally, requiring an urgent response from the clinical diagnostics community. A detailed description of the clinical validation and implementation of a laboratory-developed real-time PCR (rt-PCR) test for detecting non-variola Orthopoxvirus (OPXV) specific DNA based on the newly designed RealStar Zoonotic Orthopoxvirus (altona Diagnostics, Hamburg, Germany) assay is presented. The validation was performed using an accuracy panel (n=97) comprising skin lesion swabs in universal transport media (UTM) and from Mpox virus (MPXV) genomic DNA spiked into pooled MPXV-negative remnant UTM of lesion specimens submitted for routine clinical testing in the NewYork-Presbyterian Hospital clinical laboratory system. Accuracy testing demonstrated excellent assay agreement between expected and observed values and comparable diagnostic performance to three different reference tests. Analytical sensitivity with 95% detection probability was 126 copies/ml and analytical specificity, clinical sensitivity, and clinical specificity were 100%. In summary, the RealStar Zoonotic Orthopoxvirus assay provides a sensitive and reliable method for routine diagnosis of mpox infections.