Validation and implementation of an orthopoxvirus qualitative real-time PCR for the diagnosis of monkeypox in the clinical laboratory

BACKGROUND: The globally concerning outbreak of monkeypox virus (MPXV) in non-endemic countries in 2022 warranted an increased capacity of diagnostic clinical testing. In this study, we detail the Johns Hopkins analytical validation of a qualitative orthopoxvirus real-time PCR and characterize its analytical performance for MPXV testing. We also describe our first month of MPXV clinical laboratory diagnosis, including assay utilization and positivity. METHODS: The analytical performance of a previously characterized orthopoxvirus assay that targets the DNA polymerase gene of non-variola orthopoxviruses was determined. In silico inclusivity analysis was performed for the primer and probe sequences. The limit of detection, reproducibility, agreement, and specificity were evaluated as well as target stability at room temperature. Clinical chart reviews were performed for patients who received testing within the first month of offering the assay for diagnosis. RESULTS: Alignment of the primer and probe binding regions of 151 sequenced genomes of MPXV from 2022 showed Sequence homology of 100%. The assay was able to detect MPXV at a concentration of at least 100 copies/mL and showed no cross reactivity with other organisms tested. Targets were stable for at least one week at room temperature. During the first month of implementation, a total of 33 patients were tested, 11 of whom were positive and presented mainly with rash in the genital region. CONCLUSIONS: Increased availability of MPXV testing is essential with the progressive increase in the number of cases. The non-variola orthopoxvirus assay is a sensitive and reproducible method for diagnosing monkeypox during the current surge in cases.