A laboratory-developed test for the automated molecular detection of mpox clade I, mpox clade II, and non-variola Orthopoxvirus using the Panther Fusion® System.

In response to the ongoing clade II mpox (formerly monkeypox) outbreak and the emergence and rapid spread of clade I mpox, public health laboratories need an effective testing method for identifying and monitoring mpox cases. The currently available assays are either labor-intensive, low-throughput, and/or unable to differentiate between clade I and clade II mpox, making them less than ideal for the current global public health emergency. Additionally, the lack of the non-variola Orthopoxvirus target in an mpox assay creates the chance of missing cases due to mutations within the clade-specific target genome regions. We present the development of a fully automated nucleic acid extraction and RT-PCR laboratory-developed test (LDT) for the detection of clade I mpox, clade II mpox, non-variola Orthopoxvirus, and RNase P. This automated assay runs on the Panther Fusion® System, which has an Open Access feature that allows LDTs to utilize the platform's automated nucleic acid extraction, RT-PCR, and automated results generation features. To assess assay performance, 63 previously tested and 16 contrived specimens were tested. The mpox multiplex LDT demonstrated 100 % positive and negative agreement with the reference assays. Analytical sensitivity was 1.37 × 104 copies/mL for all targets, with amplification efficiencies of 103.8 %, 104.1 %, 96.2 %, and 98.9 % for mpox clade I, mpox clade II, NVO, and RNase P, respectively. The mpox multiplex LDT assay was successfully developed for high-throughput testing and can be rapidly adopted by other laboratories.