Evaluation of a portable multiplex real-time pcr assay for the detection of monkeypox virus and non-variola orthopoxviruses.

Monkeypox (mpox) is the most significant orthopoxvirus (OPXV) infection in humans since smallpox eradication. The World Health Organization (WHO) has twice declared public health emergency of international concern due to the expansive spread of clade I and II mpox cases, thus prompting public health scientists to develop rapid and effective diagnostic tools. Clinical presentations of monkeypox can overlap with other vesiculopustular diseases, particularly those caused by Varicella-Zoster virus (VZV), complicating accurate diagnosis in decentralized or resource-limited settings. In this study, we performed an in-lab evaluation of a multiplexed real-time PCR on a portable platform (T-COR 8) for the detection of monkeypox virus (MPXV) and non-variola orthopoxvirus (NVAR-OPXV), and differentiation from varicella-zoster virus (VZV). The T-COR 8 platform required minimal technical skill, limited hands-on time, and generated results for 8 samples within 1 h using crude specimens, without the need for nucleic acid extraction. The performance characteristics were investigated using MPXV and VZV clinical sample remainders and a panel of laboratory cultured viruses. Testing of 116 clinical remainder specimens in the MPXV/NVAR-OPXV assay yielded 100% sensitivity, 97.8% specificity, and 99.1% agreement with prior test results. The MPXV/NVAR-OPXV/VZV assay detected and correctly differentiated MPXV and VZV in a limited set of clinical samples (n = 39). The MPXV assay demonstrates specificity for MPXV, except for Akhmeta virus (AKMV), out of the 20 different species/isolates tested, including 15 orthopoxviruses and 4 non-orthopoxviruses, and VZV. These findings support the T-COR 8 platform as a rapid, accurate, and field-deployable diagnostic tool suitable for expanding monkeypox testing capacity.