Monkeypox virus (MPXV), a zoonotic virus in the genus Orthopoxvirus, is an emerging pathogen endemic within Central and Western Africa. Two clades of MPXV have been described, with clade I MPXV (formerly Congo Basin strain) being associated with a higher mortality rate than clade II MPXV (formerly West African strain). The ongoing global clade II mpox (formerly monkeypox) outbreak that began in 2022 has caused more than 100,000 reported cases of mpox [1]. While low levels of clade II mpox continue to circulate in many countries, several travel-related cases of clade I mpox have been reported in different countries, stemming from the ongoing clade I mpox outbreaks in Eastern and Central Africa [1]. Studies have shown that clade I MPXV infection is associated with more severe disease and higher case fatality rates than clade II MPXV infection [2], demonstrating the need for differentiating clades to support tracking outbreak transmission patterns, clade-specific epidemiological surveillance, and public health response strategies. Though the threat of a clade I mpox outbreak in the United States is assessed as low, the U.S. Centers for Disease Control and Prevention (CDC) has recommended that U.S. public health laboratories increase their testing capacity for both clade I and clade II mpox for emergency preparedness [3].

There are currently no U.S. Food and Drug Administration (FDA) approved or cleared test methods for clade I mpox detection. The CDC Laboratory Response Network (LRN) RT-PCR assay for the detection of non-variola Orthopoxvirus (NVO) DNA is FDA cleared, but this assay cannot differentiate between clade I and clade II mpox. To date, 2 assays have been granted emergency use authorization by the FDA: Cepheid Xpert® Mpox and Roche cobas® MPXV. The Xpert® Mpox assay runs on a rapid RT-PCR platform that comes in 2, 4, 16, 48, or 80-module configurations, allowing for a range of testing capacity. This assay specifically detects clade II mpox, as well as NVO. However, it does not specifically detect clade I mpox. The cobas® MPXV assay runs on the fully automated, high-throughput cobas 6800/8800 system, which can be useful for timely results generation and reporting during an outbreak. However, this assay detects a generic MPXV target, which does not differentiate between clade I and clade II mpox. The assay also lacks an NVO target. The inclusion of NVO as a target in an mpox assay is essential to ensuring that potential mutations that can impact clade-specific tests do not cause positive cases to be missed.

The District of Columbia Public Health Laboratory has been using the CDC LRN non-variola Orthopoxvirus RT-PCR assay, along with an additional CDC LRN RT-PCR assay for Orthopoxvirus detection (non-FDA cleared/approved), since the clade II mpox outbreak started in 2022. This method is both time- and labor-intensive and does not differentiate between clade I and clade II mpox. This paper describes the development of a laboratory-developed test (LDT) for clade I and clade II mpox, NVO, and RNase P to run as a fully automated extraction and RT-PCR assay on the Panther Fusion® (Hologic). The Panther Fusion’s Open Access capability allows users to implement LDTs that operate on the instrument’s automated nucleic acid extraction and RT-PCR platform. Result interpretation is also automated based on user-defined parameters. This is the first report of an LDT adapted on the Panther Fusion® for high-throughput automated testing that detects both clade I and clade II mpox, as well as NVO, making the assay an ideal option for the current global situation.