Comparative analytical sensitivity evaluation of five mpox point-of-care assays for mpox clades Ia, Ib, IIa and IIb

Abstract Backgrounds Following an increase in mpox clade Ib cases in several African countries, the World Health Organization recognized mpox as a public health emergency of international concern, highlighting the need for reliable and accessible diagnostic tools. As several mpox clades are co-circulating in endemic countries, the analytical sensitivity of diagnostic assays should be evaluated for all of them in a comparative manner. Methods This study evaluated the analytical sensitivity of three rapid tests (Flowflex Monkeypox virus rapid test from ACON Biotech, Monkeypox antigen rapid test from Assure Tech, and Standard Q Monkeypox Ag Test from SD Biosensor) and two point-of care molecular tests (SD Biosensor’s M10 and Cepheid’s Xpert MPOX assays) using serial dilutions of viral stocks from the four clades of Monkeypox virus (MPXV): Ia, Ib, IIa, and IIb. Findings Upon our analytical comparative benchmarking, the three rapid tests demonstrated comparable analytical sensitivity for all four clades, with a limit of detection of approximately 1,000,000 DNA copies/mL or 1,000 FFU/mL. The two molecular tests demonstrated also comparable analytical sensitivity to an in-house PCR assay for all four clades, detecting concentrations down to 10-100 DNA copies per mL, corresponding to a non-detectable titer of infectious particles. The Xpert assay detected the Clade Ib strain only through its orthopoxvirus target and not its MPXV target, and none of the assays could distinguish between MPXV clades. Interpretation No differences in sensitivity was observed across MPXV clades neither for Ag-RDTs nor for molecular POCTs. However, the potential of simple, affordable tests, such as Ag-RDTs, is limited by poor sensitivity while the use of highly sensitive POC molecular platforms remains limited by their cost. To date, the lack of accurate, affordable POC MPXV-specific assays with potential to differentiate clades, limits diagnostic capacities as well as our understanding of the virus and its epidemiology. Funding This work was supported by FIND and internal funds of the Centre for Emerging Viral Diseases. Mpox diagnostic tests were provided by FIND and the World Health Organization (WHO). MOD was supported by Schmidt Science Fellows, in partnership with the Rhodes Trust.