Wide mismatches in the sequences of primers and probes for Monkeypox virus diagnostic assays

Abstract

The current monkeypox outbreak as a global health emergency now spreads into more than 90 counties. Rapid and accurate diagnosis of the infections is fundamental for the viral disease containment. Here, we analyzed the primer and probe sequences in the CDC recommended monkeypox virus (MPV) generic real-time PCR assay by aligning against 683 reported MPV genomes worldwide. Sequence mismatches were found in more than 92% of genomes for the MPV generic forward and reverse primers. We also evaluated the specificity of seven other real-time PCR assays for MPV and orthopoxvirus (OPV) detection, and identified two assays (MPV_F3L and OPV_F8L) with the highest matching score (>99.4%) to the global MPV genome database. Genetic variations on the MPV genomes corresponding with the real-time PCR primer and probe regions were further specified and used to indicate the temporal and spatial emergence pattern of monkeypox disease. Our results show that the current MPV real-time generic assay may be unsuitable to accurately detect MPV highlighting the need to develop new detection assays.

High quality of SARS-CoV-2 molecular diagnostics in a diverse laboratory landscape through supported benchmark testing and External Quality Assessment

“…Although this method generally allows for quick and relatively simple upscaling of diagnostic capacity, when this strategy was implemented for SARS-CoV-2 in 2020 in the USA it had its challenges, namely contamination of primers/probes with synthetic template and improper primer/probe design 20 , 24 – 26 which impaired the testing system. The CDC had a similar experience when implementing an mpox assay in their laboratory network in 2022 27 , 28 . While we acknowledge that this topic is too complex to be discussed thoroughly in our paper, we feel it is worth briefly mentioning in this discussion as a way of starting or adding to pandemic preparedness systems discourse.…”

Not a waste: Wastewater surveillance to enhance public health

“…A study in Amsterdam qualitatively detected Mpox in 31% of samples from five districts, 56% samples from two WWTFs, and 26% of samples from the Schiphol airport using both assays (de Jonge et al, 2022). Primer and probe mismatches have also been identified in the CDC Mpox generic real time PCR assay and seven other Mpox and orthopoxvirus diagnostic assays (Wu et al, 2022b), suggesting the need for careful evaluation of those assays before testing. Further investigation is warranted to optimize analytical methods in Mpox detection, improve clinical/biological understanding of viral shedding dynamics, evaluate the overall effectiveness of Mpox vaccines, and quantify viral recovery efficiency in wastewater, which are crucial for using WBE data for epidemiological inference.…”