RAPID LOW TECH MOLECULAR IDENTIFICATION OF MONKEYPOXVIRUS AND OTHER ZOONOTIC ORTHOPOXVIRUSES.

The ongoing outbreak of Monkeypoxvirus (MPXV) turned the spotlight back on zoonotic poxviruses (ZPs). MPXV, Cowpoxvirus (CPXV) and Vaccinia Virus (VV) are the zoonotic species belonging to the Orthopoxvirus (OPXV) genus in the Poxviridae family. The diagnostics currently available are designed for specialised laboratories and are unsuitable for field and point of care use. Here we present a sensitive and specific low-tech workflow for the rapid identification of ZPs. An isothermal (Hyperfluux) multiple dye assay, targeting the OPXV RNA Polimerase (RPO) was designed and validated. Analytical sensitivity was assessed by using both a sintetic dsDNA fragment and MPXV field samples. Standards were assigned to absolute values by using digital PCR and a probe- based assay. LOD was estimated by fitting a probit regression model with a prediction level of 95% after running a negative sample and 5 low positive samples in 5 replicates. Specificity was assessed by using bacterial and viral DNA from herpesviruses, zoonotic parapoxviruses and orthopoxviruses. For field applications, Xample Prep tool was employed to treat MPXV clinical samples prior amplification; the results were then compared to those obtained with DNAs extracted with standard method. The assay resulted highly sensitive with estimated LOD of 18,6 DNA copies/μl and rapid being performed in 35 minutes overall from samples to results. Only Zoonotic OPXV DNA was amplified showing a high specificity and comparable results were obtained from purified viral DNA and Xample Prep treated samples. For its performances, rapidity, thermostable reagents, and ability to efficiently amplify clinical samples, without prior DNA extraction, the assay is suitable for use in low-resource settings, in the field and in community clinics. Besides clinical applications, to allow proper patients' management, the availability of such a diagnostic workflow can be critical to understand the real incidence and prevalence of zoonotic OPXV among humans and animal reservoirs. [ABSTRACT FROM AUTHOR]