Rapid adaptation of established high-throughput molecular testing infrastructure for detection of monkeypoxvirus

Background Since May 2022, a rising number of monkeypox-cases has been reported in non-endemic countries of the northern hemisphere. In contrast to previous clusters, infections seem predominantly driven by human-to-human transmission, rather than animal sources. In this study, we adapted two published qPCR assays (non-variola orthopoxvirus and monkeypoxvirus specific) for use as a lab-developed dual-target monkeypoxvirus-test on widely used automated high-throughput PCR-systems (cobas5800/6800/8800). Methods Selected assays were checked for in-silico inclusivity and exclusivity in current orthopoxvirus sequences, as well as for multiplex compatibility. Analytic performance was determined by serial dilution of monkeypoxvirus reference material, quantified by digital PCR. Cross reactivity was ruled out through a clinical exclusivity set containing various bloodborne and respiratory pathogens. Clinical performance was compared to a commercial manual RUO-kit using clinical remnant samples. Results Analytic lower limit of detection (LoD) was determined as 4.795 dcp/ml (CI95%: 3.598 - 8.633 dcp/ml) for both assays combined, with a dynamic range of at least 5 log-steps. The assay showed 100% positive and negative agreement with the manual RUO orthopoxvirus PCR test kit in clinical swab samples. Discussion While the full extend of the ongoing monkeypox outbreak remains to be established, the WHO and local health authorities are calling for increased awareness and efforts to limit further spread. For this, timely and scalable PCR tests are an important prerequisite. The assay presented here allows streamlined high-throughput molecular testing for monkeypoxvirus on existing hardware, broadly established previously for SARS-CoV-2 diagnostics. ### Competing Interest Statement ML and DN received speaker honoraria and related travel expenses from Roche Diagnostics. All other authors declare no conflict of interest. ### Funding Statement This study did not receive any funding ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This work was conducted in accordance with Paragraph 12 of the Hamburg hospital law (Paragraph 12 HmbKHG). The use of anonymized samples was approved by the ethics committee, Freie und Hansestadt Hamburg, PV5626. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors * Dcp : digital copies LoD : Limit of Detection IC : internal control IVD : in-vitro diagnostic RFI : relative fluorescence increase CI : confidence interval