International external quality assessment study for detection of monkeypox virus by PCR supporting laboratory preparedness during the 2022-2023 mpox outbreak and beyond

Background: Diagnostic capabilities and correspondent External Quality Assessments (EQA) are key for outbreak preparedness. To support diagnostic facilities with a quality assessment of newly established monkeypox virus (MPXV) molecular diagnostic workflows, Quality Control for Molecular Diagnostics (QCMD) and the Bundeswehr Institute of Microbiology (IMB) piloted an international EQA study conducting four challenges from autumn 2022 to summer 2023 during the global mpox outbreak. Objectives: To assess the performance (sensitivity/specificity) of molecular assays used by diagnostic laboratories. Study design: Inactivated EQA panels were prepared and distributed containing seven samples of clade Ia and clade IIb MPXV strains at different viral concentrations, two specificity controls with other zoonotic orthopoxviruses (vaccinia and cowpox virus) and a negative control. Assessment was based on reported qualitative testing results. Results: In this outbreak-related EQA study, a total of 192 laboratories from 37 countries reported 346 qualitative datasets. Overall, core samples were correctly detected by approximately 92 % of participants in all four challenges. While sensitivity performance was acceptable in at least 90 % of datasets correctly reported even for educational MPXV-positive samples with low viral concentration [102 genome equivalents (GE)/mL], several laboratories reported the educational specificity controls as false positives or were unable to differentiate MPXV from related orthopoxviruses. Conclusions: Mpox is now a globally occurring infection with a demand for quality-assured diagnostic capabilities. The newly established EQA scheme presented here, offers a multi-purpose panel for orthopoxviruses with a focus on MPXV which will continue to ensure diagnostic quality in clinical settings with up-to-date sample panels.