Molecular Diagnosis of Human Monkeypox Virus during 2022-23 Outbreak: Preliminary Evaluation of Novel Real-Time Qualitative PCR Assays.

In 2022-23, the human monkeypox virus (MPXV) caused a global outbreak in several non-endemic countries. Here, we evaluated the diagnostic performance of four qualitative PCR assays for the laboratory diagnosis of mpox (monkeypox) monkeypox disease. From July to August 2022, 27 positive and 10 negative specimens (lesion, crust and exudate swabs) were tested in the laboratory of the Hygiene Unit of the San Martino Hospital (Genoa, Italy) by using home-made real-time PCR to detect MPXV generic G2R_G DNA. According to the manufacturer's instructions, we also retrospectively analyzed these specimens using RealCycler MONK-UX/-GX (Progenie Molecular), STANDARD M10 MPX/OPX (SD Biosensor), Novaplex MPXV (Seegene Inc.) and RealStar Orthopoxvirus PCR Kit 1.0 (Altona Diagnostics) assays, recognized as research-use-only tests. The diagnostic accuracy and sensitivity of these assays ranged from 97.3% (95% CI: 86.2-99.5%) to 100% (95% CI: 90.6-100%) and 96.3% (95% CI: 81.72-99.34%) to 100% (95% CI: 72.2-100%), respectively. The RealCycler MONK-UX and STANDARD M10 MPX/OPX did not detect one positive sample with a cycle threshold of 36. The overall specificity was 100% (95% CI: 72.2-100%), and Cohen's Kappa values ranged from 1 (95% CI: 0.67-1) to 0.93 (95% CI: 0.61-1). As they are highly accurate, reliable and user-friendly, these tests should be recommended for the routine or rapid laboratory discrimination of mpox from other rash illnesses.