Evaluation of 5 Polymerase Chain Reaction Assays for the Detection of Mpox Virus.

Abstract

In 2022, the global dissemination of mpox virus (MPXV) outside endemic regions prompted the expansion of diagnostic testing worldwide. This study assesses the performance characteristics of 5 real-time polymerase chain reaction (PCR) assays in detecting MPXV during the 2022 outbreak. Clinical specimens collected from patients across Ontario, Canada, were tested on the following assays: RealStar Orthopoxyvirus PCR and FlexStar Monkeypox virus PCR (Altona Diagnostics), Novaplex MPXV (Seegene), VIASURE Monkeypox virus Real Time PCR Reagents (CerTest Biotec), and a laboratory-developed test. Positive percent agreement (PPA), negative percent agreement (NPA), relative limit of detection (LOD), and precision were evaluated and MPXV lineages were determined using an amplicon-based whole-genome sequencing (WGS) assay. Swabs were collected from various anatomic sites (65 positive and 30 negative). All assays demonstrated 100% NPA (95% confidence interval, 88.4%/88.1%-100.0%), with PPA ranging from 92.2% (82.7%-97.4%) to 96.9% (89.3%-99.6%). LOD and precision were comparable across assays, with coefficient of variations <3%. WGS analysis identified 6 lineages, all belonging to subclade IIb. The assays exhibited excellent PPA, NPA, LOD, and precision. Ongoing performance monitoring is essential to detect assay escape mutants and ensure universal detection of evolving MPXV strains.