A Multi-Laboratory Evaluation of Commercial Monkeypox Molecular Tests

Abstract

In this report, we describe the first national scale multi-laboratory evaluation of commercial quantitative PCR kits for detection of Monkeypox virus (MPXV) DNA. The objective of this study was to assess the performance of two kits by different diagnostic laboratories across Israel. A panel of 10 standardized samples was tested simultaneously using the Novaplex (15 laboratories) and Bio-Speedy (seven laboratories) kits. An in-house assay based on previously published tests was used as reference. Comparison of the results showed high intra-assay consistency between laboratories, with small variations for most samples. The sensitivity of the two kits was similar to that of the in-house assay, with an analytical detection limit of less than ten copies per reaction. Significant differences were observed, however, in the Cq values and relative fluorescence (RF), between the assays. The RF signal of the in-house and Bio-Speedy assay ranged between 5,000 and 10,000 RFU, while the signal in the Novaplex assay was less than 600 RFU. Due to the kit measurement protocol, the Cq values of the Bio-Speedy kit were 5-7.5 cycles lower than those of the In-house assay. On the contrary, the Cq values of the Novaplex kit were significantly higher than those of the in-house assay, with differences of 3-5 cycles per sample. Our results suggest that while all assays were similar in their overall sensitivity, direct comparison of Cq values between them may be misleading. Additionally, the low fluorescence obtained with the Novaplex kit may be problematic with marginal or high-background samples. Diagnostic laboratories should therefore consider all these aspects when choosing a specific MPX detection assay.