A Multi-Laboratory Evaluation of Commercial Monkeypox Virus Molecular Tests

Abstract

In this report, we describe the first national scale multi-laboratory evaluation of monkeypox virus (MPXV) DNA commercial PCR kits. The objective of this study was to evaluate 2 kits by different diagnostic laboratories across Israel. Ten standardized samples were tested simultaneously using the Novaplex (15 laboratories) and Bio-Speedy (seven laboratories) kits. An in-house assay based on previously published reactions was used as reference. Comparison of the results showed high intra-assay agreement between laboratories, with small variations for most samples. The in-house assay had an analytical detection limit of less than 10 copies per reaction. While the 2 commercial kits were able to detect specimens with low viral loads similarly to the in-house assay, significant differences were observed, in the Cq values and relative fluorescence (RF), between the assays. The RF signal of the in-house and Bio-Speedy assays 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 to 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 to 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. To our knowledge, this is the first methodical evaluation of commercial MPX test kits. We therefore anticipate that this study would help diagnostic laboratories in choosing a specific MPX detection assay. IMPORTANCE To the best of our knowledge, this study is the first methodical evaluation of commercial kits designed for Monkeypox virus detection. This was done by performing the same tests using the same sample set in multiple laboratories, simultaneously, on a national scale. It therefore provides important and unique information on the performance of such kits and provides a guideline for choosing the assay of choice for monkeypox virus diagnosis in a standard diagnostic laboratory. It also demonstrates potential complications when trying to compare the results of different assays, even when testing exactly the same samples, under identical conditions.

Keywords: PCR; commercial kit; comparative analysis; detection kit; molecular diagnostics; monkeypox; real-time PCR; validation.

FIG 1

Comparison of the Cq values obtained with each assay, for each MPXV standard sample. Each individual value is represented by a single dot. The mean Cq in each plot is marked with a thick dotted line. The thin lines denote the quartile values of each sample. The distribution is depicted by the colored area for each assay, as indicated in the figure legend. SGN – Seegene Novaplex MPX kit, BSP – BioSpeedy MPX kit, CVL assay – the assay used in the Central Virology Laboratory, based on the MPXV reactions described by Li et al. (7). Statistical significance of the mean Cq value difference was calculated for each sample, between the CVL assay and each of the commercial kits. The P-value for each sample is indicated as follows: *, P < 0.05, **, P < 0.01, ***, P < 0.005, ****, P < 0.001. The P-value for sample 5 was calculated excluding the sample marked with an arrow (Cq = 26.2), which is 7–10 cycles lower than all other Novaplex values obtained for this sample.