Emergence of a mutation in the nucleocapsid gene of SARS-CoV-2 interferes with PCR detection in Canada

Abstract

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) was met with rapid development of robust molecular-based detection assays. Many SARS-CoV-2 molecular tests target multiple genetic regions of the virus to maximize detection and protect against diagnostic escape. Despite the relatively moderate mutational rate of SARS-CoV-2, numerous mutations with known negative impact on diagnostic assays have been identified. In early 2021, we identified four samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing revealed a single common mutation in the N gene C29200T. Spatiotemporal analysis showed that the mutation was found in at least six different Canadian provinces from May 2020 until May 2021. Phylogenetic analysis showed that this mutation arose multiple times in Canadian samples and is present in six different variants of interest and of concern. The Cepheid testing platform is commonly used in Canada including in remote regions. As such, the existence of N gene mutation dropouts required further investigation. While commercial SARS-CoV-2 molecular detection assays have contributed immensely to the response effort, many vendors are reluctant to make primer/probe sequences publicly available. Proprietary primer/probe sequences create diagnostic 'blind spots' for global SARS-CoV-2 sequence monitoring and limits the ability to detect and track the presence and prevalence of diagnostic escape mutations. We hope that our industry partners will seriously consider making primer/probe sequences available, so that diagnostic escape mutants can be identified promptly and responded to appropriately to maintain diagnostic accuracy.

Figure 1

Canadian distribution of SARS-CoV-2 genome sequences with the C29200T mutation. Blue circles represent the number of SARS-CoV-2 sequences per province or territory per time period available on GISAID. The orange circles indicate the number of sequences including the C29200T mutation. Data were retrieved from GISAID as of May 22, 2021, and of samples collected from (A) January–April 2020, (B) May–August 2020, (C) September–December 2020, (D) January–April 2021, and (E) January 2020-April 2021. The maps were built with the geographic information system QGIS (v2.18.21, https://qgis.org).

Figure 2

Maximum-likelihood tree of 337 sequences of SARS-CoV-2 in Canada and emergence of C29200T clades. Colour codes show the province where the patient sample was collected for the strains with C29200T mutations as follows: Alberta in red, British Columbia in magenta, Manitoba in orange, Nova Scotia in light blue, Ontario in yellow, and Quebec in lime. Sequences without the mutation used as controls were left uncolored. Sequence lineages are also indicated. The sixteen clades (#1–3, 5–6, 8–18) and two singletons (#4 and 7) analysed are identified with the outer numbers.