Large scale analysis of the SARS-CoV-2 main protease reveals marginal presence of nirmatrelvir-resistant SARS-CoV-2 Omicron mutants in Ontario, Canada, December 2021-September 2023.

Background: In response to the COVID-19 pandemic, a new oral antiviral called nirmatrelvir-ritonavir (Paxlovid^TM) was authorized for use in Canada in January 2022. In vitro studies have reported mutations in M^pro protein that may be associated with the development of nirmatrelvir resistance.

Objectives: To survey the prevalence, relevance and temporal patterns of M^pro mutations among SARS-CoV-2 Omicron lineages in Ontario, Canada.

Methods: A total of 93,082 M^pro gene sequences from December 2021 to September 2023 were analyzed. Reported in vitro M^pro mutations were screened against our database using in-house data science pipelines to determine the nirmatrelvir resistance. Negative binomial regression was conducted to analyze the temporal trends in M^pro mutation counts over the study time period.

Results: A declining trend was observed in non-synonymous mutations of M^pro sequences, showing a 7.9% reduction (95% CI: 6.5%-‬9.4%; p<0.001) every 30 days. The P132H was the most prevalent mutation (higher than 95%) in all Omicron lineages. In vitro nirmatrelvir-resistant mutations were found in 3.12% (n=29/929) Omicron lineages with very low counts, ranging from one to 19. Only two mutations, A7T (n=19) and M82I (n=9), showed temporal presence among the BA.1.1 in 2022 and the BQ.1.2.3 in 2022, respectively.

Conclusion: The observations suggest that, as of September 2023, no significant or widespread resistance to nirmatrelvir has developed among SARS-CoV-2 Omicron variants in Ontario. This study highlights the importance of creating automated monitoring systems to track the emergence of nirmatrelvir-resistant mutations within the SARS-CoV-2 virus, utilizing genomic data generated in real-time.