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.

Objective: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hospital outbreaks have been common and devastating during the coronavirus disease 2019 (COVID-19) pandemic. Understanding SARS-CoV-2 transmission in these environments is critical for preventing and managing outbreaks. Design: Outbreak investigation through epidemiological mapping and whole-genome sequencing phylogeny. Setting: Hospital in-patient medical unit outbreak in Toronto, Canada, from November 2020 to January 2021. Participants: The outbreak involved 8 patients and 10 staff and was associated with 3 patient deaths. Results: Patients being cared for in geriatric chairs at the nursing station were at high risk for both acquiring and transmitting SARS-CoV-2 to other patients and staff. Furthermore, given the informal nature of these transmissions, they were not initially recognized, which led to further transmission and missing the opportunity for preventative COVID-19 therapies. Conclusions: During outbreak prevention and management, the risk of informal patient care settings, such as geriatric chairs, should be considered. During high-risk periods or during outbreaks, efforts should be made to care for patients in their rooms when possible.

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Background The World Health Organization (WHO) introduced molecular identifiers for the diagnosis and prognosis of CNS tumors including the mutational status of isocitrate dehydrogenase or IDHgenes in glial tumors. Currently used immunohistochemistry (IHC) is not capable of detecting the non‐canonical mutations, and sequencing is often required as a follow‐up. Current next‐generation sequencing (NGS) technologies used in tumor molecular marker detection introduce key challenges including high capital cost, complex infrastructure requirements, and long turnaround times. These challenges considerably limit the ability to perform NGS testing in many pathology laboratories. In this study, we aimed to use third generation nanopore sequencing technology to resolve these limitations. The Oxford Nanopore MinION, a pocket‐sized nanopore sequencing device, has minimal capital costs and infrastructural requirements, and shorter turnaround times. However, the nanopore technology has not been validated in clinical practice and has not been optimized on formalin‐fixed paraffin‐embedded (FFPE) tissue. Methods DNA extraction of selective tumor areas was performed from the corresponding FFPE tissue blocks from a cohort of gliomas with confirmed IDH1and IDH2gene statuses (n=65). A PCR amplicon‐based approach was used to amplify hot spots of the IDH1and IDH2genes, starting with 30ng DNA material. The amplicon libraries were sequenced for 2 hours in multiplex on the MinION device and IDHSNPs were called with the Nanopolish software. ASIP Abstract Mashiat Mimosa Results 26 IDH mutant samples were identified: 21 IDH1R132H, 2 IDH1R132G, 2 IDH2R172G, and 1 IDH2D177H. All cases showed concordant IDHmutational status when compared to the reference methods (IHC or NGS) and both analytical sensitivity and specificity were 100%. Precision analysis of variant allele frequency (VAF) showed the coefficient of variation was less than 5% (both inter and intra runs), and the limit of detection for VAF was 3%. The range of read depth obtained was 882X to 43,000x with an average of 20,000x. This assay revealed a $50‐$100 material cost per sample, and the time taken from extracted nucleic acid to final result generation was 1‐2 business days. Conclusion This project is the first to optimize and validate an approach to detect SNP mutations in FFPE samples using nanopore technology. It has demonstrated the feasibility and efficacy of the nanopore amplicon sequencing method in cancer FFPE tissue with excellent test performance characteristics, significantly shorter turnaround times at considerably lower costs and without any infrastructural needs. Thus, it can be used to circumvent challenges to current NGS testing platforms and can be the milestone that would make cancer NGS testing available for every laboratory.