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Background: In 2022, the global dissemination of mpox virus (MPXV) outside endemic regions prompted the expansion of diagnostic testing worldwide. This study assesses the performance characteristics of 5 real-time polymerase chain reaction (PCR) assays in detecting MPXV during the 2022 outbreak., Methods: Clinical specimens collected from patients across Ontario, Canada, were tested on the following assays: RealStar Orthopoxyvirus PCR and FlexStar Monkeypox virus PCR (Altona Diagnostics), Novaplex MPXV (Seegene), VIASURE Monkeypox virus Real Time PCR Reagents (CerTest Biotec), and a laboratory-developed test. Positive percent agreement (PPA), negative percent agreement (NPA), relative limit of detection (LOD), and precision were evaluated and MPXV lineages were determined using an amplicon-based whole-genome sequencing (WGS) assay., Results: Swabs were collected from various anatomic sites (65 positive and 30 negative). All assays demonstrated 100% NPA (95% confidence interval, 88.4%/88.1%-100.0%), with PPA ranging from 92.2% (82.7%-97.4%) to 96.9% (89.3%-99.6%). LOD and precision were comparable across assays, with coefficient of variations <3%. WGS analysis identified 6 lineages, all belonging to subclade IIb., Conclusions: The assays exhibited excellent PPA, NPA, LOD, and precision. Ongoing performance monitoring is essential to detect assay escape mutants and ensure universal detection of evolving MPXV strains., Competing Interests: Potential conflicts of interest. K. B. and L. M. M. and received PCR kits in kind from Altona Diagnostics Canada, Seegene Canada, and BD Canada. L. M. M. is the president of the board of the Canadian Foundation for Infectious Diseases. R. K. and P. M. S. are recipients of financial support from the University of Toronto EPIC Mpox grant. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Importance: Nirmatrelvir-ritonavir is an oral antiviral medication that improves outcomes in SARS-CoV-2 infections. However, there is concern that antiviral resistance will develop and that these viruses could be selected for after treatment., Objective: To determine the prevalence of low-frequency SARS-CoV-2 variants in patient samples that could be selected for by nirmatrelvir-ritonavir., Design, Setting, and Participants: This retrospective cohort study was conducted at 4 laboratories that serve community hospitals, academic tertiary care centers, and COVID-19 assessment centers in Ontario, Canada. Participants included symptomatic or asymptomatic patients who tested positive for SARS-CoV-2 virus and submitted virus samples for diagnostic testing between March 2020 and January 2023., Exposure: SARS-CoV-2 infection., Main Outcomes and Measures: Samples with sufficient viral load underwent next-generation genome sequencing to identify low-frequency antiviral resistance variants that could not be identified through conventional sequencing., Results: This study included 78 866 clinical samples with next-generation whole-genome sequencing data for SARS-CoV-2. Low-frequency variants in the viral nsp5 gene were identified in 128 isolates (0.16%), and no single variant associated with antiviral resistance was predominate., Conclusions and Relevance: This cohort study of low-frequency variants resistant to nirmatrelvir-ritonavir found that these variants were very rare in samples from patients with SARS-CoV-2, suggesting that selection of these variants by nirmatrelvir-ritonavir following the initiation of treatment may also be rare. Surveillance efforts that involve sequencing of viral isolates should continue to monitor for novel resistance variants as nirmatrelvir-ritonavir is used more broadly.

Genome-wide variation in SARS-CoV-2 reveals evolution and transmission dynamics which are critical considerations for disease control and prevention decisions. Here, we review estimates of the genome-wide viral mutation rates, summarize current COVID-19 case load in the province of Ontario, Canada (5 January 2021), and analyze published SARS-CoV-2 genomes from Ontario (collected prior to 24 November 2020) to test for more infectious genetic variants or lineages. The reported mutation rate (∼10 -6  nucleotide [nt] -1  cycle -1 ) for SARS-CoV-2 is typical for coronaviruses. Analysis of published SARS-CoV-2 genomes revealed that the G614 spike protein mutation has dominated infections in Ontario and that SARS-CoV-2 lineages present in Ontario have not differed significantly in their rate of spread. These results suggest that the SARS-CoV-2 population circulating in Ontario has not changed significantly to date. However, ongoing genome monitoring is essential for identification of new variants and lineages that may contribute to increased viral transmission., (Copyright © 2021 Sjaarda et al.)

The emergence and rapid global spread of SARS-CoV-2 demonstrates the importance of infectious disease surveillance, particularly during the early stages. Viral genomes can provide key insights into transmission chains and pathogenicity. Nasopharyngeal swabs were obtained from thirty-two of the first SARS-CoV-2 positive cases (March 18-30) in Kingston Ontario, Canada. Viral genomes were sequenced using Ion Torrent (n = 24) and MinION (n = 27) sequencing platforms. SARS-CoV-2 genomes carried forty-six polymorphic sites including two missense and three synonymous variants in the spike protein gene. The D614G point mutation was the predominate viral strain in our cohort (92.6%). A heterozygous variant (C9994A) was detected by both sequencing platforms but filtered by the ARTIC network bioinformatic pipeline suggesting that heterozygous variants may be underreported in the SARS-CoV-2 literature. Phylogenetic analysis with 87,738 genomes in the GISAID database identified global origins and transmission events including multiple, international introductions as well as community spread. Reported travel history validated viral introduction and transmission inferred by phylogenetic analysis. Molecular epidemiology and evolutionary phylogenetics may complement contact tracing and help reconstruct transmission chains of emerging diseases. Earlier detection and screening in this way could improve the effectiveness of regional public health interventions to limit future pandemics.

Due to the lack of detailed reports of Klebsiella pneumoniae carbapenemase (KPC)-producing enterobacteria in Ontario, Canada, we perform a molecular characterization of KPC-producing Enterobacteriaceae submitted to the provincial reference laboratory from 2008 to 2011. Susceptibility profiles were accessed by E-test. Molecular types of isolates were determined by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. Screening of ß-lactamase genes was performed by multiplex PCR and alleles were identified by DNA sequencing. The genetic platform of blaKPC gene was analyzed by PCR. Plasmid replicons were typed using PCR-based typing approach. KPC-plasmids were also evaluated by S1 nuclease-PFGE and Southern blot. Thirty unique clinical isolates (26 Klebsiella pneumoniae, 2 Enterobacter cloacae, 1 Citrobacter freundii and 1 Raoultella ornithinolytica) were identified as blaKPC positive: 4 in 2008, 3 in 2009, 10 in 2010 and 13 in 2011. The majority exhibited resistance to carbapenems, cephalosporins and fluoroquinolones and two isolates were also resistant to colistin. The isolates harbored blaKPC-2 (n = 23) or blaKPC-3 (n = 7). blaTEM-1 (n = 27) was commonly detected and occasionally blaOXA-1 (n = 3) and blaCTX-M-15 (n = 1). As expected, all K. pneumoniae isolates carried blaSHV-11. blaKPC genes were identified on Tn4401a (n = 20) or b (n = 10) isoforms, on plasmids of different sizes belonging to the incompatibility groups IncFIIA (n = 19), IncN (n = 3), IncI2 (n = 3), IncFrep (n = 2) and IncA/C (n = 1). The occurrence of KPC ß-lactamase in Ontario was mainly associated with the spread of the K. pneumoniae clone ST258.