Real-time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with Variants of Concern (preprint)

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of SARS-CoV2 is possessed by the three most common variants of concern - B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the COVID-19 pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription polymerase chain reaction assay using allelic discrimination of the spike gene N501Ymutation to screen for potential variants of concern and differentiate them from wild-type SARS-CoV-2. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or wild-type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real time reverse transcriptase polymerase chain reaction assay. Our assay compared to sequencing, the gold standard for SNP detection and lineage identification, demonstrated clinical sensitivity of 100% for all 57 specimens displaying N501Y mutant, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wildtype and mutant. Clinical specificity was 100% in all 103 specimens typed as wild-type, with A23063 identified as wild-type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole genome sequencing which are time-consuming, labor-intensive and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole genome sequencing.

Impact of COVID-19 pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results (preprint)

Background: Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent resolved infection with persistent RNA shedding, presymptomatic or asymptomatic infection, or a false positive test. This study assessed clinical specificity of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay. Materials and Methods: A total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. Results: Significantly less positive results in the lowest pre-test probability group (facilities with institution-wide screening having [≤]3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in all other groups combined (5/32, 15.6% vs 61/90, 68%; p <0.0001), and in each subgroup with higher pre-test probability (individual subgroup range 50.0% to 85.0%). Conclusions: A higher proportion of false-positive test results are likely with lower pre-test probability. Positive SARS-CoV-2 PCR results should be interpreted within the context of patient history, clinical setting, known exposure, and estimated community disease prevalence. Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level.

Characteristics of SARS-CoV-2 Testing for Rapid Diagnosis of COVID-19 during the Initial Stages of a Global Pandemic (preprint)

Accurate SARS-CoV-2 diagnosis is essential to guide prevention and control of COVID-19. From January 11 - April 22, 2020, Public Health Ontario conducted SARS-CoV-2 testing of 86,942 specimens collected from 80,354 individuals, primarily using real-time reverse-transcription polymerase chain reaction (rRT-PCR) methods. We analyzed test results across specimen types and for individuals with multiple same-day and multi-day collected specimens. Nasopharyngeal compared to throat swabs had a higher positivity (8.8% vs. 4.8%) and an adjusted estimate 2.9 Ct lower (SE=0.5, p<0.001). Same-day specimens showed high concordance (98.8%), and the median Ct of multi-day specimens increased over time. Symptomatic cases had rRT-PCR results with an adjusted estimate 3.0 Ct (SE=0.5, p<0.001) lower than asymptomatic/pre-symptomatic cases. Overall test sensitivity was 84.6%, with a negative predictive value of 95.5%. Molecular testing is the mainstay of SARS-CoV-2 diagnosis and testing protocols will continue to be dynamic and iteratively modified as more is learned about this emerging pathogen.

Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide (preprint)

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared on March 11, 2020 by the World Health Organization. As of the 31st of May, 2020, there have been more than 6 million COVID-19 cases diagnosed worldwide and over 370,000 deaths, according to Johns Hopkins. Thousands of SARS-CoV-2 strains have been sequenced to date, providing a valuable opportunity to investigate the evolution of the virus on a global scale. We performed a phylogenetic analysis of over 1,225 SARS-CoV-2 genomes spanning from late December 2019 to mid-March 2020. We identified a missense mutation, D614G, in the spike protein of SARS-CoV-2, which has emerged as a predominant clade in Europe (954 of 1,449 (66%) sequences) and is spreading worldwide (1,237 of 2,795 (44%) sequences). Molecular dating analysis estimated the emergence of this clade around mid-to-late January (10 - 25 January) 2020. We also applied structural bioinformatics to assess D614G potential impact on the virulence and epidemiology of SARS-CoV-2. In silico analyses on the spike protein structure suggests that the mutation is most likely neutral to protein function as it relates to its interaction with the human ACE2 receptor. The lack of clinical metadata available prevented our investigation of association between viral clade and disease severity phenotype. Future work that can leverage clinical outcome data with both viral and human genomic diversity is needed to monitor the pandemic.