S-Gene Target Failure as a Marker of Variant B.1.1.7 Among SARS-CoV-2 Isolates in the Greater Toronto Area, December 2020 to March 2021

Objectives The current study evaluates a testing algorithm for the rapid identification of SARS-CoV-2 variants that includes the use of PCR-based targeted Single Nucleotide Polymorphism (SNP) detection assays preceded by a multiplex PCR sensitive to s -Gene Target Failure (SGTF). Methods PCR SNP assays targeting SARS-CoV-2 s -gene mutations ΔH69-V70, L452R, E484K, N501Y, H655Y, and P681R using melting curve analysis were performed on 567 samples in which SARS-CoV-2 viral RNA was detected by a multiplex PCR. Viral whole genome sequencing (WGS) was performed to confirm the presence of SNPs and to identify the Pangolin lineage. Additionally, 1133 SARS-CoV-2 positive samples with SGTF were further assessed by WGS to determine the presence of ΔH69-V70. Results The N501Y-specific assay (N=567) had an overall percent agreement (OPA) of 98.5%. The ΔH69-V70- (N=178) and E484K-specific (N=401) assays had an OPA of 96.6 and 99.7% respectively. Assessment of H655Y (N=139) yielded a 100.0% concordance when applied in the proposed algorithm. The L452R- (N=67) and P681R-specific (N=62) assays had an OPA of 98.2 and 98.1% respectively. The proposed algorithm identified six variants of concern/interest (VOC/VOI) – Alpha (N=149), Beta (N=65), Gamma (N=86), Delta (N=49), Eta (N=6), Kappa (N=6) – and 205 non-VOC/VOI strains – including the variants under monitoring B.1.214.2 (N=43) and B.1.1.318 (N=18) and Epsilon (N=1). An excellent concordance was observed for the identification of all SARS-CoV-2 lineages evaluated. Conclusions We present a flexible testing algorithm for the rapid detection of current and emerging SARS-CoV-2 VOC/VOIs which can be easily adapted based on the local endemicity of specific variants.

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“…Several lineages display SGTF, which has been linked to ΔH69-V70 [ 13 ]. In total, 1133 samples with SGTF were analyzed by WGS.…”

Section: Resultsmentioning

confidence: 99%

Monitoring the SARS-CoV-2 pandemic: screening algorithm with single nucleotide polymorphism detection for the rapid identification of established and emerging variants

Mertens

Coppens

Loens

et al. 2022

Clinical Microbiology and Infection

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“…This approach may be particularly useful in settings where sequencing capacity is limited, as sequencingindependent means of stratifying samples by lineage (e.g., variant-specific primers, single gene failure etc.) can be used to then compare Ct values between variants [43]. However, we emphasize that this approach currently requires samples obtained through random crosssectional surveillance, or nearly random samples such as non-COVID patient hospital testing.…”

Section: Discussionmentioning

confidence: 99%

Viral loads observed under competing strain dynamics

Hay

Kennedy‐Shaffer

Mina

2021

Preprint

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A plausible mechanism for the increased transmissibility of SARS-CoV-2 variants of concern (VOCs) results from VOC infections causing higher viral loads in infected hosts. However, investigating this hypothesis using routine RT-qPCR testing data is challenging because the population-distribution of viral loads changes depending on the epidemic growth rate; lower cycle threshold (Ct) values for a VOC lineage may simply reflect increasing incidence relative to preexisting lineages. To understand the extent to which viral loads observed under routine surveillance systems reflect viral kinetics or population dynamics, we used a mathematical model of competing strain dynamics and simulated Ct values for variants with different viral kinetics. We found that comparisons of Ct values obtained under random cross-sectional surveillance were highly biased unless samples were obtained at times when the variants had comparable growth rates. Conversely, comparing Ct values from symptom-based testing was largely unaffected by epidemic dynamics, and accounting for the time between symptom onset and sample collection date further reduced the risk of statistical errors. Finally, we show how a single cross-sectional sample of Ct values can be used to jointly estimate differences in viral kinetics and epidemic growth rates between variants. Epidemic dynamics should be accounted for when investigating strain-specific viral kinetics using virologic surveillance data, and findings should be corroborated with longitudinal viral kinetics studies.

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“…A number of reports have performed assessments of nucleotide sequence variation within the RT-PCR primers and probes creating mismatches and potentially contributing to false-negative outcomes [47,[64][65][66]. The B.1.1.7 or United Kingdom (UK) variant (now referred to as the Alpha variant), which until recently was the dominant strain in the United States, is associated with S gene target failure (SGTF) in some RT-PCR assays [67]. Similarly, others have described nucleocapsid (N) gene and envelope (E) gene target failures associated with different variants [68,69].…”

Section: Sars-cov-2 Genome Sequence Variation and Its Impact On Diagnosis And Diseasementioning

confidence: 99%

Molecular Diagnosis of SARS-CoV-2: Assessing and Interpreting Nucleic Acid and Antigen Tests

Zimmerman

King

Ghannoum

et al. 2021

PAI

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In this review, we summarize the current status of nucleic acid and antigen testing required for diagnosing SARS-CoV-2 infection and COVID-19 disease. Nucleic acid amplification (NAAT) and antigen-detection (Ag) tests occupy a critically important frontline of defense against SARS-CoV-2 in clinical and public health settings. In early stages of this outbreak, we observed that identifying the causative agent of a new illness of unknown origin was greatly accelerated by characterizing the nucleic acid signature of the novel coronavirus. Results from nucleic acid sequencing led to the development of highly sensitive RT-PCR testing for use in clinical settings and to informing best practices for patient care, and in public health settings to the development of strategies for protecting populations. As the current COVID-19 pandemic has evolved, we have seen how NAAT performance has been used to guide and optimize specimen collection, inform patient triage decisions, reveal unexpected clinical symptoms, clarify risks of transmission within patient care facilities, and guide appropriate treatment strategies. For public health settings during the earliest stages of the pandemic, NAATs served as the only tool available for studying the epidemiology of this new disease by identifying infected individuals, studying transmission patterns, modeling population impacts, and enabling disease control organizations and governments to make challenging disease mitigation recommendations to protect the expanding breadth of populations at risk. With time, the nucleic acid signature has provided the information necessary to understand SARS-CoV-2 protein expression for further development of antigen-based point-of-care (POC) diagnostic tests. The advent of massive parallel sequencing (ie, next generation sequencing) has afforded the characterization of this novel pathogen, informed the sequences best adapted for RT-PCR assays, guided vaccine production, and is currently used for tracking and monitoring SARS-CoV-2 variants.

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S-Gene Target Failure as a Marker of Variant B.1.1.7 Among SARS-CoV-2 Isolates in the Greater Toronto Area, December 2020 to March 2021

Cited by 116 publications

References 4 publications

Monitoring the SARS-CoV-2 pandemic: screening algorithm with single nucleotide polymorphism detection for the rapid identification of established and emerging variants

Monitoring the SARS-CoV-2 pandemic: screening algorithm with single nucleotide polymorphism detection for the rapid identification of established and emerging variants

Viral loads observed under competing strain dynamics

Molecular Diagnosis of SARS-CoV-2: Assessing and Interpreting Nucleic Acid and Antigen Tests

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