Higher Relative Viral Load Excretion Determined by Normalised Threshold Crossing Value in Acute Cases infected with the B.1.1.7 Lineage VOC 202012/01 (Using S gene target failure as a Proxy) When Compared to other Circulating Lineages in Wales

  1. Anastasia Couzens
  2. Isa Murrell
  3. Laura Gifford
  4. Ben Johns
  5. Kathleen Pheasant
  6. Luke Turner
  7. Jonathan Evans
  8. Catherine Moore

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Abstract

Since the emergence of SARS-CoV-2, global monitoring of the virus using whole genome sequencing has identified mutations occurring across the viral genome. Whilst the majority have little impact on the virus, they are used effectively to monitor the movement of the virus globally and to inform locally on transmission chains.

In late 2020, a variant of SARS-CoV-2 (B.1.1.7 - VOC 202012/01) was identified in the UK with a distinct constellation of mutations, including in the spike gene that increased transmissibility. A deletion in spike also affected one of the screening qPCR tests being used in the UK outside of Wales, causing a failure to detect the target. This quickly became a surrogate marker for the variant to allow rapid monitoring of the virus as it seeded into new regions of the UK.

A screening study using this assay as a proxy marker, was undertaken to understand the prevalence of the variant in Wales. Secondary analysis of a screening qPCR that didn’t target the S gene and also included an endogenous control, was also performed to understand viral load excretion in those infected with the variant compared to other circulating lineages. Using a combination of analytical methods based on the C t values of two gene targets normalised against the endogenous control, there was a difference in the excreted viral load. Those with the variant excreting more virus than those not infected with the variant. Supporting not only increased infectivity but offering a plausible reason why increased transmission was associated with this particular variant.

Whilst there are limitations in this study, the method using C t as a proxy for viral load can be used at the population level to determine differences in viral excretion kinetics associated with different variants.

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  1. ScreenIT

    SciScore for 10.1101/2021.04.02.21254832: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    The results of which then underwent log transformations for ease of statistical analysis, and subject to one-way ANOVA and Tukey’s multiple comparisons post-hoc test in Prism ® (V 9.0.1) by GraphPad Software (San Diego, California, United States).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A limitation of this analyses is as this was performed retrospectively, the efficiency of the amplification kinetics of the PE assay was not determined. Because of this, the original iteration of the ΔΔCt method in which efficiency is assumed to be 100%, was used. Using a single assay and gene target failure to determine the presence or not of a particular variant also has limitations, as despite genomics showing that in regions where the variant might dominate 98% or more of the target failure samples are positive for the VOC, the change that leads to the failure may also be sporadic. For example, ΔH69/V70 was reported in some circulating lineages before the emergence of VOC 202012/01, In Wales however, the molecular epidemiology generated by systematic whole genome sequencing in pillar 1 and latterly in pillar 2, largely support the data generated by the proxy assay for the arrival and establishment of VOC 202012/01 across Wales. This study does demonstrate that whilst Ct values generated in a qualitative PCR test can’t be used to firmly determine infectivity, they can be used to indicate changes in viral load excretion patterns associated with emerging variants at a population level, when a common testing platform with an endogenous control is used to normalise target gene against.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.04.02.21254832v1 on medRxiv