Rapid surveillance platforms for key SARS-CoV-2 mutations in Denmark

  1. Katja Spiess
  2. Vithiagaran Gunalan
  3. Ellinor Marving
  4. Sofie Holdflod Nielsen
  5. Michelle G. P. Jørgensen
  6. Anna S. Fomsgaard
  7. Line Nielsen
  8. Alonzo Alfaro-Núñez
  9. Søren M. Karst
  10. The Danish COVID-19 Genome Consortium (DCGC)
  11. Shila Mortensen
  12. Morten Rasmussen
  13. Ria Lassaunière
  14. Maiken Worsøe Rosenstierne
  15. Charlotta Polacek
  16. Jannik Fonager
  17. Arieh S. Cohen
  18. Claus Nielsen
  19. Anders Fomsgaard

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Abstract

Multiple mutations in SARS-CoV-2 variants of concern (VOCs) may increase, transmission, disease severity, immune evasion and facilitate zoonotic or anthoprozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K and N501Y, occur in the SARS-CoV-2 spike glycoprotein in combinations that allow detection of the most important VOCs. Here we present two flexible RT-qPCR platforms for small-and large-scale screening to detect these mutations, and schemes for adapting the platforms for future mutations. The large-scale RT-qPCR platform, was validated by pair-wise matching of RT-qPCR results with WGS consensus genomes, showing high specificity and sensitivity. Detection of mutations using this platform served as an important interventive measure for the Danish public health system to delay the emergence of VOCs and to gain time for vaccine administration. Both platforms are valuable tools for WGS-lean laboratories, as well for complementing WGS to support rapid control of local transmission chains worldwide.

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    SciScore for 10.1101/2021.10.25.21265484: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Ethics: Exemption for review by the ethical committee system and informed consent was given by the Committee on Biomedical Research Ethics - Capital region in accordance with Danish law on assay development projects.
    Consent: Ethics: Exemption for review by the ethical committee system and informed consent was given by the Committee on Biomedical Research Ethics - Capital region in accordance with Danish law on assay development projects.
    IRB: Ethics: Exemption for review by the ethical committee system and informed consent was given by the Committee on Biomedical Research Ethics - Capital region in accordance with Danish law on assay development projects.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Subsequent passages to expand virus stocks were performed in 75 cm2 flasks seeded with 1.5×106 Vero E6 the day before. 25 µL of primary isolate supernatant was used as inoculum in the presence of 2 mL infection media.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Extracted Influenza virus RNA from viruses cultured in Madin Darby Canine Kidney (MDCK) cells (A/Christchurch/16/2010(H1N1), pdm09-like virus, B/Phuket/3073/2013-like virus, B/Brisbane/60/2008-like virus, were all previously confirmed by PCR to be positive at high concentration (Ct <<30) for respective pathogens.
    MDCK
    suggested: None
    Software and Algorithms
    SentencesResources
    RT-qPCR Validation: Nucleotide sequences corresponding to the Sof consensus genomes derived from WGS were aligned using the MAFFT version 7.480 (mafft.cbrc.jp), utilizing the FFT-NS-2 algorithm with a maximum of 1000 iterations40,41.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    Alignments were viewed and processed in Jalview 2.11.1.4 (jalview.org,42) and codons encoding key mutations were extracted, translated and compared to RT-qPCR results.
    Jalview
    suggested: (Jalview, RRID:SCR_006459)
    Positive and Negative Predictive Values were calculated according to the following formulas:where Sen = sensitivity, Spec = specificity and Prev = prevalence calculated from WGS consensus genomes.
    WGS
    suggested: None
    All analyses were performed in Rstudio version 1.4.1717 using R version 4.1.1 and using the packages tidyverse (1.3.1), seqinr (4.2-8), lubridate (1.7.10), ggplot2 (3.3.4), cowplot (1.1.1), zoo (1.8-9) and ggpubr (0.4.0).
    Rstudio
    suggested: (RStudio, RRID:SCR_000432)
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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.
    • Thank you for including a protocol registration statement.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.10.25.21265484v1 on medRxiv