Comparative effects of viral-transport-medium heat inactivation upon downstream SARS-CoV-2 detection in patient samples

  1. Jamie L. Thompson
  2. Angela Downie Ruiz Velasco
  3. Alice Cardall
  4. Rebecca Tarbox
  5. Jaineeta Richardson
  6. Gemma Clarke
  7. Michelle Lister
  8. Hannah C. Howson-Wells
  9. Vicki M. Fleming
  10. Manjinder Khakh
  11. Tim Sloan
  12. Nichola Duckworth
  13. Sarah Walsh
  14. Chris Denning
  15. C. Patrick McClure
  16. Andrew V. Benest
  17. Claire H. Seedhouse

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Abstract

Introduction. The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.

Hypothesis. Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.

Aim. We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).

Methodology. We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.

Results. Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically ( n =127; R 2 =0.9259).

Conclusion. Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.

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  1. ScreenIT

    SciScore for 10.1101/2020.07.30.20164988: (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
    RT-qPCR cycle numbers generated during this work were compared to those generated diagnostically by the RealStar (NUH) or the Abbott systems (ULH).
    Abbott systems
    suggested: None
    All data analysis and graph production were performed using GraphPad Prism 8.3.
    GraphPad Prism
    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:
    Alternative testing methods to the widely used two-step RNA purification and RT-qPCR, are being employed by laboratories to increase capacity and avoid limitations caused by supply constraints (9). We confirm viral inactivation using heat treatment and direct RT-qPCR, bypassing RNA extraction, can be used to detect SARS-CoV-2 in swab samples. However, we found accurate detection using this method to be dependent on the RT-qPCR assay used. We thus highlight the need for local assay validation before implementing an extraction-free workflow in a clinical laboratory. The VIASURE assay performed well on purified RNA, but was inhibited by non-purified material. Further investigation into the incompatibility of the VIASURE assay with crude swab material, using spiked RNA, revealed the reverse transcriptase step is adversely affected (data not shown). This could be explained by autofluorescence of the viral transport medium, or other contaminants within swab samples. In contrast to the VIASURE assay, the combined CDC N1, N2 Quantabio UltraPlex assay gave comparable results using both RNA purification and extraction-free methods. Similar discrepancies between assay kits have been reported in other heat-processing systems (12). Detection accuracy of viral RNA swabs in clinical laboratories varies on the swab type used, the quality of sample collection and the stage of the viral infection. The combined CDC N1, N2 Quantabio UltraPlex assay was able to detect SARS-CoV-2 in a large cohort...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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

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  2. Version published to 10.1099/jmm.0.001301 on Access Microbiology
  3. Version published to 10.1101/2020.07.30.20164988v1 on medRxiv