Pathophysiological Response to SARS-CoV-2 Infection Detected by Infrared Spectroscopy Enables Rapid and Robust Saliva Screening for COVID-19

  1. Seth T. Kazmer
  2. Gunter Hartel
  3. Harley Robinson
  4. Renee S. Richards
  5. Kexin Yan
  6. Sebastiaan J. van Hal
  7. Raymond Chan
  8. Andrew Hind
  9. David Bradley
  10. Fabian Zieschang
  11. Daniel J. Rawle
  12. Thuy T. Le
  13. David W. Reid
  14. Andreas Suhrbier
  15. Michelle M. Hill

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Abstract

Fourier transform infrared (FTIR) spectroscopy provides a (bio)chemical snapshot of the sample, and was recently used in proof-of-concept cohort studies for COVID-19 saliva screening. However, the biological basis of the proposed technology has not been established. To investigate underlying pathophysiology, we conducted controlled infection experiments on Vero E6 cells in vitro and K18-hACE2 mice in vivo. Potentially infectious culture supernatant or mouse oral lavage samples were treated with ethanol or 75% (v/v) Trizol for attenuated total reflectance (ATR)-FTIR spectroscopy and proteomics, or RT-PCR, respectively. Controlled infection with UV-inactivated SARS-CoV-2 elicited strong biochemical changes in culture supernatant/oral lavage despite a lack of viral replication, determined by RT-PCR or a cell culture infectious dose 50% assay. Nevertheless, SARS-CoV-2 infection induced additional FTIR signals over UV-inactivated SARS-CoV-2 infection in both cell and mouse models, which correspond to aggregated proteins and RNA. Proteomics of mouse oral lavage revealed increased secretion of kallikreins and immune modulatory proteins. Next, we collected saliva from a cohort of human participants (n = 104) and developed a predictive model for COVID-19 using partial least squares discriminant analysis. While high sensitivity of 93.48% was achieved through leave-one-out cross-validation, COVID-19 patients testing negative on follow-up on the day of saliva sampling using RT-PCR was poorly predicted in this model. Importantly, COVID-19 vaccination did not lead to the misclassification of COVID-19 negatives. Finally, meta-analysis revealed that SARS-CoV-2 induced increases in the amide II band in all arms of this study and in recently published cohort studies, indicative of altered β-sheet structures in secreted proteins. In conclusion, this study reveals a consistent secretory pathophysiological response to SARS-CoV-2, as well as a simple, robust method for COVID-19 saliva screening using ATR-FTIR.

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  1. Version published to 10.3390/biomedicines10020351
  2. ScreenIT

    SciScore for 10.1101/2021.12.22.21268265: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Virus stocks were prepared in Vero E6 cells as described (48) with all infectious SARS-CoV-2 work conducted in a dedicated suite in a biosafety level-3 (PC3) facility at the QIMR Berghofer MRI (Australian Department of Agriculture, Water and the Environment certification Q2326 and Office of the Gene Technology Regulator certification 3445).
    IACUC: Mouse work was approved by the QIMR Berghofer Medical Research Institute animal ethics committee (P3600, A2003-607).
    IRB: Cohort study: The project was approved by Human Research Ethics Committees of QIMR Berghofer Medical Research Institute (QIMRB, P3675), New South Wales Health Pathology (NSWHP-RPAH 2020/ETH02630) and The Prince Charles Hospital (
    Consent: All participants provided written informed consent.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cells were checked for mycoplasma using MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland)

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Virus stocks were prepared in Vero E6 cells as described (48) with all infectious SARS-CoV-2 work conducted in a dedicated suite in a biosafety level-3 (PC3) facility at the QIMR Berghofer MRI (Australian Department of Agriculture, Water and the Environment certification Q2326 and Office of the Gene Technology Regulator certification 3445).
    Vero E6
    suggested: None
    The reference cDNA was generated from a pool of SARS-CoV-2 infected VERO-E6 cell supernatant RNA and the viral copy number of reference cDNA was estimated relative to a plasmid containing the 5’UTR of SARS-CoV-2 (gift from Dongsheng Li, QIMR Berghofer MRI), using primer set 2.
    VERO-E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    K18-hACE2+/- mice were purchased from Jackson laboratories and were maintained in-house as heterozygotes by backcrossing to C57BL6/J mice (17, 48).
    K18-hACE2+/-
    suggested: RRID:IMSR_GPT:T037657)
    C57BL6/J
    suggested: None
    Software and Algorithms
    SentencesResources
    Protein identification was completed by MaxQuant using Swiss-Prot mouse proteome (version 2021_04) and default parameters.
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)

    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 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.

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

    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.

  3. Version published to 10.1101/2021.12.22.21268265v1 on medRxiv