SARS-CoV-2 viral load monitoring by extraction-free testing of saliva

  1. Yue Qiu
  2. Ling Lu
  3. Dexiang Gao
  4. Patrick McGrath
  5. Chann Han
  6. Igor Kogut
  7. Bob Blomquist
  8. Xin Yao
  9. Jose P. Zevallos
  10. Brian L. Harry
  11. Shi-Long Lu

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Abstract

Real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) remains the foundation of SARS-CoV-2 testing due to its accessibility, scalability, and superior assay performance. Variability in specimens and methods prevent standardization of RT-qPCR assays and reliable quantitative reporting to assess viral load. We developed an extraction-free RT-qPCR assay for detection of SARS-CoV-2 in saliva and monitored viral load until convalescence in COVID-19 patients. Comparison of 231 matched anterior nares swab and saliva specimens demonstrated that extraction-free testing of saliva has equivalent analytical and clinical assay performance compared to testing of RNA extracts from either anterior nares or saliva specimens. Analysis of specimen pairs revealed higher viral loads in the nasal cavity compared to the oral cavity, although this difference did not impact clinical sensitivity for COVID-19. Extraction-free testing of a combination specimen consisting of both nasal swab and saliva is also demonstrated. Assessment of viral load by RT-qPCR and parallel digital droplet PCR (ddPCR) revealed that cycle threshold (Ct) values less than approximately 30 correlated well with viral load, whereas Ct values greater than 30 correspond to low viral loads <10 copies/µL. Therefore, extraction-free saliva testing maximizes testing efficiency without compromising assay performance and approximates viral loads >10 copies/µL. This technology can facilitate high-throughput laboratory testing for SARS-CoV-2, monitor viral load in individual patients, and assess efficacy of therapies for COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.02.21261502: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Data Analysis: Data was analyzed using Microsoft Excel and GraphPad Prism Version 9.1.1
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    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:
    However, there are limitations of this study. First, this study did not include collection of matched NP swabs and compared the relative performance of nasal swabs and saliva. Second, the small cohort of community COVID-19 patients studied here is likely not representative of other patient populations, such as immunocompromised or hospitalized patients. Third, this study was performed in 2020 prior to the emergence of SARS-CoV-2 variants of concern in the U.S. and widespread COVID-19 vaccination. At that time the alpha variant predominated in the studied geographic region. The delta variant for example may display altered biology. Yet RT-qPCR remains foundational for SARS-CoV-2 testing. Simplified specimen collection using saliva and efficient laboratory testing using extraction-free methods offer an innovative approach that is attractive to resource-poor settings and high throughput laboratories alike. Viral load monitoring of individual patients by a single laboratory has the potential to predict outcomes in COVID-19, assess therapeutic response particularly in the setting of clinical trials, and personalize guidance for quarantine and repeat testing.

    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.

    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.

  2. Version published to 10.1101/2021.08.02.21261502v1 on medRxiv