Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test

  1. Qing Sun
  2. Jonathan Li
  3. Hui Ren
  4. Larry Pastor
  5. Yulia Loginova
  6. Roberta Madej
  7. Kristopher Taylor
  8. Joseph K. Wong
  9. Zhao Zhang
  10. Aiguo Zhang
  11. Chuanyi M. Lu
  12. Michael Y. Sha

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Abstract

Sensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirus ™ SARS-CoV-2 test using saliva as the testing specimens with or without pooling.

Methods

Development and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirus ™ SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus ™ SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated.

Results

The LOD for the QuantiVirus ™ SARS-CoV-2 test was confirmed to be 100–200 copies/mL. The clinical performance studies using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus ™ SARS-CoV-2 test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus ™ SARS-CoV-2 test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus ™ SARS CoV-2 test had 80% concordance rate and no significant difference ( p = 0.13) between paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from local communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples (1:6 pooling) for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%).

Conclusion

The studies demonstrated that the QuantiVirus ™ SARS-CoV-2 test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus ™ SARS-CoV-2 test offers a highly desirable testing platform during the ongoing COVID-19 pandemic.

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  1. Version published to 10.1371/journal.pone.0243183
  2. ScreenIT

    SciScore for 10.1101/2020.10.27.20219196: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: This study was approved by the institutional review board (IRB) at UCSF (UCSF IRB #11-05207) as a no-subject contact study with waiver of consent and as exempt under category 4.
    Consent: This study was approved by the institutional review board (IRB) at UCSF (UCSF IRB #11-05207) as a no-subject contact study with waiver of consent and as exempt under category 4.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    , and conserved regions in each target gene were identified using BioEditor 7.2.5.
    BioEditor
    suggested: None
    Primers and probes were designed to target the most conserved regions of each of the target genes of the viral genome, using Primer3plus software and following general rules of real-time PCR design.
    Primer3plus
    suggested: (Primer3Plus, RRID:SCR_003081)
    Statistical data analysis: Average cycle threshold (Ct), standard deviation (SD) and coefficient of variation (CV) were calculated using Microsoft Office Excel 365 software (Microsoft, Redmond, WA).
    Microsoft Office Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    Clinical sensitivity, clinical specificity, Positive Predictive Value (PPV) and Negative Predictive value (NPV) at two-sided 95% confidence interval (CI) were analyzed using MedCalc software Version 19.3.1
    MedCalc
    suggested: (MedCalc, RRID:SCR_015044)

    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: 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.
    • 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.

  3. Version published to 10.1101/2020.10.27.20219196v1 on medRxiv