Ultrasensitive assay for saliva-based SARS-CoV-2 antigen detection

  1. Annie Ren
  2. Dorsa Sohaei
  3. Antigona Ulndreaj
  4. Oscar D. Pons-Belda
  5. Amaia Fernandez-Uriarte
  6. Ioannis Zacharioudakis
  7. George B. Sigal
  8. Martin Stengelin
  9. Anu Mathew
  10. Christopher Campbell
  11. Nikhil Padmanabhan
  12. Daniel Romero
  13. Jessica Joe
  14. Antoninus Soosaipillai
  15. Vathany Kulasingam
  16. Tony Mazzulli
  17. Xinliu A. Li
  18. Allison McGeer
  19. Eleftherios P. Diamandis
  20. Ioannis Prassas

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Abstract

Objectives

Widespread SARS-CoV-2 testing is invaluable for identifying asymptomatic/pre-symptomatic individuals. There remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests suitable for frequent mass testing. Compared to nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling. Current saliva-based SARS-CoV-2 rapid antigen tests (RATs) are hindered by limited analytical sensitivity. Here, we report one of the first ultrasensitive, saliva-based SARS-CoV-2 antigen assays with an analytical sensitivity of <0.32 pg/mL, corresponding to four viral RNA copies/µL, which is comparable to that of PCR-based tests.

Methods

Using the novel electrochemiluminescence (ECL)-based immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 salivas, obtained from non-COVID-19 and COVID-19 patients. We then verified the results with a second, independent cohort of 689 patients (3.8% SARS-CoV-2 positivity rate). We also compared our method with a widely used point-of-care rapid test.

Results

In the first cohort, at 100% specificity, the sensitivity was 92%. Our assay correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were obtained for 86 cases. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (<0.32 pg/mL) and strongly positive (>2 pg/mL) N antigen concentrations. In the second cohort, at 100% specificity, sensitivity was also 92%. Our assay is about 700-fold more sensitive than the Abbott Panbio Rapid Test.

Conclusions

We demonstrated the ultrasensitivity and specificity assay and its concordance with PCR. This novel assay is especially valuable when compliance to frequent swabbing may be problematic.

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  1. Version published to 10.1515/cclm-2021-1142
  2. ScreenIT

    SciScore for 10.1101/2021.02.17.21251863: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Clinical Samples: A total of 105 retrospectively collected saliva specimens were obtained under approval of the Sinai Health System Research Ethics Board (REB#: 02-0118U).
    Consent: For saliva samples, patients were asked under informed consent to spit into a sterile 50-mL specimen container which was topped with 2.5 ml of phosphate-buffered saline.
    Randomizationnot detected.
    BlindingAll N antigen values were obtained with the S-PLEX ultrasensitive ECL immunoassay platform (Meso Scale Diagnostics) in a fully blinded fashion and according to manufacturer protocols (accessible online at https://www.mesoscale.com/en/products/S-PLEX-SARS-CoV-2-n-kit-sector-1-pl-k150adhs/).
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Statistical Analysis: All data analysis was performed in GraphPad PRISM 9.
    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, all types of nucleic acid amplification technologies can be impacted by technological caveats, especially during sudden outbreaks in rural areas. Additionally, PCR-technologies are semi-quantitative, and the resulting CT values may vary between instruments, genes, and/or operators. Hence, alternative non-competing quantitative platforms that could complement the existing PCR-based diagnostic capacity should be urgently explored to enhance total testing capacity. Direct to consumer lateral flow rapid antigen tests (RATs) are promising (and as yet largely unexplored) tools for curbing large transmission chains. However, their utility, especially for saliva-based screening, is hampered by their relatively lower sensitivity thresholds 11. For instance, in the case of our study presented here, a RAT test with an LLD of 100 pg/ml (typically seen in FDA-approved LFA-based antigen assays) would erroneously miss the majority of the potentially active COVID-19 cases (reducing the sensitivity from 90.2% to 17.1%) (as shown by drawing a horizontal dashed line at a LLD of 100 pg/ml in Figure 2). This is the first in-depth characterization of SARS-CoV-2N antigen concentration in saliva samples from COVID-19 patients. Recently, a similar assay was successfully used to describe N concentration distributions in clinical NP swabs 11. Our study reveals that N concentrations in saliva span five orders of magnitude (0.2–1,000 pg/ml). Our assay displayed absolute specificity (in non-COVID...

    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 10.1101/2021.02.17.21251863v1 on medRxiv