Accurate SARS-CoV-2 seroprevalence surveys require robust multi-antigen assays

  1. Christos Fotis
  2. Nikolaos Meimetis
  3. Nikos Tsolakos
  4. Marianna Politou
  5. Karolina Akinosoglou
  6. Vaia Pliaka
  7. Angeliki Minia
  8. Evangelos Terpos
  9. Ioannis P. Trougakos
  10. Andreas Mentis
  11. Markos Marangos
  12. George Panayiotakopoulos
  13. Meletios A. Dimopoulos
  14. Charalampos Gogos
  15. Alexandros Spyridonidis
  16. Leonidas G. Alexopoulos

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Abstract

There is a plethora of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) serological tests based either on nucleocapsid phosphoprotein (N), S1-subunit of spike glycoprotein (S1) or receptor binding domain (RBD). Although these single-antigen based tests demonstrate high clinical performance, there is growing evidence regarding their limitations in epidemiological serosurveys. To address this, we developed a Luminex-based multiplex immunoassay that detects total antibodies (IgG/IgM/IgA) against the N, S1 and RBD antigens and used it to compare antibody responses in 1225 blood donors across Greece. Seroprevalence based on single-antigen readouts was strongly influenced by both the antigen type and cut-off value and ranged widely [0.8% (95% CI 0.4–1.5%)–7.5% (95% CI 6.0–8.9%)]. A multi-antigen approach requiring partial agreement between RBD and N or S1 readouts (RBD&N|S1 rule) was less affected by cut-off selection, resulting in robust seroprevalence estimation [0.6% (95% CI 0.3–1.1%)–1.2% (95% CI 0.7–2.0%)] and accurate identification of seroconverted individuals.

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  1. Version published to 10.1038/s41598-021-86035-2
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    SciScore for 10.1101/2020.09.09.20191122: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: All samples were acquired under approved clinical protocols and informed consent (see Ethics statement).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Percentage of asymptomatic individuals (n=1,255) positive for total (IgG/IgA/IgM) SARS-CoV-2 antibodies using single antigen readouts and multi-antigen rules.
    total (IgG/IgA/IgM
    suggested: None
    SARS-CoV-2
    suggested: None
    Consensus between positive samples for total (IgG/IgA/IgM) antibodies against N, S1 and RBD and for IgG antibodies against N antigen (Abbott) in the population screen.
    antibodies against N, S1 and RBD and for IgG antibodies against N antigen
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Specifically, the S1 subunit of HCoV-HKU1, HCoV-229E and HCoV-NL63 and the S1+S2 subunits from HCoV-OC43 were used.
    HCoV-229E
    suggested: None
    Software and Algorithms
    SentencesResources
    Frozen back-up samples (n=1,225) were sent to the Immunology Laboratory of the National Public Health Organization, Athens, Greece and analyzed for the presence of IgG antibodies against the N antigen using the Abbott IgG assay with the ARCHITECT i2000SR analyzer (Abbott, Illinois, United States).
    Abbott
    suggested: (Abbott, RRID:SCR_010477)

    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:
    An important limitation of such comparisons between serological assays in population-wide surveys is the fact that there is no gold standard method to identify the truly asymptomatic SARS-CoV-2 positive cases. In conclusion, our study has demonstrated that serological assays based on single antigens, while good at diagnosing infected individuals in a clinical setting, may not be ideal in low seroprevalence, population-wide COVID19 screens, where low antibody responses from mostly asymptomatic individuals are expected. A multi-antigen approach combined with a rules-based framework for diagnostic decisions can provide a better alternative in this context, through its enhanced specificity and reduced dependency on cut-off values. We believe that such multi-antigen approaches should be performed in a single multiplex assay, thus diminishing possible differences attributed to operational issues of independent assay formats (11). An added advantage of multiplexing is the reduced usage of resources and time. The embrace of multiplex assays or multiple single antigen-based assays, by the scientific community, for epidemiological studies can eventually lead to more accurate and reliable results regarding SARS-CoV-2 spread in the population.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04408209RecruitingConvalescent Plasma for the Treatment of Patients With Sever…

    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

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  3. Version published to 10.1101/2020.09.09.20191122v2 on medRxiv
  4. Version published to 10.1101/2020.09.09.20191122v1 on medRxiv