Multiplex Antibody Analysis of IgM, IgA and IgG to SARS-CoV-2 in Saliva and Serum From Infected Children and Their Close Contacts

  1. Carlota Dobaño
  2. Selena Alonso
  3. Marta Vidal
  4. Alfons Jiménez
  5. Rocío Rubio
  6. Rebeca Santano
  7. Diana Barrios
  8. Gemma Pons Tomas
  9. María Melé Casas
  10. María Hernández García
  11. Mònica Girona-Alarcón
  12. Laura Puyol
  13. Barbara Baro
  14. Pere Millat-Martínez
  15. Sara Ajanovic
  16. Núria Balanza
  17. Sara Arias
  18. Natalia Rodrigo Melero
  19. Carlo Carolis
  20. Aleix García-Miquel
  21. Elisenda Bonet-Carné
  22. Joana Claverol
  23. Marta Cubells
  24. Claudia Fortuny
  25. Victoria Fumadó
  26. Anna Codina
  27. Quique Bassat
  28. Carmen Muñoz-Almagro
  29. Mariona Fernández de Sevilla
  30. Eduard Gratacós
  31. Luis Izquierdo
  32. Juan José García-García
  33. Ruth Aguilar
  34. Iolanda Jordan
  35. Gemma Moncunill

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Abstract

COVID-19 affects children to a lesser extent than adults but they can still get infected and transmit SARS-CoV-2 to their contacts. Field deployable non-invasive sensitive diagnostic techniques are needed to evaluate the infectivity dynamics of SARS-CoV-2 in pediatric populations and guide public health interventions, particularly if this population is not fully vaccinated. We evaluated the utility of high-throughput Luminex assays to quantify saliva IgM, IgA and IgG antibodies against five SARS-CoV-2 spike (S) and nucleocapsid (N) antigens in a contacts and infectivity longitudinal study in 122 individuals (52 children and 70 adults). We compared saliva versus serum/plasma samples in infected children and adults diagnosed by weekly RT-PCR over 35 days (n=62), and those who consistently tested negative over the same follow up period (n=60), in the Summer of 2020 in Barcelona, Spain. Saliva antibody levels in SARS-CoV-2 RT-PCR positive individuals were significantly higher than in negative individuals and correlated with those measured in sera/plasmas. Asymptomatic infected individuals had higher levels of anti-S IgG than symptomatic individuals, suggesting a protective anti-disease role for antibodies. Higher anti-S IgG and IgM levels in serum/plasma and saliva, respectively, in infected children compared to infected adults could also be related to stronger clinical immunity in them. Among infected children, males had higher levels of saliva IgG to N and RBD than females. Despite overall correlation, individual clustering analysis suggested that responses that may not be detected in blood could be patent in saliva, and vice versa.

In conclusion, measurement of SARS-CoV-2-specific saliva antibodies should be considered as a complementary non-invasive assay to serum/plasma to determine COVID-19 prevalence and transmission in pediatric populations before and after vaccination campaigns.

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  1. Version published to 10.3389/fimmu.2022.751705
  2. ScreenIT

    SciScore for 10.1101/2021.03.22.21254120: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study protocols for human subject research were approved by the Institutional Review Board and the Hospital Sant Joan de Déu Ethics Committee (Ref.
    Consent: CEIC-7455), and written informed consent was obtained from participants or guardians before starting study procedures.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Saliva samples were collected with Oracol devices (Malvern Medical Development, UK) for optimal harvesting of crevicular fluid, enriched with serum antibodies10,11.
    antibodies10,11
    suggested: None
    Measurement of antibodies: Quantitative suspension array technology (qSAT) assays to measure IgM, IgA and IgG against SARS-CoV-2 were adapted from our previous standardized serum/plasma protocols12 to a saliva matrix for SARS-CoV-2 antibody evaluation.
    IgG against SARS-CoV-2
    suggested: None
    Antibodies in serum eluted from DBS and from serum/plasma samples are shown together since no differences were observed in the available paired samples of plasma and DBS.
    DBS
    suggested: None
    After antigen-coupled beads were incubated with samples, plates were washed and phycoerythrin-labeled secondary antibodies (anti-human IgG, IgM, or IgA, Moss) added.
    anti-human IgG
    suggested: None
    IgA , Moss ) added.
    suggested: None
    Software and Algorithms
    SentencesResources
    Study design, human subjects and samples: We compared the levels of SARS-CoV-2 antibodies in subjects with a positive diagnosis by nasopharyngeal RT-PCR and/or ELISA SARS-CoV-2 IgG, IgM test (Euroimmune Architect – Abbott) independently of the COVID-19 compatible symptoms or not, and subjects with negative nasopharyngeal RT-PCR9 and serology by RDT (SureScreen)
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    The ggplot2 package was used to perform boxplot graphs14.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)

    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:
    A study limitation was that seropositivity thresholds could not be estimated with pre-pandemic saliva samples due to lack of access to them, and thus sensitivity and specificity could not be established for the saliva assays by standard methods. Using RT-PCR negative pandemic samples was somewhat useful for serum/plasma samples, but with saliva there was substantial overlap between antibody levels in infected and non-infected individuals. In addition, being pandemic samples, we cannot ascertain that they were not previously exposed at low levels and therefore this approach is not optimal. This constraint could be overcome in follow up studies by assessing seroconversion in consecutive samples calculating the fold change increase in levels (e.g. ≥4) over a given study period16 and, in future, access to pre-pandemic samples in international biobanks will be sought. Finally, there could be some imbalance between age and infection that could affect the antibody results. However, we analyzed the effect of age stratifying by infection status to take that into account. In conclusion, antibody levels in saliva measured with our high-throughput qSAT assay largely correlated with those in serum/plasma from individuals with confirmed RT-PCR diagnosis of SARS-CoV-2 infection, depending on the antigen. Higher antibody levels found in asymptomatic individuals and in children, particularly among males, could indicate protection against disease and stronger immunity. This non-invasive field ...

    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 10.1101/2021.03.22.21254120 on medRxiv