Saliva molecular testing bypassing RNA extraction is suitable for monitoring and diagnosing SARS-CoV-2 infection in children

  1. Marta Alenquer
  2. Tiago Milheiro Silva
  3. Onome Akpogheneta
  4. Filipe Ferreira
  5. Sílvia Vale-Costa
  6. Mónica Medina-Lopes
  7. Frederico Batista
  8. Ana Margarida Garcia
  9. Vasco M. Barreto
  10. Cathy Paulino
  11. João Costa
  12. João Sobral
  13. Maria Diniz-da-Costa
  14. Susana Ladeiro
  15. Rita Corte-Real
  16. José Delgado Alves
  17. Ricardo B. Leite
  18. Jocelyne Demengeot
  19. Maria João Rocha Brito
  20. Maria João Amorim

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Abstract

Adults are being vaccinated against SARS-CoV-2 worldwide, but the longitudinal protection of these vaccines is uncertain, given the ongoing appearance of SARS-CoV-2 variants. Children remain largely unvaccinated and are susceptible to infection, with studies reporting that they actively transmit the virus even when asymptomatic, thus affecting the community.

Methods

We investigated if saliva is an effective sample for detecting SARS-CoV-2 RNA and antibodies in children, and associated viral RNA levels to infectivity. For that, we used a saliva-based SARS-CoV-2 RT-qPCR test, preceded or not by RNA extraction, in 85 children aged 10 years and under, admitted to the hospital regardless of COVID-19 symptomatology. Amongst these, 29 (63.0%) presented at least one COVID-19 symptom, 46 (54.1%) were positive for SARS-CoV-2 infection, 28 (32.9%) were under the age of 1, and the mean (SD) age was 3.8 (3.4) years. Saliva samples were collected up to 48 h after a nasopharyngeal swab-RT-qPCR test.

Results

In children aged 10 years and under, the sensitivity, specificity, and accuracy of saliva-RT-qPCR tests compared to NP swab-RT-qPCR were, respectively, 84.8% (71.8%–92.4%), 100% (91.0%–100%), and 91.8% (84.0%–96.6%) with RNA extraction, and 81.8% (68.0%–90.5%), 100% (91.0%–100%), and 90.4% (82.1%–95.0%) without RNA extraction. Rescue of infectious particles from saliva was limited to CT values below 26. In addition, we found significant IgM positive responses to SARS-CoV-2 in children positive for SARS-CoV-2 by NP swab and negative by saliva compared to other groups, indicating late infection onset (>7–10 days).

Conclusions

Saliva is a suitable sample type for diagnosing children aged 10 years and under, including infants aged <1 year, even bypassing RNA extraction methods. Importantly, the detected viral RNA levels were significantly above the infectivity threshold in several samples. Further investigation is required to correlate SARS-CoV-2 RNA levels to viral transmission.

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  1. Version published to 10.1371/journal.pone.0268388
  2. ScreenIT

    SciScore for 10.1101/2021.08.11.21261899: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: This study was approved by the Ethics committees of Centro Hospitalar Universitário de Lisboa Central and Hospital Professor Doutor Fernando Fonseca, in compliance with the Declaration of Helsinki, and follows international and national guidelines for health data protection.
    Consent: All participants, or their guardians, provided informed written consent to take part in the study.
    Field Sample Permit: Samples were stored at 4ºC, sent to Instituto Gulbenkian de Ciência, and processed within 72 h from collection.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were stained with a rabbit polyclonal SARS-CoV-2 nucleocapsid antibody (1:1,000; ThermoFisher, #MA536270) and an anti-rabbit IgG Alexa-Fluor 488 secondary antibody (1:1,000; Invitrogen, #A-21206)
    anti-rabbit IgG
    suggested: (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792)
    #A-21206
    suggested: None
    For each isotype test plate, an IgG, IgM (GenScript [clone 2001]) or IgA (Absolute Antibody [clone 3022])
    IgA
    suggested: None
    Secondary antibody goat anti□Human Fc□HRP IgG, IgA or IgM (Abcam) diluted 1 in 25,000 in PBS-BSA-T was added to respective isotype plates and incubated for 1 hour at 37ºC to reveal bound IgG, IgA or IgM.
    Secondary antibody goat anti□Human Fc□HRP IgG, IgA
    suggested: None
    anti□Human Fc□HRP IgG
    suggested: None
    IgM
    suggested: (Nicholas M. Kanaan at Michigan State University Cat# TOC1, RRID:AB_2832939)
    Experimental Models: Cell Lines
    SentencesResources
    Two hundred μL of each sample were added in triplicate to Vero E6 cells (a kind gift from Rupert Beale, The Francis Crick Institute, UK) pre-seeded in 24 well plates with coverslips and centrifuged for 15 min at 3,500 g, 37ºC.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    One negative control was performed without template for the same three conditions (N1, N2 and RP).
    N2
    suggested: None
    Software and Algorithms
    SentencesResources
    23 The analytical sensitivity of SARS-CoV-2 RNA detection between saliva and nasopharyngeal swabs from SARS-CoV-2 positive adults and children was compared using a Wilcoxon matched-pairs signed-rank test, in GraphPad Prism 9.1.2.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    STATA (StataCorp LLC, USA, V16) was used for all analyses.
    STATA
    suggested: (Stata, RRID:SCR_012763)
    StataCorp
    suggested: (Stata, RRID:SCR_012763)

    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.

    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.

  3. Version published to 10.1101/2021.08.11.21261899 on medRxiv