Pediatric COVID-19 patients in South Brazil show abundant viral mRNA and strong specific anti-viral responses

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1. Version published to 10.1038/s41467-021-27120-y

   Nov 25, 2021
2. 

   Version published to 10.21203/rs.3.rs-424079/v1 on Research Square

   May 7, 2021
3. 

   ScreenIT

   Apr 17, 2021

   SciScore for 10.1101/2021.04.13.21255139: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Ethics Statement: This study was approved by the Institutional Review Board (IRB 30749720.4.1001.5330) at Hospital Moinhos de Vento and by the Ethics Committee at Universidade Federal de Ciências da Saúde (CAAE 30749720.4.3001.5345). Consent: Legal consent was obtained from all participants or their legal guardians.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After, were stained with the BD Horizon™ Fixable Viability Stain 510 together with antibodies for surface markers, as follows: anti-CD3-APC-H7 (clone SK7), anti-CD24-APC-H7 (clone ML5), …

   SciScore for 10.1101/2021.04.13.21255139: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Ethics Statement: This study was approved by the Institutional Review Board (IRB 30749720.4.1001.5330) at Hospital Moinhos de Vento and by the Ethics Committee at Universidade Federal de Ciências da Saúde (CAAE 30749720.4.3001.5345). Consent: Legal consent was obtained from all participants or their legal guardians.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After, were stained with the BD Horizon™ Fixable Viability Stain 510 together with antibodies for surface markers, as follows: anti-CD3-APC-H7 (clone SK7), anti-CD24-APC-H7 (clone ML5), anti-HLA-DR-APC-H7 (clone G46-6), anti-CD4-PerCP-Cy5.5 (clone RPA-T4), anti-CD27-PerCP-Cy5.5 (clone M-T271), anti-CD11c-PerCP-Cy5.5 (clone B-ly6), anti-CD14-PerCP-Cy5.5 (clone M5E2), anti-CD8-FITC (clone HIT8a), anti-IgG-FITC (clone G18-145), Lineage 2-FITC (cat. 643397), anti-CD16-FITC (clone 3G8), anti-CXCR5 (CD185)-BB515 (clone RF8B2), anti-CD19-APC (clone HIB19), anti-CD127-Alexa 647 (clone HIL-7R-M21), anti-CX3CR1-Alexa647 (clone 2A9-1), anti-CD69-APC (clone FN50), anti-CD38-PE (clone HIT2), anti-ICOS (CD278)-PE (clone DX29), anti-CD141-PE (clone 1A4), anti-CD66b-PE (clone G10F5), anti-CD137 (4-1BB)-PE (clone 4B4-1), anti-HLA-DR-PE-Cy7 (clone G46-6), anti-CD19-PE-Cy7 (clone SJ25C1), anti-CD25-PE-Cy7 (clone 2A3), anti-CD45RA-PE-Cy7 (clone L48), anti-IgM-BV421 (clone G20-127), anti-PD-1 (CD279)-BV421 (clone MIH4), anti-CD303-BV421 (clone V24-785), anti-CD56-BV421 (clone NCAM 16), anti-CCR7-BV421 (clone 2-L1-A) antibodies.	anti-CD3-APC-H7 suggested: None anti-CD24-APC-H7 suggested: None anti-HLA-DR-APC-H7 suggested: None anti-CD27-PerCP-Cy5.5 suggested: None anti-CD11c-PerCP-Cy5.5 suggested: None anti-CD14-PerCP-Cy5.5 suggested: None anti-CD8-FITC suggested: None anti-IgG-FITC suggested: (Miltenyi Biotec Cat# 130-093-192, RRID:AB_1036185) anti-CD16-FITC suggested: (Sigma-Aldrich Cat# F3668, RRID:AB_259555) anti-CXCR5 suggested: (BD Biosciences Cat# 552032, RRID:AB_394324) CD185)-BB515 suggested: None anti-CD19-APC suggested: (Sigma-Aldrich Cat# SAB4700110, RRID:AB_10898737) anti-CD127-Alexa suggested: None anti-CX3CR1-Alexa647 suggested: None anti-CD69-APC suggested: (Sigma-Aldrich Cat# SAB4700220, RRID:AB_10897283) anti-CD38-PE suggested: (Sigma-Aldrich Cat# P6722, RRID:AB_261136) anti-ICOS suggested: (BD Biosciences Cat# 557801, RRID:AB_396877) CD278)-PE suggested: None anti-CD141-PE suggested: None anti-CD66b-PE suggested: None anti-CD137 suggested: (Thermo Fisher Scientific Cat# EPX140-15803-901, RRID:AB_2576106) 4-1BB)-PE suggested: None anti-HLA-DR-PE-Cy7 suggested: None anti-CD19-PE-Cy7 suggested: None anti-CD25-PE-Cy7 suggested: None anti-CD45RA-PE-Cy7 suggested: None anti-IgM-BV421 suggested: None anti-PD-1 (CD279)-BV421 suggested: None anti-CD303-BV421 suggested: None anti-CD56-BV421 suggested: None
   For intracellular staining, cells were first stained for surface markers and subsequently fixed and permeabilized using the Transcription Factor Buffer Set (BD Biosciences-Pharmingen, USA), then stained with anti-Ki-67-BV421 (clone B56), anti-Perforin-Alexa 647 (clone δG9), and anti-Granzyme B-BV421 (clone GB11) antibodies.	anti-Ki-67-BV421 suggested: None anti-Perforin-Alexa suggested: None anti-Granzyme B-BV421 suggested: None
   Following in vitro stimulation assays with specific peptides, cells were stained with the BD Horizon™ Fixable Viability Stain 510 and anti-CD3-PE-Cy7 (clone SK7), anti-CD4-PerCP-Cy5.5 (clone RPA-T4), anti-CD8-APC-H7 (clone SK1), anti-CCR7-BV421 (clone 2-L1-A), and subsequently fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences-Pharmingen, USA), then stained with anti-IFNγ-FITC (clone 4S.B3), anti-TNF (clone MAb11) and anti-IL-17-PE (clone SCPL1362) antibodies.	anti-CD3-PE-Cy7 suggested: None anti-CD4-PerCP-Cy5.5 suggested: None anti-CD8-APC-H7 suggested: None anti-CCR7-BV421 suggested: None anti-IFNγ-FITC suggested: None anti-TNF suggested: None anti-IL-17-PE suggested: None
   ELISA: Plasma was tested for IgG and IgA antibodies to S-RBD protein (#RP-87678 - Invitrogen, USA) and N protein (kindly provided by Dr. Ricardo Gazinelli - Fiocruz Belo Horizonte, Brazil) using a protocol described in (16).	IgA suggested: None #RP-87678 suggested: None
   Software and Algorithms
   Sentences	Resources
   PBMCs were next isolated by density-gradient centrifugation using Ficoll–Paque™ PLUS (GE Healthcare®), and either studied directly or resuspended in FBS 5% DMSO and stored in liquid nitrogen until use.	GE Healthcare® suggested: (GE Healthcare, RRID:SCR_000004)
   All samples were analyzed using a BD Biosciences - FACSCanto II and FlowJo 10.7.1 software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   All analyses were performed either in GraphPad Prism v.9 or R and sometimes confirmed in Python. 3D analysis of PCA was plotted in Python.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) Python suggested: (IPython, RRID:SCR_001658)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   The main limitation of this study is that it is mostly an exploratory, descriptive one, and compares individuals in different age groups. We believe this was a valid approach given the magnitude of what is unknown at the moment. Usually, biomarkers in peripheral blood are only useful when highly correlated with outcomes. Yet, most studies that seek to understand how immune responses can correlate with protection compare adult s with mild and severe disease, and it is known that these are in different age groups. At present, the best age-matched controls for SARS-CoV-2 infected patients are still unknown. Certainly, the absence of a pre-pandemic healthy children control group is a limitation of this and all the other studies that focused on the general, non-specific immune profile of children with COVID-19. There is still much to be understood about immunological differences not only between pediatric and adult COVID-19 patients but also in other diseases. In this sense, we believe our work contributes significant information which was collected in a completely unbiased investigation -we did not know what differences or similarities to expect. At the time the project started, COVID-19 numbers in Brazil were still not high and the frequency of MISC patients or children with severe manifestations of the disease was still too low to include. Inclusion of patients was easiest in ASD because they were admitted to the hospital, thus their time of symptoms to sample collection is sho...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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   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.

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   Read the original source
4. 

   Version published to 10.1101/2021.04.13.21255139v1 on medRxiv

   Apr 16, 2021