Dynamics of a Dual SARS-CoV-2 Lineage Co-Infection on a Prolonged Viral Shedding COVID-19 Case: Insights into Clinical Severity and Disease Duration

  1. Nicole Pedro
  2. Cláudio N. Silva
  3. Ana C. Magalhães
  4. Bruno Cavadas
  5. Ana M. Rocha
  6. Ana C. Moreira
  7. Maria Salomé Gomes
  8. Diogo Silva
  9. Joana Sobrinho-Simões
  10. Angélica Ramos
  11. Maria J. Cardoso
  12. Rita Filipe
  13. Pedro Palma
  14. Filipa Ceia
  15. Susana Silva
  16. João T. Guimarães
  17. António Sarmento
  18. Verónica Fernandes
  19. Luisa Pereira
  20. Margarida Tavares

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Abstract

A few molecularly proven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases of symptomatic reinfection are currently known worldwide, with a resolved first infection followed by a second infection after a 48 to 142-day intervening period. We report a multiple-component study of a clinically severe and prolonged viral shedding coronavirus disease 2019 (COVID-19) case in a 17-year-old Portuguese female. She had two hospitalizations, a total of 19 RT-PCR tests, mostly positive, and criteria for releasing from home isolation at the end of 97 days. The viral genome was sequenced in seven serial samples and in the diagnostic sample from her infected mother. A human genome-wide array (>900 K) was screened on the seven samples, and in vitro culture was conducted on isolates from three late samples. The patient had co-infection by two SARS-CoV-2 lineages, which were affiliated in distinct clades and diverging by six variants. The 20A lineage was absolute at the diagnosis (shared with the patient’s mother), but nine days later, the 20B lineage had 3% frequency, and two months later, the 20B lineage had 100% frequency. The 900 K profiles confirmed the identity of the patient in the serial samples, and they allowed us to infer that she had polygenic risk scores for hospitalization and severe respiratory disease within the normal distributions for a Portuguese population cohort. The early-on dynamic co-infection may have contributed to the severity of COVID-19 in this otherwise healthy young patient, and to her prolonged SARS-CoV-2 shedding profile.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.22.20248392: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The research protocol was approved by the Ethics Committee of the CHUSJ and University of Porto Medical School (process number: 102-20).
    Consent: The patient and her close relative provided written informed consent for publication.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    RT-PCR and Antibody Testing: For detection of viral RNA by RT-PCR, each tube sample contained a nasopharyngeal and an oropharyngeal swab immersed in virus preservation solution.
    RT-PCR
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    In Vitro Culture of the Virus: To ascertain if the virus was still viable in the samples after the second hospitalization, in vitro culture in Vero cells (ATCC CCL-81; ATCC, Manassas, VA, USA) was performed, followed by SARS-CoV-2 spike antibody (GeneTex, Irvine, CA, USA) immunofluorescence detection.
    Vero
    suggested: None
    Software and Algorithms
    SentencesResources
    The serological test used was a chemiluminescent microparticle immunoassay for qualitative detection of IgG against SARS-CoV-2 nucleoprotein (Abbott Diagnostics, Chicago, USA)
    Abbott
    suggested: (Abbott, RRID:SCR_010477)
    The bioinformatic pipeline consisted in: alignment of the raw data versus the reference genome (accession number NC_045512.2) with BWA tool; variant calling with three tools, FreeBayes, BCFtools and GATK, with editing of variants identified in at least two; variant annotation with SnpEff; consensus sequence was inferred with Bcftools [12].
    BWA
    suggested: (BWA, RRID:SCR_010910)
    FreeBayes
    suggested: (FreeBayes, RRID:SCR_010761)
    GATK
    suggested: (GATK, RRID:SCR_001876)
    Phylogenetic analysis and lineage/clade affiliation were done by merging the consensus sequences with publically available SARS-CoV-2 whole genomes from ViPR [13] with Mafft [14] (first 130bp and last 50bp were masked).
    ViPR
    suggested: (vipR, RRID:SCR_010685)
    Mafft
    suggested: (MAFFT, RRID:SCR_011811)
    Plink was also used to estimate PRS values via an additive model, as the sum of the risk alleles, weighted by the effect size estimates from the genome-wide association study.
    Plink
    suggested: (PLINK, RRID:SCR_001757)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 20. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • 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.

  2. Version published to 10.3390/microorganisms9020300
  3. Version published to 10.1101/2020.12.22.20248392v1 on medRxiv