Detection and quantification of infectious severe acute respiratory coronavirus-2 in diverse clinical and environmental samples

  1. Yi-Chan Lin
  2. Rebecca J. Malott
  3. Linda Ward
  4. Linet Kiplagat
  5. Kanti Pabbaraju
  6. Kara Gill
  7. Byron M. Berenger
  8. Jia Hu
  9. Kevin Fonseca
  10. Ryan S. Noyce
  11. Thomas Louie
  12. David H. Evans
  13. John M. Conly

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Abstract

To explore the potential modes of Severe Acute Respiratory Coronavirus-2 (SARS-CoV-2) transmission, we collected 535 diverse clinical and environmental samples from 75 infected hospitalized and community patients. Infectious SARS-CoV-2 with quantitative burdens varying from 5 plaque-forming units/mL (PFU/mL) up to 1.0 × 10 6  PFU/mL was detected in 151/459 (33%) of the specimens assayed and up to 1.3 × 10 6  PFU/mL on fomites with confirmation by plaque morphology, PCR, immunohistochemistry, and/or sequencing. Infectious virus in clinical and associated environmental samples correlated with time since symptom onset with no detection after 7–8 days in immunocompetent hosts and with N-gene based C t values ≤ 25 significantly predictive of yielding plaques in culture. SARS-CoV-2 isolated from patient respiratory tract samples caused illness in a hamster model with a minimum infectious dose of ≤ 14 PFU. Together, our findings offer compelling evidence that large respiratory droplet and contact (direct and indirect i.e., fomites) are important modes of SARS-CoV-2 transmission.

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  1. Version published to 10.1038/s41598-022-09218-5
  2. ScreenIT

    SciScore for 10.1101/2021.07.08.21259744: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: All participants provided informed consent for the use of any previous clinical samples completed for COVID-19 testing, collection of additional clinical or environmental samples, and other clinical information for research purposes.
    IRB: The study was approved by the University of Calgary Conjoint Research Ethics Board (REB20-0444).
    Field Sample Permit: Cough and continuous speech samples were collected in sealable, transparent polyethylene bags (∼18×19 cm or 27×27 cm) which were opened wide enough to accommodate the mouth and lower face and held in place at a distance <2-4 cm to ensure adequate sample collection.
    IACUC: Hamsters are highly susceptible to SARS-CoV-2 (Rosenke et al. 2020) and all of the studies were conducted in BSL3 containment with the approval of the University of Alberta’s Animal Care and Use Committee under authorization AUP00001847.
    Sex as a biological variableTransmission and Virulence Studies in a Syrian Hamster Model: Eight-week-old Syrian male hamsters (Mesocricetus auratus) were purchased from Charles River Laboratories, Montreal, Quebec.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To further confirm the identity of a subset of virus isolates, the fixed and strained plates were de-stained with ethanol and then immunostained with a 1:500 diluted rabbit anti-SARS-CoV-2 spike antibody (Prosci Inc., cat#3525).
    anti-SARS-CoV-2
    suggested: None
    The plaques were then visualized with a 2° goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Invitrogen, cat#G21234) and a KPL TrueBlue peroxidase substrate (SeraCare, cat#5510-0052).
    anti-rabbit IgG
    suggested: (Thermo Fisher Scientific Cat# G-21234, RRID:AB_2536530)
    cat#5510-0052
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell Culture and Virus Titration: Vero (ATCC #CCL-81) and Vero E6/TMPRSS2 (JCRB cell bank 1819) were cultured with Modified Eagle’s Medium (MEM, Gibco) supplemented with 100 units/mL of penicillin, 100 µg/mL of streptomycin, 0.25 µg/mL of Amphotericin B (Gibco), and 10% fetal bovine serum (Gibco).
    Vero
    suggested: ATCC Cat# CCL-81, RRID:CVCL_0059)
    Ten-fold serial dilutions of the virus were plated in duplicate on Vero CCL-81 cells and cultured for 3 days at 37°C in MEM supplemented with 100 units/mL of penicillin, 100 µg/mL of streptomycin, 0.25 µg/mL of Amphotericin B (Gibco), and 1% carboxymethyl cellulose (CMC) (Sigma C4888).
    Vero CCL-81
    suggested: None
    In a few cases, detected late in the study, some slow-growing viruses that produced small plaques on Vero CCL-81 cells were also plated on Vero E6/TMPRSS2 cells (Matsuyama et al. 2020).
    Vero E6/TMPRSS2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    hospitals (Foothills Medical Centre, South Health Campus, Peter Lougheed Centre, and Rockyview General) and one Edmonton, AB hospital (Misericordia Community Hospital) between April 22
    AB
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical Analysis: Statistical analysis was performed using GraphPad Prism v9 (GraphPad Software) with additional calculations performed with Excel v16.50 (Microsoft) and appropriate tests of significance applied for continuous or dichotomous variables.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Excel
    suggested: None
    Transmission and Virulence Studies in a Syrian Hamster Model: Eight-week-old Syrian male hamsters (Mesocricetus auratus) were purchased from Charles River Laboratories, Montreal, Quebec.
    Charles River Laboratories
    suggested: (Charles River Laboratories, RRID:SCR_003792)

    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:
    We recognize our study has some limitations. Although most large-plaque forming SARS-CoV-2 strains plate equally efficiently on Vero CCL-81 cells and on TMPRSS2 transduced cell lines, we did detect a couple of specimens that were more easily detected on the latter cells. By mostly using CCL-81 cells there may have been a few other missed isolates. Whether any manner of cell culture can replicate the “plating efficiency” that is observed in the human pharynx or lung is unknown without a proper understanding of the minimal infectious dose in humans. We were unable to collect serial specimens in many cases. Our continuous speech sampling strategy was limited by the health of the patients, and we cannot rule out that more prolonged acquisition times or singing or airflow dependent droplet carriage might detect infectious virus. However, under the conditions tested, our findings suggest continuous speech for up to 5 minutes is not associated with the detection of infectious virus. We also have limited numbers of experiments on the effects of drying on the virus infectiousness and most are relegated to the hospital as opposed to the community environment. Similarly, the hand transfer experiment was not done in replicate and there remains the possibility that there was infectious virus on the cleansed hand as no cultures were obtained before the handshake to document no pre-existing virus. However, shear forces created by the friction of the cleaning and previous studies demonstrati...

    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 10.1101/2021.07.08.21259744v1 on medRxiv