The size and culturability of patient-generated SARS-CoV-2 aerosol

  1. Joshua L. Santarpia
  2. Vicki L. Herrera
  3. Danielle N. Rivera
  4. Shanna Ratnesar-Shumate
  5. St. Patrick Reid
  6. Daniel N. Ackerman
  7. Paul W. Denton
  8. Jacob W. S. Martens
  9. Ying Fang
  10. Nicholas Conoan
  11. Michael V. Callahan
  12. James V. Lawler
  13. David M. Brett-Major
  14. John J. Lowe

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Abstract

Background

Aerosol transmission of COVID-19 is the subject of ongoing policy debate. Characterizing aerosol produced by people with COVID-19 is critical to understanding the role of aerosols in transmission.

Objective

We investigated the presence of virus in size-fractioned aerosols from six COVID-19 patients admitted into mixed acuity wards in April of 2020.

Methods

Size-fractionated aerosol samples and aerosol size distributions were collected from COVID-19 positive patients. Aerosol samples were analyzed for viral RNA, positive samples were cultured in Vero E6 cells. Serial RT-PCR of cells indicated samples where viral replication was likely occurring. Viral presence was also investigated by western blot and transmission electron microscopy (TEM).

Results

SARS-CoV-2 RNA was detected by rRT-PCR in all samples. Three samples confidently indicated the presence of viral replication, all of which were from collected sub-micron aerosol. Western blot indicated the presence of viral proteins in all but one of these samples, and intact virions were observed by TEM in one sample.

Significance

Observations of viral replication in the culture of submicron aerosol samples provides additional evidence that airborne transmission of COVID-19 is possible. These results support the use of efficient respiratory protection in both healthcare and by the public to limit transmission.

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  1. Version published to 10.1038/s41370-021-00376-8
  2. ScreenIT

    SciScore for 10.1101/2020.07.13.20041632: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Twenty uL of lysate was combined with a 2X reducing sample buffer (Invitrogen), boiled and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis followed by Western blot using the antibody against SARS-CoV N protein and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam).
    SARS-CoV N protein
    suggested: None
    GAPDH
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    To quantify the virus present in each sample from the measured Ct values obtained from the rRT-PCR, a standard curve was developed using RNA extracted from a known quantity of SARS-CoV-2 virus (BEI_ USA-WA1/2020) cultivated in Vero-E6 cells (using the same method described below for environmental samples).
    Vero-E6
    suggested: None
    Cell Culture and Detection of Viral Replication: Vero E6 cells were used to culture SARS-CoV-2 virus from environmental samples.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Definitive replication was considered to occur for rRT-PCR samples in which a significant increase in RNA was detected in the supernatant (student’s t-test, Microsoft Excel, P<0.05).
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    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:
    Therefore, it can be said that infectious SARS-CoV-2 exists in particles <1 µm and may exist in particles up to 4 µm, but it cannot be said that they do not exist in particles larger than 4 microns, due to the limitations of the collection technique. A comparison of the APS and BC251 aerosol data (Table S6) is suggestive of a relationship between the measured mass distributions and viral RNA concentrations from the different stages of the BC251 sampler. However, the small number of data points (6, in all cases), does not allow the determination of a statistically relevant relationship. Despite that, examination of the observed APS modes against the size bins of the BC251 sampler and comparison to studies of human respiratory emissions does offer valuable insight into the source of RNA-containing and infectious aerosol present in these samples. SARS-CoV-2-containing aerosols collected in the submicron filter of the BC251 is likely attributable to a combination of the small and large modes that were fit to the APS data, with the dominant fraction of the aerosol mass attributable to the small mode. This would indicate that the portion of aerosol containing both RNA and infectious aerosol that were identified in three of the rooms likely had their origins in the bronchial region of those patients. The 1-4 µm BC251 stage was dominated by the large mode, in which the human associated fraction is most consistent with particles produced in the larynx, and are produced more heavily du...

    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. ScreenIT

    SciScore for 10.1101/2020.07.13.20041632: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Twenty uL of lysate was combined with a 2X reducing sample buffer (Invitrogen), boiled and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis followed by Western blot using the antibody against SARS-CoV N protein and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam).
    SARS-CoV N protein
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>GAPDH</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell Culture and Detection of Viral Replication Vero E6 cells were used to culture SARS-CoV-2 virus from environmental samples.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Furthermore, exposure of recovered samples to Vero-E6 cells demonstrated statistically significant viral growth (95% confidence based on P<0.05) after six days (five days in the case of the submicron sample from room 5A) in 3 of the 18 samples (7B, 5A and 5C; Figure 1).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero-E6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Definitive replication was considered to occur for rRT-PCR samples in which a significant increase in RNA was detected in the supernatant (student’s t-test, Microsoft Excel, P<0.05).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Microsoft Excel</b></div>
        <div>suggested: (Microsoft Excel, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016137">SCR_016137</a>)</div>
      </div>
    </td></tr></table>

    Data from additional tools added to each annotation on a weekly basis.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.07.13.20041632 on medRxiv