Multisystemic Cellular Tropism of SARS-CoV-2 in Autopsies of COVID-19 Patients

  1. Dickson W. L. Wong
  2. Barbara M. Klinkhammer
  3. Sonja Djudjaj
  4. Sophia Villwock
  5. M. Cherelle Timm
  6. Eva M. Buhl
  7. Sophie Wucherpfennig
  8. Claudio Cacchi
  9. Till Braunschweig
  10. Ruth Knüchel-Clarke
  11. Danny Jonigk
  12. Christopher Werlein
  13. Roman D. Bülow
  14. Edgar Dahl
  15. Saskia von Stillfried
  16. Peter Boor

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Abstract

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.

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  3. Version published to 10.3390/cells10081900
  4. ScreenIT

    SciScore for 10.1101/2021.06.03.21258241: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical issues: The study was approved by the local ethics committee (EK 304/20, EK 119/20, EK 092/20 and 9621_BO_K_2021) and was carried out in accordance with the declaration of Helsinki for medical research involving human subjects.
    Sex as a biological variableAt the time of death, patients were 68±9·55 years old and predominantly male (M:F = 5:3).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The slides were then incubated with primary antibody against SARS spike glycoprotein (1:1000; Abcam, Cambridge, UK; Ab272420) in 1% BSA/PBS solution.
    SARS spike glycoprotein
    suggested: (LSBio (LifeSpan Cat# LS-C19480-1000, RRID:AB_1277029)
    Software and Algorithms
    SentencesResources
    RNA isolation from FFPE specimens and SARS-CoV-2 RNA detection: We extracted RNA from FFPE tissue using a Maxwell® 16 LEV RNA FFPE Purification Kit (Promega GmbH, Walldorf, Germany) on the Maxwell® 16 IVD instrument (Promega GmbH) or with the ReliaPrep™ FFPE Total RNA Miniprep System (Promega GmbH) according to the manufacturer’s instructions.
    FFPE
    suggested: (ffpe, RRID:SCR_001307)

    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:
    Our study has several limitations. We have not found viral presence in a few of the analyzed tissues, including bone marrow, esophagus, large bowel, and spleen. This might be due to the low number of analyzed patients. We aimed to perform a detailed analysis and cellular localization, which in the absence of reliable protein or ultrastructural detection, required to perform FISH analysis. This method is time-consuming, particularly when using co-localization with consecutive sections, making it hard to apply in a large number of cases. Therefore, for more high-throughput approaches, we suggest pre-screening of the cases and tissues using RT-PCR. Our study is descriptive, provides a single time-point analysis of only fatal cases. However, these are intrinsic limitations of autopsy studies. On the other hand, there are no other approaches that would allow analyzing comprehensively all human tissues in a comparable way. The variable degrees of autolysis in some tissue samples might limit the efficacy of viral detection. This was particularly true for electron microscopy, which was not applicable in our hands. Methodologically, in some cell types with scarce cytoplasm and overlap with neighbouring cells, it was not possible to allocate the ISH signal localization to a particular cell. We found a small population of SARS-CoV-2 positive cells that were negative for ACE2 or TMPRSS2 RNA. This is most likely due to a sampling bias, given that the FISH or CISH slides are extremely thin...

    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.
    • Thank you for including a protocol registration statement.

    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.

  5. Version published to 10.1101/2021.06.03.21258241v1 on medRxiv