Review 1: "Multisystemic cellular tropism of SARS-CoV-2 in autopsies of COVID-19 patients"

Jay Rappaport; Robert V. Blair

doi:doi:10.1162/2e3983f5.e70e947e

Multisystemic cellular tropism of SARS-CoV-2 in autopsies of COVID-19 patients

by Dickson W.L. Wong, Barbara M. Klinkhammer, Sonja Djudjaj, Sophia Villwock, M. Cherelle Timm, Eva M. Buhl, Sophie Wucherpfennig, Claudio Cacchi, Till Braunschweig, Ruth Knüchel-Clarke, Danny Jonigk, Christopher Werlein, Roman D. Bülow, Edgar Dahl, Saskia von Stillfried, and Peter Boor

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dx.doi.org

Description

AbstractBackgroundMultiorgan tropism of SARS-CoV-2 has previously been shown for several major organs.MethodsWe have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n=8), using detailed 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. Finally, we confirmed these findings in an independent external autopsy cohort (n=9).FindingsSARS-CoV-2 RNA was mainly localized in epithelial cells, endothelial and mesenchymal 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. The findings were validated using in situ hybridization on external COVID-19 autopsy samples. Finally, apart from the lung, correlation of virus detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2.InterpretationSARS-CoV-2 could be observed in virtually all organs, colocalizing with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells, and viral replication was found across all organ systems. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.Research in contextEvidence before this studySARS-CoV-2 has been shown to infect the respiratory tract and affect several other major organs. However, on a cellular level, the localization of SARS-CoV-2 and its targets ACE2 and TMPRSS2 have not been described comprehensively.Added value of this studyWe have analyzed tissue SARS-CoV-2 RNA using RT-PCR and visualized its localization together with ACE2 and TMPRSS2 using in situ hybridization (ISH) in 25 different autopsy tissues. SARS-CoV-2 sense and antisense RNA were detected in 16 tissues/organs, mainly in epithelial cells and, to a lesser extent, in endothelial or stromal cells. Detection of viral protein using immunohistochemistry or viral particles using transmission electron microscopy did not yield specific results. Interestingly, apart from the respiratory tract and specifically the lungs, we have not found a specific pathology that would be associated with extrapulmonary viral spread.Implications of all the available evidenceWe provide a recommendation on using these methods in autopsy diagnostics for SARS-CoV-2. Our data extend the current hypothesis of severe COVID-19 being multisystemic diseases. Our data also provide clear evidence of infection and replication of SARS-CoV-2 in the endothelial cell across all organs, extending the hypothesis on the (micro)vascular involvement in COVID-19.

RR:C19 Evidence Scale rating by reviewer:

Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review:

Defense of SoES/Review: This manuscript is a description of autopsy findings from eight individuals and utilizes a range of detection methods (PCR, immunohistochemistry (IHC), electron microscopy (EM), and in situ hybridization (FISH)) to characterize the tissue and cellular tropism of SARS-CoV-2 in combination with ACE2 and TMPRSS2. A major strength of this study is the use of autopsy data from confirmed SARS-CoV-2-positive patients. There has been a paucity of autopsy data from COVID-19 patients, compared to data generated from basic science and animal models studies. There is a critical need for autopsy data to confirm, support, and/or refute the findings from basic science and animal model studies. This manuscript helps address this need by adding a small postmortem series to the existing COVID-19 autopsy literature. The results of this study confirm and support the growing body of literature from in vitro, animal model, and autopsy studies that demonstrate a wide tissue and cellular tropism for SARS-CoV-2 that includes epithelial, mesenchymal, and endothelial cells, and the authors do well to cite this prior work. We found the comparison between the SARS-CoV-2 detection methods (IHC, EM, and FISH) and their limitations in autopsy tissues to be very informative and was discussed in greater detail than most autopsy studies. Readers should be aware of several methodologic limitations present in this study that cause the results to be interpreted with some reservations. These include the use of autopsy patients with variable autolysis, suboptimal tissue fixation (for some of the methods used), and the use of H&E overlay of serial sections for cellular identification/ phenotyping. The authors discuss the first two which are inherent to autopsy studies but do not mention the latter which we consider to be a major limitation. This manuscript could be further strengthened by additional staining studies that combined probes of interest (SARS-CoV-2, ACE, TMPRSS2) with phenotypic markers for epithelial, mesenchymal, and endothelial cells. This would allow for definitive phenotypic identification of the cells expressing the markers of interest, remove variability generated by serial sectioning, and facilitate reporting of quantitative data for Figure 3. This manuscript supports the findings of previous work indicating that SARS-CoV-2 has a wide tissue and cellular tropism. The authors do not make any novel claims regarding tropism, but instead utilize their results to make recommendations on diagnostic methods for SARS-CoV-2 in autopsy cases, which in our opinion is an underappreciated and infrequently discussed challenges.

Point-by-point response to review questions:

Does the manuscript confirm previous work or refute the current understanding? Confirm prior work
a. Do the findings contribute to broader research understandings? Not really
b. Can the evidence and arguments presented support advancement of COVID-19 understanding within society? Not really
How well does the manuscript position the work within the current literature/understanding? Adequately
a. Does the manuscript cite current literature and discuss limitations? Yes
b. Is it steeped in reality with the potential to impact the implementation of policy and programs? It is steep in reality but the potential impact does not appear substantial in its present form.
Would you recommend this manuscript for publishing? Yes, with modifications.
Is there clarity regarding the recommended actions that result from the findings? Clear but not actionable at present.
a. Is it well-structured and well-written, with an ability to speak to key audiences? Yes
Do authors pay attention to ethics, diversity, and inclusion? N/A

a. Have the authors adequately discussed ethical concerns? N/A

b. When appropriate, have they been inclusive and taken into account equity, rights, and diversity? N/A