Using EM data to understand COVID-19 pathophysiology – Authors' reply
Carsten Dittmayer, Jenny Meinhardt, Helena Radbruch, Josefine Radke, Barbara Ingold Heppner, Frank L. Heppner, Werner Stenzel, Gudrun Holland, Michael Laue
Abstract
We fully agree with Marisa Dolhnikoff and colleagues that we should aim to understand COVID-19 pathophysiology. However, their arguments directed at our Correspondence,1Dittmayer C Meinhardt J Radbruch H et al.Why misinterpretation of electron micrographs in SARS-CoV-2-infected tissue goes viral.Lancet. 2020; 396: e64-e65Summary Full Text Full Text PDF PubMed Scopus (84) Google Scholar which should support their Case Report2Dolhnikoff M Ferreira Ferranti J de Almeida Monteiro RA et al.SARS-CoV-2 in cardiac tissue of a child with COVID-19-related multisystem inflammatory syndrome.Lancet Child Adolesc Health. 2020; 4: 790-794Summary Full Text Full Text PDF PubMed Scopus (185) Google Scholar on ultrastructural identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patient tissue, are not convincing. As in other fields of ultrastructural research, identification of subcellular structures is made on the basis of ultrastructural features, which are characteristic for each structure. The putative virus particles in the publication by Dolhnikoff and colleagues2Dolhnikoff M Ferreira Ferranti J de Almeida Monteiro RA et al.SARS-CoV-2 in cardiac tissue of a child with COVID-19-related multisystem inflammatory syndrome.Lancet Child Adolesc Health. 2020; 4: 790-794Summary Full Text Full Text PDF PubMed Scopus (185) Google Scholar lack essential and distinct ultrastructural features, such as biomembranes and surface structures, for an unambiguous identification of SARS-CoV-2. Their electron micrographs suggest poor structural preservation (eg, biomembranes are not detectable), for an unknown reason. Hence, the identification of enveloped viruses is very difficult and most likely impossible. We acknowledge the application of complementary techniques for viral detection, but their RT-PCR indicated a low viral load and immunohistochemistry was not directed to viral components.2Dolhnikoff M Ferreira Ferranti J de Almeida Monteiro RA et al.SARS-CoV-2 in cardiac tissue of a child with COVID-19-related multisystem inflammatory syndrome.Lancet Child Adolesc Health. 2020; 4: 790-794Summary Full Text Full Text PDF PubMed Scopus (185) Google Scholar Detection of SARS-CoV-2 by electron microscopy might be impossible if there is an insufficient concentration of viral particles, which is further complicated in cases of focal infections. Thus, it cannot be generally expected to find many and evenly distributed locations in RT-PCR-positive samples, which show a significant number of viral particles. The localisation of hot spots of virus particle assembly in tissue might be guided by immunohistochemistry or in-situ hybridisation.3Meinhardt J Radke J Dittmayer C et al.Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19.Nat Neurosci. 2020; (published online Nov 30.)https://doi.org/10.1038/s41593-020-00758-5Crossref PubMed Scopus (873) Google Scholar, 4Shieh WJ Hsiao CH Paddock CD et al.Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan.Hum Pathol. 2005; 36: 303-309Crossref PubMed Scopus (121) Google Scholar Identification of SARS-CoV-2 at the ultrastructural level requires characteristic morphological features (figure). Autolysis and necrosis might negatively affect these features1Dittmayer C Meinhardt J Radbruch H et al.Why misinterpretation of electron micrographs in SARS-CoV-2-infected tissue goes viral.Lancet. 2020; 396: e64-e65Summary Full Text Full Text PDF PubMed Scopus (84) Google Scholar but even in suboptimal samples, including formalin-fixed and paraffin-embedded tissue, coronavirus particle identification is possible if the characteristic ultrastructure is preserved.3Meinhardt J Radke J Dittmayer C et al.Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19.Nat Neurosci. 2020; (published online Nov 30.)https://doi.org/10.1038/s41593-020-00758-5Crossref PubMed Scopus (873) Google Scholar, 4Shieh WJ Hsiao CH Paddock CD et al.Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan.Hum Pathol. 2005; 36: 303-309Crossref PubMed Scopus (121) Google Scholar Generally, coronavirus particle profiles should show the characteristic size (∼60–160 nm without spikes), shape (round to oval), biomembrane (at least partly visible), granular or dense interior space, and surface projections (at least partly visible), whereas additional features such as grouped or regular localisation within membrane compartments or in the extracellular space might help to identify SARS-CoV-2. At least 30 publications, many also peer-reviewed, showed putative coronavirus, which lacked either sufficient image quality or ultrastructural features for clear identification as SARS-CoV-2. We only discussed two in our Correspondence1Dittmayer C Meinhardt J Radbruch H et al.Why misinterpretation of electron micrographs in SARS-CoV-2-infected tissue goes viral.Lancet. 2020; 396: e64-e65Summary Full Text Full Text PDF PubMed Scopus (84) Google Scholar as examples to precisely address our concerns. The extent of misinterpretations or insufficiently founded interpretations of putative coronavirus particles was recently shown in a detailed review.5Hopfer H Herzig MC Gosert R et al.Hunting coronavirus by transmission electron microscopy—a guide to SARS-CoV-2-associated ultrastructural pathology in COVID-19 tissues.Histopathology. 2020; (published online Sept 27.)https://doi.org/10.1111/his.14264Crossref Scopus (67) Google Scholar Similarly, we believe that electron micrographs that were published during the SARS pandemic in the early 2000s require further discussion. We agree with Dolhnikoff and colleagues that some concerns could be directly addressed to the authors. However, disputable publications spread very quickly, which can flaw the basis for a precise ultrastructural characterisation of SARS-CoV-2; thus, we are convinced that this crucial discussion required publication. We declare no competing interests. We thank the Core Facility for Electron Microscopy of the Charité for support in acquisition of the data. Why misinterpretation of electron micrographs in SARS-CoV-2-infected tissue goes viralWith interest we follow the publications that show the presence of putative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by electron microscopy (EM) in patient tissues and the debate about these results, which should have sufficiently raised attention to their correct interpretation.1,2 Full-Text PDF Using EM data to understand COVID-19 pathophysiologyThe pathophysiology of multisystem inflammatory syndrome in children is not completely understood, but it is a field in COVID-19 under extensive investigation. Evidence of the effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on extrapulmonary tissues is essential for understanding the disease's course and treatment. Full-Text PDF