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      Nachweismethoden von SARS-CoV-2 in Gewebe Translated title: Methods of SARS-CoV-2 detection in tissue

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          Abstract

          Hintergrund

          Die Analyse von SARS-CoV‑2 in Geweben von COVID-19-Patienten ist wichtig für ein besseres Verständnis der Pathophysiologie der Krankheit, die Interpretation der diagnostischen histopathologischen Befunde in Autopsien, Biopsien und Resektaten oder um ein potenzielles berufsbedingtes Infektionsrisiko einzuschätzen.

          Material und Methoden

          In dieser Übersichtsarbeit haben wir 136 publizierte Studien zu Detektionsmethoden von SARS-CoV‑2 in Gewebe in der kuratierten Literaturdatenbank LitCovid von PubMed identifiziert und bezüglich Fehlerquellen, Spezifität und Sensitivität der Methoden unter Berücksichtigung eigener Erfahrungen ausgewertet.

          Ergebnisse

          Es gibt keine ausreichend spezifischen histomorphologischen Veränderungen bzw. diagnostischen Merkmale von COVID-19. Daher werden 3 Ansätze zum SARS-CoV-2-Nachweis genutzt: Nachweis von RNA, Proteinen/Antigenen oder morphologischer Nachweis mittels Elektronenmikroskopie. In der präanalytischen Phase liegt die dominante Fehlerquelle in der Gewebequalität, insbesondere den unterschiedlichen Intervallen zwischen Probenentnahme und -aufarbeitung, sowie spezifisch in Autopsien im Intervall zwischen Tod und Probenentnahme. Diese Angaben finden sich in weniger als der Hälfte der Studien (z. B. nur in 42 % der Autopsiestudien). Eigene Erfahrungen und erste Studien belegen die deutlich höhere Sensitivität und Spezifität von RNA-basierten Nachweismethoden gegenüber Antigen- bzw. Proteinnachweis mittels Immunhistochemie oder Immunfluoreszenz. Der Nachweis mittels Elektronenmikroskopie ist zeitintensiv und die Interpretation schwierig.

          Schlussfolgerungen

          Es stehen verschiedene Methoden zum Nachweis von SARS-CoV‑2 im Gewebe zur Verfügung. Derzeit ist der RNA-Nachweis mittels RT-PCR die Methode der Wahl. Notwendige, umfangreiche Validationsstudien und Methodenharmonisierung sind derzeit noch nicht verfügbar.

          Zusatzmaterial online

          Zusätzliche Informationen sind in der Online-Version dieses Artikels (10.1007/s00292-021-00919-8) enthalten.

          Translated abstract

          Background

          Analyses for the presence of SARS-CoV‑2 in the tissues of COVID-19 patients is important in order to improve our understanding of the disease pathophysiology for interpretation of diagnostic histopathological findings in autopsies, biopsies, or surgical specimens and to assess the potential for occupational infectious hazard.

          Material and methods

          In this review we identified 136 published studies in PubMed’s curated literature database LitCovid on SARS-CoV‑2 detection methods in tissues and evaluated them regarding sources of error, specificity, and sensitivity of the methods, taking into account our own experience.

          Results

          Currently, no sufficiently specific histomorphological alterations or diagnostic features for COVID-19 are known. Therefore, three approaches for SARS-CoV‑2 detection are used: RNA, proteins/antigens, or morphological detection by electron microscopy. In the preanalytical phase, the dominant source of error is tissue quality, especially the different intervals between sample collection and processing or fixation (and its duration) and specifically the interval between death and sample collection in autopsies. However, this information is found in less than half of the studies (e.g., in only 42% of autopsy studies). Our own experience and first studies prove the significantly higher sensitivity and specificity of RNA-based detection methods compared to antigen or protein detection by immunohistochemistry or immunofluorescence. Detection by electron microscopy is time consuming and difficult to interpret.

          Conclusions

          Different methods are available for the detection of SARS-CoV‑2 in tissue. Currently, RNA detection by RT-PCR is the method of choice. However, extensive validation studies and method harmonization are not available and are absolutely necessary.

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          Most cited references51

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          Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

          Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
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            Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19

            Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (Covid-19) pandemic. Despite widespread interest in the pathophysiology of the disease, relatively little is known about the associated morphologic and molecular changes in the peripheral lung of patients who die from Covid-19.
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              Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome

              The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS. Copyright 2003 Massachusetts Medical Society
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                Author and article information

                Contributors
                pboor@ukaachen.de
                Journal
                Pathologe
                Pathologe
                Der Pathologe
                Springer Medizin (Heidelberg )
                0172-8113
                1432-1963
                1 March 2021
                : 1-8
                Affiliations
                [1 ]GRID grid.412301.5, ISNI 0000 0000 8653 1507, Institut für Pathologie, , Universitätsklinik der RWTH Aachen, ; Pauwelsstr. 30, 52074 Aachen, Deutschland
                [2 ]GRID grid.412301.5, ISNI 0000 0000 8653 1507, Medizinische Klinik II (Nephrologie und Immunologie), , Universitätsklinik der RWTH Aachen, ; Pauwelsstr. 30, 52074 Aachen, Deutschland
                Author notes
                [Schwerpunktherausgeber]

                W. Roth, Mainz

                P. Boor, Aachen

                Article
                919
                10.1007/s00292-021-00919-8
                7919251
                33646360
                ff58e3d3-1fed-4e88-a8fd-b1ce598ab90d
                © Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2021

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 14 January 2021
                Categories
                Schwerpunkt: COVID-19

                covid-19,elektronenmikroskopie,fluoreszenz-in-situ-hybridisierung,reverse-transcriptase-polymerase-kettenreaktion,präanalytische phase,electron microscopy,fluorescence in situ hybridization,reverse transcriptase polymerase chain reaction,preanalytical phase

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