Abstract
Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.
Highlights
In January 2020, a severe acute respiratory syndrome, caused by the Novel Coronavirus 2019 SARS-CoV-2, was firstly identified in China and quickly spread throughout the world
Before the infection caused by SARSCoV-2, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) are the best-known examples of large-scale epidemic coronavirus-associated involvement in severe acute respiratory syndromes [1]
The present study shows the SARS-CoV-2 RNA analysis on a series of post-mortem lung samples performed by the reverse transcription polymerase chain reaction (RT-PCR) based on an extensive panel of targets, including E, N, S, and Orf1ab genes, and the in situ hybridization (ISH) method using two different probes, such as the Spike and the Orf1ab sense RNA strand produced
Summary
In January 2020, a severe acute respiratory syndrome, caused by the Novel Coronavirus 2019 SARS-CoV-2, was firstly identified in China and quickly spread throughout the world. The World Health Organization (WHO) has officially declared the SARS-CoV-2 disease a pandemic with a public health emergency of international concern. SARS-CoV-2 belongs to the family of the coronaviruses (CoVs) that are enveloped, positive-sense, single-stranded RNA viruses. Before the infection caused by SARSCoV-2, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) are the best-known examples of large-scale epidemic coronavirus-associated involvement in severe acute respiratory syndromes [1]. The SARS-CoV-2 genome is closely related to two bat coronaviruses, bat-SLCoVZC45 and batSL-CoVZXC21 (89–96.3% sequence homology), while it has less sequence similarity (79–82%) with SARS-CoV and MERS-CoV [2, 3]. The viral structures and genome of SARS-CoV-2 showed a unique feature compared to all other coronaviruses [1]
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