Variable levels of spike and ORF1ab RNA in post-mortem lung samples of SARS-CoV-2-positive subjects: comparison between ISH and RT-PCR

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.

The Role of Toll-like Receptor-4 in Macrophage Imbalance in Lethal COVID-19 Lung Disease, and Its Correlation with Galectin-3

“…All patients who died from a lethal form of SARS-CoV-2 infection in our cohort of COVID-19 lethal disease had ARDS due to lung tissue viral persistence demonstrated by PCR as previously reported [32]. Therefore in this study we presented the percentage of Spike-1 (+) cases as evaluated by ISH compared to mean values ± Standard Error Means (SEM) of selected studied markers (TLR-4, CD68, CD163, GAL-3) (Table 1 and Figure 2).…”

“…Briefly, ISH detection of SARS-CoV-2 was been performed using V-nCoV2019-S probe to detect viral sequences Spike (S) of SARS-CoV-2 in lung tissue using RNAscope 2.5 high-definition detection kit (Advanced Cell Diagnostics). SARS-CoV-2 was been detected in lung tissues of lethal COVID-19 disease by in ISH according to methods reported in previous work of our laboratories in which we compared ISH with PCR methods [32,56,57], and in current literature [58,59].…”

Discovery of novel papain-like protease inhibitors for potential treatment of COVID-19

“…4 ) and inhibits SARS-CoV-2 replication as measured by its inhibition of mRNA expression of two different SARS-CoV-2 genes, spike (S) and open reading frame (ORF1b) ( Fig. 5 A and B) [ 50 ]. Different studies were conducted to gain more insight into the viral inhibitory mechanism of SIMR3030.…”

Visualising SARS-CoV-2 infection of the lung in deceased COVID-19 patients

“… 3 , 24 RNAscope probes for negative-sense spike RNA were applied in several studies and visualisation of intracellular puncta was reported in some of these studies, but without correlation with the disease course ( Table S1 ). 19 , 20 , 22 , 25 , 26 Visualisation of positive-sense intracellular RNA species 10 , 11 , 13 , 14 , 19 , 25 , 26 , 27 , 28 , 29 or nucleocapsid/spike-immunoreactive signal 10 , 12 , 13 , 14 , 15 , 23 , 26 , 30 , 31 , 32 , 33 has frequently been proposed to identify target cell types of SARS-CoV-2 in the lung ( Table S1 ) but does not provide robust evidence about cell tropism and ongoing viral replication. 23 , 31 , 34 …”