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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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00428-021-03262-8.

SUBMITTER: Zito Marino F

PROVIDER: S-EPMC8805427 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe emergency of new COVID-19 variants over the past three years posed a serious challenge to the public health. Cities in China implemented mass daily RT-PCR tests by pooling strategies. However, a random delay exists between an infection and its first positive RT-PCR test. It is valuable for disease control to know the delay pattern and daily infection incidences reconstructed from RT-PCR test observations.MethodsWe formulated the convolution model between daily incidences and positive RT-PCR test counts as a linear inverse problem with positivity restrictions. Consequently, the Richard-Lucy deconvolution algorithm was used to reconstruct COVID-19 incidences from daily PCR tests. A real-time deconvolution was further developed based on the same mathematical principle. The method was applied to an Omicron epidemic data set of a bar outbreak in Beijing and another in Wuxi in June 2022. We estimated the delay function by maximizing likelihood via an E-M algorithm.ResultsThe delay function of the bar-outbreak in 2022 differs from that reported in 2020. Its mode was shortened to 4 days by one day. A 95% confidence interval of the mean delay is [4.43,5.55] as evaluated by bootstrap. In addition, the deconvolved infection incidences successfully detected two associated infection events after the bar was closed. The application of the real-time deconvolution to the Wuxi data identified all explosive incidence increases. The results revealed the progression of the two COVID-19 outbreaks and provided new insights for prevention and control strategies, especially for the role of mass daily RT-PCR testing.ConclusionsThe proposed deconvolution method is generally applicable to other infectious diseases if the delay model can be assumed to be approximately valid. To ensure a fair reconstruction of daily infection incidences, the delay function should be estimated in a similar context in terms of virus variant and test protocol. Both the delay estimate from the E-M algorithm and the incidences resulted from deconvolution are valuable for epidemic prevention and control. The real-time feedback is particularly useful during the epidemic's acute phase because it can help the local disease control authorities modify the control measures more promptly and precisely.

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

Supplementary Information

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