Abstract

COVID-19 identification is routinely performed on fresh samples, such as nasopharyngeal and oropharyngeal swabs, even if, the detection of the virus in formalin-fixed paraffinembedded (FFPE) autopsy tissues could help to underlie mechanisms of the pathogenesis that are not well understood. The gold standard for COVID-19 detection in FFPE samples remains the qRT-PCR as in swab samples, contextually other methods have been developed, including immunohistochemistry (IHC), and in situ hybridization (ISH). In this manuscript, we summarize the main data regarding the methods of COVID-19 detection in pulmonary and extra-pulmonary post-mortem samples, and especially the sensitivity and specificity of these assays will be discussed.

Introduction

The diagnosis of the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently based on the clinical manifestations associated with the detection of viral RNA by Real-Time Quantitative Reverse Transcription-PCR (qRT-PCR) in samples collected by nasopharyngeal and oropharyngeal swabs. To date, according to WHO recommendations, real time-PCR represents the gold standard technique to identify SARS-CoV-2 infection on swabs.

Although COVID-19 identification is routinely performed on fresh samples, the analysis of the virus on formalin-fixed paraffin-embedded (FFPE) autopsy tissues plays a pivotal role in understanding the biological characteristics and the pathogenesis of this infection.

The identification of SARS-CoV-2 on FFPE samples can be performed by several methods, including qRT-PCR, immunohistochemistry (IHC), and in situ hybridization (ISH) ^1,2.

In the literature, various studies have analyzed SARS-CoV-2 in FFPE samples post-mortem using different methods (Tab. I). We summarize the advantages and limitations of different methods used for detection on post-mortem samples showing an overview of the main results currently reported about the sensitivity and specificity of these assays.

Real-Time Quantitative Reverse Transcription-PCR

The qRT-PCR is currently considered the gold standard approach for the identification of SARS-CoV-2 in nasopharyngeal and oropharyngeal swabs ²¹. This molecular analysis is based on the identification of viral RNA using specific primers and probes targeting viral nucleic acid sequences of interest ²² (Fig. 1).

The main viral targets analyzed by qRT-PCR include genes encoding spike (S), nucleocapsid (N), envelope (E), open reading frame 1ab (ORF1ab) and RNA proteins RNA-dependent polymerase (RdRp) ²³.

The qRT-PCR is a reliable and fast molecular test, producing results within hours with high throughput ^2,24.

The qRT-PCR is highly sensitive for the identification of COVID-19 in swab samples; however, it can be affected by the poor quality and high fragmentation index of the nucleic acid in FFPE samples ³.

The qRT-PCR kits for SARS-CoV-2 detection include generally reverse transcription and amplification enzymes, primer sets and probes for amplification of viral genome’s target regions, and various controls, such as negative, positive, internal controls ²⁵.

The qRT-PCR assay is preceded by the isolation and purification of the total RNA that is used for a reverse transcription (RT) reaction to the synthesis of complementary DNA (cDNA) of target regions used for qPCR reaction ^26,27.

The assay is based on the use of TaqMan probes that bind exclusively to the target segment allowing to quantify the cDNA amplification in real-time based on the number of amplification cycles (Ct value) ^28,29.

According to World Health Organization (WHO) guidelines, the qRT-PCR on fresh samples is considered positive if at least two viral target genes are amplified, while if only one viral gene is amplified the result is considered equivocal, and the test should be repeated.

The test is negative when all viral genes are not amplified, instead the absence of gene control amplification suggests an invalid analysis due to incorrect sampling or PCR inhibition ^25,30. These criteria have also been applied for interpretation of SARS-CoV-2 qRT-PCR in FFPE samples ^9-11,14.

The Ct value to define a sample COVID-19 positive may be variable between different laboratories according to the kit and the protocol used for the test. The equivocal Ct values ranging from 37 to 40 involve repeating the analysis in order to avoid false results ^31,32. The kits used for qRT-PCR include various primer-probe sets targeting different segments of the SARS-CoV-2 genome thus different specificities and sensitivities have been reported in FFPE series ^32,33.

Previous data showed no significant differences in qRT-PCR results in terms of Ct values between fresh tissues and FFPE ³⁴.

The qRT-PCR shows high sensitivity for the identification of SARS-CoV-2 in FFPE samples, although, the test can be affected by pre-analytical problems associated with FFPE samples, such as fixation times and high degree of nucleic acid fragmentation. Moreover, this molecular approach is not useful for the subcellular localization of the virus since the overall tissue architecture is lost.

In situ hybridization

The ISH is a method that identifies the presence of the SARS-CoV-2 in FFPE samples allowing the definition of the virus’s localization, and the types of cells infected while preserving the morphology of the samples ^35,36.

ISH is based on the use of nucleic acid probes that bind the complementary sequences that are revealed through the 3,3’-diaminobenzidine tetrahydrochloride (DAB) staining (Fig. 2).

To date, three probes for SARS-CoV-2 detection have been developed by RNAscope technology, particularly probes targeting the spike (S, nt 21,563-25,384), nucleocapsid (N, nt 28,274-29,533), and open reading frame 1ab (ORF1ab, nt 266-13,467) ^37,38 (Fig. 3).

RNAscope technology uses innovative probes consisting of two independent probes (double Z probes) that hybridize to the target sequence in tandem for signal amplification to occur.

Although previous data showed that SARS-CoV-2 ISH have low sensitivity especially in samples with a low viral load ⁹, this assay can provide information related to the cellular distribution of virus ³⁹.

The majority of the published studies have analyzed by ISH exclusively the SARS-CoV-2 S, and few data have been reported for N and ORF1ab ^1,3,7-16,19.

The studies that examined SARS-CoV-2 by ISH have analyzed different types of organs, but positivity for the virus was mainly identified in lung tissue, in particular spike RNA has been detected in pneumocytes, alveolar cells, hyaline membranes rather than in endothelial cells ^1,8,11 (Tab. II).

The sensitivity and specificity of ISH compared to qRT-PCR for detecting SARS-CoV-2 on postmortem lung specimens in two studies were 36-46% and 100%, respectively ^3,9, while Massoth et al reported a sensitivity of 86.7% and a specificity of 100% ¹¹ (Tab. III).

Immunohistochemistry

SARS-CoV-2 identification can be performed by immunohistochemistry (IHC) using specific antibodies that have been developed for the S and N proteins (Fig. 4).

The main advantage of this technique is the possibility of localizing the virus in specific cells allowing to understand the correlation with etiopathogenesis. Furthermore, the widespread adoption of IHC in the majority of the pathology unit implies easier use of this assay for SARS-CoV-2 detection in post-mortem samples.

To date, various antibodies are commercially available for the identification of SARS-CoV-2, in particular anti-S (clone 1A9), and anti-N (clone 6F10 and clone 4B21) (Fig. 3).

The anti-N antibody clone 6F10 showed high sensitivity and specificity for virus detection ⁴⁰.

The studies that analyzed SARS-CoV-2 by IHC in lung post-mortem tissue showed the following localization of the virus: intraalveolar cells, bronchiolar airways, and hyaline membranes.

The sensitivity and specificity of S IHC for detecting SARS-CoV-2 on post-mortem lung specimens compared to qRT-PCR ranging from 55.5% to 62.5% and 100%, respectively. Similarly, N IHC showed a sensitivity of 64.7% and specificity of 100% ⁷. Several studies have performed IHC SARS-CoV-2 not only in lung tissue, but also in other sites showing a sensitivity of 62-66% and a specificity of 87-100% ^7,12 (Tab. IV).

The discordant results between qRT-PCR and IHC for the detection of SARS-CoV2 in the lungs and other sites demonstrate a low sensitivity of IHC ^6,13,18 (Tab. V).

Most studies reported a concordance between IHC and ISH results both in terms of sensibility and specificity ¹⁰. Moreover, the expression of S and N SARS-CoV-2 IHC was consistent with the RNA-ISH assay regarding the topographic localization, including especially the hyaline membrane and intra-alveolar region ¹⁰.

Conclusion

COVID-19 is primarily a respiratory disease, although recent studies have shown that it can also affect multiple sites leading to the development of extra-pulmonary symptoms. In particular, the emerging literature has shown that SARS-CoV-2 can affect several sites beyond the pulmonary district, leading to numerous clinical presentations ⁴¹.

Histopathological studies have reported organ-tropism of SARS-CoV-2 beyond the respiratory tract, including renal, neurological, gastrointestinal, and myocardial tropism ⁴², suggesting an influence of disease course and aggravation of pre-existing conditions.

In this context, the evaluation of post-mortem biomaterial by different assays, i.e., IHC and ISH, improve the knowledge about the pathophysiology of COVID-19 disease. Since autopsy biomaterial can be subjected to many pre-analytical limitations, optimization of methods to identify the virus on these specimens is needed.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

FUNDING

None.

ETHICAL CONSIDERATION

None

AUTHORS’ CONTRIBUTIONS

RF: manuscript conception, IT and FZM: writing, AR, ANDN, MD, MM, CPC, GP: reviewing.

Figures and tables

Figure 1.Workflow qRT-PCR COVID-19 detection in post-mortem samples. qRT-PCR of SARS-CoV-2 RNA from FFPE post-mortem biomaterial and preparation of the mix for analysis. Detection of N, S, ORF1ab and RNase P genes. qRT-PCR, Real-Time Quantitative Reverse Transcription-PCR; ORF1ab, Open reading frame 1ab; N, Nucleocapsid; E, Envelope; S, Spike; M, Membrane; RNase P, Ribonuclease P; dsDNA, double strand DNA.

Figure 2.Workflow ISH assays to detect COVID-19 in post-mortem samples. Detection of SARS-CoV-2 N, S and ORF1ab genes using Z probes for in situ hybridization. Tissue pretreatment, hybridization of Z probe to RNA, binding of pre-amplifiers to probe and binding of amplicons to each pre-amplifier, followed by binding of chromogenic enzyme to amplicons and optical microscope visualization. FFPE, formalin-fixed paraffin-embedded; N, nucleocapsid; S, spike; ORF1ab, Open Reading Frame 1ab; ISH, In situ hybridization; DAB, 3,3′-Diaminobenzidine.

Figure 3.Representative results of immunohistochemistry (IHC) and in situ hybridization (ISH) for SARS-CoV-2 detection in pulmonary and extra-pulmonary post-mortem samples. Nucleocapsid IHC in lung tissue (A), Nucleocapsid IHC in brain tissue (B), Nucleocapsid IHC in heart tissue (C), Spike ISH in lung tissue (D), Spike ISH in liver tissue (E), Spike ISH in heart tissue (F).

Figure 4.Workflow IHC assays to detect SARS-CoV-2 in post-mortem samples. Detection of SARS-CoV-2 using antibody anti-N and anti-S for immunohistochemistry. Tissue pretreatment, binding of primary antibody to the S and N antigens, binding of a secondary antibody conjugated to horseradish peroxidase polymers (HRP) which provides an enzyme to convert DAB into a precipitate visible under optical microscope. FFPE, formalin-fixed paraffin-embedded; N, nucleocapsid; S, spike; IHC, immunohistochemistry; DAB, 3,3′-Diaminobenzidine.

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