SARS‐CoV‐2 RNA screening in routine pathology specimens

Abstract

Virus detection methods are important to cope with the SARS-CoV-2 pandemics. Apart from the lung, SARS-CoV-2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom-indicated swab testing. Here, we tested and validated several approaches and selected the most reliable RT-PCR protocol for the detection of SARS-CoV-2 RNA in patients' routine diagnostic formalin-fixed and paraffin-embedded (FFPE) specimens available in pathology, to assess (i) organ tropism in samples from COVID-19-positive patients, (ii) unrecognized cases in selected tissues from negative or not-tested patients during a pandemic peak, and (iii) retrospectively, pre-pandemic lung samples. We identified SARS-CoV-2 RNA in seven samples from confirmed COVID-19 patients, in two gastric biopsies, one small bowel and one colon resection, one lung biopsy, one pleural resection and one pleural effusion specimen, while all other specimens were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID-19 case in tonsillectomy samples. All pre-pandemic lung samples were negative. In conclusion, SARS-CoV-2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID-19, allow retrospective studies, and advance our understanding of SARS-CoV-2 organ tropism and effects.

Multisystemic Cellular Tropism of SARS-CoV-2 in Autopsies of COVID-19 Patients

“…False-negative test results cannot be excluded, neither in the RT-PCR nor the FISH analyses. This seems not to be strongly dependent on RNA quality or quantity [24]. Importantly, we performed all analyses on material that was previously selected as suitable by pathologists (documented by the figures showing the results of the FISH analyses).…”

“…The remaining IHC steps were performed as previously described [23], and the staining was developed with ImmPACT ® VIP Substrate (Vector Labs, Burlingame, CA, USA). Previously we compared two different kits for RT-PCR analysis [24]. Here, we used the TaqMan™ Fast 1-Step Master Mix (Thermo Fisher Scientific GmbH, Dreieich, Germany) for the qualitative detection of the E gene (encoding envelope protein) of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) by a primer pair (0.4 µM) and probe (0.2 µM) set labelled with fluorescent reporters and quencher dyes.…”

“…We used TaqMan ® Exogenous Internal Positive Control reagents (Thermo Fisher Scientific GmbH, Dreieich, Germany) as internal PCR controls. RT-PCR was performed as previously described [24,25]. Briefly, we reversely transcribed (50 • C for 10 min) and amplified the RNA extracted from FFPE tissues with the reaction mixture at 95 • C for 20 s and followed by 45 cycles of 95 • C for 3 s and 58 • C for 30 s. We used the Amplirun ® SARS-CoV-2 RNA control (Bestbion dx GmbH, Cologne, Germany) provided with 13,000 viral RNA copies µL −1 to calculate the viral RNA copies in the tissue samples.…”

“…Protein detection of SARS-CoV-2 spike glycoprotein is widely used in studies in FFPE tissues. We stained for the SARS-CoV-2 spike glycoprotein in the COVID-19 autopsy lung and selected the most commonly used antibodies based on previous studies [24,27], targeting the SARS spike glycoprotein (Abcam, ab272420) and SARS-CoV S∆10 within S2 domain protein (Genetex, GTX632604). We have tested these two antibodies on FFPE autopsy lung tissues with four different antigen retrieval methods, as outlined in the methods section.…”

An Approach to Cellular Tropism of SARS-Cov-2 Through Protein-Protein Interaction and Enrichment Analysis

“…Other studies have pointed to the potential infection of these tissues. Interestingly, Stillfried et al using pathological specimens, have identi ed SARS-CoV-2 by RT-PCR in different tissues, including the small intestine 21 .…”