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.

Fig. 1

Evaluation of RT‐PCR efficiency of the different primer and probe sets with the two different RT‐PCR methods by SARS‐CoV‐2 RNA standard. (A) C_t values of primer and probe sets as singleplex and multiplex approaches using TaqMan and RealStar methods in twofold dilution series of SARS‐CoV‐2 RNA standard. (B) Using linear regression of each assay, RT‐PCR efficiency was calculated according to the equation 100× (−1 + 10^−1/slope). For successful RT‐PCR assays, the efficiency ranges from 90 to 110% (dotted lines, Taylor et al., 2010). Bland–Altman plots for comparison of TaqMan singleplex and multiplex methods for the targets (C) E_Sarbeco and (D) RdRp_SARSr show a mean bias close to zero, indicating a low average discrepancy.

Fig. 2

Results of RT‐PCR‐based detection of SARS‐CoV‐2 RNA in the three cohorts studied (N = 363) and method validation using fluorescence in situ hybridization (FISH). (A) SARS‐CoV‐2 RT‐PCR detection in all three cohorts resulted in four positive samples in COVID‐19 patient samples and one positive sample in pandemic peak patient samples. (B–C) Method validation by FISH and hematoxylin‐eosin staining; (B') SARS‐CoV‐2 RNA‐positive pleural effusion sample (arrows, patient 12, Table 4) and (B'') SARS‐CoV‐2‐positive tonsil sample from pandemic patient with viral RNA in detritus‐filled crypts (arrows); (B''') lung tissue from an autopsy case with clinically confirmed SARS‐CoV‐2 infection showed a red fluorescent signal of SARS‐CoV‐2 RNA (arrows, scale bar = 5 µm), (B'''') positive control with a red fluorescent signal of Homo sapiens POLR2A gene (arrows, scale bar = 10 µm), (B''''') Negative control (dap gene from Bacillus subtilis, scale bar = 10 µm). (C') Light micrograph of pleural effusion sample showing a group of reactive macrophages (arrow, HE, scale bar = 10 µm). (C'') Light micrograph of tonsil sample in (B''); (C''') light micrograph of lung tissue in (B''') with reactive macrophages (arrows, HE, scale bar = 20 µm). (d) RT‐PCR results (individual SARS‐CoV‐2 E‐gene viral copy number µl⁻¹) of the samples.

Fig. 3

Histological findings in lungs from patients in the late phase of COVID‐19. A. Histologic images of lung tissue from a patient with lung surgery for pulmonary metastases who had recovered from mild COVID‐19 (i.e. without the need for mechanical ventilation), 66 days after initial symptoms. Note the circumscribed fibrotic areas surrounded by thin alveolar septa with open alveolar spaces (arrows, A' hematoxylin‐eosin, A'' Elastica van Gieson stain, scale bar = 200 µm). B. Histological images of lung tissue from a patient with severe COVID‐19 (i.e. impaired oxygenation and subsequent failure of mechanical ventilation with the need for extracorporeal membrane oxygenation (ECMO)) with bacterial superinfection. The patient underwent surgery on day 82 after initial symptoms for histologic evaluation of fibrotic changes in the lungs. Note disappeared macrophage‐filled alveolar spaces and multinucleated giant cells (B' arrows, hematoxylin–eosin, scale bar = 250 µm, insert: scale bar = 100 µm) and diffusely fibrotic alveolar septa (B'') arrows, Elastica van Gieson stain, scale bar = 250 µm, insert: scale bar = 250 µm.