Detection methods for SARS-CoV-2 in tissue

Abstract

Background: Analyses for the presence of SARS-CoV‑2 in the tissues of COVID-19 patients is important in order to improve our understanding of the disease pathophysiology for interpretation of diagnostic histopathological findings in autopsies, biopsies, or surgical specimens and to assess the potential for occupational infectious hazard.

Material and methods: In this review we identified 136 published studies in PubMed's curated literature database LitCovid on SARS-CoV‑2 detection methods in tissues and evaluated them regarding sources of error, specificity, and sensitivity of the methods, taking into account our own experience.

Results: Currently, no sufficiently specific histomorphological alterations or diagnostic features for COVID-19 are known. Therefore, three approaches for SARS-CoV‑2 detection are used: RNA, proteins/antigens, or morphological detection by electron microscopy. In the preanalytical phase, the dominant source of error is tissue quality, especially the different intervals between sample collection and processing or fixation (and its duration) and specifically the interval between death and sample collection in autopsies. However, this information is found in less than half of the studies (e.g., in only 42% of autopsy studies). Our own experience and first studies prove the significantly higher sensitivity and specificity of RNA-based detection methods compared to antigen or protein detection by immunohistochemistry or immunofluorescence. Detection by electron microscopy is time consuming and difficult to interpret.

Conclusions: Different methods are available for the detection of SARS-CoV‑2 in tissue. Currently, RNA detection by RT-PCR is the method of choice. However, extensive validation studies and method harmonization are not available and are absolutely necessary.

Keywords: COVID-19; Electron microscopy; Fluorescence in situ hybridization; Preanalytical phase; Reverse transcriptase polymerase chain reaction.

Fig. 1

SARS-CoV‑2 gene sequence (green) and protein sequence (red), with indication of currently used molecular detection methods (severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu‑1, complete genome. Figure modified from NCBI reference sequence: NC_045512.2). CISH chromogenic in situ hybridization, FISH fluorescence in situ hybridization, RT-PCR reverse transcriptase polymerase chain reaction, ORF open reading frame

Fig. 2

SARS-CoV‑2 detection methods in pathology. CISH chromogenic in situ hybridization, FISH fluorescence in situ hybridization, NGS next-generation sequencing, RT-PCR reverse transcriptase polymerase chain reaction, ImmunoTEM immuno-transmission electron microscopy, ISH-EM in situ hybridization electron microscopy

Fig. 3

SARS-CoV‑2 detection methods in lung tissue. a Monoclonal anti-SARS spike glycoprotein antibody (mouse, monoclonal, Abcam, Cambridge, UK, Ab272420, 1:100) with apparent specific cytoplasmic granular staining pattern (a: arrowheads) in bronchial epithelia in autopsy lung tissue from a COVID-19-positive patient (SARS-CoV‑2 E [envelope protein] gene, RdRp [RNA-dependent RNA polymerase] gene, and N [nucleocapsid protein] gene positive in reverse transcriptase polymerase chain reaction [RT-PCR], disease duration 38 days). The regular negative control shows the lack of nonspecific binding of the secondary antibody (b: biotinylated horse anti-mouse, 1:300; scale bar = 40 µm). c–e Nonspecific binding of the primary antibody with similar granular staining pattern in bronchial epithelial cells and macrophages in autopsy lung tissue: c without any lung disease, d in acute respiratory distress syndrome (ARDS), and e in H1N1 influenza (scale bar = 40 µm)

Fig. 4

SARS-CoV‑2 RNA detection by fluorescence in situ hybridization (FISH) in lung tissue from a SARS-CoV-2-positive patient (measurement bar = 50 µm), a HE lung and pulmonary vessel, b consecutive sectional step to a with positive RNA detection for SARS-CoV‑2 (red) in macrophages (I.), endothelia (II.), and capillary endothelia (III.) in the absence of signal in the negative control (neg. contr.)