Comparison of In Situ Hybridization, Immunohistochemistry, and Reverse Transcription–Droplet Digital Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Testing in Tissue

Abstract

Context: Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-detection in formalin-fixed paraffin-embedded (FFPE) tissue using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and/or RNA-in situ hybridization (RNAish). Objective: To compare droplet digital PCR (ddPCR), IHC, and RNAish to detect SARS-CoV-2 in FFPE tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. Design: ddPCR and RNAish used commercially available probes; IHC utilized clone 1A9. Twenty-six autopsies of COVID-19 patients with FFPE tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. Results: ddPCR had the highest sensitivity for SARS-CoV-2 (96%) when compared to IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between ddPCR and IHC or RNAish was fair (κ=0.23, κ=0.35, respectively). Agreement between IHC and ISH was substantial (κ=0.75). Interobserver reliability was almost perfect for IHC (κ=0.91) and fair to moderate for RNAish (κ=0.38–0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by ddPCR (P=.03, pearson corr = −0.65) and were more likely to be positive by RNAish (P=.02) than lungs from patients who died later. SARS-CoV-2 was identified in hyaline membranes, pneumocytes, and rarely in respiratory epithelium. ddPCR showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. Conclusions: ddPCR was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

Viral mapping in COVID-19 deceased in the Augsburg autopsy series of the first wave: A multiorgan and multimethodological approach

“…The subsequently performed analyses using RNA-ISH and IHC identified viral RNA or protein with a specificity of 100%, but with substantially lower sensitivities in between 23% (IHC and RNA-IHC negative-sense) and 77% (RNA-ISH positive-sense) of the PCR-positive samples. Roden et al recently reported a high sensitivity for droplet digital PCR and a considerably lower sensitivity by RNA-ISH and IHC of approximately one-third, which is comparable to our results [25]. On the basis of RT-qPCR as the gold standard, Massoth et al compared RNA-ISH and IHC for the detection of this virus in 58 autoptic FFPE samples from different tissues.…”

Afectación Respiratoria y Sistémica producida por el Virus SARS-Cov-2.

“…En el hígado se identificó esteatosis leve, áreas de necrosis hepática, hiperplasia de células de Kupffer y dilatación leve de los capilares sinusoidales de la zona 3, sin evidencias de cambios trombóticos. Un estudio realizado por Roden et al, 2021 (55) evidenciaron mediante hibridación in situ, inmunohistoquímica y reacción en cadena de la polimerasa de transcripción inversa para el diagnóstico del síndrome respiratorio agudo severo por Coronavirus 2 (SARS-CoV-2) vs pruebas en tejido, reportando que la reacción en cadena de la polimerasa tuvo la mayor sensibilidad para el SARS-CoV-2 (96%) en comparación con IHC (31%) y RNAish (36%). Las 3 pruebas tuvieron una especificidad del 100%.…”

“…Las autopsias revelan que las alteraciones del corazón han sido mínimas, no demostrándose evidencias de miocarditis viral. La investigación de Roden et al 2021 (55) mostró que la reacción en cadena de la polimerasa digital de gotitas mostró un bajo número de copias en 7 corazones de autopsia de ProteoGenex Inc. Todos los demás tejidos extrapulmonares fueron negativos.…”

SARS-CoV-2 RNA detection in Formalin-Fixed Paraffin-Embedded (FFPE) tissue by droplet digital PCR (ddPCR)

“…Current or prior infection might also be recognized by a serum test for SARS-CoV-2 IgG antibodies. A subset of patients may present without the typical upper respiratory symptoms or with concurrent unusual features that potentially represent long-term sequelae of SARS-CoV-2 [11] , [12] . Patients with current or previous SARS-CoV-2 infection who present with “unexplained” acute or acute on chronic respiratory failure or other organ-specific manifestations may undergo tissue biopsy.…”