ABSTRACT
BACKGROUND: SARS-CoV-2 is an RNA virus that primarily causes respiratory disease; however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. MATERIALS AND METHODS: A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. RESULTS: SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. CONCLUSION: This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.
Subject(s)
COVID-19 , RNA, Viral , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , COVID-19/diagnosis , Formaldehyde , Humans , Paraffin Embedding , RNA, Viral/isolation & purification , SARS-CoV-2/geneticsABSTRACT
Background SARS-CoV-2 is an RNA virus that causes primarily causes respiratory disease;however, infection of other tissue has been reported. Evaluation of SARS-CoV-2 in tissue specimens may increase understanding of SARS-CoV-2 pathobiology. Materials and Methods A qualitative test for detection of SARS-CoV-2 in formalin-fixed paraffin-embedded (FFPE) tissues was developed and validated using droplet digital PCR (ddPCR), which has a lower limit of detection than reverse transcription (RT)-qPCR. After extraction of total RNA from unstained FFPE tissue, SARS-CoV-2 nucleocapsid (N1, N2) target sequences were amplified and quantified, along with human RPP30 as a control using the Bio-Rad SARS-CoV-2 ddPCR kit. Results SARS-CoV-2 was detected in all 21 known positive samples and none of the 16 negative samples. As few as approximately 5 viral copies were reliably detected. Since January 2021, many tissue types have been clinically tested. Of the 195 clinical specimens, the positivity rate was 35% with placenta and fetal tissue showing the highest percentage of positive cases. Conclusion This sensitive FFPE-based assay has broad clinical utility with applications as diverse as pregnancy loss and evaluation of liver transplant rejection. This assay will aid in understanding atypical presentations of COVID-19 as well as long-term sequelae.
ABSTRACT
CONTEXT.: Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription-polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish). OBJECTIVE.: To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. DESIGN.: Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded 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.: Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair (κ = 0.23 and κ = 0.35, respectively). Agreement between IHC and in situ hybridization 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 droplet digital polymerase chain reaction (P = .03, Pearson correlation = -0.65) and were more likely to be positive by RNAish (P = .02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. CONCLUSIONS.: Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.