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1.
Virchows Arch ; 480(3): 597-607, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1661693

ABSTRACT

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Humans , In Situ Hybridization/methods , Lung , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity
2.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1614505

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.


Subject(s)
COVID-19/diagnosis , In Situ Hybridization, Fluorescence/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Caco-2 Cells , Cell Line, Tumor , Chlorocebus aethiops , Humans , In Situ Hybridization/methods , Microscopy, Electron/methods , RNA, Viral/ultrastructure , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sensitivity and Specificity , Vero Cells , Virus Release/drug effects , Virus Release/genetics , Virus Release/physiology , Virus Replication/drug effects , Virus Replication/physiology
3.
Arch Pathol Lab Med ; 145(7): 785-796, 2021 07 01.
Article in English | MEDLINE | ID: covidwho-1134421

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.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Immunohistochemistry , In Situ Hybridization/methods , Lung/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Adult , Aged , Aged, 80 and over , COVID-19/virology , Female , Humans , Male , Middle Aged , Observer Variation , Prospective Studies , RNA, Viral/isolation & purification , Reproducibility of Results , SARS-CoV-2/genetics , Sensitivity and Specificity
4.
Hum Pathol ; 109: 69-79, 2021 03.
Article in English | MEDLINE | ID: covidwho-1036691

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was demonstrated in the placenta; however, the data on the prevalence of placental infection and associated histopathology are limited. To identify the frequency and features of SARS-CoV-2 involvement, we performed a clinicopathologic analysis of 75 placental cases from women infected at the time of delivery and 75 uninfected controls. Placental samples were studied with anti-SARS-CoV-2 immunohistochemistry and/or in situ hybridization. Positive results were confirmed by electron microscopy and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). During delivery, only one woman had symptoms of coronavirus disease 2019, six women reported previous symptoms, and 68 women were asymptomatic. All neonates tested negative for SARS-CoV-2 as per nasopharyngeal swab PCR results. Obstetric histories were unremarkable in 29 of 75 SARS-CoV-2-positive and 8 of 75 SARS-CoV-2-negative women. Placental examination was normal in 12 of 75 infected and 3 of 75 uninfected subjects, respectively. In the remaining cases, placental pathology correlated with obstetric comorbidities without significant differences between SARS-CoV-2-positive and SARS-CoV-2-negative women. SARS-CoV-2 was identified in one placenta of an infected, but asymptomatic, parturient. Viral staining was predominantly localized to the syncytiotrophoblast (STB) which demonstrated marked damage accompanied by perivillous fibrin deposition and mixed intervillositis. A significant decrease of viral titers was detected in the attached umbilical cord compared with the villous parenchyma as per qRT-PCR. SARS-CoV-2 is seldom identified in placentas of infected women. Placental involvement by the virus is characterized by STB damage disrupting the placental barrier and can be seen in asymptomatic mothers without evidence of vertical transmission.


Subject(s)
COVID-19/virology , Placenta/pathology , SARS-CoV-2/pathogenicity , Trophoblasts/pathology , Trophoblasts/virology , Adult , Female , Humans , In Situ Hybridization/methods , Placenta/virology , Pregnancy , RNA, Viral , Trophoblasts/chemistry , Viral Load
5.
Arch Virol ; 165(10): 2373-2377, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-695405

ABSTRACT

In situ hybridization (ISH) and immunohistochemistry (IHC) are essential tools to characterize SARS-CoV-2 infection and tropism in naturally and experimentally infected animals and also for diagnostic purposes. Here, we describe three RNAscope®-based ISH assays targeting the ORF1ab, spike, and nucleocapsid genes and IHC assays targeting the spike and nucleocapsid proteins of SARS-CoV-2.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Animals , Antibodies, Monoclonal , Antibodies, Viral , Antisense Elements (Genetics)/genetics , COVID-19 , COVID-19 Testing , Chlorocebus aethiops , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Genes, Viral , Humans , Immunohistochemistry/methods , In Situ Hybridization/methods , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/virology , Polyproteins , RNA, Viral/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolism
6.
Lab Invest ; 100(11): 1485-1489, 2020 11.
Article in English | MEDLINE | ID: covidwho-638974

ABSTRACT

Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes.


Subject(s)
Betacoronavirus/isolation & purification , Immunohistochemistry/methods , In Situ Hybridization/methods , Female , Humans , Indicators and Reagents , Kidney/virology , Lung/virology , Paraffin Embedding , Placenta/virology , Pregnancy , SARS-CoV-2
7.
Ann Diagn Pathol ; 48: 151565, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-635270

ABSTRACT

Infection by SARS-CoV-2 commonly begins in the nasopharynx, and the cytologic and molecular correlates are not characterized. Fifty-eight cytologic preps (20 oral and 38 from the nasopharynx) were obtained from ten patients and analyzed in a blinded fashion for SARS-CoV-2 spike and envelope protein by immunohistochemistry and viral RNA by in situ hybridization. qRTPCR identified three positive cases and seven controls; the three cases reported mild symptoms that resolved in 2-3 days. Blinded analyses confirmed the presence of the SARS-CoV-2 spike and envelope proteins and viral RNA in the three cases and viral absence in the seven controls. A signal for the positive cases was evident in each nasopharyngeal and none of the oral samples. Viral RNA/proteins localized exclusively to glandular cells and was present in high copy number. Blinded analysis of the cytology documented that the glandular cells infected by SARS-CoV-2 showed marked degeneration with ciliocytophthoria; viral inclusions were not evident. Co-expression analysis showed viral infected cells had increased apoptosis, marked by strong expression of activated caspase 3. Weekly serial testing of two of the cases showed persistence of productive viral infection for up to 2 weeks after symptom onset. It is concluded that the target cell of SARS-CoV-2 in the head and neck region is the glandular cell of the nasal passages, that viral infection is lytic and associated with high copy number that facilitates viral spread. The method outlines a simple, rapid test for productive SARS-CoV-2 based on immunohistochemistry or in situ hybridization of the glandular cells from the nasopharynx.


Subject(s)
Coronavirus Infections/diagnosis , Cytodiagnosis/methods , Immunohistochemistry/methods , In Situ Hybridization/methods , Nasopharynx/virology , Pneumonia, Viral/diagnosis , Betacoronavirus , COVID-19 , Coronavirus Infections/virology , Humans , Pandemics , Pneumonia, Viral/virology , RNA, Viral/analysis , SARS-CoV-2 , Viral Proteins/analysis
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