Pathology and Pathogenesis of SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States

Roosecelis B. Martines; Jana M. Ritter; Eduard Matkovic; Joy Gary; Brigid C. Bollweg; Hannah Bullock; Cynthia S. Goldsmith; Luciana Silva-Flannery; Josilene N. Seixas; Sarah Reagan-Steiner; Timothy Uyeki; Amy Denison; Julu Bhatnagar; Wun-Ju Shieh; Sherif R. Zaki

Disclosures

Emerging Infectious Diseases. 2020;26(9):2005-2015. 

In This Article

Materials and Methods

Study Patients and Data Collection

As part of the public health response to COVID-19, the CDC Infectious Diseases Pathology Branch (Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases) was consulted on autopsies of 8 patients with laboratory evidence of SARS-CoV-2 by reverse transcription PCR (RT-PCR) on respiratory swab specimens collected either before or after death. We reviewed available medical records and preliminary autopsy reports for information regarding demographics, symptom history, underlying conditions, infectious disease testing, imaging study findings, treatment and advanced supportive care received, and date of death. This investigation was reviewed in accordance with CDC's human subjects review procedures and was determined to not meet the definition of research.

Histopathology and Immunohistochemistry

We performed routine hematoxylin–eosin stains for histopathologic evaluation. We conducted an IHC assay for SARS-CoV-2 using a rabbit polyclonal antibody raised against SARS-CoV nucleocapsid (Novus Biologicals, https://www.novusbio.com)[19] at 1:100 dilution and a Mach 4 Universal AP Polymer Kit (Biocare Medical, https://biocare.net) with Permanent Red Chromogen (Cell Marque/Millipore Sigma, https://www.cellmarque.com). We pretreated the slides with heat-induced epitope retrieval with a citrate-based buffer (Biocare Medical). We ran appropriate negative controls in parallel, using normal rabbit serum in place of the primary antibody. We validated cross reactivity of the anti–SARS-CoV antibody with SARS-CoV-2 by testing controls created from SARS-CoV-2–infected Vero cells embedded with normal human tissues; we used this control as the positive control for subsequent IHC assays. The SARS-CoV nucleocapsid antibody did not cross-react with influenza A(H1N1) virus, influenza B virus, respiratory syncytial virus, parainfluenza virus type 3, human coronavirus (HCoV) 229E, or MERS-CoV in PCR-confirmed tissue samples. For cases with bronchopneumonia, we performed IHC testing for bacterial agents using a mouse monoclonal antibody raised against Streptococcus pneumoniae but known to also detect other Streptococcus spp. and a rabbit polyclonal anti-Klebsiella pneumoniae antibody (both from Thermo Fisher, https://www.thermofisher.com) known to also detect other gram-negative bacteria (Escherichia coli, Haemophilus influenzae, and Pseudomonas spp.).

For double-stained assays, we used Envision G/2 Double Stain System, Rabbit/Mouse (DAB Permanent Red) from Agilent Technologies (https://www.agilent.com). We used antibodies against CD163 (Leica Biosystems, https://www.leicabiosystems.com) or surfactant apoprotein A (Dako, https://www.agilent.com), followed by the anti–SARS-CoV nucleocapsid antibody (Novus Biologicals). We performed all assays according to the manufacturer's guidelines. We used SARS-CoV-2–infected Vero cells as a positive control and used non–COVID-19 cases and normal rabbit serum in place of primary antibody as negative controls.

Electron Microscopy

We obtained upper airway and lung tissue specimens from formalin-fixed samples, cut them into cubes, rinsed them with 0.1 mmol/L phosphate buffer, postfixed them with 2.5% glutaraldehyde, and rinsed them in phosphate buffer. In addition, we removed tissue samples from areas corresponding to positive SARS-CoV-2 immunostaining from paraffin blocks with a 2-mm punch or from 4-μm sections on glass slides; we deparaffinized the samples in xylene and rehydrated. We processed tissues for transmission EM as described previously.[20] We immersed the sections embedded in epoxy resin in boiling water, removed them from the slides with a razor blade, and cut out the areas of interest and glued them onto a blank EM block. We stained EM sections with uranyl acetate and lead citrate and examined them on a Thermo Fisher/FEI Tecnai Spirit or Tecnai BioTwin electron microscope.

RT-PCR

We extracted nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues and assessed them by a conventional RT-PCR specifically targeting the nucleocapsid gene of SARS-CoV-2 (J. Bhatnagar, unpub. data) and real-time RT-PCR/PCR targeting other respiratory pathogens, including influenza viruses, respiratory syncytial virus, human parainfluenza viruses, and Streptococcus pneumoniae, as described previously.[21,22] The SARS-CoV-2 nucleocapsid assay detects SARS-CoV-2 and SARS-CoV but does not amplify MERS-CoV and other common human coronaviruses, including alphacoronavirus (HCoV-NL63) and betacoronavirus (HCoV-HKU1).

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