key: cord-309513-dleo9rpl
authors: Zhang, Huilan; Zhou, Peng; Wei, Yanqiu; Yue, Huihui; Wang, Yi; Hu, Ming; Zhang, Shu; Cao, Tanze; Yang, Chengqing; Li, Ming; Guo, Guangyun; Chen, Xianxiang; Chen, Ying; Lei, Mei; Liu, Huiguo; Zhao, Jianping; Peng, Peng; Wang, Cong-Yi; Du, Ronghui
title: Histopathologic Changes and SARS–CoV-2 Immunostaining in the Lung of a Patient With COVID-19
date: 2020-03-12
journal: Ann Intern Med
DOI: 10.7326/m20-0533
sha: 
doc_id: 309513
cord_uid: dleo9rpl

nan

Biopsy lung sections were analyzed with hematoxylineosin staining, and immunostaining for SARS-CoV-2 was conducted as reported elsewhere (1) . Throat swabs were assessed for SARS-CoV-2 by using real-time reverse transcriptase polymerase chain reaction assays (2) .

The CT scans revealed patchy bilateral ground glass-like opacifications ( Figure 1A -C, arrows). Despite antiviral therapies, respiratory and hemodynamic instability continued and the patient died 3 weeks after diagnosis. Permission for postmortem transthoracic needle biopsy, but not autopsy, was obtained from the patient's family.

Histopathologic examination of lung biopsy tissues revealed diffuse alveolar damage, organizing phase. Denuded alveolar lining cells ( Figure 2 , A-1, arrow 1), with reactive type II pneumocyte hyperplasia, were noted ( Figure 2 , A-1, arrow 2). Intra-alveolar fibrinous exudates were present ( Figure 2 ( Figure 2 , B, bottom panel, green arrows). In contrast, viral protein expression was minimally detectable on blood vessels ( Figure 2 , B, dashed black line) or in the interstitial areas between alveoli (Figure 2, B, bottom panel, blue arrows) . Immu-nostaining of Huh7 cells infected with SARS-CoV and of lung sections from an HIV-positive patient who died of fungal infection served as positive and negative staining controls, respectively (Figure 2, C) . A. Histopathologic examination revealing diffuse alveolar damage, organizing phase (A-1); denudation of alveolar lining cells (arrow 1), with presence of reactive type II pneumocyte hyperplasia (arrow 2) (A-2); intra-alveolar fibrinous exudates (arrow 3) and interstitial loose fibrosis with chronic inflammatory infiltrates (arrow 4) (A-3); and intra-alveolar loose fibrous plugs (arrow 5) (A-4). In most foci, intra-alveolar organizing fibrin is seen (arrow 6). B. Immunostaining of SARS-CoV-2 in lung sections. Images were taken under light and fluorescent conditions, respectively (×100 magnification). Merged images were also generated. Blue arrows indicate interstitial areas between the alveoli, and green arrows indicate injured epithelial cells desquamated into the alveolar spaces. The dashed black lines indicate the blood vessel. Immunostaining of SARS-CoV-2 was done by using a rabbit polyclonal antibody (made in house, 1:100) against the Rp3 NP protein, which is highly conserved between SARS-CoV and SARS-CoV-2, followed by probing with a Cy3-conjugated goat antirabbit IgG (1:50, Abcam, ab6939). C. Positive and negative controls for immunostaining. For the positive control, the Huh7 cells were infected with SARS-CoV-2 at multiplicity of infection of 0.5 for 48 hours. After extensive washes, the cells were then fixed with 2.5% (wt/vol) glutaraldehyde. The infected cells were stained in red, and nuclei were stained with DAPI (Beyotime, Wuhan, China) in blue. For the negative control, biopsy lung sections derived from a patient with HIV who died of fungal infection were stained in parallel with lung sections from the patient with COVID-19 as above.

Note: Authors indicated with an asterisk

Disclosures: Authors have disclosed no conflicts of interest. Forms can be viewed at www.acponline.org/authors/icmje/ConflictOfInterest Forms.do?

By the National Natural Science Foundation of China (grants 81974456 and 91749207); the Clinical Research Physician Program of Tongji Medical College, Huazhong University of Science and Technology (grant 5001540075); and the SARS-CoV-2 Pneumonia Emergency Technology Public Relations Project (grants 2020FCA009 and

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR