Letters5 May 2020

Histopathologic Changes and SARS-CoV-2 Immunostaining in the Lung of a Patient With COVID-19

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    Background: Although many studies have demonstrated the epidemiologic characteristics of SARS-CoV-2 disease (COVID-19), details of pathologic changes in the lung are still lacking.

    Objective: To describe the histopathologic changes in the lung of a patient with COVID-19.

    Case Report: A 72-year-old man with a history of diabetes and hypertension presented with fever and cough. His throat and pharyngeal swabs were positive for SARS-CoV-2 by day 6 after the initial symptoms. Rapidly progressive respiratory failure required endotracheal intubation and mechanical ventilation 1 week after presentation.

    Lung tissue was obtained by transthoracic 14-gauge needle biopsy from the left upper anterior segment (Figure 1, A, arrow), left upper lingular segment (Figure 1, B, arrow), and left lower lobe (Figure 1, C, arrow), coinciding with ground-glass opacities on chest computed tomography (CT). Two throat swab samples were collected from the tonsils and posterior pharyngeal wall.

    Figure 1. Computed tomographic images obtained from the patient 3 weeks after initial clinical manifestations of COVID-19 and 2 weeks before transthoracic biopsy, demonstrating ground glass–like opacifications.

    Pleural thickening and enlarged mediastinal lymph nodes were present. Arrows indicate the approximate locations of the subsequently obtained postmortem transthoracic needle biopsy samples. A. Left upper anterior segment. B. Left upper lingular segment. C. Left lower lobe.

    Figure 1. Continued

    Biopsy lung sections were analyzed with hematoxylin–eosin 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 1, A to 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, A-2, arrow 3), along with loose interstitial fibrosis and chronic inflammatory infiltrates (Figure 2, A-2, arrow 4). Intra-alveolar loose fibrous plugs of organizing pneumonia were noted (Figure 2, A-3, arrow 5), with presence of intra-alveolar organizing fibrin seen in most foci (Figure 2, A-4, arrow 6).

    Figure 2. Histopathologic examination of lung biopsy tissues and immunostaining from a patient who died of COVID-19 (×100 magnification).

    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.

    Immunostaining of lung sections with an antibody to the Rp3 NP protein of SARS-CoV-2 revealed prominent expression on alveolar epithelial cells (Figure 2, B, top panel), including damaged, desquamated cells within the alveolar space (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). Immunostaining of Huh7 cells infected with SARS-CoV-2 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).

    Discussion: The histopathologic changes seen on postmortem transthoracic needle biopsies from a patient with COVID-19 who had respiratory failure and radiographic bilateral ground-glass opacities are consistent with diffuse alveolar damage. Although such nonspecific findings may be seen in response to several conditions that result in respiratory failure, its demonstration in the setting of COVID-19 helps to inform the clinical course of disease.

    Our study is limited by our inability to obtain larger tissue specimens. The present findings warrant further study with larger tissue samples, obtained by open or thoracoscopic lung biopsy, or autopsy, for example.

    References

    • 1. Zhou P Yang XL Wang XG et alA pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature2020. [PMID: 32015507] doi:10.1038/s41586-020-2012-7 CrossrefMedlineGoogle Scholar
    • 2. Corman VM Landt O Kaiser M et alDetection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill2020;25. [PMID: 31992387] doi:10.2807/1560-7917.ES.2020.25.3.2000045 CrossrefMedlineGoogle Scholar

    Comments

    Huilan Zhang, PhD., Cong-Yi Wang, PhD., Peng Zhou, PhD., Huihui Yue, MD., Ronghui Du, MD..30 March 2020
    Authors' Response
    We would like to thank Dr. Narasaraju Teluguakula for his attention and valuable comments on our report. We believe that the resolution of these issues would provide valuable information to the understanding of the pathogenesis and pathologic mechanisms underlying COVID-19. Now we respond to the three questions raised by Dr. Teluguakula.

    First, Dr. Teluguakula noticed the gathering of neutrophils evidenced by the elongated nuclear staining in our figure image (Figure 2 A1). To address this question, our team further examined lung sections for neutrophils (MPO+) and macrophages (CD68+) by immunostaining, and found that macrophages were the predominant infiltrating cells, while neutrophils merely account for a small proportion, which is consistent with previous studies [1, 2]. Given that neutrophil extracellular traps (NETs), critical for the bactericidal function of neutrophils, are a specialized molecular structure formed by dying neutrophils, it is therefore plausible that massive neutrophil infiltration would be obvious upon the severe occurrence of secondary bacterial infection.

    Second, we do not exclude the possibility that COVID-19 patients would exhibit lung hemorrhagic changes. However, this is not obvious in our case, which could be related to the disease course and disease severity of the COVID-19 patients. Therefore, more autopsy evidence and clinical information are needed to confirm the correlation between hemorrhagic pathological changes and COVID-19, such as whether the patients had used ECOM before death and whether the patients had comorbidities like severe coagulation dysfunction.

    Finally, we co-stained SARS-Cov-2 with CD3 (T cell marker), CD68 (macrophage marker) and MPO (neutrophil marker) by immunofluorescence, and the results showed and a few of neutrophils.

    We hope that our answers to Dr. Teluguakula would shed more light on the mechanisms and pathological processes of COVID-19.

    Reference
    1. Luo, W.; Yu, H.; Gou, J. et al. Clinical Pathology of Critical Patient with Novel Coronavirus Pneumonia (COVID-19). Preprints 2020, 2020020407.
    2. Xu Z, Shi L, Wang Y, et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome [published online ahead of print, 2020 Feb 18] [published correction appears in Lancet Respir Med. 2020 Feb 25;:]. Lancet Respir Med. 2020;S2213-2600(20)30076-X. doi:10.1016/S2213-2600(20)30076-X
    Jincheng Ma30 March 2020
    Comment
    Dear Editor, I am a clinical first-line doctor from Ningxia Autonomous Prefecture General Hospital. I read the latest paper of Zhang et al. in your journal with interest1, and I would like to make the following comments.
    Firstly, the results in this study are conducive to understanding the pulmonary histopathology and virus distribution caused by SARS-CoV-2 better. Ningxia, home to a Chinese minority, is also influenced by SARS-CoV-2 with more than 100 COVID-19 patients. In the course of treating COVID-19 patients, poor absorption of lesions in bilateral lung was observed in some patients after recovery, and even fibrotic processes occurred such as increased mesh shadow, formation of subpleural line, gradual bronchial distortion and stretch bronchiectasis. The pathological report in this study (Figure 2, A2) indicated that intra-alveolar fibrinous exudates were obeserved, along with loose interstitial fibrosis and chronic inflammatory infiltrates. However, I do not think the measurement of the degree of pulmonary fibrosis by HE staining is convincing enough. Therefore, could this study provide Masson staining to further uncover the status of collagen fiber deposition in the region of lesion, which will contribute to a better understanding of whether SARS-CoV-2 causes extensive pulmonary fibrosis in this patient as previously reported2.
    Secondly, I noticed that there were some unstructured areas in red stained in the alveolar cavity from the HE images, which was similar to the pathology of alveolar proteinosis. Furthermore, for some SARS-CoV-2 infected patients , CT image of both lungs would present crazy-paving pattern, which is a typical characteristic of pulmonary alveolar proteinosis. In addition, alveolar macrophages, which might be attacked by SARS-CoV-2, are unable to complete lipid clearance or phagocytosis3. Therefore, we are wondering the lipoprotein deposition in the alveolar cavity of COVID-19 patients.
    Finally, combined with the pathological features and chest CT findings, the type of interstitial lung changes in this patient is acute fibrinous and organizing pneumonia(AFOP) or organizing pneumonia (OP) or nonspecific interstitial pneumonia(NSIP) or mixed?I believe that the resolution of these problems will contribute to revealing the relationship between SRAS-CoV-2 and pulmonary fibrosis, and provide new ideas for the treatment of COVID-19.

    Reference
    1.Zhang H, Zhou P, Wei Y, et al. Histopathologic Changes and SARS–CoV-2 Immunostaining in the Lung of a Patient With COVID-19. Ann Intern Med. 2020; [Epub ahead of print 12 March 2020]. doi: https://doi.org/10.7326/M20-0533.
    2.Luo, W.; Yu, H.; Gou, J. et al. Clinical Pathology of Critical Patient with Novel Coronavirus Pneumonia (COVID-19). Preprints 2020, 2020020407.
    3.Kumar A, Abdelmalak B, Inoue Y, Culver DA. Pulmonary alveolar proteinosis in adults: pathophysiology and clinical approach. Lancet Respir Med. 2018;6(7):554–565. doi:10.1016/S2213-2600(18)30043-2
    Huilan Zhang, PhD., Cong-Yi Wang, PhD., Ronghui Du, MD.18 April 2020
    Authors' Response to Raja
    We would like to thank Dr. RAJA for his attention on our report and now response the question raised by Dr. RAJA.
    Firstly, the methodology of postmortum needle biopsy: After the patient's death, the doctors prepared personal protective equipment, including N95 masks, goggles and protective clothing. Then, according to the CT image results, the 14-gauge needle biopsy is used to collect lung tissue near the lesions in the isolated room, and the tissue samples should be fixed in 4% formaldehyde solution for later use.
    In the biopsy of died COVID-19 patients, it is necessary to pay attention to personal protection against COVID-19 infection. More details about the method of biopsy, please refer to the previous research results.

    Reference
    1. Khankan AA, Al-Muaikeel M. Image-guided percutaneous transthoracic biopsy in lung cancer--emphasis on CT-guided technique. J Infect Public Health. 2012;5 Suppl 1:S22–S30. doi:10.1016/j.jiph.2012.09.001
    2. Barton LM, Duval EJ, Stroberg E, Ghosh S, Mukhopadhyay S. COVID-19 Autopsies, Oklahoma, USA [published online ahead of print, 2020 Apr 10]. Am J Clin Pathol. 2020;aqaa062. doi:10.1093/ajcp/aqaa062
    Yanqiu Wei, MD., Huihui Yue, MD., Fengqin Zhang, MD., Mei Lei, PhD., Ronghui Du, MD..8 April 2020
    Authors' Response to Ma
    We appreciate Dr. Jincheng Ma for his attention and valuable comments on our report. Now we respond to the three questions raised by Dr. Jincheng Ma.
    First, in the HE staining process, firstly, the nucleus and calcium salt mucus are blue after hematoxylin staining, and then using eosin to stain the cytoplasm, the various components of the cytoplasm can present different shades of pink. Specifically, when collagen fibers are deposited, the color of eosin show a trend from shallow to deep, meaning that the general morphological structure of various tissues or cell components and lesions can be displayed [1]. Therefore, we take HE staining as the first choice. Masson staining is mainly used for the differential staining of collagen fibers and muscle fibers. It is also a commonly used connective tissue staining method, in which collagen fibers are blue and muscle fibers are red [2]. In fact, we also performed Masson staining on the lung tissue of patient with SARS-CoV-2, and the result showed blue collagen fiber deposition, which is consistent with our HE staining result.
    Second, as for whether COVID-19 patient has alveolar proteinosis. SARS-CoV-2 is capable of infecting and then inactivating mononuclear macrophages, which can indeed be the cause of secondary alveolar proteinosis. And from the HE image, there are some red deposition areas in the alveolar cavity, which is similar to the pathology of alveolar proteinosis. We were also curious about this ourselves, PAS staining thus has been added [3]. However, the result of PAS staining could not support alveolar proteinosis. The explanation is as follows, there is a feature in the HE staining of patients with SARS-CoV-2, which cannot be ignored. A large number of alveolar epithelial cells are seen in the alveolar cavity. Since alveolar epithelial cells are attacked by a large number of viruses, they could not normally secrete alveolar active proteins.
    Finally, according to the CT appearance of the chest of this patient, it can be found that there are large consolidation and scattered ground glass opacities, which will be aggravated, especially in the later stages of the disease. Furthermore, the histopathological results of the patient’s lung biopsy showed that there was cellulose exudation in the alveoli. Combined with pathological features and chest CT manifestations, we believe that this patient has mixed performance of acute fibrous tissue pneumonia (AFOP) and tissue pneumonia (OP).

    We hope that our answer of these issues would provide valuable information to the understanding of the pathogenesis and pathologic mechanisms underlying COVID-19.

    Reference
    1. N. Schmitzy, S. Lavertyz, V.B. Kraus, et al. Basic methods in histopathology of joint tissues. Osteoarthritis and Cartilage 18 (2010) S113eS116.
    2. S N Asonova, N S Migalkin. et al. Use of Masson's trichrome method for staining decalcified bone tissue. Arkhiv Patologii, 1996, 58(1):66-67.
    3. Sarris A H, Palade G E. et al. The sialoglycoproteins of murine erythrocyte ghosts. A modified periodic acid-Schiff stain procedure staining nonsubstituted and O-acetylated sialyl residues on glycopeptides. 1979, 254(14):6724-31.
    RAJA6 April 2020
    METHODOLOGY OF POSTPARTUM NEEDLE BIOPSY PROCEDURE
    Dear Editor, as this COVID-19 is new to all, I just want to know the methodology of postmortum needle biopsy from deceased due to COVID-19. Method and precautions and which specialist can do this invasive procedure.
    Kindly email me the response.
    Narasaraju Teluguakula23 March 2020
    No Conflicts of interest
    Teluguakula Narasaraju, PhD
    Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma, USA, 74078.
    Correspondence: Narasaraju Teluguakula; Email: [email protected]

    Dear Editor, Zhang and colleagues (1) examined histopathology and immunostaining analysis in the lung biopsy samples from COVID-19 infected patient. These findings are critical as it shows distribution of COVID-19 in the infected lungs and also early changes in the lung pathology.
    I would like to bring two important aspects in these analysis. First, Figure 2 A1 identified diffuse alveolar damage with necrotic epithelium and epithelial denudation. In addition to these lesions, I noticed significant accumulation of neutrophil extracellular (NETs) in this histology image. The NETs are appearing as elongated nuclear staining, present as single cell origin or forming as bundles in this biopsy section. Further, NETs are prominently scene in both airways and alveoli in this section. Earlier, we observed similar pathologic lesions in lethal influenza infected mice as described in Figures 2 and 4 (2). The presence of NETs in these tissue samples can be confirmed by immunostaining analysis using antibodies against histones, myeloperoxidase or neutrophil elastase (2). I believe it is critical, as numerous clinical reports from COVID-19 infected patients have identified neutrophilia as a prominent laboratory finding in all nonsurvivors compared those recovered from infection (3, 4). In addition, high increase in blood IL-8 were found, suggesting a possibility of a massive neutrophil influx and activation in infected lungs (4).
    Another distinct pathologic finding from this study is the absence of hemorrhagic exudative lesions as described in lung autopsy analysis reported earlier (5). These findings rise a question whether the hemorrhagic and exudative vascular pathology could be an aftermath effect of early alveolar epithelial injury mediated by excessive neutrophils and NETs-related toxic components. Likewise, we observed two distinct phages in fatal influenza infected mice (6). At early stages of infection, we observed necrotic epithelia, epithelial denudation, and massive neutrophil influx/NETs. However, at late stages of infection we observed a clear hemorrhagic exudative pathology with decreased presence of immune cells, similar to pathologic lesions observed in lung autopsy studies in COVID-19 infected patients. Analysis of bronchoalveolar lavage fluids or temporal lung biopsy analysis will determine if immunopathology plays major role in pathologic manifestations of acute respiratory distress syndrome in COVID-19 infected patients.
    Finally, the immunostaining analysis shows wide distribution of the virus in different epithelial cells as well as immune cells (ex. macrophages) as seen in the bright field images. Identifying different cell types using cell-specific markers would help to understand the possible target cells for the virus. This would help for other researchers to quickly set up in vitro lung epithelial cell culture models to test efficacy of various novel drugs for their antiviral activity.
    References.

    1. Zhang H, Zhou P, Wei Y, et al. Histopathologic Changes and SARS–CoV-2 Immunostaining in the Lung of a Patient With COVID-19. Ann Intern Med. 2020; [Epub ahead of print 12 March 2020]. doi: https://doi.org/10.7326/M20-0533.

    2. Narasaraju T, Edwin Y, Ramar PS, et al. Excessive Neutrophils and Neutrophil Extracellular Traps Contribute to Acute Lung Injury of Influenza Pneumonitis. American Journal of Pathology. 2011;179:199-210.

    3. Wang D, Hu B, Hu C, et al. Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA. 2020 Feb 7. doi: 10.1001/jama.2020.1585. [Epub ahead of print]

    4. Jing Gong, Hui Dong, Song Qing Xia, et al. Correlation Analysis Between Disease Severity and Inflammation-related Parameters in Patients with COVID-19 Pneumonia
    medRxiv 2020.02.25.20025643; doi: https://doi.org/10.1101/2020.02.25.20025643.

    5. Luo, W.; Yu, H.; Gou, J. et al. Clinical Pathology of Critical Patient with Novel Coronavirus Pneumonia (COVID-19). Preprints 2020, 2020020407.

    6. Ashar HK, Mueller NC, Rudd JM etal. Role of extracellular histones in influenza virus pathogenesis. American Journal of Pathology. 2018;188:135-148.