key: cord-270462-d9l3agr0 authors: Zeng, Zhi; Xu, Li; Xie, Xiao‐yu; Yan, Hong‐lin; Xie, Bao‐jun; Xu, Wan‐zhou; Liu, Xin‐an; Kang, Gan‐jun; Jiang, Wan‐li; Yuan, Jing‐ping title: Pulmonary Pathology of Early Phase COVID‐19 Pneumonia in a Patient with a Benign Lung Lesion date: 2020-05-06 journal: Histopathology DOI: 10.1111/his.14138 sha: doc_id: 270462 cord_uid: d9l3agr0 AIMS: An ongoing outbreak of 2019 novel coronavirus (SARS‐CoV‐2) diseases (COVID‐19) has been spreading in multiple countries. One of the reasons for the rapid spread is that the virus can be transmitted from infected individuals without symptoms. Revealing the pathological features of early phase COVID‐19 pneumonia is important to the understanding of its pathogenesis. The aim of this study was to explore pulmonary pathology of early phase COVID‐19 pneumonia in a patient with a benign lung lesion. METHODS AND RESULTS: We analyzed the pathological changes of lung tissue from a 55‐year‐old female patient with early phase SARS‐CoV‐2 infection. In this case, right lower lobectomy was performed for a benign pulmonary nodule. Detailed clinical, laboratory and radiological data were also described. This case was confirmed to have preoperative SARS‐CoV‐2 infection by real‐time RT‐PCR and RNA in situ hybridization on surgically removed lung tissues. Histologically, COVID‐19 pneumonia was characterized by exudative inflammation. The closer to the visceral pleura, the more severe the exudation of monocytes and lymphocytes. Perivascular inflammatory infiltration, intraalveolar multinucleated giant cells, pneumocyte hyperplasia and intracytoplasmic viral‐like inclusion bodies were seen. However, fibrinous exudate and hyaline membrane formation, which were typical pulmonary features of SARS pneumonia, were not evident in this case. Immunohistochemical staining results showed that an abnormal accumulation of CD4+ helper T lymphocytes and CD163+ M2 macrophages in the lung tissue. CONCLUSION: The results highlighted the pulmonary pathological changes of early phase SARS‐CoV‐2 infection and suggested a role of immune dysfunction in the pathogenesis of COVID‐19 pneumonia. Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses that can infect human beings and many other vertebrates to cause respiratory, gastrointestinal, and nervous system diseases. [1] [2] [3] In December 2019, an outbreak of the 2019 novel coronavirus (SARS-CoV-2) diseases attracted great attention from all over the world. As of 24:00 on April 23, 2020, 82,804 confirmed cases and 4,632 deaths have been reported in China. 4 The epidemic has also spread rapidly in many other countries such as United States, 5 South Korea, 6 Italy, 7 Iran 7 and so on, posing a serious threat to human life and health. At present, research on the etiology, epidemiology, radiology, pathogenesis, and therapeutics of this new coronavirus pneumonia is being carried out urgently. [8] [9] [10] However, the pathological changes in the lungs caused by SARS-CoV-2, especially in the early stage of infection, have seldom been described. 11, 12 In this report, we analyzed the pathological changes of lung tissue from a COVID-19 patient. In this case, right lower lobectomy was performed for a benign pulmonary nodule. The patient was confirmed to have preoperative SARS-CoV-2 infection. The patient was a 55-year-old woman who lived in Wuhan. She was found to have a 7.5 mm Accepted Article ground-glass pulmonary nodule in her right lower lobe by chest computed tomography (CT) in May 2019. On January 14, 2020, the patient came to hospital for further treatment and the chest CT showed that the pulmonary nodule further increased to 10 mm ( Figure 1A ). The nodule was close to the hilum of the right lung. Except the pulmonary nodule, the patient was not in any other co-morbid conditions such as hypertension, diabetes, cardiovascular disease and chronic obstructive pulmonary disease, and she did not have symptoms like fever, dyspnea, cough on admission. Moreover, she never smoked. The patient underwent right lower lobectomy on January 17, 2020. On the 1 st day post operation, her blood lymphocyte count decreased to 0.82×10 9 /L (normal range: 1.1~3.2×10 9 /L). On the 2 nd postoperative day, she presented with a fever of 38.5℃ without dyspnea. On the 3 rd day, laboratory studies showed a white blood cell (WBC) count of 3.09×10 9 /L (normal range: 3.5~9.5×10 9 /L) with 0.76×10 9 /L lymphocyte. On the 4 th day, the patient experienced slight dyspnea without oxygen inhalation and her blood oxygen saturation decreased to 95% (2L/min, 29%). SARS-CoV-2 RNA was detected as positive by real-time RT-PCR in both patient's nasopharyngeal swab and sputum. Tests for influenza virus and other infectious agents were negative. Repeat chest CT showed patchy ground-glass opacity and focal consolidation with increasing density in the left and right lungs ( Figure 1B ). On the 5 th day, the counts of peripheral blood CD3+, CD4+ and CD8+ T lymphocytes, as well as the serum IgG level dropped dramatically. On the 6 th day, her blood oxygen saturation decreased to 90% (4L/min, 37%) and she developed severe dry cough. One day later, the blood oxygen saturation fell below 55% (6L/min, 45%), and patient showed serious dyspnea. She died before mechanical ventilation could be installed. Following her death, two of her family members, one surgeon and four bedside nurses were confirmed with SARS-CoV-2 infection ( Figure 2 ). We reviewed and collected electronic medical records, pathological sections, laboratory findings, and chest CT of the patient. Two authors independently re-reviewed the collected data and pathological findings to confirm their accuracy. This article is protected by copyright. All rights reserved Lung tissue was fixed with 4% neutral formaldehyde, embedded in paraffin wax, and 4 µm sections were cut. Sections were stained with hematoxylin and eosin (H&E). Real-time RT-PCR detection of SARS-CoV-2 in paraffin-embedded lung tissue was performed as described previously with minor revision. 13 In brief, deparaffinized and rehydrated tissue sections This article is protected by copyright. All rights reserved Formalin-fixed and paraffin-embedded tissue sections (4μm) were obtained for immunohistochemical staining according to the standard protocol. Briefly, deparaffinized and rehydrated sections were treated with 3% hydrogen peroxide and subjected to antigen retrieval by citrate buffer (pH 6.0). Sections were blocked with 5% BSA for 20 minutes and incubated overnight with primary antibody at 4℃. Primary antibodies against cell markers used in this study included CD3, CD4, CD8, CD19, CD20, CD56, CD68, CD163, MUM1 (multiple myeloma oncogene-1, also called IRF4), PAX5 (paired box gene 5), pan-cytokeratin (PCK), and thyroid transcription factor 1 (TTF1), which were purchased from DAKO (Carpinteria, CA, USA). Antibody binding to the cells in sections was detected using the horseradish peroxidase (HRP) reaction kits (DAKO, Carpinteria, CA, USA) according to the manufacturer's protocol instructions. The reaction products were stained with DAB and slight counterstained with hematoxylin. Baseline complete blood count, biochemistry, coagulation studies and immune cell analysis are listed in Supplemental Table 1 and Table 2 . Two fragments of lung tissues were obtained from the right lower lobectomy, measuring 12.5×5.7×3.0 cm and 11.9×9.0×3.5 cm. No area of consolidation or nodular lesions could be found on gross examination. Real-time RT-PCR performed on the lung tissue showed a Ct value of 36.18 at ROX (N gene) This article is protected by copyright. All rights reserved channel. RNA in situ hybridization performed on the lung tissue sections showed that the positive signals were brown in color and located in cytoplasm at the light microscope level ( Figure 3A ). In addition, at higher magnification of H&E staining, intracytoplasmic viral-like inclusions could be identified in a few type II pneumocyte-like cells ( Figure 3B ) or macrophage-like cells ( Figure 3C ). These viral-like inclusions were light red globules of about half size of an erythrocyte and were surrounded by clear halos. These results demonstrated the presence of SARS-CoV-2 in the lung tissue and thus confirmed that the patient was infected with SARS-CoV-2 preoperatively. Near the visceral pleura, features of viral pneumonia were noted. They were mainly characterized by exudative inflammation. The closer to the visceral pleura, the more severe the exudation of monocytes and lymphocytes ( Figure 4A ). These inflammatory cells centered on small blood vessels, which further infiltrated the surrounding alveolar septa and spaces ( Figure 4B ). The alveolar septa were widened, with obvious hyperemia, dilation of capillaries. Interstitial fibrosis was rarely present. In the alveolar spaces, there was a large number of monocytes, a few lymphocytes and variable numbers of red blood cells to form clumps ( Figure 4C ). Multinucleated giant cells were also seen in a few alveolar spaces ( Figure 4D ). Neither fibrinous exudate nor hyaline membrane formation was observed. Besides, areas of serous exudation with pulmonary edema were found. In these areas, some alveolar spaces were filled with a large amount of light red, homogenous proteinaceous fluid, admixed with variable numbers of red blood cells, lymphocytes and monocytes ( Figure 4E ). In addition, scattered large protein globules in alveolar spaces were observed ( Figure 4F ). The bronchial structure remained intact. At higher magnification, there was focal hyperplasia of type II pneumocytes, some of which showed mild cytologic atypia ( Figure 5A ). Some enlarged pneumocytes showed abundant cytoplasm with a ground-glass appearance, and prominent eosinophilic nucleoli ( Figure 5B, C) . The pulmonary nodule near the hilum detected by preoperative CT was demonstrated to be meningothelial-like nodules with extensive thick-walled vessels in lobectomy specimen. The COVID-19-related histopathologic changes as described above were mild in this area. Only capillary dilation This article is protected by copyright. All rights reserved and congestion were observed around the nodules (Supplemental Figure S1 ). The inflammatory cells infiltrated the blood vessels and alveolar wall were mainly composed of T lymphocytes (CD3+) with a few B lymphocytes (CD20+ and PAX5+) and plasma cells (MUM1+) ( Figure 6A-F) . In order to further understand the role of T lymphocytes in this viral pneumonia, we used the surface markers of T lymphocytes to identify the subtypes. Immunohistochemistry confirmed that CD4+ helper T lymphocytes were the predominant component, while CD8+ cytotoxic T lymphocytes and natural killer (NK) cells (CD56+) were scattered ( Figure 6G-I) . Subsequently, we examined the expression of PCK and TTF1, the markers of alveolar epithelial cells. Though the alveolar septa were infiltrated by lymphocytes, the alveolar structure was essentially intact as highlighted by immunostains ( Figure 7A -C). The enlarged cells with amphophilic granular cytoplasm and prominent nucleoli along the alveolar spaces recognized on H&E stain were PCK positive ( Figure 5C ). This result confirmed the presence of abnormal hyperplasia of type II pneumocytes. In addition to hyperplasia of type II pneumocytes, clusters of macrophage-like cells could be found in the alveolar spaces ( Figure 7A ), which could be attributed to the desquamation of pneumocytes and recruitment of macrophages. Figure 7D showed that CD68+ macrophages were the main source of cell clusters in the alveolar spaces. CD3+ T lymphocytes were also present ( Figure 7E ). In addition, immunostain for CD163, the marker of M2 macrophages, showed strong positivity and site-specific expression, suggesting that M2 macrophages may play a role in pathogenesis ( Figure 7F ). The mean incubation period for SARS-CoV-2 infection is 5.2 days. In its early stage, the epidemic doubles every 7.4 days, with a mean serial interval of 7.5 days. 14 Our patient had no This article is protected by copyright. All rights reserved symptoms or other underlying diseases before having lung surgery. She developed fever on day 2 post operation, and lab tests showed a decreased lymphocyte count and an increased WBC count in the peripheral blood, which could be easily confused with a stress response to surgery. However, her WBC and lymphocyte counts continued to decrease, and chest CT showed evidence of viral pneumonia. The diagnosis of preoperative COVID-19 was finally established by real-time PCR and RNA in situ hybridization for the virus in the lung tissue. Combining epidemiological characteristics, clinical presentation, nucleic acid testing and intracytoplasmic viral-like inclusions, it is reasonable to speculate that this case represents an early stage of lung injury secondary to SARS-CoV-2 infection. 8 Although they are about half size of SARS viral inclusions, they are found in enlarged cells similarly to SARS viral inclusions. [15] [16] [17] [18] Further studies are needed to confirm this observation. The primary pathology of both SARS and MERS in the lungs is diffuse alveolar damage (DAD). 15 Specifically, the SARS lungs show acute exudative DAD with extensive edema and hyaline membrane formation in the early phase. 16, 17 Subsequently, fibrous organization follows depending on the duration of SARS. 16 In our case of COVID-19, however, only serous exudate, instead of fibrinous exudate, is observed in the alveolar spaces. Feature of hyaline membrane formation is not observed, which is similar to the pulmonary pathology of early phase COVID-19 pneumonia reported by Tian et al. 11 Interestingly, Xu et al. observed features of DAD with hyaline membrane formation in the late stage of COVID-19 pneumonia. 12 Therefore, we consider that hyaline membrane formation may be a time-dependent pathological process, and the patient in our case might be in the exudative phase of DAD, too early for obvious hyaline membrane formation. Moreover, considering that our case showed sharply decreased blood oxygen saturation and serious dyspnea later, it is likely that she may have also progressed to full-blown DAD by the time of death. Unfortunately, we did not get a This article is protected by copyright. All rights reserved permission for autopsy to confirm this. The pathophysiology of SARS is related to the dysfunction of immune system, including hyperinduction of chemokines and cytokines, abnormal cellular immune response and insufficient interferon reaction. [18] [19] [20] [21] Our studies provide evidence that dysfunctional immune system may also be involved in the pathogenesis of COVID-19. Like SARS-CoV, 20,22 SARS-CoV-2 could directly attack epithelial cells that leads to the hyperplasia of type II pneumocytes. The inflammatory cells (mainly CD4+ T lymphocytes) that infiltrate the blood vessels and alveolar wall in the lungs could cause abnormal release of cytokines (cytokine storm). [18] [19] [20] [21] In addition, the decrease in the number of CD4+ T lymphocytes in the peripheral blood of this case suggests an abnormal redistribution between peripheral blood and lung tissue. SARS-CoV-2 could also trigger the chemotaxis movement of macrophages as evidenced by macrophage aggregates within alveolar spaces. In summary, we report histopathologic findings in the lung tissue from a patient with established diagnosis of COVID-19. We believe that the findings represent changes seen in the early stage of the disease in comparison to those observed at autopsy. Our data also suggest that immune dysfunction is implicated in the pathogenesis of SARS-CoV-2 infection. The study (WDRY2020-K005) was approved by the Ethics Committee of Renmin Hospital of the Wuhan University and was performed in accordance with the ethical standards laid down in the Accepted Article same committee. This article is protected by copyright. All rights reserved (arrow), confirming their epithelial origin. Streptavidin peroxidase, original magnification ×400. 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