key: cord-0980747-atf9n90o
authors: Zhou, Baoyong; Zhao, Wei; Feng, Ruixi; Zhang, Xiaohui; Li, Xuemei; Zhou, Yang; Peng, Li; Li, Yixin; Zhang, Jinyan; Luo, Jing; Li, Lingyu; Wu, Jingxian; Yang, Changhong; Wang, Meijiao; Zhao, Yong; Wang, Kejian; Yu, Huarong; Peng, Qiling; Jiang, Ning
title: The pathologic autopsy of coronavirus disease 2019 (COVID-2019) in China: a review
date: 2020-06-13
journal: Pathog Dis
DOI: 10.1093/femspd/ftaa026
sha: 0214372281f9b7339fe4021d8828d843b71c61f3
doc_id: 980747
cord_uid: atf9n90o

The coronavirus disease 2019 (COVID-2019) emerged in Wuhan, China, has rapidly spread to many countries across all six WHO regions. However, its pathobiology remains incompletely understood and many efforts are underway to study it worldwide. To clarify its pathogenesis to some extent, it will inevitably require lots of COVID-2019-associated deaths at pathologic autopsy. Pathologists from all over the world have raised concern with pathologic autopsy relating to COVID-2019. The issue of whether a person dies from COVID-2019 infection or not always is an ambiguous problem in some cases, and ongoing epidemiology from China may shed light on it. This review retrospectively summarizes the research status of pathologic autopsy in COVID-2019 in China, which will be important for the cause of death, prevention, control and clinical strategies of COVID-2019. Moreover, it points out several challenges at autopsy. We believe pathological studies from China enable to correlate clinical symptoms and pathological features of COVID-2019 for doctors and provide an insight into COVID-2019 disease.

Coronavirus disease 2019 (COVID-2019) is a seriously infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This coronavirus was firstly identified as the causative organism by Chinese facilities by deep sequencing analysis of patients' respiratory tract samples. 1 Its clinical symptoms include fever, cough, and shortness of breath. Other symptoms may include muscle pain, sputum production, diarrhea, sore throat, loss of smell, and abdominal pain. 2 Terribly, some patients associated with SARS-CoV-2 infection progress to pneumonia and multi-organ failure, which are extremely harmful to humans. Until now, the disease is rapidly spreading across all six WHO regions. As of 26 April 2020, in total, more than 2,804,796 cases of COVID-2019 have been reported in 197 countries (A total of 84,338 confirmed cases were reported in China, 195,351 cases were confirmed in Italy, and 1,094,846 cases were confirmed in the United States). 3 The virus is thought to spread mainly from human-to-human transmission. It took 67 days from the outbreak of COVID-19 to a total of 100,000 cases, the second 100,000 cases took 11 days, and the third 100,000 cases took only four days. 4 It has reported that the overall mortality is 4.6 percent; ranging from 0.2 percent to 15 percent according to age group and underlaying diseases. 5 In clinical practice, the possibility of infection is commonly confirmed by reverse transcription polymerase chain reaction (RT-PCR) from a nasopharyngeal swab. On the other hand, the infection can also be diagnosed from a combination of symptoms and radiological evaluation such as computerized tomography (CT) [6] [7] [8] [9] and chest X-ray. [10] [11] Even worse, there is currently no vaccine to prevent COVID-2019. Until recently, Chinese scholars have made a lot of progress in etiology, epidemiology, diagnosis and therapeutic strategies, etc. Although many studies on COVID-2019 epidemiology and clinical characteristics have been published, studies on the histopathological characteristics of each tissue are relatively insufficient due to barely accessible autopsy. To advance research progress of COVID-2019, pathologic autopsy is inevitable method to discover the changes of tissues and clarify its etiology and pathogenesis to some extent and finally determine whether a therapy is proper and reasonable. Thus, a comprehensive theory of pathogenesis for this newly emerged infectious disease is essential.

By April 5, professor Bian's team of the Army Military Medical University in Chongqing, China, has declared to accomplish the pathologic autopsies of 39 cases positive for COVID-2019 and established the first pathological bank of this disease in the world. However, some available pathologic data have not attracted wide attention. This review summarizes the research status of pathologic autopsy in COVID-2019 restricted from China, which will facilitate understanding for the cause of death and improve clinical strategies against COVID-2019 worldwide.

As the availability and quality of imaging techniques, CT and chest X-ray together with RT-PCR have been important modalities in assisting in the diagnosis and management of patients with COVID-2019 pneumonia. For example, Shi and colleagues 6 analysed chest CT images from 81 patients positive for COVID-2019 pneumonia and discussed temporal changes of COVID-2019 pneumonia since onset of symptoms, which provides insight in the evolution of the disease and its corresponding imaging changes.

Guan et al. 7 have investigated chest CT images from 53 patients confirmed COVID-2019 infection, by which they demonstrated pulmonary lesions in majority of patients were predominantly distributed peripherally in the subpleural area. On the other hand, Pan et al. 8 have studied the chest CT images from 21 patients with RT-PCR confirmed COVID-19 infection and they found that lung abnormalities on chest CT showed greatest severity approximately 10 days after initial onset of symptoms. Ai et al. 9 have investigated the consistency of chest CT as compared with comparison to RT-PCR assay among 1014 patients in Wuhan, China and suggested that the sensitivity of chest CT was 97% based on positive RT-PCR results. Although chest CT has been shown to be an effective imaging technique for lung-associated disease diagnosis, chest X-ray is more widely available due to its faster imaging time and considerably lower cost than CT. Zhang et al. 10 have collected 100 chest X-ray images of 70 patients confirmed with COVID-2019 and demonstrated that chest X-ray is critical for efficient and reliable COVID-2019 screening.

Meanwhile, Abbas et al. 11 have adopted their previously developed (convolutional neural networks) classification of COVID-2019 chest X-ray images and its accuracy approaches to 95.12%. CT and chest Xray are the most common and widely used diagnostic imaging techniques for COVID-2019, which greatly accelerate the speed of disease screening, especially in epidemic areas and alleviate the lack of nucleic acid kits. Whereas, CT and chest X-ray are not able to confirm COVID-2019, directly prove the distribution of the virus in tissues, or analyze the cause of death in cases. Pathologic autopsy will primarily provide critical insights into the pathogenesis of COVID-2019 in humans.

Pathologic autopsy developed rapidly in the 18 th century, which reached its peak in the middle of 20 th century was regarded as a scientific pathology to study human diseases. 12 Since then, pathologic autopsy has become a very important method to understand the pathologic basis of disease and the cause of death. As early as 2003, the pathogenesis of the severe acute respiratory syndrome (SARS), which was caused by SARS-CoV and began to break out in Guangdong province, China, initially remained poorly understood. Ding et al. 13 Meanwhile, viral particles were localized in the pneumocytes, pulmonary macrophages, renal proximal tubular epithelial cells and macrophages infiltrating the skeletal muscles. Because of the importance of autopsy to study the basic pathology, it is an indispensable research method for the study of infectious diseases.

The SARS and MERS-associated coronaviruses are both considered HG3 pathogens; Resembly, SARS-CoV-2 has recently been categorised as a HG3 organism. 20 A comprehensive theory of pathogenesis for this newly emerged infectious COVID-2019 is still lacking. Although China has made great progress in preventing and controlling infectious diseases, more effective treatment and prevention strategies are strongly required. Unfortunately, we have not been able to produce vaccines to cure infectious COVID-2019. Thus, the pathological characteristics, cause of death, distribution of pathogens in tissues remain an urgent problem worldwide, which probaly provides supports for the therapies of 

On February 16, 2020, Liu and his team from Tongji medical college, Wuhan, performed the first systematic anatomy of COVID-2019's corpse, an 85-year-old male. 22 They have estimated that the lung injury is obvious by microscopic zones, an inflammatory lesions (pale) are predominantly in the left lung as shown in Figure 1A . By naked eye, the left lung shows patchy peripheral hemorrhage of parenchyma ( Figure 1B ), and alveoli of the lungs lose their elasticity. In addition, the cut surfaces display fibous cords appearance with lots of sticky secretion exudation from pulmonary alveoli ( Figure 1C ). Foam-like mucus secretions are present in trachea and bronchus, respectively ( Figure 1D and 1E). All of these pathologic changes suggest that SARS-CoV-2 infection causes inflammation characterized by injuries of large airway and alveoli. Previously, it predicts that the pathological characteristics of COVID-2019 in lung injury resemble those caused by SARS, due to the similarities between SARS-COV-2 and SARS-CoV in its genome sequence, biological behavior and clinical manifestations. 13, [23] [24] From the observation of systematic anatomy, pulmonary fibrosis and consolidation are not as serious as those caused by SARS, but the sticky secretion is more than that of SARS. On the one hand, this anatomy is of great significance to discover the lesions of other tissues. An amount of yellow fluid is found in the pericardial cavity and epicardium involves by a slight edema. The myocardial section is grayish red ( Figure 1G ). Among the digestive system, small intestinal tract is of normal color with segmentary dilatation ( Figure 1H ). Fi-nally, they also have estimated that SARS-CoV-2 infection involves damage to the brain, liver, kidneys and other tissues. However, there is no microscopic assess of histological examination in the first pathologic autopsy from China.

In some respect, microscopic assessments enable to show general layout and distribution of cells and provides a general overview of a tissue sample's structure, which are of significance for pathological analysis. Almost at the same time, Xu et al. 25 from PLA General Hospital, Beijing, have investigated the pathological characteristics of a 50-year-old patient by minimally invasive autopsy, who died from severe infection with SARS-CoV-2. In their study, hematoxylin-eosin (H&E) staining was carried out to analy pathologic features of the lung tissues from this victim. Postmortem examination shows bilateral DAD with cellular fibromyxoid exudates (Figure 2A and B) . And it reveals evident desquamation of pneumocytes and hyaline membrane formation on the right lung, indicating acute respiratory distress syndrome (ARDS; Figure 2A ). On the other hand, the left lung tissue appears as pulmonary oedema with hyaline membrane formation, a suggestive of early-phase ARDS ( Figure 2B ). Moreover, interstitial mononuclear Besides, we have intense concern with whether the virus can infect other organs except for lung.

Tian et al. 27 Figure   3A ). It is worth mentioning that no presence of prominent inflammatory cellular infiltration is confirmed in Case 2 ( Figure 3B ), and Case 3 demonstrates focal interstitial thickening ( Figure 3C ). H&E staining in Figure 3D , E and F are carried out from Case 4. Together with hyaline membranes in some airspaces, large areas of intra-alveolar hemorrhages and fibrin cluster formation are observed nearby ( Figure 3D ). In addition, the alveolar wall contains increased stromal cells, fibrin, and infiltration by mononuclear inflammatory cells. From Figure 3E , type II pneumocyte hyperplasia results in interstitial thickening, along with fibrinoid necrosis of the small vessels ( Figure 3E, inset) . In Case 4, there is also evidence of consolidation by abundant intra-alveolar neutrophilic infiltration, consistent with bronchopneumonia of a superimposed bacterial infection ( Figure 3F ). These results demonstrates that main pathologic findings from the lungs include hyaline membrane formation, fibrin exudates, epithelial damage, and diffuse type II pneumocyte hyperplasia, which are all characterics of DAD. On the other hand, mild thickening of alveolar walls is also evident in some cases, suggesting a more advanced stage of this illness. In their conclusion, the pathologic changes in the lungs are similar to those seen in the confirmed cases of SARS. However, it is a big pity that there are not enough regions of liver and heart to observe histopathological changes because of the limited scope of puncture samples.

Zhang et al. 28 Similar to as described prviously, SARS-CoV-2 can be found on the lung tissues and the histopathologic changes are consistent with DAD. 26 On the other hand, Gao et al. 29 have conducted autopsies on 4 patients with COVID-2019 who died in the Huoshenshan Hospital, Wuhan and summarized that nucleocaspid protein is highly pathogenic for lung damage through MASP-2-induced overactivation of complement, indicating that the pathogenicity and death of SARS-CoV-2 correlates with over immune response. In this study, they have given advice that complement-activation through lectin pathway is prospective for the therapy of COVID-2019 disease.

As everyone knows, immune system is the body's defense against viral infections, which attacks pathogens and helps keep us healthy. However, it has already reported that lymphocytopenia is well established infected by the SARS-CoV-2. 30 Feng et al. 31 from Jinyintan Hospital, Wuhan, have observed that SARS-CoV-2 enables to directly infect secondary lymphoid organs from six cases with postmortem examinations. Firstly, spleens and lymph nodes (LNs) from 6 COVID-2019 patients were collected by standard examination at autopsy. In the same way, the virions in LNs were visually observed by TEM and pathological features were analyzed by H&E staining. Moreover, viral nucleocapsid protein (NP) antigen, cell apoptosis and proinflammatory cytokine expression were measured by IHC. In their study, the lymph follicles and paracortical areas in virus-infected tissues are not identifiable with wide distribution of necrotic and apoptotic lymphocytes, which results in a significant reduction of total lymphocytes as shown in Figure 5 . Moreover, interstitial blood vessels have proliferation and expansion. Moreover, the spleens are congested, hemorrhagic, and lacking lymphoid follicles. Additionally, the spleen corpuscles are atrophic, along with hyperplasia of interstitial vessels and fibrous tissue in the splenic sinus. These results demonstrate that SARS-CoV-2 infection causes severe damage in human LNs and spleen.

In the same study, they have also investigated location and distribution of SARS-CoV-2 in secondary lymphoid tissues by expression of viral NP antigen. By IHC assay, it is evident that SARS-CoV-2 NP antigens can be observed in spleens and LNs from all of 6 autopsies, but in absence of normal healthy controls (see Figure 6A and B). In the spleen, SARS-CoV-2 is primarily distributed in red pulp and blood vessels, although occasionally in presence of white pulp in Figure 6A . In lymph nodes, SARS-CoV-2 are observed within marginal sinus of lymph nodules, capillaries, germinal centers as well as cytoplasm, whereas nucleus is negative for expression of viral NP antigen ( Figure 6B ). Then they also have performed addition staining to identify the LN cell types prone to SARS-CoV-2 infection.

Immunofluorescent double staining confirmed that the majority of SARS-CoV-2 are observed in ACE2overexpressed cells and CD68-overexpressed macrophages. While, CD3-overexpressed T cells and B220overexpressed B cells are tolerant of SARS-CoV-2 infection. Moreover, these results have also illustrated that SARS-CoV-2 is observed in CD169-overexpressed macrophages in the subcapsular sinus of LNs.

Thus, SARS-CoV-2 infection enables to directly infect human LNs and spleens, leading to tissue damage and lymphocyte reduction.

Apart from the respiratory and immune system, it is vague whether SARS-CoV-2 can also directly infect other tissues such as the kidney or induce acute renal failure. Diao et al. 32 have investigated kidney tissues by postmortem examinations from 6 patients positive for SARS-CoV-2 infection in Wuhan. In their research, H&E staining demonstrates that acute renal tubular damage can be observed in all 6 cases, while the glomeruli are intact (see Figure 7) . Then, they have employed in situ IHC assay to analyze distribution of SARS-CoV-2 in kidney tissues by the expression of viral NP antigen and found that SARS-CoV-2 is restricted to the renal tubular cells of the infected tissues. This study has demonstrated that kidney tissues from 6 cases of postmortems have severe acute tubular necrosis but no evidence of glomerular pathology. And that SARS-CoV-2 was primarily accumulated in kidney tubules, which directly infects human kidney tubules and induces acute tubular damage as well as urine transmission.

COVID-2019 has spread to many countries in a very short period of time, and the number of deaths has exceeded 193,000 by now. In the past month and a half, China has carried out more than 30 autopsies, by which it demonstrates the lung is the main target organ attacked by SARS-CoV-2; however, some other organs such as liver, kidney, and spleen are also damaged to some extent. The results of those pathologic autopsies will give a preliminary insight into understanding of COVID-2019, which is helpful for pathogenesis and clinical strategies of patients, but still limited. To fundamentally find the pathogenicity and lethality of COVID-2019, a larger number of urgent autopsies are required. To date, we find that there are still many challenges: (i) there are currently more than 110,000 deaths from COVID-2019 in elderly population. One expert from China points out that COVID-2019 requires more autopsies in various patient populations, the rate of which is best to reach 10% of victims. For example, Ruan et al. have pointed out that death cause of COVID-2019 patients may be due to virus-activated "cytokine storm syndrome" or fulminant myocarditis, which requires adequate autopsy studies to confirm this point of view. 33 clinical researches, especially in pathology, to control large-scale prevalence and incidence of the epidemic disease. Although CT and chest X-ray are effective methods to roughly diagnose the COVID-2019, they do not enable to profoundly provide insight into this epidemic disease from pathological prospectives. Pathologic autopsy is an inevitable method to discover the changes of tissues and clarify its etiology and pathogenesis to some extent and finally determine whether a therapy is proper and reasonable. Thus, a comprehensive theory of pathogenesis for this newly emerged infectious disease is essential. To explore its pathogenicity and lethality of COVID-2019, we urgently need to obtain the relevant pathological knowledge of COVID-2019 through autopsy, especially from China. To fundamentally find more pathogenicity and lethality of COVID-2019, a larger number of urgent autopsies are strongly required. To date, we find that there are still many challenges. This review will force a rethink how to deal with COVID-2019 in the future. Images were taken under light field; (B) and fluorescent conditions, respectively (100× objective). Merged images were also generated. The dashed blue lines indicate the blood vessel. Immunostaining assay of SARS-CoV-2 was carried out by using a rabbit polyclonal antibody against the Rp3 NP protein, which is highly experssed on the surface of SARS-CoV-2, followed by probing with a Cy3-conjugated goat antirabbit IgG. Images adapted from reference 28, with permission. 

A novel coronavirus from patients with pneumonia in China

WHO Director-General's opening remarks at the media briefing on COVID-19-23

COVID-19) Mortality Rate. 2020, www.worldometers.info

Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study

Imaging Features of Coronavirus disease 2019 (COVID-19): Evaluation on Thin-Section CT

Time course of lung changes on chest CT during recovery from 2019 novel coronavirus (COVID-19) pneumonia. Radiology

Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19) in China: a report of 1014 cases

COVID-19 Screening on Chest X-ray Images Using Deep Learning based Anomaly Detection

Classification of COVID-19 in chest X-ray images using DeTraC deep convolutional neural network

Autopsy pathology: a manual and atlas

The clinical pathology of severe acute respiratory syndrome (SARS): a report from

Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore

Multiple organ infection and the pathogenesis of SARS

Middle East respiratory syndrome

Clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of Middle East respiratory syndrome coronavirus infection in the United Arab Emirates

Value of autopsy emphasized in the case report of a single patient with Middle East respiratory syndrome

Histopathology of Middle East respiratory syndrome coronovirus (MERS-CoV) infectionclinicopathological and ultrastructural study

COVID-2019 deaths have been dissected in China

A report on the general observation of the necropsy of a newly developed coronavirus pneumonia

Structure, function, and evolution of coronavirus spike proteins

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

A pathological report of three COVID-19 cases by minimally invasive autopsies. Chin

Pathological Study of the 2019 Novel Coronavirus Disease (COVID-19) through Post-Mortem Core Biopsies. 2020

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

Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement overactivation

Lymphopenia predicts disease severity of COVID-19: a descriptive and predictive study

The Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Directly Decimates Human Spleens and Lymph Nodes

Human Kidney is a Target for Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection

Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China