Pathogenesis Clues from the Early Clinical Presentation of 300 Hospitalized COVID-19 Patients

Background: The global spread of the 2019 coronavirus disease (COVID-19) has lasted more than half a year. Except for the gene sequence and viral structure of SARS-CoV-2, its clinical characteristics, pathological mechanisms and corresponding measures have not been fully revealed. We aimed to speculate about the possible pathological mechanism from the early clinical manifestations of patients with COVID-19. Methods: The onset symptoms, laboratory examinations and CT ndings on admission of 300 patients in two wards of Wuhan Third Hospital from January 28 to March 15 were analyzed retrospectively. Results: There was no difference in incidence between men and women, but women were hospitalized later after onset. Upper respiratory symptoms and sputum were rare. The incidence of fever was 71%. Blood lymphocyte counts were decreased signicantly on admission and were related to the severity of the disease. In moderate patients without hypoxia, thrombocytopenia occurred in 12.37%, CRP rose in 64.43%, BUN was elevated in 20.62%, creatinine rose in 17.53%, D-dimer was elevated in 74.74%, and creatine kinase and α-hydroxybutyrate dehydrogenase were elevated in 45.36% and 54.12% of patients, respectively. Early CT showed a small amount of inltration in the subpleural and lateral zones of the lung and thickening of the interlobular septum. Approximately 5 days later, inltration was worse in some of the patients, and the proportion of involvement of the affected lung was negatively correlated with the lymphocyte count. Conclusions: There was no sex difference in patients with SARS-CoV-2 infection. Alveolar cells and T lymphocytes may be the main targets of the virus, and apoptosis may be the primary mechanism of pathogenesis. The virus entering the lung may be transmitted through lymph or blood vessels rather than directly dispersing through the respiratory tract. Early damage to multiple organs may be caused by the immune response.

64.43%, BUN was elevated in 20.62%, creatinine rose in 17.53%, D-dimer was elevated in 74.74%, and creatine kinase and α-hydroxybutyrate dehydrogenase were elevated in 45.36% and 54.12% of patients, respectively. Early CT showed a small amount of in ltration in the subpleural and lateral zones of the lung and thickening of the interlobular septum. Approximately 5 days later, in ltration was worse in some of the patients, and the proportion of involvement of the affected lung was negatively correlated with the lymphocyte count.
Conclusions: There was no sex difference in patients with SARS-CoV-2 infection. Alveolar cells and T lymphocytes may be the main targets of the virus, and apoptosis may be the primary mechanism of pathogenesis. The virus entering the lung may be transmitted through lymph or blood vessels rather than directly dispersing through the respiratory tract. Early damage to multiple organs may be caused by the immune response.

Background
After badly affecting China for more than three months, the 2019 coronavirus disease (COVID- 19) outbreaks have abated in China; however, the threat from COVID-19 is not over, and COVID-19 is now prevalent all over the world. As of July 6, there were more than 11.7 million cases of infection and 539554 deaths worldwide. Before a vaccine for COVID-19 is available, quarantine is a passive defensive measure that has to be taken. However, long-term quarantine is also associated with many problems, such as social and economic problems. The standardization of epidemic prevention measures is currently the main problem. How can infected individuals be identi ed and quarantine measures be implemented as soon as possible? Although laboratory tests (nucleic acid tests and antibody tests) have been greatly improved, the clinical recognition and judgment by doctors is still very important, especially when in uenza and the common cold can occur in the same period and complicate the diagnosis.
The history of COVID-19 is quite short; it was found and identi ed in early January 2020. The virus 2019-nCoV causes a lower respiratory tract disease that was initially called novel coronavirus pneumonia by the Chinese government. The disease is now o cially named COVID-19 by the World Health Organization (WHO). Moreover, 2019-nCoV was renamed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses. On 11 March 2020, the WHO characterized COVID-19 as a pandemic. To minimize the spread of SARS-CoV-2, China locked down Wuhan and nearby cities beginning January 23, 2020. The strong measures, including suspension of all urban transportation, was apparently successful in preventing further spreading of SARS-CoV-2 to other cities.
The peak of infection occurred at the end of January 2020, and the morbidity decreased dramatically after mid-February [1]. Until March, only sporadic cases were reported. However, COVID-19 is very severe worldwide, and the WHO declared recently that COVID-19 may be present in the human population forever, which means that the ght against SARS-CoV-2 will last. Two major problems still trouble us: there is no vaccine to prevent and no drug to cure COVID-19. The present treatments are symptomatic treatments based on previous experience, but identi cation of additional measures should be of great importance, as COVID-19 is unique and the pathogenesis is quite different from severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) or others.
The recognition of disease rst comes from clinical manifestations, especially early clinical manifestations, which can better re ect the characteristics of diseases and lay a foundation for understanding the pathological mechanism and for making treatment plans. Studies on the natural history of SARS-CoV-2 infection in humans are urgently needed. The WHO and International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) designed a case report form speci c for COVID-19 that can provide clinical data anonymously and in a standardized manner to obtain accurate clinical manifestations and natural history data for this purpose. The features of the clinical manifestations of COVID-19 have been described in many reports, most of which are based on early data from January 2020 [2][3][4]. However, these reports are only the beginning of a comprehensive and systematic study.
Here, we report clinical data from a cohort of COVID-19 patients and propose suggestions based on these clinical manifestations.

Patients
This study only retrospectively reviewed a cohort of patients who were admitted to the Wuhan Third Hospital in the Guanggu District, which was a referral center for local COVID-19 patients diagnosed in the public health center in Wuhan, China. All patients were con rmed to have SAS-CoV-2 by testing before admission. The test was performed on samples from nasopharyngeal swabs using real-time reverse transcriptase PCR as described previously. Due to the sudden outbreak of COVID-19 in late January, the medical system in Wuhan was caught off guard, there were not enough infectious wards, and a large number of patients remained outside of the hospital. Our hospital was temporarily restructured as a designated COVID-19 hospital. On the day of opening (Jan 28), all beds were lled immediately. A total of Due to incomplete data from 32 patients, 300 patients were included in this study. The data collected included demographic, clinical symptom, and laboratory and chest CT ndings. The severity of pneumonia in these patients was assessed according to the Guidelines for Diagnosis and Management of COVID-19 (6th edition, in Chinese) issued by the National Health Commission of China [5].

CT image acquisition
All CT scans were obtained using one of the following scanners: SOMATOM Perspective, SOMATOM Spirit, or SOMATOM De nition AS+ (Siemens Healthineers, Forchheim, Germany) with patients in the supine position. Scans were performed from the level of the upper thoracic inlet to the inferior level of the costophrenic angle, and the following parameters were used: detector collimation widths 64 × 0·6 mm, 128 × 0·6 mm, 64 × 0·6 mm, and 64 × 0·6 mm; and tube voltage 120 kV. The tube current was regulated by an automatic exposure control system (CARE Dose 4D; Siemens Healthineers). Images were reconstructed with a slice thickness of 1-5 mm or 1 mm and an interval of 1-5 mm or 1 mm, respectively. The reconstructed images were transmitted to a workstation, and a picture archiving and communication system (PACS) was used for multiplanar reconstruction post processing.

Image interpretation
Images from patients seen at Wuhan Third Hospital were analyzed by two respiratory doctors (FY with 25 years of experience and XW with 34 years of experience). All Digital Imaging and Communications in Medicine (DICOM) images from the CT studies were analyzed without access to clinical or laboratory ndings. The evaluators independently and freely assessed the CT features using both axial CT images and multiplanar reconstruction images. After separate evaluations, any disagreements in the results were resolved by discussion and consensus.

Statistics
Statistical analysis was performed using SPSS Software version 25.0 for Windows (SPSS Inc., Chicago, IL, USA). Descriptive analyses were conducted for demographic, clinical, and laboratory data.

Results
Characteristics of sex, age and the days from onset of symptoms to rst hospital admission We did not nd a difference in morbidity between men and women, as in previous reports [6][7][8], which is also different from that in MERS-CoV [9]. However, our result is consistent with 50466 case analyses from Chinese Center for Disease Control and Prevention [2]. The average age of the patients was approximately 60 years old in both men and women. The variation of the days from onset to rst admission was quite large, usually an early onset of symptoms with a delay in hospital admission, which represented the shortage of medical resources during late January and early February. The shortage was relieved very quickly as many referral hospitals were established, and the incidence of disease also decreased. Noticeably, the median time from onset of symptoms to hospital admission was signi cantly longer for women (Table 1). An analysis of 36 families with cluster-related infection revealed that the time of onset was essentially the same for all patients (data not shown). Members of one family were often admitted to several hospitals. The main reasons for male patients being hospitalized earlier may be that women's resilience to symptoms was higher than in men, and men had more severe disease and more comorbidities [10]. Most of the patients with severe and critical disease were elderly-over 60 years old.
Because patients with mild disease were admitted to the shelter hospital, the patients in this group all had moderate, severe and critical disease based on the guidelines. Similar to previous reports, the patients with severe and critical illness were mainly elderly patients over 60 years old, and there was no difference in the sex distribution of patients ( Table 2).The clinical manifestations of COVID-19 in older patients were systemic symptoms and more severe radiological abnormalities (data not shown). Early symptoms included fewer upper respiratory symptoms and less sputum Most patients in this group had been treated in the outpatient department several times, and their symptoms at the time of onset were recorded. As shown in Fig. 1, fever, cough and fatigue were the three main symptoms at onset, while the incidence of upper respiratory symptoms (nasal obstruction 1.34%, runny nose 2.34%, and sore throat 7.67%) was lower than the incidence of diarrhea 15.67%. In all 300 cases, more than 71% of the patients had fever, 58.3% of the patients had asthenia, and many patients had chills, but headache, dizziness, muscle pain, chills and other symptoms were rare. The fever type was classi ed as uncertain, low, medium and high. Most of the patients had dry cough symptoms in the early stage, occasionally accompanied by expectoration. Hemoptysis was rare.
Abnormalities in laboratory data occurred in some nonhypoxemic patients The laboratory data of the patients at admission, including routine blood examination, C-reactive protein (CRP), liver and kidney function, coagulation and other indicators, are presented in Table 3. We also found that the lymphocyte count in patients was related to the severity of the disease. A lymphocyte count less than 0.8 × 10 9 /L was more likely to cause critical illness (Fig. 2). Moreover, thrombocytopenia, reduced renal function, hypoalbuminemia, elevated D-dimer and elevated CRP, creatine kinase and αhydroxybutyrate dehydrogenase were also found in COVID-19 patients and were linked to progressive infection (Table 3). It is very interesting to note that there were abnormal myocardial enzyme spectra, liver enzyme spectra and coagulation markers in moderately ill patients who did not have hypoxemia. Subpleural lesions were typical manifestations on lung CT in the early stage of disease According to the analysis of the patients' rst lung CT examinations, most were performed within 5 days after onset, and the images were characterized by single or multiple subpleural ground-glass opacities, consolidation, and involvement of less than 50% of the lung. After admission, the lung CT ndings showed that most of the patients had reticulation, and a few patients had signi cantly increased in ltrative shadows, even involving the whole lung (data not shown). Moreover, we observed a relationship between lymphocyte counts and lung CT imaging ndings, as shown in Fig. 3. The lymphocyte count was negatively correlated with lung injury(R = 0.426).

Discussion
In this study, we retrospectively analyzed the early clinical manifestations of 300 hospitalized COVID-19 patients. The main impressions of the early clinical manifestations are that there is no sex bias in the incidence; that rare upper respiratory symptoms, age and the lymphocyte counts relate to disease severity, peripheral pulmonary involvement and lymphopenia; and that multisystem damage occurs in some moderately ill patients without hypoxia. Although many papers have been published recently, the concrete pathogenesis of COVID- 19 has not yet been identi ed. What could we learn from the early clinical features of COVID-19? Clinical features provide many clues for pathogenesis.
The mode of transmission of the virus is thought to be largely by inhalation of respiratory droplets. Did the virus in the affected lung lesion come from airway spread? Although the nasopharynx mucosa was believed as rst infected with SARS-CoV-2, the symptoms involving the upper respiratory tract occur less often, which is consistent with previous reports [7,[11][12][13] and indicates that the upper respiratory tract in ammation was mild. Moreover, the distribution of lung lesions was characterized by subpleural and lateral zones in the early stage, which suggested that the infection did not spread along the airway.
Transmission of SARS-CoV-2 through contaminated surfaces might be possible [14], while aerosols are currently not considered the primary mode of transmission [15]. The above points suggested that the lung lesion may be infected as a result of SARS-CoV-2 moving along lymphatics or blood vessels, as well as invading other target tissues. We propose that the virus might pass through the mucous membranes of the nasal passages, larynx or skin and then enter the blood, causing viremia. The virus attacks the targets, including the lungs and T lymphocytes. It is very interesting to nd that the virus could be detected in the upper respiratory tract with no symptoms and mild in ammation within the bronchi and bronchioles, along with prominent mucosal edema within the bronchial mucosa [16]. It seems that the upper respiratory tract, trachea, bronchi and bronchioles were not the targets of the virus, although their mucous membrane expressed angiotensin converting enzyme II (ACE2).
The early pathogenesis of COVID-19 is still unclear. The primary cells affected by SARS-CoV-2 may be both alveolar cells and T lymphocytes. Apoptosis could be the main early event. The rst evidence comes from clinical studies. The lymphocyte count is negatively related to disease severity and is commonly considered a marker of prognosis [2,17,18]. This phenomenon also suggests that alveolar cells and T lymphocytes are both targets of SARS-CoV-2, and the T lymphocytes count could re ect the alveolar cell counts. The main evidence should be derived from the histopathologic features: diffuse alveolar damage (DAD) [19] and T-lymphocyte apoptosis in lymphoid organs, especially the spleen [20,21]. This is similar to MERS-CoV that can e ciently infect T cells from the peripheral blood and from human lymphoid organs and induce apoptosis in T cells, which involves the activation of both the extrinsic and intrinsic apoptosis pathways [22]. The number of CD4-positive T cells and CD8-positive T cells in the spleen and lymph nodes decreased, they were degenerated and necrotic, and macrophages proliferated in the spleen.
The immune response may explain the early clinical manifestations. The initial symptoms are mainly fever, cough, andlymphopenia. Increased values of liver enzymes, lactate dehydrogenase (LDH), muscle enzymes, coagulation factors and C-reactive protein can be found in some mildly ill patients without hypoxia in the early stage, which means that these abnormal changes were not secondary damage from hypoxia or secondary infection. The possible pathogenesis may be damage from the virus directly or by immune response. The possibility of an immune response should be greater than the direct invasion of the virus. The most direct evidence is the result of autopsy. No SARS-CoV-2 was detected in the heart, liver, kidney, spleen, pancreas, or gastrointestinal tract by immunohistochemistry and PCR [23]. Transcriptome sequencing of RNA isolated from bronchoalveolar lavage uid (BALF) and peripheral blood mononuclear cell (PBMC) specimens of COVID-19 patients revealed distinct host in ammatory cytokine pro les to SARS-CoV-2 infection in patients and highlighted the association between COVID-19 pathogenesis and excessive cytokine release, such as that of CCL2/MCP-1, CXCL10/IP-10, CCL3/MIP-1A, and CCL4/MIP1B [24].
It has also been hypothesized that the virus might use ACE2 as a receptor, similar to SARS-CoV [25]. ACE2 is also expressed on many different cell types, including cardiac, renal, intestinal, endothelial, and gut cells [26,27]. As no virus was found in these tissues [21], the receptor for SARS-CoV-2 has not been fully elucidated. SARS-CoV-2 may use ACE2 for entry and the serine protease TMPRSS2 for S protein priming [28].
Our study has several limitations inherent to its design. First, this was a retrospective study with the potential of incomplete and possible variation of data recording from the primary care providers. Second, patients with mild disease were not included. Third, much larger cohorts are needed.
COVID-19 is a new disease, our knowledge is gradually updated based on the ongoing research ndings and clinical practice experience. The current study is a start to our understanding of the clinical presentation and spectrum of the disease for greater precision of estimates and an initial step to performing prognostic and risk factor analyses. It was expected that the human-to-human transmission toxicity of COVID-19 would be reduced [29] because a genetic bottleneck of RNA viruses often occurs in the process of respiratory droplet transmission; however, the real world scenario is still serious. The spread of COVID-19 could not be stopped.  Lymphocyte count was associated with disease severity: The ndings are presented as lymphocyte counts and disease subtypes.