A Comprehensive Evaluation of Early Predictors of Disease Progression in Patients with COVID-19: A Case Control Study


 Background: The 2019 coronavirus disease (COVID-19) has become an unprecedented public health crisis with nearly 16 million confirmed cases and 630,000 deaths worldwide. Methods: We retrospectively investigated the demographic, clinical, laboratory, radiological and treatment data of COVID-19 patients consecutively enrolled from January 18 to May 15, 2020, in Taihe and Jinzhou central hospital. Results: Of all 197 patients, the median age was 66.5 years (IQR 7-76), and 120 (60.9%) patients were males. We identified 88 (44.7%) of 197 COVID-19 patients as the disease progression (aggravation) cases. The aggravation cases tend to have more medical comorbidity: hypertension (34.1%), diabetes (30.7%), and presented with dyspnea (34.1%), neutrophilia (60.2%), and lymphocytopenia (73.9%), compared with those without. And the patients with disease progression showed significantly higher level of Fibrinogen (Fbg), D-dimer, IL-6, C-reactive protein (CRP), procalcitonin (PCT), and serum ferritin, and were more prone to develop organ damage in the liver, kidney, and heart (P<0.05). Multivariable regression showed that advanced age, comorbidities, lymphopenia, and elevated level of Fbg, lactate dehydrogenase (LDH), Cardiac troponin (CTnI), IL-6, serum ferritin were the significant predictors of disease progression. Further, we investigated antibody responses to SARS-CoV-2 and found that the levels of IgM and IgG were significantly higher in the disease progression cases compared to non-progression cases from 3 weeks after symptom onset. In addition, the disease progression group tended to peak later and has a more vigorous IgM/IgG response against SARS-CoV-2. Further, we performed Kaplan-Meier analysis and found that 61.6% of patients had not experienced ICU transfer or survival from hospital within 25 days from admission.Conclusions: Investigating the potential factors of advanced age, comorbidities and elevated level of IL-6, serum ferritin and Kaplan-Meier analysis enables early identification and management of patients with poor prognosis. Detection of the dynamic antibody may offer vital clinical information during the course of SARS-CoV-2 and provide prognostic value for patients infection.


Introduction
In December 2019, the outbreak of unidenti ed pneumonia has aroused great attention all over the world, which is not only an epidemic but even a disaster (1). On January 27 of 2020, the World Health Organization (WHO) issued worldwide surveillance and vigilance of the highly contagious respiratory diseases and formulated the rst edition of prevention and control strategies (2). Sequence analysis of the coronavirus from lower respiratory tract samples has shown a structure typical to that of other coronaviruses such as SARS coronavirus and MERS coronavirus (3,4). And then the WHO named the unidenti ed Coronavirus as SARS-CoV-2 and the pneumonia of unknown origin as COVID-19 (5,6). SARS-CoV-2 belongs to a unique clade of the sarbecovirus subgenus of the Orthocoronavirinae subfamily (7,8).
Evidence has been found that SARS-CoV-2 is extremely contagious to humans, and could be transmitted through respiratory droplets, contact, and even via fecal-oral transmission (9).
The clinical spectrum of SARS-CoV-2 pneumonia ranges from mild to critically ill cases. As reported in the literature, patients with COVID-19 mainly presented with fever, cough, fatigue, myalgia, dyspnea (10). Most patients represented various degrees of abnormality on imaging, and the moderate and severe phenotypes were always associated with pronounced imaging abnormalities (9,11). Several studies have shown that the majority of patients were considered to have a favorable prognosis, however, elderly men and those with underlying diseases including hypertension, diabetes, chronic obstructive pulmonary disease (COPD), had a higher risk of developing acute respiratory distress syndrome (ARDS), which may be the leading cause of death (12,13). Therefore, investigate the risk factors associated with disease progression are therefore greatly warranted. In this study, we investigated the clinical characteristics and relevant factors associated with the outcomes of patients with SARS-CoV-2 infection, which may provide considerable value for the early identi cation of individuals who are at risk of disease progression and who are most likely to bene t from intensive care treatment. We believe our ndings will give further details to the epidemic situation and clinical characteristics of this novel coronavirus.

Study participants and design
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Taihe and Jinzhou central hospital, and all participants provided written informed consent before data were collected retrospectively. All of the patients were consecutively enrolled from January 18 to May 15, 2020, in Taihe and Jinzhou central hospital, who had been con rmed with COVID-19 infection according to World Health Organization interim guidance.

Data collection
We reviewed the clinical medical records of all patients to collect the demographic data and clinical and laboratory ndings of the patients. All data including age, sex, occupation, symptoms (fever, fatigue, dry cough, myalgia, dyspnea, etc.), underlying comorbidities (COPD, cancer, hypertension, and diabetes), laboratory results (complete blood cell counts, creatinine, liver function tests, and in ammatory markers), treatment, and the outcomes data were retrospectively extracted with standardized data collection forms. If the variable were not available from electronic medical records or needed further clari cation, we would clarify the details with the attending doctors and other health care providers. Clinical outcomes were followed up to Mar 15, 2020. To identify the con rmed case, nasal and pharyngeal swab samples were obtained from patients at admission and were tested using the real-time reverse transcriptase polymerase-chain-reaction (RT-PCR). The exact date of onset was de ned as the day when the symptom was noticed. A weekly assessment of disease progression was performed that provided details about the patients' clinical status. All study participants were divided into the disease progression group and non-progression group based on the speci c criteria: (1). Progression group (aggravation): a higher body temperature than before, aggravated symptoms, and varied progression of imaging examination manifestation; (2). Non-progression group (non-aggravation): body temperature decreasing, symptom and imaging improvement, or no signi cant change in body temperature, respiratory symptoms, and imaging manifestation. ARDS and shock were de ned according to the interim guidance of WHO for the novel coronavirus. Acute kidney injury was identi ed and classi ed based on the highest serum creatinine level or urine output criteria according to the kidney disease improving global outcomes classi cation. The cardiac injury was diagnosed if serum levels of cardiac biomarkers (e.g. Troponin I) were above the 99th percentile upper reference limit, or new abnormalities were shown in electrocardiography and echocardiography. Finally, a total of 197 subjects were enrolled in the study and divided into two groups: 88 cases with disease progression and 109 cases without disease progression.

Statistical analysis
Categorical variables were expressed as frequencies and numbers (%) and analyzed using the Chisquared test or Fisher's exact test. The continuous variables were presented as median or interquartile ranges (IQR) values and Two-sided unpaired t-test or Mann-Whitney test was used as appropriate to compare groups. All statistical analyses and graphs were generated and plotted using GraphPad Prism (version 7.0) and SPSS (Statistical Package for the Social Sciences version 21.0). A con dence level of p < 0.05 was considered statistically signi cant for all analyses.

Demographic characteristics
In this study, a total of 197 patients with COVID-19 were con rmed from Taihe Hospital and Jinzhou central hospital. The demographic and clinical characteristics are shown in Table 1. The median age of the patients was 66.5 years (IQR 7-76), and 97 (49.2%) patients were older than 60 years. 120 (60.9%) patients were males. And less than 20% of patients had a smoking history. Of the 197 patients, 72 (36.5%) had at least one underlying chronic diseases, including hypertension (25.9%), diabetes (20.8%), COPD (18.8%), cardiovascular disease (10.2%), cerebrovascular diseases (9.1%), malignancy (8.1%), chronic kidney disease (7.6%), chronic liver disease (10.2%), HIV infection (4.6%). Further, COVID-19 patients with ACEI/ARB therapy were enrolled, and we found that about 50.7% of patients had taken ACEI/ARB in the progression group, which is much higher than that of the control group (25%). In addition, of these patients, 36 (18.3%) were infected medical staff. Signs and symptoms of the patients on admission are shown in Table 1. The most common symptoms at the onset of illness were fever (81.5%), dry cough (79.7%), followed by fatigue (63.5%), chest distress (38.6%), dyspnea (23.4%). The majority of patients (76.6%) had both fever and cough. 89 (45.3%) had a fever with fatigue, and 72 (36.8%) patients had a fever with dyspnea; The less common symptoms were rhinorrhea (4.6%), runny nose (7.6%), and chest pain (7.6%). Notably, we found that 24 (12.7%) patients represented at least one gastrointestinal symptom including abdominal pain (9.6%) nausea or vomiting (11.2%). Besides, 11 (5.6%) patients were clinically characterized by conjunctival hyperemia. Though, these digestive and ocular symptoms were less common in COVID-19 patients, special attention should be paid to the care of this unique group of patients. The median time from onset to hospital admission was 5.0 days (3.0-8.0).

Laboratory ndings
The laboratory ndings of the con rmed patients are summarized in Table 2  There existed many differences in laboratory parameters between patients with disease progression and non-progression patients in Table 3. In the aggravation group, the total lymphocyte was signi cantly decreased in comparison with the non-aggravation group (P = 0.001). The cell count of the lymphocyte subtype was then further analyzed, which revealed that the count of CD3 + , CD4 + and CD8 + T cell in aggravation group was signi cantly lower than that of the non-aggravation group (P < 0.05), but there was no difference in the counts of CD16 + and CD19 + cells (P > 0.05). We also found that the number of neutrophils was notably increased in the patients of aggravation group (P < 0.001). Blood biochemical examination results suggested that patients with disease progression demonstrated higher levels of AST, Albumin, BUN, Creatinine, CK-MB, CTnI, ProBNP, LDH. At present, some researchers reported that the coagulation function would be dysregulation with the aggravation of the disease. We concluded that the levels of D-dimer, and Fbg were remarkably elevated in the aggravation patients. Table 3. also revealed the differences in in ammation immunologic related indices between the two groups. It suggested that the level of CRP, serum ferritin, procalcitonin, and IL-6 were signi cantly higher in the aggravation group. patch shadow with interstitial involvement. Compared with the patients of non-aggravation, the aggravation group showed more bilateral ground-glass opacity and subsegmental areas of consolidation as well as the lung interstitial involvement. Most of the lesions were localized in the periphery then the center follows and the last was both the periphery and center of the lung (105, 53.3% vs 18, 9.1% vs 74, 37.6%). In the non-aggravation patients, the lesions were more localized in the periphery rather than the center of the lung. However, the lesions would spread to the center of bronchus and gradually to the whole lung in aggravation patients. Therefore, the lesion was more likely located in both the periphery and center of the lung in the disease progression group (  Nucleic acid testing is the standard method for the diagnosis of COVID-19 infections. However, some research suggested that this method usually showed lower positive rates due to poor RNA stability, different specimen position, and quality. Therefore, the IgM-IgG combined assay was recommended to increase the sensitivity of COVID-19 diagnoses especially in patients with suspected SARS-CoV-2 infection. In this study, the dynamic changes of IgM-IgG antibody levels were detected. The speci c IgG and IgM antibodies can be detected 4-7 days after onset of illness. The IgM antibody titers increased sharply and notably in the initial two weeks and peaked at 1-2 week after symptom onset and signi cantly declined after 21 days. The IgG antibodies titers increased over time peaking at 4-5 week after onset of illness, and then maintained higher levels for the whole observation period (Fig. 1a). In addition, we compared the IgM-IgG levels between the two groups and found that there was no difference in the levels of IgM between the aggravation group and the non-aggravation group during the rst two weeks. After that, the aggravation group tended to have a more vigorous IgM response against SARS-CoV-2 and displayed a higher peak, which suggested that serum IgM antibody levels were signi cantly correlated with disease progression from day 3-week onward (Fig. 1b). However, the levels of IgG in aggravation group were markedly lower compared to the non-aggregation group in the early stage of infection, and then it experienced a rapid growth and exceed those of the non-aggravation group Fig. 1c.

Treatments and clinical outcomes
All of the con rmed patients were isolated and treated in a negative pressure ward with applicable protective equipment.   (25, 14%). And patients with disease progression showed higher complication rates than that of the patients without Table 5.

Risk factors for disease progression
To investigate the risk factors for disease progression, we compared the epidemiological, clinical characteristics, laboratory, and radiological ndings of COVID-19 patients between the two groups. In univariate logistic regression analysis, we found that the older age, male, underlying diseases (hypertension, diabetes), dyspnea, chest tightness were more frequently administered to patients in the progression group. It revealed that lymphopenia, neutrophilia, high levels of AST, lactic acid, Urea, Creatinine kinase, LDH, CTnI, ProBNP, Fbg, D-dimer, Serum ferritin, Procalcitonin, IL-6 were all signi cantly correlated with disease progression. Then, these variables were included in the multivariable logistic regression model, which indicated that older age, hypertension, diabetes, dyspnea, lymphocytes, Fbg, LDH, CTnI, serum ferritin, IL-6 were all independently associated with disease progression Table 6.

Discussion
The present study included a total of 197 patients who were hospitalized with COVID-19 from Jan 25, 2020, to Mar 15, 2020. All of the patients were evaluated for therapeutic e cacy after at least one week relatively high proportion of severe and disease progression cases (14)(15)(16)(17)(18). Consistent with previous studies, males were identi ed more likely to be contamination with the virus (39.1%women versus 60.9% men), and have severe progression (60.3%). Fewer cases have been identi ed among children and infants. Our study showed that advanced age is the independent risk factor for disease progression.
Of all patients, 72 (36.5%) cases had one or more underlying diseases including diabetes, hypertension, COPD. The logistic regression analysis showed a signi cantly increased number of patients with diabetes, hypertension in disease progression groups than that of the non-progression group. It has been reported that patients with hypertension or diabetes accounted for 20-30% of total infected patients and comprised of over half of patients in ICU (19)(20)(21). Recently, a retrospective cohort study also demonstrated that these comorbidities have been responsible for 60.9% of deaths caused by COVID-19 (22). A large number of studies have indicated that the Angiotensin-converting enzyme 2 (ACE2) receptor is highly expressed in the cardiovascular/cerebrovascular and lung tissue in the hypertension patients (23). In addition, ACE2 is one of the most important host receptors of H7N9, SARS and COVID-19, whose activity is closely related to the pathogenesis of in ammatory and acute injury of lung disease caused by these viruses (24)(25)(26). Given that experimental studies suggested that the spikes of SARS-CoV-2 could bind to the ACE2 receptors, which mediated virus entry into of HeLa cells and viral replication (22,27).
Some scholars have found that most of COVID-19 patients showed higher activity of angiotensin II compared to the uninfected people, and the abnormal increase in angiotensin II was related to lung failure and death (28,29). ACEIs and ARBs are recommended for the management of hypertension and renal disease associated with diabetes (30). Recent studies have reported that the application of ACE inhibitor could induce a marked increase expression of ACE2 expression, which means that ACEIs/ ARBs would increase the risk of infection of SARS-CoV-2 and disease progression of COVID-19 in hypertension and diabetes patients receiving these drugs (31,32). In the present study, COVID-19 patients with ACEI/ARB therapy were enrolled, and we found that there are more patients taking ACEI/ARB in the progression group. Therefore, taking ACEI/ARB may be another potential risk factor for disease progression of COVID-19.
In our cohort, no signi cant difference in the median days from symptom onset to hospital admission was found between the disease progression and non-progression patients. Consistent with the symptom reported before (20,33), the most common symptoms are fever, cough, fatigue, chest tightness, and myalgia or arthralgia. The proportion of patients with myalgia or arthralgia, chest distress and dyspnea was signi cantly higher in the disease progression group. Remarkably, few patients exhibited some less common symptomatology including abdominal pain, vomiting, and conjunctival hyperemia, which may result in missed diagnosis and transmission to other people. Previous studies found that virus could be detected in stool samples in the patients with symptoms of abdominal pain, vomiting, or asymptomatic (34,35). Therefore, further research is still required to determine whether fecal oral transmission exists. In our study, no signi cant difference was identi ed between the two groups regarding this uncommon symptomatology.
SARS-CoV-2 induced immune responses and infection cytokine storms are believed to play major roles in disease progression (36,37). The destruction of lung cells recruit macrophages and monocytes, trigger the adaptive T and B cell immune responses, and release cytokines to resolve the infection and even mediate widespread excessive in ammation at the same time (38,39). In the present study, 92 (46.7%) patients showed signi cantly neutrophilia and lymphopenia with pronounced reduction of peripheral blood CD3 + , CD4 + , and CD8 + cells. And, the lymphocyte subsets, especially CD3 + , CD4 + , impaired more severely in the disease progression patients. ,This is consistent with the previous study by Qin et al, which demonstrated that the percentage of memory, regulatory and effector T cells were signi cantly decreased in severe cases when compared to non-severe cases (38,40). Previous studies have shown that serum in ammation-related indices were closely related to the degree of in ammation and disease severity (41).
In this study, compared with patients without disease progression, the disease progression patients showed signi cantly increased expression of in ammation-related factors including IL6, CRP, serum ferritin, procalcitonin. And the multivariate analysis revealed that the elevated level of IL6, serum ferritin, were the risk factors of disease progression patients.
It is previously established that patients with severe SARS and MERS had a higher incidence of multiple organ dysfunction syndromes including liver damage, acute heart/kidney injury, coagulation dysfunction, and even death (42,43). And a lot of research has shown the clinical characteristics and laboratory ndings associated with different degrees of multiple organs in patients with COVID-19 (17,42,44). In our study, liver damage and acute heart/kidney injury had been considered to occur in more than a third of patients. The level of AST, ALT, direct bilirubin, LDH, Creatinine, BUN, CTnI, ProBNP seemed to be signi cantly higher in patients with disease progression. The multivariate analysis revealed that the elevated level of LDH, CTnI, ProBNP were the risk factors for patients with disease progression. Presently, there are three potential mechanisms for this observation: rstly, SARS-CoV-2 binding to ACE2 positive cells mediated direct damage; Secondly, systemic in ammatory response syndrome including cytokine storm, dysregulated immunocytes, and uncontrolled in ammation; Thirdly, exogenous drugs induced organ metabolizing burden, or worsening organ function (45,46). Recently, the coagulation dysfunction has attracted more and more attention among scholars. It has been reported that the incidence of coagulopathy in all patients showed abnormalities of varying degrees in coagulation function parameters. A previous study revealed that Fbg and D-dimer elevation were related to the severity of the disease (47). And Ji et al. demonstrated that coagulation activation and hyper-brinolysis were coexistent in patients with severe COVID-19 infection (48). And patients with elevated plasmin and Fbg may have an increased risk of ARDS and mortality (49). In this study, we found that the incidence of abnormalities in coagulation function parameters (APTT, Fbg, D-dimer) was higher in patients with disease progression compared to the improved patients. Further analysis revealed that the elevated level of Fbg was signi cantly associated with the outcome. Therefore, measurements of these coagulation function parameters may be important biomarkers of disease progression and outcome.
Until now, no specialized antiviral treatment has been identi ed for COVID-19 infection, except for meticulous supportive care. Currently available treatment approaches include symptomatic and supportive therapies, such as oxygen therapy, antivirals, prevention and treatment of infections, and combination treatment with glucocorticoids (50,51). Prior studies have suggested that patients with SARS-CoV would bene t from the combination of lopinavir and ritonavir with fewer adverse clinical outcomes (52,53). And some preclinical research suggested that remdesivir have broad-spectrum antiviral activity for MERS-CoV and SARS-CoV1/2 infections (54). However, further studies on a larger set of clinical specimens will be required to assess the e cacy and safety of antiviral drugs (52,55,56). Due to the cytokine storm associated with COVID-19, corticosteroids were widely used in the treatment of patients with severe illness to attenuate in ammation associated with lung injury. Nevertheless, some researchers refuted that the use of glucocorticoids did not reduce mortality, but could easily lead to the disease progression and increase the risk of secondary infections (57). Therefore, the rational use of an appropriate dose of glucocorticoids could suppress the excessive activation of immune system and secretion of in ammatory cytokines. In our study, most patients received combination therapy with oxygen support, antibiotics, antiviral, and glucocorticoids. Almost of patients received respiratory support including nasal cannula oxygen and continuous positive air pressure. The progression group was signi cantly more likely to receive higher levels of respiratory support. Without a doubt, patients with disease progression have a higher mortality rate than patients without disease progression. And ARDS remained the most common cause of death, followed by multiple organ failure. Therefore, the prevention and treatment of ARDS represent an important strategic objective for the reduction of mortality and morbidity.

Limitations
The study also has severe important limitations. Firstly, the inherent shortcomings belong to a retrospective observational study, small sample size and short term follow-up make it di cult to reach a rm conclusion. Secondly, our institution was only the speci ed hospital for severe patients during the early outbreak, which could have resulted in some selection bias. Therefore, a larger cohort study of patients from China and other countries may help to further investigate the clinical characteristics and risk factors for the outcome. Finally, the actual duration of viral clearance was overrated owing to the frequency of respiratory specimen collection. In addition, the viral load of the SARS-CoV-2 was not quanti ed accurately, and false-negative results for an upper respiratory sample have also been reported.
Therefore, studies on the dynamic characteristics of the viral load are still warranted.

Conclusions
The outbreak of 2019 coronavirus disease (COVID-19) has become an unprecedented global health crisis with over 6 million con rmed cases and 380,000 deaths worldwide. Investigating the potential factors of advanced age, comorbidities and elevated level of IL-6, serum ferritin enables early identi cation of patients with poor prognosis. Detection of the dynamic antibody may offer vital clinical information during the course of SARS-CoV-2 and provide prognostic value or even foreseeable therapeutic options for patients infection. However, a larger cohort study of patients from China and other countries may help to further investigate the clinical characteristics and risk factors for the outcome.

Declarations
Ethics approval and consent to participate: All participants provided written informed consent before data were collected retrospectively.
Consent for publication: Not applicable.
Availability of data and materials: This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Taihe and Jinzhou central hospital, and all participants provided written informed consent before data were collected retrospectively.