C-Reactive Protein and Lymphocyte are important indicators of severe coronavirus disease and poor prognosis in elderly patients

Objective (cid:0) To explore the value of C-reactive protein (CRP) and lymphocyte (L) in the assessment of disease severity and prognosis of elderly COVID-19 patients. Methods: A total of 194 positive COVID-19 patients were collected from Tianyou Hospital and Puren Hospital, aliated hospital of Wuhan University of Science and Technology. Their demographic characteristics were analyzed. The dynamic changes of CRP and L in peripheral blood were retrospectively studied. Results: (1) There were signicant statistical differences in CRP, L in clinical typing and clinical outcome in patients over 60 years old compared with those under 60 years old. Survival analysis showed that the risk of death was greater in patients over 60 than in those under 60. (cid:0) 2 (cid:0) In 125 patients over 60 years old, the hospitalized patients with severe or critical types had signicantly higher CRP than those with moderate type (p<0.01). In the outcome of the elderly patients, the CRP of the patients with the outcomes of discharge, improvement, aggravation and death increased successively (p<0.01). According to the analysis of Logistic regression model, the increase of CRP constitutes a risk factor for death in elderly patients. (3) In the ROC curve analysis to distinguish the death outcome and non-death outcome of COVID-19 patients, the area under the curve (AUC) of CRP and L was 0.751 and 0.720 respectively. CRP and L had good diagnostic accuracy for the death outcome of patients. (4) Changes in CRP were correlated with changes in CT imaging and were consistent with changes in the course of the disease. Conclusions: (1) COVID-19 patients over 60 years old were clinically heavy at admission and had poor prognosis, especially elderly male patients. (2) CRP and L are important monitoring indicators of COVID-19 in elderly patients. Combined with CT examination and observation of their dynamic changes, CRP and L are of important clinical guiding value for the judgment of disease severity and the evaluation of prognosis. value of CRP, L and other indicators on the death outcome was analyzed by the receiver operating characteristic curve (ROC), and the area under the curve (AUC) and its 95% CI were calculated. The optimal threshold value is determined by the maximum approximate index, providing both sensitivity and specicity. The test level selected for this statistical analysis was a=0.05, and P<0.05 indicated that the difference was statistically signicant.


Introduction
Since December 2019, novel coronavirus (SARS-CoV-2) has been identi ed as the cause of pneumonia in Wuhan, Hubei province, China [1][2] . The World Health Organization has o cially named the Disease caused by SARS-CoV-2 as Corona Virus Disease (COVID-19) [3][4] . At present, COVID -19 has been effectively controlled in China, but it is still in the global outbreak, which has become a global public health problem. SARS-CoV-2 spreads rapidly, causing a wide range of lesions with varying degrees of severity. However, the condition of some elderly patients changes rapidly, and in ammatory storms rapidly appear, leading to worsening or even life-threatening conditions [5][6][7][8] . Therefore, early diagnosis of the severity of the disease and prognosis of the elderly patients are particularly important. Absolute values of C-reactive protein (CRP) and lymphocyte (L), commonly used laboratory in ammatory indicators in clinical practice, C-reactive protein (CRP) is a protein (acute protein) that rises sharply in the plasma when the body is infected or damaged. Lymphocyte (L) count refers to the number of lymphocytes in the peripheral blood.
The normal range of lymphocyte count measured in this study is 1.1× 109 / L-3.2 × 109 / L, which is abnormal if it is lower or higher than the normal range. These indicators can assist in the diagnosis of infectious in ammation and disease severity in patients, but are they also applicable to elderly patients with COVID-19? And can we determine the early prognosis? In our previous studies [9] , we have concluded that COVID -19 patients with SAA, CRP levels increased signi cantly, L is on the decline, as the disease progression, from light, heavy to the critically ill, SAA, CRP gradually increases, while L gradually decline, but now we nd in between the elderly and young patients with the disease and prognosis of differences, therefore, this study explore the C-reactive protein and lymphocyte count in aged > 60 years COVID -19 patients with disease severity and prognosis evaluation of value. Now, we continue to increase the sample size by about 50% based on the previous study. We retrospectively analyzed the demographic characteristics of 194 COVID-19 patients in a designated COVID-19 hospital in Wuhan, China, we studied the dynamic changes of in ammatory indicators CRP and L in peripheral blood of 125 patients over 60 years old, and explored the dynamic changes of in ammatory indicators in peripheral blood of elderly patients with COVID-19, so as to determine whether it can be used to determine the severity and prognosis of COVID-19 in elderly patients. 2) Completed the laboratory data collection and clinical condition assessment at the three time points required by this study; 3) Study composite endpoint cures discharged patients and deaths before February 26, 2020; cases being treated in hospital on February 26, 2020.

Exclusion criteria
Those who failed to collect laboratory data in three times.

Criteria for evaluation of clinical conditions 1) Case classi cation
According to the COVID-19 diagnosis and treatment plan issued by the National Health Commission of China, it is divided into 4 types: mild, moderate, severe and critical. In the analysis of this study, the patients of mild type were merged into moderate type.
Mild type: mild clinical symptoms, no pneumonia on imaging Moderate type: Symptoms such as fever, respiratory tract, pneumonia can be seen on imaging.
Severe type: According with any of the following.
Critical type: One of the following conditions: (1) Have respiratory failure and require mechanical ventilation; (2) Shock; (3) Combining other organ failure requires ICU monitoring treatment.
This study mainly studied the demographic characteristics, disease severity and outcome of COVID-19 patients. To optimize the research program, we mixed light and common types into light and common types according to the research purpose and clinical practice.

2) Clinical outcome
According to the clinical characteristics, the patients were divided into four types: discharge, improvement, aggravation and death.

Treatment method
According to the COVID-19 diagnosis and treatment plan issued by the National Health Commission of China, patients generally receive effective oxygen therapy, lopinavir / ritonavir and other drug antiviral treatments, and provide respiratory and circulatory support treatment for patients with severe and critical illness. 5. Composite endpoints of the study By February 26, 2020, the number of discharge and death cases before February 26, 2020 should be counted, and the length of stay in the hospital should be more than 7 days. The changes of their conditions should be evaluated, and the outcome indicators should be improved, aggravated and stable.

Experimental data collection
The experimental data were collected at three time points: admission, 3-5 days of hospitalization, and the composite end point. At the same time, clinical typing, disease severity and outcome were determined, and the corresponding length of hospitalization was collected.
The speci c methods of blood routine test and CRP test were as follows: 1) The venous blood of the subjects was extracted in the fasting state of the patients in the morning, and the venous blood was placed in a 2ml EDTA-K2 anticoagulant tube and a 5mL procoagulant tube respectively, mixed upside-down, and sent for timely examination.
2 2ml EDTA-K2 anticoagulant whole blood samples were put into automatic hemocyte analyzer for routine blood test.
3 The 5ml procoagulant tube was put into an ordinary low-speed centrifuge and centrifuge at 3000rpm/min for 10min. After the serum was separated, the sample was put into a biochemical analyzer and the CRP level in the blood samples was detected by immunoturbidimetry.
4) The normal reference range of CRP is < 3mg/L; The normal reference range of lymphocytes is 1.1× 10 9 / L-3.2 × 10 9 / L. 7. Analytical methods SPSS25.0 software was used for statistical analysis. Non-parametric test (Mann-Whitney test) was used for quantitative data with skewed distribution and uneven variance to analyze differences between groups, and chi-square test was used for qualitative data to analyze differences between groups, if the theoretical frequency is less than 1, the Fisher exact probability test method is adopted. Survival analysis was used to determine the difference in survival time distribution of patients under different conditions during clinical observation. The risk degree of admission type and death outcome of each factor was analyzed by binary regression model and multiple regression model. The diagnostic value of CRP, L and other indicators on the death outcome was analyzed by the receiver operating characteristic curve (ROC), and the area under the curve (AUC) and its 95% CI were calculated.
The optimal threshold value is determined by the maximum approximate index, providing both sensitivity and speci city. The test level selected for this statistical analysis was a=0.05, and P<0.05 indicated that the difference was statistically signi cant.

Results
The 194 patients included in this research were divided into two groups, younger age group(<60 years old) and older age group ≥60 years old . Among them, older age group (125 patients) were more than younger age group (69 patients). The M±SD of Hospitalization days, C-reactive protein, lymphocyte count were calculated. CRP and L were grouped according to the normal reference range (CRP: <3 mg/L, 3-100mg/L, >100mg/L; L: 1.1-3.2 × 10 9 / L,<1.1× 10 9 / L,>3.2× 109 / L). For quantitative data, the average of Hospitalization days in the two age groups was 20.71 days and 20.27 days, respectively. The average CRP level of older age group is higher than younger age group, with mean values of 55.06 and 39.59. The average L level of younger age group is closer to the normal reference range than older age group. In the classified data, 90.21% of patients were above the normal level of CRP and 34.54% of patients had L levels deviated from the normal range (See Table 1). In Figure 1, patients were divided into moderate, severe & critical groups. As can be seen from Figure 1, the mean values of L and CRP in older age group were lower than younger age group in the Moderate group. In the Severe & Critical group, the mean CRP of older age group was higher than younger age group, and the mean L of older age group was lower than younger age group. Mann-whitney test was used to analyze the differences of CRP and L levels in patients of different ages in the two groups. In Figure 1A, Patients of different ages with Moderate clinical classification showed no significant difference in CRP (p > 0.05), but patients of different ages with Severe & critical clinical classification showed significant statistical difference in CRP (p < 0.01). In Figure   1B, there were significant statistical differences in L levels between younger age group and older age group in the Moderate and Severe & Critical groups (P <0.05).
In Figure 2, the Kaplan-Meier method of survival analysis was used to describe the cumulative survival rate at the end of events during hospitalization. With the increase of observation time in hospital for all patients, the survival rate curve gradually showed a step-like decline from 1-0. During in-hospital clinical observation, older age group had earlier death events compared with younger age group. The log-rank test showed that P <0.05(P=0.013).
The survival rate of patients in the two groups was significantly different and the difference was statistically significant, so the risk of death in older age group was greater than younger age group.
In Therefore, 125 samples of elderly patients (over 60 years old) were extracted in this study and the relationship between characteristics of elderly patients (demographic characteristics, clinical indicator characteristics) and clinical classification and clinical outcome was studied.  In the binary Logistic regression analysis of whether the elderly patients had severe or critical clinical type, we used univariate Logistic regression analysis to preliminarily determine the factors affecting clinical type and the degree of risk, and the results showed that CRP, L level had a significant impact on the clinical type of the patients. For each unit increase of CRP index, the risk of disease severity was increased 1.022 times, and the 95% interval of OR value was greater than 1, which constitute the conditions of risk factors. For every unit increase in L level, the risk of disease severity increased by 0.359 times. In order to correct the effects of confounding factors, we will be more variable into the multi-factor Logistic regression analysis, the results showed that CRP index for every rise in unit, 1.019 times the risk of disease severity ascension, OR 95% of the value range are greater than 1, constitute the conditions of the dangerous factors, L level for every rise in unit, the risk of disease severity increase 0.478 times. (See Table 4) In the multivariate Logistic regression analysis of hospitalization days, gender, CRP, L and clinical outcomes of elderly patients, the elderly patients discharged from hospital were compared with elderly patients who died when each unit increase in CRP, the risk of death in discharged patients was 0.981 times that of those who died.
Compared with the elderly patients who died, the increased length of stay in hospital constituted the risk factor for death, and the OR value was 1.330, 1.396 and 1.377. (See Table 5) In the analysis of the prognostic diagnostic value of CRP and L on death outcome in elderly patients, we used non-death outcome and death outcome as the basis for positive classification. With AUC=0.5 as the null hypothesis, the significance of CRP and L in ROC curve analysis was less than 0.05. It can be seen in the ROC curve that both CRP and L have good diagnostic value for death outcomes. The area under the curve (AUC) of CRP is greater than 0.7, that is 0.751, and the area under the curve (AUC) of L is 0.720. The optimal cutoff values for CRP and L as indicators to determine the outcome of death were calculated using the maximum but over activation can cause in ammatory storms [10][11] . The onset of the disease is mainly fever, most of which are light, and a few are critical [12][13][14] . Some patients may gradually develop dyspnea. In severe cases, the disease progresses rapidly, and severe in ammatory storms may occur, leading to death, especially in elderly patients.
COVID-19 is a newly discovered human infectious disease [15] . The commonly used clinical in ammatory indicators such as CRP and L are helpful in the diagnosis and evaluation of many in ammatory diseases. CRP is an acute temporal protein synthesized by liver cells in response to in ammatory stimuli, such as microbial invasion or tissue damage, in very low concentrations in serum from healthy people (<5mg/L), and its concentration signi cantly increased during bacterial infection or tissue damage [16] . L is the core of immune response, which can be divided into three types: T cells, B cells and NK cells. Chaolin H et al. believed that patients with COVID-19 had a large amount of IL-1β IFN-γ IP-10 MCP-1 and other cytokines release, which may lead to Th1 cell activation [17] . CRP and L can be used as sensitive indicators to re ect the control of infection and in ammation. But can these indicators have the same clinical value for COVID-19 patients, especially elderly patients?
The results of this study showed that there was a signi cant statistical difference between different age groups and outcome of COVID-19 patients, and elderly patients (over 60 years old) were a highly vulnerable group. From the 194 con rmed cases, we extracted 125 samples from the elderly population (over 60 years old), and made a targeted study on the relationship between the demographic and clinical characteristics of the elderly population and admission classi cation, outcome, and death outcome. The relationship between CRP and admission clinical typing in elderly patients was explored by using bilateral chi-square test or Fisher's exact probabilistic test. The results showed that there was a signi cant difference between CRP index and clinical typing in the elderly, suggesting that CRP, a laboratory in ammatory index, could be used to assist in determining the severity of COVID-19 in elderly patients.
Furthermore, binary Logistic regression analysis was carried out to investigate whether the elderly patients died or not. The study found that compared with the elderly patients who died, the risk of death was 0.981 times for each unit of decline in CRP, and the 95% interval of OR value was all less than 1. In the diagnostic relationship between CRP, L and death outcome in elderly patients, we used non-death outcome and death outcome as the basis for positive classi cation. We took AUC=0.5 as the null hypothesis, and the signi cance of CRP and L in ROC curve analysis was less than 0.05. It can be seen from the ROC curve that both CRP and L have good diagnostic values for death outcomes, and the optimal thresholds for L and CRP diagnostic death outcomes were determined by the maximum Youden index, which were 91.5 and 0.615, respectively.
CT is very important in the clinical diagnosis and typing of COVID-19. The results of this study showed that CRP was consistent with CT in judging the changes of the disease, and it could be used in combination to judge the changes of the disease.
To sum up, the results of this clinical study showed that COVID-19 patients over 60 years old had severe clinical classi cation and poor prognosis. CRP and L can be used as the key monitoring indicators of COVID-19 in elderly patients. Combined with CT examination and observation of its dynamic changes, CRP and L can play an important clinical guiding value in the assessment of disease severity and prognosis.

Declarations
Ethics approval and consent to participate Because this study was a retrospective analysis and all the data were anonymous, we did not involve patients' private information. This study was approved by the Medical Ethics Review Board of Wuhan University of Science and Technology (No. 202009).

Consent for publication
After revision and review by all authors, all authors nally agree to publish.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding: No funding
Authors' contributions QH, XFH, XCX, and SYC contributed equally to this article, they are co-rst author. The whole experimental design was completed under the guidance of professor QW and professor QMW. QH and XFH were responsible for the arrangement of data, XCX and SYC are responsible for analyzing the data, KWG and XLL participated in the interpretation of the results, QW and WXL wrote the initial draft with all authors providing critical feedback and edits to subsequent revisions. QW, KWG and QMW reviewed and revised the paper before submission, they are co-corresponding authors.

Figure 1
Analysis of differences in clinical typing between patients aged ≥60 years and < 60 years Survival analysis among patients aged ≥60 years and < 60 years The prognostic value of CRP and L in elderly patients with COVID-19 Figure 4 (A male patient aged over 60 years): serial CT scans and CRP dynamic changes