Analysis of functional indices indicates multiple organ function damage in patients with severe Coronavirus Disease 2019 (COVID-19) pneumonia

Background: This study aims to analyze the changes and signicance of organ function indices in patients with severe Coronavirus Disease 2019 (COVID-19) pneumonia for prediction of major organ damages and guiding treatment schemes. Methods: 63 patients with severe COVID-19 pneumonia were selected as the severe group and 73 patients with mild syndromes were selected as the mild group. SAS9.4 software was used for statistical analysis of the data. Results: Levels of ALT, AST, cTnI, Cr, PT, APTT and D-DIC of the severe group were signicantly higher while PLT was lower than those of the mild group. The data of all quantitative variables were converted into categorical variables. Signicantly higher levels of AST, ALB, D-DIC and higher proportion of bilateral lung involvement were observed from the severe group comparing to those in the mild group, while the difference in the other indices between the two groups was insignicant in statistical perspective. Discussion: There are signicant differences in the levels of multiple organ function indices between the severe group and the mild group of patients with COVID-19 pneumonia infection. Through examining the relevant indices, conditions of patients’ multiple organ function damage could be predicted and used as guidance of treatment.


Introduction
Since the start of the outbreak of Coronavirus Disease 2019 (COVID-19) infection from Dec 2019 in Wuhan, China, it now becomes a worldwide pandemic and affects almost everyone in the world. According to the latest statistics of National Health Commission of the People's Republic of China, there were 84522 con rmed cases and 4645 deaths being reported in China on 24 May 2020. In total, there were more than 5.1 millions con rmed cases and 333401 deaths reported from 216 countries, areas or territories in the worldwide as released by World Health Organization on 24 May 2020. (www.who.int) COVID-19 is highly infectious with high speed of transmission and high mortality rate. [1] Its routes of transmission from person to person include aerosol, fecal-oral, ocular surface and secretions and etc. [1][2][3] It has been reported that mortality rate is higher in the aged patients and those with chronic underlying diseases such as diabetes and cardiovascular diseases. [4,5] Typical symptoms of COVID-19 infection include fever, coughing, muscle ache, fatigue, chest pain and etc. [6] Various studies investigated the changes in the level of blood routine indices and sequential organ failure assessment scores to assess the infection progression in the patients. [7][8][9] Moreover, SARS-CoV-2 viral infection would damage various major organs and tissues including the liver, the kidney, the heart and the blood, and is able to develop to multiple fetal organ failures leading to the death of the patients. [6,7,10,11] Deaths of COVID-19 infected kidney transplant recipients were also reported, showing the in uence of this virus on major organ functions. [12] However, current treatment measures taken towards COVID-19 infection are still mostly supportive and unspeci c. Alternative treatment measures have been tested. Improvements of the clinical conditions could be seen from critically ill patients after receiving antibody-containing covalescent plasma transfusion. [8] However, further thorough study is still needed on the effectiveness of this treatment measure. Therefore, it is spectacularly important to monitor the relevant indices of organ functions to predict the severity of the infection for patient protection and corresponding treatments. In this study, we analyzed multiple organ function indices of 136 COVID-19 pneumonia patients and compared the differences in the index levels between the severely ill patients and the patients with mild syndromes to understand the progress and the outcome of the COVID-19 infection for better disease control and more effective treatment.

Sample collection and tests
This study is approved by the Ethics Committees from Shenzhen Third People's Hospital(2020-142). The index values of peripheral venous blood samples collected on admission from the two groups of patients were used for retrospective analysis. The biochemical indices and blood routine indices of the blood samples were examined using chemiluminescent method. Patients' lung involvement conditions and the results of colour echocardiography were also collected. Data and reference ranges are listed in Table S1. 1.3 Statistical analysis SAS9.4 software was used for the statistical analysis of the indexes. Kolmogorov-Smirnov test was adopted to check the normality of the quantitative data. The data following normal distribution is expressed as mean ± standard deviation (false) while the data not following normal distribution is expressed as median (interquartile range). Inter-group comparison of the normally distributed data was done using Student's t test while the inter-group comparison of the non-normally distributed data was done using Wilcoxon rank-sum test. Inter-group comparison of the qualitative data was done using Chi-square test. Fisher's exact test was applied for those un tted for Chi-square test. Signi cant statistical difference is indicated by p<0.05.

Patients epidemiological history and chronic diseases
Based on the information collected, patients in the severe group have higher average age of 61.3 years and more chronic diseases. In the severe group, 44 patients had residential/contact history to Wuhan/Hubei epidemic areas while 4 patients had contact history with con rmed cases. 2 patients had neither been in Wuhan/Hubei or in contact with any con rmed case. 34 patients have at least one type of chronic diseases, including hypertension, diabetes, coronary heart disease, arrhythmia, COPD and hepatitis B. 13 of them have two or more underlying diseases. Six patients turned to critical state and three of them died. Two died cases were with either hypertension or COPD while the third case had no chronic disease history. The other three critical cases had chronic disease history of hypertension or diabetes, or both. Other patients were discharged upon recovery. The average course of disease was 4.9 days while average length of hospitalization was 29 days.
In the mild group, 53 patients had residential/contact history to Wuhan/Hubei epidemic areas. 13 patients had contact history with con rmed cases. 7 patients had no epidemiological history. 13 of the patients have at least one type of chronic diseases, including hypertension, diabetes, emphysema, tuberculosis, hyperlipemia, hepatitis B and cirrhosis at decompensation stage. 2 patients have two underlying chronic diseases. All of the patients in the mild group were discharged upon recovery. The average course of disease was 3.6 days while average length of hospitalization was 19 days. (Table S1) 2.2 Inter-group comparison of the levels of multiple organ function indices From the results of biochemical test and blood routine tests, signi cant differences in the levels of multiple organ function indices were seen between the severe group and the mild group. Statistically, positive t or z value represents a higher mean/median value of the index level in the severe group comparing to the mild group, and vice versa. The higher the absolute t or z value, the more different in the index level between the two groups and the lower the p-value. (Table 1) Signi cantly lower oxygenation index was detected from the severe group comparing to the mild group. Levels of liver function indices, alanine aminotransferase (ALT) and aspartate aminotransferase(AST), are signi cantly higher, while level of albumin is lower in the severe group. Elevated concentrations of immune system function indices including C-reactive protein(CRP), procalcitonin(PCT), erythrocyte sedimentation rate (ESR), neutrophil count and interleukin-6 (IL-6) but lower lymphocyte count were also observed in the severe group. Longer plasma prothrombin time (PT) and activated partial prothrombin time (APTT), higher level of D-Dimer and lower platelet counts were detected from the severe group. Kidney function index, creatinine(Cr), and heart function index, troponin(Tnl), were also increased in patients with severe syndromes. No difference was observed from other indices between the two groups in statistical perspective. (Table 1, details in Table S1) These results are possibly indicating altered functions of major organs and systems including the heart, the liver, the kidney, the immune system and the blood coagulation system as the severity of the infection increased.

Inter-group comparison of categorical variables
By converting quantitative variables to categorical variables, the ratio of abnormalities in each category was analyzed between the two groups. Statistically, the proportion of patients with abnormal level of each index were compared between the two groups. Higher x 2 value represents larger difference between the two groups. Based on the comparison, there were more male patients in the severe group which aligned with the result of previous research study. [6] There are higher proportions of patients with altered lymphocyte count, D-dimer level, albumin level, aspartate aminotransferase level, procalcitonin level, C-reactive protein level, erythrocyte sedimentation rate and interleukin-6 level in the severe group. More patients in the severe group have bilateral lung involvement and altered heart function in the severe group comparing to the mild group. Other indexes are not signi cantly different in statistical perspective. (Table  2, details in Table S1) This showed that COVID-19 infection have more extensive in uence to organ function indices in the severe group.

Effects of underlying diseases on the blood indices
There are more patients with underlying diseases in the severe group comparing to the mild group. Thus, further analysis according to these underlying diseases was done to examine the effects of these underlying diseases on the variation of blood indices between the two groups. By comparing between the blood index levels quantitatively and qualitatively between patients with underlying diseases in the two groups, it was found that Cr, PCT, CRP, N, ESR, IL_6, PT, D_DIC were signi cantly higher in the severe group while levels of PLT and ALB were signi cantly higher in the mild group. (Table 3) This showed that underlying disease is indeed a factor affecting the severity of COVID-19 infection. Blood index levels were then compared respectively according to hypertension and diabetes which are most common underlying diseases observed from the patients. Elevated levels of PCT, CRP, N and decreased levels of PLT and ALB were observed from patients with diabetes in the severe group, where level of N increased both quantitatively and as a categorical variable. (Table 3) For those patients with hypertension in the severe group, levels of Cr, PCT, CRP, and ESR were signi cantly increased while levels of PLT and ALB were decreased, where levels of CRP, ESR and ALB were varied both quantitatively and as a categorical variable. (Table 3) Such results speci cally highlighted the possibility of the in uence of underlying diseases on the organ functions in the COVID-19 patients.

Discussion
Here we analysed the differences between the levels of organ functional indices detected from peripheral venous blood samples between two cohorts of patients with either severe or mild COVID-19 pneumonia infection. We measured the biochemical indexes and blood routine indexes in the early stage of medication in order to avoid damage the functions of patients' major organs including liver, kidney and other organs in the course of medication. Through inter-group comparison, it was found that levels of ALT, AST and Cr were signi cantly higher in the severe group (p<0.05), re ecting a higher vulnerability of severe patients to the damage of liver and kidney functions. Our results also indicated from the perspective of blood index levels the in uence of underlying diseases on the organ functions in the severe group.
It has been reported that human coronavirus is able to replicate quickly after infection while causes extensive in ltration of in ammatory cells and enhanced responses of pro-in ammatory cytokine/chemokine. [13]Another group has also described the pathophysiological changes of COVID-19 infection using "cytokine storm". This series of changes would lead to systemic in ammatory response syndrome and induce related organ damage in the patients. In the early stage of infection, some patients had respiratory distress syndrome and decreased lung ventilation ability which lead to hypoxia of organs and tissues. Hypoxia would result in oxidative stress responses of the body while the continuous increase of reactive oxygen species and its peroxides could further promote the release of proin ammatory factors, aggravating liver and kidney function damages. [14] Angiotensin-converting enzyme 2 (ACE-2) receptors are distributed over the cells surface of brain, heart, kidney, liver, lungs and etc. Being a linear single positive strand RNA virus, COVID-19 virus infects human cells by interacting with ACE-2 receptors through RBS domains of S proteins inducing a series of pathological changes and related organ function damages. [15] Our results also con rmed that patients in the severe group had signi cantly altered level of in ammatory indices including lymphocyte count, PCT, CRP, ESR, IL-6 and etc, and higher proportion of bilateral lung involvement, comparing to those in the mild group. This also indicates that severe patients are more prone to have in ammatory storm and hypoxia of respiratory distress tissue.
Moreover, patients in the severe group have higher average age and more chronic diseases which make them more vulnerable to liver and kidney function damages under the in uence of in ammatory storm comparing to the patients in the mild group. There are several studies reported on the organ damage in the COVID-19 patients. Cui et.al has reported a case of female infant with COVID-19 pneumonia presented damages on liver and heart, diagnosed by laboratory examinations. [16] Liver dysfunctions as indicated mostly by increased AST levels in the COVID-19 patients were also reported. The mechanisms of the induction of these liver dysfunctions were postulated due to mediation of ACE2, in ammatory responses, hypoxia and even medications. [17] Our group has also reported one severe patient with onset of viral hepatitis after con rmed with COVID-19 infection. [18] Kidney damages related to COVID-19 were possibly induced in the patients by cytokine storm, organ crosstalk and Systemic effects. [19] This disease is also more severe in the immunocompromised kidney transplant recipient. [20] COVID-19related myocardiac injury was due to SARS-CoV-2 invasion mediated by ACE-2. [21] One case in the severe group had de nite myocardial injury which is resulted from viral myocarditis caused by COVID-19 virus. There is no difference statistically in the levels of CK-MB and TnI in other severe patients comparing to those in the mild group (P>0.05). Further dynamic observation and analysis are still needed due to the factors of sample size and collection time of peripheral blood. In summary, patients with severe COVID-19 pneumonia infection are possibly associated with different organ damages in the early stage, such as in the lung, the liver, the kidney and etc. Therefore, it is necessary to dynamically monitor the relevant organ function indices for prompt drug intervention, prevention of aggravation of patients' conditions and improvement of prognosis.