Impact of COVID-19 on Liver Functions Tests in Egyptian Patients with or without Chronic Liver Disease

significantly more gastrointestinal tract symptoms than group 1 (p =0.033). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, direct bilirubin and international normalized ratio (INR) after infection were significantly higher than before infection in both groups (p <0.001). Group 2 had significantly higher mortality rate (47%) than group 1 (32%) (P= 0.02). MELD score was significantly higher in died patients than recovered patients in group 2 (p <0.001). After COVID-19 infection, hepatic encephalopathy (HE) and aggravation of ascites was found in 34% and 36% of patients in group 2. Conclusion: COVID-19 infection can cause liver function abnormalities in both cases with and without CLD with aggravation of ascites and HE development in cirrhotic patients.


INTRODUCTION
For the first time in Wuhan, China (December 2019), a pandemic of the novel coronavirus disease-2019 (COVID-19) started as an outbreak of unexplained pneumonia.[1] 7 410 510 confirmed cases and 418 294 mortalities have been recorded as of 12 June 2020.Based on a report from the Chinese Center for Disease.Control and Prevention (CDC), the overall mortality rate was 2.3%.[2] Multiple studies have detailed the clinical features of cases with COVID19, symptoms typically are similar to viral pneumonia including fatigue, dry cough, fever, headache and anosmia and the symptoms could evolve to respiratory failure.[3] The virus has now been named (SARS-CoV-2).
Digestive system involvement such as nausea, vomiting and diarrhea has also been reported.[4].Even though acute respiratory failure and diffuse alveolar damage are the hallmarks of COVID-19, other organs' impairment are also reported.[5].
Significant systemic signs of COVID-19 also include myocarditis, acute kidney injury, acute liver injury (LI) and thrombosis.[6] A significant proportion of COVID-19 cases have been reported to have elevated liver enzymes, even though its impact of on the liver remains poorly understood.Severe LI has been reported in COVID-19, even though the transaminases levels' elevation is usually mild [one to two times the upper limit unit of normal (ULN)].It is not entirely understood how SARS-CoV-2 affects the liver, although it is believed to be a mix of immune-mediated inflammatory response and direct virallymediated injury [7,8].
The presence of the angiotensin-converting enzyme 2 (ACE2) receptor, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) cellular receptor, in hepatic and biliary endothelial cells offers a potential mechanism for the observed injury in the liver.Other LI etiologies including congestion, shock, druginduced LI, sepsis, and extrahepatic sources of aspartate aminotransferase (AST) must be considered [7] Li et al. concluded that lymphopenia and raised C-reactive protein (CRP) are risk factors of liver injury in COVID-19 infected cases [9] Our objective was to evaluate COVID-19 effect on liver functions tests in Egyptian subjects with or without chronic liver disease (CLD).Also, we aim to evaluate the correlation between these abnormalities and COVID-19 severity.

PATIENTS AND METHODS
This retrospective research was performed on COVID-19 cases who attended the outpatient clinic of National Liver Institute (NLI) and New Mansoura General hospital (International) in the period from April 2020 to January 2021.Real time reverse transcriptase polymerase chain reaction (RT-PCR) assay was used for the detection of SARS-CoV-2 using nasopharyngeal swabs, and also if there was one or more of these symptoms of COVID-19 symptoms are present such as: fever, cough, chills, shortness of breath, fatigue, red eyes, rash, drowsiness, confusion body aches, headache, congestion or runny nose, new loss of taste and/or smell, persistent chest pressure or pain, sore throat, gastrointestinal (GI) symptoms such as nausea or vomiting or diarrhea.All studied patients were classified into two groups: group 1 (n=100) included cases without CLD and group 2 (n=100) included cases with CLD.All studied cases underwent the following laboratory investigations: Liver tests such as gamma glutamyltranspeptidase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total and direct bilirubin, alkaline phosphatase (ALP), international normalized ratio (INR), Viral markers as HBsAg and HCV Ab, complete blood count (CBC), ferritin, D-dimer, C reactive protein (CRP), abdominal ultrasound and computed tomography (CT) of the chest.The diagnosis of chronic liver disease was performed by determining the etiology (Chronic HCV infected patients were positive for HCV Ab and HCV RNA by real time PCR.Chronic HBV infected patients were positive for HBsAg and HBV DNA by real time PCR for more than 6 months) and complications of the disease (Ultrasound abdomen in case of CLD detects size and echogenicity nodularity of liver and absence or presence of ascites).Assessment of severity of liver disease was done according to Child Pugh classification and model for end stage liver disease (MELD).
The classification of the pattern of these abnormalities was: cholestatic, hepatocellular, or mixed.
Cases with hepatocyte type had high AST and/or ALT levels > 3 times the ULN.Cases with cholangiocyte type had elevated ALP or GGT twice the ULN.Cases with mixed type [Abnormality type (1)] had an elevation in both ALT/AST > 3times the ULN and ALP/GGT twice the ULN [10].
The liver abnormality patterns were assessed based on another criterion: When the activity of AST/ALT was higher than the activity of ALP/GGT, with the calculation of liver enzyme activities by multiples of their ULN, respectively, cases were classified as hepatocyte type; and were classified as cholangiocyte type [Abnormality type (2)] when the opposite occurred [10].

COVID-19 severity:
On the basis of chest radiography, symptoms, and clinical examination, all cases were categorized as severe or mild cases.Cases without abnormalities, and with mild symptoms (i.e., cough, expectoration, fever, and other symptoms of the upper respiratory tract), or with mild changes on chest radiography will be classified as non-severe cases.The presence of any of the subsequent criteria defines severe pneumonia: i) hypoxia: oxygen saturation ≤93% (while resting); ii) significantly elevated respiration rate (RR): RR ≥30 breaths/minute; iii) respiratory or other organ failure that necessitates monitoring and treatment in the intensive care unit (ICU), or shock [10].

Statistical analysis
SPSS V26 was used to conduct the statistical analysis (IBM Inc., ARMONK, NY, USA).Categorical data were displayed as frequency and percentage (%) and were analyzed utilizing the Chi-square test or Fisher's exact test when appropriate.Continuous data were displayed as mean and standard deviation (SD) and compared between the two groups utilizing unpaired Student's t-test and compared between the three groups utilizing ANOVA (F) test with post hoc test (Tukey).Kaplan Meier curve was used to show the time of mortality.A two tailed p< 0.05 was deemed statistically significant.

RESULTS
The clinical features of the two studied groups were described.Group 2 had significantly higher age than group 1 (p <0.001) and males were significantly higher in group 2 than group 1 (p= 0.048).Many of patients complained from fever, cough and GIT symptoms.Fever and cough were insignificantly different between both groups while group 2 had significantly more GIT symptoms than group 1 (p= 0.033).The main medical history was diabetes mellitus (DM) and hypertension with insignificant difference between both groups as mentioned in table 1.
Many laboratory investigations were done during hospitalization.HCV antibody and HBs Ag were negative in all patients in group 1 while HCV antibody was positive in 90 (90%) patients in group 2 and HBs Ag was positive in 10 (10%) patients in group 2. The mean± SD of CRP in group 1 was 89.02 ± 54.46 mg/dl and it was 76.27 ± 35.62 mg/dl in group 2 with insignificant variation between the 2 studied groups (p= 0.051).D-dimer was high in 67 and 71 cases in group1 and group 2 respectively with insignificant difference between them.Serum ferritin was high in 44 and 42 cases in group1 and group 2 respectively with insignificant different between them as mentioned in table 2.
Abdominal ultrasound and CT chest were done for all cases.Abdominal ultrasound findings for liver were normal for all cases of group 1 while, in group 2 all patient had cirrhotic liver, 96 patients had splenomegaly and about 91 patients had variable degrees of ascites.CT chest for all patients varied from CO-RAD 3 to CO-RAD 5 with insignificant variation between both groups (p= 0.91).
During hospitalization, liver function tests (AST, ALT, ALP and GGT) were found to be abnormal in 41 and 55 individuals in group 1 and group 2 respectively.LI abnormality type 1 was hepatocyte type in 27 individuals in group 1 and in 32 individuals in group 2, mixed type in 1 individual in both groups with no cholangiocyte type in both groups.Abnormality type 2 was hepatocyte type in 35 individuals in group 1 and in 48 individuals in group 2 and cholangiocyte type in 3 individuals in group 1 and in 2 individuals in group 2. LI, abnormality type 1 and abnormality type 2 were insignificantly different between both groups (p< 0.05).
Direct bilirubin, TBIL, INR, AST, and ALT after infection were significantly elevated than before infection in both group 1 and group 2. GGT was insignificantly different between before and after infection and ALP and serum albumin levels after infection were significantly reduced than before infection in group 1.In group 2, GGT, ALP and serum albumin after infection were significantly lower than before infection.Group 2 had significantly increased delta change of TBIL and INR than group 1 (p =0.007 and <0.001 respectively).Group 2 had significantly lower delta change of serum albumin than group 1 (p=0.002).Delta change of ALT, AST, direct Bilirubin, GGT and ALP were insignificantly different between both groups as mentioned in tables 3&4.
At admission, MELD score and Child score were calculated for the cases of group 2 and result showed that MELD score range from7 to 18 with a mean value (± SD) of 11.9 (±2.45).
Due to complications and LI happened after covid-19 infection for both groups, 41(41%) patients had elevated Liver enzymes, 33 (33%) patients had hypoalbuminemia and 23 (23%) patients had elevated total bilirubin after infection in group 1,but aggravation of ascites and hepatic encephalopathy occurred in 36 (36%) patients and 34 (34%) patients respectively in group 2. Child score was significantly higher after covid-19 infection, it ranged from 4-8 but after infection it ranged from 5-13.Regarding Child-classification, class A was significantly lower after infection than before infection (p <0.001), class B was insignificantly different between before and after infection and class C was significantly higher after infection than before infection (p <0.001) as mentioned in table 5 Hospitals stay (in days) for patients were insignificant different between the two groups.Also, the two groups of patients received the same treatments (anti-viral, anti-biotic, antiinflammatory, anti-coagulant) with insignificant difference between both groups except for anticoagulant (Heparin/ Clexan) was significantly elevated in group 1 in comparison to group 2 (p<0.001).but about 47 cases in group 2 died and only 32 cases died at group 1 so, group 2 had significantly higher mortality rate than group 1 (p =0.02) and the causes of death were respiratory failure or multi-organ failure with insignificant difference between both groups as mentioned in table 6 and figure 1.
Our study showed that PaO2/FiO2 was significantly reduced in died cases in group 1 and group 2 than recovered cases.MELD score was significantly higher in died cases (median 14; range 9-18) than recovered cases (median 10; range 7-15) in group 2 (p <0.001), but as shown in table 7: age, direct bilirubin, TBIL, AST, ALT, GGT, ALP, serum albumin, INR, hemoglobin (HB) and platelets were insignificant predictors of mortality.

DISCUSSION
Based on multiple researches, the infection of SARS-CoV-2 results in abnormal hepatic tests, including elevated levels of prothrombin time (PT), ALT, AST, and total bilirubin [11].
In addition, lower levels of platelets, lymphocytes and albumin, advanced age, and elevation in ALT, lactate dehydrogenase, leucocytes, ferritin, creatinine and PT are associated with increased mortality [12].
Our objective was to evaluate COVID-19 effect on liver functions tests in Egyptian patients with or without CLD.Also, we aimed to assess the correlation between these abnormalities and COVID-19 severity.
In our study, oxygen saturation and respiration rate were insignificantly different between both groups.
The present research revealed that cases with CLD had a significant reduction in O2 saturation, indicating that hospitalization was most likely necessitated with the decompensation of CLD.
In our work, TBIL, direct bilirubin, AST, ALT, and INR after infection were significantly elevated than before infection in both group 1 and group 2. GGT level was insignificantly different between before and after infection and ALP and serum albumin after infection were significantly reduced than before infection in group 1. GGT, ALP and serum albumin after infection were significantly reduced than before infection in group 2.
In Also, the APCOLIS trial reported that CLD cases infected with COVID-19 had an increased incidence of liver-related complications such as ascites, aggravation of jaundice, hematemesis, spontaneous bacterial peritonitis, and hepatic encephalopathy.These outcomes were more prevalent in cases with decompensated cirrhosis and diminished hepatic reserves at baseline.In the current research, aggravation of ascites and hepatic encephalopathy had happened in 36 (36%) patients and 34 (34%) patients respectively after infection in group 2 [29].
Our results ha some limitations such as its retrospective design, which may introduce selection bias and confounding factors.The relatively limited sample size may restrict the generalizability of the findings.

Conclusions
COVID-19 infection can cause abnormalities in the liver function in cases with and without CLD.Additionally; severe COVID-19 was associated with more liver dysfunction and complications, including lower serum albumin, higher bilirubin and more aggravation of ascites and HE development.However, liver function test abnormalities were not found to be significant predictors of mortality in this study. .

Table 1 :
Demographics of the studied groups.

Table 2 :
Possible markers for Covid -19 infection of the studied groups.

Table 3 :
Liver tests in each group before and after covid-19 infection.

Table 4 :
Delta change of Liver tests in each group between before and after infection.

Table 5 :
Child-score and Child-classification before and after infection of the group 2

Table 6 :
Hospital stay, prognosis and cause of death of the two studied groups

Table 7 :
Relation between PaO2/FiO2, MELD score and prognosis * Significant as P value < 0.05 PaO2/FiO2 was significantly lower in die patients in group 1 and group 2 than recovery patients.MELD score was significantly higher in die patients than recovery patients in group 2 (P value <0.001).

15, 25]. Phipps et al., reported
In our study, age was insignificant predictor of mortality in our study.Incontrast, Phipps et al. found that peak older age was independent predictor of mortality [18].A research by Mangia et al. reported that older age and cirrhosis of metabolic origin were risk factors for mortality [23].Previous meta-analyses indicated that subjects with severe COVID-19 and those hospitalized to the ICU had higher levels of transaminases [a severe LI with ALT > 5 times above the ULN in 6.4% of their cases [18].Moreover, Ponziani et al. found that changes in the levels of transaminase in COVID-19 cases were mild to moderate, while a few cases had pure cholestatic injuries and significant LI with transaminases > 3 times the ULN [26].percent of patients in group 1 and 15% of subjects in group 2 in our study had severe LI with ALT > 5 times above the ULN.Furthermore, El-Adly et al. reported that severe COVID-19 cases show signs of liver dysfunction more frequently compared to the cases with milder infection.Many ICU cases had an elevation in the levels of AST, ALT, and TBIL [27].Cai et al. mentioned that cases with abnormal liver tests were more likely to have severe pneumonia [10].Boettler et al. discovered that the risk of a severe COVID-19 did not seem to be raised due to chronic viral hepatitis [28].