Effect of Alcohol on Clinical Outcomes of Hepatitis Virus-Induced Liver Cirrhosis: A Consecutive Ten-Year Study


 BACKGROUND & AIM: Coexisting alcoholic liver disease and virus-induced liver cirrhosis (ALD+HBV and ALD+HCV) has not been thoroughly studied. This cross-sectional study aims to showcase the influence of alcohol on the laboratory values and on the clinical outcomes of patients with hepatitis B and C virus-induced liver cirrhosis.METHOD: Patients diagnosed with liver cirrhosis (n=22287) from January 2010 to December 2019 were collected from our hospital electronic database, and divided into five groups according to the etiology: ALD (1652 cases, 7.4%), HBV (18079 cases, 81.1%), HCV (682 cases, 3.1%), ALD+HBV (1594 cases, 7.2%) and ALD+HCV (280 cases, 1.3%). Laboratory results and proportion of different liver cirrhosis complications were contrasted between groups.RESULTS: ALD+HBV and ALD+HCV presented with higher proportions of poor prognosis patients and cirrhosis complications compared to HBV and HCV respectively. Multivariate logistic regression revealed that the risk of HCC and that of EGVB in the ALD+HBV group was respectively 2.14-fold and 1.47-fold that in the HBV group (HCC: OR=2.14, 95%CI: [1.71-2.69]; EGVB: OR=1.47, 95%CI: [1.17-1.84]) after adjusting for potential confounders. Furthermore, a decrease in the risk of HCC and EGVB with the duration of alcohol abstinence was observed. Similar pattern was noticed while comparing ALD+HCV group to HCV group.CONCLUSION: Alcohol significantly increased the severity of liver function impairment and the prevalence of liver cirrhosis complications such as HCC and EGVB in hepatitis virus-induced liver cirrhosis patients. Remarkably, long period of alcohol abstinence significantly decreased the prevalence of HCC and EGVB in these populations.


INTRODUCTION
HBV, HCV and alcohol abuse are the three major causes of liver cirrhosis worldwide and in China. Yearly death toll associated with HCV-related diseases including cirrhosis and hepatocellular carcinoma is estimated to approximately 700000 worldwide 1 . By date, over 350 millions people are estimated to be chronically infected by HBV in the world 2 . Due to the considerable progress achieved by the vaccination programs and treatment of hepatitis B and C, the prevalence of these two entities has noticed a significant decrease over the decades. However the actual socioeconomic development of the Chinese population participated in the rise of the prevalence of alcohol-related liver disease (ALD) in China 3 . This had led to an increase in cases of coexisting ALD and hepatitis virus B or C patients (ALD+HBV, ALD+HCV). According to WHO, over 3 million people die every year due to alcohol abuse.
This represents approximately 6% of the overall deaths worldwide. ALD is a spectrum of disease ranging from steatosis to steatohepatitis, fibrosis and even hepatocarcinoma. The conventionally used biomarkers for the diagnosis of ALD include elevated MCV and GGT, AST/ALT greater than 1.5, etc. Even though the specificity and sensitivity of these markers are limited, they still play an important role in the suspicion and diagnosis of ALD. However, the extent to which the presence of ALD together with viral hepatitis affects these laboratory findings has not been fully studied.
The interaction between alcohol and hepatitis virus B or C has been widely studied recently with nonunanimous conclusions 4 . Heavy alcohol consumption had been shown to accelerate the development of liver fibrosis, and to increase the prevalence of HCC and mortality in HCV infected patients 5 . Concerning the interaction between alcohol and HBV, less conclusive studies has been done. Some studies showed an increase risk of HCC in HBV infected patients with heavy alcohol consumption habit, whereas other estimates that small to moderate alcohol use did not increase the risk of fibrosis in HBV patients 6 . Nevertheless, few is known about the prevalence of other liver cirrhosis complications such as EGVB among these groups of patients. Therefore, this studied has been designed to assess the effect of alcohol in HBV and HCV cirrhotic patients by, first comparing disease severity between ALD, HBV and HCV patients and also between ALD+HBV and HBV, and between ALD+HCV and HCV, using commonly available biomarkers and disease severity assessment scores; then evaluate the prevalence of different liver cirrhosis complications such as HCC and EGVB between these groups.

Patients enrollment and data acquisition
Medical records of all the patients diagnosed for the first time with liver cirrhosis in our hospital from January 2010 to December 2019 were retrospectively collected from the hospital electronic database. The identification of the cirrhosis cases was made using ICD-10-CM codes of liver cirrhosis (K74.100). Patients with etiology other than ALD, HBV, HCV, ALD+HBV or ALD+HCV, and those with incomplete data were excluded. The remaining cases were divided into five groups according to the etiology of their liver condition: alcohol-induced liver cirrhosis (ALD group), hepatitis B virus-induced liver cirrhosis (HBV group), hepatitis C virus-induced liver cirrhosis (HCV group), coexisting ALD and HBV (ALD+HBV), and coexisting ALD and HCV (ALD+HCV). Information on clinical complications, laboratory data and personal history such as gender, age , history of alcohol etc. was collected.

Statistical analysis
We first assessed liver function impairment between the groups using laboratory data such as ALT, AST, MCV, Child classification, MELD score, GAHS (Glasgow alcoholic hepatitis score) and MDS (Maddrey's discriminant function Scale). The proportion of different liver cirrhosis complications was then evaluated among the groups.
Multivariate logistic regressions were conducted to evaluate the association between different etiologies and the risk of HCC and EGVB. The difference in the proportion of HCC and EGVB between males and females in each group was also evaluated. Patients from ALD, ALD+HBV and ALD+HCV groups were later divided into four subgroups according to the duration of abstinence from alcohol: no abstinence (subgroup 1), abstinent for less than five years (subgroup 2), abstinent for five to ten years (subgroup 3) and abstinent more than ten years (subgroup 4). Abstinence from alcohol was defined as restraint from any alcohol beverage for more than six months. The relation between alcohol abstinence and risk of HCC and EGVB was assessed in different groups through linear-by-linear association test and logistic regression analysis. Covariates included in the multivariate logistic regressions were identified through univariate analysis. Statistical data were expressed as the median and 25 th , 75 th percentiles or as percentage as appropriate. Kruskal-Wallis H test along with Bonferroni correction was used to compare quantitative variables between groups. Chi square (X 2 ) test was used to compare categorical variables. All statistical tests were two-sided; P values below 0.05 were considered statistically significant. All data were analyzed using SPSS 25.0 (Armonk, NY: IBM corporation,).

RESULTS
A total of 25782 cases of liver cirrhosis diagnosed from January 2010 to December 2019 were collected. Cases of congenital biliary atresia (35 cases), Wilson disease (28 cases), autoimmune liver disease (842), cases with two or more etiologies other than ALD+HBV or ALD+HCV (930 cases), cases with incomplete data, unconfirmed or unknown cirrhosis etiology (1660) were all excluded from this study. The remaining cases (22287 cases) were divided into five groups according to their etiologies: ALD group, HBV group, ALD+HBV group, HCV group and ALD+HCV group. The changing trend of the proportion of each etiology over a ten-year period is depicted in supplementary figure 1. HBV was found to be the major cause of liver cirrhosis, with an annual proportion fluctuating around 80% over the ten-year period.

Characteristics of the laboratory data
Differences in the laboratory data between the five groups are detailed in Table 1. Serum level of MCV was more elevated in the ALD group compared to that in both HBV group and HCV group. Moreover, MCV appeared higher in both ALD+HBV and ALD+HCV groups compared to HBV group and to HCV group respectively (P<0.001).
AST/ALT was higher in the ALD group compared to the HBV group and to the HCV group. This ratio was more elevated in ALD+HBV group compared to that in HBV (1. 5 Likewise, the proportions of patients with MELD score ≥18 were higher in the ALD+HBV group compared to that in the HBV group (24.1% vs 18.5%, P<0.05) and also in the ALD+HCV group compared to the HCV group (13.1% vs 7.6%, P<0.05). Similar trends were observed comparing GAHS, MDF and Child-Pugh classification between the five groups.

Assessment of different liver cirrhosis complications between the groups
HCC was less frequent in the ALD group (10.3%) compared to the HBV group (42.2%) and to the HCV group (20.8%), P<0.001. Moreover, HCC was more prevalent in the ALD+HBV group compared to the HBV group (52.2% vs 42.2%, P<0.001), and also in the ALD+HCV group compared to the HCV group (34.3% vs 20.8%, P<0.001), see Table 2. A multivariate logistic regression estimated the risk of HCC in ALD+HBV group at 2.14 times that of HBV group (95%CI: 1.71-2.69, P<0.001) after adjusting for confounders. Similarly, the risk of HCC in ALD+HCV group was estimated to be 1.87 times that of HCV group (95%CI: 0.96-3.64, P=0.066). Other independent predictors of HCC are displayed in Table 3 and Supplementary table 1.
The ALD group registered a higher proportion of EGVB compared to HBV group (34.5% vs 24.0%, P<0.001).
This difference was non-significant comparing ALD group to HCV group (34.5% vs 27.1%, P>0.05). The proportion of EGVB was also significantly higher in ALD+HBV group than that in HBV group (30.1% vs 24.0%, P<0.001); see Table 2. A multivariate logistic regression analysis evaluated the risk of EGVB in ALD+HBV group to be 1.47 times that of HBV group (95%CI: 1.17-1.84, P=0.001) after adjusting for confounders. Even though there was a rise in the proportion of EGVB in ALD+HCV group compared to HCV group, this difference was not statistically significant.
Other independent predictors for EGVB are displayed in Table 4 and Supplementary table 2. Other liver cirrhosis complications such as infection, ascites, HE, and HRS were all significantly more frequent in the ALD group (33.5%, 54.0%, 7.7%, 3.0% respectively) compared to the HBV group (23.1%, 43.3%, 5.2%, 1.5% respectively) and to the HCV group (18.9%, 37.0%, 2.9%, 1.2% respectively). Furthermore, apart from HRS, these complications were significantly more common in the ALD+HBV group compared to the HBV group, and also in the ALD+HCV group compared to the HCV group, see Table 2.

Differences in the proportion of HCC and EGVB between genders
In the ALD group, the proportion of HCC and EGVB were similar between males and (HCC: 10.4% vs 7.3% P=0.526; EGVB: 34.6% vs 30.4%, P=0.825). In the HBV group, HCC was more prevalent in male (45.6% vs 25.8%, P<0.001), whereas the prevalence of EGVB appeared similar between both genders (P=0.098). In the ALD+HBV group, HCC was similarly frequent between males and females (P=0.156). However, the proportion of EGVB in females surpassed drastically that in males (28.8% vs 84.2%, P<0.001). In the HCV group, similar rate of HCC and EGVB was found between males and females (HCC: P=0.296; EGVB: P=0.099). On the other hand, in the ALD+HCV group the proportion of HCC was remarkably higher in females (30.6% vs 77.3%, P<0.001). In contrast, no case of EGVB was registered among the females in the ALD+HCV group, as shown in Figure 1.

Effect of alcohol abstinence on the proportions of HCC and EGVB in ALD, ALD+HBV and ALD+HCV
ALD, ALD+HBV and ALD+HCV groups were each divided into four subgroups according to the duration of alcohol abstinence: "no abstinence", "abstinent less than five years", "abstinent five to ten years" and "abstinent more than ten years". A linear-by-linear association test from chi square analysis between duration of abstinence and  In patients with an abstinence period greater than ten years, this risk was estimated at 0.07 (95%CI: 0.03-0. 16, P<0.001) in ALD group, 0.11 (95%CI: 0.59-0.98, P<0.001) in ALD+HBV group and at 0.12 (95%CI: 0.01-1.03, P=0.053) in ALD+HCV group, see Table 5. Furthermore, the proportion of HCC which was higher in non-abstinent ALD+HBV patients compared to that in HBV patients (64.9% vs 42.2%, P<0.001), was significantly lower in ALD+HBV patients with over ten years of abstinence compared to that in HBV patients ( On the other hand, there was a decrease in the proportion of EGVB with the duration of alcohol abstinence in ALD and in ALD+HBV. However, this trend was not statistically significant in ALD+HCV (overall P=0.312), see

DISCUSSION
Owing to the reputation of our center in the diagnosis and management of liver diseases, the findings of this research can effectively reflect the situation of coexisting ALD and viral hepatitis in southern China. Our study successfully showed the exacerbation of liver impairment and the increase in the proportions of liver cirrhosis complications among coexisting ALD and viral hepatitis patients.
In this study, over 80% of the enrolled patients were diagnosed with HBV-induced liver cirrhosis, placing HBV as the leading cause of liver cirrhosis in our hospital. This finding is consistent with the situation in other Asia-Pacific region 10 . Elevated blood levels of MCV, GGT and AST/ALT had been identified as the main laboratory characteristics in ALD 11 . These characteristics were also reflected in our study, with levels of MCV, GGT and AST/ALT significantly higher in the ALD group compared to HCV and to HBV groups. Furthermore, these values were worse in the ALD+HBV and ALD+HCV groups. To our knowledge there has been no previous report on the synergic effect of alcohol and hepatitis virus on these laboratory markers. Our findings can therefore help clinicians suspect the presence of alcohol abuse in viral hepatic cirrhosis patients with unclear history of alcohol intake.
Disease severity was compared between ALD, HBV and HCV, between ALD+HBV and HBV, and also between ALD+HCV and HCV. ALD patients presented with more severe liver impairment compared to HBV and to HCV patients, with the highest proportion of patients with MELD≥18, GAHS≥9, MDS≥32 and child C. These findings are in line with that of Astrid Marot et al. who concluded in their study that ALD patients had worse liver function at admission compared to other liver cirrhosis patients 12 . In our study, the median value of the above-mentioned assessment scores, and the proportion of poor prognosis patients (MELD≥18, GAHS≥9, MDS≥32, Child C) were all higher in coexisting ALD+viral hepatitis compared to the corresponding viral hepatitis group (ALD+HBV vs HBV and ALD+HCV vs HCV). This edifies the negative effect of alcohol on the liver function of HBV and HCV patients.
Possible explanations that had been given in previous literatures are: the enhancement of hepatitis viral replication by alcohol 13,14 , delayed hepatitis B "e" antigen loss 15 , increased hepatocyte toxicity, increased oxidative stress and more weakened host immune response 16 .
In view of the difference in the pathogenesis of different etiologies of liver cirrhosis, it is expected that the decompensation pattern of liver cirrhosis may differ according to the etiologies. In our study, HCC was less prevalent in the ALD group compared to HBV and to HCV groups. This finding is identical to that of Astrid Marot et al 12 . Apart from HCC, other cirrhosis complications were all more prevalent in the ALD group compared to HBV and HCV groups. This high frequency of liver cirrhosis complications in ALD patients was previously pointed out in a former study on Danish population 17 . Moreover, Several researches had proven the role of alcohol in the acceleration of liver cirrhosis decompensation and the worsening of survival in HCV patient 18,19 .This is in accordance with our study which revealed that the proportion of HCC and other complications such as infection, ascites, HE, thrombus formation, HRS, and EGVB were all more prevalent in ALD+HCV group compared to HCV group. This can be explained by the fact that during the natural evolution of HCV-related liver disease, repeated regeneration of the hepatocytes due to persistent liver injury by HCV may cause hepatocyte DNA to become susceptible to mutagenesis, resulting in gene instability 20 . Furthermore, ethanol induces enzymatic activation for the conversion of procarcinogens into carcinogens and consequential induction of hepatic neoplasm 21, 22 . Likewise, recent researches had mentioned the negative impact of alcohol on the decompensation of HBV-induced liver cirrhosis patients. Our study revealed that proportion of HCC was higher in the ALD+HBV group compared to the HBV group. These findings match those of Larkin J et al. in their study on the impact of ethanol on HBV gene expression and replication in transgenic mice 13 . Similar observations were made clinically in several other studies during the last decades 19,23 .
In general, women are known to have lower rate of liver cirrhosis decompensation and malignant tumors compared to men 24 . However, studies showed that women with ALD have more rapid progression to fibrosis and HCC compared to men 25,26 . This is partly due to higher endotoxin levels, increased gut permeability to endotoxins noted in females, and also to the important role of estrogen in the activation of liver Kupffer cells 27,28 . Controversially, in HCVinduced liver cirrhosis, estrogen has been shown to inhibit stellate cells, which are responsible for fibrogenesis in the liver 29 , thus less decompensation in liver function. Studies on HBV-induced HCC are scanty, however it has been reported that the risk of HCC is higher in males with HBV compared to females 30 . This is also the case in our study where the proportion of HBV-induced HCC in males was almost double that in females. Researches on the difference in the prevalence of EGVB between genders are scarce. To our knowledge, there was no previous literature comparing the influence of alcohol on the prevalence of HCC and EGVB between genders in HBV or HCV patients. In our study, we found that the proportion of HCC was similar between both genders in HCV group, however this proportion became significantly higher in females in ALD+HCV group. On the other hand, EGVB which was in similar proportion between males and females in HBV became more prominent in females in ALD+HBV group. Therefore, more surveillance should be given to females with ALD+HBV in the follow-up of esophageal gastric varices.
Similarly, females with ALD+HCV should be more often screened for HCC during their follow-up. However, a prospective study on a larger population, with both pre-menopause and post menopause women is needed to validate these findings.
Studies done on the effect of alcohol abstinence on ALD all over the world are not unanimous. In our study, patients with ALD, ALD+HBV and ALD+HCV all noticed a significant reduction of the proportion of HCC and EGVB following alcohol abstinence. This reduction seemed to be more remarkable with longer duration of abstinence.
These findings are in accordance with a previously published research on the positive long-term effect of alcohol abstinence 31 . Moreover, Clare Verrill et al. in their study found that patients with more severe liver cirrhosis had greater benefit from alcohol abstinence compared to those with relatively mild cases of cirrhosis 32

. Evangelos
Kalaitzakis et al. in their study 33 noticed that cirrhotic patients who had abstained from alcohol showed more severe liver cirrhosis expressed as higher MELD score compared to those who had not abstained. Moreover, no difference was observed in the prevalence of HCC and that of EGVB between the abstinent and the non-abstinent patients 34 .
Nevertheless, Alcohol abstinence is undeniably the cornerstone in the management of ALD.
In conclusion, Alcohol increased significantly the severity of liver function impairment and the prevalence of liver cirrhosis complications such as HCC and EGVB in hepatitis virus-induced liver cirrhosis patients (HBV and HCV). Remarkably, longer period of abstinence from alcohol successfully decreased the prevalence of HCC and EGVB in these populations, thus the importance of stressing not only on the importance of antiviral treatment in patients with coexisting ALD and viral cirrhosis, but also on long term alcohol abstinence.
We acknowledge that our study has some limitations that are worth mentioning. Even though our center has a large number of cirrhotic cases, the proportion of HCV is still relatively small. This major disparity in the etiology encountered in our center could underestimate the actual situation of HCV patients worldwide. Moreover, the large number of patients with incomplete data that were excluded from the study may somewhat influence some of our findings. Thus, the need of a multicentered large cohort prospective study. Table 1 Liver  statistically significant compared to that in HBV+ALD. Bonferroni correction was used for "P " during pairwise comparisons. Esophageal gastric variceal bleeding, HRS: Hepatorenal Syndrome; " a " means value statistically significant compared to that in ALD; " b " means value statistically significant compared to that in HBV, " c " means value statistically significant compared to that in HCV, " d " means value statistically significant compared to that in HBV+ALD.

Tables
Bonferroni correction was used for "P" during pairwise comparisons. " †" The proportion of EGV was calculated by dividing the number of patients reported with EGV by the number of patients who underwent upper GI endoscopy or other imaging examination such as CT or MR, capable of detecting EGV. " ‡" The proportion of EGVB was calculated by dividing number of patients reported with EGVB by the number of confirmed EGV patients.       A) and EGVB (fig B) according to gender in different groups. ALD, alcohol liver disease; HBV, Hepatitis B virus; HCV, hepatitis C virus; ALD+HBV, co-existing ALD and HBV; ALD+HCV, co-existing ALD and HCV; HCC, Hepatocellular carcinoma; EGVB, Esophageal gastric variceal bleeding. " ** " indicates P<0.01, " *** " indicates P<0.001. No case of EGVB was recorded in female of the ALD+HCV group.

Supplementary tables and figure legends
Supplementary table 1 Univariate and multivariate logistic regression evaluating factors associated with HCC in ALD, HCV and ALD+HCV patients