The Histological Assessment of Hepatitis B Viral Activity in Patients with Heavy Alcohol Consumption

1Division of Gastroenterology and Hepatology, Department of Medicine, E-Da Dachang Hospital, I-Shou University, Kaohsiung, Taiwan 2Health Examination Center, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan 3School of Medicine, College of Medicine, I-Shou University, Kaohsiung, Taiwan 4Department of Pathology, University of Washington School of Medicine, Seattle, Washington, USA 5Liver Unit, Cathay General Hospital, Taipei, Taiwan 6School of Medicine, Fu-Jen Catholic University, New Taipei, Taiwan


Introduction
Asia-Pacific region is a region with a high prevalence of hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) [1,2]. Alcoholic liver disease is a major cause of chronic liver disease worldwide and can lead to fibrosis, cirrhosis, HCC, and mortality [3,4]. Economic progress in this region has led to an increase of alcohol consumption and changes in drinking behavior, which have resulted in an increased number of cases of alcoholic liver disease in Taiwan [5][6][7][8].
Alcohol abuse is not uncommon among those patients with HBV infection. The synergism and interaction between HBV infection and alcohol consumption have been reported [9][10][11][12]. Alcoholic consumption may increase viral replication and exacerbate liver injury, which results in the much more rapid progression of chronic hepatitis to cirrhosis and HCC [9][10][11][12][13]. Our recent study confirmed that the heavy alcohol consumption significantly increased the risk of HCC in HBV-related cirrhotic patients [13]. The clinical diagnosis for the role of concomitant HBV infection in alcoholic patients is difficult and remains uncertain. Furthermore, the histological assessment between chronic hepatitis B (CHB) and alcoholic liver disease has never been discussed in the literature. Thus, we investigated the impact of heavy alcohol consumption and HBV infection on histological findings and clinical diagnosis.

Patients
We prospectively collected 229 patients (33 with concomitant heavy alcoholism and HBV infection, 114 with HBV infection alone, and 82 with heavy alcoholism alone) at the Cathay General Hospital/ Fu-Jen Catholic University, Taipei, Northern Taiwan, and E-DA Hospital/I-SHOU University, Kaohsiung, Southern Taiwan, between 2009 and 2012. The evaluations commenced after approval of the study protocols by the Institutional Review Board of Cathay General hospital and E-DA hospital. The behavior of alcohol consumption was routinely evaluated by interviewing patients and family members for the duration, types, and amount of alcohol consumed per day. Heavy alcoholism was defined as consuming more than 80 g of ethanol each day for at least 5 years.

Hepatitis B marker
All patients had blood chemistry and were tested for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and anti-HBe antibody (Abbott Laboratories, Chicago, IL, USA), α-fetoprotein (AFP), and serum HBV DNA (Cobas Amplicor, Hepatitis B Virus Test; Roche Diagnostics, Branchburg, NJ, USA) with with a minimum detection limit of 300 copies/ml. CHB was defined as the positivity for serum HBsAg for more than 6 months as well as chronic liver disease based on the clinical findings of laboratory tests, sonography, and upper gastrointestinal endoscopy. All patients had blood sampling and liver biopsy collected on the same day. All patients without liver histology due to decompensated liver function, international normalized ratio>1.5 and the occurrence of ascites were excluded. All patients with other hepatitis or liver malignancies were excluded.

Statistical analysis
Data were expressed either as median (range) or percentage (%). Continuous variables were analyzed using Student's t-test. Categorical variables were analyzed using Pearson's Chi-square test or Fisher's exact test, as appropriate. All analyses were performed using the Statistical Package for Social Sciences (SPSS, version 15.0; Chicago, IL, USA).

Baseline demographic data
The clinical and biochemical features of all patients were shown in Table 1. Patients with alcoholism with and without HBV infection were male predominant. Patients with concomitant alcoholism and HBV infection were younger, less platelet, and less platelet to spleen ratio than patients with alcoholism alone or HBV infection alone. Compared with patients with HBV infection with and without alcoholism, patients with alcoholism alone had significantly lower body mass index, hemoglobin, leukocyte, and albumin but higher mean corpuscular volume, serum bilirubin, alkaline-phosphate, gamma-glutamyl transpeptidase, and AST/ALT ratio (p<0.001). For the patients with HBV infection alone, 111 of 114 (97.4%) patients with HBV infection had detectable HBV DNA. For the patients with concomitant HBV infection and alcoholism, 29 of the 32 (90.6%) patients with HBV infection had detectable HBV DNA. None of the patients with alcoholism alone had detectable viral load (p<0.001).

Histological features of viral hepatitis B
The histological features of viral hepatitis B were shown in Table  2. Compared with those patients with alcoholism alone, patients with HBV infection with and without alcoholism had much more piecemeal necrosis, confluent necrosis, focal necrosis, portal inflammation and necroinflammatory grading (p<0.001) regardless of HBV infection. However, patients with concomitant HBV infection and alcoholism or alcoholism alone had much more cases having cirrhosis with Ishak stage 5-6 fibrosis than those with HBV infection alone (p<0.001).

Histological features of alcoholic liver disease
The histological features of alcoholic liver disease are shown in Table 3. Compared with those with HBV infection alone, patients  with alcoholism with and without HBV infection had much more pericelluar fibrosis, sclerosing hyaline necrosis, NAFLD ballooning, NAFLD activity score (NAS) and NAFLD Stage 4 fibrosis (p<0.001). Patients with alcoholism alone had much more steatosis than those with HBV infection with and without alcoholism (p<0.001).

Discussion
Liver biopsy has been the most sensitive and specific method for evaluating the degree of hepatic injury and fibrosis to help the clinical diagnosis and therapeutic decision [20,21]. In the present results, the patients having concomitant alcoholism and HBV infection developed the histological features of both alcoholic liver disease (steatosis, hepatocyte ballooning, focal necrosis and fibrosis) [17] and viral hepatitis B (piecemeal necrosis, confluent necrosis, focal necrosis, portal inflammation and fibrosis) [19]. Typical histological changes of viral hepatitis B are important in the clinical differentiation from alcoholic liver disease. Furthermore, typical histological changes of alcoholic liver disease were also important in the clinical differentiation from viral hepatitis B in high HBV endemic area, such as Taiwan and Asian Pacific region. To the best of our knowledge, this result has not previously been reported in the literature.  [22,23]. In clinics, our study presents that patients with concomitant HBV infection and alcoholism have high percentages of hepatitis B viral load and high levels of HBV DNA. This could be possibly due to patients with alcoholic liver disease having poor nutrition status and relatively weaker immune status. HBV replication is mediated by immune system and is the key role of immune-mediated liver injury and disease progression [21,24].
HBV infection and heavy alcohol consumption has been recognized as major risk factors for fibrosis, cirrhosis, and HCC [10,24]. Our recent study presented that heavy alcohol consumption significantly increases the risk of HCC in HBV-related cirrhotic patients and elevated baseline serum HBV DNA is a strong risk predictor of HCC [11]. In this study, patients with concomitant HBV infection and alcoholism had much more cirrhosis with Ishak stage 5-6 fibrosis and NAFLD activity score with stage 4 fibrosis than those with HBV infection alone in histological findings. Our study demonstrates that patient with concomitant HBV infection and alcoholism have more advanced fibrosis and cirrhosis.
Alcohol intake increases the prevalence of fatty liver and hepatic steatosis is positively associated with moderate alcohol consumption [25]. Our study presented that patients with alcoholism alone had much more steatosis than those with HBV infection with and without    alcoholism. However, patients with concomitant HBV infection and alcoholism had much less steatosis than those with alcoholism alone. In some studies, HBV infection is associated with a lower prevalence of fatty liver, hypertriglyceridemia and metabolic syndrome [26]. Moreover, hepatitis B viral replication may affect lipid metabolism and the secretion of various adipokines [25][26][27]. Indeed, hepatitis B viral load is negatively associated with hepatic steatosis and may suggest a protective effect on hepatic steatosis in alcohol and non-alcohol induced hepatic steatosis and liver disease.
The current study excludes patients with decompensated liver function, who were unsuitable for liver biopsy. Most alcoholic patients with decompensated liver function developed severe degree of alcoholic hepatitis or cirrhosis clinically. The percentage of patients having alcoholic hepatitis and alcoholic cirrhosis in this study was less than our previous studies and was underestimated.

Conclusion
The patients having concomitant alcoholism and HBV infection had developed the histological features of both alcoholic liver disease and viral hepatitis B. In high HBV endemic area, the histology presents the features of alcoholic liver disease (steatosis, hepatocyte ballooning, and focal necrosis) in patients with HBV infection. These patients should provide full details of their alcohol consumption history and the disease prognosis should be handled with extra care. Furthermore, the assessment of hepatitis B viral activity in alcoholic liver disease depends on detectable viral load and histological features of viral hepatitis B in patients with concomitant HBV infection and alcoholism.