Pathological Features and Prognosis in Chronic Hepatitis B Virus Carriers

The Journal of International Medical Research 2011; 39: 71 77 Pathological Features and Prognosis in Chronic Hepatitis B Virus Carriers ZH LU, W CHEN, ZC JU, H PEI, XJ YANG, XB GU AND LH HUANG Department of Epidemiology, Wuxi Infectious Disease Hospital, Wuxi, China This retrospective study examined 220 Chinese chronic hepatitis B virus carriers over 5 years. After initial liver biopsy, liver function tests and serological analysis, patients underwent further tests of liver function and hepatitis B seromarkers at 6- month intervals. Second and third liver biopsies were performed in 56 and 23 patients, respectively. Liver pathology was classified according to inflammatory activity (G 0 G 4 ) and degree of fibrosis (S 0 S 4 ). A significantly greater proportion of hepatitis B e antigen antibody-positive patients had a more severe level of inflammation and fibrosis than patients who were hepatitis B e antigen (HBeAg)- positive. Abnormal inflammation ( G 2 ) occurred in 122 (55.5%) patients. Hepatitis B reactivation occurred in 35 (15.9%) patients: 33 had obvious liver inflammation at the initial biopsy ( G 2 ) and only two had a low level of liver inflammation (G 0 ). The hepatitis B reactivation rate was significantly related to age but not to gender. Hepatitis B surface antigen clearance was 1.55% per year and HBeAg seroconversion was 5.36% per year. In conclusion, hepatitis B reactivation was closely correlated with age and the level of liver inflammation. KEY WORDS: CHRONIC HEPATITIS B VIRUS CARRIER; HEPATITIS B REACTIVATION; LIVER BIOPSY; LIVER PATHOLOGY Introduction Chronic hepatitis B infection is a global public health problem that is estimated to cause between 500 000 and 1.2 million deaths annually. 1 3 In China, about 10% of the population is positive for hepatitis B surface antigen (HBsAg); this proportion has declined over time. Alanine amino - transferase (ALT) levels are often normal for a long time in patients who are HBsAgpositive; 3 some HBsAg-positive patients have occult hepatitis B virus (HBV) infection. 3 In 2007, reactivation of hepatitis B was defined in the American Association for the Study of Liver Diseases Practice Guidelines 4 as the reappearance of active necroinflammatory disease in the liver of a person who is known to be an inactive HBsAg carrier or who has resolved hepatitis B. Such patients have clinical symptoms and signs, such as weakness, anorexia, abnormal liver function tests and hepatosplenomegaly. Some will eventually develop hepatic failure, hepatocirrhosis and primary hepatocellular carcinoma. 5 This retrospective study examined the records of 220 chronic HBV carriers in Wuxi, China, over a 5-year period, observing their clinical symptoms, liver biopsy, liver function and viral seromarker results. 71

Patients and methods PATIENTS Chronic HBV carriers presenting as outpatients at Wuxi Infectious Disease Hospital, Wuxi, China, from January 2001 to December 2003 were identified and their records over the subsequent 5-year period were examined. All patients underwent family history and clinical symptom inquiry, laboratory assessment (serum analysis for liver function, HBV seromarkers and HBV DNA), and histopathological assessment (liver biopsy) at their initial outpatient visit. The diagnosis of chronic HBV carrier was based on the criteria set out by the Chinese Medical Association. 6 Patients with other hepadnavirus infections, toxic or druginduced liver disease, alcoholic liver disease or fatty liver were excluded from the study. The study protocol was approved by the Ethics Committee of Wuxi Infectious Disease Hospital. Patients provided verbal permission for their data and records to be used in this study. HISTOPATHOLOGICAL ASSESSMENT Liver biopsy involved the 1-second procedure using a Tru-cut needle (HS Hospital Service, Aprilia, Italy), removing at least 1.5 cm of liver tissue. The samples were fixed in 4% neutral formaldehyde, embedded in paraffin and cut into 5 µm thick sections. Haematoxylin and eosin staining, reticular fibre staining and Masson staining were then performed sequentially. Histopathological assessment was made according to the criteria established by the Chinese Medical Association in 2000: 6 level of inflammatory activity was classified as G 0 G 4 and the degree of fibrosis was classified as S 0 S 4. 7,8 All slides were examined independently by two pathologists. LABORATORY ASSESSMENT Blood samples were taken from all patients for laboratory analysis. Liver function test (measurement of ALT) was performed using a fully automated biochemical analyser (Abbott Laboratories, Chicago, IL, USA). HBV seromarkers, including HBsAg, antibody to HBsAg (anti-hbs), hepatitis B e antigen (HBeAg), antibody to HBeAg (anti-hbe) and antibody to hepatitis B core antigen (anti- HBc), were detected by enzyme-linked immunosorbent assay (ELISA) using a fully automated ELISA analyser (Abbott Laboratories). The level of HBV DNA was detected using quantitative fluorescent polymerase chain reaction (PCR) (LightCycler PCR system; Roche, Indianapolis, IN, USA); a negative result for HBV DNA was defined as < 5 10 2 copies/ml. FOLLOW UP Patients were followed up at 6-month intervals for 5 years. Each visit included symptom inquiry and serum analysis for liver function and HBV seromarkers. Some patients underwent a second or third liver biopsy to monitor changes in liver inflammation and fibrosis. Patients with inflammatory activity G 2 were advised to rest and were given vitamin and silymarin preparations. 9 Patients with high viral titres of HBV DNA (> 10 5 copies/ml) were given antiviral agents, mainly nucleoside analogues. 10,11 All other patients maintained their normal daily activities and were not given any drug treatment. STATISTICAL ANALYSIS Between-group differences were analysed using the rank sum test or the χ 2 test. A P- value < 0.05 was considered to be statistically significant. Results PATIENTS A total of 220 chronic HBV carriers were 72

included in the study. Of these, 156 (70.9%) were male and 64 (29.1%) were female. The patients ages at the first visit ranged from 12 to 62 years and their HBsAg-positive history ranged from 1 to 25 years in duration. None of the patients had received any drug treatments and 147 (66.8%) had a family history of hepatitis B. LIVER FUNCTION AND HBV SEROMARKERS A total of 168 patients (76.4%) were positive for HBeAg and all these had normal ALT levels throughout the follow-up period. The remaining 52 patients (23.6%) were positive for anti-hbe. There were also 135 patients (61.4%) who were positive for anti-hbc. HBV DNA was detected in 93 patients (42.3%) and, of these 51 (54.8%) had HBV DNA levels > 4 10 3 copies/ml and were considered to be HBV DNA-positive. Viral titres > 10 5 copies/ml were found in 14 patients and they were given antiviral agents, mainly nucleoside analogues. 10,11 LIVER PATHOLOGY The level of inflammatory activity and the degree of liver fibrosis in the initial liver biopsies in the 220 chronic HBV carriers are given in Tables 1 and 2, respectively, divided according to their HBeAg status. Inflammatory activity was abnormal ( G 2 ) in 55.5% of patients. A significantly greater proportion of anti-hbe-positive patients had a more severe level of inflammation and fibrosis than the HBeAg-positive patients (χ 2 = 8.6822, P = 0.0032 for inflammation; χ 2 = 6.8382, P = 0.0089 for fibrosis). TABLE 1: Level of liver inflammatory activity according to hepatitis B e antigen status in chronic hepatitis B virus carriers Level of inflammation Status n G 0 G 1 G 2 G 3 G 4 HBeAg-positive 168 20 (11.9) 61 (36.3) 57 (33.9) 20 (11.9) 10 (6.0) Anti-HBe-positive 52 4 (7.7) 13 (25.0) 12 (23.1) 17 (32.7) 6 (11.5) Total 220 24 (10.9) 74 (33.6) 69 (31.4) 37 (16.8) 16 (7.3) Data presented as number, n (%) of patients. Between-group statistical significance by rank sum test (χ 2 = 8.6822, P = 0.0032). HBeAg, hepatitis B e antigen; anti-hbe, antibody to hepatitis B e antigen. TABLE 2: Degree of liver fibrosis according to hepatitis B e antigen status in chronic hepatitis B virus carriers Degree of fibrosis Status n S 0 S 1 S 2 S 3 S 4 HBeAg-positive 168 17 (10.1) 57 (33.9) 54 (32.1) 25 (14.9) 15 (9.0) Anti-HBe-positive 52 5 (9.6) 10 (19.2) 13 (25.0) 14 (26.9) 10 (19.3) Total 220 22 (10.0) 67 (30.5) 67 (30.5) 39 (17.7) 25 (11.3) Data presented as number, n (%) of patients. Between-group statistical significance by rank sum test (χ 2 = 6.8382, P = 0.0089). HBeAg, hepatitis B e antigen; anti-hbe, antibody to hepatitis B e antigen. 73

When patients were divided according to age ( 40 years and < 40 years), there was no significant difference between the two groups in the proportions of patients with a G 0 versus a G 2 level of liver inflammation. The degree of liver fibrosis, however, was significantly different (χ 2 = 7.37, P < 0.01); a significantly greater proportion of patients aged 40 years had more severe liver fibrosis than those aged < 40 years (Table 3). HEPATITIS B REACTIVATION During the 5-year follow-up period, 35 of the 220 patients (15.9%) showed symptoms of hepatitis B reactivation, including clinical weakness, anorexia, abnormal liver function tests and hepatosplenomegaly. Four were amongst the 69 patients with an initial G 2 level of inflammation (5.8% of those with an initial G 2 level), 21 were amongst the 37 patients with an initial G 3 level of inflammation (56.8% of those with an initial G 3 level) and eight were amongst the 16 patients with an initial G 4 level of inflammation (50.0% of those with an initial G 4 level). Only two were amongst the 98 patients originally classified as G 0 (2.0% of those with an initial G 0 level). Analysis using the χ 2 test revealed that the incidence of hepatitis B reactivation was statistically related to the level of liver inflammation (G 0 1 versus G 2, χ 2 = 25.41, P < 0.01). Among the 35 patients who showed hepatitis B reactivation, 27 (77.1%) were aged 40 years. A χ 2 test analysis demonstrated that the relationship between age and hepatitis B reactivation was statistically significant (χ 2 = 6.72, P < 0.01). Twenty-two of the 35 patients (62.9%) with hepatitis B reactivation were male; amongst the 220 patients studied, hepatitis B reactivation was seen in 22 out of 156 (14.1%) of the male patients and in 13 out of 64 (20.3%) of the female patients. There was no statistically significant relationship between hepatitis B reactivation in chronic HBV carriers and gender. CHANGES IN LIVER PATHOLOGY Further liver biopsies were performed in a number of patients 2 4 years after the first biopsy. Fifty-six patients underwent a second liver biopsy. Of these, 30 patients had G 0 liver inflammation in the first liver biopsy and 26 still showed G 0 inflammation at the second biopsy, three patients had progressed to G 2, and one patient showed G 3 levels of inflammation. Among the 14 patients with G 2 liver inflammation in the first liver biopsy, seven patients still showed G 2 inflammation, five patients had progressed to G 3 and two patients had progressed to G 4. Among the seven patients TABLE 3: Level of liver inflammatory activity and degree of liver fibrosis according to age in chronic hepatitis B virus carriers Level of inflammation Degree of fibrosis Age n G 0 G 2 S 0 S 1 S 2 < 40 years 126 59 (46.8) 67 (53.2) 56 (44.4) 70 (55.6) 40 years 94 39 (41.5) 55 (58.5) 25 (26.6) 69 (73.4) Total 220 98 (44.5) 122 (55.5) 81 (36.8) 139 (63.2) Data presented as number, n (%) of patients. Between-group statistical significance by χ 2 test (χ 2 = 7.37, P < 0.01) for degree of fibrosis; no betweengroup statistical significance by χ 2 test for level of inflammation. 74

with G 3 liver inflammation in the first liver biopsy, six patients still showed G 3 inflammation and one patient had improved to G 2. Among the five patients with G 4 in the first liver biopsy, two patients still showed G 4 inflammation and two had improved to G 3. Twenty-three patients underwent a third liver biopsy. Of these, 10 patients had G 2 levels of inflammation in the second liver biopsy and eight still showed G 2 inflammation in the third biopsy whereas two had improved to G 1. Among the 11 patients with G 3 levels of inflammation in the second liver biopsy, two still showed G 3 levels in the third biopsy, whereas the remaining nine had improved to G 2 or G 1 inflammation levels. Among the two patients with G 4 levels of inflammation in the second liver biopsy, one patient had improved to G 3 and another to G 2 in the third liver biopsy. CHANGES IN HBV SEROMARKERS Clearance of HBsAg occurred in only 17 of the 220 chronic HBV carriers studied over the course of 5 years, giving an HBsAg clearance rate of 1.55% per year. HBeAg seroconversion occurred in 45 of the 168 patients who were HBeAg-positive at the start of the study, giving an HBeAg clearance rate of 5.36% per year. Among these 45 patients, HBeAg seroconversion occurred spontaneously in 36 patients and after nucleoside analogue treatment, given on the basis of high viral titres of HBV DNA, in the other nine patients. Discussion With raised health awareness in recent years, chronic HBV carriers are increasingly wanting treatments that are less toxic. There is also still an active debate regarding whether chronic HBV carriers need treatment at all and, if so, how. 12 Liver inflammation and fibrosis in chronic HBV carriers can be easily diagnosed using the 1-second liver biopsy, 13,14 which can help doctors to make a correct assessment of disease state and, therefore, the treatment needs of the patient. 4,15 17 In addition, the 1-second liver biopsy is easy to perform, causes little damage and has few complications, improving the likelihood that patients will be willing to undergo the procedure. There are few reports on the long-term follow up of chronic HBV carriers worldwide. Brechot et al. 18 studied the relationship between heavy use of alcohol and hepatitis B reactivation in chronic HBV carriers. They concluded that alcohol was associated with liver cell denaturalization and immune function depression, which provided an opportunity for HBV to survive and replicate. They emphasized that alcoholic liver damage was just one factor in the reactivation of hepatitis B. Some patients eventually developed hepatocirrhosis or hepatic carcinoma. Chen s research group 19,20 demonstrated that long-term high titres of serum HBV DNA but not ALT were an independent risk factor for progression from chronic hepatitis B to primary hepatic carcinoma. Gigi et al. 21 performed a liver biopsy in 35 chronic HBV carriers. In 32 patients the histological activity index was > 4; of these, 22 patients were given antiviral agents and their health improved. Among the 220 chronic HBV carriers in the present 5-year study, 35 patients (15.9%) developed clinical symptoms and changes in liver function related to hepatitis B reactivation. There was a close correlation between hepatitis B reactivation and liver inflammation, with reactivation eventually occurring in over half of the patients with severe liver inflammation ( G 3 ). Analysis of the data showed that age but not gender 75

were significantly related to hepatitis B reactivation in chronic HBV carriers. In the present study, only 10.9% of patients had inflammatory activity classified as G 0 and 10.0% had liver fibrosis classified as S 0. Although most of the chronic HBV carriers had mild or severe liver inflammation and fibrosis, 11.3% of the patients had histological cirrhosis (S 4 ) despite having no clinical symptoms, normal serology and normal appearance of the liver. More anti-hbe-positive patients than HBeAg-positive patients had higher levels of inflammation and fibrosis, indicating that the pathological changes were not markedly alleviated by seroconversion. This may be due to the induction of immune resistance by HBeAg, allowing HBV to escape immune clearance. It may be that, in anti-hbepositive patients, clearance of serum HBeAg leads to an increase in cytotoxic T-cells, which invade the liver and attack infected liver cells, aggravating liver damage, which is in agreement with other research performed in China. 22,23 When analysed according to age, there was no significant difference between patients of age 40 years and those < 40 years in the proportions of patients with a G 0 versus a G 2 level of liver inflammation, whereas a significantly greater proportion of the older group had a higher degree of liver fibrosis compared with the younger group. This may be due to occult deterioration. At the immune clearance stage and the inactive or low replication stages, liver inflammation is repaired, whereas fibrotic tissue remains, and this is consistent with the observations of Fan et al. 24 The results of subsequent liver biopsies during follow up indicated that patients with normal liver tissue in the initial liver biopsy were usually stable and their liver tissue could remain normal for several years. Once liver damage appeared, as evidenced by G 3 or G 4 levels of inflammation, liver function became abnormal; this condition could last for a long time and rarely showed recovery, although a few patients with liver inflammation at the G 3 or G 4 level appeared to show a degree of histological recovery. The G 2 level of liver inflammation was an unstable status, with five out of 14 patients progressing to G 3 and two progressing to G 4 by the time of the second liver biopsy. It is clear, therefore, that patients with G 2 liver inflammation need to be closely followed, in particular with liver biopsies, to detect the development of cirrhosis. 12,25 During follow-up, a few patients showed spontaneous clearance of HBsAg together with HBeAg seroconversion. This could be due to the effect of enhanced immune function over the course of the disease. These patients mostly only had minor abnormalities in the initial liver biopsy and the second liver biopsy usually indicated alleviation of liver inflammation though not to an absolutely normal level. In conclusion, hepatitis B reactivation in chronic HBV carriers was closely correlated with age and the level of liver inflammation. Conflicts of interests The authors had no conflicts of interest to declare in relation to this article. Received for publication 26 July 2010 Accepted subject to revision 31 August 2010 Revised accepted 21 October 2010 Copyright 2011 Field House Publishing LLP References 1 Lavanchy D: Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J 76

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