Liver International ISSN 1478-3223
Hepatitis B surface antigen levels after hepatitis B e-antigen seroclearance: a longitudinal follow-up study James Fung1,2,3, Wai-kay Seto1,2, Danny Ka-ho Wong1,2, Ching-lung Lai1,2 and Man-fung Yuen1,2 1 Department of Medicine, The University of Hong Kong, Hongkong, China 2 State Key Laboratory for Liver Research, The University of Hong Kong, Hongkong, China 3 Liver Transplant Centre, Queen Mary Hospital, Hongkong, China
Keywords chronic hepatitis B – flare – HBeAg – HBsAg level – hepatitis
Correspondence Prof Man-Fung Yuen, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Rd, Hong Kong SAR, China Tel: (852) 2255 3984 Fax: (852) 2816 2863 e-mail: [email protected]
Received 29 September 2013 Accepted 13 May 2014 DOI:10.1111/liv.12596 Liver Int. 2015; 35: 854–859
Abstract Background & Aims: The role of quantitative hepatitis B surface antigen (HBsAg) after hepatitis B e-antigen (HBeAg) seroclearance is not well defined. To determine the role of HBsAg levels in predicting significant viremia and hepatitis flares after HBeAg seroclearance. Methods: A total of 228 chronic hepatitis B patients with spontaneous HBeAg seroclearance were included. Patients were followed up regularly at 3–6 monthly intervals with routine liver biochemistry and hepatitis B serology. Levels of HBV DNA and HBsAg were measured at yearly intervals for up to 5 years after HBeAg seroclearance. Results: The median log HBsAg and HBV DNA level after HBeAg seroclearance was 3.52 IU/ml and 4.13 IU/ ml respectively, with no significant correlation observed between them (P = 0.572). The HBV DNA at HBeAg seroclearance was 4.13 log IU/ml, compared with 3.12 log IU/ml after 5 years (P < 0.001). No significant change was observed for HBsAg levels (P = 0.991). Hepatitis B flares occurred in 76 (33.3%) patients. Patients who developed hepatitic flares compared with those without hepatitic flares were older (40 vs. 36 years, P = 0.001), had a higher HBV DNA at the time of HBeAg seroclearance (4.70 vs. 3.77 log IU/ml, P =< 0.001), and more likely to be males (42.7% vs. 23.4%, P = 0.002) respectively. There was no difference in HBsAg levels between those with and without hepatitis flare (3.54 vs. 3.52 log IU/ml respectively, P = 0.555). Conclusion: HBV DNA levels, but not HBsAg levels, after HBeAg seroclearance were associated with subsequent significant viremia and hepatitic flares. Male gender and older age was associated with significant viremia.
In patients with chronic hepatitis B (CHB) infection, up to 40% may develop liver-related complications, including cirrhosis, and hepatocellular carcinoma (1). The natural history of CHB can be categorized into four phases (2). The first phase is known as the immune tolerant phase, which is characterized by a positive hepatitis B e-antigen (HBeAg), high levels of hepatitis B virus (HBV) DNA, normal alanine aminotransferase (ALT) levels and minimal histological inflammatory activity. Loss of immune tolerance marks entry into the immune clearance phase. During this phase, the HBV DNA and ALT levels tends to fluctuate, with histological evidence of inflammatory activity. After HBeAg seroconversion, the HBV DNA levels may remain low with normalization of ALT levels, which is the hallmark of the inactive phase. However, a significant proportion of subjects still have evidence of reactivation with fluctuating ALT and HBV DNA levels. In this reactivation phase, ongoing injury to the liver
occurs, and if untreated, may lead to further worsening of liver function with the development of cirrhosis and its complications. Therefore, it is evident that even after HBeAg seroclearance, when subjects are HBeAg negative, significant disease activity with progression of liver disease can still occur. Previous studies have shown that HBV DNA, older age, genotype C, presence of core promoter mutations and male gender are factors associated with adverse outcome after HBeAg seroclearance (3–5). Over the last few years, there has been an exponential increase of interest in using quantitative HBsAg to predict response to antiviral therapy and long term outcome (6–8). The presence of HBsAg has long been shown to be a risk factor for the development of HCC compared to subjects without HBsAg (9). For CHB patients who clear HBsAg, the survival is improved with lower rates of HCC and liver decompensation (10). The levels of HBV DNA Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Fung et al.
levels have been well established as important predictors of disease progression and outcome (11). In contrast, much less is known about HBsAg levels, and previous studies examining the correlation between HBsAg and HBV DNA levels have been conflicting (12–14). Most of the studies performed are cross-sectional, and there is still a paucity of studies documenting the levels of HBsAg over time in the natural history of CHB. The aim of this study was to determine the role of HBsAg levels in predicting significant viremia and hepatitic flares after HBeAg seroclearance, and to describe the trend of HBsAg levels and its correlation with HBV DNA in a longitudinal study. Patients and methods
Two hundred and twenty-eight treatment-naive CHB patients followed up regularly at the Hepatitis and Liver Clinics, Queen Mary Hospital, The University of Hong Kong were recruited. These patients were recruited based on the evidence of spontaneous HBeAg seroclearance during the follow-up, together with a minimum of 5 years of additional follow-up after HBeAg seroclearance. Patients co-infected with hepatitis C virus or human immunodeficiency viruses were excluded. Those with co-existing liver disease including autoimmune hepatitis, primary biliary cirrhosis and alcoholic liver disease were also excluded. Patients were followed up regularly at 3–6 monthly intervals (or earlier if required), and routine liver biochemistry and hepatitis B serology were performed at each visit. Assessment of liver fibrosis was performed using transient elastography. Genotyping of HBV was performed by PCRsequencing of the HBV S region, followed by phylogenetic comparison with HBV reference sequences using the NCBI HBV genotyping tools as previously described (15). Determination of HBsAg levels
Hepatitis B surface antigen levels were measured at yearly intervals for up to 5 years after spontaneous HBeAg seroclearance using stored sera. The accuracy of HBsAg quantification in stored sera has been documented previously (16). To measure the HBsAg level, the Elecsys HBsAg II assay (Roche Diagnostics, Penzberg, Germany) was used. Briefly, the samples were diluted to 1:400 using a two-step process using the Research Use Only diluents. In the initial step, 20 ll of sample and 380 ll of diluent were used. This is followed by 20 ll of the first dilution mixture and 380 ll of diluent. The diluted serum samples were measured according to the manufacturer’s protocol for HBsAg II. If the cut-off index was greater than 1000, further dilution using three steps of 1:20 dilutions was carried out. If the cut-off index was 29 upper limit of normal together with significant viremia Liver International (2015) © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
95% Confidence interval
1.05 2.88 1.39 1.69
1.01–1.09 1.41–5.91 0.47–4.07 1.32–2.17
0.019 0.004 0.554 2000 IU/ml). The normal upper limit of ALT was defined as 30 U/L in males and 19 U/L in females. In the current cohort, 76 (33.3%) patients developed hepatitic flares. Patients who developed hepatitic flares were older compared to those without flares (40 vs. 36 years respectively, P = 0.001), had a higher HBV DNA at the time of HBeAg seroclearance (4.70 vs. 3.77 log IU/ml respectively, P =< 0.001), and males had a higher proportion compared to females (42.7% vs. 23.4% respectively, P = 0.002). Higher rates of flares were observed in patients with genotype B compared with genotype C virus (50.0% vs. 30.9% respectively, P = 0.022). No difference was observed between anti-HBe positive and anti-HBe negative subjects (33.9% vs. 20.0% respectively, P = 0.502). There was no difference observed in HBsAg levels between those with and without hepatitis flare (3.54 vs. 3.52 log IU/ml respectively, P = 0.555). For those with HBV DNA