Clinical Report

Journal of International Medical Research

41(5) 1732–1739
Hepatitis B surface antigen ! The Author(s) 2013
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DOI: 10.1177/0300060513487643
with chronic hepatitis B imr.sagepub.com

infection: A clinical study

Peng Ruan1, Shao-Yong Xu2, Bo-Ping Zhou3,
Jian Huang3 and Zuo-Jiong Gong1

Abstract
Objective: To investigate the clinical characteristics of hepatitis B surface antigen (HBsAg)
seroclearance in patients with chronic hepatitis B virus (HBV) infection.
Methods: Patients with chronic HBV infection who achieved sustained virological response (SVR)
within 6 years of ceasing formal antiviral treatment were assessed for HBsAg seroclearance
(defined as loss of serum HBsAg on repeated testing for a period of >6 months), using enzyme
immunoassays. Phase of HBV infection and liver function (serum alanine aminotransferase [ALT]
and aspartate aminotransferase [AST] levels) and HBV DNA levels were also assessed.
Results: In total, 272 patients with chronic HBV and SVR were included; HBsAg seroclearance was
achieved in 42 patients and not achieved in 230 patients. Serum HBsAg and ALT levels, ratios of
HBsAg to HBV DNA and ratios of AST to ALT were significantly different between patients
achieving, and not achieving, HBsAg seroclearance. The area under the receiver operating
characteristic (ROC) curve of HBsAg levels for predicting the likelihood of HBsAg seroclearance
was 0.85; the cut-off value was 203.86 IU/ml.
Conclusions: These data demonstrate that HBsAg seroclearance was independently associated
with host immunity, serum HBsAg level, serum ALT level, serum HBsAg to HBV DNA ratio and
timing of drug therapy within the course of chronic HBV infection.

Keywords
Chronic hepatitis B infection, hepatitis B surface antigen, hepatitis B surface antigen seroclearance,
sustained virological response

Date received: 1 March 2013; accepted: 8 March 2013

1
Infectious Diseases Department, Renmin Hospital of
Wuhan University, Wuhan, China Corresponding author:
2
Gastroenterology Department, Renmin Hospital, Hubei Dr Zuo-Jiong Gong, Infectious Diseases Department of
University of Medicine, Shiyan, China Renmin Hospital of Wuhan University, Zhangzhidong Road
3
Institute of Hepatology, Shenzhen Third People’s Hospital, 99, Wuhan 430060, Hubei Province, China.
Guangdong Medical College, Shenzhen, China Email: zjgong@163.com
Ruan et al.
Introduction
Hepatitis B virus (HBV) infection is one of
the most serious global health problems,
with 350–400 million people infected world-
wide.1 Approximately 1 million deaths occur
annually, due to the long-term complica-
tions of HBV infection, which include cir-
rhosis, liver failure and hepatocellular
carcinoma.2 Among the clinical parameters
of HBV infection, hepatitis B surface anti-
gen (HBsAg) has been regarded as a poten-
tial predictor of response to chronic HBV
treatment and as a partial surrogate marker
of HBV covalently closed circular DNA
(cccDNA).3 HBV cccDNA, located in hep-
atocytes, is the intracellular template of
HBV DNA, therefore HBsAg is one of the
subviral replication products of cccDNA.
Obtaining liver tissues from patients in order
to detect intrahepatic cccDNA levels in the
clinical setting is difficult, consequently
serum HBsAg is regarded as a surrogate
marker of this molecule. HBsAg seroclear-
ance is one of the ultimate goals of anti-
HBV therapy, therefore studies have inves-
tigated the predictive value of HBsAg
seroclearance.4,5 However, to date, the clin-
ical characteristics of HBV patients who can
achieve HBsAg seroclearance have not been
fully elucidated. In the present study we
evaluated patient characteristics (including
phase of chronic HBV infection and liver
function) by investigating the differences in
clinical parameters between those who had
achieved HBsAg seroclearance and those
who had not, within 6 years of cessation of
antiviral therapy.
Patients and methods
Patients and treatments
Patients with chronic HBV infection who
had achieved a sustained virological
response (SVR; defined as undetectable
serum HBV DNA for a period of at least 6
months),6 within a 6-year time period
between July 2006 and July 2012, were
1733
eligible for study inclusion. The study was
carried out in the Infectious Diseases
Department of the Shenzhen Third People’s
Hospital, Shenzhen, Guangdong, China.
Patients were excluded if they were coin-
fected with hepatitis delta virus, hepatitis C
virus, or human immunodeficiency virus;
tests for these viruses were undertaken using
standard laboratory methods. Patients with
Wilson’s disease, autoimmune hepatitis or
primary biliary cirrhosis, and those with a
substantial intake of alcohol (20 g per day
for female patients; 30 g per day for male
patients) were also excluded.
Hepatitis B surface antigen seroclearance
was defined as loss of serum HBsAg on
repeated testing for a period of >6 months
and during all subsequent follow-ups until
time of analysis.7 During the study period,
patients were treated with nucleoside ana-
logues (adefovir, 10 mg/day or lamivudine,
100 mg/day), either as monotherapy or with
pegylated interferon-a (100 mg/week) for >6
months, according to clinical practice
guidelines.7
All patients were HBV treatment naı̈ve
before enrolment, had serum HBV DNA
levels 100 copies/ml, and had been persist-
ently HBsAg positive for >6 months.
Patients gave verbal consent to enter the
study and the study itself was approved by
the Ethics Committee of Shenzhen Third
People’s Hospital
Laboratory measurements
Serum HBsAg and Hepatitis Be antigen
(HBeAg) levels were quantified through
enzyme immunoassays (ARCHITECT plat-
form; Abbott Laboratories, Abbott Park,
IL, USA) according to the manufacturer’s
instructions.
Serum alanine aminotransferase (ALT)
was measured using the ALAT (GPT) FS kit
and aspartate aminotransferase (AST) levels
were measured using the ASAT (GOT) FS
kit (both from DiaSys Diagnostic Systems,
1734
Holzheim, Germany), according to the
manufacturer’s instructions. Serum HBV
DNA levels were quantified using the
CobasÕ TaqManÕ real time–polymerase
chain reaction analyser (Roche Diagnostics,
Indianapolis, IN, USA), as previously
described.8 As HBV genotyping can influ-
ence HBV replication in patients with
chronic HBV infection, HBV genotyping
(B/C) was performed by restriction fragment
length polymorphism9 using the restriction
enzyme Tsp509I (obtained from MBI
Fermentas, Canada) in 120 patients. Not
all patients could be tested, for various
reasons including cost and insufficient HBV
DNA samples. Patients were categorized
according to their phase of chronic HBV
infection, as follows: immune tolerance (IT);
immune clearance (IC); HBeAg negative
low-replicative (LR); HBeAg negative hepa-
titis (ENH).
Statistical analyses
Continuous variables were expressed as the
mean (range) and were analysed using
nonpaired Student’s t-test or Mann–
Whitney U-test, as appropriate. HBV
DNA (copies/ml) and HBsAg (IU/ml) were
logarithmically transformed for analysis.
Area under the receiver operating charac-
teristic (ROC) curve analysis was used to
predict the likelihood of HBsAg seroclear-
ance. The Kaplan–Meier method (using a
log-rank test) was applied for the cumulative
rates of SVR and HBsAg seroclearance.
Statistical analyses were performed using
SPSSÕ software, version 13.0 (SPSS Inc,
Chicago, IL, USA). All statistical tests were
two-sided, and statistical significance was
achieved at the 5% level (P < 0.05).
Results
Data were available from 272 patients with
chronic HBV infection. The main patient
characteristics and values for all parameters
Journal of International Medical Research 41(5)
studied are shown in Table 1. In total, 190
male and 40 female patients did not achieve
HBsAg seroclearance, and 30 male and 12
female patients (median age 32.5 years;
range 11–49 years) achieved HBsAg
seroclearance.
Patients not achieving HBsAg seroclear-
ance were categorized according to their
phase of chronic HBV infection (IT, IC,
LR, ENH), as shown in Table 1. There was a
significant difference in the distribution of
the phase of chronic HBV infection between
the group achieving seroclearance and the
group not achieving seroclearance.
(P ¼ 0.00243). HBsAg seroclearance had no
statistically significant association with age,
sex, HBV genotype, log10HBV DNA or AST
level. Patients achieving HBsAg seroclear-
ance had significantly lower baseline ratios
of AST/ALT (P ¼ 0.004), log10HBsAg/
log10HBV DNA (P < 0.0001), log10HBsAg
(P < 0.0001) and significantly higher base-
line ALT levels (P ¼ 0.016), compared with
those not achieving HBsAg seroclearance.
The area under the ROC curve of
HBsAg levels for predicting the likelihood
of HBsAg seroclearance was 0.85 (95%
CI: 0.77–0.93; P < 0.0001). These findings
indicate that patients with chronic
hepatitis B infection whose serum HBsAg
levels were <203.86 IU/ml (which was
defined as the cut-off value; HBsAg negative
was defined as < 0.05 IU/ml) may likely
achieve HBsAg seroclearance in the
following 6 years. The cut-off value had
a sensitivity of 83.9%, specificity of
81.0%, positive predictive value of 83.3%
and negative predictive value of 86.1%
(Figure 1).
Cumulative rate analysis showed that the
SVR rate was significantly higher in patients
achieving HBsAg seroclearance compared
with those not achieving seroclearance. In
the group that went on to eliminate HBsAg,
100% of patients had achieved SVR by 53
months, compared with 99.1% achieving
SVR by 60 months, in those who did not go
Ruan et al. 1735

Table 1. Clinical parameters of patients with chronic hepatitis B virus (HBV) infection, stratified according
to those who did not achieve hepatitis B virus surface antigen (HBsAg) seroclearance (HBsAg seropositive)
and those who did achieve HBsAg seroclearance (HBsAg seronegative).

Parameter HBsAg seropositive, n ¼ 230 HBsAg seronegative, n ¼ 42 P-value

Age, years 34.0 (12–59) 32.5 (11–49) NSa

Male/female 190/40 30/12 NSb
HBV genotype, B/C 60/26 28/6 NSb
HBV phase, nb

IT 73 (32.74) 6 (14.29) 0.00243

IC 49 (21.30) 20 (47.62)
LR 32 (12.92) 6 (14.29)
ENH 76 (33.04) 10 (23.81)
ALT, U/l 127.46 (8–2600) 364.18 (11–2194) 0.016c
AST, U/l 86.33 (13–1726) 177.10 (8–1370) NSc
AST/AL 0.92 (0.27–3.20) 0.70 (0.16–1.25) 0.004a
Log10HBs, IU/ml 2.87 (0.85–4.36) 1.77 (0.004–4.73) <0.0001a
Log10HBV DNA, IU/ml 5.60 (2.70–8.98) 5.38 (2.42–8.12) NSa
Log10HBsAg/HBV DNA 0.58 (0.24–1.41) 0.35 (0.001–0.64) <0.0001a

Data presented as n, n (%) or mean (range).

ALT, alanine aminotransferase (normal range 5–40 U/l); AST, aspartate aminotransferase (normal range 5–40 U/l).
Patients were categorized according to their phase of chronic HBV infection, as follows: immune tolerance (IT); immune
clearance (IC); HbeAg-negative low-replicative (LR); HbeAg-negative hepatitis (ENH). HBsAg negative: 0–0.05 IU/ml; HBV
DNA-negative: <100 copies/ml.
a
Nonpaired Student’s t-test.
b 2
 -test.
c
Mann–Whitney U-test.

on to achieve HBsAg seroclearance Newly synthesized envelope protein inter-

(P < 0.0001; Figure 2).
In patients achieving HBsAg seroclear-
ance, a serum ALT level >80 U/l was
significantly associated with HBsAg sero-
clearance: HBsAg seroclearance cumulative
percentage rates at 12, 24, 48 and 60 months
were 68.18%, 77.27%, 86.36% and 95.45%,
respectively, in patients with serum ALT
levels 80 U/l, and 15%, 30%, 60% and
95%, respectively, in patients with serum
ALT levels <80 U/l (P ¼ 0.01; Figure 3).
Discussion
Hepatitis B serum antigens are generated
from two HBV mRNA transcripts, with
subsequent translation resulting in small,
medium and large surface envelope proteins.
acts with mature HBV nucleocapsids at the
endoplasmic reticulum, prior to being
secreted from the hepatocyte.10 Studies
have documented that patients who achieve
HBsAg seroclearance before the develop-
ment of cirrhosis have a considerably more
favourable long-term prognosis compared
with those who remain HBsAg positive,
even when HBV DNA can still be detected
within their hepatocytes.11
In our study, serum HBsAg levels in
patients achieving HBsAg seroclearance
were significantly lower than those in
patients not achieving HBsAg seroclear-
ance. This finding suggests that transcrip-
tion of cccDNA for surface envelope
proteins in the former may be suppressed
by various intrinsic factors, such as degree of
1736
Figure 1. Receiver operating characteristic curve
of serum hepatitis B virus surface antigen (HBsAg)
levels for predicting the likelihood of HBsAg
seroclearance in patients with chronic hepatitis B
virus infection. Patients with chronic hepatitis B
infection whose serum HBsAg levels were
<203.86 IU/ml (defined as the cut-off value; HBsAg
negative was defined as <0.05 IU/ml) may likely
achieve HBsAg seroclearance in the following 6
years. The cut-off value had a sensitivity of 83.9%,
specificity of 81.0%, positive predictive value of
83.3% and negative predictive value of 86.1%.
cccDNA methylation or acetylation of the
surrounding histones.12–14 While our obser-
vation is consistent with other preliminary
studies, our cut-off value for the serum
HBsAg level was higher than that reported
elsewhere (203.86 IU/ml in the present
study, versus 100 IU/ml elsewhere).15,16
This may be due to our recruitment of
greater numbers of patients who went on
to achieve HBsAg seroclearance.
As HBV genotype can influence HBV
replication in patients with chronic HBV
infection, HBV genotyping was performed.9
However, no significant difference was found
in the genotyping of the two groups and we
believe this had no influence on the results.
Our data indicate that the immunity of
patients achieving HBsAg seroclearance
may be more robust than that of patients
not achieving HBsAg seroclearance. On one
hand, the percentage of patients achieving
HBsAg seroclearance who were in the IC
phase of HBV infection was significantly
Journal of International Medical Research 41(5)
Figure 2. Cumulative rates of sustained viro-
logical response (SVR) according to hepatitis B
surface antigen (HBsAg) seroclearance, in patients
with chronic hepatitis B (CHB) virus infection: 100%
of those who eliminated HBsAg achieved SVR by 53
months; 99.1% of those who did not go on to
achieve HBsAg seroclearance achieved SVR by 60
months (P<0.0001).
greater than the percentage in IC who did
not achieve HBsAg seroclearance (47.62%
versus 21.30%). In addition, the percentages
of patients in the IT and ENH phases were
also substantially higher in those in the
seropostive group (32.74% and 33.04%,
respectively) compared with those in the
seronegative group (14.29% and 23.81%,
respectively). Patients with chronic HBV
infection who transform spontaneously
into the IC phase have been described as
generally having a ‘potent’ immune
response.17 Conversely, it was confirmed
that the function of natural killer cells may
be impaired in IT phase,18 and the emer-
gence of viral mutations (such as BCP/PC
variants in ENH phase) may result in
immune escape.19 It was also suggested
that increased serum ALT levels and a low
AST/ALT ratio predict potent anti-HBV
immunity in patients with chronic HBV;20
these traits were both identified in the
Ruan et al.
Figure 3. Cumulative rates of hepatitis B virus
surface antigen (HBsAg) seroclearance according to
liver function, in patients with chronic hepatitis B
(CHB) virus infection. HBsAg seroclearance cumu-
lative percentage rates at 12, 24, 48 and 60 months
were 68.18%, 77.27%, 86.36% and 95.45%, respect-
ively, in patients with serum alanine aminotransfer-
ase (ALT) levels 80 U/l, and 15%, 30%, 60% and
95%, respectively, in patients with serum ALT levels
<80 U/l (P ¼ 0.01).
patients achieving HBsAg seroclearance in
our study.
Contrary to our data, other research has
indicated that there was no significant dif-
ference in serum ALT levels between
patients achieving, and not achieving,
HBsAg seroclearance.16 This discrepancy
may be due to the fact that other research
has focused on spontaneous HBsAg
seroclearance and did not take into
account the effects of drug therapy.
Pharmacotherapeutic choices may be influ-
ential in determining the rate of HBsAg
seroclearance in patients with chronic HBV
infection. Compared with combination ther-
apy of nucleoside analogue and interferon-
a, nucleoside analogue therapy alone has a
limited impact on HBsAg seroclearance.21
Prolonged drug use may lead to mutations
and systemic side-effects, and premature
termination of drug therapy can result in
1737
resurgence of serum HBsAg and HBV-DNA
levels. Otherwise, cccDNA is the intracellu-
lar template for HBsAg production and,
although no available drugs can completely
eliminate existing cccDNA in the nuclei of
the infected hepatocyte, there may be differ-
ential treatment effects at different hepato-
cyte proliferation phases (for example the
initial proliferation, exponential prolifer-
ation and growth confluency phases).22
Several lines of evidence have suggested
that HBV replication is highly dependent on
the growth status of hepatocytes; clinical
specimens have shown low levels of intrahe-
patic or serum HBV DNA during severe
acute hepatitis at the time of active liver
regeneration, representing a stage of rapid
cell growth.23 Cell culture data indicate that
lamivudine can significantly eliminate
cccDNA in the exponential proliferation
phase compared with other phases, possibly
due to a dilution effect.24 Another study
demonstrated that high serum ALT levels
may predict damage to hepatocytes, result-
ing in rapid cell proliferation.25 Cumulative
rate analysis in our study demonstrated that
patients achieving HBsAg seroclearance
also achieved SVR earlier than those who
did not. In the former, patients with a higher
serum ALT level (80 U/l) acquired HBsAg
seroclearance earlier than those with a lower
serum ALT level <80 U/l, indicating that
drug therapy during the rapid cell prolifer-
ation period may have a positive effect in
achieving HBsAg seroclearance.
The serum HBsAg/HBV DNA ratio
reflects the association between HBsAg pro-
duction and HBV replication in patients
with chronic HBV. Research has found that
the ratio generally ranged between 0.5 and
0.6, and was significantly higher in HBeAg-
negative patients than in HBeAg-positive
patients.10 This may be due to the fact that
HBsAg production is preserved under pref-
erential immunity pressure in HBeAg-
negative patients;3 alternatively it may be
due to HBV integration in the host
1738
genome.20 One study observed that a large
number of HBV integration events was
positively associated with serum HBsAg
levels according to whole genome sequen-
cing.26 Another paper reported that HBeAg-
negative patients usually had a longer HBV
infection history, and were expected to have
more extensive HBV DNA integration,
compared with HBeAg-positive patients.27
While the serum HBsAg to HBV DNA ratio
in patients not achieving HBsAg seroclear-
ance in our study (0.58) was similar to that
observed by others (0.5–0.6), it was signifi-
cantly lower in patients achieving HBsAg
seroclearance (0.35). This may be due to
enhanced host immunity in relation to the
control of subviral production, or because
fewer HBV integration events occurred in
patients achieving HBsAg seroclearance
than in those not achieving HBsAg sero-
clearance, as the proportion of HBeAg
negative patients was significantly higher in
the latter group, in our study.
In conclusion, our study identified several
clinical characteristics of patients with
chronic HBV infection that may help to
predict those most likely to achieve HBsAg
seroclearance after antiviral treatment.
These characteristics include having a vig-
orous immune response, having low serum
HBsAg levels (cut-off value, 203.86 IU/ml),
having high serum ALT levels and having a
low serum HBsAg to HBV DNA ratio.
Appropriate antiviral therapy during the
rapid cell-proliferation period may also be
helpful in achieving HBsAg seroclearance.
Acknowledgements
The authors thank XC Chen and M Wang from
the Institute of Hepatology, Shenzhen Third
People’s Hospital for their technical assistance.
Declaration of conflicting interest
The authors declare that there are no conflicts of
interest.
Journal of International Medical Research 41(5)
Funding
This research received no specific grant from any
funding agency in the public, commercial, or not-
for-profit sectors.
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