Journal of Antimicrobial Chemotherapy (2006) 58, 1031–1035
Advance Access publication 19 September 2006
Sequential treatment with lamivudine and interferon-a
monotherapies in hepatitis B e antigen-negative Chinese patients
and its suppression of lamivudine-resistant mutations
Ming Shi1,2, Rong Sheng Wang3, Hua Zhang3, Yu Fen Zhu3, Bei Han3, Yong Zhang2,
Li Ji Jin1, Zhi-Jun Yang4and Yong Ping Xu1*
1Department of Biotechnology, Dalian University of Technology, Dalian 116023,
Liaoning Province, China;2No. 6 Hospital of Dalian, Dalian 116001, Liaoning Province, China;
3Jiangsu Oil Field Hospital, Shaobo, Jiangdu 225261, Jiangsu Province, China;4Management School,
Shanghai Jiaotong University, Shanghai, China
Received 23 April 2006; returned 5 June 2006; revised 16 August 2006; accepted 30 August 2006
Objectives: To assess the efficacy of sequential treatment with lamivudine and interferon-a
monotherapies in Chinese patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B.
Methods: One hundred and sixty-two patients with HBeAg-negative chronic hepatitis B were included
fourwere treatedwith lamivudinealone (100mgper day)for20weeks, then combinedwithinterferon-a-2b
Results: After 48 weeks of treatment, the percentage of patients with normalization of alanine
aminotransferase (ALT) levels or hepatitis B virus (HBV) DNA levels below 1000 copies/mL was not
significantly different between the lamivudine monotherapy group (55.10% and 55.10%, respectively)
and the sequential treatment group (59.36% and 56.25%, respectively). The percentage of patients
with normalized ALT levels was significantly higher in group A (53%) than in group B (36%) at week 72
(P < 0.05). The percentage of patients with lamivudine-resistant mutations was significantly higher with
lamivudine monotherapy (22.45%) than with sequential therapy (P < 0.05).
Conclusions: Sequential treatment of chronic hepatitis B with lamivudine and interferon-a monothera-
pies is as effective as lamivudine-alone treatment in Chinese patients. However, sequential treatment
can significantly suppress the emergence of lamivudine-resistant mutations.
Keywords: chronic hepatitis B, hepatitis B virus, HBV, YMDD
It is estimated that 350 million individuals are chronically
infected with hepatitis B virus (HBV).1Carriers of HBV are at
increased risk of developing cirrhosis, hepatic decompensation
and hepatocellular carcinoma (HCC).2The long-term aim in the
treatment of these patients is to prevent or at least to decrease the
risk of the development of cirrhosis and HCC. However, this
long-term aim is unpractical for clinical use. In clinical trials with
follow-up of 1 year or less after therapy, more realistic short-term
objectives are usually used. The most widely used short-term
objectives are normalization of serum alanine aminotransferase
(ALT) levels and reduction of serum HBV DNA levels,3,4which
can reflect biochemical improvement of the liver and suppression
of the virus replication.
The agents currently used or under investigation for the
treatment of chronic hepatitis B can be broadly divided into two
main groups: immunomodulators and nucleoside analogues.5
Interferon-a is the best known of the immunomodulators and
lamivudine is the most extensively studied nucleoside analogue.
Interferon is effective in white patients but the results in Asian
tion of serum ALT levels, loss of hepatitis e antigen (HBeAg)
and reduction of serum HBV DNA.8–11However, long-term
*Corresponding author. Tel: +86-411-82718511 3083; Fax: +86-411-82714773; E-mail: firstname.lastname@example.org
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by guest on June 13, 2013
lamivudine therapy will lead to the emergence of a mutant
YMDD (tyrosine, methionine, aspartate and aspartate) motif
of the HBV polymerase gene.8,12,13With the emergence of
these mutant virus strains, there is increasing resistance to the
therapeutic effects of lamivudine. In several studies, the
combination of lamivudine and interferon-a has been used to
overcome this emergence of resistance to therapy14–17and this
strategy can reduce or delay the emergence of the YMDD
interferon-a can induce a sustained virological response in
patients with chronic hepatitis B not responding to interferon-a
alone without the selection of drug-resistant mutants.21Sequential
combination therapy can induce sustained virological response
in a subgroup of HBeAg-negative/virus-DNA-positive chronic
hepatitis B.22Compared with combination therapy, sequential
treatment will be cost-effective, which is important in low-income
countries such as China. However, limited by a small number of
studies, the effectiveness of sequential treatment with lamivudine
and interferon-a monotherapies in patients with chronic hepatitis
B needs further exploration.
Inthe present study,we
sequential treatment with lamivudine and interferon-a in
HBeAg-negative Chinese patients with chronic hepatitis B. A
sensitive TaqMan-based real-time PCR method23was used to
investigate lamivudine-resistant mutations during this sequential
Patients and methods
This study was performed between June 2002 and July 2004. A total
of 162 patients who were untreated with antiviral agents were
included in the sequential treatment study. All the 162 patients were
over 16 years of age, positive for hepatitis B surface antigen (HBsAg)
for at least 6 months, negative for HBeAg and positive for hepatitis B
e antibody (anti-HBe), and had HBV DNA levels of more than
100000 copies/mL and serum ALT levels greater than 1.5 times but
less than 10 times the normal range according to recommendations
of Chinese Experts Committee for Clinical Use of Lamividine.
Exclusion criteria included co-infection with hepatitis A, C, D and E
virus or HIV, decompensated liver diseases or HCC, a history of
alcohol or drug abuse within 1 year before entry, other possible
causes of chronic liver damage, and previous treatment of chronic
Study design and treatment protocols
The patients were randomly divided into two groups in a ratio of
2:3. Group A (n = 64) for sequential treatment and group B (n = 98)
for treatment with lamivudine alone. The study was conducted
according to the guidelines of the Declaration of Helsinki and the
principles of Good Clinical Practice. All patients gave their informed
Patients in group A received lamivudine (100 mg/day) (Glaxo
Wellcome, Suzhou, China) for 20 weeks, followed by interferon-a-2b
(5 million units three times per week) (Schering-Plough, Shanghai,
China) plus lamivudine (100 mg/day) for 4 weeks, and they were then
treated for another 24 weeks with interferon-a-2b alone (5 million
units three times per week). Patients in group B received 100 mg of
lamivudine once daily for 48 weeks. All patients were followed up
after the end treatment for 24 weeks.
Detection of YMDD mutations
YMDD mutations were detected at baseline and every 12 weeks
thereafter using a TaqMan-based real-time PCR assay established
by us.23In this assay, three different forward primers that would
selectively amplify YMDD (wild-type), YVDD (rtM204V) and
YIDD (rtM204I) variants and a common reverse primer to a highly
conserved sequence within the polymerase open reading frame were
used to discriminate different mutants. The amplicon was detected
and quantified by using real-time PCR and a TaqMan probe that
annealed to a highly conserved region between the forward and
reverse primers. A universal forward primer was used in the control
for amplification of all kinds of variants. The amplification was
performed by incubating the reaction mixture at 50?C for 2 min,
followed by 5 min at 95?C and 40 cycles of PCR amplification (94?C
for 20 s and 53?C for 30 s) on a ABI 7000 sequence detector (Applied
Biosystems, Foster City, CA, USA).
Assays for ALT, HBV markers and HBV DNA
ALT levels, HBsAg, hepatitis B surface antibody (anti-HBs), HBeAg,
anti-HBe and hepatitis B core antibody (anti-HBc) were measured
using commercially available kits at baseline and every 30 days
thereafter. HBV DNA levels were tested using real-time PCR (Fosun
Diagnostics, Shanghai, China) on an ABI 7000 sequence detector
(Applied Biosystems, Foster City, CA, USA). All reagents used in
this study were approved by the State Food and Drug Administration
of China for in vitro diagnostic use.
Efficacy analyses included all randomized patients enrolled in this
study. The study had two primary measures of efficacy assessed after
24 weeks of follow-up: the normalization of ALT levels and the
suppression of HBV DNA levels to below 1000 copies/mL.
Secondary efficacy measures assessed after 24 weeks of follow-up
included the proportion of patients with HBsAg loss or HBsAg
seroconversion (defined by the loss of HBsAg and the presence of
The significance of a difference between two groups was determined
using the c2test. The level of significance was set at P < 0.05.
Baseline characteristics of patients
The baseline characteristics of patients before treatment are given
in Table 1. Ninety-eight patients were randomly assigned to
receive lamivudine and 64 to receive sequential treatment with
lamivudine and interferon-a monotherapies. All the patients were
Chinese and well matched in terms of age, weight and laboratory
results at baseline. However, the percentage of males is lower in
group A than in group B.
At week 24, serum ALT normalized in 28 of 64 (44%) patients in
group A and 72 of 98 patients (73%) in group B. However, at
week 48, 38 of 64 (59%) patients in group A and only 54 of 98
(55%) patients in group B had a sustained biochemical response.
Shi et al.
by guest on June 13, 2013
The percentage of patients with normalized ALT levels in group
B (73%) was significantly higher than in group A (44%, P < 0.05)
at week 24 but there was no significant difference at week 48.
After 24 weeks of follow-up (week 72), the percentage of patients
with normalized ALT levels was significantly higher in group A
(53%) than in group B (36%).
At week 24, the percentage of patients with HBV DNA levels
below 1000 copies/mL was 81% in group A and 78% in group B
compared with 56% in group A and 55% in group B at week 48.
There were no significant differences in suppression of serum
HBV DNA between groups A and B at week 24 and week 48.
At week 72, suppression of HBV DNA levels to below
1000 copies/mL occurred in nine patients (14%) from group A
and 18 patients (18%) from group B (Table 2).
At week 72, HBsAg loss or seroconversion was not identified in
either group A or group B.
No patients had evidence of lamivudine-resistant mutations at
baseline and YMDD mutants were monitored in all patients every
12 weeks thereafter. Only two patients (3.13%) were found with
the YIDD variant at week 24 and no patients had evidence of
YMDD mutations at week 48 among 64 patients in group A. The
two patients who had YIDD variants at week 24 had normalized
ALT and undetectable HBV DNA at week 48 and week 72. In
contrast, YMDD mutations were found in six patients (four YIDD
variants and two YVDD variants, 6.12%) at week 24 and in 22
patients (12 YIDD variants, eight YVDD variants and two with a
mixture of YIDD and YVDD variants, 22.45%) at week 48 from
group B (P < 0.05). All the 22 patients who had YMDD mutations
at week 48 had rebounds of serum HBV DNA and 18 (82%) had
rebounds of ALT levels.
Lamivudine was well tolerated and no adverse symptoms were
identified during treatment. During the course of interferon, six
patients had serious adverse events including pyrexia, fatigue,
myalgia and headache. Depression was not observed in this study.
Mean neutrophil and platelet counts were reduced during
treatment but returned to baseline levels shortly after treatment
was stopped. All the patients completed their treatments.
Interferon-a monotherapy has little long-term benefit in the
treatment of Chinese patients with chronic hepatitis B.7,8,24This is
probably due to immunological tolerance to HBV induced by
exposure to the virus in early life.24Sequential treatment with
lamivudine and interferon-a monotherapies was effective in
patients with chronic hepatitis B not responding to interferon
alone.21The main reason might be reduction in HBV DNA and
recovery of T-cell reactivity against HBV induced by lamivudine
improving the efficacy of interferon-a.21,25In the present study,
we found that sequential treatment starting with lamivudine
monotherapy followed by interferon monotherapy resulted in an
effective response in HBeAg-negative Chinese patients with
chronic hepatitis B. In addition, lamivudine-resistant mutations
were suppressed during sequential treatment. YMDD mutations
were found in none of the 64 patients with sequential treatment
but in 22 of 98 patients (22.45%) with lamivudine monotherapy at
the end of treatment.
Long-term use of lamivudine induced the emergence of
YMDD mutations, which was occasionally associated with severe
flares of hepatitis.26,27A previous study showed that sequential
treatment with lamivudine and interferon-a monotherapies
induced a sustained virological response in non-responders to
Table 1. Baseline characteristics of the patients
(group A, n = 64)
(group B, n = 98)
Male sex (%)38 (60%) 78 (80%) (P < 0.05)
mean – SD
37 – 10.4
32 – 9.3 (P > 0.05)
mean – SD
62 – 7.5
62 – 8.4 (P > 0.05)
HBV DNA (log copies/mL)
mean – SD
6.73 – 1.16
6.85 – 0.97 (P > 0.05)
mean – SD
135.59 – 90.81
120.47 – 65.71 (P > 0.05)
ALT >1.5 times ULN64 (100%)98 (100%)
Table 2. Virological and biochemical responses at weeks 24,
48 and 72
(group A, n = 64)
(group B, n = 98)P value
Normalization of ALT
HBV DNA < 1000 copies/mL
Sequential treatment of Chinese patients with CHB
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interferon-a alone, without the selection of lamivudine-resistant
mutations.21Combination and sequential combination therapies
of chronic hepatitis B with lamivudine and interferon-a-2a were
effective and also delayed the selection of lamivudine-resistant
A recent study indicated that a three-phase
sequential treatment with lamivudine and interferon in young
patients with chronic hepatitis B reduced HBV DNA serum levels
but did not prevent the emergence of lamivudine-resistant
mutations.28In our study, 56% of patients with sequential
treatment with lamivudine and interferon and 55% of patients
with lamivudine monotherapy had negative serum HBV DNA by
real-time PCR at the end of therapy. No lamivudine-resistant
mutations were selected in the sequential treatment group but
22.45% of patients with YMDD mutations were found in the
lamivudine-alone group. These findings were consistent with
combination treatment studies.4,18,29The sequential monotherapy
appears to be effective and may also suppress lamivudine-
Two patients in the sequential treatment group were detected
with the YIDD variant at week 24 but had normalized ALT and
undetectable serum HBV DNA at week 48 and week 72. The
mutants emerged 12–24 weeks after the start of lamivudine
treatment and disappeared in 12 weeks after interferon-a
treatment. This suggests that interferon-a may be effective in
some lamivudine-resistant patients.
ALT levels were higher in group A than in group B at week 24.
This could be attributed to flare-ups of ALT in 21 patients in
group A when interferon-a was added. Among these 21 patients,
6 exhibited a sustained biochemical and virological response at
week 72. We cannot conclude that ALT flare-ups during the
initial interferon-a treatment might have a prolonged response.
However, sequential treatment resulted in a higher biochemical
response rate at week 72. This might be due to the signifi-
cant effects of interferon-a on normalization of serum ALT
The major limit of this study is the absence of histological
response data. In other studies, patients with prolonged virological
responses experienced histological improvements.21There was a
significant association between improved histological activities
and either a prolonged biochemical or virological response.4The
percentage of males is higher in group B than in group A, which
may cause bias in the results concerning treatment efficacy. In
addition, the sustained response rate was low in this study due to
the relative short treatment period. A longer treatment period
needs to be evaluated in future studies.
Compared with combination therapy, sequential therapy is
much more cost-effective. In China, interferon-a is much cheaper.
A 24 week treatment costs about 3000 CNY, whereas a 48 week
lamivudine treatment costs about 6000 CNY. China has the
greatest burden of hepatitis B in the world. Owing to limited
income, most Chinese people will benefit from low-cost
therapeutic regimens. To date, the State Food and Drug
Administration of China has approved five treatments for chronic
HBV infection in China. They are interferon-a, lamivudine,
adefovir, peginterferon-a-2a and entecavir, which are effective
agents for treatment of chronic hepatitis B.4,30–32Differences in
drug administration, duration and efficacy of the treatment,
frequency of drug resistance, and cost do exist for these drugs.33
Although entecavir has a better outcome in the absence of pre-
existing lamivudine-resistant mutants,31,32lamivudine will be
preserved in China because of its much lower price. Our results
showed that sequential treatment with lamivudine and interferon-
a monotherapies in HBeAg-negative Chinese patients with
chronic hepatitis B was as effective as single lamivudine
treatment but can significantly suppress the emergence of
YMDD mutations. However, sequential treatments and the timing
of administration for chronic hepatitis B with different drugs still
need further controlled trials.
This work was supported by grants from the National Natural
Science Foundation of China (no. 30371053) and the Foundation
for Distinguished Young Scholars from National Natural Science
Foundation of China (no. 30125034).
Dr Z.-J. Y. had been a staff member of Fosun Diagnostics.
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