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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS

Clinics and Research in Hepatology and Gastroenterology (2017) xxx, xxx—xxx

Available online at

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www.sciencedirect.com

ORIGINAL ARTICLE

Analysis of intrahepatic total HBV DNA,

cccDNA and serum HBsAg level in Chronic
Hepatitis B patients with undetectable
serum HBV DNA during oral antiviral therapy
Jie Li a,c, Xizhen Sun a, Jianting Fang a, Chuanxi Wang b,
Guoqing Han a, Wanhua Ren a,∗

a
Department of Infectious Disease, Shandong Provincial Hospital Affiliated to Shandong University,
324 Jingwu Road, Ji’nan, Shandong 250021, China
b
Department of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwu
Road, Ji’nan, Shandong 250021, China
c
Division of Gastroenterology and Hepatology, Stanford University Medical Center, Palo Alto, CA,
United States

Summary
Background: This study aimed to investigate the relationship between intrahepatic cccDNA and
serum HBsAg in chronic Hepatitis B (CHB) patients with undetectable serum HBV DNA during
antiviral therapy.
Methods: We investigated HBsAg serum levels and their relationship to intrahepatic total
cccDNA and HBV DNA in CHB patients with undetectable serum HBV DNA during oral antivi-
ral therapy. Intrahepatic cccDNA and HBV DNA quantitation were performed in the same needle
biopsy material, while serum HBsAg, HBeAg and HBV DNA levels were measured in samples
drawn on the day of the liver biopsy.
Results: A total of 90 patients who had a liver biopsy were enrolled, including 80 patients
with CHB and 10 patients with liver cirrhosis (LC). All the CHB patients were divided into
HBeAg-positive and HBeAg-negative group. By using real-time PCR detection, we found that
intrahepatic cccDNA and HBV DNA levels were higher in CHB patients than those in LC patients
(Intrahepatic cccDNA: 6.15 ± 1.19 vs. 6.12 ± 0.36, HBV DNA: 7.26 ± 0.49 vs. 5.59 ± 0.45, both

∗
Corresponding author.
E-mail addresses: lijier@sina.com (J. Li), mw50881212@sohu.com (X. Sun), sdshij@sohu.com (J. Fang), chuanxiwang@yeah.net
(C. Wang), gqinghan@126.com (G. Han), whr631212@sina.com (W. Ren).

http://dx.doi.org/10.1016/j.clinre.2017.03.004
2210-7401/© 2017 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
2 J. Li et al.

P < 0.05). Intrahepatic cccDNA level was positively correlated with serum HBsAg in HBeAg-
negative (r = 0.66, P = 0.02) and lower serum HBeAg (≤50S/CO) CHB patients (r = 0.47, P = 0.03),
but not in higher serum HBeAg (>50S/CO) CHB patients (both P > 0.05). In HBeAg negative
patients, serum HBsAg level was correlated with intrahepatic total HBV DNA level (r = 0.52,
P = 0.006). However, no relationship between HBsAg level and intrahepatic total HBV DNA level
was found in HBeAg positive patients (both P > 0.05).
Conclusions: Serum HBsAg can be used to predict intrahepatic cccDNA and HBV DNA level in
CHB patients with low serum HBeAg statues, especially in HBeAg negative patients.
© 2017 Elsevier Masson SAS. All rights reserved.

Introduction affiliated to Shandong University between October 2011 and

Chronic Hepatitis B virus (HBV) infection is the leading
cause of hepatic failure, liver cirrhosis, and hepatocellu-
lar carcinoma with a high morbidity and mortality rate [1].
Although many current antiviral therapies have dramatically
improved the long-term outcomes of patients with chronic
HBV infection (CHB), they rarely achieve a cure due to
the refractory nature of an intracellular viral replication
intermediate termed covalently closed circular (ccc) DNA.
cccDNA can reside in the nucleus of infected cells even in
the absence of detectable HBV DNA or HBsAg in the blood.
In addition, persistent intrahepatic cccDNA can result in a
viral relapse in CHB patients after discontinuation of or even
during the antiviral treatment [2—5].
A cure of chronic hepatitis B requires elimination
of cccDNA. Therefore, quantitation of intrahepatic HBV
cccDNA is suggested to be valuable in evaluating anti-
HBV therapeutic efficacy and estimating treatment endpoint
[6—11]. But the requirement for invasive liver biopsy is the
major obstacle for quantitation of cccDNA in clinic. Thus,
finding serum surrogate maker of intrahepatic cccDNA is
clinically meaningful. Serum hepatitis B virus surface anti-
gen (HBsAg) quantitation was suggested as a useful marker
for predicting the response to interferon alpha-based
therapies and oral antiviral therapies [12—14]. Recently, cor-
relation between serum HBsAg and HBV DNA levels with
intrahepatic cccDNA level has been actively investigated
in CHB patients. However, many controversial conclusions
existed [15—17]. The relationship between serum HBsAg and
intrahepatic cccDNA levels, especially in CHB patients with
undetectable serum HBV DNA during antiviral therapy has
not yet been clarified.
In the present study we utilized a set of real-time PCR
assays to study HBV replication in liver biopsies, with aim
to investigate the relationship between serum viral mark-
ers and intrahepatic HBV replication factors, including HBV
cccDNA and HBV DNA in CHB patients with undetectable
serum HBV DNA during antiviral therapy.
Materials and methods
Study subjects
In this study, we recruited 351 chronic HBV infected
patients who were admitted to Shandong Provincial Hospital
September 2013. Patients co-infected with hepatitis C virus,
hepatitis D virus or human immunodeficiency viruses were
excluded. Those with co-existing liver diseases including
autoimmune hepatitis, primary biliary cirrhosis and alco-
holic liver disease were also excluded. 280 patients had
received 1 of the 4 nucleos(t)ide analogs (NA) antiviral
treatment (such as lamivudine, adefovir, entecavir, or tel-
bivudine) according to the guideline of the Clinical Practice
Guidelines-Management of CHB in Asia-Pacific[18] and with
undetectable serum HBV DNA (<500 copies/ml) for at least
6 months prior to entry. 190 patients refused to receive
liver biopsy and 90 chronic HBV infection patients undergo
liver biopsy finally (Fig. 1). This study was approved by the
ethics committee of Shandong Provincial Hospital affiliated
to Shandong University and written informed consents were
obtained from all patients. The study protocol conformed to
the ethical guidelines of the 1975 Declaration of Helsinki.
Sample collection
Liver biopsy was obtained from all patients. The liver
biopsies were stored in −80 ◦ C until analyzed. Sera were
collected on the same day of liver biopsy for biochemical,
virological and serological analyses.
Histological evaluation
Histopathologic evaluations were performed on liver nee-
dle biopsy specimens according to the ISHAK scoring system
by the same pathologist in a single center. After fixation
with 10% formol, tissues were embedded in paraffin follow-
ing routine tissue surveillance. Slices obtained from paraffin
blocks were stained with hematoxylin—eosin and examined
under light microscope. Masson’s trichrome staining was
then used to demonstrate the development of fibrotic tissue.
Serological tests and quantification of serum HBV
DNA
Serum HBsAg, HBeAg, anti-HBs, anti-HBe, anti-HBc, and
antibodies to HCV, HDV, HIV-1 and HIV-2 were determined
by commercial enzyme immunoassay kits (AXSYM System,
Abbott, Wiesbaden, Germany) according to the manufac-
ture’s instruction. The lower limit of serum HBsAg detection
was 0.05 IU/ml. If the initial test value was higher than

Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
Analysis of intrahepatic HBV DNA, cccDNA and serum HBsAg level in Chronic Hepatitis B patients 3

Figure 1 Flowchart of patients recruitment for the study.

the upper limit of detection (250 IU/ml), the samples were
diluted (1:500) and reassessed. HBV DNA was extracted from
serum samples and quantified by using a commercial poly-
merase chain reaction (PCR) diagnostic kit with a detect
limit of 500 copies/ml (PG Biotech, Shenzhen, China).
Quantitation of intrahepatic HBV cccDNA and HBV
DNA
HBV DNA and cccDNA were amplified from genomic DNA
extracted from liver specimens obtained from all patients.
Briefly, about 30 mm formalin fixed paraffin-embedded
(FFPE) liver biopsy tissue was sectioned to 6 mm each
for DNA extraction. To prevent contamination, disposable
tweezers, brush and interleaver were used and sectioning
blades were carefully cleaned with 70% ethanol after every
sampling. The DNA was extracted using QIAamp FFPE DNA
Mini Kit (QIAGEN, GmbH, Hilden, Germany) according to the
instructions of the manufacturer. PSAD (Epicentre, Madison,
WI, USA) was used to digest HBV rcDNA, replicative dsDNA
and ssDNA. Afterwards, rolling Circle Amplification (RCA)
was conducted to selectively amplify circle DNA. Using the
RCA products as template, HBV cccDNA was further ampli-
fied and quantified with TaqMan real-time PCR mediated
by a pair of cccDNA-selective primers. Intrahepatic HBV
cccDNA detection range is between 2.50 × 102 copies/ml and
2.50 × 109 copies/ml. The detect limit of intrahepatic HBV
DNA is 500 copies/ml [19].
Statistical interpretation
Data was presented as M+ and inter-quartile range. Serum
HBV DNA and HBsAg levels were expressed in logarithm. Dif-
ferences between variables were tested by Student’s t-test
or one-way ANOVA test where it is appropriate. Regression
and Pearson’s correlation analysis were applied to compare

Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
4 J. Li et al.

Figure 2 Intrahepatic HBV cccDNA, HBV DNA and serum HBsAg levels in chronic Hepatitis B and liver cirrhosis patients. Comparison
of intrahepatic HBV cccDNA (A), HBV DNA (B) and serum HBsAg (C) levels between 80 CHB and 10 LC patients. The data were analyzed
by the Mann—Whitney U test. LC, liver cirrhosis; CHB, chronic Hepatitis B.

the data obtained by real-time PCR methods. A P-value

Table 1 Clinical and laboratory parameters of chronic HBV
(2-tailed) of 0.05 was considered statistically significant.
infected patients.
All statistical analyses were carried out in Statistical Pro-
gram for Social Sciences (SPSS 19.0 for Windows; SPSS Inc., Characteristic LC patients CHB patients
Chicago, IL). (n = 10) (n = 80)

Gender (M/F) 8/2 66/14

Results Age (years) 46 (35—60) 44 (18—54)
TBil (mmol/L) 8.4 (5.7—13.1) 12.2 (8.6—24.6)
General subject characteristics Albumin (g/L) 39 (38—41) 41 (37—43)
ALT (IU/L) 33 (17—39) 20 (17—32)
In this study, we incorporated 90 chronic HBV infected Proportion of patients 10 (100%) 80 (100%)
patients (74 male and 16 female) who fit the inclusion crite- with HBV DNA < 500
ria, including 80 CHB patients and 10 patients with liver copies/ml, n (%)
cirrhosis (LC) based on their liver pathology. The mean age of HBeAg positive 2 (20%) 32 (40%)
these patients was 30.9 ± 17.4 years (range: 18—60 years). patients, n (%)
The demographics and laboratory data are summarized in Current NA therapy, n (%)
Table 1. Lamivudine 2 (20%) 8 (10%)
Adefovir 1 (10%) 12 (15%)
Entecavir 5 (50%) 41 (51.25%)
Intrahepatic HBV cccDNA, HBV DNA and serum
Telbivudine 1 (10%) 19 (23.75%)
HBsAg levels in different chronic HBV infected Duration of NA 17.9 ± 8.3 15.3 ± 5.2
patients therapy, n (months)
Drug resistance, n (%) 0 0
Although all patients had undetectable serum HBV DNA
Data are median, unless otherwise indicated.
for at least 6 months during NA antiviral therapy, we
LC: liver cirrhosis; CHB: chronic Hepatitis B; ALT: alanine
still detected high level of HBV DNA in the liver tissues. aminotransferase; TBil: total bilirubin; NA: nucleos(t)ide
As illustrated in Fig. 2A, intrahepatic HBV DNA can be analogs.
detected both in CHB and LC patients. CHB patients had

Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
Analysis of intrahepatic HBV DNA, cccDNA and serum HBsAg level in Chronic Hepatitis B patients 5

Figure 3 Intrahepatic HBV cccDNA, HBV DNA and serum HBsAg levels in different groups of Chronic Hepatitis B patients. Com-
parison of intrahepatic HBV cccDNA (A), HBV DNA (B) and serum HBsAg (C) levels between different groups of Chronic Hepatitis
B patients divided according to the serum HBeAg level. Compared with * group, both P < 0.05. The data were analyzed by the
Mann—Whitney U test.

significantly higher intrahepatic HBV DNA than those in LC HBeAg-negative to HBeAg-positive A—C group, respectively)
patients (7.26 ± 0.49 vs. 5.59 ± 0.45, P < 0.05). In addition, (Fig. 3B and C).
intrahepatic HBV cccDNA level was higher in CHB patients
than those in LC patients (6.15 ± 1.19 vs. 6.12 ± 0.36, Correlation of intrahepatic HBV cccDNA with serum
P < 0.05) (Fig. 2B). Additionally, we found no significant HBsAg in CHB patients with different serum HBeAg
difference in the serum HBsAg level between CHB and LC
level
patients (3.33 ± 0.67 vs. 3.20 ± 0.34, P = 0.36) (Fig. 2C).
In order to further comparison, the total 80 CHB
After analyzing the different groups of chronic HBV infected
patients were divided into HBeAg-positive and HBeAg-
patients separately, we found that the significant positive
negative group. HBeAg-positive CHB patients were
correlation between intrahepatic HBV cccDNA level and
divided into 3 groups according to the serum HBeAg
serum HBsAg level was only observed in HBeAg-negative
level (group A: 1S/CO ≤ HBeAg < 50S/CO, group B:
(r = 0.66, P = 0.00), lower HBeAg level (<50S/CO) patients
50S/CO ≤ HBeAg < 100S/CO, group C: HBeAg ≥ 100S/CO).
(r = 0.47, P = 0.03) rather than in other patients (both
The intrahepatic cccDNA levels were 6.03 ± 0.71,
P > 0.05) (Fig. 4).
6.07 ± 0.85, 6.01 ± 0.60, 6.70 ± 1.00 log10 IU/ml in
these groups, respectively (from HBeAg-negative to
HBeAg-positive A—C group). HBV cccDNA level was Correlation of intrahepatic HBV DNA with serum
significantly higher in HBeAg ≥ 100S/CO patient than HBsAg in CHB patients with different serum HBeAg
those in HBeAg < 100S/CO patients (Fig. 3A). Intrahep- level
atic HBV DNA and serum HBsAg levels were significantly
higher in HBeAg-negative patients than those in HBeAg- Next, we analyzed the correlation between intrahepatic
positive patients (intrahepatic HBV DNA: 6.90 ± 0.49, HBV DNA and serum HBsAg in different CHB patients. Our
7.22 ± 0.36, 7.06 ± 0.35, 7.31 ± 0.88; HBsAg level: date indicated that intrahepatic HBV DNA and serum HBsAg
2.83 ± 0.83, 3.53 ± 0.58, 3.42 ± 0.36, 3.80 ± 0.42, from level were significantly higher in HBeAg-negative patients

Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
6 J. Li et al.

Figure 4 Correlation of intrahepatic HBV cccDNA with serum HBsAg in patients with different serum HBeAg level. Significant
positive correlation was found between the intrahepatic HBV cccDNA and serum HBsAg in HBeAg-negative (r = 0.66, P = 0.00) and
lower HBeAg level (<50S/CO) (r = 0.47, P = 0.03) patients (A and B), but not in 50S/CO ≤ HBeAg < 100S/CO (r = 0.10, P = 0.64) and
HBeAg ≥ 100S/CO (r = 0.37, P = 0.12) patients (C and D). Dates were shown as X—Y scatter plots. P values were calculated using the
Spearman rank correlation. Solid line: linear growth trend, r: correlation coefficient.

Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
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CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
Analysis of intrahepatic HBV DNA, cccDNA and serum HBsAg level in Chronic Hepatitis B patients 7

Figure 5 Correlation of intrahepatic HBV DNA with serum HBsAg in CHB patients with different serum HBeAg level. Significant
positive correlation was found between the intrahepatic HBV DNA and serum HBsAg in HBeAg-negative (r = 0.52, P = 0.006) patients
(A), but not in HBeAg-positive patients (group A: r = −1.21, P = 0.59; group B: r = 0.16, P = 0.45; group C: r = 0.43, P = 0.07) (B—D).
Dates were shown as X—Y scatter plots. P values were calculated using the Spearman rank correlation. Solid line: linear growth
trend, r: correlation coefficient.

than those in HBeAg-positive patients. Further analyzing
showed a significant positive correlation between intrahep-
atic HBV DNA and serum HBsAg level was only observed
in HBeAg-negative (r = 0.52, P = 0.006), but not in HBeAg-
positive patients (both P > 0.05) (Fig. 5).
Conclusions
The goal of current treatment of CHB is to suppress viral
replication to the lowest possible level thus halting disease
progression. NA have a suppressive effect on the transcrip-
tion of pregenomic RNA, and the administration of these
agents can induce a rapid and dramatic decrease in serum
HBV DNA [20]. However, currently NA therapy is unable to
induce a complete elimination of intrahepatic HBV DNA and
cccDNA in CHB patients. NA treatment does not directly
affect the cccDNA content of the liver. Decreases in cccDNA
levels are derived from the lack of sufficient re-entry of
viral nucleocapsids to the nucleus, due to strong inhibi-
tion of viral DNA synthesis in the cytoplasm, and fewer
incoming viruses from the blood. Consistent with previ-
ous studies [21,22], our data showed that high levels of
HBV DNA and cccDNA were still been detected in the liver
tissues although in those patients who have undetectable
serum HBV DNA during antiviral therapy (Fig. 1). In contrast,
persistent intrahepatic HBV DNA and cccDNA is the main
cause of viral relapse after NA therapy is stopped. There-
fore, the measurement of intrahepatic cccDNA and/or HBV
DNA has additional value when evaluating anti-HBV thera-
peutic efficacy and estimating treatment endpoint to CHB
patients.
The major obstacle for quantitation of intrahepatic HBV
DNA and cccDNA in clinic is the requirement for invasive liver
biopsy. Thus there has been increasing research to identify
surrogate, non-invasive markers of the amount of intrahep-
atic HBV DNA and cccDNA in the liver. Both HBeAg and HBsAg
titer have been proposed as surrogates for infected liver
cell mass. If the hepatocyte were considered in isolation,
HBsAg and HBeAg would be expected to directly corre-
late with liver cccDNA, as all are translated from separate
transcripts (Pre-S1, Pre-S2/S, precore/core, and pregenomic

Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
+Model
CLINRE-994; No. of Pages 9 ARTICLE IN PRESS
8 J. Li et al.
mRNA, respectively) and directly derived from cccDNA. In
fact, HBeAg is not a reliable marker in this setting as intra-
hepatic HBV DNA and cccDNA level were still significantly
higher in HBeAg negative patients (Fig. 2). In contrast, serum
HBsAg level appeared to be a better surrogate marker of
intrahepatic HBV DNA and cccDNA in CHB patients.
Otherwise, discrepancy in the relationship between
HBsAg titer and total intrahepatic HBV DNA/cccDNA has
been described from previous studies [15—17,23]. The
role of HBsAg as surrogate markers of intrahepatic HBV
DNA/cccDNA activity during antiviral treatment is also a
subject of debate. In fact, most of the studies examining
HBsAg quantification have not compared HBeAg positive and
HBeAg negative populations, known to be characterized by
distinct immunological milieu and different levels of virion
productivity [3,23]. As shown in Figs. 3 and 4, our data indi-
cates that the relationship between HBsAg, HBeAg and HBV
replicative intermediates are complex. HBsAg titer corre-
lated with total intrahepatic HBV DNA only in patients with
HBeAg negative CHB, but not in HBeAg positive CHB patients.
In addition, HBsAg titer correlated with intrahepatic cccDNA
in patients with low HBeAg level (<50S/CO), including HBeAg
negative CHB.
This correlation is not statistically significant in HBeAg
positive patients probably because the dynamic processes
of intrahepatic and serum HBV replication markers are sug-
gested to be complex in CHB patients [24,25]. The regulation
of HBsAg expression involves more than the amount of
cccDNA in infected cells, for example, its transcriptional
regulation and the possibility that envelope proteins could
be expressed from viral sequences integrated into the host
genome [26]. Furthermore, transcription of cccDNA can be
suppressed or enhanced by several intrinsic factors, such
as the degree of cccDNA methylation or the acetylation
of the surrounding histones, respectively [27,28]. Methyl-
ation may have the same effect not only on cccDNA related
transcription, but also on transcription of HBV fragments
integrated into the host genome and coding for viral pro-
teins, including HBsAg. Currently, it is not known whether
cccDNA methylation is enhanced during the HBeAg-negative
phase, and whether it differentially interferes with the
whole HBV genome vs. HBsAg transcription. Those factors
influence the association between intrahepatic cccDNA and
serum viral markers needs further clarification.
In summary, serum HBsAg level has been shown to reflect
active intrahepatic cccDNA and HBV DNA in low HBeAg level
CHB patients, especially HBeAg negative patients. These
findings provided a new insight on the clinical implications of
HBV replicative intermediates and the serum HBsAg in differ-
ent stages of HBV infection. In addition, serum HBsAg could
provide the only easily measurable HBV product in treated
HBeAg-negative patients, used as an on-treatment marker.
Nevertheless, one limitation of the study is the relatively
small sample size for patients, especially the liver cirrhosis
patients. Further studies are needed to determine the role
and potential prognostic values of these observations.
Disclosure of interest
The authors declare that they have no competing interest.
Please cite this article in press as: Li J, et al. Analysis of intrahepatic total HBV DNA, cccDNA and serum HBsAg level in
Chronic Hepatitis B patients with undetectable serum HBV DNA during oral antiviral therapy. Clin Res Hepatol Gastroenterol
(2017), http://dx.doi.org/10.1016/j.clinre.2017.03.004
Acknowledgments
This study was supported by grants from the National Natural
Science Foundation of China (Grants 81201288, 81473483),
China Postdoctoral Science Foundation (2014M550367),
WBE liver fibrosis foundation (CFHPC20131015), the Key
Science and Technology Program of Shandong Province
(2010GSF0271), and the Natural Science Foundation of Shan-
dong Province (BS2013YY048).
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