HBV genotype C is independently associated with cirrhosis

StephenW

http://www.wjgnet.com/1007-9327/16/379.asp

ISSN 1007-9327 CN 14-1219/R  World J Gastroenterol  2010 January 21; 16(3):
379-383

BRIEF ARTICLE
HBV genotype C is independently associated with cirrhosis in community-based
population

Jian-Hua Yin, Jun Zhao, Hong-Wei Zhang, Jia-Xin Xie, Wei-Ping Li, Guo-Zhang
Xu, Jie Shen, Hong-Jun Dong, Jun Zhang, Lin Wang, Jian-Kang Han, Hong-Yang
Wang, Guang-Wen Cao


-------------------------------------------------------------------------------
-


Jian-Hua Yin, Hong-Wei Zhang, Jia-Xin Xie, Guang-Wen Cao, Department of
Epidemiology, Second Military Medical University, Shanghai 200433, China
Jun Zhao, Department of Hepatobiliary Surgery, the 3rd Affiliated Hospital,
Second Military Medical University, Shanghai 200438, China
Wei-Ping Li, Department of Ultrasonography, the 1st Affiliated Hospital,
Second Military Medical University, Shanghai 200433, China
Guo-Zhang Xu, Hong-Jun Dong, Municipal Center for Disease Control and
Prevention of Ningbo, Ningbo 315010, Zhejiang Province, China
Jie Shen, Jun Zhang, Municipal Center for Disease Control and Prevention of
Soochow, Soochow 215007, Jiangsu Province, China
Lin Wang, District Center for Disease Control and Prevention of Yangpu,
Shanghai 200090, China
Jian-Kang Han, Municipal Center for Disease Control and Prevention of Huzhou,
Huzhou 313000, Zhejiang Province, China
Hong-Yang Wang, Laboratory for Signal Transduction, the 3rd Affiliated
Hospital, Second Military Medical University, Shanghai 200438, China


Author contributions: Yin JH, Zhao J and Zhang HW contri­buted equally to
this work; Yin JH, Zhao J and Zhang HW were involved in experimental work and
data analysis; Xie JX was involved in HBV genotyping; Li WP was responsible
for ultrasonographic examination; Xu GZ, Shen J, Dong HJ, Zhang J, Wang L, and
Han JK were responsible for field study; Wang HY provided partial financial
support; Cao GW designed the study and wrote the manuscript.

Supported by Ministry of Health of China, No. 2008ZX10002-15; National Natural
Science Foundation of China, No. 30921006; Shanghai Science & Technology
Committee, No. 08XD14001; and Shanghai Heath Bureau of Health, No. 08GWD02 and
08GWZX0201

Correspondence to: Dr. Guang-Wen Cao, Professor of Medicine, Chairman,
Department of Epidemiology, Second Military Medical University, 800 Xiangyin
Rd., Shanghai 200433, China. [email protected]
Telephone: +86-21-81871060  Fax: +86-21-81871060

Received: November 6, 2009    Revised: December 2, 2009
Accepted: December 9, 2009
Published online: January 21, 2010

Abstract
AIM: To determine the association of hepatitis B virus (HBV) genotypes with
probable cirrhosis and fatty liver in community-based populations.

METHODS: A multi-stage cluster probability sampling method was applied to
recruit 10 167 subjects aged between 6 and 72 years from our epidemiological
bases in Eastern China. After excluding the subjects co-infected with
hepatitis C or hepatitis D viruses, the hepatitis B surface antigen
(HBsAg)-positive subjects were examined for HBV genotype, serum viral load,
alanine aminotransferase (ALT), hepatitis B e antigen (HBeAg) status, and
ultrasonographic changes. Logistic regression models were used to determine
the factors associated with probable cirrhosis and fatty liver.

RESULTS: Of 634 HBsAg-positive subjects with HBV genotype determined, 82 had
probable cirrhosis (ultrasonographic score ≥ 5), 42 had ultrasonographic
fatty liver. Probable cirrhosis was only found in the HBeAg-negative subjects,
and more frequently found in the subjects with genotype C than in those with
genotype B (14.8% vs 8.0%, P = 0.018). In HBeAg-negative subjects, high viral
load was frequently asso­ciated with abnormal ALT level, while ALT
abnormality was more frequent in those with probable cirrhosis than those
without (19.5% vs 7.8%, P = 0.001). Univariate analysis showed that age, sex,
HBV genotypes, and viral load were not significantly associated with
ultrasonographic fatty liver, whereas ALT abnormality was significantly
related to ultrasonographic fatty liver (OR = 4.54, 95% CI: 2.11-9.75, P <
0.001). Multivariate analysis demonstrated that HBV genotype C, age (≥ 45
years), male sex, and ALT abnormality were independently associated with
probable cirrhosis (AOR = 2.30, 95% CI: 1.26-4.19; AOR = 1.81, 95% CI:
1.10-2.99; AOR = 1.74, 95% CI: 1.03-2.95; AOR = 2.98, 95% CI: 1.48-5.99,
respectively).

CONCLUSION: A crude prevalence of probable cirrh­osis is 12.9% in the
community-based HBV-infected subjects. HBV genotype C is independently
associated with probable cirrhosis in the HBeAg-negative subjects.

© 2010 Baishideng. All rights reserved.

Key words: Hepatitis B virus; Genotype; Viral load; Alanine aminotransferase;
Probable cirrhosis; Ultrasono­graphy

Peer reviewer: Raffaele Pezzilli, MD, Department of Internal Medicine and
Gastroenterology, Sant’Orsola-Malpighi Hospital, Via Massarenti, 9, Bologna
40138, Italy

StephenW

Yin JH, Zhao J, Zhang HW, Xie JX, Li WP, Xu GZ, Shen J, Dong HJ, Zhang J, Wang
L, Han JK, Wang HY, Cao GW. HBV genotype C is independently associated with
cirrhosis in community-based population. World J Gastroenterol 2010; 16(3):
379-383  Available from: URL:
<http://www.wjgnet.com/1007-9327/full/v16/i3/379.htm>  DOI:
http://dx.doi.org/10.3748/wjg.v16.i3.379

INTRODUCTION
Hepatitis B virus (HBV) infection is a serious public health problem.
Approximately 2 billion people have been exposed to HBV, and more than 300
million are chronically infected with HBV. Chronic HBV infection is the most
important risk factor of liver cirrhosis and hepatocellular carcinoma (HCC) in
HBV endemic areas[1]. Liver fibrosis, which is the natural wound healing
process to necroinflammation frequently caused by chronic HBV infection, is
the essential pathogenic process that leads to cirrhosis. Metabolic syndrome
is also an independent risk factor of liver cirrhosis in the patients with
chronic hepatitis B[2]. Subclinical liver cirrhosis diagnosed by
ultrasonography is significantly associated with the risk for HCC[3].
    HBV genotypes have distinct geographical distributions, and have been
shown to differ with regard to clinical outcome and prognosis[4]. Genotypes B
and C are endemic in most parts of Asia[5]. Genotype C is associated with HCC
in the aged[6,7]. Genotype B is associated with HCC in the young, relapse of
HCC, and acute hepatitis B in adults[8-10]. However, the relationship between
HBV genotypes and liver cirrhosis remains controversial. Some studies
suggested that genotype C had a higher risk of cirrhosis, whereas other
studies indicated that the progression to cirrhosis did not differ among
genotypes B- and C-related chronic liver diseases[11-13]. In addition, the
association between HBV genotypes and subclinical cirrhosis has not been
evaluated in community-based studies in the HBV endemic areas.
    Our objective was to determine the prevalence of probable liver cirrhosis
in community-based subjects who were seropositive for hepatitis B surface
antigen (HBsAg), and to evaluate the viral and demographic factors
contributing to subclinical cirrhosis.

MATERIALS AND METHODS
Study population and epidemiological survey
The study was carried out at our epidemiological bases in Eastern China, from
February to July 2009. A multistage cluster probability sampling method was
applied to select the study population. A total of 10 167 residents aged
between 6 and 72 years were involved in this study. The participants were
interviewed by the trained research assistants using a standard questionnaire
requesting information about sociodemographic characteristics. Fasting blood
samples (4 mL) were collected with vacuum blood collection tube (BD
Diagnostics, Plymouth, UK) without anticoagulant. The serum was separated by
centrifugation at 4℃ at the Centers for Disease Control and Prevention,
transported on dry ice and stored at -40℃ in the Department of Epidemiology,
Second Military Medical University. Informed consent in writing was obtained
from each participant or guardian. Each resident who agreed to participate in
the study completed a questionnaire and provided blood samples. The study
protocol conformed to the ethical guidelines of the 1975 Declaration of
Helsinki, and was approved by the Institutional Review Board of this
university.

Examination of HBV serological markers, serum viral load, and serum alanine
aminotransferase level
All participants received HBsAg examination. Those positive for HBsAg were
examined for hepatitis B e antigen (HBeAg), serum viral load, and alanine
amino­transferase (ALT). HBsAg was examined using enzyme linked immunosorbent
assay (Kehua, Shanghai, China) according to the manufacturer’s instructions.
Serological testing for HBeAg, antibody to hepatitis C virus (HCV), and
antibody to hepatitis D virus (HDV), liver function tests, and a-fetoprotein
examination were performed as previously described[9]. Upper limit of normal
ALT was 45 U/L. Viral load was measured in the LightCycler™ 480 (Roche,
Basel, Switzerland) using quantitative HBV PCR fluorescence diagnostic kits
(Fosun Dia­gnostics, Shanghai, China). The kit has a certified lower limit of
detection of 500 copies/mL, which was standardized using the Abbott reagents
(Abbott Laboratories, North Chicago, IL).

HBV genotyping
HBV DNA was extracted from 200 mL HBsAg-posi­tive serum using High Pure Viral
Nucleic Acid Kits (Roche Diagnostics, Mannheim, Germany) according to the
manufacturer’s instruction. HBV genotype was determined using a multiplex
PCR assay[9,14]. HBV genotypes of samples with low level of HBV DNA were
identified by nested multiplex PCR. Outer primers were
5'-TTTGCGGGTCACCATATTCTTGG-3' and 5'-CGAACCACTGAACAAATGGCACTAG-3'. An
Autorisierter Thermocycler (Eppendorf AG, Hamburg, Germany) was programmed to
initially denature the samples for 3 min at 95℃, followed by 35 cycles
consisting of 94℃ for 60 s, 58℃ for 60 s, 72℃ for 60 s, followed by a
final elongation step at 72℃ for 10 min. The products (2 mL) were used as
templates for multiplex PCR[14].

Ultrasonographic examination of liver cirrhosis and fatty liver
With the use of a Philips iU22 scanner (Philips Medical Systems, Best, the
Netherlands) equipped with a 2-4 MHz variable convex probe or Toshiba systems
(SSA-340; Toshiba, Tokyo, Japan) with a 3.75 MHz convex probe, probable liver
cirrhosis and fatty liver were determined. Each subject was examined by two
independent operators who were blinded to the clinical details. Discrepancies
were resolved by consensus. The ultrasonographic scoring system consisting of
liver surface, parenchyma, vascular structure, and splenic size was used to
describe the existence and the severity of cirrhosis. The scores ranged from 4
for a normal liver to 11 for advanced cirrhosis[15]. A score of 8 or more was
used as the cutoff point for ultrasonographic cirrhosis. The subjects with the
score from 5 to 7 were diagnosed as having cirrhosis-like ultrasonographic
abnormality. A score of 5 or more was defined as probable cirrhosis. The
subject with an ultrasonographic steatosis score of 2 or more was diagnosed as
having fatty liver[16].

Statistical analysis
c2 test was used to determine the differences in categorical variables, such
as HBeAg positivity and the percentage of HBV genotypes. Continuous variables,
like serum viral load and ALT level with skewed distribution, were adjusted to
normal distribution by transformation into logarithmic function, and then
tested by Student’s t test. Univariate and multivariate regression analyses
were performed to obtain the odds ratio (OR) and adjusted odds ratio (AOR) of
factors for the risk of probable liver cirrhosis or ultrasonographic fatty
liver and their 95% confidence intervals (CI). All statistical tests were
two-sided, and performed using the Statistical Program for Social Sciences
(SPSS15.0 for Windows, SPSS, Chicago, IL). A P value of < 0.05 was considered
as statistically significant.

StephenW

RESULTS
Of the 10 167 participants, 793 were HBsAg positive; 745 of the 793 subjects
were free of antibodies to HCV or HDV; and 634 of 745 subjects had HBV
genotyped. Ten of the 634 subjects (8 with genotype C, one with genotype D,
and one with genotype B) were diagnosed as having ultrasonographic cirrhosis
(score 8 or higher), while 72 had cirrhosis-like ultrasonographic
abnormalities (scores 5-7). Of the 634 subjects, one was diagnosed as having
HCC. A crude prevalence of probable cirrhosis (ultrasonographic cirrhosis and
cirrhosis-like ultrasonographic abnormalities) was 12.9%. Table 1 shows the
demographic and viral characteristics and liver abnormalities of the 634
subjects. There were no significant differences in proportions of age, sex,
ALT level, HBeAg positivity, and fatty liver between the subjects infected
with genotype B and those with genotype C. Compared with genotype C, HBV
genotype B was more frequently seen in those with a high viral load (log10
copies/mL ≥ 4). Of the 634 subjects, 39 were positive for HBeAg. The
HBeAg-positive subjects were significantly younger than the HBeAg-negative
subjects (25.4 ± 11.2 years vs 42.7 ± 12.4 years, P < 0.001). The subjects
with probable cirrhosis were significantly older than those without probable
cirrhosis (45.3 ± 10.5 years vs 41.1 ± 13.3 years, P = 0.001). Probable
cirrhosis was only found in the HBeAg-negative subjects, and more frequently
in the subjects with genotype C than in those with genotype B (14.8% vs 8.0%,
P = 0.018). Serum viral load was significantly higher in the HBeAg-negative
subjects with abnormal ALT levels than in the HBeAg-negative subjects with
normal ALT levels (4.54 ± 2.16 log10 copies/mL vs 3.31 ± 1.36 log10
copies/mL; P < 0.001). However, this association was not found in the
HBeAg-positive subjects. Serum ALT level was significantly higher in the
subjects with high viral load (≥ 1 × 104 copies/mL) than in those with low
viral load (< 1 × 104 copies/mL) (33.9 ± 2.4 U/L vs 22.4 ± 1.9 U/L, P <
0.001). Serum ALT level was significantly higher in the subjects with
ultrasonographic cirrhosis (score ≥ 8) than in the HBeAg-negative subjects
with ultrasonographic score less than 7 (41.1 ± 1.6 U/L vs 24.1 ± 2.0 U/L, P
= 0.026). ALT abnormality was more frequent in HBeAg-negative subjects with
probable cirrhosis than those without probable cirrhosis (19.5% vs 7.8%, P =
0.001).
    Table 2 shows the factors associated with probable liver cirrhosis in the
HBeAg-negative subjects by uni­variate and multivariate regression analyses.
Age (≥ 45 years vs < 45 years), sex (male vs female), HBV DNA (≥ 4 log10
copies/mL vs < 4 log10 copies/mL), ALT (> 45 U/L vs ≤ 45 U/L), and HBV
genotypes (genotype C vs genotype B) were included in the models. It was found
that age (≥ 45 years), male sex, genotype C, and ALT abnormality were
independently associated with probable cirrhosis (AOR = 1.81, 95% CI:
1.10-2.99; AOR = 1.74, 95% CI: 1.03-2.95; AOR = 2.30, 95% CI: 1.26-4.19; AOR =
2.98, 95% CI: 1.48-5.99, respectively).
    Forty-two (6.6%) of the 634 subjects had ultras­onographic fatty liver,
including 11 with abnormal ALT levels. Ultrasonographic fatty liver was not
found in the subjects with probable cirrhosis. In the subjects with high viral
load (log10 copies/mL ≥ 4), ultrasonographic fatty liver was more frequently
found in those with genotype B than in those with genotype C (12.5% vs 0.0%, P
= 0.005). Univariate analysis showed that age, sex, HBV genotypes, and viral
load were not significantly associated with ultrasonographic fatty liver,
whereas ALT abnormality was significantly associated with ultrasonographic
fatty liver (OR = 4.54, 95% CI: 2.11-9.75, P < 0.001).

DISCUSSION
This large epidemiological study for the first time described the prevalence
of probable liver cirrhosis in community-based, HBV-infected subjects who were
free of HCV or HDV infection. About 13% of HBV-infected subjects had probable
cirrhosis. The subjects with probable cirrhosis were significantly older than
the subjects without cirrhosis. Probable cirrhosis was only found in the
HBeAg-negative subjects. The HBeAg-positive subjects were significantly
younger than the HBeAg-negative subjects. These results indicate that age is
an important determinant for the development of probable liver cirrhosis. High
viral load and ALT abnormality are associated with liver fibrosis in the
HBeAg-negative patients[17]. We further demonstrated that high viral load was
associated with increased serum ALT levels in the HBeAg-negative subjects and
high ALT levels were frequently found in the subjects with probable cirrhosis,
indicating that continuing HBV replication and hepatocyte damage contribute to
the development of liver cirrhosis.
    Importantly, the occurrence of probable liver cirrhosis was significantly
higher in the subjects with genotype C than in those with genotype B.
Multivariate analysis indicated that genotype C was significantly associated
with an increased risk of probable liver cirrhosis. This was probably related
to the prolonged immune clearance and delayed HBeAg seroconversion[18,19].
Although genotype B is associated with acute hepatitis[10], it tends to be
self-limiting and short-living. However, genotype C was associated with the
longer duration of liver damage in the HBeAg-negative subjects[12,20], which
may be the main reason for the development of liver cirrhosis. In addition,
genotype C-specific viral mutations are associated with probable
cirrhosis[11,21,22]. Our recent meta-analysis has shown that PreS deletion,
C1653T, T1753V, and A1762T/G1764A are increasingly more prevalent as chronic
HBV infection progressed from the asymptomatic HBsAg carrier to cirrhosis or
HCC[23]. Further studies are needed to probe into the different mutation
patterns between genotypes B and C and their roles in the development of liver
cirrhosis.
    Since metabolic syndrome increased the risk of liver cirrhosis in the
patients infected with HBV[2], we evaluated the prevalence and possible risk
factors of ultrasonographic fatty liver in the 634 HBV-infected subjects.
Interestingly, ultrasonographic fatty liver was not found in those with
probable cirrhosis, while ultrasonographic fatty liver was more frequently
found in those with genotype B than in those with genotype C at high viral
load levels. This suggests that ultrasonographic fatty liver is unlikely to be
a late event during the development of probable cirrhosis.
    In conclusion, this study found that HBV genotype C, age (≥ 45 years),
ALT abnormality, and male sex are independently associated with an increased
risk of probable cirrhosis. Ultrasonographic fatty liver is not found in the
subjects with probable cirrhosis. Although cirrhosis-like ultrasonographic
abnormalities are not clinical liver cirrhosis, it is an early event during
the development of clinical cirrhosis. Genotype C HBV-infected male residents
at the age of 45 years or older should be routinely examined for active
hepatitis and early cirrhosis. Early intervention to the HBV-infected subjects
with high risks of cirrhosis might be effective for decreasing the overall
mortality from liver cirrhosis and subsequent HCC.

ACKNOWLEDGMENTS
We thank Dr. Hong-Xia Ni and Dr. Ting Fang, Municipal Center for Disease
Control and Prevention of Ningbo, for their great help in field study; Dr. Lei
Han, Dr. Wen-Ying Lu, Dr. Yi-Fang Han, Dr. Xiao-Jie Tan, and Dr. Wen-Jun
Chang, Department of Epidemiology, Second Military Medical University for
their technical assistance.

StephenW

COMMENTS
Background
Chronic hepatitis B virus (HBV) infection is the most important risk factor of
liver cirrhosis and hepatocellular carcinoma (HCC) in HBV endemic areas.
Metabolic syndrome has been found to be an independent risk factor of liver
cirrhosis in the patients with chronic hepatitis B. The relationship between
HBV genotypes and liver cirrhosis remains controversial. Furthermore, the
association between HBV genotypes and subclinical cirrhosis has not been
evaluated in community-based population.
Research frontiers
HBV genotypes have distinct geographical distributions and differ with regard
to clinical outcome, prognosis, and response to interferon treatment. The role
of genotype B and C, the two major HBV genotypes endemic in East Asia, in the
development of liver cirrhosis has not been unequivocally addressed. In this
study, the authors demonstrate that infection with HBV genotype C is closely
associated with subclinical cirrhosis in the community-based subjects with
increasing age.
Innovations and breakthroughs
Recent reports have highlighted the importance of HBV genotypes, alanine
aminotransferase (ALT), age, and sex in hepatocarcinogenesis and the
development of clinical liver cirrhosis. Metabolic syndrome has been found to
be independently associated with liver cirrhosis in the patients with chronic
hepatitis B. This is the first study to report that HBV genotype C, age (≥
45 years), male sex, and ALT abnormality are independently associated with
probable cirrhosis in community-based HBV-infected subjects, especially with
the subclinical liver cirrhosis. Furthermore, this study suggested that fatty
liver may not be associated with probable liver cirrhosis.
Applications
This study suggests that genotype C HBV-infected male residents at the age of
45 years or older should be routinely examined for active hepatitis and early
cirrhosis. Early intervention to the HBV-infected subjects with high risks of
cirrhosis might be effective for decreasing the overall mortality from liver
cirrhosis and subsequent HCC.
Terminology
Probable cirrhosis is referred to ultrasonographic cirrhosis (ultrasonographic
score ≥ 8) and cirrhosis-like ultrasonographic abnormalities
(ultrasonographic score from 5 to 7). Probable cirrhosis is not histologically
confirmed liver cirrhosis. Ultrasonography is an imaging examination which is
widely accepted by the community-based HBV-infected subjects without apparent
clinical manifestations.
Peer review
The results of this study provide sufficient experimental evidences or data
from which scientific conclusions can be drawn. The discussion is well
organized and an overall theoretical analysis is given.

REFERENCES <CUT>

jsmscym

看不懂，请翻译一下。