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Epidemiological study of hepatitis B virus genotypes, core promoter and precore mutations of chronic hepatitis B infection in Hong Kong
Journal of Hepatology 41 (2004) 119–125 www.elsevier.com/locate/jhep
Epidemiological study of hepatitis B virus genotypes, core promoter and precore mutations of chronic hepatitis B infection in Hong Kong Man-Fung Yuen1, Erwin Sablon2, Yasuhito Tanaka3, Takanobu Kato3, Masashi Mizokami3, Joke Doutreloigne2, He-Jun Yuan1, Danny Ka-Ho Wong1, Siu-Man Sum1, Ching-Lung Lai1,* 1
Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, People’s Republic of China 2 Innogenetics N.V., Ghent, Belgium 3 Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Kawasumi, Mizuho, Nagoya 467-8601, Japan
Background/Aims: We conducted a population study to document the prevalence of hepatitis B virus (HBV) genotypes in Hong Kong. Methods: HBV genotypes, core promoter (CP) and precore mutations were determined in 776 asymptomatic patients. Results: 92.6% patients had single genotype [B (32.5%), C (62.5%)]. 99.1% of genotype B was subtype Ba. Patients with age < 50 years had a lower prevalence of genotype B than patients with age >51 years (32.5% vs. 41%, respectively, P 5 0.028). Compared to patients with genotype C, patients with genotype B had a higher cumulative rate (P 5 0.018) and younger age (40.1 vs. 34.2 years, respectively, P 5 0.018) of HBeAg seroconversion. There were no differences in the HBV DNA levels between patients with genotypes B and C, and with wild-type and mutants of CP and precore regions. By multivariate analysis, patients with genotype C and with CP mutations had higher alanine aminotransferase (ALT) levels. Conclusions: B and C were the two most common HBV genotypes in Hong Kong. The former had a higher chance of earlier HBeAg seroconversion and lower ALT levels. The prevalence of genotype B was lower in patients with age < 50, probably related to influx of immigrants from China since 1949. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Epidemiology; Hepatitis B virus; Genotype; Precore mutation; Core promoter mutation
1. Introduction Globally, there are around 400 million people with chronic hepatitis B (CHB) infection [1]. Of these, more than 75% of CHB patients are Asians [2]. Hepatitis B virus (HBV) has been classified into 7 major genotypes according to the criterion of $ 8% differences in the complete nucleotide sequence of the viral genome [3]. The different geographical distribution of various genotypes are as follows: A—Northwest Europe, North America, Central Africa; B and C—Asia; D—Mediterranean area, Middle East, India; E—sub-Saharan Africa; F—United States of America; and G—United States of America and France. Received 22 December 2003; received in revised form 20 February 2004; accepted 16 March 2004; available online 7 April 2004 * Corresponding author. Tel.: þ 852-28554252; fax: þ 852-28162863. E-mail address: [email protected] (C.L. Lai).
Another newly discovered HBV genotype H has been found in Central America [4]. Studies suggest that there are differences between patients with different genotypes in the time of hepatitis B e antigen (HBeAg) seroconversion, development of hepatocellular carcinoma and responsiveness to interferon treatment [5 – 14]. Recently, two strains of genotype B are identified in Asia [15,16] namely, Bj (mainly found in Japan) and Ba (mainly found in other parts of Asia). Bj is probably the “original” genotype B whereas as Ba is a B/C recombinant with the precore/core region originating from genotype C. Large population studies are required to document the prevalence of HBV genotypes and the two common HBV mutants, namely core promoter and precore mutants in different parts of the world. The present study is a large epidemiological study aimed at documenting the age specific prevalence and the clinical significance of genotypes B and C in CHB patients in
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.03.004
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Table 1 Demographic data, HBeAg/anti-HBe status and liver biochemistry of the 776 asymptomatic patients
Number of patients Male: female ratio Median age, years (range) HBeAg: anti-HBe Median albumin level, U/L (range) Median bilirubin level, mmol/l (range) Median ALT level, U/L (range)
All patients
Patients with data on presentation and subsequent follow-up Patients with data on presentation
776 565: 211 37.5 (1.2–80.5) 406: 370 44 (36–54) 11 (1–36) 48 (7–1251)
498 356: 142 38.3 (1.2–80.5) 217: 281 44 (37– 53) 11 (1– 36) 43 (7– 1251)
278 209: 69 35.7 (7.8–79.6) 189: 89 45 (36–54) 11 (2– 22) 57 (9– 838)
ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg.
Hong Kong where the prevalence of CHB infection is high (10%). The prevalence of core promoter and precore mutations in patients with genotypes B and C and their clinical significance were also determined.
2. Patients and methods From January 1999 to June 2003, 776 asymptomatic patients with CHB infection seen for the first time or followed up consecutively in the Hepatitis Clinic, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong were recruited. To determine the epidemiology of HBV genotypes, core promoter and precore mutations in clinical asymptomatic CHB carriers, we excluded patients presenting with hepatocellular carcinoma (HCC) and/or cirrhosis-related complications during this period ðn ¼ 187Þ: All patients were hepatitis B surface antigen (HBsAg) positive for at least 6 months. Patients with other concomitant diseases including hepatitis C or D virus infection, autoimmune hepatitis, Wilson’s disease, primary biliary cirrhosis, alcoholic liver disease and fatty liver (diagnosed by ultrasonography) were excluded. We excluded patients with these concomitant conditions because of their possible effects on the liver biochemistry especially on the alanine aminotransferase levels. Patients who had received treatment for CHB prior to presentation or during subsequent follow-up were also excluded. Serum on presentation of all the 776 patients were taken to determine the liver biochemistry including alanine aminotransferase (ALT), albumin and bilirubin levels, HBeAg and antibody to HBeAg (anti-HBe) [by microparticle enzyme immunoassay (MEIA), Abbott Laboratories, Chicago, Illinois, USA], and HBV genotyping and core promoter and precore mutations (methodology described below). Data on the subsequent followup were analyzed for 498 out of the 776 patients. The median follow-up of these 498 patients was 66 months (range 4–376 months). These patients were followed up at intervals of 3– 6 months with monitoring of the liver biochemistry, HBeAg and anti-HBe status. Serum HBV DNA levels on presentation were measured by Cobas Amplicor HBV Monitor test, Roche Diagnostics, Branchburg, NJ (lower limit of detection of 200 copies/ml) in 351 patients (45.2%) and by Digene Hybrid Capture assay (Digene Corporation, Gaithersburg, Maryland, USA) (lower limit of detection 140,000 copies/ml) in 288 patients (37.1%). For the Cobas Amplicor HBV Monitor test, pre-assay 100,000 folds dilution was performed in samples from HBeAg-positive patients. HBV genotyping were performed by two different assays: the line probe assay (INNO-LiPA HBV Genotyping, Innogenetics NV, Gent, Belgium) and the enzyme immunosorbent assay (ELISA). To verify and confirm the results from these two assays, HBV genotypes were determined in 82 patients by both assays. The HBV genotypes of 437 patients were determined by the line probe assay and 257 patients were determined by ELISA. The methodologies for both assays were described in our previous studies [5,16]. HBV genotype B subtypes (Ba, Bj) were determined by a PCR-restriction fragment length polymorphism method as described in our previous study [16]. The core promoter and precore mutations were determined by another line probe assay, INNO-LiPA HBV Precore, Innogenetics, Belgium. The details of the methodology were described in our previous study [17].
2.1. Statistical analysis The prevalence of genotypes between different age groups were compared by Chi-square test. The differences in the median albumin, bilirubin, and ALT and HBV DNA levels between different HBV genotypes were compared by Mann–Whitney test. Linear regression analysis was use to test the independent association between the ALT levels and other variables. P values (two-tailed) of #0.05 were considered to be statistically significant.
3. Results The demographic data, HBeAg/anti-HBe status and liver biochemistry of the 776 asymptomatic patients are listed in Table 1. 3.1. Determination of HBV genotypes by both assays The results of the HBV genotypes determined by both LiPA and ELISA assays were compared in 82 patients. All the 82 patients had single HBV genotype of either B or C. Concordant results were found in 78 patients (95.1%) (19 patients with genotype B, 59 patients with genotype C). The remaining four patients showed different results determined by the two assays (genotype C by LiPA but genotype B by ELISA). Direct sequencing of the surface gene with primers spanning nt 2808– 2830 and nt 977 –996 showed that two were of genotype B and two were of genotype C. These results showed that both the assays yield highly consistent genotyping results. 3.2. Prevalence of HBV genotypes Of the 776 patients, 750 patients (96.6%) had single HBV genotype [(A—12 (1.5%), B—252 (32.5%), C—486 (62.6%)] and 26 patients had mixed genotypes (3.4%). The two most common HBV genotypes were genotypes B and C. The prevalence of HBV genotypes B and C in patients of different age groups are listed in Table 2. There were no differences in the prevalence of genotypes B and C among first three younger age groups (P ¼ NS for all). Patients with the age 31 – 40 years had a significantly lower prevalence of genotype B compared to patients with the age $ 51 years ðP ¼ 0:027Þ: When patients were subdivided
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Table 2 Prevalence of HBV genotypes according to different age groups Patients’ age (years)
HBV genotypes
#30 ðn ¼ 217Þ 31–40 ðn ¼ 269Þ 41–50 ðn ¼ 173Þ $51 ðn ¼ 117Þ
B ðn ¼ 252Þ
C ðn ¼ 486Þ
Others or mixed genotypes ðn ¼ 38Þ
71 (32.7%) 79 (29.4%)* 54 (31.2%) 48 (41.0%)*
141 (65.0%) 174 (64.7%)* 108 (62.4%) 63 (53.8%)*
5 (2.3%) 16 (5.9) 11 (6.4%) 6 (5.1%)
P ¼ 0:027: Table 3 Prevalence of HBV genotypes B and C with respect to the age and the HBeAg/anti-HBe status Patients’ age (years)
HBV genotypes
#30 ðn ¼ 212Þ 31–40 ðn ¼ 253Þ 41–50 ðn ¼ 162Þ $51 ðn ¼ 111Þ
B, HBeAg: anti-HBe
C, HBeAg: anti-HBe
P value
60: 11 (84.5%: 15.5%) 38: 41 (48.1%: 51.9%) 11: 43 (20.4%: 79.6%) 5: 43 (10.4%: 89.6%)
108: 33 (76.7%: 23.4%) 107: 67 (61.5%: 38.5%) 46: 62 (42.6%: 57.4%) 20: 43 (31.7%: 68.3%)
0.118 0.046 0.005 0.011
HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg. Table 4 Liver biochemistry of patients with genotypes B and C on presentation Total
Albumin, g/l Bilirubin, mmol/l ALT, U/L
HBeAg positive
Anti-HBe positive
B ðn ¼ 252Þ
C ðn ¼ 486Þ
P value
B ðn ¼ 114Þ
C ðn ¼ 281Þ
P value
B ðn ¼ 138Þ
C ðn ¼ 205Þ
P value
45 (35–54) 11 (3–57) 40 (7–1153)
44 (37–53) 11 (1– 96) 52 (8– 1251)
0.09 0.11 0.002
45 (36–54) 11 (4– 39) 48.5 (9–1153)
45 (37–53) 10 (3–96) 58.5 (12–1251)
0.72 0.42 0.076
44 (35– 53) 11 (3–57) 33 (7–859)
44 (37– 53) 11 (1– 60) 48 (8– 648)
0.71 0.29 0.072
Values expressed in median (range). ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg.
only into age , 50 and . 51 years, there was a significantly lower prevalence of genotype B in the former group (32.5%, 204 out of 627) compared to the latter group (41%, 48 out of 111, P ¼ 0:028). Subtypes Ba and Bj were determined in 115 patients with genotype B. 114 patients (99.1%) were of subtype Ba, whereas only one patient (0.9%) was of subtype Bj. 3.3. HBeAg seroconversion in patients with genotypes B and C Since the two most common HBV genotypes were B and C, only patients with single genotypes B or C are examined in the subsequent analyses. Patients at different age groups were further classified according to the HBeAg/anti-HBe status. The prevalence of genotypes B and C with respect to the age groups and the HBeAg/anti-HBe status are listed in Table 3. Comparing patients with genotypes B and C, patients with genotype B were associated with a higher antiHBe positivity rate at all age groups except those with age , 30 years.
Among the 498 patients with longitudinal follow-up data, 217 (43.6%) were HBeAg positive on presentation. Of these, 63 patients had genotype B and 143 patients had genotype C. Patients with genotype B had a higher cumulative rate of HBeAg seroconversion compared to patients with genotype C ðP ¼ 0:018Þ (Fig. 1). In addition, patients with genotype B had a younger median age of HBeAg seroconversion compared to patients with genotype C [34.2 (range 6.2– 78.7) vs. 40.1 (range 21.2 –71.5) years respectively, P ¼ 0:018]. 3.4. Effects of HBV genotypes on liver biochemistry on presentation The comparisons of the median albumin, bilirubin and ALT levels on presentation were made between patients with genotype B and C in Table 4. Patients with genotypes B and C had comparable median albumin and bilirubin levels. However, patients with genotype B had a significantly lower median ALT level compared to those with genotype C [40 (7 –1153) vs. 52 (8 – 1251) U/L respectively, P ¼ 0:002]. By
0.83
1.3 £ 105 (,200–1.4 £ 109) ðn ¼ 92Þ 1.2 £ 106 (,1.4 £ 105 –7.1 £ 108) ðn ¼ 85Þ
Fig. 1. The cumulative HBeAg seroconversion rate in patients with genotype B (solid line) and patients with genotype C (dotted line).
Values expressed in median (range), copies/ml. HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg.
0.40
3.6 £ 104 (,200–4.9 £ 108) ðn ¼ 63Þ 3.3 £ 105 (,1.4 £ 105 –4.8 £ 108) ðn ¼ 64Þ 0.67
1.8 £ 107 (,200 –1.1 £ 1013) ðn ¼ 141Þ 4.5 £ 106 (,1.4 £ 105 – 1.4 £ 109) ðn ¼ 97Þ Cobas Amplicor HBV Monitor test 2.5 £ 107 (,200 –4.8 £ 1010) ðn ¼ 55Þ Digene Hybrid Capture assay 2.0 £ 107 (,1.4 £ 105 –1.3 £ 109) ðn ¼ 42Þ
Genotype C P value Genotype B Genotype B
Genotype C
Anti-HBe positive HBeAg positive HBV DNA
Table 5 HBV DNA levels of patients with genotypes B and C with respect to the HBeAg/anti-HBe status on presentation
0.71
M.-F. Yuen et al. / Journal of Hepatology 41 (2004) 119–125 P value
122
subdividing patients according to the HBeAg/anti-HBe positivity, there were no differences in the median albumin and bilirubin levels between patients with genotype B and C (Table 4). However, there were trends for patients with genotype B to have a lower median ALT level than those with genotype C both at the HBeAg positive ðP ¼ 0:076Þ and anti-HBe positive ðP ¼ 0:072Þ phases (Table 4). 3.5. Effects of HBV genotypes on HBV DNA levels on presentation Serum HBV DNA levels were measured in 351 patients by the Amplicor HBV Monitor test and in 288 patients by the Digene Hybrid Capture assay. The median HBV DNA levels were compared between patients with genotypes B and C with respect to the HBeAg and anti-HBe status and the two different assays (Table 5). There were no differences in the median HBV DNA level measured by both the assays between patients with genotypes B and C both at the HBeAg positive and anti-HBe positive phases. 3.6. HBV genotypes, core promoter and precore mutations For patients with either genotypes B or C, the presence of mutants of the core promoter and precore regions were determined in 363 and 368 patients, respectively. The prevalence of these mutations with respect to HBV genotypes are listed in Table 6. Patients with genotype B had a higher prevalence of precore mutants than patients with genotype C [83.6 vs. 29.1%, respectively, P , 0:001; Odds ratio (OR) 12.4, 95% confidence interval (CI) ¼ 7.2– 21.3]. By contrast, patients with genotype C had a higher prevalence of core promoter mutations compared to those with genotype B (87.9 vs. 38.6%, respectively, P , 0:001; OR ¼ 11.5, 95% CI ¼ 6.8– 19.5).
M.-F. Yuen et al. / Journal of Hepatology 41 (2004) 119–125 Table 6 Prevalence of core promoter and precore mutations in patients with genotypes B and C Genotype B
Genotype C
P value
Core promoter region Wild-type 81 (61.4%) Mutant 51 (38.6%)
28 (12.1%) 203 (87.9%)
,0.001
Precore region Wild-type Mutant
166 (70.9%) 68 (29.1%)
,0.001
22 (16.4%) 112 (83.6%)
3.7. Core promoter, precore mutations and HBeAg/anti-HBe status There was no difference in the prevalence of core promoter mutations between HBeAg-positive patients (128 out of 177, 72.3%) and anti-HBe-positive patients (144 out of 213, 67.6%, P ¼ 0:37). Anti-HBe-positive patients had a higher prevalence of precore mutations compared to HBeAg-positive patients (122 out of 215, 56.7% vs. 75 out of 181, 41.4%, respectively, P ¼ 0:003; OR ¼ 1.9, 95% CI ¼ 1.2– 2.8). 3.8. Effects of core promoter and precore mutations on liver biochemistry on presentation The liver biochemistry of patients with core promoter and precore mutations are listed in Table 7. There were comparable median albumin and bilirubin levels between patients with core promoter wild-type and mutants and between patients with precore wild-type and mutants (P ¼ NS for all). However, patients with core promoter mutants had a significantly higher median ALT level compared to those of wild-type both at the HBeAg and anti-HBe positive phases (Table 7). 3.9. Effects of core promoter and precore mutations on HBV DNA levels The HBV DNA levels measured by Amplicor HBV Monitor test and the Digene Hybrid Capture assay with respect to the mutations of the core promoter and precore regions are listed in Table 8. There were no differences in the median HBV DNA levels measured by both assays between patients with wild-type and mutants of both the core promoter and precore regions (P ¼ NS for all). 3.10. Multivariate analysis on the factors associated with ALT levels Multivariate analysis including variables of genotypes, core promoter mutations, patients’ age and HBeAg/antiHBe status revealed that patients with genotype C ðP ¼ 0:037Þ and patients with core promoter mutations ðP ¼ 0:05Þ were both independently associated with higher ALT
123
Table 7 Liver biochemistry of patients with core promoter and precore mutants on presentation Albumin (g/l)
Bilirubin (mmol/l)
ALT (U/L)
11 (3–37) 11 (1–96)
45 (7– 624)* 60 (11– 1251)*
10 (4–28) 11 (3–96)
44 (12– 624)† 65 (12– 1251)†
11 (3–37) 11 (1–57)
46 (7– 291)‡ 57 (11– 859)‡
43 (37–53) 43 (32–53)
11 (2–96) 11 (1–38)
54 (11– 1251) 49 (7– 859)
44 (37–50) 43 (32–50)
11 (3–96) 11 (4–38)
51 (13– 1251) 70 (10– 642)
43 (35–53) 43 (36–53)
12 (2–57) 11 (1–37)
56 (11– 473) 47.5 (7–859)
Core promoter region Total WT ðn ¼ 118Þ 44 (35–53) MT ðn ¼ 272Þ 43 (37–53) HBeAg þ ve WT ðn ¼ 49Þ 44 (35–50) MT ðn ¼ 128Þ 43 (37–50) Anti-HBe þ ve WT ðn ¼ 69Þ 43 (37–53) MT ðn ¼ 144Þ 43 (35–53) Precore region Total WT ðn ¼ 199Þ MT ðn ¼ 197Þ HBeAg þ ve WT ðn ¼ 106Þ MT ðn ¼ 75Þ Anti-HBe þ ve WT ðn ¼ 93Þ MT ðn ¼ 122Þ
Values expressed in median (range). ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg; WT, wildtype; MT, mutants. *P ¼ 0:002; †P ¼ 0:024; ‡P ¼ 0:049:
levels on presentation compared to those with genotype B and those with core promoter wild-type, respectively.
4. Discussion To our knowledge, the present study is the largest epidemiologic study on HBV genotypes examining the prevalence of HBV genotypes B and C, core promoter and precore mutations in Asia. Our center accepted referrals for patients with or without symptoms of hepatitis and with normal or elevated ALT levels. There should be no referral bias of the study population. Therefore, the findings of the present study are representative to the CHB patient population in Hong Kong. The majority of the CHB patients had single genotype in which 32.5% were genotype B and 62.6% were genotype C. This pattern of distribution is unique when compared to those of other countries in Asia. The prevalence of genotype B is higher and genotype C is lower in Hong Kong compared to two cites in the Peoples’ Republic of China, namely Shanghai and Nanning where the prevalence of genotypes B and C are 14– 17% and 79 – 81%, respectively [18,19]. In contrast, the prevalence of genotype B (60 – 68%) is much higher and genotype C is lower (32 – 34%) in Taiwan [20,21]. There is a variable pattern of distribution of genotypes in Japan. Over 94% of patients on the Japanese mainland have genotype C, whereas 60% of patients in southern island and 23% of patients in the northern part of the mainland have genotype B [22].
0.64
1.1 £ 106 (,200–9.2 £ 1012) ðn ¼ 64Þ 2.4 £ 106 (,1.4 £ 105 –1.4 £ 109) ðn ¼ 128Þ 0.10
6.8 £ 106 (,200–1.1 £ 1013) ðn ¼ 60Þ 2.3 £ 106 (,1.4 £ 105 –1.3 £ 109) ðn ¼ 129Þ 0.52
Values expressed in median (range), copies/ml.
4.2 £ 106 (,200 –1.1 £ 1013) ðn ¼ 81Þ 1.9 £ 106 (,1.4 £ 105 –1.4 £ 109) ðn ¼ 178Þ Cobas Amplicor HBV Monitor test 1.1 £ 106 (,200 –9.2 £ 1012) ðn ¼ 44Þ Digene Hybrid Capture assay 5.3 £ 106 (,1.4 £ 105 – 1.3 £ 109) ðn ¼ 72Þ
Mutants P value Wild-type Wild-type
Mutants
Precore region Core promoter region HBV DNA
Table 8 HBV DNA levels of patients with core promoter and precore mutations on presentation
0.07
M.-F. Yuen et al. / Journal of Hepatology 41 (2004) 119–125 P value
124
When the prevalence of HBV genotypes B and C in Hong Kong was stratified according to different age groups at an interval of 10 years, the prevalence of HBV genotypes B and C remained at around 29 – 33% and 62 – 65%, respectively, across the age groups of , 30, 31 –40 and 41– 50 years. However, the prevalence of HBV genotype B was significantly higher (41%) and hence HBV genotype C was lower (53.8%) in the patients with age . 51 years when compared to those with age 31 –40 years ðP ¼ 0:027Þ (Table 2) and those with age , 50 years ðP ¼ 0:028Þ: Since majority of the Chinese patients are infected with HBV during perinatal and childhood period, these findings suggest that there was a change in the viral population around 40 –50 years ago but it had remained unchanged over the last 40 years. This phenomenon may be related to the influx of immigrants from the Peoples’ Republic of China to Hong Kong after the Communist takeover in 1949. Since the prevalence of HBV genotype B was lower, around 14– 17%, in the Peoples’ Republic of China [18,19], an influx of immigrants from China would result in a subsequent decrease in the prevalence of HBV genotype B in the last 40 – 50 years. The present study also showed that for patients with genotype B, 99.1% were of subtype Ba and only 0.9% was of subtype Bj. It supported the findings that Bj was nearly exclusively found in Japan and Ba was mainly found in Asia. There are suggestions from other studies that clinical differences exist between HBV subtype Bj and Ba. Sugauchi and his colleagues show that the chance of loss of HBeAg is the highest in patients with subtype Bj followed by patients with subtype Ba and is the lowest in patients with genotype C [16]. Akuta and his colleagues show that the emergence rate of lamivudine resistance is significantly higher in the Ba subgroup than in the Bj subgroup of HBV [23], though this conclusion is drawn from only 4 patients with Ba and 14 patients with Bj subtype. However, no difference in the rate of lamivudine resistance is found between patients with genotype B and C, which was also confirmed in a recent study conducted in our Center [24]. Further studies are required to compare the clinical differences, if these exist, between patients with subtypes Bj, Ba and genotype C. The higher prevalence of anti-HBe positivity in patients with genotype B compared to those with genotype C at all age groups except in the group with age , 30 years (Table 3) suggested that patients with genotype B had a higher chance of earlier HBeAg seroconversion compared to those with genotype C. The reason for the absence of this difference in the group of patients with age , 30 years is mainly due to the fact that these patients are still in the immunotolerance phase. In the Chinese population, the immunoclearance phase usually begins at the age after 30 years and the median age of HBeAg seroconversion was 34.5 years [25]. The higher chance of HBeAg seroconversion in patients with genotype B was further confirmed by the significantly higher cumulative rate (Fig. 1) and younger median age of HBeAg seroconversion in patients with genotype B
M.-F. Yuen et al. / Journal of Hepatology 41 (2004) 119–125
compared to those with genotype C. The present large-scale study confirmed this phenomenon with other studies [5 –7, 19]. It is important to note that, though the age of HBeAg seroconversion for patients with genotype B was earlier than those with genotype C, the median age of HBeAg seroconversion for patients with genotype B was still more than three decades after the initial hepatitis B infection. Whether the slightly earlier HBeAg seroconversion in these patients carries any clinical significance in term of the future development of HCC and cirrhosis related complications remains unknown to date. The present study showed that patients with genotype C and patients with core promoter mutations were independently associated with higher ALT levels on presentation compared to those with genotype B and those with core promoter wild-type respectively (Tables 4 and 7). However, there were no differences in the albumin, bilirubin and HBV DNA levels on presentation when comparisons were made between these respective groups of patients (Tables 4, 5, 7 and 8). The possible effect of the higher chance of earlier HBeAg seroconversion, and the lower ALT levels on presentation in patients with genotype B compared to those with genotype C are currently being studied in our center by liver histology in term of degree of necroinflammation and fibrosis, and by following up patients for the chance of development of HCC and cirrhosis-related complications. In conclusion, the present study showed that genotypes B and C were the two most common HBV genotypes in Hong Kong contributing 32.5% and 62.6% of CHB population respectively. The prevalence of genotype B was lower in patients below the age of 50, probably related to influx of immigrants from China since 1949. More than 99% of patients with genotype B were of subtype Ba. Patients with genotype B had a higher chance of earlier HBeAg seroconversion and lower ALT levels on presentation compared to patients with genotype C.
References [1] Yuen MF, Lai CL. Treatment for chronic hepatitis B. Lancet Infect Dis 2001;1:232–241. [2] Lai CL. Chronic hepatitis B in Hong Kong: immunisation strategies for the control of hepatitis B virus infection. In: Zuckerman, editor. Screening, diagnosis and control. Hepatitis B in the Asian-Pacific region, vol. 1. Royal College of Physicians of London; 1997. p. 79–87. [3] Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Imai M, Miyakawa Y, et al. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 1988;69:2575 –2583. [4] Arauz-Ruiz P, Norder H, Robertson BH, Magnius LO. Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America. J Gen Virol 2003;2059–2073. [5] Yuen MF, Sablon E, Yuan HJ, Wong DKH, Hui CK, Wong BCY, et al. The significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications and hepatocellular carcinoma. Hepatology 2003;37:562–567.
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[6] Chu CJ, Hussain M, Lok ASF. Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology 2002;122:1756–1762. [7] Sumi H, Yokosuka O, Seki N, Arai M, Imazeki F, Kurihara T, et al. Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease. Hepatology 2003;37:19– 26. [8] Fujie H, Moriya K, Shintani Y, Yotsuyanagi H, Iino S, Kimura S, et al. Hepatitis B genotypes and hepatocellular carcinoma in Japan. Gastroenterology 2001;120:1564– 1565. [9] Sakugawa H, Nakasone H, Nakayoshi T, Orito E, Mizokami M, Yamashiro T, et al. Preponderance of hepatitis B virus genotype B contributes to a better prognosis of chronic HBV infection in Okinawa, Japan. J Med Virol 2002;67:484 –489. [10] Orito E, Mizokami M, Sakugawa H, Michitaka K, Ishikawa K, Ichida T, et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV genotype research group. Hepatology 2001;33:218– 223. [11] Wai CY, Chu CJ, Hussain M, Lok AS. HBV genotype B is associated with a higher response rate to interferon therapy than genotype C. Hepatology 2002;36:1425 –1430. [12] Kao JH, Wu NH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes and the response to interferon therapy. J Hepatol 2000;33:998–1002. [13] Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 2000;118:554– 559. [14] Kao JH, Chen PJ, Lai MY, Chen DS. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology 2003;124:327–334. [15] Sugauchi F, Orito E, Ichida T, Kato H, Sakugawa H, Kakumu S, et al. Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene. J Virol 2002; 76:5985–5992. [16] Sugauchi F, Orito E, Ichida T, Kato H, Chutaputti A, Lai CL, et al. Epidemiologic and virologic characteristics of hepatitis B virus genotype B having the recombination with genotype C. Gastroenterology 2003;124:925 –932. [17] Yuen MF, Sablon E, Yuan HJ, Hui CK, Wong DKH, Doutreloigne J, et al. The relationship between the development of precore and core promoter mutations and HBeAg seroconversion in chronic hepatitis B. J Infect Dis 2002;186:1335–1338. [18] Ding X, Mizokami M, Yao G, Xu B, Orito E, Ueda R, et al. Hepatitis B virus genotype distribution among chronic hepatitis virus carriers in Shanghai, China. Intervirology 2001;44:43 –47. [19] Ding X, Mizokami M, Ge X, Orito E, Iino S, Ueda R, et al. Different hepatitis B virus genotype distributions among asymptomatic carriers and patients with liver diseases in Nanning, southern China. Hepatol Res 2002;22:37–44. [20] Kao JH, Chen PJ, Lai MY, Chen DS. Genotypes and clinical phenotypes of hepatitis B virus in patients with chronic hepatitis B virus infection. J Clin Microbiol 2002;40:1207 –1209. [21] Lee CM, Chen CH, Lu SN, Tung HD, Chou WJ, Wang JH, et al. Prevalence and clinical implications of hepatitis B virus genotypes in southern Taiwan. Scand J Gastroenterol 2003;38:95–101. [22] Orito E, Ichida T, Sakugawa H, Sata M, Horiike N, Hino K, et al. Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology 2001;34:590–594. [23] Akuta N, Suzuki F, Kobayashi M, Tsubota A, Suzuki Y, Hosaka T, et al. The influence of hepatitis B virus genotype on the development of lamivudine resistance during long-term treatment. J Hepatol 2003; 315–321. [24] Yuen MF, Wong DKH, Sablon E, Yuan HJ, Sum SM, Hui CK, et al. Hepatitis B virus genotypes B and C do not affect the anti-viral response to lamivudine. Antiviral Therapy 2003;8:531–534. [25] Yuen MF, Yuan HJ, Hui CK, Wong DKH, Wong WM, Chan AOO, et al. A large population study of spontaneous HBeAg seroconversion and acute exacerbation of chronic hepatitis B infection: implications for antiviral therapy. Gut 2003;53:416– 419.
Epidemiological study of hepatitis B virus genotypes, core promoter and precore mutations of chronic hepatitis B infection in Hong Kong Man-Fung Yuen1, Erwin Sablon2, Yasuhito Tanaka3, Takanobu Kato3, Masashi Mizokami3, Joke Doutreloigne2, He-Jun Yuan1, Danny Ka-Ho Wong1, Siu-Man Sum1, Ching-Lung Lai1,* 1
Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, People’s Republic of China 2 Innogenetics N.V., Ghent, Belgium 3 Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Kawasumi, Mizuho, Nagoya 467-8601, Japan
Background/Aims: We conducted a population study to document the prevalence of hepatitis B virus (HBV) genotypes in Hong Kong. Methods: HBV genotypes, core promoter (CP) and precore mutations were determined in 776 asymptomatic patients. Results: 92.6% patients had single genotype [B (32.5%), C (62.5%)]. 99.1% of genotype B was subtype Ba. Patients with age < 50 years had a lower prevalence of genotype B than patients with age >51 years (32.5% vs. 41%, respectively, P 5 0.028). Compared to patients with genotype C, patients with genotype B had a higher cumulative rate (P 5 0.018) and younger age (40.1 vs. 34.2 years, respectively, P 5 0.018) of HBeAg seroconversion. There were no differences in the HBV DNA levels between patients with genotypes B and C, and with wild-type and mutants of CP and precore regions. By multivariate analysis, patients with genotype C and with CP mutations had higher alanine aminotransferase (ALT) levels. Conclusions: B and C were the two most common HBV genotypes in Hong Kong. The former had a higher chance of earlier HBeAg seroconversion and lower ALT levels. The prevalence of genotype B was lower in patients with age < 50, probably related to influx of immigrants from China since 1949. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Epidemiology; Hepatitis B virus; Genotype; Precore mutation; Core promoter mutation
1. Introduction Globally, there are around 400 million people with chronic hepatitis B (CHB) infection [1]. Of these, more than 75% of CHB patients are Asians [2]. Hepatitis B virus (HBV) has been classified into 7 major genotypes according to the criterion of $ 8% differences in the complete nucleotide sequence of the viral genome [3]. The different geographical distribution of various genotypes are as follows: A—Northwest Europe, North America, Central Africa; B and C—Asia; D—Mediterranean area, Middle East, India; E—sub-Saharan Africa; F—United States of America; and G—United States of America and France. Received 22 December 2003; received in revised form 20 February 2004; accepted 16 March 2004; available online 7 April 2004 * Corresponding author. Tel.: þ 852-28554252; fax: þ 852-28162863. E-mail address: [email protected] (C.L. Lai).
Another newly discovered HBV genotype H has been found in Central America [4]. Studies suggest that there are differences between patients with different genotypes in the time of hepatitis B e antigen (HBeAg) seroconversion, development of hepatocellular carcinoma and responsiveness to interferon treatment [5 – 14]. Recently, two strains of genotype B are identified in Asia [15,16] namely, Bj (mainly found in Japan) and Ba (mainly found in other parts of Asia). Bj is probably the “original” genotype B whereas as Ba is a B/C recombinant with the precore/core region originating from genotype C. Large population studies are required to document the prevalence of HBV genotypes and the two common HBV mutants, namely core promoter and precore mutants in different parts of the world. The present study is a large epidemiological study aimed at documenting the age specific prevalence and the clinical significance of genotypes B and C in CHB patients in
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.03.004
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Table 1 Demographic data, HBeAg/anti-HBe status and liver biochemistry of the 776 asymptomatic patients
Number of patients Male: female ratio Median age, years (range) HBeAg: anti-HBe Median albumin level, U/L (range) Median bilirubin level, mmol/l (range) Median ALT level, U/L (range)
All patients
Patients with data on presentation and subsequent follow-up Patients with data on presentation
776 565: 211 37.5 (1.2–80.5) 406: 370 44 (36–54) 11 (1–36) 48 (7–1251)
498 356: 142 38.3 (1.2–80.5) 217: 281 44 (37– 53) 11 (1– 36) 43 (7– 1251)
278 209: 69 35.7 (7.8–79.6) 189: 89 45 (36–54) 11 (2– 22) 57 (9– 838)
ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg.
Hong Kong where the prevalence of CHB infection is high (10%). The prevalence of core promoter and precore mutations in patients with genotypes B and C and their clinical significance were also determined.
2. Patients and methods From January 1999 to June 2003, 776 asymptomatic patients with CHB infection seen for the first time or followed up consecutively in the Hepatitis Clinic, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong were recruited. To determine the epidemiology of HBV genotypes, core promoter and precore mutations in clinical asymptomatic CHB carriers, we excluded patients presenting with hepatocellular carcinoma (HCC) and/or cirrhosis-related complications during this period ðn ¼ 187Þ: All patients were hepatitis B surface antigen (HBsAg) positive for at least 6 months. Patients with other concomitant diseases including hepatitis C or D virus infection, autoimmune hepatitis, Wilson’s disease, primary biliary cirrhosis, alcoholic liver disease and fatty liver (diagnosed by ultrasonography) were excluded. We excluded patients with these concomitant conditions because of their possible effects on the liver biochemistry especially on the alanine aminotransferase levels. Patients who had received treatment for CHB prior to presentation or during subsequent follow-up were also excluded. Serum on presentation of all the 776 patients were taken to determine the liver biochemistry including alanine aminotransferase (ALT), albumin and bilirubin levels, HBeAg and antibody to HBeAg (anti-HBe) [by microparticle enzyme immunoassay (MEIA), Abbott Laboratories, Chicago, Illinois, USA], and HBV genotyping and core promoter and precore mutations (methodology described below). Data on the subsequent followup were analyzed for 498 out of the 776 patients. The median follow-up of these 498 patients was 66 months (range 4–376 months). These patients were followed up at intervals of 3– 6 months with monitoring of the liver biochemistry, HBeAg and anti-HBe status. Serum HBV DNA levels on presentation were measured by Cobas Amplicor HBV Monitor test, Roche Diagnostics, Branchburg, NJ (lower limit of detection of 200 copies/ml) in 351 patients (45.2%) and by Digene Hybrid Capture assay (Digene Corporation, Gaithersburg, Maryland, USA) (lower limit of detection 140,000 copies/ml) in 288 patients (37.1%). For the Cobas Amplicor HBV Monitor test, pre-assay 100,000 folds dilution was performed in samples from HBeAg-positive patients. HBV genotyping were performed by two different assays: the line probe assay (INNO-LiPA HBV Genotyping, Innogenetics NV, Gent, Belgium) and the enzyme immunosorbent assay (ELISA). To verify and confirm the results from these two assays, HBV genotypes were determined in 82 patients by both assays. The HBV genotypes of 437 patients were determined by the line probe assay and 257 patients were determined by ELISA. The methodologies for both assays were described in our previous studies [5,16]. HBV genotype B subtypes (Ba, Bj) were determined by a PCR-restriction fragment length polymorphism method as described in our previous study [16]. The core promoter and precore mutations were determined by another line probe assay, INNO-LiPA HBV Precore, Innogenetics, Belgium. The details of the methodology were described in our previous study [17].
2.1. Statistical analysis The prevalence of genotypes between different age groups were compared by Chi-square test. The differences in the median albumin, bilirubin, and ALT and HBV DNA levels between different HBV genotypes were compared by Mann–Whitney test. Linear regression analysis was use to test the independent association between the ALT levels and other variables. P values (two-tailed) of #0.05 were considered to be statistically significant.
3. Results The demographic data, HBeAg/anti-HBe status and liver biochemistry of the 776 asymptomatic patients are listed in Table 1. 3.1. Determination of HBV genotypes by both assays The results of the HBV genotypes determined by both LiPA and ELISA assays were compared in 82 patients. All the 82 patients had single HBV genotype of either B or C. Concordant results were found in 78 patients (95.1%) (19 patients with genotype B, 59 patients with genotype C). The remaining four patients showed different results determined by the two assays (genotype C by LiPA but genotype B by ELISA). Direct sequencing of the surface gene with primers spanning nt 2808– 2830 and nt 977 –996 showed that two were of genotype B and two were of genotype C. These results showed that both the assays yield highly consistent genotyping results. 3.2. Prevalence of HBV genotypes Of the 776 patients, 750 patients (96.6%) had single HBV genotype [(A—12 (1.5%), B—252 (32.5%), C—486 (62.6%)] and 26 patients had mixed genotypes (3.4%). The two most common HBV genotypes were genotypes B and C. The prevalence of HBV genotypes B and C in patients of different age groups are listed in Table 2. There were no differences in the prevalence of genotypes B and C among first three younger age groups (P ¼ NS for all). Patients with the age 31 – 40 years had a significantly lower prevalence of genotype B compared to patients with the age $ 51 years ðP ¼ 0:027Þ: When patients were subdivided
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121
Table 2 Prevalence of HBV genotypes according to different age groups Patients’ age (years)
HBV genotypes
#30 ðn ¼ 217Þ 31–40 ðn ¼ 269Þ 41–50 ðn ¼ 173Þ $51 ðn ¼ 117Þ
B ðn ¼ 252Þ
C ðn ¼ 486Þ
Others or mixed genotypes ðn ¼ 38Þ
71 (32.7%) 79 (29.4%)* 54 (31.2%) 48 (41.0%)*
141 (65.0%) 174 (64.7%)* 108 (62.4%) 63 (53.8%)*
5 (2.3%) 16 (5.9) 11 (6.4%) 6 (5.1%)
P ¼ 0:027: Table 3 Prevalence of HBV genotypes B and C with respect to the age and the HBeAg/anti-HBe status Patients’ age (years)
HBV genotypes
#30 ðn ¼ 212Þ 31–40 ðn ¼ 253Þ 41–50 ðn ¼ 162Þ $51 ðn ¼ 111Þ
B, HBeAg: anti-HBe
C, HBeAg: anti-HBe
P value
60: 11 (84.5%: 15.5%) 38: 41 (48.1%: 51.9%) 11: 43 (20.4%: 79.6%) 5: 43 (10.4%: 89.6%)
108: 33 (76.7%: 23.4%) 107: 67 (61.5%: 38.5%) 46: 62 (42.6%: 57.4%) 20: 43 (31.7%: 68.3%)
0.118 0.046 0.005 0.011
HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg. Table 4 Liver biochemistry of patients with genotypes B and C on presentation Total
Albumin, g/l Bilirubin, mmol/l ALT, U/L
HBeAg positive
Anti-HBe positive
B ðn ¼ 252Þ
C ðn ¼ 486Þ
P value
B ðn ¼ 114Þ
C ðn ¼ 281Þ
P value
B ðn ¼ 138Þ
C ðn ¼ 205Þ
P value
45 (35–54) 11 (3–57) 40 (7–1153)
44 (37–53) 11 (1– 96) 52 (8– 1251)
0.09 0.11 0.002
45 (36–54) 11 (4– 39) 48.5 (9–1153)
45 (37–53) 10 (3–96) 58.5 (12–1251)
0.72 0.42 0.076
44 (35– 53) 11 (3–57) 33 (7–859)
44 (37– 53) 11 (1– 60) 48 (8– 648)
0.71 0.29 0.072
Values expressed in median (range). ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg.
only into age , 50 and . 51 years, there was a significantly lower prevalence of genotype B in the former group (32.5%, 204 out of 627) compared to the latter group (41%, 48 out of 111, P ¼ 0:028). Subtypes Ba and Bj were determined in 115 patients with genotype B. 114 patients (99.1%) were of subtype Ba, whereas only one patient (0.9%) was of subtype Bj. 3.3. HBeAg seroconversion in patients with genotypes B and C Since the two most common HBV genotypes were B and C, only patients with single genotypes B or C are examined in the subsequent analyses. Patients at different age groups were further classified according to the HBeAg/anti-HBe status. The prevalence of genotypes B and C with respect to the age groups and the HBeAg/anti-HBe status are listed in Table 3. Comparing patients with genotypes B and C, patients with genotype B were associated with a higher antiHBe positivity rate at all age groups except those with age , 30 years.
Among the 498 patients with longitudinal follow-up data, 217 (43.6%) were HBeAg positive on presentation. Of these, 63 patients had genotype B and 143 patients had genotype C. Patients with genotype B had a higher cumulative rate of HBeAg seroconversion compared to patients with genotype C ðP ¼ 0:018Þ (Fig. 1). In addition, patients with genotype B had a younger median age of HBeAg seroconversion compared to patients with genotype C [34.2 (range 6.2– 78.7) vs. 40.1 (range 21.2 –71.5) years respectively, P ¼ 0:018]. 3.4. Effects of HBV genotypes on liver biochemistry on presentation The comparisons of the median albumin, bilirubin and ALT levels on presentation were made between patients with genotype B and C in Table 4. Patients with genotypes B and C had comparable median albumin and bilirubin levels. However, patients with genotype B had a significantly lower median ALT level compared to those with genotype C [40 (7 –1153) vs. 52 (8 – 1251) U/L respectively, P ¼ 0:002]. By
0.83
1.3 £ 105 (,200–1.4 £ 109) ðn ¼ 92Þ 1.2 £ 106 (,1.4 £ 105 –7.1 £ 108) ðn ¼ 85Þ
Fig. 1. The cumulative HBeAg seroconversion rate in patients with genotype B (solid line) and patients with genotype C (dotted line).
Values expressed in median (range), copies/ml. HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg.
0.40
3.6 £ 104 (,200–4.9 £ 108) ðn ¼ 63Þ 3.3 £ 105 (,1.4 £ 105 –4.8 £ 108) ðn ¼ 64Þ 0.67
1.8 £ 107 (,200 –1.1 £ 1013) ðn ¼ 141Þ 4.5 £ 106 (,1.4 £ 105 – 1.4 £ 109) ðn ¼ 97Þ Cobas Amplicor HBV Monitor test 2.5 £ 107 (,200 –4.8 £ 1010) ðn ¼ 55Þ Digene Hybrid Capture assay 2.0 £ 107 (,1.4 £ 105 –1.3 £ 109) ðn ¼ 42Þ
Genotype C P value Genotype B Genotype B
Genotype C
Anti-HBe positive HBeAg positive HBV DNA
Table 5 HBV DNA levels of patients with genotypes B and C with respect to the HBeAg/anti-HBe status on presentation
0.71
M.-F. Yuen et al. / Journal of Hepatology 41 (2004) 119–125 P value
122
subdividing patients according to the HBeAg/anti-HBe positivity, there were no differences in the median albumin and bilirubin levels between patients with genotype B and C (Table 4). However, there were trends for patients with genotype B to have a lower median ALT level than those with genotype C both at the HBeAg positive ðP ¼ 0:076Þ and anti-HBe positive ðP ¼ 0:072Þ phases (Table 4). 3.5. Effects of HBV genotypes on HBV DNA levels on presentation Serum HBV DNA levels were measured in 351 patients by the Amplicor HBV Monitor test and in 288 patients by the Digene Hybrid Capture assay. The median HBV DNA levels were compared between patients with genotypes B and C with respect to the HBeAg and anti-HBe status and the two different assays (Table 5). There were no differences in the median HBV DNA level measured by both the assays between patients with genotypes B and C both at the HBeAg positive and anti-HBe positive phases. 3.6. HBV genotypes, core promoter and precore mutations For patients with either genotypes B or C, the presence of mutants of the core promoter and precore regions were determined in 363 and 368 patients, respectively. The prevalence of these mutations with respect to HBV genotypes are listed in Table 6. Patients with genotype B had a higher prevalence of precore mutants than patients with genotype C [83.6 vs. 29.1%, respectively, P , 0:001; Odds ratio (OR) 12.4, 95% confidence interval (CI) ¼ 7.2– 21.3]. By contrast, patients with genotype C had a higher prevalence of core promoter mutations compared to those with genotype B (87.9 vs. 38.6%, respectively, P , 0:001; OR ¼ 11.5, 95% CI ¼ 6.8– 19.5).
M.-F. Yuen et al. / Journal of Hepatology 41 (2004) 119–125 Table 6 Prevalence of core promoter and precore mutations in patients with genotypes B and C Genotype B
Genotype C
P value
Core promoter region Wild-type 81 (61.4%) Mutant 51 (38.6%)
28 (12.1%) 203 (87.9%)
,0.001
Precore region Wild-type Mutant
166 (70.9%) 68 (29.1%)
,0.001
22 (16.4%) 112 (83.6%)
3.7. Core promoter, precore mutations and HBeAg/anti-HBe status There was no difference in the prevalence of core promoter mutations between HBeAg-positive patients (128 out of 177, 72.3%) and anti-HBe-positive patients (144 out of 213, 67.6%, P ¼ 0:37). Anti-HBe-positive patients had a higher prevalence of precore mutations compared to HBeAg-positive patients (122 out of 215, 56.7% vs. 75 out of 181, 41.4%, respectively, P ¼ 0:003; OR ¼ 1.9, 95% CI ¼ 1.2– 2.8). 3.8. Effects of core promoter and precore mutations on liver biochemistry on presentation The liver biochemistry of patients with core promoter and precore mutations are listed in Table 7. There were comparable median albumin and bilirubin levels between patients with core promoter wild-type and mutants and between patients with precore wild-type and mutants (P ¼ NS for all). However, patients with core promoter mutants had a significantly higher median ALT level compared to those of wild-type both at the HBeAg and anti-HBe positive phases (Table 7). 3.9. Effects of core promoter and precore mutations on HBV DNA levels The HBV DNA levels measured by Amplicor HBV Monitor test and the Digene Hybrid Capture assay with respect to the mutations of the core promoter and precore regions are listed in Table 8. There were no differences in the median HBV DNA levels measured by both assays between patients with wild-type and mutants of both the core promoter and precore regions (P ¼ NS for all). 3.10. Multivariate analysis on the factors associated with ALT levels Multivariate analysis including variables of genotypes, core promoter mutations, patients’ age and HBeAg/antiHBe status revealed that patients with genotype C ðP ¼ 0:037Þ and patients with core promoter mutations ðP ¼ 0:05Þ were both independently associated with higher ALT
123
Table 7 Liver biochemistry of patients with core promoter and precore mutants on presentation Albumin (g/l)
Bilirubin (mmol/l)
ALT (U/L)
11 (3–37) 11 (1–96)
45 (7– 624)* 60 (11– 1251)*
10 (4–28) 11 (3–96)
44 (12– 624)† 65 (12– 1251)†
11 (3–37) 11 (1–57)
46 (7– 291)‡ 57 (11– 859)‡
43 (37–53) 43 (32–53)
11 (2–96) 11 (1–38)
54 (11– 1251) 49 (7– 859)
44 (37–50) 43 (32–50)
11 (3–96) 11 (4–38)
51 (13– 1251) 70 (10– 642)
43 (35–53) 43 (36–53)
12 (2–57) 11 (1–37)
56 (11– 473) 47.5 (7–859)
Core promoter region Total WT ðn ¼ 118Þ 44 (35–53) MT ðn ¼ 272Þ 43 (37–53) HBeAg þ ve WT ðn ¼ 49Þ 44 (35–50) MT ðn ¼ 128Þ 43 (37–50) Anti-HBe þ ve WT ðn ¼ 69Þ 43 (37–53) MT ðn ¼ 144Þ 43 (35–53) Precore region Total WT ðn ¼ 199Þ MT ðn ¼ 197Þ HBeAg þ ve WT ðn ¼ 106Þ MT ðn ¼ 75Þ Anti-HBe þ ve WT ðn ¼ 93Þ MT ðn ¼ 122Þ
Values expressed in median (range). ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg; WT, wildtype; MT, mutants. *P ¼ 0:002; †P ¼ 0:024; ‡P ¼ 0:049:
levels on presentation compared to those with genotype B and those with core promoter wild-type, respectively.
4. Discussion To our knowledge, the present study is the largest epidemiologic study on HBV genotypes examining the prevalence of HBV genotypes B and C, core promoter and precore mutations in Asia. Our center accepted referrals for patients with or without symptoms of hepatitis and with normal or elevated ALT levels. There should be no referral bias of the study population. Therefore, the findings of the present study are representative to the CHB patient population in Hong Kong. The majority of the CHB patients had single genotype in which 32.5% were genotype B and 62.6% were genotype C. This pattern of distribution is unique when compared to those of other countries in Asia. The prevalence of genotype B is higher and genotype C is lower in Hong Kong compared to two cites in the Peoples’ Republic of China, namely Shanghai and Nanning where the prevalence of genotypes B and C are 14– 17% and 79 – 81%, respectively [18,19]. In contrast, the prevalence of genotype B (60 – 68%) is much higher and genotype C is lower (32 – 34%) in Taiwan [20,21]. There is a variable pattern of distribution of genotypes in Japan. Over 94% of patients on the Japanese mainland have genotype C, whereas 60% of patients in southern island and 23% of patients in the northern part of the mainland have genotype B [22].
0.64
1.1 £ 106 (,200–9.2 £ 1012) ðn ¼ 64Þ 2.4 £ 106 (,1.4 £ 105 –1.4 £ 109) ðn ¼ 128Þ 0.10
6.8 £ 106 (,200–1.1 £ 1013) ðn ¼ 60Þ 2.3 £ 106 (,1.4 £ 105 –1.3 £ 109) ðn ¼ 129Þ 0.52
Values expressed in median (range), copies/ml.
4.2 £ 106 (,200 –1.1 £ 1013) ðn ¼ 81Þ 1.9 £ 106 (,1.4 £ 105 –1.4 £ 109) ðn ¼ 178Þ Cobas Amplicor HBV Monitor test 1.1 £ 106 (,200 –9.2 £ 1012) ðn ¼ 44Þ Digene Hybrid Capture assay 5.3 £ 106 (,1.4 £ 105 – 1.3 £ 109) ðn ¼ 72Þ
Mutants P value Wild-type Wild-type
Mutants
Precore region Core promoter region HBV DNA
Table 8 HBV DNA levels of patients with core promoter and precore mutations on presentation
0.07
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124
When the prevalence of HBV genotypes B and C in Hong Kong was stratified according to different age groups at an interval of 10 years, the prevalence of HBV genotypes B and C remained at around 29 – 33% and 62 – 65%, respectively, across the age groups of , 30, 31 –40 and 41– 50 years. However, the prevalence of HBV genotype B was significantly higher (41%) and hence HBV genotype C was lower (53.8%) in the patients with age . 51 years when compared to those with age 31 –40 years ðP ¼ 0:027Þ (Table 2) and those with age , 50 years ðP ¼ 0:028Þ: Since majority of the Chinese patients are infected with HBV during perinatal and childhood period, these findings suggest that there was a change in the viral population around 40 –50 years ago but it had remained unchanged over the last 40 years. This phenomenon may be related to the influx of immigrants from the Peoples’ Republic of China to Hong Kong after the Communist takeover in 1949. Since the prevalence of HBV genotype B was lower, around 14– 17%, in the Peoples’ Republic of China [18,19], an influx of immigrants from China would result in a subsequent decrease in the prevalence of HBV genotype B in the last 40 – 50 years. The present study also showed that for patients with genotype B, 99.1% were of subtype Ba and only 0.9% was of subtype Bj. It supported the findings that Bj was nearly exclusively found in Japan and Ba was mainly found in Asia. There are suggestions from other studies that clinical differences exist between HBV subtype Bj and Ba. Sugauchi and his colleagues show that the chance of loss of HBeAg is the highest in patients with subtype Bj followed by patients with subtype Ba and is the lowest in patients with genotype C [16]. Akuta and his colleagues show that the emergence rate of lamivudine resistance is significantly higher in the Ba subgroup than in the Bj subgroup of HBV [23], though this conclusion is drawn from only 4 patients with Ba and 14 patients with Bj subtype. However, no difference in the rate of lamivudine resistance is found between patients with genotype B and C, which was also confirmed in a recent study conducted in our Center [24]. Further studies are required to compare the clinical differences, if these exist, between patients with subtypes Bj, Ba and genotype C. The higher prevalence of anti-HBe positivity in patients with genotype B compared to those with genotype C at all age groups except in the group with age , 30 years (Table 3) suggested that patients with genotype B had a higher chance of earlier HBeAg seroconversion compared to those with genotype C. The reason for the absence of this difference in the group of patients with age , 30 years is mainly due to the fact that these patients are still in the immunotolerance phase. In the Chinese population, the immunoclearance phase usually begins at the age after 30 years and the median age of HBeAg seroconversion was 34.5 years [25]. The higher chance of HBeAg seroconversion in patients with genotype B was further confirmed by the significantly higher cumulative rate (Fig. 1) and younger median age of HBeAg seroconversion in patients with genotype B
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compared to those with genotype C. The present large-scale study confirmed this phenomenon with other studies [5 –7, 19]. It is important to note that, though the age of HBeAg seroconversion for patients with genotype B was earlier than those with genotype C, the median age of HBeAg seroconversion for patients with genotype B was still more than three decades after the initial hepatitis B infection. Whether the slightly earlier HBeAg seroconversion in these patients carries any clinical significance in term of the future development of HCC and cirrhosis related complications remains unknown to date. The present study showed that patients with genotype C and patients with core promoter mutations were independently associated with higher ALT levels on presentation compared to those with genotype B and those with core promoter wild-type respectively (Tables 4 and 7). However, there were no differences in the albumin, bilirubin and HBV DNA levels on presentation when comparisons were made between these respective groups of patients (Tables 4, 5, 7 and 8). The possible effect of the higher chance of earlier HBeAg seroconversion, and the lower ALT levels on presentation in patients with genotype B compared to those with genotype C are currently being studied in our center by liver histology in term of degree of necroinflammation and fibrosis, and by following up patients for the chance of development of HCC and cirrhosis-related complications. In conclusion, the present study showed that genotypes B and C were the two most common HBV genotypes in Hong Kong contributing 32.5% and 62.6% of CHB population respectively. The prevalence of genotype B was lower in patients below the age of 50, probably related to influx of immigrants from China since 1949. More than 99% of patients with genotype B were of subtype Ba. Patients with genotype B had a higher chance of earlier HBeAg seroconversion and lower ALT levels on presentation compared to patients with genotype C.
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