Received 30 May 2004/Returned for modification 6 August 2004/Accepted 12 August 2004

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2004, p Vol. 42, No /04/$ DOI: /JCM Copyright 2004, American Society for Microbiology. All Rights Reserved. Longitudinal Study of Hepatitis Activity and Viral Replication before and after HBeAg Seroconversion in Chronic Hepatitis B Patients Infected with Genotypes B and C Man-Fung Yuen, 1 Scott K. Fung, 2 Yasuhito Tanaka, 3 Takanobu Kato, 3 Masashi Mizokami, 3 John Chi-Hang Yuen, 1 Danny Ka-Ho Wong, 1 He-Jun Yuan, 1 Siu-Man Sum, 1 Annie On-On Chan, 1 Benjamin Chun-Yu Wong, 1 and Ching-Lung Lai 1 * Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong 1 ; Department of Medicine, University of Toronto, Toronto, Canada 2 ; and Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan 3 Received 30 May 2004/Returned for modification 6 August 2004/Accepted 12 August 2004 The aims of this study were to compare chronic hepatitis B (CHB) patients with genotypes B and C for the probability of HBeAg seroconversion, hepatitis activity, and viral replication before and after HBeAg seroconversion and to compare the prevalence of core promoter and precore mutations. A total of 180 asymptomatic Chinese patients with CHB were monitored for a median of 53.8 months, and 38 patients with cirrhosis-related complications were studied. Hepatitis B virus (HBV) DNA levels were measured in 16 patients with HBeAg seroconversion at 3 months before, during, and 3 months after HBeAg seroconversion and in all patients at the last follow-up. Hepatitis B genotypes and core promoter and precore mutations were determined. Compared to patients with genotype C (n 109), patients with subtype Ba (n 69) had a higher rate of anti-hbe positivity on presentation (P 0.05). HBeAg-positive patients with subtype Ba had a higher cumulative rate of HBeAg seroconversion than patients with genotype C (P 0.02). However, there were no differences between the two groups with regard to the median HBV DNA levels before, during, and after HBeAg seroconversion; the probability of having persistently normal or elevated aminotransferase levels; and the median HBV DNA levels and liver biochemistry at the last follow-up. There was no difference in the prevalence of genotypes and core promoter and precore mutations between patients with and without cirrhosis-related complications. Though patients with subtype Ba had earlier HBeAg seroconversion, the liver biochemistry, HBV DNA levels in different phases of the disease, and the probability of development of cirrhosis-related complications were the same with genotypes Ba and C. Recently, there have been studies suggesting that disease activity of chronic hepatitis B (CHB) may be different in patients with different hepatitis B virus (HBV) genotypes (1 8, 11, 14). In Asia, which is responsible for 75% of the world population of CHB patients, the HBVs are mainly of genotype B or C. Recently, HBV genotype B has been subclassified into two subtypes, namely, Bj (subtype found exclusively in Japan) and Ba (subtype found mainly in the rest of Asia) (9, 10). HBV subtype Ba emerges as a result of the recombination of the core and precore regions of genotype C with the genuine genotype B, the subtype Bj (10). Several studies have shown that patients with genotype B have a higher probability of hepatitis B e antigen (HBeAg) seroconversion than patients with genotype C (2, 11, 14). In a previous study, patients with genotype B have the HBeAg seroconversion around 9 years earlier than those with genotype C (14). Whether this phenomenon is due to the difference in the virulence of the two genotypes, which makes the immune responses during the immune clearance phase comparatively more effective in patients with genotype B, is not known. To date, there are no studies on the severity of hepatitis (as reflected by serum alanine aminotransferase [ALT] levels) and * Corresponding author. Mailing address: Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Rd., Hong Kong. Phone: (852) Fax: (852) hrmelcl@hkucc.hku.hk. on viral replication in patients with genotypes B and C before HBeAg seroconversion. It is even more important to examine whether there are any benefits in terms of the liver biochemistry and the HBV DNA levels in patients with genotype B compared to patients with genotype C after HBeAg seroconversion. This may have important implications for the probability of the development of long-term cirrhosis-related complications. All these important aspects are best examined in longitudinal studies with serial monitoring of liver biochemistry and HBV DNA levels for both HBeAg-positive patients and patients positive for antibody to HBeAg (anti-hbe) with genotypes B and C. The present study consisted of two parts: a longitudinal follow-up study of asymptomatic CHB patients and a crosssectional analysis of patients with clinical manifestations of cirrhosis-related complications. The aims of the longitudinal follow-up study were to compare patients with genotypes B and C with regard to the following aspects: (i) the probability of HBeAg seroconversion, (ii) serial liver biochemistry for HBeAg-positive and anti-hbe-positive patients, and (iii) the change in the HBV DNA levels during HBeAg seroconversion and the HBV DNA levels in anti-hbe-positive patients. The aim of the cross-sectional study was to determine whether there were any differences in the prevalence of genotypes and core promoter and precore mutations between patients with and without cirrhosis-related complications. 5036

2 VOL. 42, 2004 ACTIVITY AND VIREMIA FOR HBV GENOTYPES B AND C 5037 TABLE 1. Prevalence of HBV genotypes in different age groups Patients age (yr) (no. of patients) Subtype Ba No. (%) of patients with: Genotype C 30 (n 29) 9 (31) 20 (69) (n 70) 27 (38.6) 43 (61.4) (n 42) 17 (40.5) 25 (59.5) (n 20) 8 (40) 12 (60) (n 17) 8 (47.1) 9 (52.9) nitrocellulose membrane. The details of the methodology were described previously (13). Statistical analysis. All statistical analyses were performed by using the Statistical Program for Social Sciences (version 10.0 for Windows; SPSS Inc., Chicago, Ill.). Continuous variables with skewed distribution were tested with a Mann-Whitney U test. Categorical variables were tested by chi-square test or Fisher s exact test. The cumulative HBeAg seroconversion was examined by Kaplan-Meier analysis with a log rank test. s of less than 0.05 were considered statistically significant. MATERIALS AND METHODS For the longitudinal follow-up study, 180 asymptomatic patients attending the Hepatitis Clinic, Queen Mary Hospital, The University of Hong Kong, Hong Kong, for at least 10 regular follow-ups were recruited during the period of January 1998 to January These patients were positive for hepatitis B surface antigen (HBsAg) for at least 6 months and had not received any form of antiviral therapy. Patients with antibodies to hepatitis C and D, a history of significant alcohol intake, evidence of autoimmune hepatitis, primary biliary cirrhosis, Wilson s disease, or drug-induced hepatotoxicity were excluded. The HBsAg and HBeAg/anti-HBe status and liver biochemistry were checked during every follow-up, which was scheduled every 3 to 6 months or more frequently if clinically indicated. The HBV DNA levels were measured in 16 patients who underwent HBeAg seroconversion at four time points (3 months before, during, and 3 months after the HBeAg seroconversion and at the last follow-up) and for all the anti-hbe-positive patients at the last follow-up. The HBV DNA levels were measured by the Cobas Amplicor HBV Monitor test, Roche Diagnostics, Branchburg, N.J. (with a lower limit of detection of 200 copies/ml). HBV genotypes were determined for all the 180 patients. For the cross-sectional analysis, a cohort of 38 consecutive patients who were seen at Queen Mary Hospital during the period of January 2003 and May 2003 for the management of the cirrhosis-related complications including ascites, spontaneous bacterial peritonitis, encephalopathy, and esophageal variceal bleeding were recruited. HBV genotypes and core promoter and precore mutations were determined in these 38 patients. Thirty-six patients (94.7%) were older than 40 years old at the time of the development of the cirrhosis-related complications. In order to compare the prevalence of HBV genotypes and core promoter and precore mutations between patients with and without the complications, the HBV genotypes and core promoter and precore mutations were determined for all the asymptomatic patients above 40 years of age (n 79) from the longitudinal study cohort. Determination of HBV genotypes. The HBV genotyping was determined by an enzyme-linked immunosorbent assay using monoclonal antibodies against seven distinct epitopes (b, m, k, s, u, f, and g) on the pres2 region products of HBsAg (12). HBV genotype B subtypes (Bj and Ba) were determined by a PCR-restriction fragment-length polymorphism method as described previously (9). Determination of core promoter and precore mutations. The core promoter and precore mutations were determined by a line probe assay (INNO-LiPA HBV Precore; Innogenetics, Ghent, Belgium). It contained specific probes covering the following motifs: (i) a wild-type core promoter (A/G) at nucleotides (nt) 1762/1764, (ii) a core promoter mutation (A/A) at nt 1762/1764, (iii) a core promoter mutation (A/T) at nt 1762/1764, (iv) a core promoter mutation (T/A) at nt 1762/1764, (v) a wild-type precore codon 28 (TGG) (nt 1896), and (vi) a precore codon 28 mutation (TAG) (nt 1896). The probes were applied onto a RESULTS Longitudinal follow-up study. (i) Demographics. The median age of the 180 patients was 38.7 (range, 14.7 to 80) years. The male-to-female ratio was 129:51. Sixty-two patients (34.4%) were HBeAg positive and 118 patients (65.6%) were anti-hbe positive on presentation. The median follow-up duration was 53.8 (range, 29.5 to 171.3) months. (ii) Prevalence of HBV genotypes. The prevalence of various HBV genotypes among patients was as follows: 1 had genotype A (0.6%), 69 had genotype B (38.3%), 109 had genotype C (60.6%), and 1 had genotype F (0.6%). All the HBV genotype B isolates belonged to subtype Ba. Among the 178 patients with either subtype Ba or genotype C, 62 (34.8%) were HBeAg positive and 116 (65.2%) were anti-hbe positive. In the subsequent analysis, only the two most common genotypes (Ba and C) were considered. There were no differences in the prevalence of these two genotypes between different age groups (Table 1). The demographic features of patients with subtype Ba (n 69) and genotype C (n 109) are listed in Table 2. There were no differences in any parameter between patients with subtype Ba and those with genotype C except that patients with subtype Ba tended to have a higher prevalence of anti-hbe positivity than patients with genotype C (51 out of 69 [73.9%] versus 65 out of 109 [59.6%], respectively; P 0.05; odds ratio [OR], 1.9; 95% confidence interval [CI], 1.0 to 3.7). (iii) HBeAg seroconversion. There were 16 patients out of 62 HBeAg-positive (25.8%) patients on presentation undergoing HBeAg seroconversion during the follow-up period. Of these 16 patients, 8 had subtype Ba and 8 had genotype C. The probability of HBeAg seroconversion was significantly higher in patients with subtype Ba than in patients with genotype C (8 out of 18 [44.4%] versus 8 out of 44 [18.2%], respectively; P 0.032; OR, 3.6; 95% CI, 1.1 to 12). The cumulative HBeAg seroconversion rate was significantly higher in patients with subtype Ba than that of patients with genotype C (P 0.02) (Fig. 1). TABLE 2. Demographic features of patients with subtype Ba and genotype C Patients with subtype Ba (n 69) Value for group Patients with genotype C (n 109) Median age, yr (range) 40.0 ( ) 38.1 ( ) 0.08 Male/female 49:20 78: HBeAg/anti-HBe 18:51 44: Median albumin level, g/liter (range) 45 (35 53) 45 (36 53) 0.77 Median ALT level, U/liter (range) 31 (9 1153) 43 (8 648) 0.20 Median bilirubin level, mol/liter (range) 11 (3 57) 10 (1 30) 0.31 Median follow-up duration, mo (range) 53.9 ( ) 53.4 ( ) 0.65

3 5038 YUEN ET AL. J. CLIN. MICROBIOL. FIG. 1. Cumulative rate of HBeAg seroconversion of patients with subtype Ba (solid line) and genotype C (dotted line). Patients with genotype Ba had a younger median age at HBeAg seroconversion than patients with genotype C (33.9 [range, 16.2 to 78.7] versus 38 [range, 30.1 to 53.7] years, respectively), although this difference was not statistically significant (P 0.44). (iv) ALT levels in HBeAg-positive patients and patients with HBeAg seroconversion. There were no differences in the median ALT level on presentation (81.5 [range, 18 to 1,153] versus 48 [range 14 to 316] U/liter; P 0.2) and the median peak ALT level (117.5 [range, 18 to 1,153] versus 132 [range, 19 to 1,413] U/liter; P 0.25) during subsequent follow-up testing between HBeAg-positive patients with subtype Ba and those with genotype C, respectively. Of the HBeAg-positive patients, 11 of 18 patients with subtype Ba (61.1%) and 32 of 44 patients with genotype C (72.7%) had peak ALT levels of more than two times the upper limit of normal (ULN) upon follow-up testing. Of these 43 patients with peak ALT levels more than two times the ULN, patients with subtype Ba had a higher probability of HBeAg seroconversion than patients with genotype C (6 out of 11 [54.5%] versus 6 out of 32 [18.8%], respectively; P 0.022; OR, 5.2; 95% CI, 1.2 to 22.9). However, in the 16 patients with HBeAg seroconversion, there was no difference in the median peak ALT level before the HBeAg seroconversion between patients with subtype Ba and those with genotype C (210.5 [range, 46 to 344] versus 159 [range, 33 to 316] U/liter, respectively; P 0.33). (v) HBV DNA before, during, and after HBeAg seroconversion. In the 16 patients with HBeAg seroconversion, the HBV DNA levels were measured 3 months before, during, and 3 months after HBeAg seroconversion as well as at the last follow-up. The median HBV DNA levels at these four time points in patients with subtype Ba and genotype C are listed in Table 3. There were no differences in the HBV DNA levels at all the four time points between patients with subtype Ba and those with genotype C. (vi) HBV DNA level and liver biochemistry in anti-hbepositive patients. The HBV DNA levels and the liver biochemistry at the last follow-up for the 116 anti-hbe positive patients (on presentation) with subtype Ba and genotype C are listed in Table 4. In these 116 patients, the median HBV DNA level was 8,830 (range, 200 to 10 8 ) copies/ml. Twenty-seven patients (23.3%) had undetectable HBV DNA levels, i.e., below 200 copies/ml. Thirty-eight patients (32.8%) had HBV DNA levels higher than 10 5 copies/ml (only 2 [1.7%] had HBV DNA levels higher than 10 7 copies/ml). There was no difference in the median HBV DNA levels between anti-hbe-positive patients with subtype Ba and patients with genotype C at the last follow-up (Table 4). There were also no differences in the proportions of patients with an HBV DNA level below 200 copies/ml and above 10 5 copies/ml between patients with subtype Ba and those with genotype C (Table 4). There were no differences in the liver biochemistry at the Viral replication TABLE 3. HBV DNA levels of 16 patients with HBeAg seroconversion Patient HBV DNA level (copies/ml) a Subtype Ba (n 8) Genotype C (n 8) Before HBeAg seroconversion (3 mo) ( ) ( ) 1.0 At the time of HBeAg seroconversion (1, ) ( ) 0.38 After HBeAg seroconversion (3 mo) (6, ) ( ) 0.76 At the last follow-up ( ) ( ) 0.38 a Ranges are shown in parentheses.

4 VOL. 42, 2004 ACTIVITY AND VIREMIA FOR HBV GENOTYPES B AND C 5039 TABLE 4. HBV DNA level and liver biochemistry at the last follow-up of anti-hbe positive patients Patients with genotype Ba (n 51) Group Patients with genotype C (n 65) Median HBV DNA level (copies/ml) a 9,400 ( ) 6,170 ( ) 0.23 No. of patients (%) with HBV DNA level (copies/ml) of: (17.6) 18 (27.7) (33.3) 18 (27.7) (17.6) 7 (10.8) (31.4) 22 (33.8) 0.93 Liver biochemistry a Albumin (U/liter) 44 (36 47) 43 (36 51) 0.93 Bilirubin ( mol/liter) 11 (5 39) 12 (4 37) 0.64 ALT (U/liter) 28 (13 243) 37 (11 256) 0.25 a Ranges are shown in parentheses. last follow-up between anti-hbe-positive patients with subtype Ba and those with genotype C (all p NS) (Table 4). Among the 116 anti-hbe-positive patients, ALT levels that were persistently elevated by more than two times ULN were observed in 23 patients. In these 23 patients, there was a comparable proportion of patients with subtype Ba (10 out of 51 [19.6%]) and patients with genotype C (13 out of 65 [20%]; P 0.96). Sixty-four anti-hbe-positive patients had persistently normal ALT levels. In these 64 patients, there was also no difference in the proportion of patients with subtype Ba (34 out of 51 [66.7%]) and patients with genotype C (34 out of 65 [52.3%]; P 0.12). There was also no difference in the median peak ALT level between anti-hbe-positive patients with genotype Ba and those with genotype C (52 [range, 16 to 1,138] versus 74 [range, 14 to 648] U/liter; P 0.36). Cross-sectional study on genotypes and cirrhosis-related complications. Of the 38 patients with cirrhosis-related complications, 11 had ascites (6 also had spontaneous bacterial peritonitis), 16 had esophageal varices, and the remaining 11 had a combination of the above (3 also had hepatic encephalopathy). The demographics and the prevalence of HBV genotypes and core promoter and precore mutations of the patients with (n 38) and without (n 79) cirrhosis-related complications are listed in Table 5. There were no significant differences in all the demographic parameters as well as the prevalence of HBV genotypes and core promoter and precore mutations between patients with and without cirrhosis-related complications (all p NS). DISCUSSION The present study confirmed the findings from our previous studies that patients with genotype B had a higher probability of HBeAg seroconversion than patients with genotype C (2, 11, 14). The present study is, however, the first longitudinal study to show that this phenomenon held true when a comparison was made between patients with subtype Ba and patients with genotype C. It has been shown in our previous cross-sectional study that the probability of the loss of HBeAg was highest in patients with subtype Bj, followed by patients with subtype Ba, and was the lowest in patients with genotype C (9). Although HBV subtype Ba has more similarities in terms of genetic sequence to HBV genotype C than to HBV subtype Bj, the difference in the HBeAg seroconversion rate was still observable between patients with subtype Ba and patients with genotype C. The cytotoxic T-cell and the T-helper (Th) epitopes are located mainly in the precore and core regions. The production of antibody against HBeAg is Th dependent. It is possible that the decreasing chance of early HBeAg seroconversion from subtype Bj to subtype Ba to genotype C is related to the acquisition of the precore and core sequences of genotype C, making HBeAg seroconversion more difficult. Further in vitro studies are required to determine whether the precore and core sequences of HBV genotype C are less immunogenic than those of HBV subtype Bj. However, in spite of the higher probability of HBeAg seroconversion in patients with subtype Ba compared to patients TABLE 5. Demographics and prevalence of hepatitis B genotypes and core promoter and precore mutations in patients with and without cirrhosis-related complications Patients with cirrhosis-related complications (n 38) Group Patients without cirrhosis-related complications (n 79) Male/female 31:9 56: Median age, yr (range) 53.6 ( ) 48.7 ( ) 0.11 HBeAg/anti-HBe (%) 9:29 (23.7 vs. 76.3) 18:61 (22.8 vs. 77.2) 1.0 HBV genotypes (Ba/C) (%) 11:27 (28.9 vs. 71.1) 33:46 (41.8 vs. 58.2) 0.26 No./total core promoter mutations (%) 22/30 (73.3) 47/72 (65.3) 0.58 No./total precore mutations (%) 14/33 (42.4) 41/72 (56.9) 0.24

5 5040 YUEN ET AL. J. CLIN. MICROBIOL. with genotype C, there was no difference in the subsequent liver biochemistry between these two groups of patients after HBeAg seroconversion. This was evidenced by three parameters. First, there was no difference in the median peak ALT levels during subsequent follow-up tests after HBeAg seroconversion (52 versus 74 U/liter, respectively; P 0.36). Second, there were no differences in the probabilities of having ALT levels persistently more than twice the ULN (19.6 versus 20%, respectively; P 0.96) and having ALT levels persistently normal (66.7 versus 52.3%, respectively; P 0.12) after HBeAg seroconversion. Third, the median albumin, bilirubin, and ALT levels at the last follow-up test after HBeAg seroconversion were comparable (Table 4). The HBV DNA levels 3 months before and during HBeAg seroconversion were comparable between the eight patients with subtype Ba and the eight patients with genotype C who underwent HBeAg seroconversion during the follow-up period (Table 3). This finding suggests that patients with HBV subtype Ba and genotype C had a similar viral load to trigger the immune response during the immune clearance phase. More importantly, the viral load after HBeAg seroconversion (3 months after HBeAg seroconversion and at the last follow-up) was also similar between patients with subtype Ba and patients with genotype C (Table 3). Furthermore there was also no difference in the median HBV DNA levels of the 116 patients who were anti-hbe positive on presentation at the last follow-up (Table 4). These comparable HBV DNA levels over different time points suggested that there was no difference in the viral load between patients with subtype Ba and those with genotype C in different phases of CHB infection. However, since the viral load decreased after HBeAg seroconversion in both patients with subtype Ba and those with genotype C (Table 3) and since patients with subtype Ba had a higher probability of HBeAg seroconversion, it was anticipated that subtype Ba should be associated with a milder disease. Nevertheless, this postulation was not supported by the crosssectional analysis of the present study in that there were no differences in the prevalence of HBV subtype Ba and genotype C between patients with and without cirrhosis-related complications (Table 5). The absence of beneficial effects in patients with subtype Ba compared to patients with genotype C despite the earlier HBeAg seroconversion may be due to the fact that CHB infection in the Chinese population starts at birth or during the first 1 to 2 years of life. The incidence of cirrhosisrelated complications and/or hepatocellular carcinoma peaks at the sixth decade of life. The effect of HBeAg seroconversion taking place one decade earlier in patients with genotype Ba may not have sufficient beneficial influence on the long-term disease process. Finally, the present study detected the most common core promoter mutations at nt 1762 and It is known that other mutations can occur in the core promoter regions. It should be noted that there might be an adverse impact on the hybridization of the probes in the LiPA assay adopted in the present study. Similarly, other mutations in the precore region beside that at nt 1896 have not been fully studied in the present study, as these infrequent mutations may also abolish the synthesis of HBeAg. Ideally, the mutation analyses should be corroborated by direct sequencing in future studies. In conclusion, patients with HBV subtype Ba had a higher probability of HBeAg seroconversion than patients with HBV genotype C. 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