Hepatitis B Virus Genotype C Is Associated With More Severe Liver Fibrosis Than Genotype B

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2009;7:1361 1366 Hepatitis B Virus Is Associated With More Severe Liver Fibrosis Than HENRY LIK YUEN CHAN, GRACE LAI HUNG WONG, CHI HANG TSE, ANGEL MEI LING CHIM, KAREN KAR LUM YIU, HOI YUN CHAN, JOSEPH JAO YIU SUNG, and VINCENT WAI SUN WONG Department of Medicine and Therapeutics, Institute of Digestive Disease, The Chinese University of Hong Kong, Hong Kong SAR, China See related article, Buster EHCJ et al, on page 2002 in Gastroenterology. BACKGROUND & AIMS: Histologic analyses of liver fibrosis have been limited by small sample sizes and the predominance of samples from patients with active hepatitis. METHODS: We performed a prospective study of transient elastography in treatment-naive patients with chronic hepatitis B, to investigate the relationship between hepatitis B virus (HBV) genotype and liver fibrosis. A validated liver stiffness measurement algorithm was used to define insignificant fibrosis and advanced fibrosis. RE- SULTS: Of 1106 patients, 711 (64%) were older than age 40, 370 (34%) had positive test results for hepatitis B e antigen (HBeAg), and 386 (35%) had increased serum levels of alanine aminotransferase. Of the patients, 524 (49%) had genotype B and 582 (51%) had genotype C HBV infection. Patients with genotype C infection had insignificant fibrosis less often (42% vs 55%; P.0001) and advanced fibrosis more often (25% vs 19%; P.015) than those infected with genotype B HBV. The difference in the severity of liver fibrosis between the 2 HBV genotypes was most marked among patients older than age 40 and those who tested negative for HBeAg. The mean age of patients infected by genotype C was greater than that of patients infected by genotype B HBV (41 vs 36 y). Among patients who were older than age 40 and tested negative for HBeAg, those with genotype C infection had higher levels of HBV DNA and alanine aminotransferase than those with genotype B HBV. CONCLUSIONS: HBV was associated with more severe liver fibrosis than genotype B HBV, probably because of delayed HBeAg seroconversion and prolonged active disease. Chronic hepatitis B virus (HBV) infection is the most common cause of cirrhosis and hepatocellular carcinoma in Asia. 1 Liver fibrosis, which is the natural wound healing process to necroinflammation, is the essential pathogenic process that leads to cirrhosis. Liver biopsy has been the gold standard to assess liver fibrosis. However, its invasiveness has limited its use in large-scaled clinical studies. For example, liver biopsy in young, hepatitis B e antigen (HBeAg)-positive patients with persistently normal alanine aminotransferase (ALT) levels rarely is indicated outside clinical trial settings. 2,3 Liver biopsy in HBeAg-negative patients with persistently normal ALT levels and low HBV DNA levels again rarely is indicated clinically. 4 Hence, most studies on the natural history of chronic hepatitis B had a biased sampling including mainly patients with active and advanced liver diseases. In recent years, transient elastography (Fibroscan; Echosens, Paris, France) has been proven a reliable noninvasive tool to assess liver fibrosis, especially in the diagnosis of advanced liver fibrosis. 5,6 It is therefore a more acceptable method than liver biopsy to perform large-scaled studies on liver fibrosis including patients with different severities of liver disease. HBV genotypes B and C are predominant in most parts of Asia. 7 Based on longitudinal series in Hong Kong and Taiwan, genotype C HBV was found to be an independent risk factor of hepatocellular carcinoma. 8 10 HBV is associated with a delayed HBeAg seroconversion and more active disease than genotype B HBV. 11 14 However, the relationship of HBV genotype and liver fibrosis has been studied inadequately in the past. Although most previous histologic studies suggested genotype C HBV has a higher risk of cirrhosis, the sample size of all these studies was small and most patients had increased ALT levels at the time of liver biopsy. 15 17 Each HBV genotype can be subclassified into different subgenotypes. Most genotype B HBV outside Japan belongs to subgenotype Ba. 18 The 2 major subgenotypes of genotype C HBV are subgenotype Cs (predominant in Southeast Asia) and Ce (predominant in East Asia). 19 Subgenotype Ce HBV is associated with a higher risk of hepatocellular carcinoma as compared with subgenotype Cs HBV, 9,20 but whether it is related to an increased risk of liver fibrosis/cirrhosis is uncertain. In our study including 139 HBeAg-negative chronic hepatitis B patients from 3 cities in China in a clinical trial, we could not show any difference in the severity of histologic fibrosis between patients infected by the 2 subgenotypes C HBV. 21 In this study, we aimed to study the association of different HBV genotypes and subgenotypes on the severity of liver fibrosis based on a large cohort of chronic hepatitis B patients undergoing transient elastography assessment. Factors attributing to the difference in liver fibrosis among the HBV genotypes/subgenotypes also were explored. Methods Study Population Stored serum samples among a prospectively recruited cohort of chronic hepatitis B patients for transient elastography between July 2006 and November 2008 were studied for HBV Abbreviations used in this paper: ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; LSM, liver stiffness measurement. 2009 by the AGA Institute 1542-3565/09/$36.00 doi:10.1016/j.cgh.2009.08.004

1362 CHAN ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 12 genotyping. 22 These patients had positive hepatitis B surface antigen for at least 6 months and other liver diseases were excluded. None of these patients had decompensated liver disease, complications of cirrhosis, hepatocellular carcinoma, previous liver surgery, or liver transplantation. None of the patients had received any antiviral treatment. The study protocol was approved by the Joint Chinese University of Hong Kong New Territories East Cluster Clinical Research Ethics Committee of Hong Kong. Definition of Liver Fibrosis by Transient Elastography Liver stiffness measurement (LSM) was determined using transient elastography (Fibroscan) according to the instructions and training provided by the manufacturer. 23 Only patients who had reliable LSM, which was defined as 10 successful acquisitions obtained with a success rate of 60% or higher and an interquartile ratio of 30% or less, were analyzed. Because serum ALT level significantly could affect the diagnostic value of LSM on liver fibrosis, we adopted the ALT-based algorithm of LSM as validated by our previous study involving 161 chronic hepatitis B patients with liver biopsy. 24 Insignificant fibrosis was defined as a LSM of 6.0 kpa or less among patients with normal ALT levels and of 7.5 kpa or less among patients with ALT levels greater than 1 to 5 times the upper limit of laboratory normal. Advanced fibrosis was defined as a LSM greater than 9.0 Ka among patients with normal ALT levels and greater than 12.0 among patients with ALT levels greater than 1 to 5 times the upper limit of laboratory normal. Under these definitions, the 2 LSM cut-off levels had greater than 90% accuracy to exclude or diagnose bridging fibrosis (Ishak stage 4 or METAVIR F3) on histology, respectively. Patients with ALT levels greater than 5 times the upper limit of normal probably had unreliable LSMs and were excluded from the analysis. 25 The proportion of patients with histologic bridging fibrosis among a subgroup of patients who also had adequate liver biopsy (specimen length, 1.5 cm) also was analyzed. Laboratory Assays Hepatitis B surface antigen was tested by commercially available enzyme-linked immunosorbent assay kits (Abbott GmBH Diagnostika, Wiesbaden-Delkenheim, Germany). HBeAg and antibodies to hepatitis B e antigen were measured by enzyme-linked immunosorbent assay (Sanofi Diagnostics, Pasteur, France). HBV DNA was quantified by TaqMan real-time polymerase chain reaction assay as described previously. 26 This assay was standardized by serial dilution of EUROHEP genotype D HBV standard, which contained 2.7 10 9 viral copies/ml. The range of HBV DNA detection was from 10 2 to 10 9 copies/ml, with correlation coefficient of the standard curve routinely greater than 0.990. Hepatitis B Virus Genotyping HBV genotyping was determined by restriction fragment length polymorphism using primers franking the HBV surface gene (nucleotide 256 796) as described previously. 17 Among the genotype C HBV samples, subgenotyping for Cs and Ce was determined by the 5=-nuclease technique based on the polymorphism at nucleotide 2733 as described previously. 27 Samples with uncertain restriction pattern or negative signal on 5=-nuclease assay were sequenced bidirectionally at the HBV surface gene to determine the HBV genotype and subgenotype. Statistical Analyses Statistical analysis was performed by using the Statistical Package for Social Science (SPSS version 15.0; SPSS, Chicago, IL). Continuous variables were expressed in mean standard deviation. The Pearson chi-square test was used to compare categoric variables, and the Student t test was used for continuous variables. Ishak scores were expressed as median (interquartile range) and compared by the Mann Whitney U test. The independent effect of HBV genotype on liver fibrosis was assessed by multiple logical regression analysis adjusting the effect of age ( 40 y), HBeAg (positive), HBV DNA ( 4 log copies/ml), and ALT ( upper limit of normal). The clinical characteristics of patients infected by different HBV genotypes were assessed by categorizing into different age groups and HBeAg status. All statistical tests were 2-sided. Statistical significance was taken as a P value of less than.05. Results Patients A total of 1531 chronic hepatitis B patients underwent transient elastography during the study period. A total of 394 patients had undetectable HBV DNA levels and failed HBV genotyping. In the remaining 1136 patients, 30 patients (14 patients infected by genotype B HBV and 17 patients infected by genotype C HBV) had an unreliable LSM as defined per protocol. A total of 1106 patients were eligible for this study. The clinical characteristics of the studied patients are shown in Table 1. Compared with the studied patients, the excluded patients (n 425) generally were older (age, 47 12 y; P.015); had a lower proportion of patients positive for HBeAg (32 patients; 8%); had lower ALT levels (median, 35 IU/mL; interquartile range, 22 64 IU/mL; P.0001); and had lower log HBV DNA levels (4.02 2.05 copies/ml; P.0001). There was no difference in sex between the excluded (265 male patients, 63%) and studied patients. Among the studied patients, 711 (64%) patients were older than age 40. A total of 370 (34%) patients were HBeAg positive. A total of 720 of 736 (98%) HBeAg-negative patients had hepatitis B e antigen antibodies. A total of 386 (35%) patients had an increased ALT level. A total of 529 (48%) patients had insignificant fibrosis as defined by transient elastography, including 363 patients with normal ALT levels and 166 patients with an increased ALT level. A total of 240 (22%) patients had advanced liver fibrosis, including 142 patients with a normal ALT level and 98 patients with increased ALT levels. A total of 337 (31%) patients, including 215 patients with a normal ALT level and 122 patients with an increased ALT level, had a LSM in the gray zone and the severity of liver fibrosis could not be determined. Hepatitis B Virus Genotype and Liver Disease A total of 524 (47%) patients were infected by genotype B HBV and 582 (53%) patients were infected by genotype C HBV (Table 1). There was no difference in the age and gender ratio between patients infected by the 2 HBV genotypes. Patients infected by genotype C HBV had a higher proportion of patients positive for HBeAg, higher HBV DNA levels, and higher

December 2009 HEPATITIS B GENOTYPE AND LIVER FIBROSIS 1363 Table 1. Clinical Characteristics of Chronic Hepatitis B Patients Infected by and C HBV Overall (N 1106) (N 524) (N 582) P value a Age, y 45 12 46 13 45 12.40 Age, 40 y 711 (64%) 341 (65%) 370 (64%).60 Male sex 705 (64%) 343 (66%) 582 (62%).26 HBeAg positive 370 (34%) 126 (24%) 244 (42%).0001 Log HBV DNA, copies/ml 5.77 1.79 5.56 1.84 5.96 1.71.0001 Log HBV DNA 4, copies/ml 901 (82%) 411 (78%) 490 (84%).014 Log HBV DNA 6, copies/ml 489 (44%) 190 (36%) 299 (51%).0001 ALT level, IU/L 59 46 55 45 63 46.006 Increased ALT level 386 (35%) 164 (31%) 222 (38%).017 LSM, kpa 8.4 5.8 7.8 5.4 8.7 6.1.002 Insignificant fibrosis 529 (48%) 286 (55%) 243 (42%).0001 Gray zone 337 (31%) 141 (27%) 196 (34%) N/A Advanced fibrosis 240 (22%) 97 (19%) 143 (25%).015 N/A, not applicable. a Comparing patients infected by genotype B and C HBV. ALT levels. The LSM of genotype C HBV-infected patients (8.7 6.1) was significantly higher than that of genotype B HBVinfected patients (7.8 5.4 kpa) (P.002). Significantly fewer genotype C HBV-infected patients had insignificant liver fibrosis than genotype B HBV-infected patients (42% vs 55%; difference, 13%; 95% confidence interval, 7% 19%). Similarly, a significantly higher proportion of genotype C HBV-infected patients had advanced liver fibrosis than genotype B HBV-infected patients (25% vs 19%; difference, 6%; 95% confidence interval, 1% 11%). On multivariate analysis, genotype C HBV was associated less often with insignificant fibrosis independent of age, ALT level, HBeAg level, and HBV DNA level (odds ratio, 0.60; 95% confidence interval, 0.47 0.78; P.0001). For advanced fibrosis, the association of genotype C HBV just fell short of statistical significance (odds ratio, 1.33; 95% confidence interval, 0.98 1.80; P.069). One hundred patients had adequate liver biopsy performed. Thirty-five (35%) patients had genotype B HBV and 65 (65%) patients had genotype C HBV infection. The median Ishak score among patients infected by genotype C HBV was 4 (interquartile range, 3 5), and the median Ishak score among those infected by genotype B HBV was 3 (interquartile range, 1 5) (P.016). Histologic bridging fibrosis was seen significantly more often among patients infected by genotype C HBV (50 patients, 77%) than those infected by genotype B HBV (16 patients, 46%) (P.001). Relationship of Hepatitis B Virus Genotype and Liver Disease With Respect to Patient Age Among patients age 40 and younger there was no significant difference in the severity of liver fibrosis between the 2 HBV genotypes (Table 2). HBV was associated with a higher proportion of patients positive for HBeAg and higher ALT levels, which might reflect a trend of prolonged immune clearance and delayed HBeAg seroconversion. Among patients older than age 40, those infected by genotype C HBV tended to have higher LSMs and more severe liver fibrosis than those infected by genotype B HBV (Table 2). HBV infection also was associated with a higher proportion of HBeAg positivity, higher ALT level, and higher HBV DNA level, signifying more active liver disease in this patient population. Relationship of Hepatitis B Virus Genotype and Liver Disease With Respect to Hepatitis B e Antigen Status There was a weak trend of more significant liver fibrosis associated with genotype C HBV infection in HBeAg-positive Table 2. Comparison of Clinical Characteristics Among Patients in Different Age Categories Infected by HBV and C Age, 40 y Age, 40 y (N 183) (N 212) P value (N 341) (N 370) P value HBeAg positive 88 (48%) 124 (59%).039 38 (11%) 120 (32%).0001 Log HBV DNA, copies/ml 6.21 2.21 6.45 1.93.26 5.21 1.49 5.68 1.50.0001 Log HBV DNA 4, copies/ml 145 (79%) 177 (84%).28 266 (78%) 313 (85%).024 Log HBV DNA 6, copies/ml 93 (51%) 128 (60%).056 97 (28%) 171 (46%).0001 ALT level, IU/L 55 47 65 48.031 55 44 61 44.076 Increased ALT level 57 (31%) 88 (42%).033 107 (31%) 134 (36%).17 LSM, kpa 6.6 3.7 7.3 4.9.12 8.5 6.0 9.8 6.6.004 Insignificant fibrosis 124 (68%) 127 (60%).11 162 (48%) 116 (31%).0001 Advanced fibrosis 18 (10%) 24 (11%).63 79 (23%) 119 (32%).008

1364 CHAN ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 12 Table 3. Comparison of Clinical Characteristics Among Patients With Different HBeAg Status Infected by HBV and C HBeAg positive HBeAg negative (N 126) (N 244) P value (N 398) (N 338) P value Age, y 36 11 41 12.0001 49 12 48 11.48 Age, 40 y 38 (30%) 120 (49%).0001 303 (76%) 250 (74%).50 Log HBV DNA, copies/ml 7.76 1.45 7.20 1.29.0001 4.86 1.33 5.07 1.39.045 Log HBV DNA 4, copies/ml 124 (98%) 243 (99.6%).23 287 (72%) 247 (73%).77 Log HBV DNA 6, copies/ml 107 (85%) 198 (81%).37 83 (21%) 101 (30%).005 ALT level, IU/L 66 54 70 48.46 52 41 57 43.075 Increased ALT level 53 (42%) 116 (48%).32 111 (28%) 106 (31%).30 LSM, kpa 8.3 5.7 9.3 6.7.13 7.7 5.3 8.6 5.7.021 Insignificant fibrosis 69 (55%) 106 (43%).039 217 (55%) 137 (41%).0001 Advanced fibrosis 24 (19%) 63 (26%).15 73 (18%) 80 (24%).076 patients (Table 3). The older age of HBeAg-positive patients infected by genotype C HBV further supported a delayed HBeAg seroconversion associated with this HBV genotype. In HBeAg-negative patients, genotype C HBV was associated with a higher LSM and more severe liver fibrosis than genotype B HBV despite similar age ranges (Table 3). The more active liver disease in the HBeAg-negative phase was supported further by a trend of higher HBV DNA and ALT levels among genotype C HBV-infected patients. Subgenotype C Hepatitis B Virus Among 582 patients infected by genotype C HBV, 111 (19%) had subgenotype Ce and 466 (81%) had subgenotype Cs (Table 4). There was no difference in the severity of liver fibrosis associated with the 2 HBV C subgenotypes. Subgenotype Ce HBV was associated with slightly higher HBV DNA levels than subgenotype Cs HBV. Discussion In this large-scaled study of liver fibrosis using transient elastography among 1106 treatment-naive chronic hepatitis B Table 4. Clinical Characteristics of Patients Infected by HBV Subgenotypes Cs and Ce Subgenotype Ce (N 111) Subgenotype Cs (N 466) P value Age, y 44 11 45 12.33 Age, 40 y 67 (60%) 298 (64%).48 Male sex 60 (54%) 298 (64%).054 HBeAg positive 54 (49%) 190 (41%).13 Log HBV DNA, copies/ml 6.40 2.00 5.89 1.61.013 Log HBV DNA 4, 98 (88%) 391 (84%).25 copies/ml Log HBV DNA 6, 63 (57%) 236 (51%).25 copies/ml ALT level, IU/L 59 45 64 46.28 Increased ALT level 38 (34%) 184 (40%).31 LSM, kpa 9.0 5.8 8.9 6.2.88 Insignificant fibrosis 45 (41%) 195 (42%).80 Gray zone 35 (32%) 159 (34%) N/A Advanced fibrosis 31 (28%) 112 (24%).39 N/A, not applicable. patients, we have shown that genotype C HBV was associated with more severe liver fibrosis than genotype B HBV. The difference was mainly among patients who were older than age 40 and who were HBeAg negative. This probably was related to the prolonged immune clearance, delayed HBeAg seroconversion, and active disease in the HBeAg-negative phase associated with genotype C HBV. The study of liver fibrosis has been a major challenge in the field of hepatology. Studies using liver histology as the outcome measure included mostly patients with increased ALT levels. 28 Because patients with inactive disease were less likely to be biopsied and reported, the impact of the HBV genotype that was associated with less severe disease might be underrepresented. In our previous study including 42 HBeAg-negative patients undergoing liver biopsy, only 11 (26%) patients had genotype B HBV infection. 17 In another study of 101 HBeAgnegative patients recruited into a treatment trial with baseline histologic assessment, 36 (36%) patients had genotype B HBV. 21,29 Both studies failed to show any difference in the severity of liver fibrosis between genotype B and C HBV-infected patients. One possible explanation was that with inactive hepatitis, which more likely was related to genotype B HBV, were not recruited for histologic assessment. In the current study, a much higher proportion of genotype B HBV infection (49%) was reported, and most of the patients in this cohort had normal ALT levels (65%) without indication for liver biopsy. Careful and unbiased sampling is therefore important to show the true impact of HBV genotype on the development of liver fibrosis, and this can be achieved only by a noninvasive assessment acceptable to the low-risk patients. Transient elastography has been validated by several groups as a reliable noninvasive tool to assess liver fibrosis in chronic hepatitis B. 24,30 32 However, different LSM cut-off values were proposed by different investigators for different stages of liver fibrosis. 29 This was partly related to the confounding effect of ALT because patients with the same fibrosis staging would have higher LSMs if their ALT level were increased. 23 The different study designs and recruitment of patients with different disease severities would affect the optimal LSM cut-off level for the respective patient cohorts. This problem can be solved only if different algorithms of interpreting LSM are used for patients with normal and increased ALT levels. In this study, we have

December 2009 HEPATITIS B GENOTYPE AND LIVER FIBROSIS 1365 adopted the validated ALT-based algorithm with more than 90% accuracy to exclude or diagnose advanced liver fibrosis. 24 As the gold standard, liver biopsy has intrinsic error of sampling bias; however, this was probably the best accuracy a noninvasive test could achieve. 33 There is ample evidence suggesting that genotype C HBV is associated with more aggressive liver disease, 14 delayed HBeAg seroconversion, 11,13 and more reactivation of hepatitis in the HBeAg-negative phase 12 than genotype B HBV. The results of our study supported these findings. HBV was associated with higher HBeAg positivity in patients younger than age 40 and also was associated with older age among HBeAg-positive patients, both reflecting a delayed HBeAg seroconversion. The general trend of more active viremia and higher ALT levels associated with genotype C HBV, particularly among HBeAg-negative patients and patients older than age 40, reflected prolonged and unsuccessful immune clearance. The consequence was more severe liver fibrosis in genotype C HBVinfected patients, and the difference between the 2 HBV genotypes was most evident in patients older than age 40 and after losing HBeAg. In other words, although genotype B HBV is sometimes found in severe hepatitis reactivation, 34 it tends to be self-limiting and shorter lived. On the other hand, genotype C HBV was associated with longer duration of liver damage, which may be the main reason for more advanced liver fibrosis. In this study, we could not show any difference in the proportion of patients with insignificant fibrosis or advanced fibrosis between those infected by subgenotype Ce and Cs HBV. This finding was in line with our previous histologic series among HBeAg-negative chronic hepatitis B patients in a clinical trial. 21 The only difference between these 2 HBV subgenotypes was the HBV DNA levels, but whether this difference could be extrapolated to any clinical significance warranted further investigation. One possibility was that a higher HBV DNA level associated with subgenotype Ce HBV could partly account for its higher risk of hepatocarcinogenesis. 9,20 This study had a few limitations. As in all studies for HBV genotypes, patients who were in the low replicative phase with low HBV DNA levels, negative HBeAg, and low ALT levels could not be assessed. The cross-sectional nature of the study did not provide information on the change in disease activity over time as in previous longitudinal series. Our large sample size with stratified analysis based on different age groups and HBeAg status could partly compensate for this limitation. The use of transient elastography might impose some error in the assessment of liver fibrosis. The algorithm we used has tried to minimize this error by eliminating those patients with LSM placed into the gray zone after analysis. This was a large-scaled study with low bias sampling on the association of HBV genotypes and liver fibrosis. In conclusion, our results have confirmed genotype C HBV was associated with more severe liver fibrosis as compared with genotype B HBV. More studies are needed to define the role of HBV genotyping in the clinical management of chronic hepatitis B. Whether a lower threshold for liver fibrosis assessment and antiviral treatment should be recommended among genotype C HBV-infected patients, particularly those with negative HBeAg and older than age 40, warrants future investigation. References 1. Chan HLY, Sung JJY. Hepatocellular carcinoma and hepatitis B virus. Semin Liver Dis 2006;26:153 161. 2. Hui CK, Leung N, Yuen ST, et al. 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1366 CHAN ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 12 21. Zhu L, Tse CH, Wong VWS, et al. A complete genomic analysis of hepatitis B virus genotypes and mutations in HBeAg-negative chronic hepatitis B in China. J Viral Hepat 2008;15:449 458. 22. Wong GLH, Wong VWS, Choi PCL, et al. Metabolic syndrome increases the risk of liver cirrhosis in chronic hepatitis B. Gut 2009;58:111 117. 23. Wong GL, Wong VW, Choi PC, et al. Assessment of fibrosis by transient elastography compared with liver biopsy and morphometry in chronic liver diseases. Clin Gastroenterol Hepatol 2008; 6:1027 1035. 24. Chan HLY, Wong GLH, Choi PCL, et al. Alanine aminotransferasebased algorithms of liver stiffness measurement by transient elastography (Fibroscan) for liver fibrosis in chronic hepatitis B. J Viral Hepat 2009;16:36 44. 25. Wong GLH, Wong VWS, Choi PC, et al. Increased liver stiffness measurement by transient elastography in severe acute exacerbation of chronic hepatitis B. J Gastroenterol Hepatol 2009;24: 1002 1007. 26. Chan HLY, Chui AKK, Lau WY, et al. Factors associated with viral breakthrough in lamivudine monoprophylaxis of hepatitis B virus recurrence after liver transplantation. J Med Virol 2002;68:182 187. 27. Chan HLY, Tse CH, Ng EYT, et al. Phylogenetic, virological and clinical characteristics of genotype C hepatitis B virus with TCC at codon 15 of the precore region. J Clin Microbiol 2006;44:681 687. 28. Chan HLY, Wong GLH, Wong VWS. A review of the natural history of chronic hepatitis B in the era of transient elastography. Antivir Ther 2009;14:489 499. 29. Chan HL, Wang H, Niu J, et al. Two-year lamivudine treatment for hepatitis B e antigen-negative chronic hepatitis B: a double-blind, placebo-controlled trial. Antivir Ther 2007;12:345 353. 30. Marcellin P, Ziol M, Bedossa P, et al. Non-invasive assessment of liver fibrosis by stiffness measurement in patients with chronic hepatitis B. Liver Int 2009;29:242 247. 31. Chang PE, Lui HF, Chau YP, et al. Prospective evaluation of transient elastography for the diagnosis of hepatic fibrosis in Asians: comparison with liver biopsy and aspartate transaminase platelet ratio index. Aliment Pharmacol Ther 2008;28:51 61. 32. Kim DY, Kim SU, Ahn SH, et al. Usefulness of FibroScan for detection of early compensated liver cirrhosis in chronic hepatitis B. Dig Dis Sci 2009;54:1758 1763. 33. Mehta SH, Lau B, Afdhal NH, et al. Exceeding the limits of liver histology markers. J Hepatol 2009;50:36 41. 34. Chan HLY, Tsang SWC, Wong ML, et al. hepatitis B virus is associated with severe icteric flare-up of chronic hepatitis B virus infection in Hong Kong. Am J Gastroenterol 2002;97: 2629 2633. Reprint requests Address requests for reprints to: Henry L. Y. Chan, MD, Department of Medicine and Therapeutics, 9/F Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, Hong Kong SAR, China. e-mail: hlychan@ cuhk.edu.hk; fax: (852) 26373852. Conflicts of interest The authors disclose no conflicts.