Genotype B hepatitis B virus is associated with severe icteric flare-up of chronic hepatitis B virus infection in Hong Kong

THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2002 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.

Vol. 97, No. 10, 2002 ISSN 0002-9270/02/$22.00 PII S0002-9270(02)04423-4

Genotype B Hepatitis B Virus Is Associated With Severe Icteric Flare-Up of Chronic Hepatitis B Virus Infection in Hong Kong Henry Lik-Yuen Chan, M.D., Steven Woon-Choi Tsang, M.D., May-Ling Wong, B.Sc., Chi-Hang Tse, M.Phil., Nancy Wai-Yee Leung, M.D., Francis Ka-Leung Chan, M.D., and Joseph Jao-Yiu Sung, M.D., Ph.D. Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, Hong Kong; and Department of Medicine, Tseung Kwan O Hospital, Hong Kong

OBJECTIVE: We aimed to investigate the association of viral genotype and the development of icteric flare-up (IF) in chronic hepatitis B virus (HBV) infection. METHODS: Twenty-one consecutive patients suffering from IF of chronic HBV infection, defined as elevation of ALT over five times the upper limit of normal, together with either bilirubin ⬎50 IU/L or elevated bilirubin plus PT ⬎3 s prolonged, were studied. Patients from three stages of HBV-related chronic liver disease were studied as controls: 1) asymptomatic carriers (31 patients), defined as persistent normal ALT for at least 2 yr; 2) active early cirrhosis (49 patients), defined as Child’s A liver cirrhosis plus HBV DNA ⬎106 Eq/ml; and 3) decompensated cirrhosis (31 patients), defined as Child’s B or C liver cirrhosis with complications. Restriction fragment length polymorphism was used for genotyping. RESULTS: Only genotype B and C HBV were identified in our studied cohort. Ninety-one percent of patients suffering from IF were infected by genotype B HBV (p ⬍0.001 vs asymptomatic carriers, early cirrhosis patients, and decompensated cirrhosis patients). On the contrary, genotype C HBV was the predominant strain at different stages of chronic liver disease; no statistical difference was found on the relative prevalence of genotype B/C HBV among asymptomatic carriers, early cirrhosis patients, and decompensated cirrhosis patients. CONCLUSIONS: Genotype B HBV is associated with IF among chronic HBV-infected patients in Hong Kong, whereas genotype C HBV is more prevalent at all stages of chronic liver disease. Our findings suggested that the two different HBV genotypes might have different pathogenic mechanisms of liver damage. (Am J Gastroenterol 2002;97: 2629 –2633. © 2002 by Am. Coll. of Gastroenterology)

Elevated liver enzymes during exacerbation of chronic hepatitis B are usually asymptomatic or associated with mild symptoms such as malaise or abdominal pain (1– 4). Some patients develop spontaneous hepatitis B e antigen (HBeAg) seroconversion and remain in quiescent disease thereafter (5–7). For others, the progressive, silent liver damage eventually leads to liver cirrhosis followed by the development of complications including ascites, varices, and hepatic encephalopathy (8 –10). In Hong Kong and Taiwan, 23–38% of patients have been reported to develop jaundice and hepatic decompensation during biochemical exacerbation of chronic hepatitis B (11, 12), and these exacerbations are associated with significant mortality without treatment (13). Most of these exacerbations were not related to superinfection by other hepatitis viruses (11), and it is unclear whether any viral factor is associated with more severe hepatitis exacerbation. Seven different genotypes of HBV (A–G) have been described according to the homogeneity of their respective genome nucleotide sequence (14, 15). Different HBV genotypes have distinct geographical distribution (14, 16). In Western countries, genotypes A and D HBV are prevalent, whereas in Southeast Asia, genotypes B and C HBV are the predominant viral strains. A recent study from Taiwan suggested that genotype B HBV was associated with young hepatocellular carcinoma, whereas genotype C HBV was associated with more advanced liver disease (17). These findings hinted that these different HBV genotypes might have different pathogenic mechanisms on hepatic damage. We aimed to conduct a case-control study to investigate whether there is any association between HBV genotypes and severe icteric flare-up (IF) among chronic HBV-infected patients in Hong Kong.

PATIENTS AND METHODS INTRODUCTION The majority of patients suffering from chronic hepatitis B virus (HBV) infection has relatively asymptomatic disease.

Patients Serum samples from consecutive patients suffering from IF of chronic HBV infection admitted to Prince of Wales

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Hospital in 14 months from July 1999 to August 2000 were studied. The definition of IF was modified from Chien et al. as elevation of ALT over five times the upper limit of normal accompanied by either bilirubin ⬎50 IU/L or elevated bilirubin ⬎15 IU/L plus PT ⬎3 s prolonged (13). Chronic HBV-infected status was confirmed by positive hepatitis B surface antigen testing at least 1 yr ago, or negative anti-HBc IgM during the IF episode if hepatitis B surface antigen was not checked in the past. Coinfection by hepatitis A virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus was excluded. Informed consent was obtained from the patients for the study. We used stored residual serum samples from patients presenting to the Prince of Wales Hospital with different stages of chronic HBV infection (asymptomatic carriers [AC], active early cirrhosis [EC], and decompensated cirrhosis [DC]) between 1993 and 1998 as controls. AC was defined as patients who had been followed up at 3– 6 monthly intervals for at least 2 yr with persistently normal ALT levels and no evidence of liver cirrhosis. Sera were retrieved from eligible patients who gave informed consent to our previous studies (18, 19). EC was defined as Child’s grade A cirrhosis confirmed on histology with HBV DNA ⬎106 Eq/ml. These patients were recruited for a trial of lamivudine in HBV-related EC, and the protocol was approved by the local Ethics Committee. None of the patients in the AC or EC groups had history of jaundice or IF of hepatitis in the past. DC was defined as patients suffering from Child’s grade B and C liver cirrhosis with complications including ascites, varices, and/or hepatic encephalopathy. This group included all end-stage cirrhotic patients who were referred for liver transplantation in our unit and DC patients who were seen in the ward and regularly followed up in the liver clinic (19, 20). Serological Assays and HBV DNA Assay Anti-hepatitis A virus IgM antibodies, hepatitis B surface antigen, anti-HBc IgM antibodies, anti-hepatits C virus antibodies (third-generation assay), anti-hepatitis D virus antibodies, and anti-hepatitis E virus antibodies were tested by commercially available ELISA kits (Abbott GmBH Diagnostika, Wiesbaden-Delkenheim, Germany). HBeAg and anti-HBe were measured by ELISA (Sanofi Diagnostics, Pasteur, France). Serum HBV DNA was quantified by the DNA cross-linking assay (NAXCOR XLnt, NAXCOR, Menlo Park, CA), which has a detection limit of 0.5 Meq/ml according to the instruction of the manufacturer as described previously (21, 22). Genotyping DNA extraction was performed as previously described by Qiaquick spin columns (Qiagen, Chatsworth, CA) according to the instructions of the manufacturer (23). Genotyping polymerase chain reaction (PCR) and restriction enzyme treatment were performed as described by Lindh et al. (16). Of the extracted DNA, 10 ␮l was used for PCR in a reaction

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volume of 50 ␮l containing 1.5 mmol/L MgCl2, 50 mmol/L KCL, 10 pmol of primers flanking the HBV genome between nucleotide 256 to 796 (sense primer 5⬘-GTGGTGGACTTCTCTCAATTTTC and antisense primer 5⬘-CGGTA(A/T)AAAGGGACTCA(A/C)GAT), and 0.75 U of Taq polymerase. PCR was performed with an initial 3-min denaturation at 94°C, 40 cycles of amplification, including denaturation for 45 s at 94°C, annealing for 60 s at 53°C, and extension for 90 s at 72°C (prolonged by 3 s per cycle), and a final 7-min extension at 72°C. PCR product (10 ␮l) was then mixed with 1 ␮l (5 U) of Tsp 5091 (New England Biolabs, Beverly, MA), 1.5 ␮l of 10⫻ buffer, and 2.5 ␮l of water, and incubated at 65°C for 3 h. In a separate reaction, 10 ␮l of PCR product was mixed with 0.5 ␮l (5 U) of HinfI (Boehringer Mannheim, Mannheim, Germany), 1.5 ␮l of 10⫻ buffer, and 3 ␮l of water, and incubated at 37°C for 3 h. After incubation, the samples were run on a composite gel containing 2% NuSieve agarose (FMC BioProducts, Rockland, ME) and 1% standard agarose. PCR product (10 ␮l) with buffer and water but no restriction enzyme was run parallel as negative controls. DNA was visualized by ethidium bromide staining, and the restriction pattern was read according to the description by Lindh et al. (16). Statistics Categorical data were compared by two-tailed Pearson ␹2 test or Fisher exact test as appropriate. Continuous variables were studied by two-tailed Student’s t test. Bonferroni correction was applied in case of multiple comparisons. Statistical significance was taken as p ⬍0.05.

RESULTS Serum samples were obtained from 21 patients and 91 controls (31 AC, 49 EC, and 31 DC) for analyses. Among the 21 patients suffering from IF, the median (range) ALT and HBV DNA levels on admission were 1146 (484 –1958) IU/L and 442.24 (⬍0.5–5564.24) ⫻106 MEq/ml, respectively. Only two patients had low titer viremia ⬍106 MEq/ ml. Eleven of the 21 patients had underlying liver cirrhosis. The demographic data and HBeAg status of the four patient groups are shown in Table 1. Comparing patients suffering from IF versus the three control groups, there was no difference in the gender ratio (IF vs AC, p ⫽ 0.69; IF vs EC, p ⫽ 0.33; IF vs DC, p ⫽ 0.74) and HBeAg status (IF vs AC, p ⫽ 1.00; IF vs EC, p ⫽ 0.71; IF vs DC, p ⫽ 0.16). Patients suffering from EC and DC were generally older than patients with IF (IF vs AC, p ⫽ 0.32; IF vs EC, p ⫽ 0.032; IF vs DC, p ⬍0.001). Only genotype B and C HBV were identified in our patient population (Table 1). Genotype B HBV was predominant among patients suffering from IF (91%), but genotype C was the predominant HBV strain among patients in the three control groups (IF vs AC, vs EC, and vs DC, p ⬍0.001). There was no statistical difference in the distribu-

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Genotype B HBV and Severe Icteric Flare-Up

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Table 1. Demographic Data, HBeAg Status, and HBV Genotype Among Patients With IF, AC, EC, and DC

Age (mean ⫾ SD) M:F HBeAg ⫹ve:⫺ve Albumin (mean ⫾ SD) g/dL Bilirubin (mean ⫾ SD) ␮g/dL PT (mean ⫾ SD) s ALT (mean ⫾ SD) IU/L Genotype B Genotype C

IF (n ⫽ 21)

AC (n ⫽ 31)

EC (n ⫽ 49)

DC (n ⫽ 31)

39 ⫾ 11 17:4 11:9 33.0 ⫾ 5.5 108.1 ⫾ 72.5 15.7 ⫾ 3.4 1349 ⫾ 394 19 (91%) 2 (9%)

35 ⫾ 12 22:9 18:13 39.6 ⫾ 3.1 11.4 ⫾ 7.0 10.8 ⫾ 0.9 46 ⫾ 20 12 (39%) 19 (62%)

43 ⫾ 8 45:4 31:18 39.6 ⫾ 3.5 11.3 ⫾ 5.0 11.4 ⫾ 0.8 104 ⫾ 80 10 (20%) 39 (80%)

54 ⫾ 9 22:9 10:21 26.5 ⫾ 6.7 66.0 ⫾ 75.8 17.3 ⫾ 7.0 83 ⫾ 95 10 (32%) 21 (68%)

tion of genotype B and C HBV among the three control groups. Genotype distribution was also studied as a function of age (Table 2) and gender (Table 3). Genotype B HBV was shown to be predominant in both genders and all age groups among patients with IF. On the contrary, genotype C HBV was predominant in both genders and all age groups among patients at different stages of chronic HBV infection.

DISCUSSION In this study, we have shown that 91% of IF of chronic HBV infection in our locality was associated with genotype B HBV. On the contrary, genotype C HBV was predominant in all stages of liver disease, from AC to EC and DC. The contrasting distribution of genotype B and C HBV among patients with IF versus other chronic HBV-infected patients highlighted the potential difference in the pathogenic mechanism of the two viral genotypes. Patients suffering from IF represented those who had severe biochemical exacerbation of the underlying chronic hepatitis B to the extent of hepatic decompensation. The hepatitis exacerbation is associated with accentuation of the T-cell response for the immune clearance of the virus (24), which at the same time causes severe hepatic inflammation. Patients with liver cirrhosis and therefore diminished hepatic reserve have a higher tendency of decompensation at acute exacerbations. In this study, 52% of our patients who had IF had underlying liver cirrhosis. In contrast, none of the Table 2. Prevalence of Genotypes B and C HBV Among Patients With IF, AC, EC, and DC With Reference to Different Age Groups

Age ⬍30 30–40 40–50 ⬎ 50 Total

IF

AC

EC

DC

No. of Patients With Genotype

No. of Patients With Genotype

No. of Patients With Genotype

No. of Patients With Genotype

B 4 5 8 2 19

B 6 3 1 2 12

B 1 3 4 2 10

B 0 1 1 8 10

C 1 0 0 1 2

C 7 6 5 1 18

C 1 14 18 6 39

C 1 1 4 15 21

AC and EC patients had previous IF, although it was difficult to exclude previous IF among patients with DC as some of them already had jaundice and hepatic decompensation. Nevertheless, patients in our control groups represented different stages of chronic HBV infection. AC represented those patients who were in the immune tolerance phase (HBeAg-positive patients) or those who had successful immune clearance of the virus (HBeAg-negative patients) (25). Based on the findings of previous studies, patients with active EC and DC represented those patients who have prolonged active liver disease that eventually resulted in liver cirrhosis (8, 9, 25). This was probably related to the weak, abortive immune response that failed to eradicate the virus but contributed to viral persistence and chronic liver disease (26). Previous studies have suggested that genotype C HBV is associated with higher prevalence of HBeAg positivity, higher necroinflammatory scores on histology, and higher prevalence of liver cirrhosis than genotype B HBV (17, 27, 28). These results suggest that genotype C HBV tends to associate with delayed HBeAg seroconversion and more prolonged, low-grade necroinflammation causing liver cirrhosis. On the other hand, genotype B HBV may be associated with more effective immune clearance and hence less chronic sequel of chronic HBV infection. Our findings, therefore, agree with the previous studies that genotype B HBV might be associated with more vigorous immune response. However, the underlying mechanism that accounts for the difference between these two HBV genotypes is unclear.

Table 3. Prevalence of Genotypes B and C HBV Among Patients With IF, AC, EC, and DC With Reference to Gender of Patients

M F Total

IF

AC

EC

DC

No. of Patients With Genotype

No. of Patients With Genotype

No. of Patients With Genotype

No. of Patients With Genotype

B 16 3 19

B 9 3 12

B 9 1 10

B 7 3 10

C 1 1 2

C 13 6 19

C 36 3 39

C 15 6 21

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Because most of our patients suffering from IF had high viral titer, this might represent viral reactivation that resulted in vigorous immune response and clinical exacerbation. Owing to the cross-sectional nature of this study, we might have missed the peak of the HBV DNA surge in some of our patients who had very effective immune clearance. Therefore, some patients have low or undetectable HBV DNA levels during the IF. Our study has several limitations. First, the case and control sera were collected at different periods. However, our results were unlikely to be explained by an age cohort effect because the progression of chronic hepatitis B is not influenced by environmental factors in different generations. Second, the control sera were not derived from consecutive patients because not all stored samples were available. Nevertheless, they represented randomly selected patients at different stages of HBV-related liver disease. This is supported by the predominance of the same HBV genotype (genotype C) in all the three control groups. Third, patients in the AC and IF groups were in general younger than the EC and DC patients. We could not exclude the possibility that some AC and IF patients might progress into EC and DC with time. However, none of the patients in the AC and EC groups had previous episodes of jaundice. Fourth, only genotypes B and C HBV are present in our population as in the case of most Southeast Asian countries. We, therefore, cannot comment on the disease pattern of other viral genotypes. One study in Switzerland suggested genotype A and D might have different tendency in the development of chronic active hepatitis and acute self-limiting hepatitis (29). Another study from the United States found that genotype A and D HBV were the predominant viral strains associated with acute liver failure, probably because these two genotypes were the predominant strains in the United States (30). Therefore, a multicenter study involving patients from both the East and the West is required for a more detailed examination of the relationship of HBV genotype and disease pattern in chronic HBV infection. In conclusion, IF of chronic HBV infection reflects severe hepatic damage and can lead to further decompensation and even death. The results of our study suggest that genotype B but not genotype C HBV is associated with IF. This provides insight into the relationship between different HBV genotypes and the pathogenesis of disease. Further studies are warranted to investigate the underlying mechanisms that account for this difference.

ACKNOWLEDGMENT The study was supported by Cheng Suen Man-suk Foundation for the Study of Viral Hepatitis. Reprint requests and correspondence: Henry Lik-Yuen Chan, M.D., Department of Medicine and Therapeutics, 9/F Prince of

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Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong. Received Aug. 30, 2001; accepted May 6, 2002.

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