Genotypes and Clinical Phenotypes of Hepatitis B Virus in Patients with Chronic Hepatitis B Virus Infection

Genotype C of hepatitis B virus (HBV) has been shown to be associatedwith a poor clinical outcome, compared to genotype B. To explorethe clinical phenotypes, with special reference to the seroconversionof hepatitis B e antigen (HBeAg) and frequency of acute exacerbationbetween patients infected with HBV genotypes B and C, a cohortof 272 Taiwanese patients with chronic HBV infection was analyzed.According to the status of HBeAg at enrollment and frequencyof acute exacerbation during the follow-up period, five groupsof patients with distinct clinical phenotypes were categorized.Of the 272 HBV carriers, 185 (68%) were infected with HBV genotypeB and the remaining 87 (32%) were infected with genotype C.Among them, 150 (55%) were positive for HBeAg and patients withgenotype C infection tended to have a higher positive rate ofHBeAg than those with genotype B infection (63 versus 51%).Genotype B was more prevalent than genotype C in different groupsof HBV carriers. However, the prevalence of genotype C in patientswith multiple episodes of acute exacerbation who failed to haveHBeAg seroconversion was significantly higher than in all 272patients (50 versus 32%, P = 0.025), in those with HBeAg seroconversionafter only one episode of acute exacerbation (50 versus 12%,P = 0.01), or in those negative for HBeAg at enrollment andwithout acute exacerbations (50 versus 23%, P = 0.002). In conclusion,patients with genotype C infection have a more aggressive clinicalphenotype than do those with genotype B infection, which contributesto the former group's progressive liver disease and poor clinicaloutcomes.

INTRODUCTION

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Introduction

Hepatitis B virus (HBV) infection is a global health problem,and more than 350 million people are chronic carriers of thevirus (3). The infection is associated with a wide spectrumof clinical manifestations, ranging from acute or fulminanthepatitis to various forms of chronic infection, including asymptomaticcarrier, chronic hepatitis, cirrhosis, and hepatocellular carcinoma(1, 3). Although serological and genotypic classifications ofHBV have been well documented (11), the clinical significanceof HBV genotypes in terms of clinical outcomes and therapeuticresponse to antiviral therapy in patients with chronic HBV infectionremains largely unknown until recently. Several studies suggestedthat HBV genotype C is associated with severer liver diseaseand a lower response rate to alpha interferon therapy than isgenotype B (5, 6, 9, 13). Taken together, these data suggestthe possible pathogenic and therapeutic differences among HBVgenotypes. However, the pathogenic link between genotype C andthe progression of liver disease remains unclear. We thereforestudied the clinical phenotypes, with special reference to theseroconversion of hepatitis B e antigen (HBeAg) and frequencyof acute exacerbation, for patients infected with HBV genotypeB and genotype C.

MATERIALS AND METHODS

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Materials and Methods

Patients. Between 1990 and 1995, a cohort of 272 patients (198 men and74 women; ages, 16 to 70 years) with chronic HBV infection wasfollowed at the gastroenterological clinic of National TaiwanUniversity Hospital. Chronic HBV infection was defined as apersistent seropositivity for HBV surface antigen (HBsAg) forat least 6 months before enrollment. All patients were negativefor antibodies to hepatitis C virus (anti-HCV) and hepatitisD virus and had no serological markers suggestive of autoimmunedisease. None had a history of alcohol abuse (>50 g/day),parenteral drug use, or hepatotoxin exposure. No specific antiviralsor immune modulators were given in the study period. These patientsreceived liver tests including serum alanine aminotransferase(ALT) activity every 3 months, more frequently if indicated.Serial serum samples taken from each patient were stored at-20°C until use.

According to the status of HBeAg at enrollment and frequencyof acute exacerbation during the follow-up period, five groupsof patients with distinct clinical phenotypes were categorized.Group I consisted of 63 patients who had positive HBeAg at enrollmentbut without acute exacerbations or seroconversion of HBeAg duringthe entire follow-up period. Group II consisted of 41 patientswho were positive for HBeAg at enrollment and had episode(s)of acute exacerbations before seroconversion of HBeAg. Amongthem, 16 had only one episode of acute exacerbation (group II-1)and the remaining 25 had multiple ( $>=$ 2) episodes of acute exacerbationbefore seroconversion of HBeAg (group II-2). Group III consistedof 46 patients who were positive for HBeAg at enrollment andhad multiple episodes of acute exacerbation but without seroconversionof HBeAg at the end of follow-up. Group IV consisted of 23 patientswho were negative for HBeAg at enrollment and had episode(s)of acute exacerbation during the follow-up period. Group V consistedof 99 patients who were negative for HBeAg at enrollment anddid not manifest acute exacerbations during their follow-upperiod. The positive rates of anti-HBe in group IV and groupV patients were 86 and 95%, respectively. Acute exacerbationwas defined as an abrupt increase of serum ALT (upper limitof normal, 40 U/liter) to a level greater than five times theupper limit of normal (200 U/liter) as previously described(4).

Hepatitis virus markers. Serum HBsAg and HBeAg were tested by Ausria-II and IMx HBe 2.0(Abbott Laboratories, North Chicago, Ill.), respectively. Anti-HCVand anti-hepatitis D virus were tested by commercially availableassays (HCV EIA II and Anti-Delta; Abbott Laboratories).

Genotyping of HBV. HBV genotypes were determined by using PCR-restriction fragmentlength polymorphism of the surface gene of HBV as previouslydescribed (5, 9). Six genotypes (A to F) of HBV could be identifiedby the restriction patterns of DNA fragments. To avoid false-positiveresults, instructions to prevent cross-contaminations were strictlyfollowed, and results were considered valid only when they wereobtained in duplicate. The sensitivity of our PCR assay was10 copies of HBV DNA per specimen by testing serial 10-folddilutions of HBV DNA transcripts with known amounts (10⁸ copies/ml).

Statistical analysis. Fisher's exact test, chi-square test with Yates' correction,and Student's t test were used where appropriate. A P valueof <0.05 was considered statistically significant.

RESULTS

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Results

The demographic and clinical data of chronic hepatitis B patientswith distinct clinical phenotypes are shown in Table 1. Patientsin groups IV and V had a significantly higher mean age thanthose in other groups (P < 0.01 and < 0.001, respectively).Of the 272 HBsAg carriers, 185 (68%) were infected with HBVgenotype B and the remaining 87 (32%) were infected with genotypeC. Among them, 150 (55%) were positive for HBeAg at enrollment,and those with genotype C infection tended to have a higherpositive rate of HBeAg than those with genotype B infection(63 versus 51%, P = 0.1).

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TABLE 1. Demographic and clinical data of 272 chronic hepatitis B patients with distinct clinical phenotypes

The distribution of HBV genotypes in different groups of chronichepatitis B patients with distinct clinical phenotypes duringthe entire follow-up period was analyzed (Table 2). In general,genotype B was more prevalent than genotype C in different groupsof HBV carriers. However, the prevalence of genotype C in patientswith multiple episodes of acute exacerbation who failed to haveHBeAg seroconversion (group III) was significantly higher thanthat in all 272 patients (50 versus 32%, P = 0.025) and thosewith HBeAg seroconversion after only one episode of acute exacerbation(group II-1) (50 versus 12%, P = 0.01). A similar situationwas also found in those negative for HBeAg at enrollment andwithout acute exacerbations (group V) (50 versus 23%, P = 0.002).

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TABLE 2. Distribution of HBV genotypes B and C in different groups of chronic hepatitis B patients with distinct clinical phenotypes

DISCUSSION

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Discussion

In addition to the serological classification of HBV isolatesinto nine subtypes according to the antigenic determinants oftheir HBsAg (2), a genetic classification based on the comparisonof complete genomes has defined seven genotypes of HBV (A toG) recently (11, 15). Like HBV serological subtypes, HBV genotypesalso have distinct geographical distributions (12). In general,genotypes B and C are prevalent in Asia, whereas genotypes Aand D prevail in Western countries. Genotype E is restrictedto Africa, while genotype F is found in Central America. GenotypeG has been identified in France and North America very recently(15). Our results indicated that 68% of the Taiwanese hepatitisB carriers were infected with genotype B, confirming that genotypeB is the most predominant HBV genotype in Taiwan (5, 7).

The clinical, virological, and therapeutic implications of HBVgenotypes in patients with chronic HBV infection have been partiallyclarified. Although several studies have suggested that HBVgenotype C is associated with the severity of liver disease(4, 5, 9, 13), the pathogenic link between genotype C and theprogression of liver disease remains largely unknown. In thenatural course of chronic HBV infection, early seroconversionfrom HBeAg to anti-HBe (immune clearance phase) usually indicatesa favorable outcome, because it is usually associated with thecessation of virus replication and nonprogressive liver disease(1). In contrast, late seroconversion of HBeAg after multipleepisodes of reactivation and remission may accelerate the progressionof chronic hepatitis B and thus have a poor clinical outcome(14). Our previous data implied that genotype C seems to staylonger in the immune clearance phase of persistent HBV infectionand shifted to stages of severer liver inflammation, and genotypeB may be associated with a faster transition through the immunoreactivestage and evolve into the residual phase in which the serumHBV DNA becomes barely detectable (5). Although not statisticallysignificant, our results suggested that patients with genotypeC infection tended to have a higher frequency of HBeAg positivitythan those with genotype B infection (63 versus 51%, P = 0.1).In addition, when the seroconversion of HBeAg and frequencyof acute exacerbation were used to define the clinical phenotypesof patients with chronic HBV infection, our data showed thatthe prevalence of genotype C in HBV carriers with multiple episodesof acute exacerbation who failed to have HBeAg seroconversionwas significantly higher than in all 272 patients (50 versus32%, P = 0.025 [Table 2]), in those with HBeAg seroconversionafter only one episode of acute exacerbation (50 versus 12%,P = 0.01 [Table 2]), or in those negative for HBeAg at enrollmentand without acute exacerbations (50 versus 23%, P = 0.002 [Table2]). These findings suggested that genotype C is associatedwith persistently HBeAg-positive chronic HBV infection withmultiple episodes of acute exacerbation and further supportedour prior observations (5). Previous studies also indicatedthat the frequency and severity of acute exacerbation of chronichepatitis B are closely associated with the development of livercirrhosis in HBsAg carriers (8, 10). In the meantime, doublemutations of the core promoter have been claimed to be associatedwith increased viral replication, active liver histology, andmore advanced liver disease (6, 9, 13, 14), and such mutationsare frequently found in genotype C strains. Taken together,it is therefore reasonable to speculate that the higher frequencyof HBeAg positivity, the longer stay in immune clearance phase,and the more frequent core promoter mutation of HBV genotypeC may contribute to the poor clinical outcomes of patients withgenotype C infection compared to those of patients with genotypeB infection.

In summary, our results suggest that patients with genotypeC infection have a more aggressive clinical phenotype than dothose with genotype B infection, which contributes to genotypeC patients' progressive liver disease and poor clinical outcomes.

ACKNOWLEDGMENTS

This work was supported by grants from the Department of Healthand the National Science Council, Executive Yuan, Taipei, Taiwan.

FOOTNOTES

* Corresponding author. Mailing address: Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, 7 Chung-Shan South Rd., Taipei 100, Taiwan. Phone: 886-2-23123456, ext. 7307. Fax: 886-2-23317624. E-mail: kjh{at}ha.mc.ntu.edu.tw.

REFERENCES

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