The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan
Address reprint requests to: Masashi Mizokami, M.D., Ph.D., Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1, Konodai, Ichikawa 272-8516, Japan. E-mail:email@example.com; fax: +81-(0)47-375-4766.
Potential conflict of interest: Nothing to report.
Supported by Japanese Ministry of Health, Labor and Welfare grant H21-003 and Japanese Ministry of Education, Culture, Sports, Science and Technology grant 22790679.
The proportion of patients who progress to chronicity following acute hepatitis B (AHB) varies widely worldwide. Moreover, the association between viral persistence after AHB and hepatitis B virus (HBV) genotypes in adults remains unclear. A nationwide multicenter study was conducted throughout Japan to evaluate the influence of clinical and virological factors on chronic outcomes in patients with AHB. For comparing factors between AHB patients with viral persistence and those with self-limited infection, 212 AHB patients without human immunodeficiency virus (HIV) coinfection were observed in 38 liver centers until serum hepatitis B surface antigen (HBsAg) disappeared or a minimum of 6 months in cases where HBsAg persisted. The time to disappearance of HBsAg was significantly longer for genotype A patients than that of patients infected with non-A genotypes. When chronicity was defined as the persistence of HBsAg positivity for more than 6 or 12 months, the rate of progression to chronicity was higher in patients with genotype A, although many cases caused by genotype A were prolonged cases of AHB, rather than chronic infection. Multivariate logistic regression analysis revealed only genotype A was independently associated with viral persistence following AHB. A higher peak level of HBV DNA and a lower peak of alanine aminotransferase (ALT) levels were characteristics of AHB caused by genotype A. Treatment with nucleotide analogs (NAs) did not prevent progression to chronic infection following AHB overall. Subanalysis suggested early NA initiation may enhance the viral clearance. Conclusion: Genotype A was an independent risk factor for progression to chronic infection following AHB. Our data will be useful in elucidating the association between viral persistence after AHB, host genetic factors, and treatment with NAs in future studies. (Hepatology 2014;58:89–97)
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Hepatitis B virus (HBV) infection is one of the most common persistent viral infections in humans. Approximately 2 billion people worldwide have been exposed to HBV and 350 million of them remain persistently infected. The incidence of HBV infection and the patterns of transmission vary greatly worldwide among different population subgroups. In Western countries, chronic HBV infection is relatively rare and acquired primarily in adulthood by way of sexual transmission or the use of injectable drugs. Meanwhile, in Asia and most of Africa, the majority of infections are the result of transmission from an infected mother to her newborn. However, very few studies of acute hepatitis B (AHB) in adults have been reported.
HBV genomic sequences vary worldwide and have been classified into at least eight genotypes (A through H) based on an intergroup divergence of 8% or more over the complete nucleotide sequence.[3, 4] These genotypes have distinct geographic distributions.[5-7] In particular, genotype A is predominant in Northwestern Europe, the United States, Central Africa, and India.[8, 9] The Japanese have been infected with genotypes B and C since prehistoric times. Recently, many lines of evidence have revealed among the Japanese an increase in acute infection with HBV genotype A following sexual transmission.[11, 12] As a result of this increasing transmission of genotype A, the distribution of HBV genotypes in Japan clearly differs among patients with acute and chronic infections. Moreover, recent studies suggest that acute infection with HBV genotype A may be associated with an increased risk of progression to persistent infection. Indeed, the prevalence of HBV genotype A in chronic hepatitis B patients doubled in Japan between 2000-2001 and 2005-2006 (1.7% versus 3.5%).
Human immunodeficiency virus (HIV)-1 infection results in an immunodeficient state, with the virus sharing routes of transmission with HBV. HIV-related immunodepletion influences the natural history of HBV infection, and epidemiological studies have revealed that HIV-positive patients are more likely to have a prolonged acute illness following HBV infection and lower rates of hepatitis B e-antigen (HBeAg) clearance. Therefore, in this study patients with coinfection of HIV were excluded to examine the influence of HBV genotype directly without the confounding influence of HIV.
From 2005 to 2010, a multicenter cohort study was conducted throughout Japan on 212 patients with AHB. The aim of this cohort study was to assess the influence of clinical and virological factors, including HBV genotypes and treatment with nucleotide analogs (NAs), on AHB patients who became persistently infected.
Patients and Methods
Patients With AHB
The multiple-source cohort included 212 randomly selected AHB patients without coinfection of HIV. From 2005 through 2010, the study participants were recruited from 38 liver centers throughout Japan. The cohort included patients who were admitted to the hospital because of AHB and who visited the hospital every month after being discharged. The diagnosis of AHB was contingent on the rapid onset of clinical symptoms accompanied by elevated serum alanine aminotransferase (ALT) levels, the detection of serum hepatitis B surface antigen (HBsAg), and a high-titer antibody to hepatitis B core antigen (anti-HBc) of the immunoglobulin M (IgM) class. Patients with initial high-titer anti-HBc (>10.0 S/CO) were diagnosed as having an exacerbation of chronic hepatitis B and were excluded. If the patient had been tested previously, the absence of serum HBsAg and anti-HBc before admission was verified from the medical record to discriminate a new infection from an acute exacerbation of a persistent infection. Patients with acute hepatitis A, hepatitis C, and drug- or alcohol-induced acute hepatitis were also excluded; hepatitis D virus infection was not determined because of its extreme rarity in Japan. The study protocol conformed to the 1975 Declaration of Helsinki and was approved by the Ethics Committees of the institutions involved. Every patient gave informed consent for this study.
Serological Markers of HBV Infection
HBsAg, HBeAg, antibodies to HBsAg (anti-HBs), HBeAg (anti-HBe), and HBcAg, and anti-HBc of the IgM class were tested by a chemiluminescent enzyme immunoassay (CLIA) by ARCHITECT (Abbott Japan, Tokyo, Japan). HBV DNA measurements were performed using a real-time polymerase chain reaction (PCR) assay (Cobas TaqMan HBV Auto; Roche Diagnostics, Tokyo, Japan).
Genotyping of HBV
The six major HBV genotypes (A through F) were determined serologically by enzyme immunoassay (EIA) using commercial kits (HBV GENOTYPE EIA; Institute of Immunology, Tokyo, Japan). This method is based on the pattern of detection by monoclonal antibodies of a combination of epitopes on preS2-region products, which is specific for each genotype.[17, 18] Samples for which EIA could not determine the genotype were examined by direct sequencing of the pre-S2/S gene, followed by phylogenetic analysis.
Treatment With NAs
Treatments with NAs were performed using lamivudine or entecavir for more than 3 months. The individual clinicians determined if NAs were administered to patients, and when the treatment was to be started. The time to onset of treatment with NAs was measured in days from onset of AHB.
Categorical variables were compared between groups by the chi-squared test and noncategorical variables by the Mann-Whitney U test. A P value less than 0.05 was considered significant. Multivariate analysis was performed using a backward stepwise logistic regression model to determine independent factors for viral persistence following AHB. Variables in the multivariate analysis were selected based on variables that were marginally significant with P < 0.1 in univariate analysis. Maintenance of HBsAg positivity was analyzed using the Kaplan-Meier method and significance was tested with the log-rank test. STATA Software (StataCorp, College Station, TX) v. 11.0 was used for analyses.
Comparison of Characteristics Between Genotype A and Non-A Genotype AHB Patients
A total of 107 AHB patients (50.5%) were infected with genotype A while 105 AHB patients (49.5%) were infected with non-A genotypes, including genotypes B (25 [11.8%]), C (76 [35.8%]), D (1 [0.5%]), F (1 [0.5%]), and H (1 [0.5%]). Compared to those infected with non-A genotypes, genotype A patients were significantly younger (36.3 ± 12.0 versus 40.7 ± 14.3 years, P = 0.032), predominantly men (95.3% versus 71.4%, P < 0.001), and more frequently positive for HBeAg (97.2% versus 75.2%, P < 0.001). Moreover, genotype A patients had a lower peak ALT levels (1,210 ± 646 versus 2,225 ± 2,851 IU/L, P = 0.045) and a higher peak level of HBV DNA (6.7 ± 8.5 versus 3.4 ± 6.5 log copies/mL, P < 0.0001). A significantly higher percentage of genotype A patients were treated with NAs (57% versus 40%, P = 0.013). These data are summarized in Table 1.
Table 1. Characteristics of Patients With Genotype A or a Non-A Genotype Acutely Infected With Hepatitis B Virus
Cumulative Maintenance of HBsAg Positivity During Follow-up in Patients With Genotype A and Non-A Genotypes
In the patients infected with genotype A and non-A genotypes, the mean durations of HBsAg positivity maintenance were 6.7 ± 8.5 and 3.4 ± 6.5 months, respectively (P < 0.0001; Table 1, Fig. 1). For 6 months after AHB onset, the number of patients with genotype A and non-A genotypes maintaining HBsAg positivity were 39/107 (36.4%) and 10/105 (9.5%), respectively (P < 0.001). However, in many patients HBsAg disappeared between 7 and 12 months after AHB onset; that is, HBsAg disappeared in 31/107 (29.0%) of patients with genotype A and in 9/105 (8.6%) of patients with non-A genotypes during this time period. However, in some patients HBsAg never disappeared after persisting for more than 12 months following AHB onset. When chronicity after AHB was defined as the persistence of HBsAg for more than 12 months, chronicity developed in 7.5% (8/107) of patients with genotype A and in 0.9% (1/105) of patients with non-A genotypes (P = 0.018).
Comparison of Characteristics Between Patients in Whom HBsAg Persisted More Than 6 or 12 Months and Those With Self-Limited AHB Infection
Table 2 compares the demographic and clinical characteristics between patients in whom HBsAg disappeared within 6 months and those in whom HBsAg persisted for more than 6 months from AHB. The peak ALT levels (1,882 ± 2,331 versus 1,018 ± 696 IU/L, P = 0.0024) and peak HBV DNA levels (6.3 ± 1.6 versus 7.4 ± 1.6 mg/dL, P = 0.0004) were significantly higher and lower in the former group than in the latter group, respectively. Moreover, marked differences were present in the distribution of genotypes between the two groups. The percentage of the HBV genotype A (46.1% versus 73.5%, P = 0.003) was significantly higher among patients in whom HBsAg was persistent for more than 6 months. In addition, we compared the demographic and clinical characteristics between patients in whom HBsAg disappeared within 12 months and those in whom HBsAg persisted for more than 12 months from AHB. Peak ALT (1,787 ± 2,118 versus 775 ± 513 IU/L, P = 0.0089) and peak total bilirubin (8.7 ± 8.2 versus 3.8 ± 6.6 mg/dL, P = 0.0039) levels were significantly higher in the former group than in the latter group. In contrast, the peak HBV DNA levels (6.4 ± 1.6 versus 7.9 ± 1.4 mg/dL, P = 0.0046) were significantly lower in the former group than in the latter group. The percentages of HBV genotype A (48.8% versus 88.9%, P = 0.018) and NAs treatment (+) (48.3% versus 88.9%, P = 0.017) were significantly higher among patients in whom the HBsAg persisted for more than 12 months.
Table 2. Comparison Between Patients With Chronicity Following Acute Hepatitis B and Those With Self-Limited Acute Infections Determined by the Persistence of HBsAg for More Than 6 or 12 Months
Disappearance of HBsAg Within 6 Months (n = 178)
Persistence of HBsAg for More Than 6 Months From AHB (n = 34)
Disappearance of HBsAg Within 12 Months (n = 203)
persistence of HBsAg for More Than 12 Months From AHB (n = 9)
Data are presented as n (%) and mean ± SD. HBsAg, hepatitis B surface antigen; AHB, acute hepatitis B, HBeAg, hepatitis B e-antigen; ALT, alanine aminotransferase; HBV, hepatitis B virus; NAs, nucleotide analogs.
Factors Independently Associated With Viral Persistence Following AHB
A stepwise logistic regression model was used to perform multivariate analysis which explains relationships between some factors and persistence of HBsAg positivity more than 6 months following AHB. Peak ALT level, peak HBV DNA level, genotype A, and treatment with NAs were retained in the final multivariate logistic model in a backward stepwise manner (P < 0.1). For predicting the persistence of HBsAg for more than 6 months, only genotype A was independently associated with progression of AHB to the persistence of HBsAg (odds ratio [OR]: 4.224, P = 0.001, Table 3).
Table 3. Multivariate Analysis of Factors Independently Associated With Persistence of HBsAg Positivity Following Acute Hepatitis B
Characteristics of Patients Who Progressed to Chronicity That Was Defined as the Persistence of HBsAg for More Than 12 Months Following Acute Hepatitis B
Table 4 shows the clinical and virological characteristics of nine patients who progressed to chronicity defined as the persistence of HBsAg for more than 12 months following AHB. Among the nine patients who progressed to chronicity from AHB, eight (88.9%) were men and eight (88.9%) were HBeAg-positive. In general, among the patients who progressed to chronicity following AHB, the peak HBV DNA levels were high, and the peak total bilirubin and ALT levels were low. In eight (88.9%) patients, entecavir was administered; however, the duration until the onset of NA treatment from AHB onset was long (75-570 days).
Table 4. Characteristics of Patients Who Progressed to Chronicity Following Acute Hepatitis B
HBV DNA (log copies/mL)
Total Bilirubin (mg/dL)
Observation Period (Months)
Duration Until NAs Treatment (Days)
HIV, human immunodeficiency virus; HBeAg, hepatitis B e-antigen; HBV, hepatitis B virus; ALT, alanine aminotransferase; NAs, nucleotide analogs; ETV, entecavir.
Early Onset of Treatment With NAs Was Able to Prevent Viral Persistence After AHB Caused by Genotype A
The cumulative proportion maintaining HBsAg positivity during follow-up, expressed in terms of time after AHB onset, were significantly longer in patients with NAs treatment than in those without NAs treatment (P = 0.046, Fig. 2A). Table 5 shows the percentages of patients in whom HBsAg persisted for more than 6 or 12 months among patients categorized based on the period of time (i.e., duration) until the onset of NAs treatment. For patients in whom the onset of NAs treatment was less than 4 weeks from the onset of AHB, 12.7% of the patients showed persistent HBsAg for more than 6 months, while none showed HBsAg positivity for more than 12 months. For patients in whom the onset of NAs treatment was at 5-8 weeks, 37.5% of the patients showed persistent HBsAg for more than 6 months, whereas none showed persistent HBsAg for more than 12 months. For all groups, the period of HBsAg positivity in patients starting NAs treatment within 8 weeks from AHB onset was significantly shorter than that in patients beginning NAs treatment after more than 8 weeks from AHB onset (P < 0.0001, Fig. 2B). Patients starting NAs treatment within 8 weeks from AHB onset never progressed to chronicity after AHB caused by genotype A.
Table 5. Proportion of Patients in Whom HBsAg Persisted for More Than 6 or 12 Months Among Patients Categorized Based on the Number of Weeks Until the Onset of NAs Treatment
Duration Until Onset of NAs Treatment (Weeks)
Persistence of HBsAg for More Than 6 Months
Persistence of HBsAg for More Than 12 Months
HBsAg, hepatitis B surface antigen; NAs, nucleotide analogs.
<4 weeks (n, %)
5-8 weeks (n, %)
9-12 weeks (n, %)
13-16 weeks (n, %)
>17 weeks (n, %)
A multicenter nationwide study was conducted throughout Japan to evaluate the influence of clinical and virological factors on chronic outcomes in Japanese patients who contracted AHB in adulthood. The study was feasible in Japan, where a universal vaccination program for HBV has not been implemented because of the extremely high efficacy of the immunoprophylaxis that is given to babies born to carrier mothers. The implementation of this program has resulted in a decrease in the persistent HBV carrier rate from 1.4% to 0.3%. Selective vaccination means that Japanese are more likely to be infected with HBV by way of horizontal transmission since the percentage of the population possessing anti-HBs is much lower than that in countries in which universal vaccination programs have been established. In addition, Japan is faced with the ever-increasing impacts of globalization: as many as 17 million Japanese travel abroad and over 7 million people visit Japan from overseas each year. This “population mixing” may help to explain the increased prevalence in Japan of AHB due to genotype A, which is transmitted through indiscriminate sexual contact. Consequently, Japan may be the only country in the world where the influences of HBV genotypes, including genotype A (as is predominant in Western countries) and genotypes B and C (as are predominant in Asian countries), on chronic outcomes after AHB can be compared.
Currently, the persistence of HBsAg in serum for more than 6 months is considered to represent a progression to chronic infection. However, our data showed that HBsAg frequently disappeared between 7 to 12 months after the onset of AHB in patients with genotype A (31/107 [29.0%]) and non-A genotypes (9/105 [8.6%]) (Fig. 1). These patients were considered to exhibit prolonged cases of AHB, rather than persistent infection. This finding reflects the higher sensitivity of the most up-to-date assays for HBsAg as compared with previous methods. In the present study, HBsAg was measured by CLIA, which has been reported to be about 150 times more sensitive in the detection of HBsAg than reverse passive hemagglutination (RPHA)-HBsAg, which has been used for the last 30 years in Japan. The use of a more sensitive assay for HBsAg results in a longer period during which HBsAg may be detected. In this study, HBsAg did not disappear in nine patients after remaining continuously detectable for more than 12 months. Therefore, the persistence of HBsAg for more than 12 months, as measured with a highly sensitive method for detecting HBsAg, may be suitable for defining the progression of AHB to chronicity; however, further study is necessary to determine whether this definition is appropriate worldwide.
It has been reported that ∼10% of patients who contract HBV as adults do not clear HBsAg from their serum and become carriers. Meanwhile, a wide variation has been seen in the rate of persistence after AHB infection in adults. For example, viral persistence following AHB was seen in 0.2% (1/507) of adults in Greece, 7.7% (5/65) of adult Alaskan Eskimos, and 12.1% (7/58) of adults in Germany. The difference in the proportion of patients progressing from AHB to chronicity in different regions may be attributable to virological and host factors. In this study, 4.2% (9/212) of patients progressed to chronicity after AHB: 7.5% (8/107) of those infected with genotype A and 0.9% (1/105) of those infected with non-A genotypes. The non-A genotypes included genotypes B, C, D, F, and H (n = 25, 77, 1, 1, and 1, respectively). Genotypes B and C are predominant in eastern Asian countries, where the majority of those infected with HBV acquired the virus during the perinatal period by way of vertical transmission. On the other hand, genotype A is predominant in Western countries, where the main route is horizontal transmission later in life.[26, 27] Because HBeAg persists long after the infection in the genotype C as compared to other genotypes, this genotype has been shown to be a risk factor for perinatal and horizontal transmission in newborns and children. The predominance of genotype A in Western countries may be attributable to a higher chronicity rate following AHB by way of horizontal transmission in adults.
In this study the characteristics of AHB associated with genotype A were a higher peak level of HBV DNA and a lower peak level of ALT. These findings were similar to those for patients with HBV-HIV coinfection. Such characteristics of genotype A or coinfection with HIV are assumed to be attributable to milder hepatitis associated with weaker cellular immune responses. More slowly replicating viruses have been reported to evoke weaker cellular responses, enhancing the likelihood of persistence. Indeed, our prior study showed that the replication of genotype A was significantly slower than that of genotype C in immunodeficient, human hepatocyte chimeric mice. Moreover, variation among genotypes in the expression pattern of HBeAg may affect the progression of AHB to chronicity. Another previous study of ours revealed that a single form of HBeAg was detected by western blot analysis in serum samples from patients infected with genotypes B through D, but that two additional larger forms of HBeAg were detected in patients with genotype A. Milich and Liang reported that HBeAg may modulate the host immune response as a tolerogen to promote chronicity. Therefore, the different expression pattern of HBeAg by genotype A HBV may contribute to chronicity following AHB.
Early NAs initiation appeared to enhance the viral clearance across genotypes, although treatment with NAs did not show any overall benefit in duration of HBsAg. Previous studies examining the efficacies of NAs for preventing progression to chronic infection after AHB have reported conflicting results. Some small-scale studies have suggested the efficacy of lamivudine and entecavir in preventing the progression of AHB to chronic hepatitis.[34, 35] Another study showed a lower seroconversion rate of HBsAg in lamivudine users. Further, a randomized placebo-controlled trial showed no significant difference in clinical outcomes. However, these previous studies did not mention the prevalence of HBV genotypes in the respective study populations. Although this was a retrospective study, our study included data on the prevalence of HBV genotypes. Additionally, our findings suggested that larger prospective randomized studies for every HBV genotype should be performed to determine whether early treatment with NAs prevented the progression of AHB to a chronic state.
In conclusion, in Japan genotype A was an independent risk factor for progression to chronic infection following AHB in adults. Confirmation of this association in patients with AHB in other countries is desirable and may provide insight into the pathogenetic mechanisms underlying this association. Early NA treatment appeared to reduce the likelihood of chronicity but this potentially important intervention needs to be prospectively studied before recommendations can be made.
Members of the Japanese AHB Study Group include Yasuharu Imai (Ikeda Municipal Hospital), Norie Yamada, Hideaki Takahashi (St. Marianna University School of Medicine), Koji Ishii (Toho University School of Medicine), Hideyuki Nomura (Shin-Kokura Hospital), Jiro Nishida (Tokyo Dental Collage Ichikawa General Hospital), Shigeru Mikami (Kikkoman Hospital), Tsuneo Kitamura (Juntendo University Urayasu Hospital), Akihito Tsubota (Kashiwa Hospital Jikei University School of Medicine), Noritomo Shimada (Shinmatsudo Central General Hospital), Tetsuya Ishikawa (Nagoya University Graduate School of Medicine), Yoshiyuki Ueno (Tohoku University Graduate School of Medicine), Tomoyoshi Ohno (Social Insurance Chukyo Hospital), Etsuro Orito (Nagoya Daini Red Cross Hospital), Michihiro Suzuki (Kawasaki Municipal Tama Hospital), Hitoshi Takagi (Gunma University Graduate School of Medicine), Eiichi Tomita (Gifu Municipal Hospital), Kumada Takashi (Ogaki Municipal Hospital), Toshihiko Mizuta (Saga University Faculty of Medicine), Tetsuya Mine (Tokai University School of Medicine), Jong-Hon Kang (Teine-Keijinkai Hospital), Katsuji Hirano (Juntendo University Shizuoka Hospital), Hirohito Tsubouchi (Kagoshima University Graduate School of Medical and Dental Sciences), Akito Nozaki (Yokohama City University Medical Center), Akito Sakai (Kanazawa University Graduate School of Medical Science), Shuhei Nishiguchi (Hyogo College of Medicine), Akihiro Tamori (Osaka City University Graduate School of Medicine), Satoru Hagiwara (Kinki University School of Medicine), Takahide Nakazawa (University of Kitasato East Hospital), Michio Sata (Kurume University School of Medicine), Toshiro Kamoshida (Hitachi General Hospital) Atsushi Takahashi (Fukushima Medical University School of Medicine), Satoshi Kakizaki (Gunma University Graduate School of Medicine), Yoshimasa Kobayashi (Hamamatsu University School of Medicine), Shigeru Sasaki (Sapporo Medical University), Tadashi Ikegami (Tokyo Medical University Ibaraki Medical Center), Yoichi Hiasa (Ehime University Graduate School of Medicine), Kenji Nagata (University of Miyazaki), Tomoyuki Kubota (Saiseikai Niigata Daini Hospital), Hiroshi Mitsui (Tokyo Teishin Hospital), Norihiko Yamamoto (Mie University School of Medicine), Masataka Tsuge (Hiroshima University), Shuichi Sato (Shimane University Hospital), Yoshito Ito (Kyoto Prefectural University of Medicine), Wataru Sato (Akita University School of Medicine), Shigeharu Uchida (Japanese Red Cross Society), Yuki Tada (National Institute of Infectious Diseases), Toshiaki Mizuochi (National Institute of Infectious Diseases), Norihiro Furusho (Kyushu University), and Shuhei Hige (Hokkaido University Graduate School of Medicine).