Usefulness of radiofrequency ablation with micro-convex probe for hepatocellular carcinoma.

BACKGROUND/AIMS: It was aimed to assess whether a micro-convex probe is superior to the present conventional probe for ultrasonography from the points of safety and efficacy during percutaneous radiofrequency ablation therapy for hepatocellular carcinoma. METHODOLOGY: Twenty-one patients with 23 hepatocellular carcinoma lesions who had one or 2 lesions, each 4 cm or less in diameter, and liver function of Child-Pugh class A or B were enrolled. All the patients except for 2 patients were seropositive for hepatitis C virus. Radiofrequency ablation was carried out under a real-time US guidance. The cooled-tip electrodes used were single and clustered. RESULTS: It was possible to perform safe and accurate percutaneous radiofrequency ablation procedure using micro-convex probes for the treatment of all hepatocellular carcinoma nodules. It was also possible to treat hepatocellular carcinoma located in the right subphrenic region without artificial pleural effusion under intercostal ultrasonography guide. Improved clustered needles were successfully applied to treat the nodules more than 3 cm in diameter with less resistance for penetration compared with the conventional needle. The findings of advanced dynamic flow image on ultrasonography to assess the therapeutic efficacy indicated the consistency with those of dynamic CT which was done 3 to 5 days later radiofrequency ablation. Major complication of radiofrequency ablation procedure was noted in none. CONCLUSIONS: These results suggest that micro-convex probe with clustered tips is superior to conventional probe for ultrasonography from the points of safety and efficacy during radiofrequency ablation for hepatocellular carcinoma nodule located in the right subphrenic region and for larger sized nodule more than 3 cm.

Distribution of hepatitis B virus genotypes among patients with chronic infection in Japan shifting toward an increase of genotype A.

Acute hepatitis B virus (HBV) infection has been increasing through promiscuous sexual contacts, and HBV genotype A (HBV/A) is frequent in patients with acute hepatitis B (AHB) in Japan. To compare the geographic distribution of HBV genotypes in patients with chronic hepatitis B (CHB) in Japan between 2005 and 2006 and between 2000 and 2001, with special attention to changes in the proportion of HBV/A, a cohort study was performed to survey changes in genotypes of CHB patients at 16 hospitals throughout Japan. Furthermore, we investigated the clinical characteristics of each genotype and examined the genomic characteristics of HBV/A isolates by molecular evolutionary analyses. Of the 1,271 patients, 3.5%, 14.1%, and 82.3% were infected with HBV/A, -B, and -C, respectively. In comparison with our previous survey during 2000 and 2001, HBV/A was twice as frequent (3.5% versus 1.7%; P = 0.02). The mean age was lower in the patients with HBV/A than in those with HBV/B or -C. Based on phylogenetic analyses of 11 full-length genomes and 29 pre-S2/S region sequences from patients, HBV/A isolates were imported from Europe and the United States, as well as the Philippines and India. They clustered with HBV/A from AHB patients and have spread throughout Japan. HBV/A has been increasing in CHB patients in Japan as a consequence of AHB spreading in the younger generation through promiscuous sexual contacts, aided by a tendency of HBV/A to induce chronic hepatitis. The spread of HBV/A infection in Japan should be prevented by universal vaccination programs.

Inflammatory cytokines modulate chemokine production patterns of HepG2 cells toward initially inclined direction.

AIM: Human hepatocytes are known to express an array of inflammatory cytokines and chemokines. In this study, we examined the potential roles of hepatocytes in regulating immune responses in the liver, by assessing the induction of Th1- or Th2-specific chemokines in HepG2 cells after various inflammatory stimulations. METHODS: HepG2 cells were stimulated with IL-1alpha, IFN-gamma, IL-4, IL-10, and/or CCL2, harvested at several time points, and served for the analyses of cytokine/chemokine mRNA expressions by semi-quantitative RT-PCR. RESULTS: (i) IL-1alpha up-regulated mRNA levels of CXCL8, CXCL10, and CCL2. IFN-gamma increased those of CXCL9, CXCL10, and CCL5, while IL-4 or IL-10 had no effect. (ii) Addition of IL-4 to the culture of IFN-gamma-stimulated cells, down-regulated CXCL9 and CXCL10 mRNA levels. (iii) Addition of IFN-gamma to the culture of IL-1alpha-stimulated cells, further up-regulated CXCL9 and CXCL10 mRNA levels. Addition of IL-4 decreased CXCL8 and CXCL10 levels, and increased CCL2 level in IL-1alpha-stimulated cells. (iv) CCL2 induced IL-4 mRNA expression. CONCLUSIONS: IFN-gamma augmented mRNA expression of Th1-specific chemokines (CXCL9 and CXCL10) in HepG2 cells. IL-4 had no effect on those of Th2-spesific chemokines (CCL17 and CCL22); however, it was supposed to augment Th2 response indirectly through the induction of CCL2 under the inflammatory condition. The findings suggest that hepatocytes have ability to promote immune responses in the liver toward the direction, initially determined by the cytokine balances in the local inflammatory region.

Hepatocellular Carcinoma 11 and a Half Years after the Resolution of Chronic Hepatitis C Virus Infection Successfully Treated with Interferon.

A 41-year-old Japanese man had received successful interferon (IFN) therapy against chronic hepatitis C in 1994. Since then, serum hepatitis C virus (HCV) RNA had been negative, and aminotransferase levels had been continuously normal. He had abstained from alcohol. However, his serum aminotransferase levels showed slight elevation as his body weight increased gradually. He was diagnosed as having fatty liver and diabetes mellitus. In January 2006, 11 and a half years after the resolution of HCV infection, he was found to have a hepatic nodule 4.0 cm in diameter at liver S4/8 region by plain abdominal CT at an annual follow-up examination. He was diagnosed as having hepatocellular carcinoma (HCC) by angiography. The tumor was curatively resected and its histological diagnosis was moderately differentiated HCC. Noncancerous lesion of the liver revealed fibrosis of stage F2 and mild inflammation of grade A1 with mild steatosis. This case suggests that all patients with chronic HCV infection should be followed as long as possible for the potential development of HCC even after clearance of the virus.

Intrahepatic gene expression profiles in chronic hepatitis B and autoimmune liver disease.

BACKGROUND: DNA microarray technology has enabled genomewide analysis of gene transcript levels, yielding insight into the molecular nature of liver disease. METHODS: We compared gene expression of liver biopsy specimens in 16 patients with different stages of chronic hepatitis B, five with autoimmune hepatitis (AIH), five with primary biliary cirrhosis (PBC), and six with druginduced hepatitis. RESULTS: Of 21 073 genes, 424 showed different expression in a particular disease group on analysis of variance. Genes associated with extracellular matrix, cell growth, and DNA repair were noted in the advanced fibrotic stage of chronic hepatitis B (B-3), while gene expression regarding complement activation and the innate immune response decreased. When we compared gene expression at the relatively early stage in each disease group with pathway analysis, pathways relating to chemotaxis and cell homeostasis were selected in chronic hepatitis B. In PBC, gene expression relating to structural constituents and contractions of muscle such as actin and myosin were enhanced, in contrast to the downregulation of genes relating to protein binding in AIH. A hierarchical clustering analysis of hepatitis B genes defined five clusters. Generally, the transcripts upregulated according to disease progression were associated with signaling pathway/transcription, including tumor-associated calcium signal transducer 1 and chemokine ligand 19, and with cell communication, such as collagen. In two groups, all transcripts were downregulated; transcripts related to chemokine ligands and metallothionein were further depressed in B-3. CONCLUSIONS: Analysis of gene expression in liver may be useful for understanding features of distinct liver diseases and for guiding disease progression, particularly in chronic hepatitis B.

Deficiency of forkhead box P3 and cytotoxic T-lymphocyte-associated antigen-4 gene expressions and impaired suppressor function of CD4(+)CD25(+) T cells in patients with autoimmune hepatitis.

AIM: Recently, forkhead box P3 (Foxp3), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR), and CD28 were identified as the key molecules that control the development and activation of CD4+CD25+ regulatory T cells (T-reg). We investigated the expression pattern of these molecules on T-reg, and investigated the ability of T-reg to produce cytokines in patients with autoimmune hepatitis (AIH). METHODS: Fifteen patients with AIH and nine healthy patients were included. To determine the frequency of T-reg, a two-color flow cytometry analysis was performed. T-reg were isolated using immunomagnetic beads, and the mRNA levels of Foxp3, CTLA-4, GITR, and CD28 were quantified by real-time polymerase chain reaction (PCR). The ability of T-reg to produce interferon-gamma, interleukin (IL)-10, transforming growth factor-beta, and tumor necrosis factor-alpha after stimulation by OKT3 was evaluated by measuring the levels of mRNA in T-reg by real-time PCR. RESULTS: The frequency of T-reg was increased in AIH. The mRNA levels of Foxp3 and CTLA-4 were significantly lower in AIH. The ability of T-reg to produce IL-10 was impaired in AIH. CONCLUSION: We speculate that the inferiority of the Foxp3 and CTLA-4 gene expressions on T-reg results in the impaired suppressor function of T-reg, and eventually in the breakdown of self-tolerance.

Role of Valpha14+ NKT cells in the development of Hepatitis B virus-specific CTL: activation of Valpha14+ NKT cells promotes the breakage of CTL tolerance.

CTLs are thought to be major effectors for clearing viruses in acute infections including hepatitis B virus (HBV). Persistent HBV infection is characterized by a lack of or a weak CTL response to HBV, which is thought to reflect tolerance to HBV antigens. In the present study, we found that alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14-positive NKT cells, strongly enhanced the induction and proliferation of HBV-specific CTLs by HBsAg. In HBsAg transgenic mice, which are thought to be tolerant to HBV-encoded antigens, administration of HBsAg or alpha-GalCer alone failed to induce HBsAg-specific CTLs, but they were induced by co-administration of both compounds. Furthermore, by limiting dilution analysis, we confirmed the existence of HBsAg-specific CTL precursors in the HBsAg transgenic mice immunized with HBsAg and alpha-GalCer. A blocking experiment using antibodies to cytokines and CD40 ligand showed that IL-2 and CD40-CD40L interaction mediate the enhancement of CTL induction caused by alpha-GalCer through NKT cell activation. Our results may open up a new method for clearing the virus from patients with persistent HBV infection.

Changes in intrahepatic gene expression profiles from chronic hepatitis to hepatocellular carcinoma in patients with hepatitis C virus infection.

AIM: DNA microarray technology has enabled genome-wide analysis of gene transcript levels, which has yielded insight into the molecular nature of hepatitis C virus infection. However, little insight into the molecular nature of the early to advanced stages of chronic liver disease has as yet been obtained. METHODS: We compared the gene expression profiles of liver biopsy specimens from 14 patients at different stages of chronic hepatitis C. We also evaluated the liver tissue of hepatocellular carcinoma and its surrounding region obtained surgically in seven patients with hepatitis C virus infection. RESULTS: Of 21 073 genes, 582 genes showed significant changes in expression levels across the disease group. Twenty-eight samples from six disease groups clustered according to the histological classification except for 4 samples. A heat map produced by hierarchical clustering revealed nine clusters where gene expression profiles were changed in abundance. Among 44 genes which changed twofold or more in transcript abundance, transcripts from chronic hepatitis tended to be upregulated, and gradually downregulated according to disease progression toward hepatocellular carcinoma in five of nine clusters. In chronic hepatitis, transcripts relating to metabolism and immune response were upregulated, while in hepatocellular carcinoma, transcripts associated with cell cycle, growth, proliferation, apoptosis and signaling pathway were upregulated. CONCLUSION: Disease progression in hepatitis C virus-infected patients appeared to be associated with changes in gene expression profiles in the liver consistent with plausible functional categories, although we should confirm these findings using larger samples.

Early decline of hemoglobin can predict progression of hemolytic anemia during pegylated interferon and ribavirin combination therapy in patients with chronic hepatitis C.

AIM: Ribavirin, used to treat chronic hepatitis C, can induce hemolytic anemia, forcing the discontinuance of treatment. To establish a predictive measure to help circumvent this, we evaluated the relationship of hemoglobin (Hb) decline with the discontinuance of treatment during the progression of ribavirin-induced anemia. METHODS: One hundred and sixteen patients (71% male) with genotype 1 chronic hepatitis C were treated with pegylated interferon (PegIFN) alpha-2b and ribavirin. The mean age was 50.6 years and 55% were IFN naïve. A decline of Hb concentration by 2 g/dL at two weeks from the start of the treatment ("2 by 2" standard) was adopted as the predictive factor for the progression of anemia. RESULTS: By applying the "2 by 2" standard, with DeltaHb >/= 2 g/dL (34%, n = 39), treatment was discontinued in 12 cases (31%), three of which (8%) because of severe anemia. ForDeltaHb < 2 g/dL (64%, n = 76), treatment was discontinued in 11 (14%) cases; none due to severe anemia. Ten percent (4/39) of patients showed the minimum Hb /= 2 g/dL group, with none in the DeltaHb < 2 g/dL group (P = 0.001). Furthermore, the patients with minimum Hb

Use of hepatitis B vaccine for the treatment of chronic hepatitis B.

Current therapeutic approaches to control chronic hepatitis B (CH-B), such as administrations of interferon or nucleoside analogs, are still unsatisfactory. Vaccination with conventional hepatitis B (HB) vaccine is another therapeutic approach with lower cost and potentially long-lasting beneficial effect. However, a response rate to vaccination therapy is not necessarily high. Therefore, combination therapy of interferon, nucleoside analogs and vaccination, would be the promising therapeutic approach that improves therapeutic effect and solves the problems of individual therapies. Herein, we report the results of the clinical trial, the combination therapy of lamivudine (LAM) and HB vaccine in patientswith B-CH as one of the candidates for the combination therapies. The results indicate that the combination therapy of LAM and HB vaccine was more effective in regulating viral replication than the LAM monotherapy was. In addition, no adverse effect was observed in the patients given HB vaccine. This novel therapy should be further examined for the improvement of its efficacy and achievement of continuous suppression of HB virus replication.

Expression of Toll-like receptors in chronic hepatitis C virus infection.

BACKGROUND: Toll-like receptors (TLRs) are involved in innate immunity. Certain viruses interact with TLRs and mediate antiviral effects as well as immune responses. The aim of this study was to investigate the effect of TLRs on pathogenesis in hepatitis C virus (HCV)-infected patients. METHODS: Peripheral blood mononuclear cells (PBMC) and CD14+ (monocytes) or CD14- cells from 25 patients with chronic liver disease and 15 healthy subjects were studied for expression of TLRs 2-9 and cytokines of extracted RNA using real-time PCR. Then TLR expression was examined in HepG2 cells transfected with entire or parts (core-NS3, NS3-NS5B) of the HCV open reading frame. TLR expression was calculated as the relative mRNA levels. RESULTS: Expression of TLRs 4, 7 and 8 in CD14+ cells of PBMC was increased in patients. Levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p35 for PBMC were also increased in patients. When PBMC were incubated with HCV core protein, enhancement of TLR2 expression and suppression of TLR4 and TLR7 were noted in patients. Similar alteration of TLRs expression was observed in controls. Among HepG2 transfectants, only TLR3 expression was changed; it was suppressed in entire gene transfectant and enhanced in core-NS3 transfectant. Expression of some proteins related to the TLR signaling pathway was suppressed in the entire gene transfectant. CONCLUSIONS: The results suggest a correlation between expression levels of TLRs and cytokines, and chronic HCV infection. TLR3 recognizes double-stranded RNA and induces type 1 interferon synthesis. Collectively, suppressed expression of TLR3 in cells transfected with entire HCV may be responsible for continuous HCV infection, although a part of the HCV gene enhances its expression.

Combination therapy with lamivudine and HB vaccine on chronic hepatitis B.

BACKGROUND AND AIM: Lamivudine (LAM) has problems of breakthrough hepatitis (BTH) and post-treatment relapse despite its significant effect for suppressing hepatitis B virus (HBV) replication. In order to find solutions for the problems, the efficacy of combination therapy of LAM plus hepatitis B (HB) vaccine in patients with chronic HBV infection was assessed. PATIENTS AND METHODS: Fifty-three patients with chronic hepatitis B, 33 hepatitis B e-antigen positive (HBeAg+), and 20 HBeAg negative (HBeAg-) patients, were enrolled in the study, and randomized to receive either LAM monotherapy or combination therapy of LAM and HB vaccine. In the combination therapy group, 100 mg/day of LAM was administered as a baseline therapy, and 10 mug of HB vaccine was injected subcutaneously every month starting at 2 months after LAM administration, six times in total. RESULTS: HBeAg negative patients responded well to LAM therapy, and there were no significant differences in short-term effects between the two therapy groups. With regard to the ratio of developing BTH, there was no difference betweenthe two groups. In HBeAg+ patients, HBV replication was suppressed more efficiently in the combination therapy group than in the monotherapy group. The ratio of developing BTH was significantly lower in the combination therapy group than in the monotherapy group. Regardless of HBeAg serologic status or therapy protocols, post-treatment relapse was seen in most patients when the administrations of LAM were discontinued. No adverse effect with the use of HB vaccine was observed in all the patients treated with the combination therapy. CONCLUSION: Combination therapy of LAM and HB vaccine is a safe and effective way to control HBV replication and prevent the development of BTH especially in patients with high viral load. However, further study is required in order to achieve the continuous suppression of HBV replication even after cessation of LAM.

Usefulness of live 3D echocardiography during radiofrequency ablation in a case of hepatocellular carcinoma.

We report a case of hepatocellular carcinoma (HCC) with chronic hepatitis C virus (HCV) infection, which was successfully treated with percutaneous radiofrequency ablation (RFA) under live three-dimensional (3D) echocardiography guidance. Recently, it was reported that live 3D echocardiography was able to enhance the efficacy of catheter-based endomyocardial injection since the 3D images made it possible to observe the target from multiple directions so that it guided more accurately. A 63-year-old Japanese man had an HCC nodule of 3.0 cm in diameter at the S8 region of the liver. Here we applied live 3D echocardiography during RFA therapy with a LeVeen needle electrode. The echocardiography guidance allowed for easier and accurate approach for needle puncture. The guidance was also effective to confirm whether an enough safety margin for the nodule was obtained. Thus, live 3D echocardiography appears to safely guide RFA needles by accurate targeting for HCC nodule, providing real-time visualization when combined with echocardiography contrast.

Double filtration plasmapheresis and interferon combination therapy for chronic hepatitis C patients with genotype 1 and high viral load.

AIM: The efficacy and safety of double filtration plasmapheresis (DFPP) plus interferon (IFN) combination therapy were compared with those of IFN therapy alone in 193 chronic hepatitis C patients having a high hepatitis C virus ribonucleic acid load of difficult-to-treat genotype 1b. METHODS: All patients received either interferon alpha-2b (IFN-alpha-2b) monotherapy or combination therapies with ribavirin and IFN-alpha-2b or pegylated interferon alpha-2b (PEG-IFN-alpha-2b). Each patient individually decided whether to receive concomitant DFPP. DFPP was immediately followed by IFN treatment, and up to five sessions were given during the first week. RESULTS: Sixty patients decided to receive DFPP. In the DFPP plus PEG-IFN-alpha-2b therapy group (n = 30), viral load reduction at 4 weeks after the start of treatment was greater than innon-DFPP (n = 74) (2.47 vs 1.52, log, P = 0.010), and the sustained virus response was also higher (77.8% vs 50.0%), even in cases of re-treated patients (relapsers or non-responders to previous IFN therapies). Adverse events, mild and transient, were observed in 38.3% of all DFPP-treated patients. CONCLUSION: DFPP plus IFN combination therapy produced a great reduction of viral load during the early stage of treatment and achieved a high sustained virus response, suggesting that this combination therapy may be a new modality for chronic hepatitis C patients at difficult-to-treat states.

Noninvasive assessment of liver disease - measurement of hepatic fibrosis using tissue strain imaging.

The development of hepatic fibrosis in patients with liver disease is associated with an increased risk of liver cancer. Assessing the degree of hepatic fibrosis is therefore one of the most important factors in treatment planning. Liver biopsy is commonly performed to assess hepatic fibrosis, but this method is associated with complications such as hemorrhage. Recently, a number of studies on the noninvasive assessment of hepatic fibrosis have appeared in the literature. The present study was conducted to determine whether an easily performed myocardial examination technique can also be applied to the assessment of hepatic fibrosis. The statistical software Minitab, which performs hypothesis testing based on the P value, was used for statistical analysis. The mean strain values were 0.26 in the normal adult group, 0.155 in the chronic hepatitis group, and 0.058 in the cirrhosis group. Statistically significant differences were observed between the groups. The results of the present study suggest that noninvasive tissue strain imaging may become the method of choice for assessing hepatic fibrosis in routine clinical practice.

Effect of vitamin K2 on the recurrence in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Vitamin K2 (VK2) appears to have a potent inhibitory activity for cell growth including HCC cells. We investigated whether VK2 could reduce incidence of tumor recurrence after treatment of HCC. Forty-five patients with cured or possibly cured HCC were randomly selected, assigning patients to treatment (n=21) or control group (n=24) with randomization list. METHODOLOGY: For the treatment group, forty-five mg of Glakay was given orally every day after therapy for HCC. No patients complained of adverse effects. Abdominal ultrasonography and dynamic CT were performed at 3-month intervals. Recurrence was confirmed by abdominal angiography. RESULTS: Recurrence of HCC occurred in 7 cases (33.3%) for the treatment group and 12 cases (50.0%) for the control group during mean observation periods of 19.5 and 16.5 months, respectively. Administration of VK2 was not an independent variable for the recurrence on univariate analysis. Cumulative incidence of HCC recurrence did not differ between the two groups, and the cumulative survival rate tended to be high in treatment group (p =0.054). Cox regression analysis revealed that serum albumin concentration alone was an independent factor affecting the recurrence. CONCLUSIONS: These findings suggest that VK2 does not appear to prevent recurrence of HCC after curative treatment. Our study is preliminary and large-scale trials are needed to determine whether VK2 is of benefit to decrease the recurrence of HCC.

Four-dimensional ultrasonography for therapeutic radiofrequency ablation for hepatocellular carcinoma.

BACKGROUND/AIMS: We examined whether four-dimensional real-time flow imaging on ultrasonography (US) is valuable to display the accurate position of percutaneous radiofrequency ablation (RFA) needle in the nodule of hepatocellular carcinoma (HCC). METHODOLOGY: Ten patients with 12 HCC nodules were studied; nine were infected with hepatitis C virus (HCV) and one was diagnosed as non-B non-C. Diagnosis was done by helical dynamic CT and/or celiac angiography. Tumor vascularities in the early arterial and post-vascular phases after injection of a microbubble contrast agent were assessed by real-time US scanning of coded harmonic imaging and intermittent interval-delay scanning with a wide-band power Doppler technology. Percutaneous RFA was performed with four-dimensional real-time flow imaging under US to display the accurate position of cool-tip needle. RESULTS: It was possible to obtain accurate position of the needle during RFA procedure in all 12 nodules. The needle was confirmed to be inserted into the center of the tumor nodule by various angles. The simultaneous study before RFA therapy showed the inflow of arterial blood and tumor staining in all nodules at early arterial phase of coded harmonic angio on contrast-enhanced US scan. Posttreatment study to evaluate the therapeutic efficacy showed no blood flow at both early vascular and post-vascular phases. No residual blood flow was noted on early phase of CT scan with adequate safety margin. There was no discrepancy in the finding at early phase between contrast-enhanced US and dynamic CT. CONCLUSIONS: It appeared that four-dimensional real-time US provided more perceptible information on the spatial relationship between RFA needle and the target lesion, and resulted in accurate therapeutic efficacy for percutaneous RFA procedure.

Spatial and chronological differences in hepatitis B virus genotypes from patients with acute hepatitis B in Japan.

Genotypes of hepatitis B virus (HBV) were determined in 485 patients with acute hepatitis B from all over Japan. They were A in 92 (19%), Ba in 26 (5%), Bj in 32 (7%), C in 330 (68%) and D in 5 (1%). Sexual contacts were the main route of transmission in them. Overall, HBV persisted in only 5 of the 464 (1%) followed patients. Genotypes C accounted for more than 68% in northern as well as southern areas, contrasting with genotype A accounting for 34% in and around the Metropolitan areas. During 24 years from 1982 to 2005, genotype A increased from 5% to 33%, while genotype B gradually decreased from 26% to 8%. Fulminant hepatitis was significantly more frequent in infection with genotype Bj (41%) than those with the other genotypes (p<0.01). The core-promoter double mutation (T1762/A1764) and precore stop-codon mutation (A1896) were more frequent in patients with fulminant than acute self-limited hepatitis (57% versus 15% and 58% versus 10%, respectively, p<0.01 for both). In conclusion, genotype A distributes unevenly over Japan, prevails in younger patients through sexual transmission and has increased with years. Furthermore, fulminant outcome was more frequent in patients with genotype Bj than those with the other genotypes.