Prevalence, characteristics, and virologic correlations of hepatitis delta (D) among patients with hepatitis B surface antigen in Mongolia.

Clinical and biochemical features of hepatitis delta virus (HDV) infections in Mongolia remain largely unknown. We aimed to investigate the clinical characteristics of HDV patients in Mongolia using several markers. The 143 hepatitis B surface antigen (HBsAg)-positive patients were divided into 122 HDV-positive and 21 HDV-negative patients by HDV RNA positivity. Subgroup analysis was performed between hepatitis B e antigen (HBeAg)-positive and -negative HDV-positive patients. Liver function, quantitative HBsAg (qHBsAg), anti-HDV Immunoglobulin (Ig) M, Mac-2 binding protein glycosylation isomer (M2BPGi), hepatitis B virus (HBV) DNA level, and HDV RNA level were tested. HDV RNA was positive in 85.3% (122/143) of patients showing anti-HDV IgG. Liver disease activity was higher in HDV-positive patients than in HDV-negative patients. The HDV-positive group included a higher proportion of patients with high qHBsAg and M2BPGi levels (p < 0.001). The positivity rate for anti-HDV IgM was significantly higher in the HDV-positive group (p < 0.001). HDV RNA levels showed an inverse correlation with qHBsAg levels in HBeAg-positive-HDV-positive patients (r = -0.49, p = 0.034), and a positive correlation with qHBsAg levels in HBeAg-negative patients (r = 0.35, p < 0.001). Hepatitis B virus (HBV) DNA and HDV RNA levels did not show any correlation. M2BPGi levels likewise did not correlate with HDV RNA levels. A high positivity rate for HDV RNA was observed for HBV patients in Mongolia using the highly sensitive HDV RNA assay. The positivity rate for anti-HDV IgM was high in HDV RNA-positive patients. Severity of liver disease and M2BPGi levels were both high in the HDV RNA-positive group.

Hepatitis B virus RNA is measurable in serum and can be a new marker for monitoring lamivudine therapy.

BACKGROUND: Changes in the serum hepatitis B virus (HBV) RNA level during lamivudine therapy were compared to those in the serum HBV DNA and HBV core-related antigen (HBVcrAg) levels in 24 patients with chronic hepatitis B. METHODS: For measurement of HBV RNA, total nucleic acid was extracted from serum samples and treated with RNase-free DNase I. After cDNA synthesis from extracted RNA, HBV RNA was measured by real-time detection polymerase chain reaction. RESULTS: The peak fraction of HBV RNA in serum samples was consistent with peak fractions of HBV DNA and HBV core protein in a sucrose gradient analysis, indicating that HBV RNA was incorporated into virus particles. All levels of HBV DNA, HBV RNA, and HBVcrAg decreased gradually during lamivudine therapy (P < 0.001 for all). The amount of decrease from the start of lamivudine therapy was significantly higher for HBV DNA than for HBV RNA or HBVcrAg during 6 months of lamivudine therapy (P < 0.001 for all). However, a similar difference was not seen between HBV RNA and HBVcrAg levels during that period. The HBV RNA level was significantly correlated (P < 0.001 for all) with levels of HBV DNA and HBVcrAg both at the beginning and 2 months after the start of lamivudine therapy. CONCLUSIONS: HBV RNA is detectable in serum in a form indicating incorporation into virus particles, and its serum level might serve as a new viral marker with a significance different from that of HBV DNA in lamivudine therapy.

Fulminant hepatitis after allogenic bone marrow transplantation caused by reactivation of hepatitis B virus with gene mutations in the core promotor region.

Under immunosuppressive conditions after hematopoietic stem cell transplantation (HSCT), even if hepatitis B virus (HBV) antigen is negative but hepatitis B surface antibody (HBsAb) or hepatitis B core antibody (HBcAb) is presented, HBV reactivates and sometimes causes fulminant hepatitis. However, it remains unclear which patients will develop fulminant hepatitis, or whether fulminant hepatitis is caused by host-related factors or by virus-related factors. A 30-yr-old man with a history of aplastic anemia since 3 yr of age underwent allogenic BMT, when HBsAb and HBcAb were positive but HBs antigen (HBsAg) was negative. The donor was negative for HBsAg, HBsAb and HBcAb. After transplantation, the patient was complicated by acute graft-vs.-host disease (GVHD), cytomegalovirus infection, intestinal thrombotic microangiopathy and aspergillus colitis. Chronic GVHD was well controlled by FK506 and prednisolone. Twenty months after transplantation, the patient was admitted with general fatigue and liver dysfunction and was found to be positive for HBsAg and HBeAg. His serum HBV-DNA level was >8.8 log of the genome equivalent (LGE)/mL. Therefore, he was diagnosed as having hepatitis B caused by HBV reactivation and 100 mg/d lamivudine treatment was started. However, jaundice and hepatic failure deteriorated and became fatal. On analysis of the HBV-DNA, two adjacent gene mutations in the core promoter region (T1762/A1764) were detected. Increased replication of the mutated HBV might have caused HBV reactivation which progressed to fulminant hepatitis.

Patients with and without loss of hepatitis B virus DNA after hepatitis B e antigen seroconversion have different virological characteristics.

The characteristic differences between patients with and without loss of hepatitis B virus (HBV) DNA after achieving hepatitis B e antigen seroconversion were analyzed by comparing changes in HBV DNA and HBV core-related antigen levels during a period from 3 years before to 3 years after the seroconversion. Of the 24 seroconverters, 6 (inactive replication group) showed continuous loss of HBV DNA in serum after the seroconversion and the remaining 18 did not lose HBV DNA (active replication group). The HBV DNA level was similar between the two groups, while the HBV core-related antigen level was significantly lower in the active replication group than in the inactive replication group before the seroconversion. The levels of both HBV DNA and HBV core-related antigen decreased remarkably around the time of seroconversion in the inactive replication group, while these levels did not change or decreased slightly in the active replication group. After the seroconversion, the HBV DNA level was significantly higher in the active replication group than in the inactive replication group, while the HBV core-related antigen level was similarly low between the two groups. Because the serum level of HBV core-related antigen mainly reflects that of HBe antigen, the low level of HBV core-related antigen seen after seroconversion in both groups might have contributed to the occurrence of seroconversion. The precore and core promoter mutations which cause diminished excretion of hepatitis B e antigen were significantly more frequent in the active replication group than in the inactive replication group. It was therefore considered that the seroconversion was caused mainly by a decrease in viral replication in the inactive replication group, and mainly by a decrease in HBe antigen production in the active replication group.

Hepatitis B virus core and core-related antigen quantitation in Chinese patients with chronic genotype B and C hepatitis B virus infection.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) core-related antigen (HBcrAg) and HBV core antigen (HBcAg) assays were developed for the measurement of serum HBV load. The aim of this study was to assess the clinical utility of these assays in Chinese patients with chronic genotype B and C HBV infection. METHODS: One hundred and ninety-three chronic hepatitis B patients were enrolled. Serum HBcrAg and HBcAg were measured by chemiluminescence enzyme immunoassay, and HBV-DNA was measured by using a sensitive polymerase chain reaction assay. The data were analyzed in patients with HBV genotype B (HBV/B) and genotype C (HBV/C). The HBcrAg/HBcAg ratio was calculated and compared between patients with and without hepatitis B e antigen (HBeAg). RESULTS: The concentrations of HBcrAg and HBcAg showed significant positive correlation with the HBV-DNA concentration in both HBV/B (r = 0.79, P < 0.001, and r = 0.77, P < 0.001, respectively) and HBV/C (r = 0.87, P < 0.001, and r = 0.90, P < 0.001, respectively). The cut-off for a positive HBcAg corresponded to approximately 4.5 log copies/mL, and that for a positive HBcrAg result corresponded to 3-4 log copies/mL. The HBcrAg/HBcAg ratio was higher in patients with HBeAg than in those without HBeAg. CONCLUSIONS: The HBcrAg assay and HBcAg assay are clinically useful in viral quantitation of HBV/B and HBV/C. A combination of these assays would be a valuable tool for analyzing the clinical status of HBV infection.

Efficacy of lamivudine for preventing hepatocellular carcinoma in chronic hepatitis B: A multicenter retrospective study of 2795 patients.

A retrospective survey of Japanese patients histologically diagnosed with chronic hepatitis B was conducted to determine the effectiveness of lamivudine in preventing hepatocellular carcinoma (HCC). Of the 2795 patients who satisfied criteria for analysis after treatment from any of 30 medical institutions, 657 had received lamivudine and the remaining 2138 had not. A Cox regression model with liver biopsy as the starting point revealed seven factors related to HCC: lamivudine therapy, gender, family clustering of hepatitis B, age at liver biopsy, hepatic fibrosis stage, serum albumin level, and platelet count. In a matched case-controlled study, 377 patients in a lamivudine-treated group and 377 matched patients in a non-treated group were selected based on their propensity scores. The mean follow-up period was 2.7 years in the lamivudine group and 5.3 years in the control group. In the lamivudine group, HCC occurred in four patients (1.1%) with an annual incidence rate of 0.4%/(patient/year), whereas in the control group HCC occurred in 50 patients (13.3%) for a rate of 2.5%/(patient/year). A comparison of the cumulative HCC incidence between the two groups by the Kaplan-Meier method showed a significantly lower incidence of HCC in the lamivudine group (p<0.001). These findings suggest that lamivudine effectively reduces the incidence of HCC in patients with chronic hepatitis B.

Nucleotide mutations associated with hepatitis B e antigen negativity.

One hundred and forty four patients with chronic hepatitis B were tested to identify new mutations associated with hepatitis B e antigen (HBeAg) negativity, using a full genome sequence analysis. All the patients were Chinese and had hepatitis B virus infection of genotype C. Patients with none of the pre-core or core promoter mutations were significantly (P < 0.001) less common in the group with anti-HBe (13%) than in the group with HBeAg (56%). The complete nucleotide sequence was determined in four anti-HBe-positive patients who had neither pre-core nor core promoter mutations and in five HBeAg-positive patients who also had neither of these mutations (the groups were matched for age and sex). Six mutations were found to be significantly more common in the former group than in the latter: G529A (3/4 vs. 0/5), C934A (4/4 vs. 1/5), A1053G (4/4 vs. 1/5), G1915T/A (4/4 vs. 0/5), T2005C/A (4/4 vs. 0/5), and C3026T (3/4 vs. 0/5). Three of the six mutations were significantly more common in the four anti-HBe-positive patients who had neither pre-core nor core promoter mutations, compared to 11 HBeAg-positive patients who had pre-core and core promoter mutations, and also compared to 15 anti-HBe-positive patients who had pre-core and core promoter mutations, suggesting further the specificity of these mutations. Of the six mutations, two resulted in amino acid substitution in the polymerase protein, and one is located near the enhancer I region. The results suggest that the six newly discovered mutations are associated with HBeAg negativity.

Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain.

DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.

A case-control study for early prediction of hepatitis B e antigen seroconversion by hepatitis B virus DNA levels and mutations in the precore region and core promoter.

Factors influencing and predictive of seroconversion from hepatitis B e antigen (HBeAg) to antibody (anti-HBe) were sought in a case-control study of 61 patients with chronic hepatitis B who had been observed from 5 years before to 1 year after seroconversion, and 32 patients who did not seroconvert during the entire 6-year period. Almost all of the patients (96%) were infected with HBV genotype C. HBV DNA levels began to decrease 3 years before seroconversion in the seroconverters, while they remained high in the non-converters. The frequency of precore mutation and the loss of HBeAg (A1896) started to increase 1 year before in the converters, and became significantly higher at seroconversion (23 vs. 3%, P = 0.030) than that in the non-converters. Double mutation in the core promoter (T1762/A1764) was more common in the seroconverters than in the non-converters 5 years before seroconversion (48 vs. 28%), and became significantly more frequent at seroconversion (65 vs. 41%, P = 0.046). Seroconversion occurred in 75% of the patients with at least HBV DNA levels <5.5 logarithmic equivalents/mL; precore mutation in 20% or more of HBV DNA; or core promoter mutation. Seroconversion occurred in 50% of those patients within 1 year, 88% within 2 years, and 93% within 5 years. These results indicate that a decrease in HBV DNA levels and mutations in the precore region and the core promoter were associated significantly and complementarily with seroconversion, and each of them or a combination thereof was predictive of seroconversion years ahead.

Factors predisposing to the occurrence of cryoglobulinemia in two cohorts of Egyptian and Japanese patients with chronic hepatitis C infection: ethnic and genotypic influence.

The association between cryoglobulinemia and hepatitis C virus (HCV) infection has been reported. However, the factors underlying its wide variation of occurrence have not yet been well identified. To investigate this, cryoglobulinemia was studied in four cohorts of Egyptian and Japanese patients. Fifty Egyptian patients with chronic hepatitis C, infected with genotype 4 (the predominant HCV genotype in Egypt), were compared with 50 age- and sex-matched Japanese patients, infected with HCV genotype 1b (the predominant HCV genotype in Japan). Thirty-two Egyptian and 30 age- and sex-matched Japanese patients with chronic hepatitis B were included as controls. All patients were noncirrhotic. Antinuclear antibody (ANA), immunoglobulins (Ig), and cryoglubulins were assessed. Results showed a significantly higher prevalence of cryoglobulinemia in chronic hepatitis C Japanese genotype 1b (40%) as compared with Egyptian genotype 4 (14%), P = 0.003, while no difference was found between Japanese (17%) and Egyptian chronic hepatitis B controls (13%). Symptomatic cryoglobulinemia was more prevalent in the Japanese than in the Egyptian chronic hepatitis C group (10% vs. 4%), but the difference was not statistically significant. Univariate analysis showed no association between cryoglobulinemia and either age, sex, alanine aminotransferase level, or HCV viral load in Japanese or Egyptian patients, while the mean IgM level was significantly higher in the cryoglobulin-positive than in the cryoglobulin-negative chronic hepatitis C patients in each group (P = 0.003 and 0.017, respectively). Cryoglobulinemia was found to be significantly associated with both high IgG level (P = 0.020), and positive ANA (P < 0.001) in Japanese patients with chronic hepatitis C, genotype 1b but not in Egyptians with genotype 4. Multivariate analysis showed that the only factors predisposing to cryoglobulinemia were Japanese ethnicity with HCV genotype1b (P = 0.002, OR = 2.56), high IgM level of >245 mg/dl (P = 0.018, OR = 2.05) and female gender (P = 0.040, OR = 1/0.66). In conclusion, cryoglobulinemia is prevalent in Japanese patients with chronic hepatitis C infected with genotype 1b, but cryoglobulinemia is not common in Egyptians with HCV genotype 4. Although it was not possible to evaluate ethnicity and HCV genotype separately in this study, HCV genotype 1b appears to predispose more to cryoglobulinemia than does genotype 4. Female gender and high serum IgM level were also related.

New enzyme immunoassay for detection of hepatitis B virus core antigen (HBcAg) and relation between levels of HBcAg and HBV DNA.

A new enzyme immunoassay specific for hepatitis B virus (HBV) core antigen (HBcAg) was developed. In order to detect HBcAg, specimens were pretreated with detergents to release HBcAg from the HBV virion and disassemble it to dimers, and simultaneously, the treatment inactivated anti-HBc antibodies. HBcAg detected by the assay peaked with HBV DNA in density gradient fractions of HBV-positive sera. The assay showed a wide detection range from 2 to 100,000 pg/ml. We observed no interference from anti-HBc antibody or blood components, but the assay was inhibited by very high concentrations (>1 microg/ml; corresponding to 80 signal/cutoff) of HBeAg. When the cutoff value was tentatively set at 4 pg/ml, all healthy control (HBsAg and HBV DNA negative, n = 160) and anti-hepatitis C virus-positive (n = 55) sera were identified as negative. HBcAg concentrations correlated very closely with HBV DNA (r = 0.946, n = 145) in 216 samples from 72 hepatitis B patients. In seroconversion panels, HBcAg concentrations changed in parallel with HBV DNA levels. The assay, therefore, offers a simple method for monitoring hepatitis B patients. With a series of sera during lamivudine therapy, HBV DNA levels fell sharply and the HBcAg concentration also decreased, but the change in HBcAg was smaller and more gradual. The supposed mechanism of these changes and their clinical significance are discussed.

Development of small hepatocellular carcinoma in a patient with chronic hepatitis C after 77 months of a sustained and complete response to interferon therapy.

We report a case of hepatocellular carcinoma (HCC) that developed 77 months following sustained and complete response to interferon (IFN) therapy for chronic hepatitis C. A 67-year-old Japanese woman presented with a small mass in the liver that was diagnosed as HCC, 77 months after having completed IFN therapy and having shown a complete response to the therapy with sustained normalization of serum aminotransferases and eradication of serum hepatitis C virus (HCV). Hepatitis C virus RNA was also not detected in the resected tumorous and non-tumorous liver tissues by polymerase chain reaction. This suggests that all patients with chronic HCV infection should be followed closely for as long as possible for the potential development of HCC, even after a complete and sustained response to IFN treatment.

Sensitive enzyme immunoassay for hepatitis B virus core-related antigens and their correlation to virus load.

A sensitive enzyme immunoassay (EIA) specific for hepatitis B virus core antigen (HBcAg) and hepatitis B e antigen (HBeAg) was developed. We designated the precore/core gene products as hepatitis B virus (HBV) core-related antigens (HBcrAg). In order to detect HBcrAg even in anti-HBc/e antibody-positive specimens, the specimens were pretreated in detergents. The antibodies are inactivated by this pretreatment and, simultaneously, the antigens are released and the epitopes are exposed. The assay demonstrated 71 to 112% recovery using HBcrAg-positive sera. We observed no interference from the tested anticoagulants or blood components. When the cutoff value was tentatively set at 10(3) U/ml, all healthy control (HBsAg/HBV-DNA negative; n = 108) and anti-HCV antibody-positive (n = 59) sera were identified as negative. The assay showed a detection limit of 4 x 10(2) U/ml using recombinant antigen. Detection limits were compared in four serially diluted HBV high-titer sera. The HBcrAg assay demonstrated higher sensitivity than HBV-DNA transcription-mediated amplification (TMA) or HBeAg radio immunoassay (RIA) in the dilution test. HBcrAg concentrations correlated well with HBV-DNA TMA (r = 0.91, n = 29) and in-house real-time detection-PCR (r = 0.93, n = 47) in hepatitis B patients. On HBeAg/anti-HBe antibody seroconversion panels, the HBcrAg concentration changed in accordance with HBV-DNA levels. HBcrAg concentration provides a reflection of HBV virus load equivalent to HBV-DNA level, and the assay therefore offers a simple method for monitoring hepatitis B patients.

Clinical significance of T.T. virus infection in maintenance hemodialysis patients of an endemic area for hepatitis C infection.

Clinical significance of TTV infection was analyzed in Egyptian hemodialysis (HD) patients. Forty-seven Egyptian patients on maintenance HD and 50 age-matched volunteer blood donors were investigated. TT virus (TTV) DNA detection and genotyping were performed using a semi-nested polymerase chain reaction with specific primers. The prevalence of TTV DNA in patients on HD (66%) was significantly (P<0.001) higher than in blood donors (24%) with genotype 1b predominance (89%) in both. Clinical background including mean age, sex, history of blood transfusion, and positive markers for either hepatitis B virus (HBV) or hepatitis C virus (HCV) did not differ between TTV DNA positive and negative HD patients. However, the mean duration of HD was significantly (P=0.032) shorter in the TTV positive patients (28+/-19 months) than in the negative ones (45+/-34 months). Mean alanine aminotransferase level in patients with HCV infection alone (41+/-24 IU/l) did not differ from that in patients with both co-infection (33+/-28 IU/l), but was significantly higher than that in patients with TTV infection alone (26+/-10 IU/l). Occurrence of chronic hepatic changes in patients with TTV infection alone (7%) was significantly less common than those with HCV infection alone (100%, P<0.001) or those with both co-infection (100%, P<0.001). Serum level of HCV core protein was similar between patients with HCV infection alone and those with co-infection with TTV. In conclusion, the prevalence of TTV infection is high in Egyptian patients on regular HD, especially with shorter duration on HD. No clinical significance of TTV virus could be elicited in HD Egyptian patients; neither it showed any clinical impact as a co-infection with HCV.