Precore Mutant Hepatitis B Virus Research Articles

Distinct disease features in chimpanzees infected with a precore HBV mutant associated with acute liver failure in humans.

Transmission to chimpanzees of a precore hepatitis B virus (HBV) mutant implicated in acute liver failure (ALF) in humans did not cause ALF nor the classic form of acute hepatitis B (AHB) seen upon infection with the wild-type HBV strain, but rather a severe AHB with distinct disease features. Here, we investigated the viral and host immunity factors responsible for the unusual severity of AHB associated with the precore HBV mutant in chimpanzees. Archived serial serum and liver specimens from two chimpanzees inoculated with a precore HBV mutant implicated in ALF and two chimpanzees inoculated with wild-type HBV were studied. We used phage-display library and next-generation sequencing (NGS) technologies to characterize the liver antibody response. The results obtained in severe AHB were compared with those in classic AHB and HBV-associated ALF in humans. Severe AHB was characterized by: (i) the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies with a significantly shorter incubation period, compared to classic AHB; (ii) earlier HBsAg clearance and anti-HBs seroconversion with transient or undetectable hepatitis B e antigen (HBeAg); (iii) limited inflammatory reaction relative to hepatocellular damage at the ALT peak with B-cell infiltration, albeit less extensive than in ALF; (iv) detection of intrahepatic germline antibodies against hepatitis B core antigen (HBcAg) by phage-display libraries in the earliest disease phase, as seen in ALF; (v) lack of intrahepatic IgM anti-HBcAg Fab, as seen in classic AHB, but at variance with ALF; and (vi) higher proportion of antibodies in germline configuration detected by NGS in the intrahepatic antibody repertoire compared to classic AHB, but lower than in ALF. This study identifies distinct outcome-specific features associated with severe AHB caused by a precore HBV mutant in chimpanzees, which bear closer resemblance to HBV ALF than to classic AHB. Our data suggest that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease.

Hepatitis B virus depicts a high degree of conservation during the immune-tolerant phase in familiarly transmitted chronic hepatitis B infection: deep-sequencing and phylogenetic analysis.

When intrafamilial transmission of hepatitis B virus (HBV) occurs, a virus with the same characteristics interacts with diverse hosts' immune systems and may thus result in different mutations to escape immune pressure. In this study, the HBV genomic characterization was assessed longitudinally after intrafamilial transmission using nucleotide sequence data of phylogenetic and mutational analyses, including those obtained by deep-sequencing for the first time. Furthermore, HBeAg-anti-HBe profile and variability of HBV core-derived epitopes were also evaluated. Strong evidence was obtained from intrafamilial transmission of HBV genotype D1 by phylogenetic inferences. HBV isolates exhibited high degree (~99%) of genomic conservation for almost 20 years, when patients were persistently HBeAg positive with normal amino transferase levels. This identity remained high among immune-tolerant siblings. In contrast, it diminished significantly (P = 0.02) when the mother cleared HBeAg (immune clearance phase). By deep-sequencing, the quantitative analysis of the dynamics of basal core promoter (BCP) (A1762T, G1764A; A1766C; T1773C; 8-bp deletion; and other) and precore (G1896A) variants among HBV isolates from family members exhibited differences during the follow-up. However, only those from the mother showed amino acid variations at core protein that would impair their MHC-II binding. Hence, when intrafamilial transmission occurs, HBV was highly conserved under the immune-tolerant phase, but it exhibited mutations more frequently during the immune clearance phase. The analysis of the HBV BCP and precore mutants after intrafamilial HBV transmission contributes to a better understanding of how they evolve over time.

Detection of Hepatitis B Virus Pre-core Mutant by Allele Specific Polymerase Chain Reaction

Introduction : Mutation in pre-core region is characterized by negative HBeAg and positive anti-HBe despite active replications of the virus. The mutation has diagnostic and prognostic implications. Therefore, detection of pre-core mutant is important. Standard diagnosis approach for detecting pre-core mutant is through DNA sequencing of hepatitis B virus (HBV) pre-core region. Unfortunately, DNA sequencing is not available in most centers. Hence, a simpler diagnostic approach is necessary. Method : An observational-analytic design study was performed. Detection of pre-core mutant was conducted in individuals with positive HBsAg and HBV DNA that had various patterns of HBeAg and anti HBe. HBsAg, HBeAg and anti-HBe was detected using immunochromatography technique. The HBV DNA was evaluated by using qualitative polymerase chain reaction (PCR) testing. PCR was done by three rounds of amplification with primers derived from wild type pre-core and mutant pre-core. Results : Of 25 sera with HBeAg negative, anti-HBe positive and HBV DNA positive, allele specific (AS) PCR pre-core mutant was detected in 20 (80%) sera. Two sera with HBeAg negative, anti HBe negative and HBV DNA positive were negative for pre-core mutant. Of 8 sera with HBeAg positive, anti HBe negative and HBV DNA positive, pre-core mutant was detected in 2 (25%) sera. Conclusion : Most of individuals with HBV DNA positive, HBeAg negative and anti-HBe positive have harbored pre-core mutant. The finding indicated that all patients with HBsAg positive, HBV DNA positive and HBeAg negative, but anti-HBe positive should be examined for the presence of pre-core mutant. Pre-core mutant is also found in HBeAg positive individual. Keywords : HBV, pre-core mutant, polymerase chain reaction

Molecular analysis of hepatitis B virus associated with vaccine failure in infants and mothers: A case–control study in Thailand

Perinatal transmission of hepatitis B virus (HBV) has been controlled incompletely despite adequate immunoprophylaxis in infants. The aim of this study was to characterize virological factors of HBV associated with vaccine failure in Thailand. Sera of 14 infected infants (13 HBeAg-positive and one HBeAg-negative) with vaccine failure and their respective mothers (group M1) were tested quantitatively for HBV DNA by real-time PCR, HBV genotypes and mutations were characterized by direct sequencing. Sera collected from 15 HBeAg-positive (group M2) and 15 HBeAg-negative (group M3) mothers whose infants had been vaccinated successfully served as controls. The results showed that group M1 and group M2 mothers had equal titers of HBV DNA but higher titers than group M3. All infected infants and their respective mothers had the same HBeAg status and HBV genotypes. DNA analysis in a pair of HBeAg-negative infant and mother revealed that both were infected with an HBV precore mutant (G1896A). Escape mutants in the "a" determinant region (residues 144 and 145) were detected in two (14%) infected infants. The prevalence of BCP mutations/deletions in groups M2 and M3 was higher significantly than in group M1 (P = 0.022 and P < 0.001, respectively). In conclusion, instead of the HBeAg status, a high titer of HBV DNA in mothers was the major contributor to perinatal transmission of HBV. Escape mutants might be associated with vaccine failure in some infants. BCP mutations/deletions in mothers might contribute to the prevention of mother-to-infant transmission of HBV.

HBV genotypes prevalence, precore and basal core mutants in Morocco

The study of hepatitis B virus (HBV) genomic heterogeneity has become a major issue in investigations aimed at understanding the relationship between HBV mutants and the wide spectrum of clinical and pathological conditions associated with HBV infection. The objective of the current study was to find out the pattern of HBV genotypes circulating in Morocco and to investigate the precore (PC) and basal core promoter (BCP) mutants’ status in Moroccan chronic hepatitis B patients. Viral genotypes were determined in 221 chronic carriers using INNO-LiPA HBV assay and hemi-nested PCR. Phylogenetic analysis was performed in 70 samples, and multiplex PCR method was used to confirm some genotyping results. PC and CP mutants were determined using Inno-Lipa. All isolates were successfully genotyped. The genotype distribution was D in 90.45% of cases, A (5.9%), E (1 case), and mixed genotypes (5 A/D and 2 D/F) in 3.17% patients. HBV carried in the HBV/D samples could be assigned to D7 (63.3%), D1 (32.7%) and 2% of strains to each D4 and D5, all HBV/A belonged to A2 subgenotype and HBV/E strain could not be sub-genotyped. In 70 studied strains, HBV mutants were detected in 88.6% of cases; PC mutants were detected in (40%) of patients and 21.5% present a mixture of wild type and G1896A mutation. BCP mutants were observed in 65.7% of cases, 22.9% were found to have the T1762/1764A double mutation, 18.6% had A1762/1764T mutation and 22.9% of patients showed the A1762T/G1764A double mutation with either A1762T/G1764T mutation. Co-infection by PC and BCP mutants was detected in 52.9% of cases.Movement from place to place most likely shapes the observed genotype distribution and consequent prevalence of genotypes other than A2 or D7 in this population. High circulation of PC and BCP mutants is common in chronic hepatitis B infection in Morocco.

Quantification of complex precore mutations of hepatitis B virus by SimpleProbe real time PCR and dual melting analysis

Background Hepatitis B virus (HBV) precore G1896A mutation is associated with Hepatitis B e antigen (HBeAg) seroconversion. This mutation and the adjacent G1899A mutation also appear to associate with increased risk of hepatocellular carcinoma. Quantitative mutant dynamics may help determine the potential of these mutants as clinical biomarkers. However, a reliable method to quantify either mutant is not available, partly because the viral genome has polymorphisms in general and the precore mutations are complex. Objectives (1) To develop a reliable and ultrasensitive assay for the quantification of HBV G1896A and/or G1899A mutants. (2) To obtain preliminary data on the quantities of the precore mutants in patients. Study design A SimpleProbe real time PCR assay was developed to quantify the HBV precore mutants. Dual melting analysis and a primer-probe partial overlap approach were used to increase detection accuracy. A wild-type selective PCR blocker was also developed to increase mutant detection sensitivity. Results The assay correctly identified the precore sequence from all 62 patient samples analyzed. More than 97% of precore sequences in the GenBank can be recognized. Mutant detection sensitivity reached 0.001% using a wild type-selective PCR blocker. At least one precore mutant can be detected from all 20 HBeAg-positive individuals who were negative for precore mutations by DNA sequencing. Conclusions The reliability of this ultrasensitive mutation quantification assay was demonstrated. The same approaches may be useful for the detection of other clinically significant mutations. Evolution of the precore mutants warrants further studies.

Nosocomial outbreak of hepatitis B virus infection involving two hospitals in the Republic of Ireland

The routes of nosocomial hepatitis B virus (HBV) transmission have changed over the years. Initiatives to prevent transfusion-associated HBV and healthcare worker-to-patient transmission have had a positive impact on these transmission routes. Recent reports of outbreaks of nosocomial HBV have implicated breaches in standard precautions as important causes of HBV transmission. This report describes a nosocomial outbreak of HBV infection in the Republic of Ireland, which occurred between January 2005 and March 2006. The outbreak was detected following identification of a case of acute HBV infection in a patient whose only risk factor was a recent surgical procedure. The extensive multi-agency investigation that followed revealed that the patient was one of five cases of acute HBV infection and that four separate transmission events between infectious cases had occurred in two different hospitals over a 15-month period. A definitive cause for each transmission event was not identified, although lapses in adherence to standard precautions, safe injection and phlebotomy practices could not be ruled out. Two secondary cases of acute HBV infection in community contacts of two of the nosocomial cases were identified. Phylogenetic analysis proved a useful tool in confirming infection with a pre-core HBV mutant and viral transmission between the seven patients. Apatient notification exercise involving 1028 potentially exposed patients found no evidence of additional cases of nosocomial HBV infection. These findings highlight the importance of consistent application of standard precautions.

Analysis of carriers of hepatitis B virus from a tertiary referral hospital: Does the viral load change during the natural course of infection?

This study was designed to determine the prevalence, forms of transmission, mutational profile and viral load at baseline of hepatitis B virus (HBV) carriers in Delhi. HBV surface antigen (HBsAg)-positive patients were enrolled and evaluated clinically for liver function, serological markers for hepatitis B and HBV DNA quantitation. Tests were carried out again 1 year later and the results were compared. Liver biopsy was carried out on all carriers with active viral replication. HBV DNA-positive samples were subjected to polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) to screen mutations in the Precore, core, and the X-gene prior to sequencing analysis. Among the 100 patients examined, HBeAg was detected in 23% and 40% were HBV DNA-positive. Of the 40 HBV DNA-positive cases, 8 had precore/core mutations, [G1896A (10%), T2066A (12.5%), T2050C (10%), and G1888A (7.5%)]. No X gene mutants were detected. Reduction in viral load was higher in HBeAg-positive patients, as compared to HBeAg-negative patients, over 1 year. At follow-up, 2/8 HBV mutants corresponded with altered liver function and morphological changes suggestive of chronic hepatitis. One patient was re-designated as DNA-negative on follow-up and had wild-type virus infection with a relatively low viral load. The predominant route for HBV transmission was determined to be parenteral. Twenty percent of the HBV carriers were infected with precore and core mutant HBV. Although the clinical and biochemical profiles of these HBV carriers remained largely stable on follow-up, there was evidence of spontaneous reduction in the mean viral load over the 1-year study period.

Downregulation of HLA Class II Molecules by G1896A Pre-Core Mutation in Chronic Hepatitis B Virus Infection

Over the past decade, increasing attention has been focused on the contribution of naturally occurring mutations in the hepatitis B virus (HBV) genome to the clinical course of the chronic infection. The aim of this study was to investigate the effect of the HBV pre-core mutation G1896A on the expression of HLA class II molecules and the core protein of hepatitis B in liver biopsies of chronic hepatitis B (CHB) infection. In 30 HBeAg-negative CHB patients the pre-core region of the HBV genome was amplified and sequenced to determine the presence of the mutation G1896A. Liver biopsies were scored based on the Histology Activity Index (HAI) system and immunohistochemistry (IHC) was performed to study the expression of HLA class II molecules on the antigen-presenting cells and the core protein in hepatocytes. We found that 19 of the 30 patients (63%) harbored the G1896A mutation. Compared to the patients without this mutation, those with G1896A had lower HAI scores (5.0 +/- 2.8 versus 7.9 +/- 4, p = 0.03). The study of the expression of HLA-II molecules in our patients revealed that subjects with the G1896A mutation had lower expression of HLA-II compared to wild-type infected subjects (1.87 +/- 0.6 versus 3.27 +/- 1.5, p < 0.01). Core protein expression was present in four patients (13.3%) who had higher HBV DNA levels than patients without core protein expression (3.81 +/- 0.78 versus 2.02 +/- 0.16, p < 0.001). These results suggest that the G1896A pre-core mutation may directly interfere with antigen presentation, most likely by decreasing the availability of HLA class II molecules, and this may result in less aggressive liver disease in HBeAg-negative CHB infection.

Long-term (7-year-) treatment with lamivudine monotherapy in HBV-associated glomerulonephritis

Glomerulonephritis (GN) is an uncommon but well-described complication of chronic hepatitis B virus (HBV) infection. HBV-related membranous nephropathy (MN) may lead to renal failure in 1/3 of the patients and spontaneous remission is rare. There is no standard therapy for HBV-associated MN. We report a case of a 28-year-old man with HBV-associated MN due to pre-core HBV mutant with complete remission under an ongoing 7-year lamivudine monotherapy.

Role of genotype and precore/basal core promoter mutations of hepatitis B virus in patients with chronic hepatitis B with acute exacerbation

Objctive. The results of long-term, follow-up studies show that the severity and frequency of acute exacerbation of chronic hepatitis B virus (HBV) are associated with the development of liver cirrhosis in chronic HBV infection. The aim of this study was to investigate the relationship between virological factors of HBV and the severity of acute exacerbation. Material and methods. Fifty-one chronic hepatitis B patients with symptomatic acute exacerbation without antiviral therapy were enrolled in the study. Genotype of HBV was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Precore (A1896) and basal core promoter (BCP) mutations (T1762 & A1764) were determined by PCR and direct sequencing. Results. Thirty-nine patients had genotype B, 11 patients had genotype C, and 1 patient had an unclassified genotype. Thirty-two patients had precore mutation and 24 patients had BCP mutation. After adjusting for age, gender, aspartate aminotransferase (ASAT) level, albumin level, and platelet count by multiple logistic regression test, precore mutation had a protective effect on the occurrence of hepatic decompensation (p=0.046), and genotype and BCP mutations were not associated with the occurrence of hepatic decompensation. Conclusions. HBV precore mutation may confer less severe liver disease during acute exacerbation of chronic HBV. Genotype and BCP mutations did not have a significant association with the occurrence of hepatic decompensation.

Navigating the Maze of Hepatitis B Treatments

Navigating the Maze of Hepatitis B Treatments

Hepatitis B virus mutation in children

Due to the lack of proof reading activity of hepatitis B virus (HBV) polymerase, mutation/variation of the viral sequence is frequently found during long term follow-ups. In the majority of children with chronic HBV infection, wild type HBV is the dominant viral strain during the natural course of chronic HBV infection. During long-term follow-up, HBV precore mutants developed spontaneously in approximately 10 to 24% of children before HBeAg seroconversion and in around 50% of children after HBeAg seroconversion mutants. Occasionally, children may be infected primarily by mutant strains of HBV. Approximately 36% of children with fulminant hepatitis and 30% of children with acute hepatitis B were infected by precore mutants of HBV transmitted by their mothers or blood donors. In addition, after universal HBV vaccination, HBV surface gene variants emerge or are selected under the immune pressure generated by the host or by administration of hepatitis B immune globulin and hepatitis B vaccination. In HBV DNA positive children from four sequential surveys in Taiwan, the prevalence of hepatitis B surface gene a determinant mutants increased from 7.8% before the vaccination program, to 19.6%, 28.1% and 23.1% at 5, 10 and 15 years after the program. Nucleoside analogue may also induce mutant strains, which reduces the antiviral effects. The most common example is the YMDD mutation of the HBV polymerase gene after antiviral therapy with lamivudine. It developed in 19% of the treated children. In conclusion, children may be infected primarily by mutant strains of HBV either naturally during acute HBV infection. Those infected with wild type HBV initially may develop mutant strains gradually during the course of chronic infection under the host immune pressure. Vaccine escape mutants may develop after immunoprophylaxis. In addition, antiviral therapy with nucleoside analogues may also induce drug resistant mutant strains. Understanding the viral mutation status will help to design accurate strategies of immmunoprophylaxis and antiviral therapy against HBV infection.

Performance of sequence analysis, INNO‐LiPA line probe assays and AFFIGENE assays in the detection of hepatitis B virus polymerase and precore/core promoter mutations

In this study, we compare results obtained by sequences analysis and commercial kits in the detection of hepatitis B virus (HBV) polymerase and precore (PC) and core promoter mutations. A total of 23 serum samples from lamivudine treated patients were tested for polymerase mutations by direct sequencing, INNO-LiPA HBV DR and AFFIGENE HBV DE/3TC. Full concordance among the three assays was observed in 63% of the total analysed codons. Concordant results were obtained between sequencing and LiPA in 80%, between sequencing and AFFIGENE in 73% and between LiPA and AFFIGENE in 74% of all tested codons. All discrepancies were observed in mixed population samples in which AFFIGENE and LiPA detected additional viral variants not revealed by sequence. In two patients, with serial samples, LiPA detected earlier than sequence and AFFIGENE an emerging mutate strain. PC and core promoter viral variants were detected in 28 serum samples collected from 14 HBV inactive carriers and from 14 hepatitis B patients with chronic liver disease. Direct sequencing, INNO-LiPA HBV PreCore and AFFIGENE HBV MUTANT VL 19 showed fully coincident results in 88% of tested positions. These findings showed that all assays evaluated were sensitive and accurate tools to analyse HBV genomic variability. Sequence analysis is essential to study new emerging mutations as LiPA and AFFIGENE assays are more easily useful in clinical laboratories to detect the appearance of well-characterized HBV variants.

Significance of hepatitis B virus genotypes A to E in a cohort of patients with chronic hepatitis B in the Seine Saint Denis District of Paris (France)

The aim of this study was to examine the genetic variability of the hepatitis B virus (HBV) and its significance. HBV genotypes, core promoter and precore mutants were characterized in 109 consecutive patients with biopsy-proven HBV chronic hepatitis. Genotypes A (26.6%), B (12.8%), C (18.3%), D (18.3%), and E (14.7%) indicate a wide genotypic distribution. Patients were from Asia (30.3%), Europe (28.4%), Sub Saharan Africa (23.9%), the Caribbean (10.1%), North Africa (5.5%), and Madagascar (1.8%). HBV genotypes A and D (HBV/A and /D) infected all subgroups except Asian patients. HBV/B or /C were found in 97% of Asian patients, whereas HBV/E only infected sub-Saharan African and Caribbean patients. Differences according to genotypes were: an increased prevalence of anti-HBe antibodies in patients infected with HBV/D (P = 0.003), higher serum transaminases in patients infected with HBV/A and/D (P = 0.043), more severe liver fibrosis in patients infected with HBV/A, /C and/D (P = 0.02). Precore and core promoter mutants were found in 87% of anti-HBe positive patients, and were associated with HBV/D (P = 0.04) and severe liver fibrosis (P = 0.002). It is concluded that HBV genotypes A, B, C, D, and E circulate in the Seine Saint Denis District, reflecting the geographical origin of patients. HBV/A, /C and/D seem to be associated with more severe hepatic disease.

P.024 Analysis of surface promoter mutants of HBV: its relation to hepatitis B surface antigen production and secretion

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAg], anti-hepatitis B e antigen [HBeAg], and HBV DNA) who underwent orthotopic liver transplantation for endstage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G → A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCH phase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S1, pre-S2, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrence of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.

Diagnosis, prevention and management of hepatitis B virus reactivation during anticancer therapy

Diagnosis, prevention and management of hepatitis B virus reactivation during anticancer therapy

Hepatic necroinflammation and fibrosis in patients with genotypes Ba and C, core‐promoter and precore mutations

The role of infection with hepatitis B virus (HBV) genotypes on liver histology is largely unknown. The aim of study was to investigate the relationships between HBV genotypes (B, C), core-promoter (CP) and precore mutants and liver histology in 66 patients. Liver biopsies were scored by histologic activity index (HAI). HBV genotypes were determined by enzyme-linked immunosorbent assay (ELISA). Eighteen (27.3%) and 48 patients (72.7%) had genotype B (all were subtype Ba) and C, respectively. Forty-seven (71.2%) and 27 (40.9%) had CP and precore mutations, respectively. Patients with genotype C compared with subtype Ba had higher median scores of HAI-necroinflammation (HAI-NI) (7 vs 3), HAI-fibrosis (HAI-F) (1 vs 0) and total HAI (8.5 vs 3) (all P < 0.03). Patients with CP mutations compared with wild-type had higher median scores of HAI-NI (7 vs 3), HAI-F (3 vs 0) and total HAI (9 vs 3) (all P < 0.03). Forty patients (83.5%) with genotype C had CP mutations. Age and alanine aminotransferase levels were positively correlated with HAI scores while albumin levels were negatively correlated (P < 0.01 for all, except albumin levels and HAI-F, P = 0.08). There was no association between precore mutations and HAI scores. Multivariate analysis indicated that higher alanine aminotransferase (ALT) levels were associated with higher HAI scores (P < 0.04) and CP mutations were associated with higher HAI-NI (P = 0.034), but not with HAI-F score (P = 0.3). CP mutations were associated with more severe necroinflammation. The association between genotype C and poor histology was probably because of the association between genotype C and CP mutations.

Hepatitis B virus genotypes and extrahepatic manifestations

This study aimed at correlating the presence of extrahepatic manifestations with hepatitis B virus (HBV) genotypes in patients with chronic HBV infection. This was a national (France), multicenter, retrospective, cross-sectional study. HBV genotypes were determined in 190 patients HBsAg-positive for at least 6 months and documented before any treatment. Patients were aged 42+/-15 years and mainly male (77%). Alcohol intake was high in 6% of them, ALT elevated in 73%; 27% were cirrhotic. All HBV genotypes were found, mainly A (24%), D (29%), C (11%), and E (10%). Thirty (16%) patients had clinical extrahepatic manifestations, mainly sensory-motor deficiency, sicca syndrome, myalgia, glomerulonephritis, and arthralgia-arthritis. Their presence was not related to any epidemiologic, viral (including genotypes) or hepatic factor, but to a higher platelet count (P=0.004). Twenty-nine (15%) patients had biological extrahepatic manifestations, mainly anti-smooth muscle, antinuclear, and anti-nucleosome antibodies. Their presence was related only to anti-HBe antibodies positivity (P=0.007) or elevated platelet count (P=0.003). Carrying precore mutant HBV increased by 2.8 folds the risk to have at least one extrahepatic biological manifestation. No relationships between HBV genotypes and the presence of extrahepatic manifestations were evidenced in patients with chronic HBV infection.

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.