RESUMO
BACKGROUND: Hepatitis B virus (HBV) infection is a public health problem in developing countries. HBV genotypes play major role in the evolution of infection since they were involved in different clinical presentations and response to treatment. OBJECTIVES: This study was conducted to evaluate the efficiency of restriction fragment length polymorphism (RFLP) analysis for HBV genotyping. PATIENTS AND METHODS: We investigated 98 samples collected from patients chronically infected with HBV. HBV genotypes were determined by analysis of patterns obtained after amplification in Pre-S region and digestion of the amplicon by two endonucleases AvaII and DpnII. Obtained results were confirmed by partial sequencing in the same region. RESULTS: Two different HBV genotypes were detected in this study, Genotype D (in 95. 9%) and Genotype A (in 4.1%). Seventy-four samples (75.5%) were successfully genotyped with RFLP analysis and all classified as genotype D. The remaining 24 samples (24.5%) which were un-genotyped by RFLP analysis, were classified by partial sequencing of the pre-S region as HBV genotype D (20 samples, 20.4%) and genotype A (4 samples, 4.1%). Atypical profiles were significantly associated with advanced liver disease (P = 0.001) as well as older age (P < 0.05). CONCLUSIONS: Several previous studies used PCR-RFLP to genotype HBV; however, we showed the high risk to obtain atypical profiles, especially in advanced stages of chronic infection, with as results difficulties to genotype the virus. These profiles resulted from the accumulation of mutations during natural course of infection resulting in a modification in restriction sites for enzymes. So, we recommended completing the investigation by partial sequencing to confirm obtained results.
RESUMO
Genetic variability of hepatitis B virus (HBV) in the C gene and its association with the different stages of chronic liver disease has been studied inadequately with controversial results. The objectives of the current study were to determine the frequency of core promoter and precore mutations in chronic hepatitis B in Tunisia and to evaluate their impact on viral replication and disease progression. Sequencing was performed in upstream regulatory sequence (URS), pre-core (PreC) and basal core promoter (BCP) regions for 123 chronic infected patients by HBV genotype D at different status of disease. Mutations were detected in 98.4% of cases, affecting URS, BCP and Pre-C in 95.1%, 95.9% and 87.8% respectively. Multi-mutations increased significantly from asymptomatic carrier to advanced liver disease status. G1896A (74.8%), G1764A/T/C (71.5%), G1899A (54.4%) and T1678C (52%) were the most common. Special attention should be paid to A1703T, T1678C/G-A1703T, and A1652G-A1679G mutations probably specific of Tunisians sequences; they were observed in 40.6%, 41.5% and 30.1% respectively. A1679G/C, T1753C/G/A, A1762T/G and A1762T-G1764A were more prevalent in older patients. High DNA levels were associated with G1899A or G1764T/C-C1766G-C1799G and advanced liver disease with mutations at positions 1762, 1764 and/or 1899 alone or in double or triple mutations. It was also shown that substitutions at nucleotides 1762, 1764 and 1899 have an impact on the disease progression. It is the first report for specific mutations in the URS region for genotype D. It should be completed by studying eventual correlation with clinical progression and the response to treatment.
