Title of article :
Evaluation of PCR-RFLP in the Pre-S Region as Molecular Method for Hepatitis B Virus Genotyping
Author/Authors :
Ouneissa, Rim Institute Pasteur de Tunis - Laboratory of Clinical Virology, Tunisia , Bahri, Olfa Institute Pasteur de Tunis - Laboratory of Clinical Virology, Tunisia , Yahia, Ahlem Ben Institute Pasteur de Tunis - Laboratory of Clinical Virology, Tunisia , Touzi, Henda Institute Pasteur de Tunis - Laboratory of Clinical Virology, Tunisia , Azouz, Mohamed Msaddak , Mami, Nabyl Ben , Triki, Henda Institute Pasteur de Tunis - Laboratory of Clinical Virology, Tunisia
Abstract :
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.
Keywords :
Hepatitis B Virus , Genotype , Restriction Fragment Length Polymorphism , Direct SequencingImplication for health policy , practice , research , medical education:The important objective of this work is to evaluate the efficacy of PCR , RFLP which was largely used for epidemiological studies because it’s easy to use especially in large , scale surveys. However, different points remain unclear , its real efficacy was not already proven. So, the impact of this study is very important while, HBV genotype is considered, nowadays, as an important marker to survey the evolution of chronic hepatitis B with or without treatment, and is also recommended for researchers who are interested in public health issues.Copyright © 2013, Kowsar Corp. , Published by Kowsar Corp. This is an open , access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.1. BackgroundHepatitis B virus (HBV) infection is the most common cause of chronic hepatitis disease with high risk of developing cirrhosis and hepatocellular carcinoma (HCC) (1). Compared to other conventional DNA viruses, HBV is characterized by complexity of its replication and high degree of genetic variability caused by an intermediate reverse , transcription step and a high level of viral releasing (1011 virions , day). Because of the lack of a 3 , 5 exonuclease activity, HBV DNA polymerase generates multiple and uncorrected errors with as results multiple mutations in the entire genome and particularly in S gene. This genetic variability promotes identification of eight genotypes (A to H) based on a sequence divergence more than 8% in the entire genome, or than 4% when only the S region is considered (2 , 4). In addition to their different geographical distribution, HBV genotypes are also associated with different clinical
Journal title :
Hepatitis Monthly
Journal title :
Hepatitis Monthly
