Detection of hepatitis c virus (HCV) genotypes by pyrosequencing in chronic HCV patients

Chronic viral hepatits are one of the most important public health problems. It has been predicted that 170-210 million people have been infected with Hepatitis C virus (HCV) in the world. HCV, which is one of the most important risk factors for diseases including acute and chronic hepatitis, chirose, and hepatocellular carcinoma is responsible for 25-40% of the liver diseases. HCV is divided into 6 main genotypes, nucleotide sequences of which can be different up to 30%, and into more than 80 subtypes. Genotypes can not be used for predicting how the HCV infection will progress but can be an indicator of response to the treatment and can be used for determining the treatment period. HCV genotypes are determined using various methods such as Direct sequence analysis, Reverse Hybridization, Restriction Fragment Length Polymorphism (RFLP), type-specific Polymerase Chain Reaction (PCR), Pirosequencing, and serotyping. Among those, pyrosequencing is a real time quantitative sequencing technique based on the detection of pyrophospates released during the synthesis of DNA. In this thesis, it was aimed to determine HCV genotypes of the patients with prediagnosis or diagnosis of chronic hepatitis C in Elazığ by using pyrosequencing method. Serum samples of 50 patients with prediagnosis/diagnosis of chronic hepatitis C were used in this study. HCV-RNA levels of all samples that were positive for anti-HCV were determined using real time PCR (Taqman 48, Roche). RNA isolation from the samples were carried out using an automated isolation system (EZ1, Qiagen) as described by the manufacturer. HCV genotype analysis of the samples were determined by using pyrosequencing method. 46 (92%) and 1(2%) of the samples were determined as genotype 1 and genotype 3, respectively. Of 46 samples that were genotype 1, 41 were found as genotype 1b and 5 as genotype 1a whereas 3 of those were not be able to subtyped. As a result, genotype 1 was found as the most common genotype in patients infected with HCV. Genotyping HCV by pyrosequencing takes quiete shorter time compared to other DNA sequencing methods. In this case, pyrosequencing can be a method of choice in routine analysis laboratories due to ease of use. Unit cost of pyrosequencing is comparable to that of DNA sequencing based methods. In fact, it is cheaper than the cost of reverse hybridization method. It is concluded that, due to the cost and required-time advantages, pyrosequencing can be used for genotyping HCV, which is an important criteria for determining the treatment response and the treatment period. Key Words: Hepatitis C Virus, Chronic hepatitis C, genotype, pyrosequence