الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Egypt has one of the highest prevalence rates of hepatitis C virus (HCV) infection in the world .The HCV epidemic appears to have been initiated by vigorous public-health campaigns using intravenous tartar emetic from the 1950s until 1982 to eradicate schistosomiasis. Among the six major HCV genotypes found worldwide, genotype 4 is the most predominant in Egypt, with 4a as the dominant subtype.
Genotype identification has been done using type-specific primers for PCR, restriction enzyme digestion of the amplified PCR product, or type-specific probes to hybridize with HCV RNA after PCR. Genotyping is accomplished by analysis of parts of the genome, such as the 5’ UTR, the core, the E1, and the NS5 region.
A reverse hybridization line probe assay (LiPA) has been developed for genotyping of HCV isolates by 5’UTR analysis that shows significant sequence variation, which allows the use of this region for HCV genotyping.
The aim of this study was to evaluate a new set of primers designed for HCV genotyping by comparison with the FDA approved PCR based reverse hybridization INNO-LiPA test and to evaluate the role of genotypes in relation to clinical, biochemical, histopathological and virological data.
This study was carried on twenty anti-HCV positive patients presented to the Alexandria Armed Forces Hospital and the Medical Research Institute .
All relevant informations were collected from each patient.
Blood samples were collected from all patients, divided into three tubes, one on citrate for Prothrombin activity, another tube on EDTA for CBC and the last was plain left to clot for serum was separated by centrifugation and stored in small aliquots
at -20°C for further investigation.
1-Detection of antibodies against hepatitis C virus by ELISA (Abbott Murex Diagnostic Division) was carried to confirm the presence of HCV antibodies in these patients
2- Detection of HCV-RNA by Real Time PCR (ABI: Applied Biosystem)
3- Determination of genotypes by several methods.
a-Determination of HCV genotypes by INNO-LiPA
b-Determination of HCV genotype using the primer-extension method
c-Determination of HCV genotypes using HCV genotype specific primers
d-Determination of HCV genotype using direct sequencing of the 5`UTR
4-Determination of serum ALT, AST, Albumin, Prothrombin activity and Platelets count.
In the present study 19 out of the 20 cases were in their fourth to sixth decades, 80% of them were males.
Nineteen out of the 20 patients were subjected to liver biopsy, 9 (47.4 %) out of them showed a metavir score of A1F0 to A1F1 while 10 (52.7%) had a higher score of A2F1 to A2 F2.On the other hand, 4 (20%) out of our cases had a viral load less than 105 IU/ml while 7 (35%) had a viral load higher than 106 IU/ml.
No significant relationship was found between the disease severity as shown by Metavir score and viral load since 5 (71.4%) out of 7 patients with a viral load of 106 IU/ml had a metavir score of A1F1 while 1(50%) out of 2 HCV patients with a low viral load 103and 104 IU/ml had a metavir score of A2F2.
Moreover, no correlation was found between serum HCV viral load and liver parameters including liver enzymes, albumin,, prothrombin time and platelet count was not affected by viral load since the majority of the 7 cases with a viral load higher than 106 IU/ ml had normal platelet count (57.1 %), ALT (71.4%), AST, albumin and prothrombin time (85.7%) compared to 50% of the 4 cases with a viral load ranging between 103 -104 IU/ ml.
In the present study HCV genotyping and sub-genotyping methods were tried by different methods including type specific primers, primer specific and mispair extension, a commercial reverse hybridization assay INNO-LiPA HCV2.0 and direct sequencing all were targeting the 5`UTR.
The twenty HCV RNA positive cases included in this study were tested for HCV genotyping by Versant HCV genotype assay INNO-LIPA 2.0
Genotype (1a) was diagnosed in one case depending on the presence of core band 25 in addition to bands 3, 4, 5 of the 5`UTR.
On the other hand, 3 bands (16,17and 18) specific for genotype 4 were detected among 18 of our 20 HCV isolates which were therefore classified easily as genotype 4.
Sub-genotyping was possible only in 1(5.2%) out of the 19 HCV genotype 4 (4e) with only 2 characteristic bands 5and 16.
Thirteen (65%) out of the 19 HCV genotype 4 could not be further sub-genotyped while five (25%) isolates were classified under the grouped subgenotypes (4a/4c/4d).
11 (73.3%) out of 15 cases have been successfully genotyped and sub-genotypes by sequencing the 5`UTR region 9 (60%) out of them previously diagnosed by INNO-LiPA as genotype 4 or (4a,4c,4d) were found to be 4a, 1 (6.6%) was sub-genotyped as 4f and the other (6.6%) was 4o.
Four (26.7%) isolates belonging to genotype 4 could not be further sub- genotyped.
In the present study, 12 (66.6%) out of the 18 HCV-4 genotyped isolates showed no or mild fibrosis and 8 (44.4%) had a low metavir grading grade (A1).
As the majority (60%) of our 15 isolates subjected to sequencing was sub-typed as HCV 4a, no relation between HCV-4 subtypes and the degree of liver fibrosis could be deduced from this study.
A trial to design type specific primers included in the 5`UTR to amplify regions specific to the corresponding genotypes was unsuccessful. Non specific amplification of genotype 4 isolates was obtained using the presumably HCV genotype 4 specific primers designed in Germany the reason might be that primers were not tailored to our Egyptian isolates.
In the present study, HCV genotyping was tried using the HCV primer extension based on specific nucleotide at the 3’end of the primer to prevent amplification of other genotypes. No amplification of our HCV genotype 4 isolates could be detected by Syber green technique using specific HCV genotype 4 primers.
HCV genotype 4 being a very heterogeneous genotype showing significant genetic divergence, design of primer-specific extension should be based on sequencing of the 5`UTR of our Egyptian isolates.
from this study it can be concluded that sequencing of the 5`UTR was more successful than INNO-LiPA for HCV genotyping and even HCV-4 sub-genotyping.
Successful designing of type specific primers for HCV-4 genotyping and sub-genotyping should be based on sequencing of Egyptian isolates due to HCV-4 well known heterogeneity.