الفهرس 
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Abstract
Hepatitis C virus (HCV) infection remains one of the most pressing health
emergencies worldwide, with an estimated global prevalence of more than 180 million
people. Egypt reports the highest prevalence of HCV in the world, with an average of 20%
(approximately 17 millions).
HCV is characterized by genetic heterogeneity. At least 7 major HCV genotypes are
identified. Egypt has the highest prevalence of genotype 4(GT-4), which is responsible for
almost 90% of infections. However, GT-4 is a very heterogeneous genotype showing
significant genetic divergence and more subtypes compared with other genotypes. To date,
18 subtypes have been identified.
The infecting HCV strain is known to be one of the main independent factors that
influence the outcome of antiviral therapy. Genotype should be determined in all HCV-infected persons prior to treatment in order to determine the necessary duration of therapy
and likelihood of response.
Up to 2011, major achievements in hepatitis C treatment have been accomplished
with a standard protocol for treatment comprising a combination therapy of pegylated-interferon (PEG-IFN) and ribavirin. However, new therapeutic approach will rely on
specifically targeted antiviral therapies (STAT-C) against essential HCV viral proteins.
This will be of great importance especially to patients who had no response to or had
experienced relapses after receipt of PEG-IFN and Ribavirin treatment.
Directly acting agents (DAA), like inhibitors of the NS3 protease and NS5B
polymerase, coupled with or without currently used PEG-IFN and Ribavirin therapies may
well provide the backbone for future treatment of chronic hepatitis C infections.
NS3 protease inhibitors target an essential step in HCV replication by blocking the
NS3/4A protease-dependent cleavage of the HCV polyprotein. Thus, NS3 protease is one
of the most promising targets for specific anti-HCV therapy.
Monitoring HCV treatment will focus on selection of resistant variants which have
not been an issue with the PEG-IFN plus Ribavirin treatment. Along with the definition of
HCV mutations associated with decreased susceptibility to the different protease inhibitors,
sequence analysis of the HCV NS3 genome region may become an integral part of the
management of patients who are under treatment or are candidates for treatment.
Given the heterogeneities in viral sequence, not all DAA will be equally effective for
all HCV genotypes, subtypes and strains. Most data on NS3 protease polymorphism
concerns HCV GT-1, sparse data are available concerning GT-4.
The aim of this study was to determine hepatitis C viral genotypes and subtypes
among chronically infected Egyptian HCV patients, to sequence NS3 protease region
coding for NS3 protease enzyme and to investigate the prevalence of possible mutations
expected to induce resistance to the protease inhibitors by comparing the obtained
sequences with that of NS3 protease reference sequences obtained from HCV worldwide
database.
Summary and Conclusion
115
The present study included 40 HCV patients; 20 (50%) treatment-naïve patients and
20 (50%) patients who failed to achieve sustained virological response (SVR) to interferon
(IFN) and Ribavirin therapy. Patients were selected from the outpatient clinic of the
Medical Research Institute, Alexandria University.20 (50%) out of the 40 HCV cases were
males and 20 (50%) were females. Their age ranged from 9 to 69 years, with the highest
percentage (57.5%) of patients in the age range between 41-60 years.
All relevant information were collected from each patient including personal data,
habits and medical data.
Blood samples were collected from all patients and prothrombin activity (INR) and
platelet count were estimated. Blood samples were then centrifuged at 1500 rpm and sera
separated and distributed in aliquots and stored at –20
o
C for further investigations. Sera
were tested for the following investigations:
1-Determination of Liver alanine transaminase (ALT)
2-Virological studies:
a- Determination of HCV viral load by real-time PCR. (Artus HCV QS-RGQ-PCR
Kit).
b- HCV genotype determination by direct sequencing of NS5B.
c- Amplification of NS3 using pairs of specifically designed primers.
d- Sequence analysis of NS3 gene coding for the NS3 protease enzyme used for HCV
genotyping .
e- NS3 amino acid sequence analysis and alignment to detect the presence of
mutations and polymorphism that may confer resistance to protease inhibitors.
In the present study, the main risk factor for acquiring HCV was dental interventions,
where 77.5% of cases had history of dental procedures. History of previous hospital
admission and surgeries were high in this study, 60.0% and 55.0% respectively.
Viral load was not significantly associated with liver functions, namely alanine
transaminase (ALT) and prothrombin activity. Platelet count was normal among all HCV
cases.
NS5B domain was used for genotyping and subgenotyping, 38(95%) of cases were
genotype 4a and 2 (5%) cases were genotype 4o, while by sequencing of NS3 domain
39(97.5%) of cases were genotype 4a and 1 (2.5%) case was genotype 4o. NS5B and NS3
sequencing gave similar genotyping and subgenotyping results in 37(92.5%) out of the 40
HCV isolates.
Analysis of the HCV NS3/4A protease-coding region in 40 HCV genotype 4 isolates
was carried out. Amino acid sequence variations and signature patterns were analyzed
using the VESPA online program. 30 amino acid positions were identified as signature
pattern for the 40 HCV cases in comparison with other HCV genotypes, including
positions 16 and 36 that have been described to be associated with resistance to protease
inhibitors.
Summary and Conclusion
116
Nucleotide and amino acid sequences of the NS3 region were aligned with Clustal X
and Bioedit version 7.2.5 software and analyzed for the presence of previously identified
substitutions and polymorphisms in HCV-4 conferring resistance to NS3 protease
inhibitors.
Polymorphism at position D168H/E known to be associated with high-level
resistance to Danoprevir, Ciluprevir, Asunaprevir and Simeprevir PIs was detected in
3(7.5%) of our cases.
Amino acid substitutions associated with Vaniprevir resistance (D168 to T/I/A/V)
were not detected among our cases, while a D168E associated with Asunaprevir resistance
was found in only 1 (2.5%) case.
T54A mutation associated with low-level resistance to Telaprevir and Boceprevir
was detected in only 1 (2.5%) case, while V170A substitution a major resistance mutation
for Boceprevir was detected in 2 (5%) cases.
V158I substitution previously reported to be associated with low-level resistance to
Boceprevir was not detected among our cases. A156T and R155K mutations known to
confer high level of resistance to all PIs were not detected in our study as well. Sites
associated with resistance to Danoprevir (F43S, Q41R, S138T and A156S/V) were also not
detected.
Substitution Q80K/R responsible for resistance to Simeprevir common among
genotype 1 patients was not detected among our cases since they were all of genotype 4.
Our results confirm the high genetic diversity of NS3 in genotype 4. This could
impair the use of protease inhibitors as well as hinder the development of new NS3
targeted drugs effective against genotype 4 infected patients. Nevertheless, large
therapeutic trials in HCV-4 patients are highly recommended to evaluate the effect of genetic polymorphism on clinical response to therapy.