الفهرس 
Introduction and aim of the workOnly 14 pages are availabe for public view
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Abstract
NS5B polymerase inhibitors represent the cornerstone of the present therapy of Hepatitis C virus infection (HCV). Naturally occurring substitution mutations to NS5B inhibitors have been recorded. The current study intended to demonstrate probable natural DAA - mutations of the HCV NS5B region in HCV patients in Minia governorate, Egypt.
In this study, a total of 35 blood specimens have been gathered from 27 treatment-naïve HCV infected cases and 8 non-responders’. Out of 27 treatment-naïve patients, 17 NS5B sequences (amino acids 221 - 345) from treatment-naïve patients and one sample of non-responders were successfully amplified. All relevant information were collected from each patient including personal data (age, sex, residence), habits (smoking, alcohol consumption), as well as medical data (history of blood transfusion, history of Schistosomiasis and anti-schistosomiasis treatment, previous surgical interference, dental interventions and pegylated-interferon and ribavirin therapy).
Sera were tested to determine HCV viral load by real-time PCR, amplification of NS5B gene by Nested PCR, HCV genotype determination by direct sequencing of NS5B region, sequence analysis of NS5B gene coding for the NS5B polymerase enzyme and the aa sequence of NS5B have been compared with that of worldwide isolates for the presence of mutations conferring resistance to polymerase inhibitors.
Among 35 HCV infected cases involved in the study, 24 (68.60 %) have been males, and 11 (31.40 %) have been females. Their age ranged from 23 to 75 years, the highest percentage (40.0%) of the infected cases age is between 51 to 60 years. The least percentage was in the age group 41-50 and 71-80 Years (8.6%) for each.
Regarding to risk factors, 80.0% of cases gave a history of dental intervention, 65.7% show history of hospital admission and 57.1% were subjected to previous surgeries. 48.6% of cases had a family history of a relative suffering from HCV infection. The least percentage (22.9%) was found between patients who received blood transfusion.
The results of viral quantification showed that (34.3%) of the HCV patients had viral loads < 104 and < 105 for each and 11 patients (31.4%) their viral loads were <106. 25 (71.4%), 27 (77.1%) and 28 (80.0%) of HCV cases had normal ALT, platelet count and prothrombin levels, subsequently. Viral load was not significantly associated with elevated ALT and Prothrombin activity.
The study revealed that 13 strains of the 18 HCV cases were of genotype 4a. Two cases were diagnosed as 1g and only one case of genotype 4o, 4l and 3a subsequently.
NS5B amino acid sequence analysis insure several novel NS5B mutations existence (more than 40 substitution mutations) that have not been previously documented to be correlated with a resistant phenotype. It was found that K304R (82.4%), E327D and P300T (76.5% each) substitutions were the most distributed in the tested samples, respectively. S282T, the major resistance mutation that induces high sofosbuvir-resistance level in addition to other reported mutations (L320F/C) and (C316Y/N) were not recognized. Q309R mutation is a ribavirin-associated resistance, which was recognized in one strain (5.9%) of genotype 1g sequences. In addition, one substitution mutation (E237G) was identified in the successfully amplified non-responder sample.
Conclusion
Our study reported that there are many combinations of multiple aa substitutions in the analyzed NS5B genes which could enhance therapy failure possibility in HCV patients treated with regimens containing DAA. Overall, it may be difficult to fully understand the role of some of these substitutions, the high virological success rate of SOF-based regimens, the rare incidence of RAS in non-responsive patients, and the minimal data developed for RAS in genotype 4 patients compared to other genotypes.
Further studies are needed to test the relation between the reported substitutions in our study and HCV resistance.