الفهرس 
Introduction
Discovery and Historical backgroundOnly 14 pages are availabe for public view
 |  | from | 312 |  |  |
| | | from | 312 | | |
Abstract
Hepatitis C virus (HCV) infection in Egypt constitutes the most dangerous and worst health problem because of its devastating effect on the society as well as its enormous negative impact on the economy and future development. Chronic hepatitis C (CHC) infection is the main cause of liver cirrhosis and liver cancer in Egypt, and indeed, one of the top five leading causes of death. The virological markers that are currently used for the clinical assessment of CHC patients include: antibody to HCV, detected by ELISA, Particle agglutination (PA) and immunoblot assays, serum HCV-RNA, which can be measured by highly sensitive qualitative and quantitative assays and the HCV genotype. More recently, a new marker of HCV, the quantitative HCV core antigen (Q HCVcAg) assay has been evaluated. Antibody detection tests are considered important tools for screening the magnitude of HCV infection among a general population. Although, HCV RNA detection is widely accepted as a gold standard test in the diagnosis of HCV infection, the wide spread use of molecular techniques may be limited by availability, the need for meticulous specimen handling and concerns regarding reproducibility. A highly sensitive enzyme immunoassay, to detect and quantify total core protein in serum, has been developed. The Q HCVcAg assay is an interesting alternative to PCR based assays. There is a high positive correlation between the quantitation of core antigen and HCV RNA. Previously, the Q HCVcAg assay among Egyptian blood donors of genotype 4 showed high sensitivity, specificity and accuracy in comparison to PCR. The aim of the current study is to evaluate the Q HCVcAg assay in large cohort of CHC patients and comparing the results with those obtained from quantitative HCV PCR (Quant HCV PCR), and to clarify the assay performance in follow up among both treated and liver transplanted patients. In addition, to analyze the value of Q HCVcAg assay as one of the predictive factors associated with response to pegylated interferon/ ribavirin (Peg/Rib) therapy. Also, to sequence the interferon sensitivity determining region (ISDR) and protein kinase R (PKR) binding domain region of non structural 5A gene in a trial to elucidate a possible role of mutations in relation to viral load levels and treatment response. This study was carried out on 101 hepatitis C patients and 20 apparently healthy individuals. The patients were positive for anti HCV antibodies and HCV PCR and were divided into 3 main groups (Gps); Gp I, II and III. Gp I included 57 untreated patients who were subdivided according to HCV viral load into; low viraemia (LV), moderate viraemia (MV) and high viraemia (HV). Gp II included 32 treated CHC patients with Peg/Rib who were subdivided according to their virological response into; sustained responder (SR), non responder (NR) and responder relapser (RR). Gp III includes 12 patients underwent orthotropic liver transplantation (OLT). In addition, 20 apparently healthy individuals were enrolled as a control group and designated as Gp IV. The patients were 73 males (72.3%) and 28 females (27.7%) with age ranging from 29-65 years. The control group (Gp IV) were 12 males (60%) and 8 females (40%) with age ranging from 30-61 years. The anti-HCV antibodies were evaluated by 2 different serological methods; ELISA method and PA assay. The ELISA results were calculated according to optical density ratio (ODR) which is obtained by dividing the patient’s OD to cut off. The HCV titers by PA were graded as follow: 212 or more: high titer, 26 ~ 211: moderate titer, 25 or less; low titer. In our study, there were co-ordination between high positive ELISA and high PA titers (93.2%) while among low positive ELISA antibodies, the majority (61.5%) were of moderate PA titer with only 1 patient (7.7%) was of low titer and 4 (30.8%) were negative. Regarding the performance, 7 samples out of 101 samples (6.9%), who were positive for anti HCV by ELISA, were negative by PA. Interestingly, there was a good correlation between PA antibodies titer and HCV viral load by both Quant HCV PCR and HCV antigenaemia by Q HCVcAg assay. The specificity and the sensitivity of PA in this study were 100% and 93.1% respectively. As, Schistosomiasis is a major public health problem, we tested for the anti-schistosomal antibody profile against crude adult worms Ag (AWA) and soluble egg Ag (SEA). There was a high significant correlation between anti-SEA and anti-AWA as (P; 0.001). The frequency of positivity for both antibodies is quite similar (positive anti-AWA; 64.5% and positive anti-SEA; 62.8% respectively) with statistical intra group significance between both antibodies (P; 0.000 for both tests). In the present study, the viral load was assessed by both qualitative and quantitative PCR, and Q HCVcAg. Genotyping by primer specific region and sequencing of ISDR and PKR regions of NS5A gene were also performed. The results showed that Q HCVcAg assay is an alternative to PCR-based assays, as it needs no specialized equipments and with a low risk of sample-to-sample contamination. Also, the results were available within 3 hours. The specificity of the assay was 100% and its sensitivity was 97 %. In the current study, few discordant results of Q HCVcAg assay were detected among the studied groups. Three patients (3%) had undetectable HCV core Ag at basal samples. This could be the result of relative differences in sensitivity of both assays or to different clearance kinetics of free core protein versus virion associated RNA. We postulated 3 levels of HCV antigenaemia; Low antigenaemia (≤900 fmol/L), Moderate antigenaemia (>900 fmol/L - <4500 fmol/L) and High antigenaemia (≥4500 fmol/L). This grading is highly correlated with the different levels of HCV viraemia (LV; ≤4×104 U/mL, MV; >0.4 -<8×105 U/mL and HV; ≥8×105 U/mL). There was a significant increase in viraemia and antigenaemia one month after OLT using both Quant HCV PCR (P; 0.050) and Q HCVcAg (P; 0.023). The follow up study using Q HCVcAg, the antigenaemia was significantly different in samples collected before, 1, 3 and 6 months after OLT (P; 0.023, 0.031 and 0.028 respectively) and between samples 1 and 3 months (P; 0.021). For evaluation of Q HCVcAg in the studied Gps (I, II, III) a correlation study with other parameters was performed. A good correlation between Q HCVcAg and HCV RNA levels among all the basal samples was found (P; 0.001). Moreover, an excellent correlation of the Q HCVcAg with Quant HCV PCR was observed among all the studied groups. These observations are independent of the type of the patient or the HCV genotype. There was a significant correlation between levels of Q HCVcAg and the progress in the liver state in GpI and with both ALT and AST levels in SR subgp. On the other hand, no correlation was recorded between Q HCVcAg levels with either fibrosis or histological activity index. Results of genotyping showed that genotype 4 is the predominant genotype in Egypt (87/101, 86.1%), followed by genotype 1b (3/101, 3%) and (11/101, 10.9%) of mixed infections (genotypes 4 and 1b). In this study, we analyzed the amino acid (a.a.) mutations in the PKR binding domain (62 a.a.) of which the first 40 a.a. constitute an ISDR. We aimed to explore a possible role of mutations in the viral load and response to therapy. The resultant sequences (n=40) were amplified from pre-treatment sera of the following patients: 37 CHC patients of genotype 4 (LV; n=9, MV; n= 19 and HV; n=9) (including Gp II; 32 CHC treated patients). 3 CHC patients of genotype 1b with their viral load were; 1.51×105, 7.36×105and 8.32×105 IU/mL. Sequencing of genotype 4 samples highlighted three important observations. First, as regard the levels of viraemia and antigenaemia, there were significant increases in the number of mutations in ISDR and PKR regions that inversely proportional to levels of viraemia and antigaenemia. Second, as regard the response to therapy, most of the SR (n=9/11) patients showed higher number of mutations in ISDR (≥4 mutations) isolated from pre treatment sera, compared to NR subgp (n=17) where 15 patients showed ≤3 mutations and 2 patients were of ≥4 mutations. Third, there was no specific substitution pattern observed among the sequenced data. On the other hand, HCV genotype 1b samples showed no specific mutations in ISDR and PKR regions. The underlying mechanisms of different virological responses to treatment in CHC patients with genotype 4 are still unclear. It was noticed from this study that the SR prevalence among genotype 4 patients was low (34.4%) in comparison to NR (53.1%) while RR constituted (12.5%). This could be attributed to the high prevalence of anti-shistosomal antibodies, high fibrosis score, presence of cirrhosis and the inverse relation with the number of mutations in ISDR and PKR of NS5A gene. from this study, we could conclude that: * The Q HCVcAg assay is a reliable marker in quantification of viral load, monitoring the response to therapy with early prediction of sustained response and follow up of liver transplanted patients. * A coordination does exist between high positive ELISA and high PA titers (93.1%). The specificity and the sensitivity of PA in this study were 100% and 93.1% respectively. * Highly comparable results were evident in using both anti AWA and anti SEA antibodies in diagnosis of shistosomal infection. * During treatment, the early response predictor depends mainly on the 2-log decrease of HCV RNA level at W12-24 than the basal level and negative HCV core antigenaemia. * A close association was found between numbers of amino acid substitution in ISDR and PKR binding domain that might affect the function of NS5A and both the levels of viraemia and antigenaemia and efficacy of Peg/Rib therapy. * The main pretreatment predictors are the reported selection criteria, anti-schistosomal Ab, cirrhosis, extent of fibrosis and the number of amino acid substitution among ISDR and PKR regions.