The hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. HCV infects more than 170 million people worldwide with an estimated 500,000 deaths per year due to complications of end stage liver disease. In Pakistan, about 10% of the general population is infected with HCV. Of those acutely infected with HCV, approximately 80% develop chronic infection. Chronic hepatitis C can lead to liver cirrhosis and hepatocellular carcinoma in a substantial number of patients. The mechanism of viral persistence is largely unknown, although the high genetic variability is thought to playa key role. This study is aimed at determining the various viral and host factors responsible for disease detection, severity, clearance, spread and persistence. The present study was started in May 2000 to address seven objectives. A summary of the research results obtained for each objective is given below:
The first objective of this study was to sequence the 5â€™Untranslated Region (5â€™UTR) region of HCV -RNA isolated from HCV positive patients from different parts of Pakistan. During the course of this study, total 100 HCV RNA PCR positive serum samples collected from different parts of the country were used for sequence analysis of 5â€™UTR of HCV. Four primers were designed based on highly conserved 5â€™UTR of HCV genome for the amplification and sequencing of 5â€™NCR. The sequence from nt: -35 to-319 (285 nt) was taken for analysis. Pakistani isolates sequenced in the present study were aligned with the representative number of sequences for each major genotype and subtype selected from the GenBank database. The results of the phylogenetic analysis of the sequenced samples showed that genotype 3 and type I are the predominant genotypes circulating in Pakistan, with an overall prevalence of 62% and 12%, respectively. Type 2 viruses were found 8%. Type 4 viruses (3%) were seen only in NWFP.
The second objective of the study was to investigate and isolate novel HCV isolates from Pakistan. On the basis of phylogenetic analysis, the 100 Pakistani isolates were classified as follows: 38% (n = 38) were type 3a, 21 % (n =21) were type 3b, 3% (n = 3) were type 3c, 7% (n = 7) were type 1a. 5% (n = 5) were type 1b/1c, 5% (n = 5) were typ,2a/2c, 3% (n = 3) were type 2b, 3% (n = 3) were type 4, and 14% (n = 14) were novel genotypes. One isolate, PP10, still remained nontypeable. The overall nucleotide similarity among different Pakistani isolates was 92.50%Â±0.50%. The percent nucleotide identity (PNI) was 98.11 %Â±0.50% within Pakistani type 1 sequences, 98.10%Â±0.60% for type 2 sequences, 97.97%Â±0.50% for type 3 sequences, and 99.80%Â±0.20% for type 4 sequences. The PNI between different genotypes was 93.90%Â±0.20% for type 1 and type 3, 94.80%Â±0.12% for type 1 and type 4, and 94.40%Â±0.22% for type 3 and type 4. There was a stretch of hypervariable region from n1: 166 to 86 in the 5â€™NCR that had motifs for different genotypes. Pakistani isolates showed 7.5%Â±0.50% nucleotide variability in this region. The comparatively conserved strctd1 from nt 241 to 165 showed only 3.30%Â±1.06% variation. It was not possible to differentiate between type 1 band 1 c isolates further into different subtypes. Both types clustered together. Similarly in the case of 2a and 2c, no differentiation was possible in the tree. In the case of the type 3 isolates, there was a Clear clustering of isolates into subtypes 3a, 3b and 3c. This study showed that type 3 and type 1 seem to be the predominant genotypes circulating in different parts of Pakistan. More or less equal proportions of type 1 and type 3 isolates were seen in Balochistan.
The third objective of the study was to establish a simple, rapid and reliable genotyping system for well-characterized Pakistani HCV isolates and to determine the suitability of this assay for the routine determination of HCV genotypes. So, a new genotyping system was established for the specific detection of HCV genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4a-h, 5a and 6a during the course of this study. Total 15 primers were designed for this new genotyping system. Both the outer sense and anti-sense primers and inner sense primer were designed on the basis of the conserved nature of these sequences. Genotype-specific primers were designed on the basis of 116 HCV isolates. The antisense primers were designed on the basis of the conserved nature of those sequences within a genotype and their poor homology with the sequences derived from other HCV genotypes. Extensive experiments were performed to optimize the genotyping PCR. Serum samples having known genotypes were used as positive controls to validate the developed assays and to generate PCR band patterns. Band patterns generated from the clinical serum samples from HCV patients were compared to the patterns produced from these control samples. In addition, the type-specific bands were sequenced from the test patients and control clinical samples to further validate the nested type-specific PCR test results. Sensitivity and specificity of the system was determined by analyzing 260 samples simultaneously by this HCV genotyping system, Ohnoâ€™s genotyping system and commercially available serotyping kit. The system showed 79.2% concordance with Ohnoâ€™s system and 65.38% with Serotyping system. Samples with discordant results were sequenced and their genotypes were determined by molecular evolutionary analysis. In all the discordant samples the assigned genotype of the present system was correct.
The fourth objective of the present study was to establish a genotyping system for mixed HCV infections as to date the true prevalence of HCV mixed-genotype infections has not been established mainly because currently available methods are not suitable for the detection of mixed genotypes in a viral population. A new genotyping method, primer-specific PCR followed by polyacrylamide gel electrophoresis (TSPCR-PAGE), which is more reliable than other genotyping assays was developed for the detection of HCV mixed-genotype infections. A genotype present at levels as low as 8.3% in a defined mix of HCV genotypes was detected, showing a 20-fold increase in sensitivity over that of direct DNA sequencing. A total of 50 HCV isolates from patients who received multiple blood transfusions were genotyped and analyzed for a comparative study of the accuracy between the present system and two other genotyping methods. The results showed that viruses in approximately 42% of the samples from this group determined to be infected with mixed genotypes by TSPCR-PAGE. Serotyping assay and restriction fragment length polymorphism analysis performed poorly, being able to identify only 18% and 10% of mixed-genotype infections, respectively. The data lead us to conclude that HCV mixed-genotype infections are more common than previously estimated and that TSPCR-P AGE may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections is required due to its higher level of sensitivity.
The fifth specific objective of the study was to find out the common genotypes of HCV present in well-characterized Pakistani HCV isolates. Total 3351 serum samples were tested by type-specific genotyping assay. All the tested serum samples were HCV-RNA positive by PCR. Out of the total 3351 tested serum samples, type-specific PCR fragments were observed in 3150 (94.00%) serum samples. The distribution of genotypes of the 3150 typable samples as determined by this assay, was as follows: 280 (8.35%) .patients were infected with HCV genotype la, 101 (3.01%) patient were infected with genotype 1 b, 5 (0.15%) with 1c, 252 (7.52%) patient were infected with subtype 2a, 27 (0.80%) were infected with subtype 2b, 3 (0.09%) were with type 2c, 1664 (49.05%) patients were infected with subtype 3a, 592 (17.66%) were infected with genotype 3b, 25 (0.75%) were with 3c, 50 (2%) patient were infected with genotype 4, 6 (0.18%) were infected with subtype 5a, 4(0.12%) were infected with subtype 6a and 161 (4.80%) patient was infected with mixed infection. Two hundred and one (6.00%) serum samples were found undetermined by the present genotyping system.
The aims of the 6th objective of the study were to determine the rate of sustained virological response (SVR) and the factors associated with SVR in chronic hepatitis C patients treated with interferon alpha and ribavirin combination therapy. Four hundred consecutive patients were prospectively evaluated and treated with combination of Interferon Alfa 2 (a or b) three million units subcutaneously three injection weekly and ribavirin 800-1200 mg orally daily for 24 weeks or 48 weeks and followed for another 6 months. End of the treatment response, sustained viral response and side effects were noted. Of four hundred patients, 394 completed the treatment. Over 67% responded at the end of treatment and 16% relapsed. Out of treated, 48.55% males and 57.62% females had sustained viral response with a total combined sustained viral response rate of 51.56%. Patients with cirrhosis had no sustained viral response. Thirty percent patients took longer than three months to show HCV RNA negativity. Side effects were usual and tolerable and only 1.5% discontinued the treatment. Non-responders were mostly males above age 50 years. In conclusion, twenty-four weeks combination treatment with Interferon alpha 2a or 2b and ribavirin has given 51.56% sustained viral response in patients and treatment was well tolerated.
The seventh and last objective of this research was to assess the association between chronic hepatitis C infection with different genotypes and hepatocellular carcinoma (HCC) in a population or patients presenting to a various hospitals. Eighty three cases or histologically confirmed liver cancer due to HCV presenting to the various hospital of Pakistan were genotyped. HCV genotype 3a was most frequently represented (34 of 83 patients). In the HCC patient population, broader distributions of genotypes were present (genotype 1 a: 8; genotype 1 b: 2; genotype 3a: 34; genotype 3b: 13; mixed genotypes 3a & 3b: 10; 1 a & 3a: 8; and la & 3b: 6). Two of the cases were untypable as no type-specific bands were obtained for them. A strong association between chronic HCV infection and hepatocarcinogenesis in the Pakistani population was observed. The study was unable to distinguish whether the high prevalence of genotype 3a in the HCC population reflected increased oncogenicity in itself, or whether 3a was simply the most prevalent genotype in Pakistan when these patients were infected.