Molecular Characterization of HCV Genotypes and Response rates of various Anti-viral Therapies among Patients with HCV Infection in Pakistan

Abstract

The Hepatitis C virus (HCV) is a major cause of hepatitis C (hep C) which is a global problem with strong evidence of endemicity in several countries including Pakistan where more than 10% of the general population are suffering from the condition according to recent investigations. HCV infection reported to cause various phenotypes of the condition including acute infection; which progresses to chronicity among 80% of the infected individuals, fibrosis, cirrhosis as well as hepatocellular carcinoma (HCC). As an RNA virus, HCV is prone to mutate frequently giving rise to variants and new genotypes which may pose challenges to the existing diagnostic and therapeutic regimens. Currently, seven major genotypes and around 100 subtypes have been described prevailing in different parts of the world. The evolution of HCV genotypes depend upon a number of factors including ethnicity, infection rate and the use of different antivirals etc. The pattern of HCV genotypes distribution in a population is important with respect to understanding dynamics of the infection as the evolution of or introduction of variants or novel types have implications for diagnostic and therapeutic strategies. In Pakistan very few small scale studies have investigated the distribution pattern of HCV genotypes and response rates of various antiviral regimens while some newly approved therapeutics such as DAAs (Direct Acting Antivirals) have never been investigated against the prevalent HCV types. This study was therefore designed to investigate the existing pattern of HCV genotypes distribution and the pan-genotypic response rates of various antivirals in Pakistan. The first objective of the study was to investigate the distribution pattern of hepatitis C virus genotypes in different geographical regions of Pakistan by using Type-specific PCR assay. The Type-specific results were further validated by sequencing of a representative number of HCV isolates at their 5' UTRs. In the current study, 5704 randomly selected subjects( from a larger pool of people seropositive for HCV infection) of both genders, different ethnicity, age groups and belonging to 24 geographical locations in different cities of Pakistan were included, out of which 3061 (53.6%) females were found with a relatively higher frequency of active HCV infection than male subjects 2198 (38.5%). In male subjects the genotype distribution pattern demonstrated that HCV 3a was abundantly found (33.4%) followed by the Untypable genotypes (3.2%), HCV 3a3b (2.9%), HCV 3b, (1.4%), HCV 2a, (0.8%), HCV 2b, (0.1%) and HCV 1b (0.001%). In female subject, HCV 3a was slightly more frequent (46.5%) in comparison to male subjects followed by the Untypable genotypes (4.7%), HCV 3a3b (3.9%), HCV 3b (2.0%), HCV 2a (1%), HCV 2b (0.1%) and HCV 1b (0.001%). HCV genotype 4a (0.001%) was found in only one female subject. The subjects were divided into 6 age groups. Mean age of subjects enrolled was 44.23 ± 11.71 yrs. In age groups (26-40 and 41-55 yrs), the active infection was documented to be 36% and 43% respectively which was higher compared to rest of the age groups. HCV genotyping results indicated that out of the 5259 samples with active infection, Typable genotypes were identified for 92.12% subjects. Furthermore, HCV genotype 3a was found to be the most frequent one n=4203 (79.9%), secondly the Untypable HCV genotypes n=415 (7.9%) were found most abundant, similarly mixed infections of HCV 3a3b were n=355 (6.7%), HCV 3b were n=182 (3.5%), HCV 2a were n=92 (1.7%), HCV were 2b n=10 (0.2%) and HCV were1b n=2 (0.03%). Interestingly a single case of HCV 4 was also observed. The pattern of HCV genotypes distribution in 24 different cities of Pakistan reflected the same trend as detected in the entire study area with majority of infections due to HCV 3a, HCV Untypable genotype and HCV 2a while HCV 2b, 1a, 1b and HCV 4 were rare. Moreover, the overall HCV genotypes distribution pattern was the same in the case of both the genders and age groups indicating a uniform pattern of HCV genotypes distribution in different parts of the country. The second objective of the study was to characterize the Untypable HCV isolates from various geographical areas of Pakistan by sequencing and phylogenetic analysis of their 5′UTRs. The emergence of large number of HCV Untypable isolates among chronically infected Pakistani subjects pose challenges to accurate diagnosis, optimal regimen, dose and duration of antiviral therapy as well as for estimating the response rate. During the course of this study, a total of 415 Untypable HCV strains were detected from different parts of the country in which 50 (12%) randomly selected serum samples were used for sequence analysis of 5′UTR of HCV. The derived consensus sequences in case of all the 50 isolates were later used for genotype prediction using NCBI BLAST (ncbi.nlm.nih.gov) and online HCV genotype prediction tools. The results indicated that all the 50 samples (100%) were very close to HCV 3a. Self-alignment of all the 5' UTR sequences identified 10 diverse types circulating in Pakistan. Most common sequence variations were conserved (*), deletion (-) and transversion. Phylogenetic analysis of HCV Untypable isolates based on the 5' UTR sequences indicated that most of these isolates were genetically closer to Pakistani HCV 3a isolates with high bootstrap value as compared to some regional isolates. However, One Untypable isolate PK3 clustered with isolates from other regions and was distant from the Pakistani isolates which indicates that HCV 3a of different origins are distinctly evolving in Pakistan. The genetic diversity and phylogenetic analysis point towards the rise of variants of HCV 3a in this region in the form of the Untypable strains which need through characterization. The third objective of the study was to evaluate the response rates of DAAs (Daclastavir/Sofosbuvir) or their combination with previous standard of care (SOC) which included Ribavirin and Pegylated Interferon) for the treatment of hepatitis C subjects. A total of 4411 subjects with chronic HCV infection were enrolled for this study out of which n=3779, (85.6%) completed the therapy in which n=2204 (58.3%) were females and n=1575 (41.7%) were males while rest of the patients with HCV were not treated because of either losing the follow up or discontinuation of therapy due to side effects. The subjects were divided into four treatment groups each receiving a different combination therapy and the response rates were assessed via monitoring the viral load, blood and liver enzyme profiles. Group-I included 1446 HCV infected subjects out of which 847 (58.5%) were females and 599 (41.5%) male subjects belonging to six age groups who were given SOFO-RBV combination therapy for 24 weeks. The End of treatment response rate (ETR) was found to be (91.7%) and SVR24 of (91.5%) among all subjects. The response rate in the age range of 26-70 years was comparatively higher than subjects older than 71 years. Response rate of the combination therapy among subjects infected with different HCV types were slightly variable, however there was no significant difference (p value >.05). Subjects infected with various HCV genotypes exhibited that the response rates of the double combination therapy against HCV 3a, HCV Untypable, HCV 3a3b mixed infections and HCV 3b were better than other HCV genotypes.In Group-I, 120 (8.2%) non-responders and 3 (0.2%) relapsers were reported in the case of HCV 3a, 3a3b and HCV Untypable. In terms of gender wise response, high SVR24 rate (92.1%) was achieved in female subjects as compared to male subjects (88.8%) mostly among subjects in the age range of 26-70 years. Group-II consisted of 473 HCV infected subjects out of which 262 (55.3%) were females and 211 (44.7%) were males belonging to six age groups. In Group-II, the overall ETR and SVR of the DCV-SOFO combination therapy administered for 24 weeks duration were (93.2%) and (92.6%) in treated subjects. Subjects infected with various HCV genotypes exhibited that the response rates of DCV-SOFO combination against HCV 3a, HCV 3a3b and the Untypable HCV were better than the other HCV genotypes. In the age range of 26-70 years, the response rate was higher in treated subjects. SVR24 was achieved among (91.7%) female subjects and (92.3%) in male subjects. In Group-II, 32 (6.7%) non-responders and 3(0.6%) relapsers were reported who were infected with HCV 3a, 3a3b and the Untypable HCV. In Group-III, a triple combination therapy of DCV-SOFO-RBV was administered for 24 weeks. The group included 747 subjects infected with different HCV genotypes, out of which 449 (60.1%) were females and 298 (39.9%) males belonging to five age groups. The overall ETR and SVR rates of the combination in Group-III turned out to be (93.9%) and (93.8%) in treated subjects. Interestingly, in the age range of 26-70 years, the response rate was higher than the rest of the age range as noted in the case of DCV-SOFO combination therapy as well. Response rate of the combination turned out to be more favorable against HCV 3a, HCV Untypable, HCV3a3b and HCV 3b than other HCV genotypes. Similarly SVR24 was achieved among (93.0%) female subjects and (93.9%) male subjects. In Group-III, 45(6.0%) non-responders and 1(0.1%) relapse patients were reported in the cases of HCV3a 3a3b, 3b and the Untypable HCV infections. In Group-IV a triple combination therapy of SOFO-RBV-PEG INF-α-2a was administered for 24 weeks. The group-IV included 1113 subjects infected with different HCV genotypes, out of which 649 (58.3%) were female and 464 (41.7%) male subjects. The overall ETR and SVR rate of the combination in Group-IV turned out to be (93.3%) and (91.1%) among all the treated subjects. SVR rate of (90.3%) was achieved in female subjects and (90.0%) in male subjects.In the age range of 26-71 years, the response rate was higher than the other age groups. Subjects infected with various HCV genotypes exhibited that the response rates of the triple combination against HCV 3a, HCV Untypable and HCV 3a3b mixed infections were higher than other HCV genotypes detected in the group. In Group-IV, 74 (6.6%) non-responders and 24 (2.3%) relapsers were reported against the SOFO-RBV-PEG INF-α-2a, combination therapies including HCV 3a and 3a3b and Untypable. This study concludes that there is a uniform pattern of HCV genotypes distribution across the country with HCV 3a and its subtypes a/b as the most dominant types followed by 2a while other HCV genotypes are rarely prevalent in different areas. Although partial genetic information reveal that the Untypable HCV may be slight variants of HCV 3a, yet whole genome sequencing on a representative scale could help reveal their entire genetic divergence and their true identity as variants or novel types. Response rates of the combination therapies used against the Untypable HCV also indicated variations with respect to response rates achieved in the case of HCV 3a thus further re-inforcing the need for through full length characterization of such strains. Although combination of the newly approved DAAs or their combinations with RBV and PEG IFN exhibited higher response rates (>90%) against various HCV subtypes, DCV-SOFO-RBV with SVR (93.8%) and DCV-SOFO with SVR (92.6%) combination therapy proved relatively more effective against the most abundant HCV 3a and 3b infection in Pakistani population