Abstract
Hepatitis C virus (HCV) genotyping is a critical parameter that acts as predictor of treatment response. According to previously reported findings, about 11 million population of Pakistan are HCV infected. Accumulating data suggest that genotype is the most prevalent genotype and mixed and untypable genotypes are the least prevalent genotypes of HCV. We observed that overall prevalence of mixed genotype (5.03%) and untypable genotype (3.3%) of HCV is on constant rise. This study highlights that the emergence of novel quasispecies could be reason of treatment failure in patients receiving direct-acting antiviral drugs.
Hepatitis C virus (HCV) is the major cause of life-threatening liver disorders with an estimated global bioburden of >71 million and 399,000 die of HCV-induced hepatocellular carcinoma and liver cirrhosis, every year (12). HCV genotyping is the key element that determines the outcome of antiviral therapy and the distribution pattern of both HCV and hepatitis B virus vary worldwide (4).
The spontaneous mutation rate is relatively high in HCV because of which the virus exists as closely related genomes (quasispecies) in infected patients. Under the selective pressure of host's immune system, this quasispecies contain group of heterogeneous RNA sequences centered around dominant nucleotide sequence that changes. It has been studied extensively that this genetic drift helps HCV escape immune surveillance of host (2). Researchers have classified HCV into different genotypes based on sequence analysis of HCV E1, core, NS5, and 5′ UTRs. There are four different types of genetic variabilities observed in HCV isolates: quasispecies (90.8–99% nucleotide sequence identities of full-length sequences), subtypes (76.9–80.1% nucleotide sequence identities of full-length sequences), genotypes (15–25% nucleotide sequence identities of full-length sequences) and types (65.7–68.9% nucleotide sequence identities of full-length sequences) (5).
HCV has been classified into six major genotypes and different subtypes based on nucleotide sequence homology. Genotypes 7 and 8 have also been reported recently (6). Previous studies have shown genotype 3 as the most prevalent genotype in Pakistani population followed by genotype 1 and genotype 2, whereas overall combined prevalence of genotypes 4, 5, and 6 is <3% (1). The reported cumulative rate of mixed and untypable genotype in Pakistan has been reported <5%. The studies on clinical significance of HCV genotypes have shown that characterization of genetic groups will contribute toward the development of effective vaccine against HCV, and these multiple genotypes also act as predictor of treatment outcome (13).
In this study, we present the first evidence that highlights changing patterns of genotypic distribution in Pakistan. Samples collected from different rural and urban settings of Pakistan were delivered to Genome Centre for Molecular Based Diagnostics and Research, Lahore, Pakistan. In total 9,932 participants were recruited for this study. Qualitative and quantitative analysis of HCV was performed following the method described by Wahid et al. (10), and genotyping was done according to method previously reported by Idrees et al. (3). In contrast to previously reported findings, we observed an increase in mixed genotype and untypable genotype in Pakistani population. The overall prevalence of HCV was found higher in Punjab province as compared with other provinces. Previous studies have shown that unsafe medical practices act as major source of transmission of HCV in Pakistan (9), whereas this study suggests that multiple parameters such as sexual transmission, tattooing, percutaneous, and mucocutaneous exposures contribute toward the spread of HCV all across Pakistan. Analysis of demographic characteristics showed that the overall prevalence of HCV was higher in women (5,719 [57.18%]) as compared with men, and mean age of patients was 43. Migrations of different ethnicities to Pakistan followed by Afghan war, increased global travelling, formation of resistant-associated variants after treatments, and new modes of transmission might lead to diversity of genotypes.
Our findings suggest that genotype 3 (72.7%) had the highest prevalence followed by untypable genotype (13.1%), mixed genotype (9.56%), genotype 1 (3.94%), and genotype 2 (0.6%). We did not observe any case of genotype 4, genotype 5, and genotype 6 (Table 1).
Geographical Distribution of Hepatitis C Virus Genotypes in Pakistan
Previous studies report that genotype 3 (78.96%) is followed by genotype 1 (7.03%), mixed genotype (5.03%), genotype 2 (3.81%), untypable genotype (3.3%), genotype 4 (1.59%), genotype 6 (0.13%), and genotype 5 (0.10%) (1). Several evidences report combined prevalence of mixed genotype and untypable genotype <7%, whereas some studies have reported mixed genotype as least prevalent HCV genotype in Pakistan (6).
Patterns of HCV mixed genotype and untypable genotype are completely different and significantly higher than those previously reported from Pakistan, India, Iran, Bangladesh, and other Asian countries (9). Previously used interferon-based regimens exhibited low response rate in patients infected with mixed genotype of HCV, whereas the potential outcome of recently developed direct-acting antiviral agents on mixed HCV infections have not been studied properly.
Further studies are needed to establish the correlation of increasing prevalence of quasispecies with recently reported cases of HCV relapse and treatment failure in patients treated with direct-acting antiviral (DAA) drugs (8,11).
In conclusion, the increasing prevalence of mixed HCV genotype and untypable genotype highlights the need for further clinical research to analyze the DAA drugs' efficacy and optimize the treatment regimen for patients infected with either mixed genotype or untypable genotype.
Footnotes
Acknowledgments
Special thanks to Genome Centre for Molecular Based Diagnostics and Research, Lahore, Pakistan.
Author Disclosure Statement
No competing financial interests exist.