Molecular epidemiology and viral load analysis of hepatitis C virus genotypes from Sindh, Pakistan

The present study was aimed to assess the molecular epidemiology of Hepatitis C Virus (HCV) genotypes and viral load (VL) of HCV infected patients. A total of 261 anti-HCV positive patients were enrolled for VL analysis using COBAS AmpliPrep/COBAS TaqMan HCV and Abbott Real Time HCV Genotype II Assay Kit to determine the genotypes. The data demonstrated that 60.15% (n=157) samples were from females and 39.85% (n=104) from male patients. Serum markers such as Bilirubin, SGPT, PT, APTT, and α-fetoproteins were analyzed by commercial kits and Cobas-C 501, Sysmex Ca500 and Cobas-C 601 automatic analyzers as per manufacturer’s guidelines. All patients were above 15 years age and their mean age was 39 years. Male to female ratio was 0.66% (104/157). Age-wise ddistribution revealed that 31-45 years age group comprised majority of the patients, whereas lowest number belonged to age group 61 and above. HCV genotyping data revealed five different genotypes/subtypes. Genotype 3a was most frequent accounting 76.24% (n=199) followed by Genotype 1a (2.29%), 1b (2.68%), 2a (4.98%). Moreover, 13.79% (n=36) samples revealed untypable genotype. Subtype 1b had highest mean VL of 6.84 log10 IU/ml among all other genotypes. The data of serum markers analysis revealed a slight fluctuation but appeared under the normal range. However, Bilirubin was slightly lower in Genotype 1a as compared with other genotypes while elevated α-fetoprotein was found in patients infected with untypable genotypes. In summary, Genotype 3a was predominant genotype throughout Sindh and subtype 1b had highest viral load compared with subtypes 3a and 2a.


Introduction
Hepatitis C virus (HCV) is major cause of hepatitis C with significant clinical problems in humans worldwide. Comprehensive knowledge of HCV genotypes is essential to understand the clinical relevance as the effectiveness of treatment and vaccine development are impacted by genotypes and subtypes. A great genetic variability in HCV has been reported regionally [1]. Moreover, spread of HCV has been shown to occur through blood transfusion and parenteral exposures with contaminated medical equipments [2,3]. HCV has a single stranded positive polarity genome of 9. 6  and lowest from Pano Aqail (0.38%). All patients were above 15 years in age. All HCV positive samples were analysed based on age and gender (Figure 1). Majority of the samples 60.15% belonged to females whereas 39. 85% were from male subjects. Females patients were more affected in all age groups. However, age group 31-45 years comprised highest number of patients followed by 46-60 while the age group 61 and above had lowest number of patients.

Distribution of HCV genotypes
The HCV genotyping data revealed five different genotypes/subtypes in which the genotype 3a was most frequent genotype infecting 76.24% (n=199) of the patients. The second most common variety found in this study was untypable 13.79% (n=36). The prevalence of other genotypes included: Genotype 1a (2.29%), 1b (2.68%), 2a (4.98%) (Figure 2). Whilst three genotypes namely 4, 5 and 6 and their subtypes were not identified from any samples investigated in this study.

Association of HCV genotypes with viral load
Viral Load analysis revealed that patients infected with HCV subtype 1b had higher VL (mean VL 6.84 log10 IU/ml) followed by subtype 3a and 2a ( Figure 3)    little fluctuation, but they were in the normal range. However, Bilirubin was slightly lower in Genotype 1a as compared with other genotypes while elevated α-fetoprotein was found in serum of patients infected with untypable genotypes (Table  1).

Discussion
The present study reports the molecular epidemiology of the HCV genotypes and their influence on viral loads among HCV infected patients from Sindh. Anti-HCV positive patient's serum was used for VL analysis followed by genotyping analysis to determine the frequency of genotypes circulating at Sindh. A few of the studies have been carried out in Sindh, regarding the genotype distribution; however, no recent study has focused on viral load analysis and its association with genotypes. The data demonstrated that females were higher (60.15%) than that of male (39.85%) patients. Among the HCV patients, Genotype 3a accounted for 76.25% of the total infected patients. These results are consistent with one of our recent report that genotype 3 was most prevalent genotypes circulating at Hyderabad, Sindh [19] as well as with other previously published reports across Pakistan demonstrating that the genotype 3 is more common in Pakistan . The second most common subtype in this study was 2a followed by 1b and 1a. Detection of HCV genotype and the viral loads have paramount importance as they aid in prediction of therapeutic outcomes among patients treated with antiviral therapy i.e. interferon plus ribavirin. The patients infected with genotype 1 have been reported to demonstrate higher VL as compared to those patients infected with genotypes 2 and 3 [20, 21]. The present study has shown that the patients infected with genotype 1a yielded higher VL as compared to genotype 2 and 3. Our finding are in agreement with published data [22], however a Chinese study has reported that besides Genotype 1, the genotype 6 was also significantly associated with higher VL as compared to genotype 2 and 3 [23].

Conclusion
In summary, five different HCV genotypes/subtypes were found circulating in the Sindh. Females were higher than male patients infected with HCV and the most commonly affected age group was 31-45 years age. HCV genotype 3a was highly prevalent followed by 2a, 1b and 1a. Viral load analysis revealed highest VL in patients infected with subtype 1b. Periodical surveillance of HCV genotypes, VL analysis and effective therapeutic options for designing the best preventive strategies for HCV infections are needed for clinical management of HCV.