Determination of HCV genotypes and viral loads in chronic HCV infected patients of Hazara Pakistan

Hepatitis C Virus (HCV) genotype and viral load are two significant predictive variables knowledge of which might persuade treatment decisions. The objective of the present study was to identify the distribution of different HCV genotypes circulating in the study area and to estimate viral load in chronically HCV infected patients. Out of total 305 HCV positive patients, 177 (58%) were males and 128 (42%) were females. Frequency breakup of the HCV positive patients was 169, 69, 38 and 29 from Abbottabad, Mansehra, Haripur and Battagram districts respectively. Out of the total 305 tested serum samples, 255 (83.06%) were successfully genotyped whereas 50 (16.4%) samples were found with unclassified genotypes. Among typable genotypes, 1a accounted for 21 (6.8%) 1b for 14 (4.6%), 2a for 4 (1.31%) 3a for 166 (54.42%) and genotype 3b for (8.19%). Twenty five (8.19%) patients were infected with mixed HCV genotypes. Viral load distribution was classified into three categories based on its viral load levels such as low (< 60, 0000 IU/mL), intermediate (60,0000-80,0000 IU/mL) and high (> 80,0000 IU/mL). The baseline HCV RNA Viral load in HCV genotype 3 infected patients was 50 (26.17%), 46 (24.08%) and 95 (49.73%) for low, intermediate and high categories respectively. For genotypes other than 3, these values for low, intermediate and high viral load categories were 50 (43.85), 35 (30.70) and 29 (25.43) respectively. Pre-treatment viral load in patients with untypable genotype was 19 (38.00%), 5 (20.00%) and 11 (44.00%) for low, intermediate and high viral load categories. Viral load distribution was also categorized sex wise; for males it was 58 (32.76%), 26 (14.68%) and 93 (52.54%) whereas for females it was 40 (31.25%), 34 (26.56%) and 54 (42.18%) for low, intermediate and high viral load respectively. In conclusion HCV genotype 3a is the most prevalent genotype circulating in Hazara Division like other parts of pakistan. Pre-treatment viral load is significantly high (p 0.014) in patients infected with HCV genotype 3 as compared to other genotypes.


Background
Hepatitis C virus (HCV) is a principal cause of chronic liver diseases including liver fibrosis, liver cirrhosis and hepatocellular carcinoma [1,2]. Nearly 170-200 million individuals infected globally including 10-17 million persons in Pakistan [1,[3][4][5]. HCV is an enveloped virus having positive single stranded RNA as its genome that was firstly discovered in 1989 belonging to a virus family Flaviviridae [3,6,7]. HCV genome is approximately 9.6 kb in length with single open reading frame and encodes a polypeptide of 3000 amino acids [8,9]. Total six major HCV genotypes and multiple subtypes have been identified from around the world [10]. Identification of HCV genotype/subtype is extremely important clinically before prescribing therapy because genotypes 1 & 4 show more resistance as compared to genotypes 2 and 3 to PEG-IFN plus ribavarin therapy, therefore different types of HCV genotypes require different duration and dose of anti viral therapy [11]. Treatment durations for genotypes 1 and 4 are 48 weeks where as for 2 and 3 is 24 weeks with PEG-IFN plus ribavarin therapy [12]. Prevalence of viral genotypes has been documented in three different patterns to date [1]. First pattern differentiate by high genetic heterogeneity involves different geographical regions of West Africa with types 1 and 2 [13], Central Africa with type 4 [14] and Asia with types 3 and 6 [15]. Second pattern entails regions with a few subtypes circulating in an intravenous drugs abusers groups, e.g., subtype 3a [16]. The last pattern involves areas where a single subtype is circulate, such as in Egypt with subtype 4a [17] and South Africa with subtype 5a [18].
* Correspondence: idrees.khan96@yahoo.com 4 Division of Molecular Virology, National Centre of Excellence in Molecular Biology University of Punjab Lahore, Pakistan Full list of author information is available at the end of the article Beside HCV genotypes, pre-treatment viral load has also been shown to be prognostic indicator of response to antiviral therapy [19] as increased pre-treatment viral load has been linked with low rates of response to standard interferon therapy [20][21][22]. Several studies have shown that patients with lower pre-treatment viral load (< 80,0000) are more likely to positively respond to currently available antiviral therapy as compared to high pre-treatment viral load (> 80,0000) [23][24][25]. The findings of several studies have also been indicated that a decline in HCV viral load during the first 2-12 weeks of therapy with antiviral treatment indicate to be prognostic of therapeutic efficacy [26,27]. Hence HCV genotype, baseline viral load and decrease of viral load in the stage of initial therapy play important roles in modifying and optimizing antiviral treatment [28].
In Pakistan few large scale studies are available that have been conducted in different districts, cities and towns for the identification of HCV genotypes in chronic HCV infected patients circulating in that particular areas [5,[29][30][31]. No study is available from Pakistan that has correlated pre-treatment viral load with HCV genotypes. Therefore, this study was conducted to identify the distribution of different HCV genotypes/Subtypes and pretreatment HCV RNA viral load in HCV infected patients belonged to different parts of Hazara Division of Pakistan.

Sampling
Total of 305 serum samples were collected from different chronically HCV infected patients for HCV genotyping along with specifically designed data sheets from patients visiting/attending various secondary and tertiary collection centers situated in different cities/towns of Hazara Division of Khyber Pakhtoonkhaw (KPK), Pakistan. Informed consent was taken in written form from each participated patient including, demographic characteristic, age, district, and estimated time of infection along with complete address and phone number of the patients. Written informed consent was taken from each patient.

HCV Qualitative test
Samples were subjected firstly for the detection of HCV RNA qualitatively as previously describe by Idrees [32]. Reverse transcription PCR (RT-PCR) was done for the identification of HCV RNA. RNA was extracted from 100 μl patient's sera using Quigen RNA extraction kit according to the kit protocol. Nested PCR were performed using Taq DNA polymerase enzyme (Fermentas Technologies USA) in a volume of 20 μl reaction mix. The nested PCR products were visualized on 2% agarose gel under "UV" light using "Uvitec" gel documentation system.

HCV Quantitative test
HCV RNA quantification was done by using SmartCycler II Real-time PCR (Cepheid, Sunnyvale, Calif. USA) with HCV RNA quantification kits (Sacace Biotechnologies, Italy). The SmartCycler II system is a PCR system by which amplification and diagnosis were accomplished at same time with TaqMan technology (Applied Biosystems, Foster City, Calif) using fluorescent probes to investigate amplification after each replicating cycle. The lower and upper detection limits of the used assay were 250 and 5.0 × 10 8 IU/mL, respectively. Specimens yielding values above the upper limit were diluted 100-fold, retested and the obtained values were multiplied by this dilution factor to get the actual HCV RNA concentration in international units (IU) per mL.

HCV Genotyping
All qualitative PCR positive sera were subjected to HCV genotyping by using type-specific HCV genotyping procedure as described previously in detail [33]. Briefly, 10 μl (about 50 ng) of HCV RNA was reverse transcribed to cDNA using 100 U of M-MLV RTEs at temperature of 37°C for 50 minutes. Two μl of synthesized cDNA was used for PCR amplification of 470-bp region from HCV 5'NCR along with core region by first round PCR amplification. The amplified first round PCR product were subjected to two second rounds of nested PCR amplifications. One with mix-I primers set and the second with mix-II primers set in a reaction volume of 10 μl. Mix-I had specific genotype primers set for 1a, 1b, 1c, 3a, 3c and 4 genotypes and mix-II contain specific genotype primers set for 2a, 2c, 3b, 5a and 6a.

Discussion
Hazara Division is a historical and beautiful locality of the province Khyber Pakhtoonkhaw (KPK) province of Pakistan. This part of the world has been mapped such that on the North and East lies the Northern Areas and Azad Kashmir, to the South are the Islamabad Capital Territory and the province of Punjab, while the rest of KPK lies to its West. The river forms majority of the western border of the Division by running in a North-south fashion. Hazara Division is one of the most diverse regions based on the ethnic groups it is comprised of, such as Awan, Dhund Abbassi, Gabari, Ghakkar, Gujjar, Jadoon, Karlal, Karlugh, Kohistanis, Maddakhel, Mishawani, Paracha, Shilmani, Syed, Swati, Tahirkheli, Tanoli, Tareen, Turks, Kashmiri, Dilazak and the Isazai Pashtoon respectively [34].
In this study we have studied the distribution of HCV genotypes and associated these genotypes with gender. The results of this study clearly showed that there is no variation among the HCV genotypes and gender as different HCV genotypes were distributed with same ratio between males and females. In agreement with this observation of our study, no significant difference was seen by Idrees and Riazuddin [5] in gender and variation among genotypes in this part of the world. On the other hand in contrast to our observation, in Libya HCV genotypes were not distributed with same pattern between males and females as seen in Pakistan. In Libya, the prevalence of HCV genotype 1 was found to be significantly associated with males while genotype 4 was seen frequently in females [35]. Our results indicated that high prevalence rate 40.32% of HCV infection was found between age group of 30-40. These findings are in agreement with the findings of Ahmad et al, [29]  that highest rate of prevalence was observed in age group of ≤ 40 years and similar findings were also observed by Inamullah et al, [30]. But our findings contradict from that of Muhammad et al, [36] that high HCV prevalence rate in Pakistan was found in old age group people. So these results suggest that early diagnosis of HCV might be due to the awareness of public about HCV infection in this part of the world. Currently a number of studies have been carried out to find the frequencies of various HCV genotypes in different geographical regions of Pakistan and the most prevalent genotype was 3a [5,28,31,37]. Data analysis of the present study showed that genotype 3a (54.42%) has high prevalence rate followed by 3b and 1a in chronic HCV infected patients. These results confirmed the findings of other studies reported from Pakistan that the most prevalent genotype is 3a followed by genotype 3b and 1a, respectively [27,6,5,33,38]. Identified HCV genotypes in our study showed no regional difference and was distributed with same fashion predominant genotype was 3a followed by 3b as has also been reported by Ali et al., [27] from various regions of Pakistan where the authors concluded that there is no regional difference in HCV genotype distribution. Our pattern of HCV genotypes distribution is similar to that reported from neighboring countries like in Iran and India where predominant genotype is 3 [39,40], and is different from South Asian countries such as Japan and Thailand where genotype 1 is the common HCV genotype [41,42].
In the present study 8.19% of the studied patients were found infected by two different genotypes at the same time, majority of these were thalassaemic patients who had received unsafe blood in past. Our results are supported by other studies that show that mixed genotypes are more frequent where blood transfusion is common especially in thalassaemic patients [27]. Franciscus [21] states that mixed genotypes in a single patient may affect the antiviral therapy response and disease succession. We were unable to identify HCV genotypes 4, 5 and 6 in the current study. This observation confirmed the previously reported arguments that these three genotypes are not existing in this region or these are partially absent from Pakistan [27,28].
It has already been reported that patients with higher viral load show lower response rates to standard antiviral therapy as compared to patients having lower viral load [21]. Our findings carry some important implication for the therapeutic hindrance that genotype 3 is the most common genotype in Pakistan [5] and also in our studied patients. Untimely detection and treatment are significant to achieve a high level of sustained virological response (SVR) [43]. Early time detection involves the identification of low HCV RNA level [11]. As determined by Von et al., [21] and Dalgard et al, [22], that shorter therapy schedules told that genotype 3 HCV infected patients with low baseline viral load (HCV-RNA, < 600, 000-800, 000 IU/mL) had more likely to attained a sustained   [21] further recommend that extensive information about HCV genotype and basal RNA viral load is necessary when planning therapy strategies against HCV at national level. These results also help to modify antiviral therapy individually for infected HCV patients with genotype 3 that will reduce economic burden, side effects of antiviral therapy, and also may promote optimize response rates. Finally the results of this study show that more than one genotype of HCV circulates in Hazara Division of KPK, Pakistan. Pre dominant genotype was 3a followed by 3b and 1a.

Conclusions
In the present study, we conclude that HCV genotype 3a is the most prevalent genotype circulating in this region of the world. Regional difference do exists in HCV genotypes. Majority of the infected patients are young ages between 31-40 years. Baseline viral load is significantly high in patients infected by HCV genotype 3 (subtypes a & b) as compared to other genotypes such as 1 (subtypes a, b, c), 2 (subtypes a, b) and untypeable genotypes.