Molecular prevalence of hepatitis C virus genotypes in district Kohat, Khyber Pakhtunkhwa, Pakistan

Hepatitis C Virus is a causative agents of liver cirrhosis, having highly diversified genome. Genotype study has important role in clinical setup and prognosis of hepatitis C virus infection. The aim of this was to investigate the prevalence and risk factors associated with hepatitis C virus genotypes in district Kohat, Khyber Pakhtunkhwa, Pakistan. Hepatitis C virus pre-infected patients (n = 600) were analyzed to assess circulating HCV genotypes, using reverse transcriptase approach. Moreover serum ALT levels were determined followed by correlation with genotypes. In current study cohort, majority of patients (62%) were, having elevated ALT (> 50 U/L). similarly patients with mixed HCV genotypes, 3a/3b have relatively higher ALT levels (~62) compared to 2a/3a (~43) and others. We found significant correlation (p = 0.002) of genotype 2a in connection with ALT (49.48) level. Similarly 3a, 3b, mixed as well as untypeable genotypes correlations were also found highly significant (p <0.001) with their mean ALT levels (47.14, 31.58, 46.69 and 43.68) respectively. In our studied population, most prevalent genotypes were 3a (25%) followed by 3b (20%) and 2a (15%). Fifteen percent of patient’s infections were untypeable while in 10% patients mixed genotype were observed. Among total 30 (5%) were blood transfusion cases, 90(15%) surgical, 540 (90%) dentistry, 360 (60%) Barber, 492 (82%) Pricks, whereas 180 (30%) cases were those having early type of HCV/HBV infection in their family. In current study, genotypes 3a and 3b were more prevalent, with two potential risk factors; dentistry and barbers. Moreover, genotypes and ALT investigations were found to be more fruitful in prognosis as well as management of HCV infection.


Introduction
Hepatitis C Virus (HCV) is a leading causative agent of liver cirrhosis and hepatocellular carcinoma.HCV is a single stranded positive sense RNA virus belongs to the genus hepacivirus of Flaviviridae family [1].It has a single open reading genome of ~9.6 kb, encoding a polypeptide of 3010 amino acids, flanked by untranslated region at both 5ʹ and 3ʹ terminus [2].This polypeptide is post-translationally modified into various structural (C, E1, E2, and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins by viral and cellular proteins [3].HCV possessed extreme genetic heterogeneity due to poor RNA-dependent RNA polymerase (NS5B-protein) activity [4].The route of transmission of HCV shown very complexity but it is more intended on futile neutralizing immune responses including unscreened blood transfusions, unsterilized dental and surgical instruments, unhygienic barber conditions and tattooing [5].Patients carrying HCV infection shows symptoms like acute/chronic liver disease, elevated serum ALT level that finally may lead to hepatocellular carcinoma [6].There are ~200 million HCV patients which account for ~3.3% of the total population of the world [7].Among them, ~10 million people having HCV infection accounting ~4.7% of the total population of Pakistan [8].The first report about HCV was reported in 1992 by screening 45 patients from the northern part of Pakistan [9].There are six major genotypes of HCV and more than one hundred subtypes reported throughout the world [10].Out of these, the genotype-1, genotype-2 and genotype-3 are most popular genotypes in terms of prevalence [10].The prevalence rate varies from region to region and have variation geographically.The prevalence of genotype 1 is a bit high (7.04%),followed by genotype 2 (3.82%).However, the prevalence rate of genotype 3 is very high 78.96%, while it subtype 3a reported 58.01%, followed by 3b (9.76%).The other genotypes such as 4, 5, 6 are very seldom reported in Pakistan.This revealed that the genotype 3a have high prevalence rate at Khyber Pakhtunkhwa, and Punjab.
The Baluchistan province of Pakistan is reported to have high prevalence of genotype 1a and the least prevalence is for genotype 3a [11].There are very high numbers of patients having HCV infection in Pakistan and this number is increasing on daily basis.Therefore, it is the need of today and tomorrow to investigate the risk factors, occurrence and molecular level of HCV genotypes and search for the possible effective treatment in order to stop the transmission and save the lives of the patients having this chronic disease [12].District Kohat is a multicultural and highly populated regions of Khyber Pakhtunkhwa having different communities of the local FATA zones in addition to local people.There is still very few report from district Kohat and need updated information in order to plan and eradicate this infection from local population.

Collection of samples from patients
Total 600 blood samples were collected from HCV positive patients through predesigned questionnaire including patient details i.e. age, socioeconomic status, risk factors, duration of infection, racial origin, and liver function test.Written consents were signed from patients and the study was initiated with proper approval from ethical committee.Samples were transported to the lab immediately and stored for onward analysis.

Antibody screening and ALT analysis
Immuno-chromatographic method was used for the screening of HCV antibodies utilizing the kit (Accurate Diagnostics™ Canada) and the sera of patients were tested by ELISA using commercially available kits (Axen Diagnostic™, Germany).ALT, as hepatocytes marker was investigated according the manufacturer's protocol by using biochemical analyzer (Microlab 200, Merck USA).

Genotyping of HCV patients
Approximately 200 µL of the blood was taken and HCV genomic RNA was extracted according the manufacturer's protocol by using RNA extraction kit of Ultrascript TM (Anagen, USA).The molecular analysis was done with little modification using the Ohno et al., (1997)  Again, 4µL of the first PCR product was further amplified by genotype specific primers (1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a) in two mixtures.Mixture-1 contained antisense primers 1b, 1.D-8 (2a), 2b, 2a and 3b while Mixture-2 composition included the antisense primers 1a, 3a, 4, 5a and 6a.The reaction mixture of both mixtures composed of one unit of Taq polymerase and 3mM MgCl2 having PCR conditions as mentioned for the first round of PCR.PCR products were separated by agarose gel electrophoresis on 2% agarose gel along with 100 bp DNA ladder and later on stained with ethidium bromide and checked under UV illuminator (Figure 1).

Frequencies of ALT levels
All the patients under study were followed for measuring their ALT levels up to six months with specific interval.Significant ALT fluctuations were reported.Moreover, 62% of patients had high ALT levels (≥ 50 U/L) and 38% patients were reported with normal ALT level (≤ 50 U/L).In this study, we have found significant increased (61.79 U/L; mean value) in ALT level.Moreover, we analyzed the ALT level in patients circulating mixed HCV genotypes in their sera, and were found that patients infected with 3a/3b genotypes have significantly high ALT level (~62%), followed by 2a/3a (~43%) compared to other co-infected HCV patients.These elevated ALT level signify a drastic effect of HCV among individuals (Figure 2).

General prevalence of HCV genotype
The high prevalence rate among different HCV genotypes was reported for 3a (25%), followed by 3b (20%), 2a (15%), 1a (5%) and 4 (5%).However, the genotypes such as 1b, 6a, and 5a were not reported among the patients under investigation.There was 15% patients which indicated no genotype and 10% of the patients showed mixed infection of HCV genotypes.The mixed genotypes were reported in decreasing order as followed; 2a/3a (45%), 3a/3b (35%) and 5% in cases of 2a/2b, 3b/1a, 1a/2a and 4/2a (Table 2).The genotypes prevalence in terms of age groups is reported as: genotype 1a was with high prevalence rate in age group of ≤20 years followed by 41-60 years; genotype 2a had high prevalence in age groups 21-40 years, followed by age group of ≤20 years, then 41-60 years with lowest rate in 61-80 years; genotype 2b was only reported in age group of 61-80 years; genotype 3a was found in all age groups with relatively equal frequency among all age groups (Table 3); genotype 3b was also high in age groups of 41-60 years followed by 21-40 years and 61-80 years with prevalence rate of 30% each; genotype 4 was only reported in age group of 61-80 years; The occurrence of mixed genotype was reported high in age groups 41-60 years and 61-80 years with 30% prevalence rate in each cases, whereas 6% rates were observed from the cohort of age 21-40 years and ≤20 years; Moreover, the prevalence rate of untypeable genotype was high in the age groups of 21-40 years followed by 61-80 years and 41-60 years with least frequency rate (9%) in ≤20 years among the cohort (Table 3 and Figure 3).

HCV genotypes and alanine amino transferase correlation
Alanine amino transferase (ALT) is the most potent hepatocytes marker to detect the degree of liver cirrhosis as a result of ongoing HCV infection.We analyzed serum ALT level for a period of six months and correlate with the respective HCV genotypes.A significant correlation (p = 0.002) of genotype 2a with ALT (49.48) level was reported.Similarly 3a, 3b, mixed as well as untypeable genotypes were found highly significant (p <0.001) with their mean ALT levels (51.33, 47.14, 46.69 and 43.68), respectively.However genotypes 1a, 2b and 4 were observed with lower prevalence to meet statistics criteria (Table 3).In similar fashion, different risk factors studied has shown high prevalence 90%, 82%, 60%, 30%, 15% and 5% in case of dental procedure, unhygienic pricks, barbers intervenes, family history, surgical cases and blood transfusion, respectively.The unsterilized instruments at barber shop, operational theater, and dental surgery are the main causes of HCV infection.In addition, the unscreened blood transfusion and family history have a major role in HCV

Discussion
[13] method.Then cDNA was made from viral mRNA by using 10µL of RNA with 1µL (200U) of RevertAid TM M-MuLV reverse transcriptase enzyme (Fermentas) at 42 o C for 30 min followed by 92 o C for 2 min in thermocycler (Techne, USA).The cDNA was amplified in first round of PCR using forward and reverse primers, one unit of Taq polymerase and 3mM MgCl2 at 94 o C for 3 min followed by 92 o C (45 Sec), 52 o C (45 Sec) and 72 o C (1 min) with final extension at 72 o C (10 min) in PCR.

Figure 2 .
Figure 2. Alanine amino transferase (ALT) level (mean) in patients infected with HCV mixed genotypes

Figure 3 .
Figure 3. Age-wise distribution of HCV genotypes among recruited HCV patients Hepatitis C virus become asymptomatic at the beginning of infection, called as silent killer.HCV has no clear clinical feature and varies depending on the age, sex, genotype and viral load of the one individual to another individual [14].The main objectives of the study were to investigate the risk factors and prediction for early diagnosis in different age, sex and genotypes based on the serum marker evaluation.Most of the patients were reported as illiterate (~62%) and of lower income class (77%).This revealed that mostly poor and uneducated people have high exposure to HCV virus due to their unawareness of the risk factors and transmission routes of HCV infection.

Table 1 . Frequency of HCV related risk factors among recruited patients Risk Factors Status Frequency of Patients Percentage Pricks (needle type)
176bp (Mix A) 232bp (Mix B) 3a & 3b (Mixed Genotypes)