Hepatitis C infection has been considered to be the major cause of most of the chronic liver diseases worldwide. The mortality rate because of hepatitis C virus (HCV) infection from primary liver diseases encompassing liver cirrhoses and hepatocellular carcinoma has been steadily increasing over the years 1,2. HCV was identified in 1989, belonging to the family Flaviviridae and the genus Hepacivirus 3. It is an enveloped RNA-containing virus with a genome size of 50 nm 4. The HCV genome is a single open reading frame with 9600 nucleotides 5. About 15–40% of HCV-infected individuals achieve clearance of the virus from their bodies during the acute phase of infection and the rest of the 60–85% of infected individuals develop chronic hepatitis 6. Almost 170 million individuals are infected by HCV worldwide and 3–4 million new cases are diagnosed each year 7. Because of limited resources and facilities, the prevalence of HCV in developing countries is higher compared with the developed world 8 Pakistan is a developing country with a population of about 170 million and has an alarmingly high rate of HCV infection 9. HCV transmission may occur because of contaminated blood transfusion, surgical instruments, dental procedures, drug abuse, and unsafe sex 10. It is also transmitted through sharing of razors, tooth brushes, and shaving in the barber shops 10,11. Blood transfusion is another chief route of HCV transmission in Pakistan. This is because of lack of resources, weak infrastructure, ill-equipped resources, untrained medical staff, and unreliable screening of blood donors for anti-HCV 12. Six HCV genotypes (major) and hundreds of subtypes have been identified worldwide so far 13,14. The genotype distribution of HCV is different in different geographical areas 15,16. HCV genotypes are clinically very important and have been used widely for epidemiological studies, vaccine development, response rates to anti-HCV therapy, and medical administration of the infection 17. HCV genotypes are distributed worldwide; genotypes 1, 2, and 3 are prevalent worldwide, whereas the other types of HCV genotypes differ in distribution from one geographic region to another. HCV-1b and HCV-1a subtypes are mostly prevalent in Europe and America, respectively 14,18–20; in Japan, subtype 1b is responsible for about 73% cases of HCV 21, and 2b and 2a subtypes are frequently found in Europe, North America, and Japan 21. Genotype 4 is most commonly prevalent in the Middle East and North Africa 22,23. Very few studies have been carried out in all provinces of Pakistan on the epidemiology of HCV genotypes 24,25. The majority of studies on determination of HCV genotypes have shown that 3a is the most common genotype present in Pakistan, with a prevalence ranging from 75 to 90, and also the most responsive to interferon therapy 26,27. The aim of this study was to examine the incidence of various HCV genotypes prevalent in the different districts of Punjab and to determine the pattern of HCV genotypes in various age groups and sexes.
Materials and methods
A total of 542 HCV-positive samples were obtained from various collection centers situated in different regions of Punjab (the most populated province of Pakistan, with almost 45% of the country’s total population) for HCV genotyping during the study period of July 2011–March 2012. All the research procedures were performed in the Genome Centre for Molecular Based Diagnostics & Research (GCMBDR; Lahore, Pakistan). GCMBDR is a state-of-the-art molecular-based diagnostic laboratory with responsive, precise, and consistent detection tests on a cost-to-cost basis utilizing state-of-the-art PCR and real-time PCR procedures. The serum samples were stored at −20°C for further analysis. Of the total of 542 HCV patients, 300 were men and 242 were women. Serological and biochemical data of these patients were recorded. When samples were categorized according to the origin/geographical regions, it was found that 245 patients were from Lahore, 61 from Sargodha, 56 from Multan, 56 from Toba Tek Singh, 53 from Faisalabad, 27 from Rawalpindi, 20 from Mandi Bahauddin, 16 from Gujranwala, and eight from Sahiwal districts (Fig. 1). A written informed consent was not required for this study from our patients as this investigation was a part of the reliable procedure in a regular workup of Molecular Diagnosis of the GCMBDR.
Viral RNA isolation and hepatitis C virus RT-PCR amplification
From a 100 μl serum sample, HCV RNA was isolated using the Anagen RNA Isolation Kit (Anagen Technologies Inc., Atlanta, Georgia, USA) following kit protocol procedures. Qualitative nested PCR was performed for the detection of HCV RNA as described previously 7. Briefly, cDNA was synthesized using M-MLV reverse transcriptase (Fermentas Thermo Fisher Scientific, Vilnius, Lithuania) for 50 min at 37°C using 1 μmol/l of the outer antisense primer. First-round and nested PCRs were performed with Taq polymerase. PCR-amplified products were subjected to electrophoresis on a 1.2% agarose gel stained with ethidium bromide and evaluated under an ‘ultraviolet’ gel documentation system.
Hepatitis C virus genotyping
HCV genotyping was performed for all the qualitative PCR-positive samples using a specific HCV genotyping assay as described earlier 28. Viral RNA was extracted and reverse transcribed to cDNA using 100 U of M-MLV reverse transcriptase for 50 min at 37°C. From the synthesized cDNA, 2 μl was used for PCR amplification of the 470-bp fragment from HCV 5′UTR besides the core region by first-round PCR amplification. HCV has 12 major genotypes; thus, the genotype-specific primers were divided into two sets on the basis of differences in the sizes of the various required bands so that no type-specific bands were of similar size in a similar set on the agarose gel. PCR products of the first round were subjected to two second-round nested PCR amplifications; first with mix-I primers that were genotype specific for 1a, 1b, 1c, 3a, 3c, and 4 genotypes, and second with mix-II primers that were type specific for 2a, 2c, 3b, 5a, and 6a genotypes. PCR second-round amplified products were electrophoresed on a 2% agarose gel for the identification of genotype-specific bands.
All the statistical analyses were carried out using the statistical software SPSS version 16.0 for windows (IBM SPSS Statistics Inc., Chicago, Illinois, USA). Findings for all the parameters were presented in the form of rates percentage. To test for a significant relationship among the categorical parameters, Student's t-tests and χ 2-tests were used. The data are presented as mean values. P-value less than 0.05 was considered to be significant.
In the present study, a total of 542 HCV-positive patients ranging in age from younger than 10 years to older than 60 years had maximum viral loads and were thus appropriate for HCV genotyping.
Distribution pattern of hepatitis C virus genotypes in the population studied
Table 1 shows the prevalence pattern of HCV genotypes in enrolled patients. Of the 542 patients, the prevalence rate was higher in male patients [55% (n=300)] than in female patients [45% (n=242)]. Of the total 542 processed serum samples, genotype-specific PCR bands were found in 476 (87.82%) patients, whereas 50 (16.4%) samples were found with undetermined genotypes as no genotype-specific bands were observed in these patients. In our results, 3a was found to be the most prevalent genotype. It was detected in 386 (71%) patients, followed by 1a in 37 (7%), 1b in 18 (3%), and 3b in five (1%). Thirty (6%) patients were found to have mixed genotypes and 66 (12%) patients had undetermined genotypes.
Age distribution of hepatitis C virus genotypes in infected patients
The prevalence of HCV genotypes in patients of various age groups is presented in Table 2. The most frequent incidence of 45 (68.18%) was found between the ages of 21 and 40 years compared with younger and old age groups.
Distribution of hepatitis C virus genotypes in various districts of Punjab
Table 3 shows the distribution of HCV genotypes that were documented from HCV patients from different districts of Punjab. Of the 542 genotypes, 245 (45.20%) were from Lahore. Among 245 (45.20%) genotypes from Lahore, 181 (73.9%) were 3a, 17 were 1a (7%), seven were 1b (3%), three were 3b (1%), mixed genotype was found in 14 (6%), and the remaining 23 (9%) patients had an untypable genotype. A total of 27 (5%) patients were investigated from Rawalpindi district and 20 (74%) were found to have 3a, four (15%) to have 1a, two (7%) to have a mixed genotype, and one patient (4%) had an encompassing untypable genotype. Among the total of 61 (11.25%) patients from Sargodha district, 48 (79%) had genotype 3a, five (8%) had 1a, two (3%) had 1b, two (3%) had mixed genotype, and four (7%) patients had an untypable genotype. Among the 20 (3.79%) analyzed samples from Mandi Bahauddin, nine (45%) were found to have 3a genotype, two individuals had 1a and 1b genotypes, respectively, two (10%) patients had a mixed genotype, and seven (35%) patients had an untypable genotype. Of the total of 56 (10.33%) patients from Multan district, 39 (70%) had the 3a genotype, one (2%) had genotype 3b, six (11%) had genotype 1a, two (4%) had genotype 1b, two (4%) had a mixed genotype, and the remaining six (11%) had an untypable genotype.
From Faisalabad district, 53 (9.77%) genotypes were identified; of these, genotype 3a was identified in 31 (58%) patients, 1b in a single individual (2%), four (8%) individuals had a mixed genotype, and 17 (32%) had an untypable genotype. From Sahiwal district, a total of eight (1.47%) patients were analyzed and all (100%) were found to have genotype 3a. From Toba Tek Singh district, 56 (10.33%) genotypes were examined; the predominant genotype identified was 3a in 37 (66%) patients, one (2%) had 3b, four (7%) had genotype 1a, four (7%) had genotype 1b, four (7%) had a mixed genotype, and the remaining six (10.71%) had an untypable genotype. A total of 16 (3%) genotypes were detected from Gujranwala district; 13 (81%) had 3a genotype, one (6%) had genotype 1b, and two (13%) had an untypable genotype. Genotypes 3a (P=0.0001), 1a (P=0.001), and untypable genotypes (P=0.03) were circulating significantly high in all the studied districts, whereas 1b (P=0.06), 3b (P=0.05), and mixed (P=0.09) genotypes were not distributed significantly in the studied area.
Prevalence of hepatitis C virus mixed genotype in hepatitis C virus-infected patients
In total 30 patients, mixed genotypes were detected as these patients had two genotypes at the same time in their blood. Of these 30 patients, 14 were from Lahore, two from Rawalpindi, two from Sargodha, two from Mandi Bahauddin, two from Multan, four from Faisalabad, and four from Toba Tek Sing districts, respectively. Twelve of the HCV samples were found to be infected by 3a+1a, followed by 3a+3b detected in six (20%), 3a+1b in six (20%), 1a+1b in four (13%), and 1a+3b in two (7%) as shown in Table 4.
Several epidemiological studies have been carried out worldwide to determine the incidence of different HCV genotypes. Although these studies have helped resolve epidemiological issues, they are also useful in therapeutic assessments when prescribing therapy to HCV patients; thus, it has been found that the severity of disease, response to treatment, and prognosis depend on different factors, out of which genotype is the principal factor 7,29. Worldwide, the socioeconomic burden of HCV infectivity is increasing very rapidly; thus, there is a need to obtain better and reliable information on viral pathogenesis to develop new HCV therapeutic strategies. Considerable local differences were identified in the prevalence of HCV genotypes in Pakistan 7; thus, information on the determination of different HCV genotypes in this country is very important for the predictive implications in chronic HCV patients. In the present study, the distribution of different HCV genotypes in various districts of the Punjab was determined. Data analysis showed that the prevalence of HCV genotype 3a (71%) was significantly high (P=0.0001) in patients from Punjab province of Pakistan. These results confirmed the findings of previous studies carried out in different parts of the country showing HCV-3a as being the most prevalent genotype distributed in different parts of the country 30,31. Similar to our findings, almost the same pattern of HCV genotypes has been reported from other Asian countries including India and Nepal, where the most frequent genotype was 3a 32,33, but different from South Asian countries such as Vietnam, Thailand, and Japan, where mostly HCV genotype 1 is prevalent 34,35. Next, a significantly increasing genotype in our data interpretation was 1a (7%, P=0.001), and these findings were in agreement with those of a previously published report of Idrees and Riazuddin 7 from Pakistan that suggested that the incidence of HCV genotype 1 is increasing without an increase in any other genotype in Pakistan, and in the next 10–15 years, the currently most common 3a genotype may be replaced by the less common 1 (a or b) genotype. This changing pattern of the HCV genotype may lead to a more complicated situation in terms of HCV management for our country in upcoming episodes due to weak response against current treatment for genotype 1 as compared to genotype 3. The trend toward identification of more than one genotype at the same time is also significantly increasing in Pakistan 36. In our study, 6% of the patients showed more than one genotype of HCV, with the predominant combination of 3a+1a (40%), followed by 3a+3b (20%) and 3a+1b (20%), 1a+1b (13%), and 1a+3b (7%). Franciscus 37 reported that dual subtypes in a single patient may influence the anti-HCV therapy response and disease progression. In our results, undetermined/untypable genotypes (912%) were significantly distributed in all the studied districts of Punjab (P=0.03). A large number of undetermined genotypes have also been reported previously by other studies carried out in Pakistan 7. Sequencing of these genotypes is required for future treatment of the disease.
Results were further analyzed to determine the incidence of HCV infection with different genotypes in different age groups. The highest prevalence was found in individuals between 21 and 40 years of age. Our results were in agreement with the observations of Ahmad et al. 38; they reported a high frequency of HCV in individuals younger than 40 years of age, and the same observations were also made by Ali et al. 39. However, our results were different from the previously published report that the most common HCV infection in Pakistan was found in the elderly 40. This might be because of lack of awareness programs and untimely diagnosis of HCV infection in the general population of this area.
In this study, the prevalence pattern of HCV genotypes and the relationship of these genotypes with sex were also investigated. These findings evidently showed no variation among the HCV genotypes and sex, as all HCV genotypes were distributed in similar ratios in men and women. In agreement with the observation of our study, no significant difference was reported by Ali et al. 39; all the HCV genotypes studied were distributed equally in male and in female patients. However, our results were in contrast to the findings in Luxembourg, where genotype 3 was statistically more prevalent in men, whereas genotype 2 was more prevalent in women 41. Several limitations exist in our interpretation of the results. We do not have any data on various risk factors to correlate the circulated genotypes in our studied population to possible routes of transmission, and the facilities to sequence the untypable genotypes are unavailable.
The most prevalent genotype in the studied population was 3a, followed by 1a. The prevalence of genotype 1a is increasing significantly in our country and it will make the present situation difficult in terms of antiviral therapy. Undetermined genotypes are also increasing statistically in patients. The most affected age group was 21–40 years. All the HCV genotypes were equally prevalent in men and women.
The authors acknowledge and express our heartfelt gratitude to the GCMBDR for providing the HCV-positive samples and all the clinicians and patients (involved in this study) for their cooperation. The study was financially supported by GCMD.
Muhammad Waqar and Asad U. Khan collected epidemiological data; Asad U. Khan and Bibi N. Murtaza gave a critical view of manuscript writing. Zeeshan Niaz, Mujaddad U. Rehman, Muhammad Idrees, Muhammad Waqar, Amjad Ali, Zobia Ismail, Muhammad Tariq, Muhammad Wasim, and Bibi N. Murtaza analyzed and arranged the data. All authors read and approved the final manuscript.
Conflicts of interest
There are no conflicts of interest.
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