|Year : 2014 | Volume
| Issue : 1 | Page : 53-56
Distribution of Hepatitis C virus genotypes in city of Mashhad, North-east of Iran
M Rastin1, M Mahmoudi1, SA Rezaee1, MA Assarehzadegan2, N Tabasi1, S Zamani1, R Nosratabadi1, D Haghmorad1, A Sheikh1, M Khazaee1, HR Panah1
1 Immunology Research Center, BuAli Research Institute, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
2 Department of Immunology, Ahvaz Joundishapur University of Medical Sciences, Ahvaz, Iran
|Date of Submission||16-Mar-2013|
|Date of Acceptance||16-Sep-2013|
|Date of Web Publication||4-Jan-2014|
Immunology Research Center, BuAli Research Institute, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad
Source of Support: None, Conflict of Interest: None
Purpose: Six major hepatitis C virus genotypes have been characterised, which vary in their geographical distribution. The prevalence of hepatitis C virus (HCV) in an area is not constant, and depends on the changes in route of infection, which may change over time. In this study, the distribution of HCV genotypes in Mashhad, the capital of Razavi Khorasan province in north-east of Iran was investigated. Mashhad is a holy city of Shiate Moslems, which attracts more than 20 million tourists and pilgrims every year. Materials and Methods: Two hundred and seventy-eight HCV infected subjects (227 males and 51 females) were included in this study. HCV genotypes were analysed by type specific reverse transcriptase polymerase chain reaction (RT-PCR). Results: Genotype 3a was detected in 49.6%, 1a in 36.3%, 1b in 12.6% and 2a in 0.4%. Two HCV genotypes were detected in 1.1% cases; 1a +3a in 1%, 3a + 1b in 0.4%. Genotypes 2b and 3b were not detected in any samples. Conclusion: We demonstrated that despite the previous reports on the frequency of HCV genotypes in Iran, 3a is the predominant genotype in Mashhad.
Keywords: Geographical distribution, hepatitis C virus genotype, hepatitis C virus, hepatocellular carcinoma
|How to cite this article:|
Rastin M, Mahmoudi M, Rezaee S A, Assarehzadegan M A, Tabasi N, Zamani S, Nosratabadi R, Haghmorad D, Sheikh A, Khazaee M, Panah H R. Distribution of Hepatitis C virus genotypes in city of Mashhad, North-east of Iran. Indian J Med Microbiol 2014;32:53-6
|How to cite this URL:|
Rastin M, Mahmoudi M, Rezaee S A, Assarehzadegan M A, Tabasi N, Zamani S, Nosratabadi R, Haghmorad D, Sheikh A, Khazaee M, Panah H R. Distribution of Hepatitis C virus genotypes in city of Mashhad, North-east of Iran. Indian J Med Microbiol [serial online] 2014 [cited 2020 Dec 3];32:53-6. Available from: https://www.ijmm.org/text.asp?2014/32/1/53/124306
| ~ Introduction|| |
More than 170 million people worldwide are chronically infected with Hepatitis C virus (HCV).  HCV is a positive-sense single-stranded RNA virus, which may develop into hepatocellular carcinoma and cirrhosis of the liver.  HCV genome shows a high rate of mutations; therefore a considerable genetic heterogeneity of HCV exists in infected individuals from all over the world. 
It has been demonstrated that HCV genotypes vary in their distribution,  which may indicate the route of acquisition  and affect the clinical outcome and response to therapy.  Before any treatment, the genotype of the virus should be determined to estimate the duration and the dose of the antiviral agents and the virological monitoring procedure. 
So far, at least 6 major genotypes and 12 subtypes of HCV have been characterised, which are different in their geographical distribution.  Genotypes 1, 2 and 3 are the most frequently encountered genotypes worldwide.  The most common subtype in south and north-west of Iran are genotypes 1a and 3a, respectively,  while genotype 1a predominates in all parts of Iran. 
The prevalence of HCV genotypes in a geographic area is not constant, and depends on changes in route of infection which may alter over the time.  Mashhad, the capital of Razavi Khorasan in North-east of Iran is a city with about 3 million population. Razavi Khorasan shares borders with Afghanistan in the east and Turkmenistan in the north. The shrine of the eighth Shiat Imam is located in Mashhad and more than 20 million people make the pilgrimage every year to this city. Therefore, investigating the HCV genotypes in infected individuals in Mashhad might help to understand the effect of tourism on genotype distribution in this region.
Several methods including restriction fragment length polymorphism, differential hybridisation, single-strand conformation polymorphism, probe melting curve, type specific polymerase chain reaction (PCR), and serological typing have been used for HCV genotyping. Although direct sequencing,  however, molecular biology-based techniques such as type specific PCR for core region of HCV is also a reliable and accessible method for genotyping of HCV-infected subjects. ,
In the present study, type specific PCR was carried out for HCV genotypes in the city of Mashhad.
| ~ Materials and Methods|| |
Plasma samples from 278 subjects with chronic HCV infection were collected during 2000-2010. The study was approved by ethic committee of Mashhad University of Medical Sciences, Mashhad Iran. Informed written consent was taken from all participants. The mean age of patients was 41.3 (± 12.1) years. The samples were sent to the molecular section of Immunology Research Center of Mashhad University of Medical Sciences for HCV genotyping. The study group comprised of 51 (18%) females with mean age of 43.9 (± 11.6) years and 227 (82%) males with mean age of 40.96 (± 12.44) years. All samples were primarily tested for the presence of antibodies to HCV with a commercial ELISA kit (Radim, Italy) according to the manufacture's instruction.
Viral RNA was extracted from plasma samples using QIAmp Viral RNA mini Kit (QIAGEN, the Netherlands) according to the manufacturer's instructions. Ten microlitre of extracted RNA was utilised for cDNA synthesis using random hexamer primers and M-MuLV reverse transcriptase (Roche Diagnostics GmbH, Germany). A qualitative nested reverse transcriptase PCR (RT-PCR) was performed to confirm the presence of HCV RNA. Then HCV genotyping was performed for every sample by two different approaches; a type specific PCR method, which was developed in our laboratory, and also a commercial kit (AmplySens, Russia).
HCV genotyping using in-house developed type specific PCR method
HCV genotyping by type specific nested RT-PCR was performed using primers specific for the core region of HCV genome as previously described by Okamoto et al. with some modifications [Table 1]. The outer primers amplified a 271 bp in the first round of the PCR. The second round PCR was performed using a universal sense primer and 6 type specific antisense primers designed to amplify 1a, 1b, 2a, 2b and 3a genotypes. The primers were modified on the basis of available data in Gene Bank.
|Table 1: Primers used in household PCR for HCV genotyping in this study. In fi rst run of PCR Fout-1 and|
Fout-2 were used as outer forward primers and Rout was used as reverse primer. In second run of PCR Fin-1 and Fin-2 were used as internal forward primers and R-1a, R-1b, R-2a, R-2b, and R-3a were used as reverse primers
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First round PCR was carried out on 25 μl reaction; 3 of each dNTPs (Roche Diagnostics GmbH, Germany), 1 unit Taq DNA Polymerase (Roche Diagnostics GmbH, Germany), 1.5 mM MgCl and 0.4 μM of each primer (Fout-1 + Fout-2 as forward and Rout as reverse). The thermal cycler (Biometra, Germany) was set for 35 cycles as follow: 94°C for 1 min, 61°C for 1 min and 72°C for 1 min. The second PCR round was performed with a mixture of Fin-1 and Fin-2 as forward and 6 antisense primers [Table 1] for 30 cycles with the following parameters: 94°C for 1 min, 61°C for 1 min and 72°C for 1 min. The type specific PCR products were then run in a 3% agarose gel containing ethidium bromide and photographed by an IMAGO gel documentation system (B and L systems, The Netherlands).
The expected size of PCR products for 1a, 1b, 2a, 2b and 3a genotypes were 57, 144, 174, 123 and 87 bp, respectively.
HCV genotyping using a commercial kit
A commercial (AmplySens, Russia) kit with hot start protocol was used for HCV genotyping according to the manufacturer's instructions. PCR products were then separated by 2% agarose gel electrophoresis. The expected size of PCR products for 1a, 1b, 2 and 3a genotypes were 338, 395, 286 and 227 bp, respectively.
The results are expressed in form of mean ± SD or percentages. The Chi-square and Mann-Whitney U tests were used for data analysis. A P value less than 0.05 was considered significant.
| ~ Results|| |
All samples were primarily tested for the presence of HCV antibodies and positive samples were confirmed by qualitative nested RT-PCR.
HVC genotyping was confirmed in all the 278 samples by two different methods as described earlier. HCV genotype 3a was detected in 138 (49.6%), 1a in 101 (36.3%), 1b in 35 (12.6%) and 2a in 1 (0.4%) samples [Figure 1]. Three patients presented co-infection with different genotypes; two with 1a + 3a (1.1%) and one with 3a + 1b. In two cases of co-infection, the predominant genotype was 1a and in one case it was 3a. Genotypes 2b and 3b were not detected in any samples.
Patients who were infected by HCV genotype 1a have higher viral load than patients with genotypes 3a and 1b (1.331.790 copy/ml versus 894.657, and 385.842 copy/ml for 1a, 3a and 1b genotypes, respectively). Female HCV-infected participants in this study had lower viral load in all detected genotypes in comparison to male participants (571.125, 424.280 and 155.682 copy/ml in female, versus 1.383.152, 981.763 and 481.742 copy/ml in male for 1a, 3a, and 1b, respectively) [Table 2].
Most participants were drug abusers with a history of using shared needles. Out of the 278 participants, 16 were patients with thalassemia or haemophilia, which were likely infected by transfusion of infected blood or blood products. Nine of these patients were infected with HCV genotype 1a (56.2%). Genotype 3a was present in six (37.5%) of these patients. The HCV viral load in these patients was similar to total participants (1.215.956, and 868.433 copy/ml in thalassemia or haemophilia patients versus 1.331.790, and 894.657 copy/ml in total participants for 1a and 3a, respectively). The mean age of participants who were infected with HCV genotype 3a was significantly lower (37.9 ± 11.0 years) in comparison to those who were infected with genotype 1a (44.4 ± 12.4 years) (P = 0.02). [Table 3] shows the relevant data.
There was no significant difference between the results of HCV genotyping by household PCR and commercial methods.
| ~ Discussion|| |
HCV genotyping has epidemiological importance, and influences the response and duration of antiviral therapy, thus it affects the outcome of disease.  HCV genotypes vary in their geographical distribution, and the prevalence of HCV genotypes in one area may change over the time, because of the differences in the route of acquisition,  and immigration patterns. 
City of Mashhad is a pilgrimage region with a population of 3 million, which is close to the borders of Afghanistan and Turkmenistan. This holy city for Shia Moslems attracts more than 20 million tourists and pilgrims every year from inside and abroad of Iran (mostly Afghanistan, Pakistan, Azerbaijan, Turkmenistan and Middle-East countries). Therefore, we expected to have different HCV genotypes in Mashhad in comparison to other parts of Iran.
At least six major genotypes of HCV have been characterised worldwide.  In north European countries and in North America, the most prevalent HCV genotype is 1a,  while in south and east Europe, genotype 1b is dominant.  In central and South Africa, genotypes 4 and 5 are prevalent.  In South Asia, Pakistan , and India, , genotype 3 is more common. In Arab countries in the Middle-East, genotype 4 is most common,  while in non-Arab countries, genotype 1 predominates. 
Some previous studies in south of Iran have demonstrated that the predominant genotype is 1a  while in north-west region, genotype 3a have been observed with a higher prevalence.  In most parts of Iran, genotype 1a is predominant while in northern parts, genotypes 3a and 1a have nearly the same distribution (38.9% vs 44.4%, respectively).  Thus it seems that the sample size, except for the samples collected from the central parts is not adequate, and simply not enough reliable evidence to get to firm conclusions. In a more recent study,  which was performed on 206 samples from 16 provinces of Iran, it was reported that genotype 3a is dominant in all parts of Iran. In a study in Isfahan (a large city in the centre of Iran), which was performed on 97 HCV-positive participants, genotype 3a was the predominant genotype in this part of Iran.  In another HCV genotype study in Fars province (in central southern of Iran) on 188 HCV positive subjects, genotype 1a was observed in 44.1%, while 3a was seen in 42.0% of cases. 
It seems that by increasing the sample size and/or by concentrating on smaller regions in HCV genotype studies more reliable results may be achieved. Our study was performed on 278 (51 female and 227 male) HCV positive samples from city of Mashhad. We demonstrated that in this population, the most predominant genotype were 3a (49.6%), followed by 1a (36.3%) and 1b (12.6%).
Our data showed that despite high number of visitors from neighbouring countries, such as Arab countries (Iraq, Qatar, Bahrain, Saudi Arabia, Kuwait, Syria, UAE and Lebanon) with predominant HCV genotype 4, , and from northern countries such as Tajikistan and Turkmenistan with the predominant 1b genotype; in Mashhad, 3a is the dominant HCV genotype.
In the past 30 years, due to political situation in Afghanistan, a considerable number of Afghan immigrants were added to Mashhad population. These immigrants usually do a lot of travelling between Iran, Afghanistan and Pakistan. There are no reliable reports regarding HCV genotypes in Afghanistan; however, several studies have shown that the most predominant HCV genotype in Pakistan is 3a. ,
In conclusion, our study showed that the high rate of HCV 3a genotype is a unique genotype in this region. Thus, further studies with large sample size in other parts of Iran might change the current concept of HCV genotypes frequency.
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[Table 1], [Table 2], [Table 3]
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