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  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 2  |  Page : 192-196
 

Distribution of hepatitis C virus genotypes in Istanbul, Turkey


Department of Medical Microbiology, Division of Virology and Fundamental Immunology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey

Date of Web Publication7-Aug-2018

Correspondence Address:
Nuran Karabulut
Department of Medical Microbiology, Division of Virology and Fundamental Immunology, Istanbul Faculty of Medicine, Istanbul University, 34000 Istanbul
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_381

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 ~ Abstract 

Purpose: The hepatitis C virus (HCV) has seven main genotypes and multiple subtypes. The distribution of HCV genotypes varies across geographical regions worldwide. Updated estimates of HCV genotype distributions have a critical importance for developing strategies to manage or eliminate HCV infection. The aim of this study was to determine the distribution of HCV genotypes in patients with HCV admitted to a university hospital in Istanbul, Turkey. Materials and Methods: A total of 412 HCV RNA positive patients with 46.6% of males and 53.4% of females between January 2013 and September 2016 were included in the study. Genotyping of HCV of the study population was performed by a commercial reverse hybridisation line probe-based assay. Results: Genotype 1 (82.5%) was dominant genotype, followed by genotype 3 (10.7%), genotype 2 (4.6%) and genotype 4 (2.2%). Among patients with genotype 1, subtype 1a, 1b and undetermined subtype were 6.3%, 38.8% and 37.4%, respectively. It was observed that genotype proportion was dependent on gender and age of the patients. Genotype 1 and genotype 2 were more prevalent in females, whereas genotypes 3 and 4 were more prevalent in males. Genotype 1 in the older patients and genotype 3 in the younger patients were more prevalent. Conclusion: The majority of patients with HCV infection had genotype 1 (82.5%), followed by genotype 3, 2 and 4. Monitoring the change in HCV genotype distribution is critical for the development of effective strategies for HCV elimination.


Keywords: Hepatitis C virus, hepatitis C virus genotype, hepatitis C virus subtype, line probe-based assay


How to cite this article:
Karabulut N, Alacam S, Yolcu A, Onel M, Agacfidan A. Distribution of hepatitis C virus genotypes in Istanbul, Turkey. Indian J Med Microbiol 2018;36:192-6

How to cite this URL:
Karabulut N, Alacam S, Yolcu A, Onel M, Agacfidan A. Distribution of hepatitis C virus genotypes in Istanbul, Turkey. Indian J Med Microbiol [serial online] 2018 [cited 2018 Nov 17];36:192-6. Available from: http://www.ijmm.org/text.asp?2018/36/2/192/238680



 ~ Introduction Top


Globally, chronic hepatitis C virus (HCV) infection affects about 71 million people and contributes to more than 399,000 deaths annually.[1] Chronic hepatitis C infection is often related to liver cirrhosis, hepatocellular cancer, hepatic failure and even death.[2] The World Health Assembly targets to eradicate HCV infection by 2030. It has been suggested that the incidence of hepatitis C infection is significantly reduced in the developed countries, but it has been the mortality rate due to HCV infection-related liver disease will continue to increase in the next years.[3]

HCV, an enveloped, has single-stranded, positive-sense RNA containing a single large open reading frame.[4] It is classified into the family Flaviviridae, genus Hepacivirus.[5] HCV genome has both highly conserved and highly variable regions. The 5' non-coding region (5'UTR), core (C), envelope 1 (E1) and non-structural protein 5B (NS5B) sites of the HCV genome are relatively well-conserved regions and used as the basis for classification. In contrast, the envelope 2 (E2) glycoprotein site of HCV is the most variable region into the genome.[4] HCV genome has high genetic heterogeneity due to the lack of proofreading capability of the RNA-dependent RNA polymerase and high replicative activity of the virus.[6],[7] Based on the sequence divergence, till date, HCV strains are divided into seven main genotypes and multiple subtypes, 67 confirmed and 20 provisional subtypes.[3] HCV genotypes display the sequence divergence at 30%–33% of nucleotide sequence while the different HCV subtypes belonging to a genotype differ by 20%–25% of the nucleotide sequence. Each HCV subtype is comprised of many different quasi-species and these variants have been estimated to differ by <10% of the nucleotide sequence.[8]

The distribution of HCV genotypes varies across geographical regions worldwide. Genotype 1 (GT1), genotype 2 (GT2) and genotype 3 (GT3), particularly subtypes 1a, 1b, 2a and 3a, are extensively distributed globally.[2] In the 70s and 80s, these subtypes termed 'epidemic subtypes' were likely spread through transfusion of contaminated blood and blood products and from drug abusers. Many other HCV subtypes termed 'endemic subtypes' are relatively uncommon and are circulation for a longer time in more restricted areas.[3],[9] Genotype 4 (GT4) is prevalent in Central and North Africa, and genotype 5 (GT5) is found mainly in South Africa. Genotypes 6 (GT6), which is responsible for 1.4% of all HCV cases worldwide, is predominant in Southeast Asia. A new genotype, genotype 7 was identified from a Central African migrant in Canada.[3],[10] Globally, the distribution of HCV genetic variations has probably been affected by global travel increasing, migration between countries and historical events.[6]

New direct-acting antiviral therapies, which target HCV gene products, such as the NS5B RNA-dependent RNA polymerase, the NS5A phosphoprotein and NS3 protease, were revolutionised with minimal side effects, shortened duration of therapy and sustained virological response rates of 60%–100%.[9],[11],[12] At present, selection of a new direct-acting antiviral regimen, duration of treatment and sustained virological response still depend on the HCV genotype and subtype.[12] For this reason, the development of specific national treatment strategies requires knowledge of regional HCV genotype distribution. Until pan-genotypic treatments are developed, sustained virological response, treatment duration and treatment cost will be impacted by the genotype distribution. In addition, curing a greater proportion of patients with specific genotype by the new direct-acting antiviral therapies can affect the distribution of genotypes in the future. For this reason, the relative prevalence of the genotypes that are more difficult to treat might increase.

There are limited data that reflect the epidemiological changes of HCV in Istanbul, Turkey. Therefore, the aim of this study was to determine the distribution of HCV genotypes in patients with HCV admitted to a university hospital in Istanbul, Turkey and to evaluate the changes in the distribution of genotypes during 4 years. An additional purpose was to determine whether there is an association of genotype with gender and age.


 ~ Materials and Methods Top


Study population

This study included 412 HCV RNA positive patients who were performed HCV genotyping at the Division of Virology and Fundamental Immunology, Department of Medical Microbiology, Istanbul University, Istanbul Faculty of Medicine, Turkey between January 2013 and September 2016. Ethical approvals were obtained from the Ethics Committee of Istanbul Faculty of Medicine (reference number: 11/09.06.2017).

Hepatitis C virus RNA extraction, reverse transcriptase polymerase chain reaction and amplification of hepatitis C virus 5' UTR region

HCV RNA was extracted from plasma sample by the Qiagen extraction kit (Germany) according to the manufacturer's protocol. The RNA pellet was reverse-transcribed to complementary DNA (cDNA) and polymerase chain reaction (PCR) was used in the amplification of the 5' noncoding region of the viral genome that is very well protected for genotyping. cDNA and PCR reaction was performed by a one-step reverse transcriptase PCR (RT-PCR) using HCV PM BIO kit (AB Analitica, Italy). To determine the amplified PCR products, agarose gel electrophoresis was applied.

Hepatitis C virus genotyping

Genotyping of HCV of the study population was performed by a commercial reverse hybridisation line probe-based assay (AMPLIQUALITY HCV-TS, AB Analitica, Italy). Six main HCV genotypes and major HCV subtypes (genotype 1, 2, 3, 4, 5, 6 and subtype 1a, 1b, 1a/1b, 2a/2c, 2b, 3a, 4a, 5a, 6a or 6b) can be identified by this assay. Hybridisation of PCR products to membrane-bound genotype-specific HCV sequences is enzymatically revealed. The resulting bands were interpreted according to the manufacturer's instructions. In addition, our laboratory has participated the external quality assessment programme for genotyping assay.

Statistical analysis

Statistical analysis was performed using Software SPSS 21 (SPSS Inc., Chicago, Illinois, USA). Chi-squared and Fisher's exact tests were used for qualitative variables. The Mann-Whitney U test was used for variables with non-normal distribution, which was determined by the Kolmogorov–Smirnov test. Comparison of HCV RNA levels among genotypes was performed by Kruskal–Wallis test. P < 0.05 was considered as statistically significant.


 ~ Results Top


The study population consisted of 412 patients, with a mean ± standard deviation age of 52.84 ± 15.69 years, range 2–82 years. As shown in [Figure 1], GT1 (82.5%) was the most common genotype, followed by GT3 (10.7%), GT2 (4.6%) and genotype 4 (2.2%). Among GT1 patients, subtype 1b was 38.8% and subtype 1a was 6.3%, but subtyping in 37.4% of the samples could not be determined by the current genotyping method. Distribution of HCV genotypes according to demographic and virological characteristics of the patients is shown in [Table 1]. Within GT2, subtype 2a, 2a/2c and undetermined subtype were 0.2%, 1.5% and 2.9%, respectively. Among GT3 patients, subtype 3a and undetermined subtype were found 8.7% and 1.9%, respectively. Subtype 4a and undetermined subtype within GT4 were detected 0.5% and 1.7%, respectively. Genotypes 5 and 6 were not found in the study population. The infection caused by more than one genotype at a time was not detected.
Figure 1: Distribution of HCV genotypes in the study population

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Table 1: Distribution of hepatitis C virus genotypes according to demographics and virological characteristics of the patients

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The study population consisted of 192 (46.6%) males and 220 (53.4%) females. The distribution of genotypes between genders of the patients was significantly different (P = 0.006). GT1 and GT2 were more prevalent in females whereas GT3 and GT4 were more prevalent in males. The median age of the patients among genotypes groups is shown in [Table 1]. The median age of the patients in GT3 group was significantly lower than GT1 and GT2 groups (P < 0.001 and P = 0.019, respectively). The median age of the patients with subtype 1b was significantly higher than the patients with GT2, GT3 and GT4 (P = 0.031, P < 0.001 and P = 0.020).

Distribution of genotypes according to years is shown in [Figure 2]. The frequency of non-1 genotypes was found higher in 2013 and 2014 years than in 2015 and 2016 [Figure 3].
Figure 2: The distribution of the genotypes during 2013–2016

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Figure 3: Distribution of genotype 1 and other genotypes during 2013–2016

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 ~ Discussion Top


The distribution of HCV genotypes shows differences worldwide. Globally, GT1 is the most common genotype accounting for 49.1% of all HCV infections among adults, followed by GT3 (17.9%), GT4 (16.8%), GT2 (11.0%), GT5 (2.0%) and GT6 (1.4%).[3] Since the discovery of HCV, GT1 has become most common genotype worldwide due to subtypes 1a and 1b are associated with the global dissemination as a result of using infected blood and blood products. The international dissemination of GT3 is believed due to subtype 3a is associated with injection drug use and migration from the countries such as India and Pakistan where subtype 3a is dominant.[13]

GT1, the most prevalent genotype in developed countries, is also the most prevalent worldwide and well respond to the second generation direct-acting antiviral therapies with the viral eradication rates of >90%.[3],[9] In Turkey, GT1 has been reported to account for the vast majority of HCV infection, with prevalence ranging 51.7%–97.1%.[14],[15] As similar to other data in our country, in the present study, GT1 was the most common genotype (82.5%). Subtype 1b is the most prevalent subtype in Turkey, with prevalence ranging 56.5%–100%.[5],[16],[17],[18] In this study, it was determined 38.8% subtype 1b, 6.3% subtype 1a and 37.4% undermined subtype within GT1. Along with high undetermined subtypes in GT1, the low rate of subtype 1b in this study reflected the lowest rate in Turkey. Although DNA sequence analysis for HCV genotyping is the gold standard, multiplex real-time PCR with genotype-specific primers and commercial tests based on the line probe reverse hybridisation assays are preferred in the routine diagnostic laboratories, since DNA sequence analysis is laborious and time-consuming.[17] The subtyping analysis based on 5' UTR, on a short part of the genome, may not discern subtypes due to insufficient sequence variation in these highly conserved regions.[4],[9],[19] This information may explain the high rate of undetermined subtype within GT1 in the current study. Kabakçı Alagöz et al.[4] could not determine HCV genotypes of twenty patients by phylogenetic analysis of 5' UTR sequences. In the study, it was revealed that the samples that remained undetermined by analysis of the 5' UTR sequences belonged to subtype 1b by analysis of NS5B and E1 sequences. It was suggested that this result supports the limited success of subtyping analysis based on 5' UTR.

The events causing social changes such as war and migration, the increasing population mobility and various transmission routes affect the epidemiology of infections.[20] GT3, the second most prevalent genotype, particularly in Europe, more common among intravenous drug users, is not susceptible to the first generation of direct-acting antiviral protease inhibitors and also is less susceptible to sofosbuvir, which the most advanced second-generation direct-acting antiviral therapy to date, than other genotypes.[3],[9],[15] There are also important differences in GT3 distribution in Turkey. In a study carried out with samples obtained from seven regions of Turkey, GT3 rate was detected 6.7%.[5] In a study in Antalya, it was reported that the GT3 rate was 11.1%.[17] In the current study, GT3 was the second most common genotype, with 10.7%. This present study was similar to the study in Antalya, but it was observed that it was higher in our country compared to other data. Istanbul is a cosmopolitan city and has intensive touristic activity and migration to this city can explain the differences in HCV genotypes. However, in a study conducted in Kahramanmaras, in the Mediterranean region, the authors reported that the prevalence of GT3 was 46%, obtained the results differed from the country data.[15] Hence, their result has represented the highest rate about GT3 in Turkey.

Although GT1 and GT3 infections are the most common globally, other genotypes are particularly prevalent in low-income countries. GT2 in West Africa and GT4 in Central and North Africa are predominant. The country with the highest prevalence of HCV in the world is Egypt.[10] The especially high prevalence of subtype 4a in Egypt could be the association with unsafe injections during the past schistosomiasis vaccine campaigns,[12] while the dissemination of subtype 4d in Europe likely could be a result of an increase in the number of injection drug users in these areas.[9] In the current study, rates of GT2 and GT4 were found 4.6% and 2.2%, respectively. The GT2 rate in the current study was higher than other studies conducted in most regions of our country. While the genotype 4 rates in our country were usually below 2.2%, it was observed extraordinary high of GT4 rate (32%) in Kayseri, Turkey.[14],[15],[17],[18],[21],[22],[23]

This study showed that genotype proportion was related to gender and age of the patients. It was revealed that GT1 and GT2 were more common in females, whereas GT3 and GT4 were more prevalent in males. In a study conducted in chronically HCV-infected 48595 patients in Spain, it was revealed that HCV GT3 and GT4 were closely associated with male gender; in contrast, GT1 and GT2 were associated with female gender.[2] Another study conducted in West European, Russian and Israeli regions displayed also similar results.[7] The reason may be ascribed that female may be more inclined to blood transfusion within the transmission routes and the main genotypes are GT1 and GT2; male may be more inclined to drug use and high-risk sexual practices and the main genotypes are GT3 and GT4.[3] In this present study, it was observed that GT1 was prevalent in the older patients, while GT3 was dominated in the younger patients. These results were found to be consistent with various studies on age and genotype.[7]

There are insufficient data about the change of HCV genotypes distribution in Turkey over the years. This present study emerged that although GT1 was still dominant, non-genotype 1 rates were higher in 2013 and 2014 than in 2015 and 2016. Istanbul, is a cosmopolitan and the most crowded city of Turkey, has intensive touristic activity and migration to this city can explain the continuous changes in HCV genotypes.

The limitations of the current study included that transmission route/risk group could not be documented of the patients. It could not be performed subtype classification within GT1 for 37.4% of the samples due to the limited success of subtyping analysis based on 5' UTR. Finally, since it was a retrospective study, it could not be performed nucleotide sequencing.


 ~ Conclusion Top


In the studied population, the majority of patients with HCV infection had GT1 (82.5%), followed by GT3, GT2 and GT4. The epidemiologic trends of HCV infection may be influenced within the next years; the role of the past and present immigration, the increase in drug consume and selection of certain genotypes by the current direct-acting antivirals may lead to changes in HCV genotype distribution.

Acknowledgment

The authors thank Murvet Bozacı who routinely serves in the laboratory.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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WHO. Hepatitis C Fact Sheet. Available from: http://www.who.int/mediacentre/factsheets/fs164/en/. [Last accessed on 2017 Oct 27].  Back to cited text no. 1
    
2.
Aguilera A, Navarro D, Rodríguez-Frias F, Viciana I, Martínez-Sapiña AM, Rodríguez MJ, et al. Prevalence and distribution of hepatitis C virus genotypes in Spain during the 2000-2015 period (the GEHEP 005 study). J Viral Hepat 2017;24:725-32.  Back to cited text no. 2
    
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Petruzziello A, Marigliano S, Loquercio G, Cacciapuoti C. Hepatitis C virus (HCV) genotypes distribution: An epidemiological up-date in Europe. Infect Agent Cancer 2016;11:53.  Back to cited text no. 3
    
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Kabakçı Alagöz G, Karataylı SC, Karataylı E, Celik E, Keskin O, Dinç B, et al. Hepatitis C virus genotype distribution in Turkey remains unchanged after a decade: Performance of phylogenetic analysis of the NS5B, E1, and 5'UTR regions in genotyping efficiency. Turk J Gastroenterol 2014;25:405-10.  Back to cited text no. 4
    
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Altindis M, Dal T, Akyar I, Karatuna O, Gokahmetoglu S, Ulger ST, et al. Six-year distribution pattern of hepatitis C virus in Turkey: A multicentre study. Biotechnol Biotechnol Equip 2015;30:335-40.  Back to cited text no. 5
    
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Gong T, Zhao X, Luo Y, Hong Y, Li S, Fu X, et al. Distribution of hepatitis C virus genotypes in volunteer blood donors from Chengdu, China. Arch Virol 2016;161:1873-82.  Back to cited text no. 6
    
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Kartashev V, Döring M, Nieto L, Coletta E, Kaiser R, Sierra S, et al. New findings in HCV genotype distribution in selected West European, Russian and Israeli regions. J Clin Virol 2016;81:82-9.  Back to cited text no. 7
    
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Welzel TM, Bhardwaj N, Hedskog C, Chodavarapu K, Camus G, McNally J, et al. Global epidemiology of HCV subtypes and resistance-associated substitutions evaluated by sequencing-based subtype analyses. J Hepatol 2017;67:224-36.  Back to cited text no. 12
    
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Caliskan A, Kirisci O, Ozkaya E, Ozden S, Tumer S, Caglar S, et al. Distribution and predominance of genotype 3 in hepatitis C virus carriers in the province of Kahramanmaras, Turkey. Hepat Mon 2015;15:e25142.  Back to cited text no. 15
    
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Sağlik İ, Mutlu D, Öngut G, İnan D, Öğünç D, Can Sarinoğlu R, et al. Distribution of hepatitis C virus genotypes among patients with chronic hepatitis C infection in Akdeniz university hospital, Antalya, Turkey: A five-year evaluation. Mikrobiyol Bul 2014;48:429-37.  Back to cited text no. 17
    
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