Background

Hepatitis C virus is an infectious disease that is a kind of a single-strand positive RNA virus and the main cause of liver cirrhosis [1]. It is one of the known agents that affect the normal functioning of the liver and causes acute and chronic infection [2, 3]. Hepatitis C is one of the issues posed by the World Health Organization and threatens about 3% of the world’s total population (approximately 170 million people) [4]. Genotype plays an important role in pathogenesis and hepatitis infection [5]. According to the latest studies, the hepatitis C virus had seven genotypes and at least 67 subsets [6]. In many studies, there is a close relationship between the hepatitis C genotype and the response to interferon (IFN) and pegylated interferon (Peg-INF), which can be effective in combination with ribavirin [7]. Hepatitis C genotypes are very important because they can provide valuable information and accordingly, can determine the duration of antiviral treatment, tolerance, or resistance to the hepatitis C virus [7, 8]. It should be noted that the prevalence of hepatitis C genotypes can be different and may show different sensitivities to viral treatment (4). Based on previous studies, it had been showed that genotypes 2 and 3 have a higher sensitivity to antiviral therapy, while the success rate of these genotypes was higher than other genotype 1 [9]. The prevalence of the hepatitis C genotype is different between countries and geographical areas [10, 11]. Worldwide, the prevalence of genotypes 1, 2, and 3 is higher than the other genotypes [12]. In Iran, the prevalence of hepatitis C infection is significantly lower than in neighboring countries. Distribution and abundance of genotypes 4 to 6 varied geographically, so genotype 4 was more distributed in North Africa and the Middle East, genotype 5 in South Africa, and genotype 6 in Southeast Asia [13]. In Europe and America, predominant subtypes 1a and 1b have been observed and have caused disease (10, 11). According to reports in Iran, there are 1a subscales (47%) and 3a (36%) [14]. Genotype 1 was more prevalent in other countries such as Russia and Belarus [15,16,17,18,19].

Material and Method

Study Population

This is a cross-sectional study in which studied the patients with confirmed hepatitis C and the selected patients simultaneously had hepatocellular carcinoma. These 235 patients were selected among the patients who were admitted at Tabriz hospitals between June 2013 and June 2019. The clinicopathological features of patients are summarized in Table 1. To prove that patients with chronic hepatitis C also have hepatocellular carcinoma, a pathologist with specific diagnostic tests and/or biopsy confirmed it. An informed consent was obtained from each patient, and the ethical approaches were approved by the ethics committee of Tabriz University of Medical Sciences.

Table 1 Clinicopathological features of patients
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Collection and RNA Extraction of Samples and Genotyping

Blood samples were collected from each patient and 5 ml of them were poured into the EDTA-containing tubes, and then, the plasma was kept at 80 °C. RNA extraction was done according to the manufacturer protocol. cDNA synthesis and HCV genotyping were done according to manufacturer protocol too (Qiagen, Netherland).

Statistical Analysis

Statistical analyses were performed using SPSS version 17 software and analyzed by T test and square tests. The mean (P < 0.05) was statistically significant.

Results

In this study, 235 patients with chronic hepatitis C who had hepatocellular carcinoma were selected prior to the use of antiviral drug treatment. The genotypes were as follows: genotype 1b was found in 71.1% of patients (165), genotype 3a in 17% of patients (40), genotype 2 as the most common genotype by affecting 6.8% of patients (16 subjects), genotype 1a in 1.7% of patients (4 people), and genotypes 1b and 3a which were infectious in 62.5%. It is noteworthy that in 6 cases, the combination of HCV infection in blood compounds was detected statistically significant (P value = 0.031). The HCV genotypes of these patients were determined based on nucleotide sequencing (Tables 2 and 3).

Table 2 Demographic data and prevalence of HCV genotype in Azerbaijani patients in health centers of Iran-Tabriz
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Table 3 Demographic data and the prevalence of HCV genotypes among male and female Azerbaijani patients of health centers of Iran-Tabriz
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The mean age of the patients was 38/3 ± 11.8 (range 2–63). Among 235 Azerbaijani patients, 139 (59.1%) were male. Statistically, the mean number of women was more than men (T test, P < 0/05).

In patients over 40 years old, genotype 1b was 70%, whereas in patients less than 40 years, it was 71.6%, which was not statistically significant. Also, the incidence of serotype 3a was higher among the patients younger than 40 years old (18.1% vs. 15%), and this serotype was prevalent among men (18.7% vs. 14.6%), which was statistically significant (T test, P < 0.05).

Discussion

The HCV genotyping is one of the epidemiological parameters. Among 235 patients with hepatic carcinoma who were confirmed to have chronic hepatitis C, the most frequent genotypes of HCV in these patients were as follows: genotype 1b was the highest one (71.1%) followed by genotype 3a with 17%, genotype 2 with 6.8%, genotype 1a with 1.7%, and HCV hybrid genotype was 3.4%.

It is little known about the prevalence of HCV genotypes in different regions such as the Soviet Union, where hepatitis C is an endemic disease (19). Although this research was carried out for the first time in Eastern Azerbaijani, it was impossible to compare the results of this study with other cases. According to related articles, there were reports of the prevalence of hepatitis C genotype in the Soviet Union, which was consistent with the results of this study, so that this genotype is prevalent among the Soviet population, such as genotype 1b in this study. The dominant genotypes in the Soviet Union are as follows: Russia 76% (19), Belarus 53.8% [20], Estonia 71% [21], Moldova 89% (19), Uzbekistan 64.2% [22], Tajikistan 84% [23], Lithuania 54% [24], Latvia 85% [25], and Georgia 59% [26]. Table 4 shows the comparison between the results of our study with other countries. Also, the prevalence of HCV genotypes, and their serotype in Arab countries [24, 27,28,29,30,31], Middle East, and non-Arab countries [28, 32], is presented in Table 4.

Table 4 Distribution of hepatitis C virus genotypes and subtypes in the former Soviet Union and the Middle East
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It is thought that the differentiation of various serotypes including 1b which occurred about 70 to 80 years ago [37] is due to the Soviet separation and then the Bolshevik revolution in 1917 which makes it be more than 80 years old. Serotype 1b is mainly from blood and blood products have been transmitted (19), while in one of the centers in Russia, the incidence of serotype 3a among drug users and the general population was very high (18, 20).

The prevalence of HCV genotypes varied over the years. For example, in 1997 (19), strains 1b (90%) and 3a (10%), and in 2003 (22), strains 1b (64.2%) and 3a (25%) have been reported in Uzbekistan (22). This distribution of HCV genotypes in these countries indicates changes that need further study. On the other hand, genotype 1b was found to be 71.1%; 3a (17%) and 2 (6.8%) were the dominant genotypes. Therefore, most studies focusing on this issue should be used to determine the prevalence of HCV genotypes among the Azerbaijani population.

Also, in patients younger than 40 years, the incidence of serotype 3a was 18.1% vs. 15.0% (P < 0.05). These results are consistent with the recent findings, indicating a high prevalence of serotype 3a among young people in Germany [38], Iran [39], and Slovenia [40].

As a final remark of this research, dominant genotypes of HCV among the Azerbaijani patients with chronic hepatitis C were as follows: serotype 1b approximately 71.1%, serotype 3a about 17% and serotype 2 approximately 6.8%. This study was performed to determine the frequency of HCV genotypes in patients who had hepatocellular carcinoma; however, it should be done using larger sample size and a large population to determine the HCV genotype in different populations of the Republic of Azerbaijan.