Introduction

Hepatitis C has been a global health problem with a well-known importance since the identification of the Hepatitis C virus (HCV) in 1989. During the last 15 years seroprevalance of HCV has been increasing and more than 185 million people are tought to be infected with this virus all around the world (1,2,3). Initially, HCV was thought to be the most common cause of post-transfusion related hepatitis and almost not changing the life expectancy in infected individuals but later on studies shown that 85% of the HCV cases become chronic and HCV is one of the most common cause of deaths due to liver cirrhosis and the reason for liver transplantation. In our country, HCV is the second most common cause of chronic viral hepatitis after Hepatitis B virus (4).

After the full-length genome sequences of HCV strains up to date 7 genotype and more than 100 subtypes have been defined based on on phylogenetic and sequence analyses (5,6). HCV genotypes are expressed in numbers from 1 to 7 and subtypes are expressed in lowercase letters such as a, b, c, etc. Each HCV genotype differs from each other by at least 20% at the nucleotide level and by more than 15% at the amino acid level (6). Moreover, base sequence changes within the same genotype can be observed at a rate of 5-8% in the nucleotide sequence and at a rate of 4-5% in the amino acid bases. The global geographical distribution of HCV genotypes is various. Some genotypes appear to be seen in a particular region of the world. In North America, genotype 1a predominates, whereas genotype 1b, which is more commonly associated with aggressive liver disease, is more common in Western Europe and Japan. Genotype 2 is less common in Europe than Asian countries such as China, Japan and Taiwan. Genotype 3 is usually found in the UK and Thailand, while genotype 4 is seen in Middle East region and Central Africa. Genotype 5 is observed in South Africa and genotype 6 in Hong Kong (7). Geneotype 7 has been reported to from Democratic Republic of Congo in Central Africa (6,8). The most common observed genotype in Turkey is the genotype 1b, followed by genotype 1a (9,10).  Besides 7 genotypes, there are 67 confirmed and 20 possible subtypes of HCV identified with at least 15% genomic variation; genotypes 5 and 7 have a single subtype, while at least seven subtypes are observed in other genotypes (11).

The most important problem of post-mortem serological analysis is that its validity. Higher positive results was observed in some studies more than expected (12,13,14). For this reason, it is recommended to use sensitive kits for post-mortem serological examinations (15). Unfortunately, most of the kits used in routine microbiology laboratory for post-mortem serological examinations were not validated for post-mortem samples. Due to loss of specific reactions, the post-mortem samples show decreased sensitivity and increased false-negative results (15). For these reasons, anti-HCV antibody tests was not used in the blood samples of autopsy cases. The success of post-mortem microbiological investigations depends on the adequacy of the post-mortem sampling protocol and strategy (16).  Firstly, it is recommended to perform the autopsy preferably within the first 24 hours after death. Microbiological samples should be obtained as soon as possible. In particular, it is emphasized that blood samples should be drawn at the beginning of autopsy (17). As a general principle, the samples should be obtained at room temperature as soon as within 2 hours, stored in suitable transport and storage environments, and if samples was needed to transferred to the study laboratory should be done within 48 hours at 2 to 8 °C (18). The most common drawbacks of post-mortem serological and molecular examinations occurs because of insufficient quantity or bad quality of blood samples, or samples obtained at inappropriate time intervals (19,20).

The aim of this study was to determine the distribution of HCV genotypes and subtypes in blood samples that were determined by polymerase chain reaction (PCR) in autopsy cases in Turkey.

Materials and Methods

This study was done in Turkish Ministry of Justice Council of Forensic Medicine Post-mortem Microbiology Laboratory, in İstanbul. Forty HCV positive autopsy cases were included in this study between years 2014 to 2018. This study was approved by the Ethics Commitee and Research and Scientific Research Commission of the Ministry of Justice Council of Forensic Medicine (approval number: 21589509/2019/125).

Blood samples were drawn from large vessels (femoral artery, femoral vein, jugular vein) for serological and PCR evaluations of autopsy cases. The blood samples were transferred to EDTA tubes and sent to the laboratory as fast as possible. After centrifuging the post-mortem blood samples at 10.000 rpm for 10 minimum, the resulting plasma samples were aliquoted and stored at -80 °C until the assay. 400 µl of the plasma samples were removed and RNA isolation was performed on the QIA symphony device with the QIA symphony DSP Virus/Pathogen Midi kit, and the amplification of the RNA was performed on the Rotor-Gene® Q device (Qiagen, Germany) by the RT PCR method using the Artus® HCV-PCR kit. For determination of the HCV genotypes (RTA) HCV Genotyping qPCR Kit [targeting NS5b and NS3 of viral 5’-UTR region obtained by real time (RT)-PCR] (RTA, Kocaeli, Turkey) was used. The Kit identifies the six major and most common HCV genotypes (1, 1a, 1b, 2, 3, 4, 5, 6). Analysis was carried out on the CFX C1000 Touch instrument (Bio-Rad, Hercules, USA) and genotypes were determined.

Statistical Analysis

The statistical software program of SPSS (Version 16) was used in the data analysis of the study. The descriptive statistics were expressed as numbers and percentages.

Results

Of the 40 cases included in the study, 35 (87.5%) were male and 5 (12.5%) were female, and the mean age was 43.1±11.8 years (range: 23-69 years). Among the 40 cases, 18 (45%) were Turkish citizens and 16 (40%) were foreign nationals. We could not reach nationality of 6 (15%) cases (Table 1). Demographic features, HCV genotype distribution, clinical history, laboratory results and the autopsy results of the study population were shown in Table 2.

By using the RT-PCR, HCV genotype 3 was determined in 11 (27.5%) of the samples, HCV genotype 1a in 9 (22.5%) samples, HCV genotype 1b in 7 (17.5%) samples, HCV genotype 2 in 2 (5%) samples, and HCV genotype 4 in 2 (5%) samples. HCV genotype could not be determined in 9 (22.5%) samples. The most common HCV genotype in the study cases was genotype 3, while the most common HCV genotype in Turkish citizens was genotype 1a. The genotype distribution of Turkish citizens and foreign nationals was shown in Table 3.

We found that 22 (55%) autopsy cases had a history of intravenous drug usage. All of the 22 cases were male, 10 of them were Turkish citizens. The HCV genotype distribution of 22 samples was as follows: genotype 3 in 7 (31.8%), genotype 1a in 6 (27.3%), genotype 2 in 2 (9.1%), genotype 1b in 1 (4.5%) and genotype 4 in 1 (4.5%) sample. The genotype could not be determined in 5 samples (22.7%). Among the 10 Turkish citizens with history of intravenous drug usage; genotype 1a was found in 5 (50%) of them, genotype 3 in 2 (20%) of them, and genotype 1b in 1 (10%) of them, and genotype could not be detected in 2 (20%) of them.

Study Limitations

There are also several limitations to our study, the rate of genotype 3 was found to be which is higher than the rates reported previously from our country. So, it is not possible to reach a definitive conclusion due to the low number of cases in our study. The second limitation, in our study, we used an automated PCR based method. Although the RT-PCR method has advantages such as being user independent, standard, automatic and yielding fast results, but in our study, the genotype of HCV subtypes could not be determined in 22.5% of cases.

Conclusion

The most common detected HCV genotype has been reported to be genotype 1, at a rate of 46% all around the world (21). In our country, while the most common HCV genotype in hepatitis C patients between years 1995 and 2014 was found to be genotype 1b (22,23), but most recently, genotype 3 was found to be the most common genotype in intravenous substance addicts, and in prisoners in different two studies (24,25). Although genotype 3 has been reported at low rates (0-4.5%) in studies conducted in different centers in Turkey, after year 2010, genotype 3 detection rates has been increasing significantly (26). In our study, the most common genotype detected in study samples was genotype 3 (27.5%), and the genotype 1a (44%) took the first place in samples from Turkish citizens. Our findings show that the presence of foreign nationals in the HCV genotype distribution conflicts with our country data. Events that cause social changes such as war, migration and tourism affect the epidemiology of infections (23). In our study, the heterogeneity among genotypes may be related to demographic changes in our country due to its geographical location. We thought that the presence of high number of foreign nationality in our cases may be responsible for excessive detection of genotype 3 (27.5%). On the other hand, the rate of genotype 3 was found to be 22.2% in Turkish nationality cases which is higher than the rates reported previously from our country (27,28). Although it is not possible to reach a definitive conclusion due to the low number of cases in our study, it may be concluded that genotype 3 is becoming more common in our country. The main route of transmission of HCV is parenteral. After initiation of screening programs in blood and blood products, intravenous drug usage has become the main parenteral route of transmission. In European countries such as France, Germany, Italy and Sweden 30-59% of all HCV infections are associated with intravenous drug usage. In America, this rate reaches 68% (29). The anti-HCV antibody positivity prevalence among intravenous drug users in Turkey has been reported to be 28.9% (30). In another study, HCV infection was determined in 47% of intravenous drug users that were mostly adolescents and young adults (31). In addition, the lack of attention to sterilization and disinfection during the medical procedures is also one of the most common causes of HCV transmission in our country (32). In a study conducted in our country in young people with intravenous drug addiction, the most common genotype was determined to be genotype 1a (26). In our study, 22 (55%) patients had a history of intravenous drug use and all were male. Ten (45%) of these drug users were Turkish citizens. In our cases among the IV drug users genotype 3 (7/22; 31.8%) was found to be the most common genotype. On the other hand, among the Turkish IV drug users genotype 1a (5/10; 50%) was found to be the most common genotype in our study. Of the all our autopsy cases cause of death was found to be related drug intoxication in 20 (50%) cases. For those reasons, much more prospective studies should be focused on people with HCV infection who are intravenous drug addicts.

There are various methods available for HCV genotyping. Techniques such as restriction fragment length polymorphism, allele-specific PCR and line probe assay are widely used to identify the HCV major and subtypes that are very common in North America, Europe and Japan. However, it has been stated that the standard reference and gold standard method comprise sequence analysis of HCV NS5, core, E1 and 5’-UTR regions and subsequent phylogenetic analysis (12). In our study, we used an automated PCR based method. Although the RT-PCR method has advantages such as being user independent, standard, automatic and yielding fast results, but in our study, the genotype of HCV subtypes could not be determined in 22.5% of cases. Like our study result, the genotype could not be determined in 9%, 25% and 27.3% of study population done in our country (13,14,33).

To our knowledge, this is the first HCV study with post-mortem cases in our country. More comprehensive molecular epidemiological studies are needed to understand the ways in which HCV enters our country and how it spreads. We think that import cases may affect the HCV biodiversity, and the detection of genotype 3 frequency in our country. Therefore, an effective HCV surveillance system should be established.

Ethics

Ethics Committee Approval: This study was approved by the Ethics Commitee and Training and Scientific Research Commission of the Ministry of Justice Council of Forensic Medicine (approval number: 21589509/2019/125).

Informed Consent: It wasn’t obtained.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Concept: N.Z., N.E., Design: N.Z., Ö.Y., M.N.A., Data Collection or Processing: N.Z., Ö.Y., Analysis or Interpretation: N.Z., N.E., Ö.Y., Literature Search: N.Z., N.E., Writing: N.Z., Ö.Y., N.E., M.N.A.

Conflict of Interest: The authors declare no conflict of interest.

Financial Disclosure: The authors declare that this study has not received any financial support.