Prevalence of Hepatitis C virus genotypes in nine selected European countries: A systematic review

Background Hepatitis C virus (HCV) infection is a global health problem especially for its increasing level of mortality. Detailed knowledge of HCV genotypes prevalence has clinical relevance since the efficacy of therapies is impacted by genotypes and subtypes distribution. Moreover, HCV exhibits a great genetic variability regionally. To date, there are no published studies assessing HCV genotypes distribution in specific countries of the Mediterranean basin. The aim of this study was to review data published from 2000 to 2017 with the purpose to estimate genotypes distribution of HCV infection in nine European countries all located in the Mediterranean basin. Methods A systematic research of peer‐reviewed journals indexed in PubMed, Scopus, and EMBASE databases selected if containing data regarding distribution of HCV genotypes in nine selected European countries (Albania, Bosnia, Croatia, France, Greece, Italy, Montenegro, Slovenia, and Spain) was performed. Results Genotype 1 is the most common (61.0%), ranging from 80.0% in Croatia to 46.0% in Greece, followed by genotype 3 (20.0%), varying from 38.0% in Slovenia to 7.0% and 8.0%, respectively, in Italy and in Albania and by genotype 4 (10.0%) that shows an increase of 1.1% with respect to data obtained till 2014 probably due to the increasing migrants arrivals to Southern Europe. G2, the fourth most frequent genotype (8.5%), particularly common in Italy (27.0%) and Albania (18.0%) might be probably introduced in Southern Italy as a result of Albanian campaign during Second World War and more and more increased by the migration flows from Albania to Italy in the 90s. Conclusion Epidemiology of HCV infection shows a high variability across the European countries that border the Mediterranean Sea. HCV genotyping is a relevant tool to monitor the dynamic process influenced by both evolving transmission trends and new migration flows on HCV scenario.


Hepatitis C virus (HCV) with about 3-4 million people infected every
year and over 350 000 deaths is one of the main leading cause of liver-related death worldwide. [1][2][3][4] It has been estimated that over 71 million people have chronic hepatitis C infection mainly among populations of WHO Eastern Mediterranean and European Regions. 3,5 Its high mortality rate seems to be essentially due to the fact that persistent HCV infection is often associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). [6][7][8][9][10][11] Seven HCV genotypes have been up to now identified, each comprising multiple subtypes (1a, 1b, and so on) differing from each other by 31%-33% over the whole viral genome. [12][13][14][15] This high genetic diversity poses an obstacle not only for vaccine development but also for an effective antiviral therapy since its duration and response rate may be greatly influenced by the different isolated viral strains.
Detailed knowledge of HCV genotype has a great clinical relevance since the efficacy of therapies measured through the rate of sustained virological response (SVR), defined as the rate of persistent viremia 24 weeks after the end of antiviral therapy, is greatly impacted by genotypes and subtypes distribution. 16 Previously, the standard therapy for the treatment of HCV infections was based on PEG-IFNα/RBV, [16][17][18][19][20] that if compared to other antiviral drugs, seemed to positively influence the rates of SVR. 21 However, it has been widely described that the IFNα/RBV treatment may cause many adverse events (ie, poor tolerability, suboptimal efficacy, and prolonged treatment course) unlike new and expensive direct-acting antivirals DAAs, 21 introduced recently. DAAs, which specifically inhibit viral proteins essential for viral replication, seem to improve the rates of SVR. 22 Thus, it is clear that a better survey of HCV epidemiology, especially focused on the knowledge of different genotypes distribution worldwide, could essentially help to reduce the effects of this severe pandemic disease. 6,23 The geographic distribution of HCV genotypes is heterogeneous and characterized by a distribution of the "epidemic subtypes" (1a, 1b, 2a, and 3a) in high-income countries and of the "endemic" strains in restricted areas, as West Africa, Southern Asia, Central Africa, and Southeastern Asia. 5,24 Regarding Europe, as previously reported, the Global Burden Diseases project subdivides it into three main areas: Central, Eastern, and Western. 25 Europe (4.9% and 5.8%, respectively). [24][25][26][27][28] The European Mediterranean basin is a particularly interesting area regarding this context not only because its shores are close to different geographical areas (Northern Africa and Middle Eastern), each characterized by a greatly different HCV epidemiology and healthcare systems, but especially for the influence thatmigration flows might have had in the last decades. 24

| MATERIAL S AND ME THODS
We conducted a systematic research of peer-reviewed journals indexed in PubMed, Scopus and EMBASE databases selected if containing data regarding distribution of HCV genotypes in nine selected European countries. Among the WHO European countries, we considered only those that geographically shore the Mediterranean Sea, specifically Albania, Bosnia, Croatia, France, Greece, Italy, Montenegro, Slovenia, and Spain. Malta and Cyprus were excluded since no updated data were available.
References were identified through indexed articles found by searching in the abovementioned databases using the following

terms: ''[Country Name] and [hepatitis C or HCV] and [genotypes].''
Articles were scored based on the study sample size (>200 subjects) and the age of the studied population (>20 years old). Studies in nonrepresentative populations (eg, people who inject drugs -PWID's-, hemophiliacs, blood donors, etc) were excluded.
In order to assure a formal evaluation of the methodological quality of the included studies, we decided to use as selection method the PRISMA system (preferred reporting items for systematic reviews and meta-analyses). 29 The PRISMA statement is an evolution of the original QUOROM guideline and consists of a 27-item checklist and a four-phase flow diagram. The checklist includes items deemed essential for the evaluation of a systematic review and referred to every section of the article (title, introduction, methods, and so on). Following this methodology, articles were selected only if responsive of the following inclusion criteria: (a) studies in which the sample population was enrolled from one of the selected countries between January 2000 up to December 2017; (b) English full-text articles concerning the HCV genotypes prevalence from the nine selected countries; and (c) studies considering only HCV-RNA-positive patients in which genotyping has been performed through a standard genotyping method. Sequencing and phylogenetic analysis of the core/E1 or NS5B region are nowadays considered to be the gold standard for HCV genotyping since it allows to accurately identify the subtype, 30,31 we chose only articles in which these methods were used. The currently available commercial techniques for HCV subtyping fail in 2%-10% of samples because of inaccurate genotyping, failure to amplify or to categorize. The majority of commercial assays are based on the amplification of short HCV RNA regions from clinical specimens, followed by a type-specific assay, such as line probe reverse hybridization, 32,33 or sequence analysis. 34,35 On the contrary, we excluded the following articles: (a) stud- Since the majority of selected articles described HCV cases at the genotype level, we preferred to use this classification as proposed by Simmonds et al. 14 . Moreover, we decided to report only data regarding the most common genotypes in the studied area (genotypes 1, 2, 3, 4), while less common ones, as genotypes 5 or 6, or mixed infections were classified as "others." Genotype 7 was not included in the analysis since we did not found any article reporting this genotype in the selected area.
HCV genotypes distribution was investigated through Stata software version 14 (Stata Corp, College Station, TX, USA), and statistical heterogeneity was explored using the I-square at the 5% significance level. Pooled mean proportions were estimated for each genotype and by country using DerSimonian-Laird random-effects meta-analyses.  Table 1.

Hepatitis C virus (HCV) is one of the most common pathogen in
Europe, whose epidemiology greatly varies regionally due to the different role of risk factors, adopted screening programs and antiviral treatment rates. 16,[40][41][42][43][44] In this study, we provide a comprehensive review of HCV epidemiology studies throughout nine selected European countries that  Although the introduction of DAAs has solved most of adverse effects of IFN-based therapy, increasing the rates of SVR the high costs and restricted accessibility of DAA drugs are still the main drivers in the treatment decisions, especially in low-and middle-income countries. Thus, it is clear that in this context, pathogenicity and the duration and cost of treatment are still influenced by different HCV genotypes. 47,[49][50][51] According to our results, G1 is the predominant genotype accounting for 61.2% (95% CI; 53.6-68.7) of all anti-HCV infections among adults, ranging from 80.0% in Croatia to 46.0% in Greece.

Comparing our data with those collected until march 2016 by The
Polaris Observatory, G1 seems to highly increase its prevalence in Croatia (+20.0%) and Italy (+4.0%) while shows a decrease in Slovenia (−14.0%), Spain (−10%), and France (−2.8%). 28 Figure 2). Comparing our data with those collected by The Polaris Observatory, 42 G3 seems to increase its prevalence in Slovenia (+8.0%) and Spain (+9.0%) while shows a decrease in Croatia (−24.0%) and Italy (−3.0%). As previously described, G3 is particularly common among drug abusers especially in West European countries and this may explain its high percentage in the Balkan area. [52][53][54][55] Matching where we reported also an increase in Italy (+3.0%). 24 It may be hypothesized that this increase is related to the increasing migrants arrivals in these countries, especially from Middle Eastern countries in war.
G2 is the fourth most frequent genotype ( Then, the heterogeneity among the studies in terms of size of studied populations could make the results more difficult to clarify.
Furthermore, a certain proportion of patients in several studies is described as mixed infection, preventing to clarify which was the primary genotype.
In conclusion, the epidemiology of HCV infection shows a high variability across the selected European countries, exhibiting a dynamic process influenced by both the changes of transmission trends and the influence of new migration flows in the last years.
Indeed, the 70s and 80s epidemics mainly related to HCV geno-