Back to the origin of HCV 2c subtype and spreading to the Calabria region (Southern Italy) over the last two centuries: A phylogenetic study
Introduction
Infection with hepatitis C virus (HCV) affects more than 170 million people worldwide, and it is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (Hoofnagle, 1997, Lauer and Walker, 2001). According to ECDC estimates, Italy is the European Country with the highest prevalence of HCV positive subjects (ECDC, 2010). Importantly, prevalence is not homogeneous. Higher prevalence rates (12.6–26%) have been reported in Southern Italy and major islands (Ansaldi et al., 2005, Mele et al., 2006). These rates approximate those found in a bordering hyper-endemic area, the World Health Organization Eastern Mediterranean region, where 17 million HCV positive patients reside (World Health Organization, 2012).
HCV has been divided into seven major genotypes and a number of subtypes (Simmonds, 2013). In Mediterranean Countries subtype 1b is the most common followed by genotypes 2–4 (Ramia and Eid-Fares, 2006). In Italy, genotype 2 is the second most prevalent after 1b, and it is mainly found in Southern Italy, particularly in the Calabria region (Ansaldi et al., 2005, Matera et al., 2002). Indeed, over the last decade, among 2153 HCVRNA positive patients, 22.4% were infected with genotype 2a/2c (Marascio et al., 2012). However, the genotype classification was only based on 5’-UTR information provided by INNO-LiPA assay, which is not reliable for correctly identifying the actual diversity within genetic groups (Bouchardeau et al., 2007). Thus, more studies using reliable techniques based on genotyping are warranted.
In the last years, circulation of different strains of genotypes 2 has been described in other European Countries, such as France and The Netherlands. In these Countries, genotype 2 was characterized by a high variability as shown by the presence of numerous subtypes and distinct HCV-2 lineages. It has been proposed that genotype 2 was introduced in Western Countries from Africa during slave trade and colonialism in 17th–18th centuries and then mainly transmitted through blood transfusions and injection drug use (IDU) (Cantaloube et al., 2008, Jordier et al., 2013, Markov et al., 2009, Markov et al., 2012, Thomas et al., 2007).
In Italy, transmission of genotype 2 occurred mainly by a distinct route due to multiple-use of glass syringes up to 1970s (Guadagnino et al., 1997). Dynamics of HCV genotype distribution during eleven years showed that subtypes 2a/2c did not vary significantly over time (Marascio et al., 2012). However, introduction path of genotype 2 still remains unknown, as well as transmission hypothesis and the current epidemiological pathways. Better knowledge of transmission chains is necessary for preventative and screening strategies.
With this objective in mind, phylogeny of HCV 2c was investigated by analyzing a fragment of the NS5B viral genomic region. A Bayesian coalescent-based framework was used to estimate origin and evolution of the HCV 2c epidemics in the Calabria region.
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Sample collection
To conduct this phylogenetic study, we selected only 2a/2c HCV subtypes determined by INNO-LiPA sampled from 2007 to 2011. All patients had to be born and resident in the Calabria region. In order to increase representativeness of the study population, patients were sampled from different areas of the Calabria region. Sixteen isolates were obtained in a population of 1012 inhabitants of the Sersale village located 30 miles from Catanzaro as previously described (Guadagnino et al., 1997,
Patient characteristics
Main characteristics of study patients are illustrated in Table 1. Median age of the 54 patients was 67.3 years (range 26–85). Percentage of females was 61.2%. Surgery and cohabitation with HCV positive individuals were the first (33.4%) and second (3.7%) most frequent risk factors for HCV acquisition reported by patients, followed by dental therapy.
Likelihood mapping
Phylogenetic noise of each dataset was investigated by means of likelihood mapping (Fig. S2. The percentage of dots falling in the central area of
Discussion
In this study, we analyzed 54 HCV-2c samples from Calabria region. Although, samples were originally typed as 2a/2c by INNO-LiPA (Marascio et al., 2012), 100% of them were finally classified as 2c by sequence analysis. Thus, the molecular analysis showed a homogeneous pattern characterized by the unique circulation of subtype 2c and absence of other subtypes. This observation was also reported in a previous study showing that subtype 1b and 2c were the most prevalent genotypes in Italy followed
Conclusions
The present study indicates, for the first time, that HCV 2c epidemic in the Calabria region initiated around 1889, possibly as a result of population movements between Italy and African Countries during Italian colonialism. Since then, it was maintained by the unsafe use of glass syringes until the late 1970s followed by other transmission routes still persisting until 1990s such as surgery and unsafe blood transfusion. So, HCV 2c became one of the most prevalent subtypes in our region, at
Acknowledgement
The authors are grateful to Vincenzo Guadagnino for conducting the original epidemiological survey in the Sersale Village (Guadagnino et al., 1997, Guadagnino et al., 2013).
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