Hepatitis B virus (HBV) infection is one of the most important human diseases with about two billion people infected worldwide, including 240–280 million with chronic hepatitis B.1 HBV-infected individuals can develop cirrhosis, hepatocellular carcinoma and other hepatic injuries.2 More than 600,000 people die each year due to clinical hepatic complications by HBV infection.3

Taxonomically, HBV belongs to the Hepadnaviridae family and presents a partial double strand DNA genome of approximately 3200 base pairs.4 Ten HBV genotypes (A to J) have already been described, with nucleotide sequences divergences greater than 8% in the entire viral genome. In Brazil, the most frequent genotypes are A (58.7%), D (23.4%), and F (11.3%), with A more frequent in Southeast, North and Northeast regions, while D predominates in the South region.5 Most genotypes present subgenotypes that may lead to different diagnostic and clinical profiles.4

HBV transmission occurs via contact with infected blood and body fluids (WHO 2016). Infection via contacts with medical/dental devices,6,7 piercings use,8 tattoo,9 and illicit drugs use (principally injected) have been reported.10 Further, this transmission may also occur in the family context11,12 and sharing of personal objects with family members is strongly associated with HBV transmission.13 In this sense, recent studies have reported important associations between HBV transmission in intrafamilial environmental.13–15

HBV vaccine has been used in Brazil in the last 20 years.16 Immunization programs started in the infant population in the 1990s and gradually covered the rest of the population. Vaccination coverage for HBV reaches approximately 50% of the population and the effectiveness is estimated at 60%.10,16 Despite elderly patients have higher rates of HBV infection (most of them were infected before the availability of the vaccine), contamination has been demonstrated in all age groups.16

HBV therapy is also largely used and aims to prevent progression to more severe clinical conditions as cirrhosis and hepatocellular carcinoma. The most used drugs in the last years were immunomodulators, such as interferon-alpha and pegylated interferon alpha, and nucleos(t)ide analogues, such as lamivudine (LAM), adefovir (ADV), entecavir (ETV), telbivudine (LdT) and tenofovir (TDF).17 LAM was the main antiviral drug used in the HBV treatment, but this drug was associated with high rates of drug resistance-related mutations in the HBV polymerase.18 Currently, the treatment of choice is TDF (disoproxil or alafenamide) since it has a high genetic barrier for drug resistance. The combination of therapies is not commonly recommended.17

Laboratory diagnosis is usually based on serology. Previous studies demonstrated markers for HBV infection in 12.7% (anti-HBc positive) and 0.7% (HBsAg positive) of the Brazilian population.16 The highest HBV detection rate in the country (17.2 cases per 100,000 inhabitants) was observed in South Region, but even there the HBV prevalence was low. In this same geographic region, HBV frequency also increases with age: 1.6% anti-HBc positive in the range of 10–19 years old versus 11.3% among those aged 20–69 years.10 In this whole region, some areas have been more studied because of the high HBV prevalence. It is noteworthy that these areas are generally of Italian descendants and with a high prevalence of genotype D.19–21

The present report is a cross-sectional study aimed to assess epidemiological risk factors for HBV infection, as well as to determine HBV genotypes/subgenotypes and resistance mutations in a sample from Bento Gonçalves, a city with a high prevalence of this disease in southern Brazil.

HBV infection is a serious health problem in Brazil. Recent data demonstrated a prevalence rate higher than the average in some cities and regions in South Brazil.19,21 Bento Gonçalves is one of these cities, with an incidence of 41.5 cases per 100,000 inhabitants.29 The whole city is geographically located in the mountains of the Rio Grande do Sul state, a region colonized by European immigrants, mainly Italians. Therefore, socio-demographic characteristics and risk factors were evaluated to identify the frequency of epidemiological characteristics associated with HBV infection in this population.

In this study, the socio-demographic characteristics of HBV infected patients were having elementary school or less (53.9%), be married (77.5%), living in rural area (66.7%), having ≥ 5 siblings (45.1%), of white skin color (74.5%), and of Italian ancestry (four grandparents) (64.9%). Further, considering risk factors, the patients evaluated had 41.2% of siblings, 27.5% of mothers, and 10.8% of fathers HBV infected. STI, blood history of transfusion, tattoo, body piercing, and previous use of glass syringe were identified in 15.7%, 21.6%, 7.8%, 4.9%, and 49.0% of the sample, respectively. Smoked drugs use was the most frequently reported (6.9%). Additionally, sharp objects use (19.6%) and vertical transmission (15.7%) were the most reported means of HBV infection. Importantly, there was a high frequency of HBV-infected family members among the study sample. In a previous study, conducted in a city geographically and culturally close to Bento Gonçalves (Caxias do Sul), important associations were detected between intrafamilial transmission of HBV,30 a fact that justifies our findings. Evidence also corroborates this type of transmission in Northern Brazil.13 Currently, phylogenetic approaches reinforce the context of intrafamilial transmission in different studies.14,15,31

Over 50% of patients had at least one relative infected with HBV demonstrating the importance of HBV transmission in the family environment, mainly in childhood as previously observed in southern Brazil.30 In addition, there is evidence of a high frequency of HBV infection in siblings (75%) born to HBsAg-positive mothers (p <0.01) as observed in other study.13

Alcohol consumption was also investigated in the present study. We observed that 13.7% of patients had alcohol use disorder by AUDIT scores. Considering the additional damage that alcohol ingestion may produce in patients with chronic hepatitis B, these findings highlight the importance of identifying the pattern of alcohol ingestion in patients with chronic liver diseases.32 In this study, frequencies for illicit drugs use of 6.9% for smoked drugs, 1.9% for sniffed drugs, and 0.9% for injected drugs were observed. Alcohol consumption and drugs use (especially injected) had already been identified as relevant risk factors for HBV infection.10 In Brazil, association between heavy alcohol consumption and HBV infection was previously seen in one region (Southeast), but not in others (Central-West, North, Northeast, and South).10 Further, in our country, illicit drugs use has also not been associated with HBV infection in a nationwide study.33 However, sniffed and inhaled drugs was shown to be associated with HBV in South Brazil in a more recent investigation.10 Finally, in the Southern region of the country (Caxias do Sul City), HBV infection was not associated with neither illicit drugs or alcohol consumption.30

In this study, 15.7% of the sample had a history of STI. This classical risk factor (STI) was not shown to be associated to HBV infection in Southern Brazil in a recent study.30 On the other hand, other studies found an association between the history of STI and infection for HBV in a multicentric population-based study in Central-West, Northeast and Federal Districts of Brazil,33 and in population-based survey in North, Southwest, and South of Brazil.10

The frequency of body piercing and tattoo were 10.6% and 7.8%, respectively. Although these esthetic features have been associated with HBV infection,8,9,33 studies in Southern Brazil have not detected any association.10,30 In this study, 49.0% and 21.6% of HBV-infected people had previous use of glass syringe and blood transfusion, respectively. These factors were shown to be associated with HBV infection in a previous study in Southern Brazil.10,33 Further, in this study, the frequency of share of personal objects in the family environment was of 50.0%. This variable was associated with HBV in other previous studies.13,30,34 Generally, sharing razor, cutlery, face towels, and toothbrush with one infected individual is strongly associated with HBV transmission.10,13,30

Additionally, we evaluated the frequency of HBV genotypes among chronically infected patients in Bento Gonçalves, Southern Brazil. Previous studies reported the major prevalence of genotypes A, D, and F among Brazilians.5,19,21 Our results revealed marked predominance of genotype D, with a minority of patients infected with genotype A. Moreover, no other genotype was detected in the present study. The high prevalence of genotype D is in agreement with studies conducted in Southern Brazil.19,21 However, this high prevalence of genotype D in Southern Brazil differs from the findings of the whole country (A is the most frequent genotype).5 This regional epidemiological difference must be taken into account because of the continental extent of the country.

Phylogenetic studies have been able to trace geographic routes and speed of dissemination of HBV genotypes and subgenotypes.35 Socio-demographic characteristics influence these processes, with the population profiles defining the migration and speed of HBV dissemination.19 These variables are useful for the characterization of risk factors for hepatitis B and definition of the clinical evolution of the disease and responses to antivirals.15,31,36 In the present study, the subgenotype D3 (81.0%) was the most frequent, followed by D2 (14.3%), and only one (4.7%) case of D1. The same scenario of D3 subgenotype was observed in cities with wide Italian immigration in southern19,21 and southeastern Brazil.15 In addition, the predominance of D3 and D2 subgenotypes was reported in the Missiones region of Argentina, which has a high frequency of Eurodescendant inhabitants.36

The results obtained also allow us to observe that D subgenotype samples were divided into seven branches in the phylogenetic tree, of which they have a high genetic identity, probably having the same evolutionary ancestry of South Europe. In this sense, studies have reported that D3 subgenotype is largely prevalent in Italy.37,38 One could than hypothesize that this subgenotype probably emerged from this region to the Southern of Brazil, especially in the historical period of immigration.

HBV genotyping and sequencing studies provide a better understanding of the risk factors for infection, prognosis and treatment for hepatitis B.15,31,35,36 Phylogenetic analyses conducted in different regions of the world have demonstrated that migratory processes influence the distribution of HBV genotypes and subgenotypes.35 In addition, phylogenetic inferences allow the identification of intrafamilial transmission of HBV, due to the similarities of the viral sequences found in relatives sharing the same domestic environments, as observed in different studies.14,15,31 However, future studies that evaluate HBV phylodynamics and phylogeographic will be useful for the understanding of viral migration and transmission routes in Southern Brazil.

In the present study, the most frequently detected amino acid substitutions in the RT enzyme were Y135S (80.9%) and I266V (66.7%). However, there is no evidence in the literature that Y135S and I266V mutations are related to antiviral resistance. Further, LAM-resistant mutation rtM204I was observed in only one patient, but this patient also presented the compensatory mutation rtL180M. The rtA181V, defined as the signature of ADV-resistant mutations, was also observed in another patient. In this sense, is important to identify antiviral-resistant mutations when using nucleos(t)ide analogues therapy.39 Some HBV mutants have the capacity of exhibiting resistance to antivirals, enhanced virulence, facilitated cell attachment or alteration of epitopes which are important in host immune response.40

Particularly, in our study, ethnicity and familial issues seem to have influence in the distribution of genotype D. Approximately 60% of patients reported that the four grandparents were of Italian ancestry, while 18% reported having at least two grandparents of Italian ancestry. These findings support the hypothesis that genotype D may have been disseminated in this region by Italian immigrants who arrived in Rio Grande do Sul in the 19th century,19,21 since there are studies that show high prevalence of this genotype in Southern Europe including Mediterranean countries, especially in Italy.41,42 Different studies suggested that the high prevalence of genotype D in the Southeast of Brazil is associated with Mediterranean immigrants.19,21 Novel studies are necessary to observe if this landscape is indeed a specific epidemiological characteristic of the other counties in the South region of the country.

In this study, there was a predominance of HBV genotype D (mainly subgenotype D3) among chronically infected patients in the city of Bento Gonçalves, suggesting an association between Italian immigration and the spread of this infection. Moreover, our results suggest an important role of intrafamilial transmission of HBV infection in the study sample.

J. Paoli, A.C. Wortmann, D. Simon, N.J.R. Fagundes, and V.R. Lunge designed the study and wrote the protocol. J. Paoli, M.G. Klein, A. Cirolini, and B. Godoy managed to recruit participants and performed the lab work. J.M. Wolf, D. Simon, V.R. Lunge, and V.R.Z.B. Pereira performed the data statistical analyses. J.M. Wolf, D. Simon, V.R. Lunge wrote the first draft of the manuscript and contributed to literature review and discussion of results. All authors contributed to and have approved the final manuscript.

This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, research grant 559598/2009-2).

The authors declare no conflicts of interest.