Hepatitis B and C virus infection among Brazilian Amazon riparians Infecção pelo virus da hepatite B e C em ribeirinhos da Amazônia brasileira

Introduction: Viral hepatitis is a major public health concern in Brazil. There are few past studies on this issue, especially among riparian communities. This study aims at determining the seroprevalence of viral hepatitis B and C in the riparian community of Pacuí Island, within the Cametá municipality of Pará State, Brazil. Moreover, this study aims to investigate the principal risk factors that this community is exposed to. Methods: The current study has accessed blood samples from 181 volunteers who have answered an epidemiological questionnaire. Analyses on serological markers have been tested with commercial ELISA kits for detecting HBsAg, total anti-HBc, anti-HBs, and anti-HCV. Within seroreactive patients for HCV, RT-PCR and line probe assay have been performed to identify the viral genotype. Results: In the serological marker analysis for hepatitis B, no reactivity for HBsAg, rate of 1.1% for total anti-HBc, and rate of 19.3% for anti-HBs have been observed. On hepatitis C, 8.8% seroprevalence has been found, in which 62.5% have gotten viral RNA. Among the risk factors studied, the following have been highlighted: non-use of condoms, sharing of cutting instruments, use of illicit drugs, and reports of family disease with HBV or HCV. Conclusions: The vaccination coverage against HBV is low, and the high prevalence of HCV within this community has been observed.


Article/Artigo
It may be reported that in Brazil, there is a major variation within the terms of prevalence of HBV and HCV, which is related to both groups studied and to the region itself.In the groups studied, the majority of information, regarding those forms of viral hepatitis, involves risk groups (patients on hemodialysis, health professionals, injectable drug users, prison inmates) or blood donors [1][2][3][4] .
Seroepidemiological studies related to HBV and HCV in indigenous people and riparians in the Brazilian Amazon are scarce due to access difficulties in those communities.Furthermore, the Amazon riparians have gotten their own features, mostly related to environmental, cultural, and socioeconomic factors 1,5,6 .
Notwithstanding the few studies carried out within the Amazon region, it is characterized as having a high rate of infection by HBV and intermediate rate to HCV and its consequences 7,8 .
The objective of this paper is to describe the seroepidemiological profile of viral hepatitis B and C in the Pacuí Riparian community, located in the Northern region, State of Pará, Brazil.The major risk factors to which the community is exposed are also analyzed to obtain knowledge about the epidemiology of these viruses within the population.

Site of the study
Pacuí is one of the 90 islands belonging to the Cametá municipality, located in the Northeast of Pará, Brazil.Pacuí is an island on the Tocantins River and is 180km from the state capital.It has an area of around 12.34km 2 and a population of 150 registered families, with 398 grown-up inhabitants according to the Municipality Health Secretariat (Figure 1).The island has two schools, two community agents, and no health center.Its economy is based on extraction and sale of regional fruits, as well as small-scale fishing.The residents live in wooden houses located along the river (Figure 2).

Patients and procedures
With support from the Municipality Health Secretariat and community agents, all adult inhabitants (18 years old or up) who live in the island and do not present any mental illness were made aware of the days that the team would be present and invited to take part in the study.From February 15-16, 2010, a professional health team collected biological samples and epidemiological forms from the residents in the island who agreed to take part in the study.
Based on spontaneous demand, 181 residents of Pacuí island who were 18 years old and above of both sexes took part in this study.They were from 80 families and comprised 45% of the grown-up island residents.
A standard and individual questionnaire was used to gather information about each patient, such as: age, sex, marital status, profession, education, family income, basic sanitation, and possible risk factors for transmitting viral hepatitis including the number of sexual partners within the last two years, use of condom, history of sexually transmitted diseases, blood transfusion, use of illicit drugs, shared cutting instruments, tattoo, and family history of HBV or HCV.
Approximately 8mL of blood were collected from each subject through peripheral venipuncture.The collected blood was centrifuged at 3,000rpm for 10 min, and the serum was separated and stored at -70ºC for further analysis of viral markers.

Serological and molecular diagnosis
The samples were tested to detect the markers of hepatitis B virus infection (HBsAg, anti-HBc, and anti-HBs) and were assessed by ELISA (Hepanostika Uni-form Organon Teknica BV, Boxtel, Holland).
The presence of anti-HCV antibodies were tested using a thirdgeneration enzyme immunoassay (ELISA) (Abbott Laboratories, Abbott Park, IL, USA).Positive samples were retested for confirmation using a line immunoassay (INNO-LIA HCV Ab III, Innogenetics).The entire samples were subjected to RNA extraction, reverse transcription, and a nested PCR with primers complementary to the conserved area of the 5' NC region of HCV, essentially as described by Ginabreda et al. 9 HCV genotyping was carried out for the entire HCV-RNA-positive samples.A line probe assay (Inno-LiPA HCV II, Innogenetics) was used to establish the genotype of the amplicons of the 5' NC region according to the procedure described by the manufacturer.

Statistical analysis
The statistical program used was BioEstat 5.0 10 .The frequency distribution for the variables investigated was expressed in percentages, and the Chi-square test was used in comparing the proportions observed.To associate the risk factors with the serological markers, the odds ratio (OR) test was performed.The accepted statistical significance level was 95%.

Ethical considerations
The current research has been approved by the Ethics Committee for Research with Human Beings at the Tropical Medicine Center (TMC), Belém, State of Pará, Brazil.
The study was able to come up with a young population, with an age range of 32 years.The population characteristics were low level of schooling, predominantly female, 7% (12/181) were illiterate, 63% (114/181) had not finished elementary school, and 30% (55/181) already finished elementary school.Regarding the family income, 86% (156/181) were earning up to one minimum salary, and 14% (25/181) were earning two minimum salaries.The majority of the inhabitants in this community were earning their living from fishing and/or farming.The region had precarious basic sanitation, with 99% (179/181) of the interviewees using water from rivers and lakes and 97% (175/181) not having have septic tanks.
In serological marker analysis for hepatitis B, no HBsAg viral antigen was observed.As for the anti-HBs marker, it was found in 19.3% (35/181), and the anti-HBc was seen in 1.1% (2/181) of the individuals.
Only two (1%) individuals were anti-HBc-and anti-HBspositive.Both were negative for IgM anti-HBc, indicating former infection with natural immunity.
Based on serological data for anti-HBs, low vaccination coverage against HBV was noted for the community, characterizing the inhabitants as a risk group for acquiring HBV.
The serological marker for hepatitis C (specific IgG HCV) was reactive in 8.8% (16/181) of the individuals (Table 1).Of those individuals, viral RNA was detected in 62.5% (10/16), and they all presented genotype 1 of HCV.
Regarding the risk factors, it was highlighted that 87% (157/181) of the volunteers were not using condom during sexual intercourse and 72% (131/181) were having a habit of sharing cutting instruments among relatives and/or neighbors.
When the risk factors were analyzed among the groups that were reactive for HCV with those who were not reactive, the reports showed that sexually transmitted diseases and the use of illicit drugs were more prevalent among those who were reactive (Table 2).
Furthermore, the reports showed that families with HBV or HCV infection were more prevalent among those who were positive (Table 2).Sixteen patients who were reactive for HCV were from eight families.In four families, two patients with HCV were found.There were two families with one patient, and two families had three patients.

DISCUSSION
This is the first study regarding the prevalence of markers for viral hepatitis B and C in the Riparian community of Pacuí, Cametá municipality, State of Pará, Brazil.
The outcomes come out with a low prevalence of hepatitis B among the riparians investigated.The negative outcome observed for the HBsAg antigen earmarks the absence of hepatitis B in the population of the study.The presence of anti-HBc and anti-HBs markers in two individuals suggests that they have had contact with HBV.The concomitantly presenting anti-HBs and anti-HBc antibodies earmark post-hepatitis B immunity, probably a serological scar.
Notwithstanding, the presence of isolated anti-HBs marker in 18% (33/181) of the individuals has been noticed, which suggests a low rate of immunity acquired after vaccination.It is observed that the low level of vaccination reinforces the impression that this population is vulnerable to HBV infection.In Brazil, studies have demonstrated that infection with HBV is greater in 20-to 40-yearold individuals, which involve the sexually active population.Thus, vaccine coverage should be emphasized as the most rapid means of dynamic socio-biological intervention against transmission in needy areas, such as the site of the present study [6][7][8] .
As for the marker for hepatitis C, a rate of 8.8% has been found for anti-HCV, which characterizes the community studied as a region of high prevalence for HCV.
It is supposed that in Riparian communities, the prevalence of infections caused by HBV and HCV may be directly related to genetic, demographic, socioeconomic, cultural, and historical features among the population studied, as well as to other unknown factors 13,17 .
Regarding the comparison of the serological marker for HCV and the risk factors studied, this study has come up with the conclusion that the family environment is one of the principal reservoirs for HCV and, thus, contributes toward its dissemination and propagation.
Intrafamilial transmission of HCV is considered as a nonsignificant route of transmission, and sexual transmission does not seem to play a role in the intrafamilial spread of HCV infection 18,19 .However, same studies have demonstrated significant anti-HCV prevalence rates in family members of patients with chronic HCV and/or in household contacts of anti-HCV-positive individuals 20 .Although with low efficiency, sexual transmission should also be considered as one of the routes of intrafamilial transmission of HCV 21 .
The authors declare that there is no conflict of interest.

REFERENCES
Considering the high prevalence of HCV observed in this small community and the associated risk factors having been verified, further studies exploring these data should be carried out to confirm and assess the potential risk of family environment in the transmission of HCV infection.
HCV is primarily transmitted via parenteral routes.The use of illicit injectable or non-injectable drugs is currently considered as the most important risk factor for HCV 21,22 .This was also an important factor in our study.
For the population in this study, 72% of the individuals shared manicure tools among family members.The shared use of manicure and pedicure material should be taken into account by local and regional health authorities and by those of other countries with cultures similar to that of northern Brazil to establish political and public strategies to control HCV transmission.
The risk factors that have been verified are parenteral procedures that may be avoided if the population is made aware of the risks of transmitting microorganisms.Shaving or trimming nails may generate trauma or microtrauma on the skin surface, resulting in exposure to HCV on blades or nail scissors contaminated by asymptomatic infected family members.
In Italy, Pakistan, and Nigeria, HCV transmission by sharing razor blades and other cutting instruments as well as other forms of viral dissemination due to a lack of public awareness have been reported as risk factors for infection [23][24][25] .
Various transmission mechanisms have been studied in the Brazilian Amazon region; however, there some transmission factors that are yet to be determined 26,27 .
The high values for infection by HCV and the low vaccine coverage against HBV point to the need for preventive programs directed to this population at risk, which should be put in place based on knowledge of the epidemiological situation and transmission dynamics for those viruses.

TABLE 2 -Distribution of Hepatitis C virus serological marker and principal risk factors.
OR: Odds ratios, CI: confidence interval, STD: sexual transmitted diseases.