Prevalence and risk factors for HBV , HCV and HDV infections among injecting drug users from Rio de Janeiro , Brazil

Viral hepatitis constitutes a major health issue, with high prevalence among injecting drug users (IDUs). The present study assessed the prevalence and risk determinants for hepatitis B, C and D viruses (HBV, HCV and HDV) infections among 102 IDUs from Rio de Janeiro, Brazil. Serological markers and HCV-RNA were detected by enzyme immunoassay and nested PCR, respectively. HCV genotyping was determined by restriction fragment length polymorphism analysis (RFLP). HBsAg, anti-HBc and anti-HBs were found in 7.8, 55.8 and 24.7% of IDUs, respectively. In the final logistic regression, HBV infection was independently associated with male homosexual intercourse within the last 5 years (odds ratio (OR) 3.1; 95% confidence interval (CI) 1.1-8.8). No subject presented anti-delta (anti-HD). AntiHCV was detected in 69.6% of subjects, and was found to be independently associated with needle sharing in the last 6 months (OR 3.4; 95% CI 1.3-9.2) and with longer duration of iv drug use (OR 3.1; 95% CI 1.1-8.7). These data demonstrate that this population is at high risk for both HBV and HCV infection. Among IDUs from Rio de Janeiro, unprotected sexual intercourse seems to be more closely associated with HBV infection, whereas HCV is positively correlated with high risk injecting behavior. Comprehensive public health interventions targeting this population and their sexual partners must be encouraged. Correspondence


Introduction
Viral hepatitis constitutes a major health issue and can be caused by different etiologic agents (1).These infections are spread worldwide, although with varying prevalence in different regions.It is estimated that there are about 350 million and 10 million carriers of hepatitis B virus (HBV) and hepatitis D virus (HDV) in the world, respectively, with 1 million deaths per year as a consequence of hepatitis B (1)(2)(3).More than 150,000 people are infected with hepatitis C virus (HCV) each year in the United States, and approxi-mately 20 to 30% of these patients are at risk for developing cirrhosis (4).
Injecting drug users (IDUs) constitute a group of frequent exposure to many viral infections, since they usually engage in high risk sexual and injecting behavior (8).Furthermore, these subjects play a role as a reservoir and source of viral transmission in the inter-and extra-exposure categories.The prevalence of blood-borne hepatitis is usually higher among IDU than in other comparable non-IDU population strata (9).Epidemiological data indicate that IDUs represent the largest risk group for HCV infection (10).
The aim of the present study was to assess the frequency of HBV, HCV and HDV infections and their respective risk factors among IDUs from Rio de Janeiro, Brazil.

IDU population
The sample analyzed here was part of an epidemiological multicenter project about human immunodeficiency virus (HIV) risks among IDUs in five Brazilian cities (Projeto Brasil).A survey was carried out on 102 volunteers selected at drug use treatment centers and from the drug scene (streets) of the city.The injecting users recruited from the drug scene were enrolled from different sources, based on previous experience of the research team in a former multicity study (11).Many different places were assessed such as public places, nightclubs and bars, scattered through various neighborhoods of the city of Rio de Janeiro.The technique used was chain (or successive) referral -usually called snowball samplingrecently shown to be reliable as a recruiting strategy in a case-control study carried out in Rio de Janeiro (12).Recruiters were specially trained for this task and all efforts were made to guarantee the privacy of the interviewees.
IDUs recruited from drug use treatment centers came basically from three reference centers in Rio de Janeiro, located respectively in the northern and western regions of the city, and in a neighboring municipality, the latter enrolling basically patients from the city and its surroundings (Greater Rio).According to ethical guidelines, formal consent was obtained from each individual, who was interviewed using a standardized questionnaire including questions about sociodemographic status, pattern of iv drug use and sexual behavior.All subjects were tested for the presence of anti-HBc, HBsAg, antidelta (anti-HD) and anti-HCV.Because of insufficient blood sample, only 89 sera could be tested for the presence of anti-HBs.Except for 3 samples, detection of HCV-RNA was performed in all anti-HCV-positive subjects (N = 68).For HCV genotyping, sera from 39 IDUs were submitted to restriction fragment length polymorphism analysis (RFLP).

Characterization of HBV, HDV and HCV infections
Plasma samples were tested for the presence of HBV serological markers by enzyme immunoassays.Tests for HBsAg, anti-HBc and anti-HBs were carried out using the Hepanostika HBsAg Uniform II kit, Hepanostika anti-HBc Uniform kit and Hepanostika anti-HBs kit (Organon Teknika, Boxtel, Holland), respectively.HBV infection was defined by the presence of anti-HBc with or without other HBV markers.Anti-HD were tested by a commercial EIA kit (Abbott Laboratories, Diagnostic Division, Chicago, IL, USA).Anti-HCV testing was performed using a third-generation recombinant assay (Abbott HCV EIA 3.0, Abbott Laboratories).
For the detection of HCV-RNA, viral RNA was extracted from 200 µl of plasma using the QIAmp Blood and QIAmp Tissue kit (QIAGEN GmbH, Hilden, Germany).The extracted RNA was eluted in 100 µl of DEPCtreated water.Reverse transcription was carried out with 2.5 µl of RNA in a final volume of 10 µl.The mixture was incubated at 42 o C for 60 min and then at 95 o C for 5 min.In the first and second PCR rounds for HCV 5NCR detection, we used 2.5 µl of reverse transcription (RT) and first PCR product, respectively, in a final volume of 25 µl.The cycling protocol consisted of 35 cycles of 94 o C for 1 min, 54 o C for 1 min and 72 o C for 2 min, followed by a final cycle of 72 o C for 5 min.The sequences of the outer and inner primers were 1b (5GGTGCACGGTCTAC GAGACC3) and 2a (5GGCGACACTCC RCGAT3), 56 (5CGCAAGCACCCTAT CAGGGCAGT3) and 4 (5GAGGAACTA CTGTCTTCACGCAGAA3), respectively.The product of the second round of PCR, composed of 260 base pairs, was subjected to electrophoresis using a 2% agarose gel in TBE buffer and visualized by ethidium bromide staining under ultraviolet light.HCV genotyping by RFLP was performed as described elsewhere (13) and genotypes were determined according to Simmonds classification (14).

Statistical analysis
A database consisting of socio-demographic characteristics, pattern of iv drug use, sexual behavior and test results was set up using Epiinfo version 6.2 (Centers for Diseases Control and Prevention, Atlanta, GA, USA).Statistical analysis and data merging were performed using the SPSS 8.0 statistical package for IBM-PC.Serological markers and HCV genotypes were correlated with the data described above.Contingency table analysis (chi-square and Fisher exact test, Yates corrected) and t-test for means were employed.Results were regarded as significant when P<0.05.To further assess the independent role of the different variables associated with HBV and HCV infection, stepwise multiple logistic regressions with backward elimination were carried out.

Demographic characteristics, pattern of iv drug use and sexual behavior
Of the 102 IDUs investigated, 88 were males (85.4%), 15 were females (14.6%), and the mean age was 33.7 ± 7.6 years.The majority was employed (44.7%) or had a temporary job (45.6%), while 9.7% had mainly illegal sources of income like drug dealing, prostitution or others.About 54% of the IDUs had lower than high school education.Most individuals reported a history of former imprisonment (50% with 1 to 4 occurrences and 23.5% with more than 5 occurrences).Among the subjects, only 4 (5%) reported sharing injection equipment while in prison.Respectively 53.5 and 46.8% of the individuals lived in middle class and low-income districts.
The most frequently injected drug was cocaine (92.2%), followed by heroin (11.6%), the latter generally injected during trips abroad.Other drugs such as injectable or non-injectable amphetamines, solvents, tranquilizers and cannabis were also consumed.
The mean age at first injection was 19.6 ± 5.2 years, and the mean duration of iv drug use was 13.9 ± 8.8 years.With respect to the frequency of injection, 24.7% of subjects injected 1 to 3 times a month, 35.5%, 1 to 3 times a week and 39.8%, more than 3 times a week.We found a very high frequency of needle sharing (64.3%) in the last six months prior to the interview (Table 1).
Individuals presented high levels of sexual risk behaviors.Despite sexual orientation (hetero-or homosexual) or the nature of the partnership (principal or occasional partners, or clients), the vast majority of subjects reported irregular or no use of condoms (over 70% for all partnerships under analysis) (Table 1).

HBV, HDV and HCV infections
Results for HBV serological markers (HBsAg, anti-HBc and anti-HBs), anti-HCV, HCV-RNA and HCV genotypes and anti-HD are presented in Table 2 and associations between socio-demographic characteristics, parenteral and sexual risk behaviors and infection rates are shown in Table 3.
HBV infection was significantly associated with a history of imprisonment (P<0.04) and male homosexual intercourse within the last 5 years (P<0.04)(Table 3).Conversely, no association was found between HBV in-fection and parenteral exposure parameters, suggesting that HBV was more efficiently transmitted by the sexual route.Associations between the response variable and the variables shown to be significantly associated with it by univariate analysis were controlled for potential confounders and assessed by multiple logistic regression procedures.The final multiple logistic regression model (chi-square model = 4.5; P = 0.03) identified male homosexual intercourse within the last 5 years as the exclusive independent risk factor (odds ratio (OR) 3.1; 95% confidence interval (CI) 1.1-8.8)for HBV infection in this sample.
The variables associated with HCV genotype 3 infection (as compared to HCV genotype 1 infection) were younger age at first injection (P<0.03), a longer duration of iv drug use (P<0.05) and a higher frequency of injection (P<0.04)(Table 3).Although not statistically significant (probably due to the small size of the subsample), a tendency toward higher levels of HCV genotype 3 infection among individuals with a history of imprisonment and male homosexual intercourse was also observed.For evaluation of multiple infection, we considered anti-HBc and anti-HCV as markers of HBV and HCV exposure, respectively.We found a 38.2%rate (39/102) of HBV/HCV co-infection.

Discussion
The present study shows a high prevalence of blood-borne viral hepatitis among IDUs from Rio de Janeiro.IDUs are more exposed to many viral agents by the sexual and/or parenteral routes, as shown by the high rate of needle sharing (64.3%) and the low rate of condom use.Additionally, secondary risks such as sharing injection paraphernalia other than needles and syringes should be considered (16).
Rio de Janeiro is a region of low endemicity for HBV infection (<2% HBsAg prevalence) (17).The frequency of HBsAg found in this study (7.8%) is comparable with that observed in areas of high endemicity, with more than 8% of HBsAg prevalence, and agrees with results from other studies of IDUs in Santos, SP, Brazil (18), and in other countries (9).HBV infection was significantly associated with male ho- mosexual intercourse, but no association with parenteral exposure variables was found.This profile suggests that unsafe sex represents the major risk determinant for HBV transmission among IDUs in Rio de Janeiro.The history of imprisonment did not remain in the final logistic regression model, and seems to be a confounder.
In our study, anti-HCV was detected in Table 3 -Main associations between socio-demographic characteristics, parenteral and sexual risk behaviors and infection rates for HBV a and HCV b infections and HCV genotypes among IDUs from Rio de Janeiro, Brazil (1994)(1995)(1996).
a HBV infection was defined by the presence of anti-HBc with or without any other HBV serological marker.b HCV infection was defined by the presence of anti-HCV.c Data were analyzed statistically by the t-test for means and chi-square/Fisher's exact test for frequencies.d Fourteen and 3 subjects were infected with HCV genotype 1 and HCV genotype 2, respectively.ns, Not significant.69.6% of the subjects, in agreement with other studies carried out among IDUs (19,20).It is noteworthy that the majority of these subjects (70.6%) are able to transmit HCV to other IDUs, since HCV-RNA was found in their sera.

Socio
HCV infection was significantly associated, both by univariate and multivariate analyses, with longer duration of iv drug use, in agreement with previously reported data (21).Likewise, a high rate of needle sharing in the last 6 months (64.3%) was observed, and this variable constituted another risk determinant for HCV infection, as previously observed (19).In the final multiple logistic regression model only these two associations, among the six associations previously identified by univariate analysis, remained as independent risk factors for HCV infection.Probably the variables age and age at first injection are, in fact, confounders for the other variables associated with higher levels of exposure to parenteral risks of transmission.
HBV and HCV infections were both associated with prison history in the univariate analysis (P<0.04 and P<0.006, respectively), but these associations did not remain in the final multivariate analysis.We should remark that in our sample only 5% of individuals reported needle-sharing while imprisoned.The international literature also highlights the risks of HIV and HBV transmission due to unprotected sexual intercourse while imprisoned, a fact that should be reassessed in future studies to be carried out in Brazil (22,23).
The observed association between higher levels of HCV infection and male gender (P<0.04),although not confirmed by multivariate analysis, was mentioned in previous studies, which showed that women eliminate HCV more frequently than men (24,25).No association was found between HCV infection and sexual risk behavior, confirming previous findings that hepatitis C among IDUs seems to be a consequence of repeated exposure to contaminated injection equipment (21).The distribution of HCV genotypes found here parallels the patterns observed in non-IDU populations from Brazil, with a higher prevalence of genotype 1 (26).However, the frequency of HCV genotype 3 among IDUs was relatively high in comparison with other populations (15), as also observed among German IDUs (27).In our investigation, HCV genotype 3 infection was positively associated with younger age at first injection (P<0.03),longer duration of iv drug use (P<0.05) and frequency of injection (P<0.04).These results suggest that the dissemination of HCV genotype 3 among IDUs could be a consequence of cumulative and repeated parenteral exposure to this genotype.
Infections by HDV were not detected in this study.This profile is remarkable since even in areas of low endemicity for HDV, 17-98% of HbsAg-positive IDUs present HDV antibodies (9).
In our sample, the most frequently injected drug was cocaine (92.2%).This seems to be an important aspect of the Brazilian drug scene, since cocaine injectors are under higher risk for blood-borne pathogen infections when compared with opiate injectors.Cocaine addicts repeatedly inject drugs over short periods of time, in binges, increasing the chance of viral exposure through the common use of injection equipment (28)(29)(30).A significant association between iv cocaine use and risky sex behavior has been pointed out in previous studies (31).
In the Southeast of Brazil, especially in Rio de Janeiro, cocaine is mainly administered by snorting (32), although the use of crack cocaine seems to be increasing (33)(34)(35).Former studies on the transition from non-injecting to injecting illicit drugs indicate that a period from months to years generally elapses between consuming by noninjecting routes and injecting (36), highlighting the need for preventive programs directed at both injecting and non-injecting cocaine users.
IDUs constitute an important source of viral infections and therefore can play an important role in the transmission of viruses to the general population.Hence, a public health intervention with the implementation of comprehensive prevention programs including information, face-to-face education, empowerment strategies, distribution or exchange of clean injecting equipment and distribution of condoms must be encouraged.These measures are especially relevant in developing countries like Brazil, where Public Health programs are underbudgeted and understaffed, and frequently lack expertise and political support to implement these programs (37).
Moreover, hepatitis B vaccination of all IDUs should be mandatory, not only as a direct strategy of prevention of new HBV infections, but also as a way to contact hardto-reach IDUs and engage them in different preventive activities.Current vaccination programs attest that this is a feasible and practical initiative, with a high cost/benefit ratio both in terms of human lives to be saved and financial resources to be spared (38,39).

Table 2 -
Percent frequency and numerical data for samples testing positive (+)/samples (N) tested for different viral hepatitis serological markers among IDUs from Rio de Janeiro, Brazil(1994- 1996).