Detection and Genetic Characterization of Hepatitis B and D Viruses: A Multi-Site Cross-Sectional Study of People Who Use Illicit Drugs in the Amazon Region.

Hepatitis B (HBV) and delta (HDV) viruses are endemic in the Amazon region, but vaccine coverage against HBV is still limited. People who use illicit drugs (PWUDs) represent a high-risk group due to common risk behavior and socioeconomic factors that facilitate the acquisition and transmission of pathogens. The present study assessed the presence of HBV and HBV-HDV co-infection, identified viral sub-genotypes, and verified the occurrence of mutations in coding regions for HBsAg and part of the polymerase in HBV-infected PWUDs in municipalities of the Brazilian states of Amapá and Pará, in the Amazon region. In total, 1074 PWUDs provided blood samples and personal data in 30 municipalities of the Brazilian Amazon. HBV and HDV were detected by enzyme-linked immunosorbent assay and polymerase chain reaction. Viral genotypes were identified by nucleotide sequencing followed by phylogenetic analysis, whereas viral mutations were analyzed by specialized software. High rates of serological (32.2%) and molecular (7.2%) markers for HBV were detected, including cases of occult HBV infection (2.5%). Sub-genotypes A1, A2, D4, and F2a were most frequently found. Escape mutations due to vaccine and antiviral resistance were identified. Among PWUDs with HBV DNA, serological (19.5%) and molecular (11.7%) HDV markers were detected, such as HDV genotypes 1 and 3. These are worrying findings, presenting clear implications for urgent prevention and treatment needs for the carriers of these viruses.

Hepatitis B Virus Infection among People Who Use Illicit Drugs: Prevalence, Genotypes and Risk Factors in the State of Amapá, Northern Brazil.

Background: Several records of high prevalence of HBV have been made in northern Brazil. Among risk groups for viral infection, people who use illicit drugs (PWUDs) stand out, especially if they inject drugs and engage in risky sexual behavior. Objectives: In this study, the prevalence, genotype distribution and factors associated with hepatitis B virus (HBV) exposure in PWUDs were estimated. Methods: This cross-sectional study used snowball sampling from nine different sites. Socio-demographic, economic, drug use and health-related information were collected of 308 PWUDs from nine municipalities in the state of Amapá, northern Brazil. Blood samples were tested for the presence of HBV using enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Logistic regressions were run to identify factors independently associated with HBV exposure. Results: In total, seventy (22.7%) PWUDs were exposed to HBV, and 12 (3.9%) had DNA HBV. Genotypes A (58.4%), D (33.3%) and F (8.3%) were identified. Age ≥35 years, use of injectable drug, daily use of drugs, use of drugs over 12 years, unprotected sex, exchange sex for money/drugs, and >12 sexual partners in the last 12 months were associated with exposure to HBV. Conclusions: This study identified important information on the epidemiological scenario of HBV infection in PWUDs, highlighting the high prevalence of HBV exposure and the urgent need for measures for control and prevention, especially vaccination against this hepatotropic virus.

Prevalence and risk factors for HIV-1 infection in people who use illicit drugs in northern Brazil.

BACKGROUND: People who use illicit drugs (PWUDs) have a high risk of viral infections. To date, there is a paucity of information on HIV infection among PWUDs in remote Brazilian regions. This study determined the prevalence and factors associated with HIV-1 infection among PWUDs in northern Brazil. METHODS: Sociodemographic, economic, drug use and health-related information were collected through interviews from a community-recruited, multi-site sample of 1753 PWUDs. The blood samples collected were tested for the presence of HIV-1 using chemiluminescence immunoassay and PCR or western blotting. Logistic regressions identified factors independently associated with HIV-1 infection. RESULTS: In total, 266 (15.2%) PWUDs were HIV-1 positive. Hepatitis B virus and/or hepatitis C virus nucleic acid was detected in 65 (3.7%) PWUDs infected by HIV-1. The factors associated with HIV-1 infection were male gender, older age, a lower educational level and a lower income, crack cocaine use, a longer drug use history and a history of drug injection and engagement in unsafe sex, sex work and a higher number of sexual partners. CONCLUSIONS: The current study provides unique, initial insights into HIV and co-infection status and pertinent risk factors among PWUDs in northern Brazil, with clear and diverse implications for urgently improved prevention and treatment intervention needs.

Hepatitis C Virus among Female Sex Workers: A Cross-Sectional Study Conducted along Rivers and Highways in the Amazon Region.

BACKGROUND: Previous studies found a high prevalence of pathogens among female sex workers (FSWs) in the Amazon region, and established their parenteral and sexual transmission. This study estimated the prevalence of hepatitis C virus (HCV) infection and associated risk factors, and the frequency of HCV genotypes and resistance-associated substitutions (RASs) in this vulnerable group. METHODS: Distinct sampling methods were used to access 412 FSWs in cities and riverside communities in the Amazon region from 2015 to 2018. Three methods for HCV diagnosis were used to determine infection status. HCV genotypes and RASs were identified by sequencing and nucleotide fragment analysis. An association between HCV infection and exposure factors was determined by bivariate and multivariate analysis. RESULTS: In total, 44 (10.7%) FSWs were exposed to HCV, and 32 (7.8%) of them had active infection. Nine socioeconomic characteristics and risky sexual behaviors were associated with HCV exposure, particularly unprotected sex and condom exemption for the clients who paid extra money. Genotype 1 (81.3%) and 3 (18.7%) were detected. The frequency of FSWs with RASs was 23.1% (6/26) for grazoprevir related to the occurrence of substitutions Y56F and S122G. CONCLUSIONS: HCV infection among FSWs is highly prevalent and dominated by genotype I. Urgent preventive and treatment measures are required to reduce HCV infection in FSWs and the general population.

Human T-lymphotropic virus 1 and 2 among people who used illicit drugs in the state of Pará, northern Brazil.

People who used illicit drugs (PWUDs) represent an important population group for acquisition and viral dispersion. In Brazil, high rates of the human T lymphotropic virus 1 (HTLV-1) and 2 (HTLV-2) have been reported in epidemiological studies. However, the epidemiological scenario of HTLV-1/2 infections in PWUDs is still poorly understood. Thus, this cross-sectional study determined the prevalence, frequency of subtypes and factors associated with HTLV-1/2 infections among PWUDs in the Brazilian state of Pará, an area considered endemic for this virus and with poor health services. Among 826 PWUDs, 53 (6.4%) presented anti-HTLV-1/2 antibodies by EIA and 44 (5.3%) presented proviral DNA by PCR. HTLV-1 and HTLV-2 were detected in 25 (3.0%) and 19 (2.3%) PWUDs, respectively. Subtypes 1a (25/44), transcontinental (23/44) and Japanese subgroups (2/44), 2b (6/44) and 2c (13/44) were identified. Involvement in illicit/criminal activity, daily use of illicit drugs, illicit drug use over 12 years, unprotected sex with other PWUDs, changes in genitalia (including ulcers and wounds), and more than 12 sexual partners were associated with HTLV-1/2 in PWUDs. This high prevalence and intense circulation of subtypes and subgroups of HTLV-1/2 is very worrying, and indicate the need for urgent actions for its control, prevention and treatment.

Hepatitis C virus infection status and associated factors among a multi-site sample of people who used illicit drugs in the Amazon region.

BACKGROUND: Elevated rates of Hepatitis C Virus (HCV) infection have been reported in epidemiological studies with people who used illicit drugs (PWUIDs) in different Brazilian regions. In Brazil's Amazon region, studies have already identified the common use of illicit drugs among adolescents and the high prevalence of HCV infections among PWUIDs. However, all studies done with PWUIDs were conducted with small samples and within limited geographic coverage. This study determined the prevalence and risk factors for HCV infection in PWUIDs in the Amazon region, northern Brazil, as well as estimating the prevalence and factors associated with the HCV spontaneous clearance (HSC). METHODS: This cross-sectional study accessed 1666 PWUIDs from multiple municipalities of the Amazon region. Socio-demographic, economic, drug use and health-related information were collected through interviews. Blood samples collected were tested for the presence of anti-HCV antibodies and RNA-HCV. HCV genotypes were identified by real-time polymerase chain reaction (PCR). Logistic regressions were run to identify factors independently associated with HCV infection status and HSC. RESULTS: In total, 577 (34.6%) featured HCV antibodies, of which 384 (23.1%) had active HCV infection and 193 (11.6%) indicated HSC. Genotypes 1 (80.2%) and 3 (18.8%) were detected. HCV infection status was associated with the length of illicit drug use history, factors related to parenteral and sexual transmission, and factors of socio-economic marginalization leading to potential risk activities for HCV. HSC was associated with the ethnic (including indigenous) background of participants. CONCLUSIONS: High levels of HCV infection were detected in PWUIDs. Genotype 1 was predominant. Intense use of illicit drugs, unprotected sexual intercourse, high number of sexual partners and social marginalization were associated with all HCV infection. HSC was associated with origin (Amazonian-born) and non-white (e.g., Black or Indigenous) of PWUIDs. These findings emphasize the need for improve HCV prevention and control services and care for PWUIDs in the Brazilian Amazon region.

Hepatitis C virus infection among illicit drug users in an archipelago of the Amazon.

Hepatitis C virus (HCV) infection causes acute and chronic liver disease and may lead to cirrhosis, liver failure or hepatocellular carcinoma. The behavior of illicit drug users (DUs) typically exposes them to risks of viral infection. In the Brazilian Amazon region, a number of studies have identified high rates of drug use among adolescents, and a high prevalence of HBV infection in DUs, disseminated by sexual and parenteral activities. However, the epidemiological scenario of HCV infection in the region is still poorly understood. This study determined the prevalence, genotypes, and risk factors for HCV infection among DUs of the Marajó Archipelago. This cross-sectional study collected samples and epidemiological information from DUs in 11 municipalities. The diagnosis was established by EIA and real-time PCR, and the samples were genotyped by multiplex real time PCR. The data were analyzed by simple and multiple logistical regression. In 466 DUs, 28.3% had anti-HCV antibodies, and 25.5% had HCV-RNA. In 92 injecting drug users, 88.0% had anti-HCV antibodies, and 80.4% had HCV-RNA. Genotypes 1 and 3 were detected, with three cases of mixed infections. The multivariate analysis indicated associations of HCV infection with age (≥ 35 years), tattoos, intravenous drug use, shared use of injection equipment, and the daily and long-term (> 3 years) use of illicit drugs. These findings will contribute to the development of effective measures for the prevention of HCV infection among Brazilian DUs, as well as its general population.

Characterization of hepatitis B virus infection in illicit drug users in the Marajó Archipelago, northern Brazil.

South America is considered to have a low prevalence of hepatitis B virus (HBV) infection, although areas with a relatively high prevalence have been identified in northern Brazil. Few epidemiological studies of populations at risk of HBV infection are available for this region. Given this, in the present study, we investigated the prevalence of HBV and the factors associated with infection among illicit drug users (DUs) in the Marajó Archipelago, northern Brazil. In this cross-sectional study, we collected samples and epidemiological information from DUs in 11 municipalities of the Marajó Archipelago. The diagnosis was established by ELISA and real-time PCR; and genotyping was done by multiplex real-time PCR. Statistical modeling was based on simple and multiple logistical regressions with the Hosmer-Lemeshow test. The mean age of the 466 DUs was 28.4 years, and most were male. The most-consumed illicit drugs were crack cocaine and marijuana. In all, 171 DUs were exposed to HBV, with genotypes A, D and F being identified. The factors associated with higher frequencies of HBV infection were (i) male gender, (ii) age above 35 years, (iii) anti-HIV positivity, (iv) tattoos, (v) the use of injected drugs, (vi) the use of illicit drugs for more than 3 years, (vii) sexual relations without protection, (viii) sexual relations with another DU, and (ix) more than 10 sexual partners in the past 24 months. In summary, this study provides important insights into the dynamics of HBV infection among DUs in the Marajó Archipelago. We hope that these findings will contribute to the development of strategies, actions and public health policies aimed at preventing and controlling this viral infection more effectively.

Epidemiological aspects of HCV infection in non-injecting drug users in the Brazilian state of Pará, eastern Amazon.

BACKGROUND: Currently, sharing of drug paraphernalia is the main form of HCV transmission worldwide. In South America, consistent findings indicate that shared sniffing equipment is an important factor in the spread of HCV among non-injecting drug users. Epidemiological data on the status of HCV infection in illicit drug users in the Amazon region are scarce, although reports of clinical cases of hepatitis or pathologies associated with HCV infection in other population groups are numerous. Thereby, this study investigated the prevalence, genotype frequency, and epidemiological factors associated with HCV infection in non-injecting drug users in the state of Pará, eastern Amazon. RESULTS: During 2008-2011, 300 non-injecting drug users attending drug-treatment centers participated in this study. Most non-injecting drug users were male (63.7%). The mean age was 32.5 years. The non-injecting drugs most consumed were: cannabis (15.6%), cocaine paste (21.3%), and oxi cocaine (25.7%). Tobacco (60.9%) and alcohol (79.4%) were also commonly consumed. One hundred six (35.1%; CI 95%: 29.8 - 41.1) non-injecting drug users presented anti-HCV antibodies by EIA. The HCV-RNA prevalence was 28.0% (95% CI: 20.6 - 35.8). Genotypes 1 (76.9%) and 3 (23.1%) of HCV have been identified. A multivariate analysis demonstrated that HCV infection was independently associated with the following factors: "age (≥ 35 years)", "tattoos", "use of a needle or syringe sterilized at home", "shared use of drug paraphernalia", "uses drugs for more than 5 years", and "use of drugs everyday". CONCLUSIONS: This study revealed a high prevalence of HCV infection in non-injecting drug users, and most infections are occasioned by genotype 1. Likely, HCV transmission is associated with the tattoos, the use of needle or syringe sterilized at home by people over the age of 35 years, and sharing, time and frequency of use of non-injecting drugs. These findings should serve as an incentive for the establishment of a program of Hepatitis C prevention and control by the local public-health authorities in order to develop effective policies and strategies for contain the spread of HCV infection.

HBV infection in HIV-infected subjects in the state of Piauí, Northeast Brazil.

In this study, the prevalence, genotype frequency, and risk factors for HBV infection in 768 HIV-infected subjects living in Piauí were determined. Forty-six (6.0 %) HIV-positive subjects were reactive for HBsAg and positive for HBV-DNA. Genotypes A (71.8 %), F (23.9 %) and D (4.3 %) were identified. Multivariate analysis revealed an association between HIV-HBV coinfection and male gender, older age groups, unprotected sex, reporting more than ten sexual partners throughout life, STD, and tattooing. This study shows the importance of monitoring sites and professionals who perform tattooing and practice safe sex to prevent the spread of HIV and HBV infections.

HCV infection among cocaine users in the state of Pará, Brazilian Amazon.

In this study, the prevalence, genotype frequency, and risk factors for HCV infection in 384 cocaine users were determined. One hundred twenty-four (32.3 %) cocaine users had anti-HCV antibodies and 120 (31.3 %) had HCV-RNA. Genotyping results indicated the predominance of genotypes 1 (73.3 %) and 3 (26.7 %). Multivariate analysis showed an association of HCV infection with tattoos, shared use of paraphernalia, daily cocaine use, and a long history of cocaine use. The epidemiological aspects of HCV infection among cocaine users presented here should serve as an incentive for the establishment of a program of hepatitis C prevention and control by the local public-health authorities in the Amazon.

Epidemiological aspects of HCV infection in HIV-infected individuals in Piauí State, Northeast Brazil.

In this study, the prevalence, genotypic frequency, and risk factors for HCV infection in 768 patients infected with HIV were determined. Fifty-two (6.77 %) HIV-positive individuals had anti-HCV antibodies and 26 (3.39 %) had HCV-RNA. Genotyping results indicated that all RT-PCR samples from patients infected with HCV belonged to genotype 1. Multivariate analysis revealed an association of HIV-HCV coinfection with drug use and having received blood transfusions before 1994. The relatively low prevalence of HCV infection in the HIV-positive population in that region may be a consequence of the small number of drug users in the sample, despite a strong association between HCV infection and drug use.

Prevalence and genotyping of hepatitis C virus in blood donors in the state of Pará, Northern Brazil.

Given the scarcity of epidemiological information on hepatitis C virus (HCV) infection in Northern Brazil, we determined the prevalence and genotypic frequency in blood donors in the state of Pará (PA). Blood samples from all of the blood donors at the Fundação HEMOPA (blood bank of PA) from 2004-2006 were screened for the presence of antibodies to anti-HCV and samples seroreactive to anti-HCV were further tested for HCV RNA using real-time PCR. In total, 116 HCV-RNA samples were genotyped, based on maximum likelihood phylogenetic analyses, using BioEdit, Modelgenerator, PHYML and FigTree software. The population consisted of 242,726 volunteers who donated blood from 2004-2006; the most common subgroup was males between the ages of 18-29 years old (37.30%). Within the whole group, 1,112 blood donors (0.46%) had indeterminate or positive serology; among these, 28.78% were males whose ages ranged from 18-29 years. A diagnosis of chronic HCV infection was confirmed for 304 donors (60.20% males; 66.45% were 30-49 years old), resulting in a prevalence of HCV RNA in 0.13% of the samples (304 of 242,726). HCV genotyping revealed a high frequency of genotype 1 (108/116) followed by genotype 3 (8/116). This study found HCV infection to be relatively infrequent in PA; genotype 1 was most commonly isolated. This information can help guide prevention and control policies aimed at efficient diagnosis and control measures.

Prevalence and genotyping of hepatitis C virus in blood donors in the state of Pará, Northern Brazil

Given the scarcity of epidemiological information on hepatitis C virus (HCV) infection in Northern Brazil, we determined the prevalence and genotypic frequency in blood donors in the state of Pará (PA). Blood samples from all of the blood donors at the Fundação HEMOPA (blood bank of PA) from 2004-2006 were screened for the presence of antibodies to anti-HCV and samples seroreactive to anti-HCV were further tested for HCV RNA using real-time PCR. In total, 116 HCV-RNA samples were genotyped, based on maximum likelihood phylogenetic analyses, using BioEdit, Modelgenerator, PHYML and FigTree software. The population consisted of 242,726 volunteers who donated blood from 2004-2006; the most common subgroup was males between the ages of 18-29 years old (37.30 percent). Within the whole group, 1,112 blood donors (0.46 percent) had indeterminate or positive serology; among these, 28.78 percent were males whose ages ranged from 18-29 years. A diagnosis of chronic HCV infection was confirmed for 304 donors (60.20 percent males; 66.45 percent were 30-49 years old), resulting in a prevalence of HCV RNA in 0.13 percent of the samples (304 of 242,726). HCV genotyping revealed a high frequency of genotype 1 (108/116) followed by genotype 3 (8/116). This study found HCV infection to be relatively infrequent in PA; genotype 1 was most commonly isolated. This information can help guide prevention and control policies aimed at efficient diagnosis and control measures.

Molecular epidemiology of HIV type 1 in northern Brazil: identification of subtypes C and D and the introduction of CRF02_AG in the Amazon region of Brazil.

The molecular epidemiology of HIV-1 strains circulating in Belem-PA and Macapa-AP, in the Northern region of Brazil, is described using sequences of the C2V3 segment of the env and the pro gene of HIV-1 from patients of the Reference Unit for Special Infectious and Parasitary Diseases (URE-DIPE) in Belem-PA and the Central Laboratory (LACEN) in Macapa-AP. Subtype B was the most frequently found in relation to pro (88.3%) in Belem and in Macapa (97.1%) and env (88.3% in Belem and 100% in Macapa). Subtype F was also described in Belem (9.3% pro and 8.3% env) and Macapa (2.8% pro). Subtype D was described for the first time in the Northern region of the country as well as the recent entry of CRF02_AG. Furthermore, several possible recombinant forms among the various subtypes were found in both cities. The results support the hypothesis that HIV-1 infection is associated with the epidemic of the virus in the Southeast region of the country and that the city of Belem is the most important route for HIV-1 entry in the Northern region of Brazil.

Mannose-binding lectin gene polymorphisms are not associated with susceptibility to hepatitis C virus infection in the Brazilian Amazon region.

The present study compares the genotype frequencies between two population groups composed by 73 hepatitis C virus (HCV)-infected patients and 92 seronegative controls and investigates the role of allele variants as a possible factor in the susceptibility to HCV infection and the influence on disease progression. The identification of MBL*B and MBL*C alleles was performed by restriction fragment length polymorphism analysis of the 349-bp product using BanI and MboII restriction enzymes, respectively, and a polymerase chain reaction-sequence-specific polymorphism for discrimination of MBL*D. The analysis of allele and genotype frequencies between an HCV-infected group and seronegative controls did not indicate significant differences. The comparison of chronically infected subjects with and without liver cirrhosis was also not statistically significant. The odds ratio estimations were not significant, and the values obtained cannot suggest that the presence of allele variant MBL*B could have some influence in the risk of HCV infection progression to liver cirrhosis and that the presence of allele MBL*D could confer some protection against disease progression, but a larger sample size is necessary to confirm the present results.

Discriminação alélica do fator V da coagulação por PCR em tempo real: diagnóstico simples e preciso / Allelic discrimination of coagulation factor V by real time PCR: a simple and accurate diagnosis

Dentre as doenças cardiovasculares, a trombose venosa (TV) destaca-se pela associação entre fatores de riscos adquiridos e fatores genéticos. A resistência hereditária à proteína C ativada tem sido identificada como a principal causa dos casos de trombose venosa, sendo frequentemente associada à mutação fator V Leiden (G1694A). Em indivíduos homozigotos, o risco de trombose venosa é 50 a 100 vezes maior que em pacientes homozigotos normais, enquanto em pacientes heterozigotos o risco é de 5 a 10 vezes. Baseado na necessidade de avaliação e acompanhamento de pacientes com casos de trombose venosa e prevenção de seus respectivos familiares, foi desenvolvido um método simples de discriminação alélica do fator V da coagulação utilizando PCR em tempo real. Foram selecionados 67 pacientes com histórico de TV e 51 indivíduos sem histórico de TV. Primeiramente, a discriminação alélica do fator V foi realizada através de PCR convencional seguida de digestão enzimática (Mnl). Posteriormente, o diagnóstico foi realizado por PCR em tempo real. Ambos os métodos foram baseados no polimorfismo G1691A, sendo no segundo utilizado fluoróforos VIC e FAM para marcar os nucleotídeos G e A, respectivamente. A técnica de PCR-RFLP foi utilizada para diagnosticar 95 indivíduos homozigotos normais, 21 heterozigotos e 2 homozigotos FVL. Utilizando PCR em tempo real foram obtidos os mesmos resultados. A máxima similaridade entre os resultados obtidos por PCR em tempo real e PCR-RFLP indicou precisão significativa do novo método de discriminação e visualização alélica do fator V.

Among cardiovascular diseases, venous thrombosis is important due to the association between acquired and genetic risks factors. Hereditary resistance to activated protein C has been identified as the main cause of venous thrombosis, and is frequently associated to the factor V Leiden mutation (G1694A). In homozygotic individuals, the risk of venous thrombosis is 50 to 100 times higher that in normal patients, while in heterozygotic patients the risk is 5 to 10 times higher. Based on the need of evaluation and follow up of patients with venous thrombosis and prevention in their respective families, a simple method of allelic discrimination of coagulation V factor was developed using real time PCR. Sixty-seven patients with a history of venous thrombosis and 51 individuals without venous thrombosis were selected for this study. First, identification of the factor V allele was achieved through conventional PCR followed by enzymatic digestion (Mnl). Subsequently, diagnosis was attained by real time PCR. Both the methods investigated the G1691A polymorphism using VIC and FAM fluorophores to mark nucleotides G and A, respectively. By PCR-RFLP, 95 individuals were diagnosed as normal homozygotes, 21 as heterozygotes and 2 as homozygotic factor V Leiden individuals. The same results were obtained using real time PCR. Maximum similarity between the results of real time PCR and PCR-RFLP indicates high precision of the new method for allelic identification and visualization of factor V Leiden.