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

This was a cross-sectional study conducted in private and public DTC in nine municipalities in the Brazilian state of Pará (Abaetetuba, Ananindeua, Belém, Benevides, Bragança, Castanhal, Marituba, São Francisco do Pará, and Vigia), eastern Amazon. Twenty-five (78.1%) of the 32 DTC that existed during the study period collaborated with the research. These included DTC located within 350 km by road from the Centro de Hematologia e Hemoterapia do Pará (HEMOPA), Belém, PA, Brazil. Each DTC was visited at a time scheduled by the research team. The DTC had an average capacity of 15 patients. All patients who were at the center on the day when the team visited participated voluntarily. This study included participants who were using only non-injectable drugs, and excluded participants aged below 18 years. All data were collected from February 2008 to January 2011. The study was approved by the Research and Ethics Committee of the Núcleo de Medicina Tropical of the Universidade Federal do Pará, Brazil.

Blood (5 ml) was collected in sterile tubes, allowed to clot, and centrifuged at room temperature. The resulting plasma samples were collected and stored at a temperature below 10°C. After the end of the collection period at each DTC, the samples were transported to HEMOPA. The plasma was then tested for the presence of anti-HCV antibodies by enzyme immunoassay (EIA). The Murex anti-HCV version 4.0 (Kyalami, Gauteng, South Africa) was used to measure anti-HCV antibody levels. Nonreactive samples were considered negative for HCV. Reactive sample by EIA were submitted to RNA extraction, reverse transcription, and real-time PCR with primers complementary to the conserved area of the 5’ UTR of HCV, as described elsewhere[17]. Furthermore, the HIV/HCV co-infection was evaluated. All plasma collected was tested for HIV antibody by EIA. The Murex HIV-1.2.O (Saluggia, Vercelli, Italy) was used to measure anti-HIV antibodies. All tests for diagnosis of viral infections were performed at the Laboratory of Cellular and Molecular Biology of the HEMOPA.

Following the molecular screening for HCV infection using real-time PCR, samples that were positive for HCV-RNA were selected for the amplification of 5’ UTR using nested PCR[39]. The amplified fragment was sequenced in both directions using a Big Dye Cycle Sequencing standard kit and the dideoxynucleotide chain terminator method in an ABI Prism 3130 system (Applied Biosystems - Foster City, California, USA). All the nucleotide sequences obtained were edited and aligned using the BioEdit program[40]. The final alignment was entered into the DnaSP program[41] for the identification of identical nucleotide sequences. Nucleotide sequences obtained from the NCBI were added to the alignment and used for the construction of the phylogenetic tree and the differentiation of the HCV genotypes, as described elsewhere[18]. Nucleotide sequence data obtained in this study are available in GenBank under the accession numbers JN243906-JN243997.

The identification of the factors associated with HCV infection was based on the use of structured questionnaire (face-to-face interviews), in which the following variables were determined: gender, age, close family or friends with hepatitis C, recipient of blood transfusion, had surgery during the subject’s lifetime, had invasive dental treatment (root canal or tartar removal) during the subject’s lifetime, tattoo, shared used of razor blades in the domestic environment, shared used of blades in a barbershop, beauty salon or similar environment, use of a needle or syringe sterilized at home, shared nail clippers, types of legal drugs used during the subject’s lifetime, total time of drug use, frequency of drug use, shared use of drug paraphernalia (tubes, cans, pipes and similar), have already been arrested in police station or prison, use of drugs during detention in jails or prison, sexual orientation, unprotected sex, sexual intercourse with another drug user, involvement in prostitution, and multiple sexual partners. Furthermore, DTC structured admission records were reviewed to assess drug-use histories. The records include data on drug preference, length of drug use, sexual risk behavior, the main drugs used and their routes of administration. When clients enter treatment, each major part of the body - head, trunk, extremities and so on is examined. If needle use indications are identified, they are recorded in the client’s records. The information provided by participants through the questionnaire was compared with the records stored in DTC to identify and delete injecting drug users.

Wilson confidence intervals were constructed for the infection prevalence estimates[42]. Simple and multiple logistic regressions were done to assess the independent effect of variables[33]. The fit of the final model was assessed using the Hosmer-Lemeshow goodness-of-fit test. Two definitions of HCV infection cases were used: (i) anti-HCV positivity shown by EIA, and (ii) HCV-RNA detection by real-time PCR. All statistical analyses were carried out using PASW Statistics version 18.0.