RESUMO
BACKGROUND: This study aims to compare the efficiency of four oral fluid collection methods (Salivette, FTA Card, spitting and DNA-Sal) to detect HBV DNA by qualitative PCR. MATERIALS AND METHODS: Seventy-four individuals (32 HBV reactive and 42 with no HBV markers) donated serum and oral fluid. In-house qualitative PCR to detect HBV was used for both samples and commercial quantitative PCR for serum. RESULTS: HBV DNA was detected in all serum samples from HBV-infected individuals, and it was not detected in control group. HBV DNA from HBV group was detected in 17 samples collected with Salivette device, 16 samples collected by FTA Card device, 16 samples collected from spitting and 13 samples collected by DNA-Sal device. Samples that corresponded to a higher viral load in their paired serum sample could be detected using all oral fluid collection methods, but Salivette collection device yielded the largest numbers of positive samples and had a wide range of viral load that was detected. CONCLUSION: It was possible to detect HBV DNA using all devices tested, but higher number of positive samples was observed when samples were collected using Salivette device, which shows high concordance to viral load observed in the paired serum samples.
Assuntos
DNA Viral/análise , Vírus da Hepatite B , Hepatite B/diagnóstico , Saliva/química , Manejo de Espécimes/métodos , Adolescente , Adulto , Idoso , DNA Viral/sangue , DNA Viral/isolamento & purificação , Feminino , Hepatite B/sangue , Vírus da Hepatite B/genética , Humanos , Masculino , Pessoa de Meia-Idade , Saliva/virologia , Carga Viral , Adulto JovemRESUMO
AIM: Dental health professionals, including dental students, are at high risk of exposure to infection with the hepatitis C virus (HCV) through occupational percutaneous injuries and eye exposure. Further, fear of HCV infection is associated with discriminatory attitudes. The current study aimed to evaluate the knowledge about HCV infection amongst dental students and their attitudes towards patients infected with HCV. METHODS: A cross-sectional survey was conducted amongst 340 Brazilian dental students from two public universities using an instrument containing information regarding demographic characteristics, knowledge of HCV and attitudes towards patients with HCV infection. Descriptive statistics, Fisher's exact test, Student's t-tests, Mann-Whitney U-test and multiple logistic regression (MLR) were carried out (P < 0.05 was considered significant). RESULTS: Response rate was 90% (n = 306), and more than half (54%, n = 165) of participants had high knowledge level (above the mean); 97.7% (n = 299) demonstrated positive attitudes. MLR showed that high knowledge of dental students regarding HCV was substantially influenced by advancement in year of study (last year; P < 0.001) and type of university (federal; P = 0.049). Positive attitude towards HCV-infected patients was mainly influenced by age (P = 0.004) and male gender (P = 0.022). CONCLUSIONS: These results demonstrated a satisfactory knowledge about HCV infection amongst dental students, but some gaps were observed, suggesting the importance of continuous education about HCV in this population to prevent HCV infection as well as discrimination and prejudice towards patients with hepatitis C.
Assuntos
Atitude Frente a Saúde , Comportamento , Conhecimentos, Atitudes e Prática em Saúde , Hepatite C , Estudantes de Odontologia/psicologia , Adolescente , Adulto , Brasil , Estudos Transversais , Feminino , Humanos , Masculino , Autorrelato , Adulto JovemRESUMO
BACKGROUND: Enzyme immunoassays (EIA) designed to detect hepatitis C virus (HCV) core antigen and anti-HCV antibodies (HCV AgAb) simultaneously can improve the early detection of HCV infection when molecular diagnostic methods are not widely available. OBJECTIVES: To evaluate the suitability of dried blood spot (DBS) samples for detecting HCV AgAb using commercial EIAs. STUDY DESIGN: Paired serum and DBS samples were assayed using two commercial EIAs for HCV AgAb (Monolisa™ HCV AgAb ULTRA and Murex HCV AgAb). Manufacturer's recommendations were followed for sera while sample volume, incubation time and cut-off (CO) determination were evaluated for the DBS samples. The values of sensitivity, specificity, inter-rater agreement, detection limit, assay precision and stability of DBS samples at different conditions (22-26°C, 2-8°C and -20°C) were determined. RESULTS: It was necessary to increase the DBS sample volume fourfold compared to the sera samples to approximate the DBS Optical Density (OD) values to the sera OD values. Using ROC curve to recalculate CO values for the DBS samples, sensitivity was 97.5% for both EIAs, while the specificity was 99.71% for Monolisa™ HCV AgAb ULTRA and 95.95% for Murex HCV AgAb. Accurate testing results were obtained with DBS samples for 60 days at all conditions evaluated; storage at -20°C resulted in low OD variation. Both EIAs demonstrated the same limit of detection among DBS samples [estimated viral load of 3.1 International Units per millilitre (IU/mL)] and low OD value variability in repetitivity and reproducibility studies. CONCLUSION: DBS samples can be used for the detection of HCV AgAb by EIA as they present comparable performance characteristics and excellent stability among various storage conditions.
Assuntos
Teste em Amostras de Sangue Seco/métodos , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Antígenos da Hepatite C/sangue , Hepatite C/diagnóstico , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Hepatite C/sangue , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/imunologia , Humanos , Técnicas Imunoenzimáticas/métodos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
This study was undertaken to optimize and compare the efficiency of two commercial EIAs for anti-HCV detection (HCV Ab Radim, Pomezzia, Italy and ETI-AB-HCVK-4 DiaSorin, Vercelli, Italy), in dried blood spot (DBS) samples. The long-term stability of anti-HCV on DBS samples stored at three environmental conditions was also evaluated at: 2-8 °C, 20-25 °C, and -20 °C. Paired DBS and serum samples were obtained from individuals with or without anti-HCV. The type of elution buffer, sample and conjugate volume, sample incubation time and cut-off values were evaluated. For both EIAs, a larger sample volume was used, and the cut-off value determined by the manufacturer was employed for Radim EIA; however, ROC curve analysis was used for the DiaSorin EIA. The sensitivity and specificity of Radim EIA on DBS were 97.5% and 99.5%, respectively, and of DiaSorin EIA were 88.9% and 98.9%, respectively. Accurate results were obtained for a period of 117 days using DBS samples stored at all storage conditions, but storage at -20 °C resulted in the lowest variation among the absorbance values. Both EIAs demonstrated the same limit of detection (until dilution of 1:10(4) with estimated viral load of 3.1 × 10(-1) UI/ml), but the Radim EIA was associated with the best performance because a low coefficient of variation was observed in the repetition and reproducibility studies. In conclusion, commercial EIAs can be optimized for anti-HCV detection in DBS samples that are extremely stable at different conditions for more than 100 days.
Assuntos
Sangue/imunologia , Dessecação , Anticorpos Anti-Hepatite C/sangue , Hepatite C/diagnóstico , Manejo de Espécimes/métodos , Adulto , Técnicas de Laboratório Clínico/métodos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Temperatura , Adulto JovemRESUMO
BACKGROUND: Antibody responses to standard regimens of hepatitis B (HBV) vaccination are lower in HIV-infected subjects and the best hepatitis B vaccine schedule in this population is not known. OBJECTIVE: To assess the immunogenicity and to evaluate predictors of serologic response of a modified regimen of a HBV recombinant vaccine in a cohort of HIV-infected subjects. METHODS: HIV-infected subjects received 4 doses (40 µg) of a recombinant HBV vaccine at 0, 1, 2 and 6 months. Demographic information as well as CD4 cell count and plasma viral load were assessed at baseline. Protective and strong responses were defined as an anti-HBs titer ≥10 mIU/mL and ≥100 mIU/mL, respectively and were evaluated one month after the third and the fourth doses. RESULTS: 163 HIV-infected individuals were evaluated 67 (40%) were male and median age was 37 years. Median CD4 cell count was 385 cells/mm(3) and 113 (70%) had undetectable HIV-1 viral load. Protective antibody response was observed in 83 and 91% and a strong antibody response was observed in 62 and 80% of the subjects after 3 and 4 doses, respectively. In a multivariate logistic model undetectable HIV-1 viral load and higher CD4 cell counts were independent predictors of a strong antibody response after 4 doses. Patients with undetectable HIV viral load were almost 3 times more likely to have anti-HBs titers above 100 mIU/mL than those with detectable viral load. CONCLUSIONS: A 4-double-dose regimen of a recombinant HBV vaccine increased response rates and determined higher antibody titers which may translate in prolonged protection against HBV. Inclusion of a fourth dose of HBV vaccine for HIV-infected subjects should be considered in the public health setting.
Assuntos
Infecções por HIV/fisiopatologia , Anticorpos Anti-Hepatite B/sangue , Vacinas contra Hepatite B/administração & dosagem , Hepatite B/prevenção & controle , Vacinação/métodos , Adulto , Formação de Anticorpos/imunologia , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Relação Dose-Resposta Imunológica , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Hepatite B/imunologia , Hepatite B/virologia , Anticorpos Anti-Hepatite B/imunologia , Humanos , Esquemas de Imunização , Masculino , Vacinas Sintéticas/administração & dosagem , Carga ViralRESUMO
BACKGROUND: Saliva samples can be used as an alternative fluid for against hepatitis C virus (anti-HCV) detection owing to the ease of collection and excellent acceptability. This study was conducted to optimize a commercial enzyme immunoassay (EIA) to detect anti-HCV in saliva samples. METHODS: Ninety-six individuals donated paired serum and saliva samples that were obtained, using a commercial device (Salivette) and spitting into a sterile container. Initially, elution buffer for the Salivette samples, sample volume, incubation time and temperature, and two different anti-HCV EIAs were evaluated. Using the optimized assay, three methods for cut-off calculation were also evaluated. RESULTS: A 20-fold increase in the sample volume for both collection methods was needed. Moreover, the Radim assay was the most appropriate assay for anti-HCV detection in saliva samples, and the quality parameters were increased when a ROC curve was used to determine the cut-off value. Using this optimized assay, the sensitivities, specificities, accuracies, positive and negative predictive values were above 90% for saliva obtained using both the Salivette and spitting methods. Using this assay, discordant false-negative results were obtained for only two Salivette samples and five spitting samples. The concordance kappa was 93% for the Salivette method and 86.1% for the spitting method, demonstrating excellent performance. CONCLUSIONS: Saliva samples obtained for both methods can be employed for anti-HCV detection among HCV-infected or HCV-suspected cases, but several modifications must be performed on commercial EIAs to obtain good results. Moreover, samples obtained with commercial devices are more appropriate for anti-HCV detection in saliva samples.
Assuntos
Anticorpos Anti-Hepatite C/análise , Saliva/imunologia , Proteínas e Peptídeos Salivares/imunologia , Manejo de Espécimes/métodos , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Técnicas Imunoenzimáticas/métodos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Reprodutibilidade dos Testes , Saliva/química , Sensibilidade e Especificidade , Adulto JovemRESUMO
AIMS: A one-year survey was conducted to examine hepatitis A virus (HAV) prevalence, distribution of genotypes and their relationship to bacterial indicators in raw and treated sewage samples. METHODS AND RESULTS: Fifty sewage samples (raw = 25 and treated = 25) were collected twice monthly from one sewage treatment plant in Rio de Janeiro. Virus concentration was performed by adsorption to an electronegative membrane followed by ultrafiltration. Viral RNA was detected by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR and positive products were directly sequenced. Total and faecal coliform concentrations were also determined. By nested RT-PCR, HAV RNA was detected in 16/50 (32%) and eight (16%) of them were found in treated sewage samples. By real-time PCR, HAV RNA was detected in 46/50 (92%) samples and 24 were from treated sewage. Phylogenetic analyses classified nine isolates (56%) as subgenotype IA and seven (44%) as IB. CONCLUSIONS: Real-time PCR was more sensitive than nested RT-PCR; the presence of subgenotypes IA and IB was described and bacterial indicators cannot be used to predict HAV presence in sewage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrated that HAV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV infection.
Assuntos
Vírus da Hepatite A , Reação em Cadeia da Polimerase/métodos , Esgotos/virologia , População Urbana , Brasil/epidemiologia , DNA Viral/análise , Hepatite A/epidemiologia , Hepatite A/transmissão , Vírus da Hepatite A/classificação , Vírus da Hepatite A/genética , Vírus da Hepatite A/isolamento & purificação , Humanos , Filogenia , Análise de Sequência de DNARESUMO
Hepatitis A virus (HAV) is a significant waterborne human pathogen. Of the global supply of potable water, Brazil retains 13%, of which 75% resides in the Amazon Basin. Although hepatitis A morbidity has declined progressively in Brazil as a whole, it remains high in the Amazon region. We used nested and real-time reverse-transcription polymerase chain reaction (RT-PCR) to detect and quantify the viral load in water samples from the Amazon Basin. Most samples tested positive (92%), with viral loads varying from 60 to 5500 copies /L, depending on sanitary conditions and the degree of flooding. Nested RT-PCR of the VP1-2A region detected HAV RNA in 23% of the samples. In low viral load samples, HAV was detected only with real-time RT-PCR, suggesting that this technique is useful for monitoring HAV contamination. The presence of HAV in water samples constitutes a serious public health problem.
Assuntos
Vírus da Hepatite A/isolamento & purificação , Microbiologia da Água , Brasil , Monitoramento Ambiental , Vírus da Hepatite A/classificação , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Estruturais Virais/genética , Abastecimento de ÁguaRESUMO
The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21% of the samples, respectively. Nucleotide sequencing was carried out in 46% of VP1/2A and in 53% of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0%. Phylogenetic analysis of the VP1/2A sequences showed that 65% belong to sub-genotype IA and 35% to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2% identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.
Assuntos
Regiões 5' não Traduzidas/genética , Vírus da Hepatite A/genética , Hepatite A/virologia , RNA Viral/análise , Proteínas Estruturais Virais/genética , Adulto , Sequência de Bases , Brasil , Feminino , Genoma Viral , Genótipo , Vírus da Hepatite A/classificação , Vírus da Hepatite A/isolamento & purificação , Humanos , Masculino , Técnicas de Amplificação de Ácido Nucleico , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21 percent of the samples, respectively. Nucleotide sequencing was carried out in 46 percent of VP1/2A and in 53 percent of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0 percent. Phylogenetic analysis of the VP1/2A sequences showed that 65 percent belong to sub-genotype IA and 35 percent to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2 percent identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.