Livia Melo Villar

Detection of hepatitis A, B, and C virus-specific antibodies using oral fluid for epidemiological studies

In this report, we examine the adaptability of commercially available serological kits to detect antibodies markers for viral hepatitis in oral fluid samples. We also assessed the prevalence of hepatitis A, B, and C virus-specific antibodies, and related risk factors for these infectious diseases through sensitivity of the tests in saliva samples to evaluate if oralfluid can be an alternative tool to substitute serum in diagnosis of acute viral hepatitis and in epidemiological studies. One hundred and ten paired serum and saliva specimens from suspect patients of having acute hepatitis were collected to detect antibodies to hepatitis A (total and IgM), hepatitis B (anti-HBs, total anti-HBc and IgM anti-HBc), and hepatitis C (anti-HCV) using commercially available enzyme-linked immunossorbent assay (EIA). In relation to serum samples, oral fluid assay sensitivity and specificity were as follows: 87 and 100% for total anti-HAV, 79 and 100% for anti-HAVIgM, 6 and 95% for anti-HBs, 13 and 100%for total anti-HBc, 100 and 100% for anti-HBc IgM, and 75 and 100% for anti-HCV The consistency observed between antibodies tests in saliva and expected risk factors for hepatitis A and C suggests that the saliva method could replace serum in epidemiological studies for hepatitis A and C.

Molecular detection of hepatitis A virus in urban sewage in Rio de Janeiro, Brazil.

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.

Assessment of dried blood spot samples as a simple method for detection of hepatitis B virus markers

Detection of hepatitis B virus (HBV) serological markers in dried blood spot (DBS) samples by enzyme immunoassay (ELISA) has not yet been fully optimized. In this study, the ability to detect three HBV markers (HBsAg, anti‐HBc, and anti‐HBs) was evaluated in DBS samples using a modified commercial ELISA. Matched serum and DBS samples were obtained from individuals with or without a past history of HBV infection. Sera samples were tested according to the manufacturers instructions, but for DBS testing, paper diameters, elution buffer, volume of input sample, and cut‐off values were evaluated to optimize the assay. Stability studies were done on DBS stored at for up to 180 days at different temperatures. The absorbance values that yielded the maximum sensitivity and specificity were determined based on the area under the ROC curve (AUROC) and chosen as the cut‐off value. Using this parameter, sensitivity was 90.5%, 97.6%, and 78% for anti‐HBc, HBsAg, anti‐HBs assays, respectively. Specificity was 92.6%, 96.7%, and 97.3% for anti‐HBc, HBsAg, and anti‐HBs assays, respectively. HBV markers could be detected in DBS samples until 63 days after sample collection at most temperatures, but storage at −20°C yielded more consistent results. These results indicate that modified ELISA can be used to detect HBV markers in DBS samples, particularly if the samples are stored appropriately. J. Med. Virol. 83:1522–1529, 2011.

Association between vitamin D and hepatitis C virus infection: A meta-analysis

AIM To evaluate the association between 25-hydroxyvitamin D [25(OH)D] and sustained virological response (SVR) in hepatitis C virus (HCV) infected individuals. METHODS Relevant studies were identified by systematically searching MEDLINE databases up to March 2012 and abstracts of the European and American Congress of Hepatology conducted in 2011. Studies must provide information on SVR and the levels of 25(OH)D₃ and/or 25(OH)D₂ [henceforth referred to as 25(OH)D] in sera samples from HCV infected individuals. The inclusion criteria were: clinical studies that included HCV infected patients aged older than 18 years regardless of HCV genotype or ethnic group; provided information on SVR rates; and were reported in the English language as full papers. Due to the heterogeneity of studies in categorizing serum vitamin D levels, a cut-off value of 30 ng/mL of serum 25(OH)D was used. Heterogeneity was assessed using I² statistics. The summary odds ratios with their corresponding 95%CI were calculated based on a random-effects model. RESULTS Overall, 11 studies (8 observational and 3 interventional) involving 1575 individuals were included and 1117 HCV infected individuals (71%) showed low vitamin D levels. Most of the studies included mono-infected HCV individuals with the mean age ranging from 38 to 56 years. Four studies were conducted in human immunodeficiency virus/HCV infected individuals. Regarding vitamin D measurement, most of the studies employed radioimmunoassays (n = 5) followed by chemiluminescence (n = 4) and just one study employed high performance/pressure liquid chromatography (HPLC). Basal vitamin D levels varied from 17 to 43 ng/mL in the studies selected, and most of the HCV infected individuals had genotype 1 (1068/1575) with mean viral load varying from log 4.5-5.9 UI/mL. With regard to HCV treatment, most of the studies (n = 8) included HCV individuals without previous treatment, where the pooled SVR rate was 46.4%. High rates of SVR were observed in HCV individuals with vitamin D levels above 30 ng/mL (OR = 1.57; 95%CI: 1.12-2.2) and those supplemented with vitamin D (OR = 4.59; 95%CI: 1.67-12.63) regardless of genotype. CONCLUSION Our results demonstrated high prevalence of vitamin D deficiency and high SVR in individuals with higher serum vitamin D levels or receiving vitamin D supplementation.

Investigação de acidentes biológicos entre profissionais de saúde

Resumen 1Aluna do Curso de Pós-Graduação em Análises Clínicas, Faculdade Redentor, Itaperuna, RJ. Bióloga, Laboratório de Referência Regional de Campos dos Goytacazes, Hospital Geral de Guarus, Campos dos Goytacazes, RJ. Brasil. E-mail: jazevedo_silva@hotmail.com, 2 Pesquisadora em Saúde Pública, Doutora em Ciências. Laboratório de Desenvolvimento Tecnológico em Virologia, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ. Brasil. E-mail: vdepaula@ioc.fiocruz.br, 3 Professor Adjunto, Doutor em Ciências. Setor de Hematologia da Clínica Médica “B” do Hospital Universitário Gaffrée e Guinle, Escola de Medicina e Cirurgia, UNIRIO, Rio de Janeiro. Brasil. E-mail:adilsonjoal@ig.com.br, 4 Tecnologista em Saúde Pública, Doutora em Ciências. Laboratório de Hepatites Virais, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ. Brasil. E-mail: lvillar@ioc.fiocruz.br PESQUISA RESEARCH INVESTIGACIÓN The aims of this study were to identify the major professional category exposed to biological risk and the principal type of accident occurred among health care workers in Campos dos Goytacazes, RJ. Based on the analysis of 183 records filled out by the workers who suffered biohazardous accidents between January 2005 and September 2005, we found the nursing auxiliaries and technicians as the professional category more exposed to biohazardous accidents (54.1%), followed by undergraduate medical and dental students (10.4%). The occurence of acidents with piercing-cutting materials was related to frequent handling of these instruments, and to the behavior of workers who maintain practices providing risks of needlestick injuries, such as inappropriate disposal of piercing-cutting materials.Os objetivos deste trabalho foram identificar a principal categoria profissional exposta a risco biologico e os principais tipos de acidentes ocorridos entre trabalhadores da area de saude, em Campos dos Goytacazes, RJ. A partir da analise das fichas de notificacao de acidentes biologicos dos 183 profissionais acidentados entre janeiro de 2005 e setembro de 2005, observamos que a categoria profissional mais exposta foi a dos auxiliares/tecnicos de enfermagem (54,1%), seguida pela dos academicos de medicina e odontologia (10,4%). A ocorrencia de acidentes com materiais perfurocortantes foi relacionada a manipulacao frequente desses objetos e ao comportamento dos profissionais que utilizam praticas que oferecem riscos de acidentes com agulhas, tais como o descarte inadequado de objetos perfurocortantes.

Genetic variability of hepatitis A virus isolates in Rio de Janeiro: implications for the vaccination of school children

The epidemiology of hepatitis A virus (HAV) infection is shifting from high to intermediate endemicity in Brazil, resulting in increased numbers of susceptible individuals and a greater potential for the emergence of outbreaks. Universal vaccination against HAV has been recommended for children, but updated sero-epidemiological data are necessary to analyze the level of natural immunity and to identify candidates for preventive measures. In addition, more molecular studies are necessary to characterize the genotypes involved in HAV infections and outbreaks. Sera from 299 school children (5-15 years old) and 25 school staff members, collected during an outbreak of HAV at a rural public school in June 2000, were tested for IgM and total anti-HAV antibodies (ELISA). Viral RNA was amplified by RT-PCR from anti-HAV IgM-positive sera and from 19 fecal samples. Direct nucleotide sequencing of the VP1/2A region was carried out on 18 PCR-positive samples. Acute HAV infection was detected by anti-HAV IgM in 93/299 children and in 3/25 adult staff members. The prevalence of total anti-HAV antibodies in IgM-negative children under 5 years of age was only 10.5%. HAV-RNA was detected in 46% IgM-positive serum samples and in 16% stool samples. Sequence analysis showed that half the isolates belonged to subgenotype IA and the other half to IB. On the basis of these data, mass vaccination against HAV is recommended without prevaccination screening, especially for children before they enter school, since nearly 90% of the children under 5 years were susceptible. Molecular characterization indicated the endemic circulation of specific HAV strains belonging to subgenotypes IA and IB.

Vaccination against hepatitis B with 4-double doses increases response rates and antibodies titers in HIV-infected adults

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.

Detection of hepatitis A virus RNA in serum during the window period of infection

BACKGROUND Hepatitis A virus (HAV) infection is the leading cause of clinically apparent viral hepatitis in many parts of the world, including developed and developing countries. Only limited information is available regarding the seronegative viremic window that follows HAV infection, and no systematic search has been reported for HAV RNA positive, IgM anti-HAV negative serum samples during hepatitis A outbreaks. OBJECTIVES To determine the proportion of HAV infected individuals among (i) children who were tested negative for anti-HAV antibodies during hepatitis A outbreaks which occurred in a public school (n = 157) and a child care center (n = 38); (ii) subjects (n = 46) initially classified as acute non-A-C hepatitis patients after clinical examination and serological tests (sporadic cases). STUDY DESIGN Reverse transcription (RT)-PCR was performed to detect the presence of HAV genome in serum samples collected from anti-HAV negative, susceptible subjects. RESULTS HAV RNA was detected in 19/157 (12%) and 5/38 (13%) anti-HAV negative children from the public school and child care center, respectively. Twelve (26%) out of the 46 acute hepatitis patients (sporadic cases) were also HAV RNA positive. From nine of these 12 patients, a second blood sample was obtained 18-34 days after the first one: all nine had seroconverted to IgM anti-HAV, and their serum transaminases had reached elevated levels (mean ALT, 418; mean AST, 241). CONCLUSIONS Detection of HAV RNA before IgM anti-HAV seroconversion may be used as an early diagnosis method during hepatitis A outbreaks. HAV RNA testing should also help to elucidate acute hepatitis cases of unknown etiology.

Hepatitis A Outbreak in a Public School in Rio de Janeiro, Brazil

From June 1 to July 1 1999, an outbreak involving 25 cases of hepatitis A occurred in a public school in Rio de Janeiro, Brazil. Since these cases were notified to the State Health Department, the National Reference Center for Hepatitis Viruses (CNRHV) was required to investigate the extent of hepatitis A virus (HAV) dissemination. Blood samples from all students were tested for IgM and total anti-HAV antibodies using a commercial enzyme-linked immunoassay (ELISA). At the same time, a questionnaire was completed in order to identify possible risk factors for HAV infection. The environmental investigation showed that there was no fecal contamination of the water supply. The epidemiological investigation demonstrated that almost 50% of this population was susceptible to HAV infection and probably person-to-person transmission was the principal mode of virus dissemination. In this situation, a massive vaccination campaign could control the HAV infection.

Update on hepatitis B and C virus diagnosis.

Viral hepatitis B and C virus (HBV and HCV) are responsible for the most of chronic liver disease worldwide and are transmitted by parenteral route, sexual and vertical transmission. One important measure to reduce the burden of these infections is the diagnosis of acute and chronic cases of HBV and HCV. In order to provide an effective diagnosis and monitoring of antiviral treatment, it is important to choose sensitive, rapid, inexpensive, and robust analytical methods. Primary diagnosis of HBV and HCV infection is made by using serological tests for detecting antigens and antibodies against these viruses. In order to confirm primary diagnosis, to quantify viral load, to determine genotypes and resistance mutants for antiviral treatment, qualitative and quantitative molecular tests are used. In this manuscript, we review the current serological and molecular methods for the diagnosis of hepatitis B and C.