Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4 percent) and 3 (7.6 percent) were found. Subtype 1a (65.7 percent) was the most prevalent, followed by subtypes 1b (26.7 percent) and 3a (7.6 percent). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4 percent) were classified as genotype 1, subtypes 1a (72.6 percent) and 1b (19.8 percent), and 8 sequences (7.6 percent) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2 percent of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1 percent for subtype 1a and 95.2 percent for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

Co-circulation of genotypes IA and IB of hepatitis A virus in Northeast Brazil

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.

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 percent. HAV-RNA was detected in 46 percent IgM-positive serum samples and in 16 percent 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 percent of the children under 5 years were susceptible. Molecular characterization indicated the endemic circulation of specific HAV strains belonging to subgenotypes IA and IB.

Human antibodies to dengue and yellow fever do not react in diagnostic assays for hepatitis C virus

Hepatitis C virus (HCV) is a recently described causative agent of the great majority of post-transfusion non A-non B hepatitis and is classified within the Flaviviridae family. Due to a high prevalence of anti-HCV and other flaviviruses circulating in Brazil, such as dengue and yellow fever, we investigated the possibility of serological cross-reactivity between these viruses. Different panels of human sera positive for dengue type 1 (9 cases) and type 2 (7 cases) from 6 patients naturally infected with yellow fever and from 94 adults vaccinated against the 17D strain of yellow fever were tested against HCV antigens used in diagnostic assays. Two enzyme immunoassay systems were tested: one, an in-house test using recombinant antigens from core, NS3 and NS5 regions of the HCV genome (Research Foundation for Microbial Disease of Osaka University, Japan); and another, using synthetic peptides representing immunodominant epitopes of structural core and non-structural NS4 and NS5 HCV regions (INNOTEST HCV Ab, Innogenetics, Belgium). A line immunoassay (INNO-LIA HCV Ab, Innogenetics, Belgium) was used as a confirmatory test. In this, HCV antigens are coated as discrete lines on a nylon strip with plastic backing. Besides 4 control lines on each strip, a total of 6 HCV lines are present: line A consists of several NS4 epitopes, line B consists of several NS5 epitopes and lines C-F contain several core epitopes. This test not only confirms but differentiates antibodies to hepatitis C virus. No positive results were detected with these tests, indicating that hepatitis C infection can be evaluated by current assays in regions where flaviviruses are endemic

Human rotavirus detection in infantile acute diarrhoea: I. A comparative study of inmunodiffusion in agarose gel (ID) and electron microscopy (EM)

Com o intuito de detectar rotavírus em fezes diarréicas, as técnicas de imunodifusäo em gel de agarose (ID) e microscopia electrônica (ME) foram usadas comaprativamente. O estudo foi realizado com 89 amostras fecais diarréicas colhidas em hospital local, originárias de crianças com até dois anos de idade acometidas de diarréia aguda. O teste de ID foi realizado utilizando-se soro de coelho anti-rotavírus. As observaçöes ao ME foram feitas após ultracentrifugaçäo ou imunoconcentraçäo (IEM) dos homogenatos fecais. Das 89 amostras fecais o antígeno de rotavírus foi detectado em 55% (49% ) dos materiais por ID e em 45% (40) pela ME. Os resultados de ME consistiram no estudo de 71 amostras fecais analisadas após ultracentrifugaçäo com uma positividade de 45% (32) e de 18 amostras estudadas por IEM com positividade de 44% (8). Todas as amostras (40) positivas pela ME foram positivas po ID, entretanto ID detectou antígeno em nove materiais consistemente negativos pela ME. A análise estatística (teste de McNemar) do resultado é favorável à ID em relaçäo à ME (0.01 > 0.0027)

Immunofluorescent antibody to rotavirus and antigen excretion in infantile acute diarrhoea

Realizamos este estudo para avaliar a relaçäo causal diarréia aguda e rotavírus em crianças abaixo de cinco anos de idade. A infecçäo viral foi caracterizada pela reaçäo de imunofluorescência indireta (IF) nos soros dos pacientes. Utilizamos como substrato para a reaçäo células MA-104 infectadas com rotavírus bovino cepa UK. De 80 amostras de soros pareados, verificamos que 23 amostras (28,75%) apresentaram soroconversäo, 19 amostras (23,75%) tiveram elevaçäo no título de 2 vezes e em 38 amostras (47,5%) näo observamos elevaçäo no título de anticorpos anti-rotavírus. Estes resultados säo confrontados com os resultados previamente obtidos na detecçäo do antígeno de rotavírus nas fezes, dos mesmos pacientes, pela técnica de contraimunoeletroforese (CIEOP)

Rotavirus: agente de diarreia infantil. / Rotavirus:agent of infantile diarrhea

E feita uma revisao da principal literatura sobre os Rotavirus, sendo analisadas as caracteristicas das particulas virais do ponto de vista morfologico e sob outros aspectos. A acao patogenica desses agentes e considerada e analisados os aspectos clinicos das perturbacoes pelos mesmos produzidos. As diferentes tecnicas para visualizacao dos Rotavirus por microscopia eletronica sao avaliadas, bem como as de natureza imunologica.Finalmente, sao mencionados os resultados obtidos em 200 exames em que os Rotavirus foram pesquisados