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.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4 percent), followed by genotype 3 (21.4 percent), and genotype 2 (7.2 percent). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4 percent), mild (57.2 percent), and moderate (21.4 percent). Viral RNA was detected in liver cells from nine patients (64.3 percent). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Prevalence and risk factors for HBV, HCV and HDV infections among injecting drug users from Rio de Janeiro, Brazil

Viral hepatitis constitutes a major health issue, with high prevalence among injecting drug users (IDUs). The present study assessed the prevalence and risk determinants for hepatitis B, C and D viruses (HBV, HCV and HDV) infections among 102 IDUs from Rio de Janeiro, Brazil. Serological markers and HCV-RNA were detected by enzyme immunoassay and nested PCR, respectively. HCV genotyping was determined by restriction fragment length polymorphism analysis (RFLP). HBsAg, anti-HBc and anti-HBs were found in 7.8, 55.8 and 24.7 percent of IDUs, respectively. In the final logistic regression, HBV infection was independently associated with male homosexual intercourse within the last 5 years (odds ratio (OR) 3.1; 95 percent confidence interval (CI) 1.1-8.8). No subject presented anti-delta (anti-HD). Anti-HCV was detected in 69.6 percent of subjects, and was found to be independently associated with needle sharing in the last 6 months (OR 3.4; 95 percent CI 1.3-9.2) and with longer duration of iv drug use (OR 3.1; 95 percent CI 1.1-8.7). These data demonstrate that this population is at high risk for both HBV and HCV infection. Among IDUs from Rio de Janeiro, unprotected sexual intercourse seems to be more closely associated with HBV infection, whereas HCV is positively correlated with high risk injecting behavior. Comprehensive public health interventions targeting this population and their sexual partners must be encouraged

Distribution of HCV genotypes among different exposure categories in Brazil

Hepatitis C virus (HCV) infection is widespread and responsible for more than 60 percent of chronic hepatitis cases. HCV presents a genetic variability which has led to viral classification into at least 6 genotypes and a series of subtypes. These variants present characteristic geographical distribution, but their association with different responses to treatment with interferon and severity of disease still remains controversial. The aim of this study was to investigate the patterns of distribution of HCV genotypes among different exposure categories in Brazil. Two hundred and fifty anti-HCV positive samples were submitted to HCV-RNA detection by RT-PCR and their genotype was determined by restriction fragment length polymorphism (RFLP) analysis. In addition, the genotype/subtype of 60 samples was also determined by a reverse hybridization assay. HCV 1 was the most prevalent (72.0 percent), followed by type 3 (25.3 percent), HCV 2 (2.0 percent) and HCV 4 (0.7 percent). The HCV genotype distribution varied among the different exposure categories, with HCV 1 being more frequent among blood donors, hemophiliacs and hemodialysis patients. A high frequency of HCV 3 was observed in cirrhotic patients, blood donors from the South of Brazil and injecting drug users (IDUs). The general distribution of the HCV genotype in Brazil is similar to that in other regions of the world

The use of non-human primates as animal models for the study of hepatitis viruses

Hepatitis viruses belong to different families and have in common a striking hepatotropsin and restrictions for propagation in cell culture. The transmissibility of hepatitis is in great part limited to non-human primates. Enterically transmitted hepatitis viruses (hepatitis A virus and hepatitis E virus) can induce hepatitis in a number of OLD World and New Worls monkey species, while the host range of non-human primates susceptible to hepatitis viruses transmitted by the parenteral route (hepatitis B virus, hepatitis C virus and hepatitis delta virus) is restricted to few species of Old World monkeys, especially the chimpanzee. Experimental studies on non-human primates have provided an invaluable source of information regarding the biology and pathogenesis of these viruses, and represent a still indispensable tool for vaccine and drug testing.

Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5'NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1 per cent). The sequence of another isolate presented a three-nucleotide deletion at codon 329.

Intragastric infection induced in marmosets (Callithrix jacchus) by a Brazilian hepatitis A virus (HAF-203)

Several specied of non-human primates have been used in studies on experimental infection with hepatitis A virus (HAV). Attempts to infect a South-American marmoset (Callithrix jacchus) with a Brazilian HAV isolate (HAF-203) are described here. Four seronegative animals were inoculated intragastrically and one was sacrificed on day 11,20,47 and 62 after infection. One uninfected animal was included as control. Liver, small intestine, lymph node, spleen and kidney samples were collected for histological diagnosis and immunocytochemistry studies. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum enzymes and anti-HAV antibodies were monitored by a colorimetric procedure (Abbott) and an enzyme immunoassay (ELISA), respectively. Feces were collected daily for HAV antigen (HAVAg) detection by ELISA. Increased levels of HAVAg were detected in hepatocytes 11 days after infection, with a gradual decrease during the course of infection. Shedding of HAVAg in feces was observed from the late incubation to the early acute phase (20th day to 47th day after infection). The end of the incubation period was indicated by the initial increases in serum ALT and AST. Severe hepatic lesions such as piecemeal necrosis and bridging necrosis were detected during the acute phase, coinciding with the maximum transaminase levels and the appearance of anti-HAV antibodies. On the 62nd day (convalescent phase), the hepatic tissue showed evidence of regeneration and the transaminase values had returned to baselines. The serological, biochemical, antigenic and histological evidence of hepatitis A was similar to that observed in several primate models inoculated with other HAV isolates. The data suggest that C. jacchus can be a valuable model for the study of hepatitis A and for the evaluation of HAV vaccines

High prevalence of hepatitis C infection among brazilian hemodialysis patients in Rio de Janeiro a one year follow-up study

Aproxidamente 400 pacientes de hemodialise tratados em 5 diferentes unidades no Rio de Janeiro foram acompanhados durante 1 ano para presenca de marcadores virais de hepatite B e C. Durante o mesmo periodo, amostras foram tambem de 35 pacientes ambulatoriais de dialise peritonial continua (CAPD) e de 242 funcionarios das unidades. Dependendo da unidade em estudo foram detectadas prevalencias de anti-HCV variando de 47 por cento a 82 por cento (media 65 por cento ). Pacientes de prevalencia de anti-HCV em funcionarios foi de 2,9 por cento . Observamos uma taxa de ataque de hepatite C de 11,5 por cento por ano na populacao paciente de hemodialise anti-HCV negativo. Uma media de 9,4 por cento de pacientes de hemodialise eram portadores cronicos do virus da hepatite B (VHB) (taxa de 1.8 por cento a 20.4 por cento ), enquanto 48.9 por cento apresentaram marcadores de infeccao passada de HBV. A taxa de ataque de HBV foi de 4.5 por cento por ano (taxa de 0 por cento a 6 por cento ). Esses resultados indicam uma alarmante prevalencia alta anti-HCV em pacientes de hemodialise dessa regiao estudada.

Hepatitis B and C prevalences among blood donors in the south region of Brazil

The prevalence of hepatitis B and C infection has been determined in a seroepidemiological survey among blood donors from the south of Brazil (Florianópolis, State of Santa Catarina). These markers has also been correlated with the levels of alanine aminotransferase (ALT), a surrogate marker to prevent post-transfusion hepatitis. Sera from 5000 donors were randomly collected in the period of April to November 1991. The prevalences of HBsAg, anti-HBs and anti-HBc were respectively 0.78, 7.02 and 13.98. The anti-HCV prevalence after confirmation testing with line immunoassay (LIA), was 1.14. Normal values of ALT ( < = 32 U/ml) were found in 59.78, values slightly above the mean (ALT between 32-70 U/ml) in 37.74 and high values of ALT ( > = 70 U/ml) in 2.48. The positivity of anti-HCV antibodies increased with the elevation of ALT levels. This correlation was not observed in relation to HBsAg. There exists a diversity in the recognition of HCV epitopes among HCV positive donors. Via the confirmation test used, we could observe that 94.7 of donors recognize the structural core antigen. Besides that, we observed that 5.26 of the HCV reactive sera recognized only epitopes located in the NS4 and/or NS5 region, indicating the importance of these epitopes for the improvement of assays.

Anti-HCV related to HCV PCR and risck factors analysis in a blood donor population of central Brazil

Ainda sao raros os casos de infeccao por hepatite C (HCV)na regiao central do Brasil. Neste estudo, 2.350 doadores voluntarios de sangue foram avaliados, resultando em prevalencias para o anti-HCV de 2,2 (por cento), pelo ELISA de segunda geracao, e de 1,4 por cento, apos o ensaio confirmatorio "line immunoassay". Anticorpos contra os antigenos "core", NS4 e NS5 do HCV foi observada em 76,6 (por cento) dos doadores anti-HCV positivos. A positividade da reacao em cadeia da polimerase (PCR) mostrou-se relacionada a reatividade aos diferentes antigenos do HCV no "line immunoassay". A maioria dos doadores positivos tiveram historia previa de exposicao parenteral. A combinacao de ALT>50 UI/1 e positividade ao anti-HBc parece nao ser eficaz como marcadores indiretos para a infeccao pelo HCV, entretanto a dosagem do ALT e a deteccao de anti-HCV sao indicadas na triagem de doadores de sangue brasileiros.

Antibodies against non-structural c100/3 and structural core antigen of hepatitis C virus (HCV) in hemodialysis patients

Two groups of patients undergoing hemodialysis (HD) maintenance were evaluated for their antibody response to non-structural c100/3 protein and structural core protein of hepatitis C virus (HCV). Forty-six patients (Group 1) never presented liver abnormalities during HD treatment, while 52 patients (Group 2) had either current or prior liver enzyme elevations. Prevalence rates of 32.6 and 41.3 were found for anti-c100/3 and anti-HCV core antibodies, respectively, in patients with silent infections (Group 1). The rate of anti-c100/3 in patients of Group 2 was 71.15 and reached 86.5 for anti-HCV core antibodies. The recognition of anti-c100/3 and anti-core antibodies was significantly higher in Group 2 than in Group 1. A line immunoassay composed of structural and non-structural peptides was used as a confirmation assay. HBV infection, measured by the presence of anti-HBc antibodies, was observed in 39.8 of the patients. Six were HBsAg chronic carriers and 13 had naturally acquired anti-HBs antibodies. The duration of HD treatment was correlated with anti-HCV positivity. A high prevalence of 96.7 (Group 2) was found in patients who underwent more than 5 years of treatment. Our results suggest that anti-HCV core ELISA is more accurate for detecting HCV infection than anti-c100/3. Although the risk associated with the duration of HD treatment and blood transfusion was high, additional factors such as a significant non-transfusional spread of HCV seems to play a role as well. The identification of infective patients by more sensitive methods for HCV genome detection should help to control the transmission of HCV in the unit under study.

Fast growth of a Brazilian hepatitis A virus (HAF-203) in a primate cell line

A hepatitis A virus (HAV, HAF-203) isolated in Brazil was submitted to 8 serial passages through fetal Rhesus kidney cells (FRhK-4). The kinetics of replication were monitored by enzyme immunoassay (EIA-HAVAg) and cDNA-RNA dot blot hybridization. The maximum level of RNA, which was observed 21 days post-infection (p.i.) during the 3rd passage, when HAVAg was still undetectable by EIA, served as a basis to establish subsequent passages every 21 days p.i. This schedule of passage resulted in a progressive reduction of time between culture infection and HAVAg and RNA production, together with an enhancement in antigen titer content of cell lysates. During the 7th passage, maximum HAVAg and RNA levels were detected at 7 days. Fourteen days after the 8th passage, clear morphological modifications appeared, suggesting a good adaptation of HAF-203 to FRhK-4 cells. Obtaining a fast-growing Brazilian HAV is very important for the development of vaccines

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

Primary isolation of a brazilian strai9n of hepatitis A virus (HAF-203) and growth in a primate cell line (FRhK-4)

Although hepatitis A is endemic in Brazil, this is the first report describing the isolation of a Brazilian strain of hepatitis A virus (HAV). Fecal specimens obtained from patients in the acute phase of hepatitis A were iunoculated into fetal Rhesus kidney cell cultures (FRhK-4). Only one inoculum, denoted HAF-203, could be propagated serially. Both cell lysates and tissue culture fluids of infected cells were used as inocula and evaluated for viral antigen and RNA content by enzyme immunoassay and cDNA-RNA hybridization, respectively. Cell lysates gave better yields when used as viral inocula. After three passages, viral RNA and antigen were detected in cell lysates 4 and 14 days post-infection, respectively. Using tissue culture fluid as inoculum, the incubation period was decreased from 49 to 7 days after 4 serial passages, reflecting the adaptation of HAF-203 to growth in FRhK-4 cells. FRhK-4 cells can now be used for HAV antigen production for diagnostic assays and molecular characterization

Resposta imune à vacina da hepatite B (Hevac B, França) em unidade de diálise / Immuneresponse to B hepatitis vaccine (Hevac B, France) in a dialysis service

Foi feita uma avaliaçäo da resposta imunológica a vacina da hepatite B (Hevac B, Instituto Pasteur, França) nos pacientes e pessoal de uma unidade de hemodiálise (HD). 88,4% (23/26) desenvolveram anticorpos contra o antígeno de superfície da hepatite B (anti-HBs) entre os elementos do pessoal da HD; 75% /12) dos pacientes adultos e 87,5% (7/8) dos pacientes infantis também desenvolveram o anticorpo, todos após seis meses da primeira dose de vacina. Nenhum dos recipientes da vacina apresentou o antígeno de superficie da hepatite B (AgABs) no sangue em qualquer momento. Ficou demonstrado alto índice de resposta a vacinaçäo contra a hepatite B no pessoal e nos pacientes da HD, particularmente nas crianças, com bons títulos de anti-HBs após quatro injeçöes da vacina Hevac B

Antibody response to heat-inactivated hepatitis B vaccine (CLB-3 microng) in hemodialysis patients and occupational risk personnel: a one year follow-up

A resposta imune contra vacina de hepatite B (CLB-3microng) foi avaliada em 59 pacientes de hemodiálise e 20 funcionários de risco em adquirir infecçäo. A soroconversäo foi observada em 52,5% e 70,0%, respectivamente. Um ano após a primeira dose, os níveis de anticorpos anti-HBs foram determinados em 37.5% dos pacientes e 60.0% dos funcionários. Os níveis de anticorpos foram expressos em unidades de radioimunoensaio (SRU =contagem da amostra sobre o controle negativo). Considerando apenas os indivíduos que responderam à vacina, no grupo de pacientes, 38.7% tiveram resposta baixa de anticorpos (2,1 - 9,9 SRU), 32,3% resposta média e 29,0% uma resposta elevada (>SRU), enquanto que no passado de risco, os valores foram 14,3%, 64,3% e 21,4%, respectivamente. Os autores sugerem o uso de vacinas de HBV com concentraçöes de HBsAg mais elevada ou um reforço no esquema de imunizaçäo para melhorar a resposta de anti-HBs, näo só para pacientes como também para pessoas sadias