Diagnóstico da hepatite por vírus C em doadores de sangue brasileiros, usando a reação de transcrição reversa e a reação em cadeia da polimerase "nested": comparação com os ensaios imunoenzimáticos e imunoblot recombinante

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA >; or = 3, ALT; or = 3, ALT>;1), group C (1Na prevenção da transmissão de Hepatite por Vírus C (HCV) em transfusões de hemocomponentes, utiliza-se rotineiramente, como testes de triagem de doadores de sangue, ensaios que detectam anticorpos anti-HCV e dosagens da enzima alanina-aminotransferase (ALT). O presente estudo tem como objetivo principal avaliar a eficiência do ensaio imunoenzimático de segunda geração (EIA-2) como teste de triagem, na identificação de doadores de sangue potencialmente infectados, e portanto, capazes de transmitir hepatite C pelos hemocomponentes. Nós utilizamos o ensaio de transcrição reversa (RT) e a reação em cadeia da polimerase "nested" («nested PCR») para investigar a presença do RNA do vírus da hepatite C (HCV) em doadores de sangue. A prevalência do RNA-HCV em indivíduos positivos foi comparada com os resultados do ensaio complementar imunoblot recombinante de segunda geração (RIBA-2) com o intuito de avaliar a utilidade de ambos como testes confirmatórios. Estes dois testes também foram comparados com a razão DO/C (valores de densidade óptica das amostras dividida pelo valor de corte da reação) no EIA-2. Os resultados das dosagens da ALT foram expressos como uma razão unitária denominada qALT, que representa o cálculo do valor do ALT da amostra dividido pelo valor máximo considerado normal para o teste (53UI/L). Os doadores de sangue foram divididos em cinco grupos de acordo com os resultados do EIA-2 e o qALT: grupo A (EIA >; ou = 3, ALT; ou = 3, ALT>;1), grupo C (

Hepatitis C virus in monozygotic twins

É relatado o caso de paciente grávida, com hepatite C crônica que deu à luz dois gêmeos monozigóticos. Um recém-nascido apresentou positividade para o RNA do vírus da hepatite C (RNA-VHC), no sangue venoso, coletado de veia periférica doze horas após o parto. O outro recém-nascido apresentou-se negativo para o RNA-VHC logo após o nascimento, porém tornou-se RNA-VHC positivo na amostra coletada aos três meses de idade. Os resultados permitem supor que um dos gêmeos provavelmente foi contaminado no período intra-uterino, enquanto o outro adquiriu a infecção no período perinatal. Ambos foram negativos para a presença do RNA-VHC e para os anticorpos anti-HCV em todas as amostras séricas coletadas após os nove meses de idade. Os exames laboratoriais dos gêmeos não mostraram a presença de infecção crônica pelo VHC durante o acompanhamento de 29 meses .A case of a pregnant patient with chronic hepatitis C who gave birth to monozygotic twins that were infected with HCV is reported. One of the newborns was positive for HCV-RNA in blood sample collected 12 hours after delivery. The other newborn was negative for HCV-RNA at birth, but was detected HCV viremia at three months of age. The results have led to the conclusion that one of the twins was probably contaminated in the intrauterine period, while the other acquired the infection in the perinatal period. Both were negative for HCV-RNA and for anti-HCV in the serum samples collected at nine months of age. The report describes the changes in the laboratory tests conducted in mother and twins until 29 months after delivery

Avaliação clínica, epidemiológica, laboratorial, histológica e ultrassonográfica de doadores de sangue anti-HCV EIA-2 positivos

Between 1992 and 1997, 790 blood donors with anti-HCV EIA-2 strongly reagent (relationship between the sample optical density/cut-off >; 3) detected at the blood bank serological screening, were evaluated in ambulatory environment. They were all negative for Chagas disease, syphilis, hepatitis B (HBsAg) and AIDS. Blood samples were collected at the first ambulatorial evaluation, for hemogram, biochemical tests and new serological tests for HCV (anti-HCV EIA-2). In blood samples of 226 repeatedly reagent anti-HCV EIA-2 blood donors, supplementary "immunoblot" test for HCV (RIBA-2) was used. In 209 donors, the presence of HCV-RNA was investigated by the PCR test. The abdominal ultrasonography was realized in 366 donors. In 269 patients liver biopsy was performed for the histopathological study. The follow-up of blood donors showed that 95.6% were repeatedly EIA-2 reagent, 94% were symptomless and denied any hepatitis history, with only 2% mentioning previous jaundice. In 47% of this population at least one risk factor has been detected for the HCV transmission, the use of intravenous drugs being the main one (27.8%). Blood transfusion was the second factor for HCV transmission (27.2%). Hepatomegaly was detected in 54% of the cases. Splenomegaly and signs of portal hypertension have seldom been found in the physical examination, indicating a low degree of hepatic compromising in HCV. Abdominal ultrasound showed alterations in 65% of the subjects, being the steatosis the most frequent (50%). In 83.5% of the donors submitted to the liver biopsy, the histopathological exam showed the presence of chronic hepatitis, usually classified as active (89%) with mild or moderate grade in most of the cases (99.5%). The histopathological exam of the liver was normal in 1.5% of blood donors. The RIBA-2 test and the HCV-RNA investigation by PCR were positive in respectively 91.6 and 75% of the anti-HCV EIA-2 reagent donors. The HCV-RNA research was positive in 82% of the RIBA-2 positive subjects, in 37.5% of the indeterminate RIBA-2 donors and in 9% of the negative RIBA-2 donors. Chronic hepatitis has also been observed in 50% of the histopathological exams of the anti-HCV EIA-2 reagent donors which were indeterminate RIBA-2. Among 18 blood donors with minimal changes histopathological exam 11 (61%) were HCV-RNA positive. Our blood donors anti-HCV reagent generally had clinical, laboratorial and histopathological features observed in patients with chronic HCV hepatitis and a high proportion could be identified in interviews and medical evaluation realized in blood blanks. Generally, these HCV infected donors are identified and discharged only by the serological tests results.Entre 1992 e 1997 foram avaliados, ambulatorialmente, 790 doadores de sangue com teste anti-HCV EIA-2 fortemente reagente (relação entre a densidade ótica da amostra / "cut-off" >; 3), que haviam sido detectados na triagem sorológica do banco de sangue. Todos eram negativos para doença de Chagas, sífilis, hepatite B (HBsAg) e AIDS. Amostras de sangue foram coletadas, na primeira consulta ambulatorial, para a realização de hemograma, exames bioquímicos e novos testes sorológicos para a HVC (anti-HCV EIA-2). Em 226 doadores anti-HCV EIA-2 repetidamente reagentes, realizou-se o teste suplementar de "immunoblot" para a HVC (RIBA-2). Em 209 doadores, pesquisou-se a presença do RNA do VHC pelo teste do PCR, através de exame automatizado (HCV-AMPLICOR, ROCHE). A ultra-sonografia abdominal foi realizada em 366 doadores e a biópsia hepática em 269 concordantes. Notou-se que 95,6% eram EIA-2 repetidamente reagentes, 94% eram assintomáticos e que apenas 2% referiram icterícia pregressa. Em 47% detectou-se, pelo menos, um fator de risco para a transmissão do VHC, sendo o uso de drogas E.V. o principal deles (27,8%). A transfusão de sangue foi o segundo fator na transmissão da HVC (27,2%). Hepatomegalia foi encontrada em 54%. Esplenomegalia e sinais de hipertensão portal foram raramente encontrados no exame físico, denotando o baixo grau de comprometimento hepático na HVC. A ultra-sonografia abdominal mostrou-se alterada em 65% dos indivíduos, sendo a esteatose a alteração mais freqüentemente observada (50%). Em 83,5% dos doadores submetidos à biópsia hepática, diagnosticou-se hepatite crônica, geralmente classificada como ativa (89%) e de grau leve ou moderado na maioria dos casos (99,5%). O histopatológico foi normal em 1,5% dos doadores. O teste de RIBA-2 e a pesquisa do RNA do VHC pelo PCR foram positivos em, respectivamente, 91,6 e 75% dos doadores anti-HCV EIA-2 reagentes. A pesquisa do RNA do VHC foi positiva em 82% dos indivíduos RIBA-2 reagentes, em 37,5% dos doadores RIBA-2 indeterminados e em 9% dos RIBA-2 negativos. Hepatite crônica foi observada em 50% dos doadores RIBA-2 indeterminados. Entre 18 doadores com alterações mínimas, ao exame histopatológico, 11 (61%) eram positivos para o RNA do VHC. Nossos doadores de sangue anti-HCV reagentes geralmente apresentam alterações clínicas, laboratoriais e histopatológicas próprias de pacientes com hepatites crônicas pelo VHC e uma elevada proporção destes podem ser identificados em entrevistas e avaliação médicas rotineiramente realizadas em bancos de sangue. Geralmente estes doadores infectados pelo VHC somente são identificados e bloqueados pelos resultados dos testes sorológicos

Hepatitis B virus: molecular genotypes and HBeAg serological status among HBV-infected patients in the southeast of Brazil

<p>Abstract</p> <p>Background</p> <p>Knowledge of HBV genotype is very important for clinical treatment. Studies have suggested possible pathogenic and therapeutic differences among HBV genotypes. The aim of this study was to determine HBV subtypes and genotypes in HBV-infected patients in our region (southeast Brazil) and to correlate results with clinical and histopathological data.</p> <p>Methods</p> <p>One hundred and thirty-nine HBsAg-positive patients were included in the study. All patients were anti-HCV and anti-HIV negative (64% male; mean age 42 ± 14.5 years; range 7-80 years; 84% Caucasian) and were followed up at the University Hospital. A method for genotyping and subtyping HBV by partial HBsAg gene sequencing with primers common to all known genotypes was used. The viral load was measured by Amplicor Monitor assay (Roche).</p> <p>Results</p> <p>HBV genotype A was the most prevalent (55%), while genotypes C, D and F were found in 3%, 38% and 4% of HBV-infected patients, respectively. Among the patients infected by genotype A, 18.3% (14/76) were African descendents and, among the patients infected by genotype D, 11.3% (6/53) were also African descendents. In the four patients infected with genotype C, 2 were Asian descendents and 2 were Caucasians. All (7) genotype F infected patients were Caucasians. Seventy percent of our HBsAg-positive patients were HBeAg negative (62% genotypes A; 26.2% D; 7.1% C and 4.7%F). The viral load of HBV-DNA was about 5 times higher in HBeAg-positive than in HBeAg-negative patients. About 40% of these patients had alanine aminotransferase of up to 1.5 times the normal level. The mean stage of fibrosis in genotype A patients (2.8) was significantly higher than the mean stage of fibrosis in genotype D patients (2.0) (P = 0.0179).</p> <p>Conclusion</p> <p>The genotypes encountered in our HBV-infected patients were apparently a consequence of the types of immigration that occurred in our region, where European and African descendents predominate. The HBeAg-negative status predominated, possibly due to the length of time of infection. The viral load in HBeAg-positive patients was higher than in HBeAg-negative individuals. The fibrosis grade in genotype A-infected patients was more advanced than genotype D-infected patients.</p

Diagnóstico da hepatite por vírus C em doadores de sangue brasileiros, usando a reação de transcrição reversa e a reação em cadeia da polimerase nested: comparação com os ensaios imunoenzimáticos e imunoblot recombinante

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT or = 3, ALT>1), group C (11) and group E (EIA or =3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.Na prevenção da transmissão de Hepatite por Vírus C (HCV) em transfusões de hemocomponentes, utiliza-se rotineiramente, como testes de triagem de doadores de sangue, ensaios que detectam anticorpos anti-HCV e dosagens da enzima alanina-aminotransferase (ALT). O presente estudo tem como objetivo principal avaliar a eficiência do ensaio imunoenzimático de segunda geração (EIA-2) como teste de triagem, na identificação de doadores de sangue potencialmente infectados, e portanto, capazes de transmitir hepatite C pelos hemocomponentes. Nós utilizamos o ensaio de transcrição reversa (RT) e a reação em cadeia da polimerase nested («nested PCR») para investigar a presença do RNA do vírus da hepatite C (HCV) em doadores de sangue. A prevalência do RNA-HCV em indivíduos positivos foi comparada com os resultados do ensaio complementar imunoblot recombinante de segunda geração (RIBA-2) com o intuito de avaliar a utilidade de ambos como testes confirmatórios. Estes dois testes também foram comparados com a razão DO/C (valores de densidade óptica das amostras dividida pelo valor de corte da reação) no EIA-2. Os resultados das dosagens da ALT foram expressos como uma razão unitária denominada qALT, que representa o cálculo do valor do ALT da amostra dividido pelo valor máximo considerado normal para o teste (53UI/L). Os doadores de sangue foram divididos em cinco grupos de acordo com os resultados do EIA-2 e o qALT: grupo A (EIA > ou = 3, ALT ou = 3, ALT>1), grupo C (11) e grupo E (EIA ou = 3 e a positividade do RNA-HCV por RT-nested PCR. Nós também pudemos observar que os doadores de sangue com RIBA-2 indeterminados apresentaram um alto grau de positividade no RT-nested PCR (75%), especialmente quando está presente a banda correspondente ao antígeno c22.3.26326

Comparative Study Of Patients With Chronic Hepatitis C Virus Infection Due To Genotypes 1 And 3 Referred For Treatment In Southeast Brazil.

The progression of liver disease in patients with chronic hepatitis C virus (HCV) infection is influenced by host and viral factors. Distinct clinical outcomes in patients infected with different HCV genotypes have been described in the literature. However, the association between specific HCV genotype and clinical outcome remains unclear. We set out to study the natural history of HCV genotype 1 and 3 infections in Campinas, São Paulo state, Brazil, focusing on epidemiological, clinical, biochemical, and histological characteristics. Patients with HCV infection referred for treatment between January 2003 and December 2006 were included in this study. We collected epidemiological, clinical, and laboratorial data using standard forms. A total of 283 patients were included; genotype 1 was identified in 163 (57.6%) patients, genotype 3 in 112 (39.6%), genotype 2 in 7 (2.5%), and genotype 4 in 1 (0.35%). Patients with genotype 2 and 4 were excluded from analysis. Multivariate analysis showed that intravenous energetic drug, positive cryoglobulin, and cirrhosis were independently and significantly associated with HCV genotype 3 (p < 0.05). Genotype 3 currently seems to be associated with intravenous energetic drug, high frequency of cryoglobulinemia, and advanced liver disease in our region. Understanding the distribution of the different HCV genotypes can elucidate transmission of HCV and support optimal prevention strategies.816

Hepatite pelo vírus C em gêmeos monozigóticos

A case of a pregnant patient with chronic hepatitis C who gave birth to monozygotic twins that were infected with HCV is reported. One of the newborns was positive for HCV-RNA in blood sample collected 12 hours after delivery. The other newborn was negative for HCV-RNA at birth, but was detected HCV viremia at three months of age. The results have led to the conclusion that one of the twins was probably contaminated in the intrauterine period, while the other acquired the infection in the perinatal period. Both were negative for HCV-RNA and for anti-HCV in the serum samples collected at nine months of age. The report describes the changes in the laboratory tests conducted in mother and twins until 29 months after delivery.É relatado o caso de paciente grávida, com hepatite C crônica que deu à luz dois gêmeos monozigóticos. Um recém-nascido apresentou positividade para o RNA do vírus da hepatite C (RNA-VHC), no sangue venoso, coletado de veia periférica doze horas após o parto. O outro recém-nascido apresentou-se negativo para o RNA-VHC logo após o nascimento, porém tornou-se RNA-VHC positivo na amostra coletada aos três meses de idade. Os resultados permitem supor que um dos gêmeos provavelmente foi contaminado no período intra-uterino, enquanto o outro adquiriu a infecção no período perinatal. Ambos foram negativos para a presença do RNA-VHC e para os anticorpos anti-HCV em todas as amostras séricas coletadas após os nove meses de idade. Os exames laboratoriais dos gêmeos não mostraram a presença de infecção crônica pelo VHC durante o acompanhamento de 29 meses .16316