Assuntos
Anticorpos Antibacterianos/sangue , Borrelia burgdorferi/imunologia , Imunoglobulina G/sangue , Doença de Lyme/epidemiologia , Adulto , Segurança do Sangue , Reservatórios de Doenças , Feminino , Humanos , Doença de Lyme/sangue , Masculino , Pessoa de Meia-Idade , População Rural/estatística & dados numéricos , Estudos de Amostragem , Escócia/epidemiologia , Estudos Soroepidemiológicos , População Urbana/estatística & dados numéricosRESUMO
Serological screening assays have greatly reduced, but not eliminated, the risk of transmission of viral infections by transfusion of blood and blood products. In addition, the 1999 regulation of the European Agency for the Evaluation of Medicinal Products requiring all plasma for fractionation to have tested negative for hepatitis C virus (HCV) RNA (CPMP/BWP/390/97, 1998) led many blood transfusion services to introduce nucleic acid amplification technology (NAT) to screen blood donations for HCV, and in some services for human immunodeficiency virus (HIV) and hepatitis B virus (HBV). BioMérieux's second-generation system, the NucliSENS easyMAG, was evaluated as a suitable platform for the automated extraction of nucleic acids for use with the existing SNBTS NAT assays. Two nucleic acid extraction protocols were examined, either lysis on the easyMAG (on board) or a 30-min pre-incubation of the sample with lysis buffer at 37 °C (off board). Off board lysis was found to extract nucleic acid more efficiently for both HCV and HIV NAT assays although the improvement was more marked with HIV. The 95% limit of detections (LODs) were 10.11 IU/ml (on board) and 7.21 IU/ml (off board) for HCV and 55.11 IU/ml (on board) and 34.13 (off board) for HIV. Using the more sensitive off board lysis, nucleic acid extraction specificity, robustness and reliability of the easyMAG were examined and over 10,000 Scottish blood donations (in 107 pools of 95 donations) were tested for HCV and HIV in parallel with the existing assay. The results indicate that the easyMAG is a suitable and flexible nucleic acid extraction system, providing high quality nucleic acids and a rapid response alternative to commercial, fully automated, approved blood screening platforms.
Assuntos
Automação/métodos , Sangue/virologia , HIV-1/isolamento & purificação , Hepacivirus/isolamento & purificação , Ácidos Nucleicos/isolamento & purificação , Manejo de Espécimes/métodos , Virologia/métodos , Doadores de Sangue , Infecções por HIV/diagnóstico , Infecções por HIV/virologia , HIV-1/genética , Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/virologia , Humanos , Escócia , Sensibilidade e EspecificidadeRESUMO
BACKGROUND AND OBJECTIVES: Positive samples identified during routine serological screening for HCV (hepatitis C virus), HBV (hepatitis B virus) and HIV (human immunodeficiency virus) are confirmed by nucleic acid testing in the SNBTS (Scottish National Blood Transfusion Service) PCR Reference laboratory. Serological screening for HTLV-I (human T-cell lymphotropic virus type I) and -II was implemented in Scotland in November 2002, at which time a PCR assay was not available for confirmation. Our aim was to develop a real-time PCR assay that could be used for the confirmation of samples showing HTLV-I serological positive or indeterminate reactivity and to investigate whether a serologically silent carrier status exists ('Tax' only) in the Scottish donor population. MATERIALS AND METHODS: A real-time HTLV PCR was devised using a lymphoblastoid cell line which has HTLV-I sequence integrated in the genome (C8166 cells). These were spiked into peripheral blood mononuclear cells. The assay was evaluated on archived serologically confirmed HTLV-positive samples and new positives identified since implementation of screening. RESULTS: HTLV-I and -II were detected in cells and plasma from stored donations and a serological positive donation identified in routine screening. HTLV DNA can also be amplified from the plasma obtained from plasma preparation tubes. There was no evidence of a carrier status ('Tax' only) in 100 serologically negative blood donors tested. The PCR assay developed is reliable and sensitive, capable of identifying one copy of HTLV-I. CONCLUSIONS: The HTLV PCR is a useful addition for HTLV confirmation, especially in serologically indeterminate samples and for look-back studies. HTLV PCR confirmation will provide additional useful information for donor medical staff for counselling donors.
Assuntos
Doadores de Sangue , Transfusão de Sangue , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 2 Humano/genética , Reação em Cadeia da Polimerase/métodos , Genes pX/genética , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-II/diagnóstico , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Vírus Linfotrópico T Tipo 2 Humano/imunologia , Humanos , Programas de Rastreamento/métodos , Escócia , Testes Sorológicos/métodosRESUMO
BACKGROUND AND OBJECTIVES: To reduce the risk of transfusion-transmissible viruses entering the blood supply, the nucleic acid amplification testing (NAT) was implemented to screen Scottish and Northern Irish blood donations in minipools. After 5 years of NAT for hepatitis C virus (HCV) and 2 years for human immunodeficiency virus-1 (HIV-1), the yield of serologically negative, nucleic acid positive 'window donations' and cost-benefit of NAT is under review. MATERIALS AND METHODS: When the Scottish National Blood Transfusion Service (SNBTS) implemented NAT in 1999, a fully automated 'black box' system was not available. Therefore, an 'in-house' assimilated NAT assay was developed, validated and implemented. The system is flexible and allows testing for additional viral markers to be introduced with relative ease. RESULTS: The HCV and HIV NAT assays have 95% detection levels of 7.25 IU/ml and 39.8 IU/ml, respectively, as determined by probit analysis. One HCV (1 in 1.9 million) and one HIV (1 in 0.77 million) window donation have been detected in 5 and 2 years, respectively, of NAT. CONCLUSION: The SNBTS NAT assays are robust and have performed consistently over the last 5 years. The design of the in-house system allowed HIV NAT to be added in 2003 at a relatively small additional cost per sample, although for both assays, the royalty fee far exceeds the cost of the test itself. Clearly NAT has a benefit in improving the safety of the blood supply although the risks of transfusion-transmitted viral infections, as reported in the Serious Hazards of Transfusion (SHOT) report, are extremely low. Also, in UK the yield of HCV antibody negative, NAT positive donations is far lower than predicted although the early detection of an HIV window period donation and the increase of HIV in the blood donor and general populations may provide a stronger case for HIV NAT. SUMMARY SENTENCE: The yield of HCV and HIV NAT in UK is significantly less than that anticipated from statistical models.
Assuntos
Doadores de Sangue , Transfusão de Sangue/normas , Infecções por HIV/diagnóstico , Hepatite C/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/normas , Adulto , Transfusão de Sangue/economia , Transfusão de Sangue/métodos , Análise Custo-Benefício , Feminino , Soronegatividade para HIV , Humanos , Irlanda , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Escócia , Sensibilidade e Especificidade , Testes SorológicosAssuntos
Doadores de Sangue , Técnicas de Amplificação de Ácido Nucleico/métodos , Bancos de Sangue/normas , Coleta de Amostras Sanguíneas/métodos , HIV/genética , Hepacivirus/genética , Humanos , Controle de Infecções/métodos , Controle de Infecções/normas , Controle de Infecções/estatística & dados numéricos , Programas de Rastreamento/métodos , Programas de Rastreamento/normas , Técnicas de Amplificação de Ácido Nucleico/normas , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , RNA Viral/sangue , Escócia , Armazenamento de Sangue/métodosRESUMO
BACKGROUND AND OBJECTIVES: In most Western countries, blood donations are routinely screened for hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR) or other nucleic acid tests. We describe the development of a multiplexed assay for human immunodeficiency virus type 1 (HIV-1) and HCV in an internally controlled PCR suitable for large-scale blood donor screening. MATERIALS AND METHODS: The HIV/HCV multiplexed PCR used primers from highly conserved regions in the long terminal repeat region. The National Institute for Biological Standards and Controls (NIBSC) International HIV-1 RNA standard, run control and HIV-1 subtype panel were used for assay evaluation. RESULTS: The HIV-1 PCR showed a sensitivity of 24 IU/ml for HIV-1 RNA (a dilution where 95% of replicate reactions were positive), which was at least five times more sensitive than the Roche Monitor version 1.5 (using the ultrasensitive extraction protocol) and Organon NASBA assays. The assay was capable of detecting all subtypes of HIV-1 (A to H), as well as the more divergent group N and O variants. The sensitivity of the PCR was unaffected by multiplexing with HCV primers and by the presence of a bovine viral diarrhoea virus (BVDV) internal control. CONCLUSION: We have developed a highly sensitive multiplexed PCR for HIV-1 and HCV RNA screening that can be introduced into current PCR-based blood donor screening at minimal cost and without significant operational changes.
Assuntos
HIV-1/genética , Hepacivirus/genética , Reação em Cadeia da Polimerase/métodos , RNA Viral/sangue , Sequência Conservada/genética , Primers do DNA/genética , Primers do DNA/normas , Estudos de Viabilidade , Repetição Terminal Longa de HIV/genética , Humanos , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
BACKGROUND AND OBJECTIVES: The infrastructure established for screening blood donations for hepatitis C virus has enabled large-scale population testing for other viruses which are potentially transmissible by transfusion of blood components and plasma-derived blood products. We have measured the frequency of viraemia of enteroviruses and parechoviruses in 83 600 Scottish blood donors to allow an initial assessment of their risk to blood safety. MATERIALS AND METHODS: Plasma samples collected from blood donors over 7 calendar months were tested anonymously in mini-pools of 95 donations, by polymerase chain reaction (PCR) for human enterovirus and parechovirus sequences. RESULTS: A total of 19 mini-pools, from the 880 that were tested, were PCR-positive for enterovirus RNA, predicting a donor prevalence of 0.023%. Enterovirus sequences were not detected in factor VIII or IX clotting factor concentrates. None of the 230 mini-pools or concentrates contained detectable parechovirus RNA. CONCLUSIONS: The prevalence of enterovirus viraemia detected in this study predicts that at least 1000 enterovirus-contaminated blood components are transfused per year in the UK. The frequency of transmission and clinical outcome after exposure to enterovirus-contaminated blood components in recipients is unknown.
Assuntos
Doadores de Sangue , Infecções por Enterovirus/diagnóstico , Enterovirus/isolamento & purificação , Sequência de Bases , Enterovirus/genética , Infecções por Enterovirus/sangue , Humanos , Dados de Sequência Molecular , ViremiaRESUMO
The diagnosis of cirrhosis in patients with hepatitis C virus (HCV) infection is currently made using a liver biopsy. In this study we have trained and validated artificial neural networks (ANN) with routine clinical host and viral parameters to predict the presence or absence of cirrhosis in patients with chronic HCV infection and assessed and interpreted the role of the different inputs on the ANN classification. Fifteen routine clinical and virological factors were collated from 112 patients who were HCV RNA positive by reverse transcriptase-polymerase chain reaction (RT-PCR). Standard and Ward-type feed-forward fully-connected ANN analyses were carried out both by training the networks with data from 82 patients and subsequently testing with data from 30 patients plus performing leave-one-out tests for the whole patient data set. The ANN results were also compared with those from multiple logistic regression. The performance of both ANN methods was superior compared with the logistic regression. The best performance was obtained with the Ward-type ANNs resulting in a sensitivity of 92% and a specificity of 98.9% together with a predictive value of a positive test of 95% and a predictive value of a negative test of 97% in the leave-one-out test. Hence, further validation of the ANN analysis is likely to provide a non-invasive test for diagnosing cirrhosis in HCV-infected patients.
Assuntos
Hepatite C Crônica/complicações , Cirrose Hepática/complicações , Redes Neurais de Computação , Adulto , Feminino , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Humanos , Cirrose Hepática/patologia , Cirrose Hepática/virologia , Modelos Logísticos , Masculino , Valor Preditivo dos TestesRESUMO
Infection with hepatitis G virus (HGV) or GB virus-C (GBV-C) is widely distributed in human populations. Viruses related to GBV-C/HGV have been recovered from several New World primate species, including tamarins, owl monkeys and marmosets. To understand more about the relationship between GB viruses and their hosts, we used primers from the 5' non-coding (5'NC), non-structural 3 (NS3) and NS5 regions in nested polymerase chain reactions to screen for related viruses infecting non-captive chimpanzees (Pan troglodytes, troglodytes and verus subspecies). Sequences from the 5'NCR and NS5 regions were amplified from samples taken from 3 of 39 chimpanzees, and from one chimpanzee in the NS3 region. Sequence comparisons of each region revealed that the GB virus infecting chimpanzees was distinct from both GBV-C/HGV and from any of the known GBV-A sequences, but was more closely related to human viruses. GB viruses recovered from different chimpanzees were more diverse than variants of GBV-C/HGV found in humans, with 25% sequence divergence in the 5'NCR and 20% (9.5% amino acid) sequence divergence in NS5 between variants recovered from the troglodytes and verus subspecies, compared with 7.4% and 10.4% (1.9% amino acid) divergence amongst GBV-C/HGV variants infecting humans. Finding GBV-C/HGV-related viruses in an Old World monkey species suggests that GB-like viruses may be widely distributed in simians, and suggests a close evolutionary relationship with their natural hosts.
Assuntos
Flaviviridae/classificação , Flaviviridae/isolamento & purificação , Hepatite Viral Animal/virologia , Doenças dos Macacos/virologia , Pan troglodytes/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Viral , Flaviviridae/genética , Humanos , Dados de Sequência Molecular , Filogenia , RNA Helicases , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Serina Endopeptidases , Proteínas não Estruturais Virais/genéticaRESUMO
BACKGROUND: A newly discovered DNA virus, transfusion-transmitted virus (TTV), has been implicated as a cause of post-transfusion hepatitis. We investigated the frequency of TTV viraemia in UK blood donors, and the extent to which TTV contaminates blood products such as factor VIII and IX clotting factors. We also investigated the possible aetiological role of TTV in cryptogenic fulminant hepatic failure (FHF). METHODS: We extracted DNA from plasma of blood donors and patients with FHF, and from blood products (factor VIII and IX clotting-factor concentrates, immunoglobulin preparations). We detected TTV by PCR using primers from a conserved region in the TTV genome. FINDINGS: TTV viraemia was detected in 19 (1.9%) of 1000 non-remunerated regular blood donors. Infection occurred more frequently in older donors (mean age 53 years), compared with the age prolife of donors infected with hepatitis C virus and other parenterally-transmitted viruses. TTV contamination was found in ten (56%) of 18 batches of factor VIII and IX concentrate manufactured from such non-remunerated donors, and in seven (44%) of 16 batches of commercially available products. Whereas solvent or detergent treatment had little effect on the detection of TTV in factor VIII and IX by PCR, this virucidal step seemed to inactivate TTV infectivity. TTV infection was detected in four (19%) of 21 patients with FHF; in three cases, infection was detected at the onset of disease and could thus not be excluded from its aetiology. INTERPRETATION: TTV viraemia is frequent in the blood-donor population, and transmission of TTV through transfusion of blood components may have occurred extensively. Clinical assessment of infected donors and recipients of blood and blood products, and assessment of TTV's aetiological role in hepatic and extra-hepatic disease, are urgently needed.
Assuntos
Transfusão de Componentes Sanguíneos/efeitos adversos , Doadores de Sangue , Vírus de DNA/isolamento & purificação , Encefalopatia Hepática/virologia , Hepatite Viral Humana/virologia , Viremia/epidemiologia , Adulto , Idoso , Criança , Vírus de DNA/genética , Feminino , Hemofilia A/virologia , Encefalopatia Hepática/epidemiologia , Hepatite Viral Humana/epidemiologia , Hepatite Viral Humana/transmissão , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Reino Unido/epidemiologia , Viremia/virologiaRESUMO
Although it is established that infection with GB virus C (GBV-C) or hepatitis G virus (HGV) can be transmitted parenterally, the prevalence of GBV-C/HGV viremia in the general population (2-5%) is relatively high compared with other parenterally borne viruses such as hepatitis C virus. To investigate the possibility of sexual transmission of GBV-C/HGV, we determined the frequency of viremia by the polymerase chain reaction and serological reactivity to the E2 protein by ELISA in samples collected from individuals at risk for sexually transmitted diseases attending a city genitourinary medicine clinic. GBV-C/HGV viremia was detected in 27 of 87 male homosexuals (31%) and 9 of 50 prostitutes (18%), frequencies significantly greater than those in matched controls (2/63) and local blood donors (2.3%). Among nonviremic individuals, a high frequency of serological reactivity to the E2 protein of GBV-C/HGV was also observed in the risk groups (male homosexuals: 14/60; prostitutes: 11/41), although these figures are likely to be underestimates of the frequency of past infection as detectable anti-E2 reactivity may attenuate rapidly over time following resolution of infection. Infection with GBV-C/HGV was more frequent among those coinfected with human immunodeficiency virus type 1. Among male homosexuals from whom retrospective samples were available, evidence for de novo infection was found in 9 of 22 individuals over a mean sampling time of 2.9 years, predicting an annualized incidence of GBV-C/HGV infection of approximately 11% in this group. The high prevalence and incidence of GBV-C/HGV infection in these individuals and prostitutes provides strong evidence for its spread by sexual contact. Further studies are required to investigate the mechanism of its transmission and the clinical significance of acute and persistent infection in these risk groups.
Assuntos
Flaviviridae , Hepatite Viral Humana/transmissão , Doenças Virais Sexualmente Transmissíveis/transmissão , Adolescente , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Flaviviridae/genética , Flaviviridae/imunologia , Flaviviridae/isolamento & purificação , Infecções por HIV/complicações , Anticorpos Anti-Hepatite/sangue , Hepatite Viral Humana/complicações , Hepatite Viral Humana/diagnóstico , Hepatite Viral Humana/epidemiologia , Homossexualidade Masculina , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Prevalência , RNA Viral/sangue , Estudos Retrospectivos , Escócia/epidemiologia , Trabalho Sexual , Doenças Virais Sexualmente Transmissíveis/complicações , Doenças Virais Sexualmente Transmissíveis/epidemiologia , Proteínas do Envelope Viral/imunologia , Viremia/diagnósticoRESUMO
BACKGROUND: The clinical significance of a single assessment of circulating hepatitis C virus (HCV) RNA and its relation to the level of intrahepatic HCV RNA remains unclear. AIMS: To investigate the relation between intrahepatic HCV levels and clinicopathological characteristics of chronic HCV infection. PATIENTS: Ninety eight consecutive patients with chronic HCV infection were studied; none had received alpha interferon therapy. Of these, 12 patients were repeatedly negative for HCV RNA in serum by reverse transcriptase polymerase chain reaction (RT-PCR). METHODS: After diagnostic laparoscopy and liver biopsy, semiquantitative analysis of intrahepatic HCV RNA levels was carried out by limiting dilution of HCV cDNA. HCV genotypes were assessed in 96 patients by restriction fragment length polymorphism analysis of HCV cDNA. RESULTS: Ten out of 12 patients who were RT-PCR negative for HCV RNA in serum were RT-PCR positive in liver; however, this group had a significantly lower intrahepatic HCV level and serum aminotransferase level than the remaining 86 patients. Histological severity (cirrhosis: n = 10); histological activity index; HCV genotype (genotype 1: n = 41; genotype 2: n = 12; genotype 3: n = 36; genotype 4: n = 7); mode of infection (intravenous drug abuse: n = 58; post-transfusion: n = 10; haemophiliac: n = 4; sporadic: n = 26) and alcohol abuse did not affect the intrahepatic virus level. There was no correlation between patient age, duration of infection, and intrahepatic HCV level. CONCLUSIONS: Intrahepatic virus levels were not determined by host factors (age of patient, mode or duration of infection) or by virus factors (HCV genotype). Repeatedly negative RT-PCR for HCV RNA in serum does not indicate absence of HCV from the liver.
Assuntos
Hepacivirus/genética , Hepatite C Crônica/virologia , Fígado/virologia , Adulto , Feminino , Genótipo , Hepatite C Crônica/enzimologia , Hepatite C Crônica/patologia , Humanos , Fígado/patologia , Masculino , Polimorfismo de Fragmento de Restrição , RNA Viral/análise , RNA Viral/sangue , Estatísticas não Paramétricas , Transaminases/sangueRESUMO
BACKGROUND: Hepatitis G virus (HGV) or GBV-C is frequently detected in patients co-infected with hepatitis C virus (HCV). This study investigated host and virologic factors influencing the response to HGV/GBV-C to alpha-interferon treatment. METHODS: HGV/GBV-C was detected and quantified by nested polymerase chain reaction. The influence of variables such as liver biopsy appearance, liver function abnormalities, and response of HCV to interferon treatment was monitored. RESULTS: Fourteen of the 25 HGV/GBV-C-infected patients treated with interferon (3-6 MIU three times a week for 6 months) became non-viraemic during treatment, although all relapsed after treatment withdrawal at 6 months, with no net change in virus load between 0 and 12 months. CONCLUSIONS: Predictive factors for clearance of HGV/GBV-C viraemia by interferon were pre-treatment severity of liver disease (median Knodell score of 4, compared with 7 for non-responders; P = 0.030) and alanine aminotransferase levels (median, 114, 182 for non-responders; P = 0.039). Clearance was associated with the treatment response of HCV. Nine of 13 who cleared HGV/GBV-C also cleared HCV, compared with 3 of 11 HGV/GBV-C non-responders; P = 0.05). The shared susceptibility of HGV/GBV-C and HCV to interferon treatment suggests a link between the mechanism of clearance of the two viruses.
Assuntos
Flaviviridae/efeitos dos fármacos , Hepatite Viral Humana/terapia , Interferon-alfa/uso terapêutico , Viremia/terapia , Adulto , Idoso , Alanina Transaminase/sangue , Feminino , Flaviviridae/isolamento & purificação , Hepatite C/sangue , Hepatite C/complicações , Hepatite C/patologia , Hepatite Viral Humana/sangue , Hepatite Viral Humana/complicações , Hepatite Viral Humana/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Viral/análise , Resultado do Tratamento , Carga ViralRESUMO
Hepatitis G virus (HGV), a novel flavivirus, has been implicated as a cause of posttransfusion hepatitis. We have performed a longitudinal study in a cohort of haemophiliacs (n = 68) who previously received non-virus inactivated coagulation factor concentrates to assess both patterns of HGV viraemia and any associated liver disease. Hepatitis C virus (HCV) RNA was present in 58/68 and co-infection with human immunodeficiency virus (HIV) was present in 15/68. HGV RNA was detected in 17/68 (25%) samples from the mid-1980s. There was no association between either HIV infection (p = 0.74) or co-infection with a particular HCV genotype (p = 0.62). However, there was a relationship between HGV viraemia and the severity of haemophilia (p = 0.0004) with HGV RNA detected in 5/19, 9/16 and 3/32 patients with mild, moderate and severe haemophilia respectively. A longitudinal study was performed in 15/17 haemophiliacs with HGV viraemia using stored serum samples from the 1980s and 1990s. HGV viraemia persisted in 8/15 and cleared in 7/15 over a variable period of time. A Weibull model was constructed to estimate the duration of HGV viraemia in the study group. The 75th and 90th percentiles for the duration of HGV were estimated to be 8.7 years (95%, confidence interval 4.8-15.7) and 23.6 years (95% confidence interval 11.8-47.1) respectively. Laparoscopic liver inspection/biopsy was performed in 25/68. There was no association between severity of liver disease and HGV viraemia (p = 0.43). This study demonstrates considerable variation in patterns of HGV viraemia in haemophiliacs. We found little evidence to implicate HGV as a major cause of chronic liver disease in haemophiliacs.
Assuntos
Fatores de Coagulação Sanguínea/efeitos adversos , Flaviviridae/isolamento & purificação , Hemofilia A/terapia , Hemofilia B/terapia , Hepatite Viral Humana/etiologia , Doenças de von Willebrand/terapia , Adolescente , Adulto , Fatores de Coagulação Sanguínea/uso terapêutico , Criança , Pré-Escolar , Contaminação de Medicamentos , Humanos , Pessoa de Meia-Idade , ViremiaRESUMO
The objective of this study was to compare the sensitivity of three different reverse transcriptase-polymerase chain reaction (RT-PCR) based tests, for detection of GB virus C (GBV-C) RNA. One commercial and two 'in house' RT-PCR assays were employed in the testing of serum samples from 114 chronic hepatitis C infected individuals. A part of the 5' non-coding region (5'NCR) of the GBV-C genome was amplified by the GBV-C LCx assay (Abbott) and one of the 'in house' RT-PCR tests. In the other 'in house' RT-PCR a segment of the NS5B region was amplified. The 'in house' assays included the use of internal controls that were co-amplified with use of the same outer PCR primers as the virus targets. The GBV-C LCx from Abbott and 5'NCR 'in house' PCR tests detected 28 and 27 GBV-C positive individuals, respectively. The sample positive only in the LCx test was confirmed by the 'in house' 5'NCR RT PCR using an increased virus input. In comparison, the NS5B 'in house' PCR test detected 24 of the GBV-C positive samples. One sample showed no amplification of internal controls/virus target in the 5'NCR 'in house' PCR and another samples was amplification negative in the NS5B PCR. The PCR assays with primers from the 5'NCR of the virus genome e.g. the GBV-C LCx, were more sensitive compared with RT-PCR using primers from the NS5B region. The GBV-C LCx seemed to be the most sensitive and robust assay. Internal controls included in the 'in house' assays identified two samples with failure of the amplification.
Assuntos
Flaviviridae/genética , Flaviviridae/isolamento & purificação , Hepatite C Crônica/virologia , Hepatite Viral Humana/virologia , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Regiões 5' não Traduzidas/genética , Primers do DNA , Genoma Viral , Humanos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genéticaRESUMO
We have investigated the ability of different subgenomic fragments to reproduce the phylogenetic relationships observed between six complete genome sequences of GBV-C/hepatitis G virus (HGV). While similar relationships were observed following analysis of part of the 5' non-coding region (5'NCR), for the coding region they were not accurately reproduced for some large fragments or for the majority of fragments of 300 or 600 nucleotides. Analysis of 5'NCR sequences from a large number of isolates, including newly obtained sequences from Pakistan, Zaïre and Scotland, produced separate groupings of Asian, African and European/North American variants. These groupings are associated with specific polymorphisms in the 5'NCR, many of which were covariant and consistent with a proposed secondary structure for this region. The relatively low level of amino acid sequence variation observed between these geographically and phylogenetically defined groups of variants suggests that they are unlikely to display significant biological differences.
Assuntos
Flaviviridae/genética , Variação Genética , Sequência de Bases , DNA Viral/química , DNA Viral/genética , Feminino , Flaviviridae/classificação , Genoma Viral , Hepatite Viral Humana/virologia , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA ViralRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. The geographical prevalence varies considerably in different countries and Scotland is regarded as an area of low risk for the disease. AIMS: To assess the association between chronic hepatitis C infection (HCV) and HCC in a population of patients presenting to a single hospital. PATIENTS: One hundred and fourteen cases of histologically confirmed liver cancer presenting to the Royal Infirmary of Edinburgh between 1985 and 1994 were examined. METHODS: Of 114 cases of HCC, 80 samples of stored sera were available. Samples positive for HCV Ab were genotyped by restriction fragment length polymorphism analysis of HCV c-DNA. A population of 29 cirrhotic patients (diagnosed between 1985 and 1994) with chronic HCV infection was also genotyped. RESULTS: Chronic HCV infection was a major risk factor (30% of tested HCC patients) identified. HCV genotype 1b was predominant (16 of 20 patients). The time from HCV transmission to development of cancer ranged from 10 to 50 years (median 30). In the cirrhotic patient population, a broader distribution of genotypes was present (genotype 1a: 7; genotype 1b: 8; genotype 2b: 3; genotype 3a: 8 and genotype 4: 2). However, this population was significantly younger. (Mean (SD) 52 (14.5) years) (p = 0.0002) and demonstrated a significantly shorter duration of infection: range 10-40 years (median: 19). CONCLUSION: There is a strong association between chronic HCV infection, cirrhosis, and hepatocarcinogenesis in this Scottish population. The study was unable to distinguish whether the high prevalence of genotype 1b in the HCC population reflected increased oncogenicity in itself, or whether 1b was simply the most prevalent genotype in Scotland when these patients were infected.
Assuntos
Carcinoma Hepatocelular/virologia , Hepatite C/complicações , Neoplasias Hepáticas/virologia , Idoso , Carcinoma Hepatocelular/epidemiologia , Doença Crônica , Feminino , Genótipo , Anticorpos Anti-Hepatite C/sangue , Humanos , Técnicas Imunoenzimáticas , Cirrose Hepática/virologia , Neoplasias Hepáticas/epidemiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Fatores de Risco , Escócia/epidemiologiaRESUMO
In a significant number of cases of fulminant (presumed viral) hepatitis worldwide, no aetiological agent has been identified. Recently, it has been suggested that a newly described flavivirus, GBV-C, is responsible for some of these cases. This study aimed to assess the clinical significance of GBV-C RNA, demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR), in the serum of patients with fulminant non-A to E hepatitis. Twenty-three consecutive cases of non-A to E fulminant hepatitis were included in the study. GBV-C RNA was reverse transcribed and amplified using two RT-PCR based detection methods. Medical records were examined to assess clinical history, duration and mode of infection, transfusion history, liver histology and clinical outcome. Five (three female, two male; mean age 21.2 years) of 23 patients had GBV-C RNA detected in their serum by RT-PCR: all five patients were RT-PCR positive following amplification by primers specific for the 5' non-coding region (NCR), whilst four were positive by primers for the NS3 region. Prior to the onset of illness, two patients had risk factors for transmission of an infectious agent; however, all five patients had been transfused during their illness, prior to testing for GBV-C. Of these, two (of two in whom serum was available) were negative for GBV-C after the onset of fulminant hepatitis but before their first transfusion. This study does not support the hypothesis that the detection of hepatitis G virus (HGV)/GBV-C RNA in the serum of patients with fulminant hepatitis indicates a causal association. However, it does demonstrate that a careful transfusion history and screening of blood products is vital before the importance of GBV-C in the aetiology of fulminant hepatitis can be established.
Assuntos
Flaviviridae/isolamento & purificação , Encefalopatia Hepática/virologia , Hepatite Viral Humana/virologia , RNA Viral/sangue , Adolescente , Adulto , Transfusão de Sangue , Criança , Feminino , Flaviviridae/genética , Encefalopatia Hepática/sangue , Encefalopatia Hepática/fisiopatologia , Hepatite Viral Humana/sangue , Hepatite Viral Humana/fisiopatologia , Humanos , Masculino , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
BACKGROUND: Hepatitis G virus (HGV or GBV-C) is a newly discovered human flavivirus distantly related to hepatitis C virus (HCV). Little information is available on its natural history or routes of transmission, although it can be transmitted parenterally. We investigated the prevalence of persistent infection of HGV and HCV in patients exposed to non-virus-inactivated pooled blood products associated with transmission of HCV. METHODS: RNA was extracted from the plasma of 112 patients with haemophilia and 57 with hypogammaglobulinaemia, as well as from 64 different batches of archived coagulation-factor concentrates and immunoglobulins. RNA was reverse transcribed and amplified with primers from the 5' non-coding region of HCV and HGV by a nested polymerase chain reaction (PCR). Viral RNA was quantified by titration of complementary DNA before amplification. FINDINGS: Among non-renumerated UK blood donors HGV infection (detected by PCR) was more common than HCV infection (four [3.2%] of 125 compared with 137 [0.076%] of 180658 in southeast Scotland). Testing of batches of factor VIII and factor IX concentrates prepared without viral inactivation procedures showed high frequencies of contamination with HGV (16 of 17 factor VIII batches positive; six of six factor IX batches positive), with no difference between renumerated and non-renumerated donors. However, among 95 haemophiliacs who had received non-virus-inactivated concentrates, 13 (14%) were positive for HGV compared with 79 (83%) who were positive for HCV. Two of 37 recipients of long-term immunoglobulin replacement therapy were positive for HGV. Virus inactivation of blood products substantially reduced or eliminated contamination by HGV RNA sequences. INTERPRETATION: Despite the extremely high level of HGV contamination of non-virus-inactivated blood products, their use was not associated with high rates of persistent infection in recipients. The infectivity of HGV in blood products may be lower than that of HCV, or the virus may be less able to establish persistent infection in humans. Whatever the case, the high prevalence of active HGV infection in the general population remains difficult to explain.