by Lap-Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 90-101). / Abstract also in Chinese. / Abstract --- p.i / Contents --- p.iv / List of tables --- p.ix / List of figures --- p.x / Abbreviations --- p.xi / Acknowledgements --- p.xii / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- The biology of hepatitis A virus --- p.1 / Chapter 1.1.1 --- History --- p.1 / Chapter 1.1.2 --- General characteristics of HAV --- p.2 / Chapter 1.1.3 --- Stability and disinfection of HAV --- p.3 / Chapter 1.1.4 --- Molecular biology of HAV --- p.5 / Chapter 1.1.4.1 --- Genomic organization of HAV --- p.5 / Chapter 1.1.4.2 --- Antigenic sites on the capsid of HAV --- p.8 / Chapter 1.1.5 --- Laboratory diagnosis and methods of study for HAV --- p.8 / Chapter 1.1.5.1 --- Cell-culture propagation and antigen detection --- p.8 / Chapter 1.1.5.2 --- Nucleic acid detection --- p.10 / Chapter 1.1.6 --- Epidemiology of HAV --- p.12 / Chapter 1.1.6.1 --- Distribution of HAV infection --- p.12 / Chapter 1.1.6.2 --- Seasonal pattern of HAV infection --- p.13 / Chapter 1.1.6.3 --- Mode of transmission --- p.13 / Chapter 1.1.6.4 --- Molecular epidemiology --- p.15 / Chapter 1.1.7 --- Epidemiology of HAV infection in Hong Kong --- p.15 / Chapter 1.2 --- Transmission of viruses through contaminated food --- p.18 / Chapter 1.2.1 --- Active accumulation of water contaminants by shellfish --- p.20 / Chapter 1.2.2 --- Retention of viruses by shellfish in contaminate water --- p.21 / Chapter 1.2.3 --- Elimination of viruses in contaminated shellfish --- p.21 / Chapter 1.2.4 --- Indicators for contamination by enteric viruses --- p.21 / Chapter 1.3 --- Detection of viruses from foods --- p.23 / Chapter 1.3.1 --- Recovery of viruses from foods --- p.23 / Chapter 1.3.2 --- Detection of viral nucleic acid --- p.23 / Chapter 1.4 --- Objectives of the study --- p.26 / Chapter Chapter 2- --- Materials and methods / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Sera from patients with acute viral hepatitis --- p.27 / Chapter 2.1.2 --- Collection of shellfish samples --- p.28 / Chapter 2.1.3 --- Purified HAV preparations as positive control --- p.30 / Chapter 2.1.4 --- Control samples for the virion capture method --- p.30 / Chapter 2.1.5 --- Preparation of dissecting instruments and processing of shellfish samples --- p.30 / Chapter 2.1.6 --- Plasticwares and glasswares --- p.30 / Chapter 2.1.7 --- "Chemicals, reagents and commercial kits" --- p.31 / Chapter 2.1.7.1 --- Samples processing --- p.31 / Chapter 2.1.7.2 --- Reagents for RNA extractions --- p.31 / Chapter 2.1.7.3 --- Oligonucleotide primers synthesis --- p.33 / Chapter 2.1.7.4 --- Primers purification after synthesis --- p.34 / Chapter 2.1.7.5 --- Gel electrophoresis --- p.34 / Chapter 2.1.7.6 --- Reagents for hybridization --- p.36 / Chapter 2.2 --- Methods --- p.37 / Chapter 2.2.1 --- Samples processing --- p.37 / Chapter 2.2.2 --- Artificially seeded HAV in shellfish --- p.37 / Chapter 2.2.3 --- RNA extraction methods --- p.37 / Chapter 2.2.3.1 --- Acid phenol method --- p.37 / Chapter 2.2.3.2 --- Spin cartridge method --- p.38 / Chapter 2.2.3.3 --- Virion capture method --- p.39 / Chapter 2.2.4 --- "Oligonucleotides used for RT, PCR and hybridization" --- p.40 / Chapter 2.2.4.1 --- Oligonucleotides used in HAV RT-PCR --- p.40 / Chapter 2.2.4.2 --- Primer set used for the evaluation of inhibitors of PCR in shellfish homogenates --- p.41 / Chapter 2.2.4.3 --- Preparation of oligonucleotide primers --- p.41 / Chapter 2.2.4.4 --- Detachment of the primer from the column --- p.42 / Chapter 2.2.4.5 --- Purification of the oligonucleotides --- p.42 / Chapter 2.2.4.6 --- Confirmation of synthesed oligonucleotide --- p.43 / Chapter 2.2.5 --- Reverse transcription of HAV genomic RNA template and PCR --- p.43 / Chapter 2.2.6 --- Human β-actin gene PCR for the evaluation of shellfish homogenates --- p.45 / Chapter 2.2.7 --- Analysis of PCR products --- p.46 / Chapter 2.2.7.1 --- Agarose gel electrophoresis for the analysis of PCR products --- p.46 / Chapter 2.2.7.2 --- Dot blot hybridization for the confirmation of PCR products --- p.46 / Chapter 2.2.7.3 --- Southern blot hybridization for the confirmation of PCR products --- p.47 / Chapter 2.2.7.4 --- 5'-end DNA labelling of oligonucleotide probe --- p.47 / Chapter 2.2.7.5 --- Hybridization in sodium chloride / sodium citrate --- p.48 / Chapter Chapter 3- --- Results / Chapter 3.1 --- Epidemiology of acute HAV infection in Hong Kong --- p.52 / Chapter 3.2 --- Synthesis and yields of oligonucleotide primers --- p.54 / Chapter 3.3 --- Development of reverse-transcription polymerase chain reaction (RT-PCR) for HAV --- p.55 / Chapter 3.4 --- Sampling of shellfish from different markets in Hong Kong --- p.59 / Chapter 3.5 --- Quantitation of HAV RNA in stock virus preparations --- p.62 / Chapter 3.6 --- Comparison of RNA extraction methods and the detection limit of the established RT-PCR method for HAV --- p.62 / Chapter 3.7 --- Specificity of the RT-PCR in combination with virion capture method for the detection of HAV --- p.65 / Chapter 3.8 --- Detection of HAV RNA by RT-PCR in shellfish in Hong Kong / Chapter Chapter 4- --- Discussion / Chapter 4.1 --- Epidemiology of acute HAV infection in Hong Kong --- p.76 / Chapter 4.2 --- Development of RT-PCR method for the detection of HAV --- p.78 / Chapter 4.3 --- Evaluation of RNA extraction methods for the detection of HAV in shellfish sample by RT-PCR --- p.78 / Chapter 4.4 --- Application of the established RT-PCR method for the detection of HAV contamination in locally available shellfish --- p.79 / Chapter Chapter 5- --- References --- p.90 / Appendix I Figures of agarose gel electrophoresis of RT-PCR products for all samples (I1 -I23 ) --- p.101 / Appendix II Figures of dot-blot hybridization assay (II2 -II4 ) --- p.114

O presente estudo avaliou materiais de 50 pacientes com cirrose hepática pelo vírus da hepatite C (VHC) que foram submetidos ao transplante hepático no Hospital das Clínicas da Faculdade de Medicina de São Paulo no período de 1993 a 2004, sendo divididos em dois grupos: Grupo 1 (33 pacientes com cirrose pelo VHC) e Grupo 2 (17 pacientes com cirrose pelo VHC com carcinoma hepatocelular). Nosso objetivo foi estudar a ocorrência da infecção oculta pelo VHB em pacientes com cirrose pelo VHC com ou sem CHC através do estudo molecular do genoma viral (DNA do VHB) no soro, tecido hepático tumoral e não tumoral pela utilização da técnica de Reação em Cadeia da Polimerase (PCR), pelos métodos in house e em tempo real. Todos os pacientes eram HBsAg negativos, possuíam soroteca e bloco de explante hepático em parafina, não apresentando concomitância com doenças hepáticas colestáticas, metabólicas e autoimunes. Foram avaliados os prontuários por um único pesquisador no sentido de coletar informações tais como: sexo, idade, dados de exames laboratoriais bioquímicos, sorológicos, ?fetoproteína e coagulação; além de dados clínicos tais como ascite e encefalopatia hepática para cálculos de índices prognósticos da cirrose (Child e MELD). Todo o material de explante hepático teve o Escore de Ishak e a Classificação das Sociedades Brasileiras de Patologia e Hepatologia para hepatites crônicas avaliados, assim como a Classificação de Edmondson e Steiner para os que apresentassem CHC. A técnica de PCR in house para detecção do DNA do VHB no soro e em tecido hepático tumoral e não tumoral apresentou negatividade em todas as amostras. Na técnica de PCR em tempo real apenas um caso do grupo 2 foi positivo no soro (sexo masculino, 66 anos, Anti-HBC total isolado e CHC); no tecido hepático tumoral no grupo 2 tivemos dois casos com resultados indeterminados e no tecido hepático não tumoral também do grupo 2, tivemos dois casos indeterminados. O grupo 1 não apresentou positividade para nenhuma das técnicas utilizadas. Concluímos que entre nossos pacientes com ou sem carcinoma hepatocelular associados à cirrose hepática pelo VHC, a infecção oculta pelo VHB foi muito baixa, provavelmente devido à baixa prevalência da infecção pelo VHB na nossa população / This study evaluated serum and liver tissue samples from 50 patients with liver cirrhosis due to hepatitis C virus (HVC) that underwent liver transplant at the Hospital das Clínicas - University of São Paulo School of Medicine during the period of 1993 to 2004, divided into two groups: Group 1 (33 cirrhotic patients due to HCV) and Group 2 (17 cirrhotic patients due to HCV with hepatocellular carcinoma - HCC). Our aim was to study the occurrence of occult HBV0 infection in cirrhotic patients due to HCV with or without HCC through the molecular study of HBV DNA in the serum, tumoral liver tissue and non tumoral liver tissue by the polymerase chain reaction (PCR) techniques using in house and real time PCR. All the patients were HBsAg negative, having previous serum samples frozen at -20ºC and liver tissue explanted in paraffin, without presenting concomitant cholestatic, metabolic and autoimmune liver diseases. The following variables were collected: gender, age, biochemical and coagulation laboratory tests and HBV serology (HBsAg, anti-HBc total, anti-HBs). Among the clinical data, ascites and encephalopathy were collected for the Child and MELD prognostic indexes. In the explanted liver tissue the Ishak\'s Score, The Brazilian Society of Pathology and Hepatology Classification for chronic hepatitis, and Edmondson and Steiner Classification for HCC were applied in the liver tissue. All samples with or without tumoral liver tissue and serum were negative for HBV DNA using in house PCR technique. By the real time PCR technique only one case from Group 2 was HBV DNA positive in serum (male, 66, isolated anti-HBc total positive and HCC). In the tumoral and non-tumoral liver tissues there were two indeterminated HBV DNA cases among Group 2 patients. All samples for Group 1 patients were negative for HBV DNA using both techniques. In conclusion, our study has shown the extremely low occult hepatitis B virus infection among the HCV cirrhotic patients with or without HCC, maybe due to the low HBV past infection among the Southeastern Brazilian population

Title from PDF of title page. / Document formatted into pages; contains 57 pages. / Thesis (M.S.P.H.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: The Centers for Disease Control and Prevention estimated 23,000,000 cases of viral gastroenteritis caused by Norovirus in 2000, 40% of which were transmitted by food including: a variety of fresh produce, cake, deli meats, fruit salad, cheeses and ice. (CDC, 2003). An estimated 83,391 cases of Hepatitis A virus was reported in 2000, of which 5% was attributed to foodborne transmission (CDC, 2003). These figures underscore an urgent need for a method that can isolate virus from a variety of food matrices. The aim of this study was to develop an overall assessment of the inhibitory effects of a variety of food matrices on Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). / ABSTRACT: Additionally, to compare a sequence specific hybridization probe amplification format to a non sequence specific SYBR Green format using the Roche LightCycler. The secondary aim was to evaluate the effectiveness of a food virus concentration and isolation protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa. Three food specimens consisting of prepackaged smoked ham, fresh cilantro, and Thompson's green grapes were seeded with three dilutions of poliovirus 3 (Sabin strain). A viral concentration procedure under development at the Florida Department of Health Bureau of Laboratories, Tampa was used to isolate the virus. Real Time RT-PCR was carried out on the Roche LightCycler in SYBR Green and Hybridization probe formats. Spiking the virus-negative samples of each matrix with a dilution series of poliovirus 3 created post flocculation spikes. / ABSTRACT: This post-flocculation dilution series amplification allowed a standard curve to be created unique to each food matrix. The flocculation and concentrations specimens were then amplified and the standard curves from the post-flocculation seed were used to calculate the loss associated with the concentration procedure. This study reports significant differences (p[0.05) in recovery detected between the various matrices, and Real Time RT-PCR formats. The concentration protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa, demonstrates a 12-78% recovery of seeded virus in a simulated "real world" virus contamination event among the various matrices. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

O presente estudo avaliou materiais de 50 pacientes com cirrose hepática pelo vírus da hepatite C (VHC) que foram submetidos ao transplante hepático no Hospital das Clínicas da Faculdade de Medicina de São Paulo no período de 1993 a 2004, sendo divididos em dois grupos: Grupo 1 (33 pacientes com cirrose pelo VHC) e Grupo 2 (17 pacientes com cirrose pelo VHC com carcinoma hepatocelular). Nosso objetivo foi estudar a ocorrência da infecção oculta pelo VHB em pacientes com cirrose pelo VHC com ou sem CHC através do estudo molecular do genoma viral (DNA do VHB) no soro, tecido hepático tumoral e não tumoral pela utilização da técnica de Reação em Cadeia da Polimerase (PCR), pelos métodos in house e em tempo real. Todos os pacientes eram HBsAg negativos, possuíam soroteca e bloco de explante hepático em parafina, não apresentando concomitância com doenças hepáticas colestáticas, metabólicas e autoimunes. Foram avaliados os prontuários por um único pesquisador no sentido de coletar informações tais como: sexo, idade, dados de exames laboratoriais bioquímicos, sorológicos, ?fetoproteína e coagulação; além de dados clínicos tais como ascite e encefalopatia hepática para cálculos de índices prognósticos da cirrose (Child e MELD). Todo o material de explante hepático teve o Escore de Ishak e a Classificação das Sociedades Brasileiras de Patologia e Hepatologia para hepatites crônicas avaliados, assim como a Classificação de Edmondson e Steiner para os que apresentassem CHC. A técnica de PCR in house para detecção do DNA do VHB no soro e em tecido hepático tumoral e não tumoral apresentou negatividade em todas as amostras. Na técnica de PCR em tempo real apenas um caso do grupo 2 foi positivo no soro (sexo masculino, 66 anos, Anti-HBC total isolado e CHC); no tecido hepático tumoral no grupo 2 tivemos dois casos com resultados indeterminados e no tecido hepático não tumoral também do grupo 2, tivemos dois casos indeterminados. O grupo 1 não apresentou positividade para nenhuma das técnicas utilizadas. Concluímos que entre nossos pacientes com ou sem carcinoma hepatocelular associados à cirrose hepática pelo VHC, a infecção oculta pelo VHB foi muito baixa, provavelmente devido à baixa prevalência da infecção pelo VHB na nossa população / This study evaluated serum and liver tissue samples from 50 patients with liver cirrhosis due to hepatitis C virus (HVC) that underwent liver transplant at the Hospital das Clínicas - University of São Paulo School of Medicine during the period of 1993 to 2004, divided into two groups: Group 1 (33 cirrhotic patients due to HCV) and Group 2 (17 cirrhotic patients due to HCV with hepatocellular carcinoma - HCC). Our aim was to study the occurrence of occult HBV0 infection in cirrhotic patients due to HCV with or without HCC through the molecular study of HBV DNA in the serum, tumoral liver tissue and non tumoral liver tissue by the polymerase chain reaction (PCR) techniques using in house and real time PCR. All the patients were HBsAg negative, having previous serum samples frozen at -20ºC and liver tissue explanted in paraffin, without presenting concomitant cholestatic, metabolic and autoimmune liver diseases. The following variables were collected: gender, age, biochemical and coagulation laboratory tests and HBV serology (HBsAg, anti-HBc total, anti-HBs). Among the clinical data, ascites and encephalopathy were collected for the Child and MELD prognostic indexes. In the explanted liver tissue the Ishak\'s Score, The Brazilian Society of Pathology and Hepatology Classification for chronic hepatitis, and Edmondson and Steiner Classification for HCC were applied in the liver tissue. All samples with or without tumoral liver tissue and serum were negative for HBV DNA using in house PCR technique. By the real time PCR technique only one case from Group 2 was HBV DNA positive in serum (male, 66, isolated anti-HBc total positive and HCC). In the tumoral and non-tumoral liver tissues there were two indeterminated HBV DNA cases among Group 2 patients. All samples for Group 1 patients were negative for HBV DNA using both techniques. In conclusion, our study has shown the extremely low occult hepatitis B virus infection among the HCV cirrhotic patients with or without HCC, maybe due to the low HBV past infection among the Southeastern Brazilian population