Submitted by ANA CLAUDIA SILVA BRAGA null (anabragga@gmail.com) on 2017-08-31T16:15:53Z No. of bitstreams: 1 Tese Doutorado Ana Claudia Silva Braga.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-09-01T14:16:37Z (GMT) No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) / Made available in DSpace on 2017-09-01T14:16:37Z (GMT). No. of bitstreams: 1 braga_acs_dr_sjrp.pdf: 4552779 bytes, checksum: 456de4a6fbfd60347292d8755d6c8c47 (MD5) Previous issue date: 2017-08-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O vírus da hepatite C (HCV) causa a doença da Hepatite C e estima-se que cerca de 3% da população mundial esteja infectada com o vírus. A infecção por HCV promove a alteração na expressão de várias proteínas celulares. Estudos têm demonstrado que muitas proteínas de choque térmico (HSPs) possuem um perfil de expressão alterado na presença do vírus e algumas HSPs interagem diretamente com proteínas do HCV. Assim, o presente estudo teve como objetivo avaliar in vitro os níveis de expressão de proteínas de choque térmico na presença e ausência de HCV. Com este propósito, células de hepatoma humano Huh7.5 e células Huh7.5 infectadas com o vírus (HCV JFH-1) foram submetidas à extração de RNA e síntese de cDNA. A expressão diferencial de 84 HSPs e chaperonas foi avaliada por qPCR Array. Os resultados demonstram que cinco genes apresentaram expressão aumentada (em Log2 2), enquanto outros cinco apresentaram expressão reduzida. Para validar estes resultados os 10 genes diferencialmente expressos foram testados por qPCR em três modelos celulares para o HCV: células contendo replicon subgenômico do HCV (SGR-JFH-1), células infectadas com JFH-1 (ambos do genótipo 2a) e células contendo o replicon subgenômico S52 (genótipo 3). O gene HSPB8 mostrou expressão aumentada nos três modelos testados, condizente com os resultados obtidos por qPCR Array. Em seguida, promovemos o silenciamento de HSPB8 e foi observado um aumento na replicação viral. Em contraste, quando aumentamos a expressão de HSPB8, o HCV teve uma diminuição na taxa de replicação. O mesmo procedimento foi adotado para o gene DNAJC5B, validado no modelo viral genótipo 3, e o HCV mostrou padrão de replicação semelhante ao observado para o gene anterior. Esses resultados sugerem que HSPB8 pode atuar como um fator intracelular contra a replicação do vírus da hepatite C e DNAJC5B apresenta a mesma função, mas específico para o genótipo 3. Também avaliamos interações diretas com proteínas do HCV e os resultados demonstraram uma interação física entre a proteína NS4B de HCV e HSPB8. Esses resultados podem contribuir para uma melhor compreensão dos mecanismos envolvidos na replicação do HCV. / Hepatitis C virus (HCV) causes Hepatitis C disease and it is estimated that about 3% of world population are infected with the virus. HCV infection promotes alteration in the expression of several cellular proteins. Studies have shown that many heat shock proteins (HSPs) have an altered expression profile in the presence of the virus and some HSPs interact directly with HCV proteins. Thus, the present study aimed to evaluate in vitro the expression levels of heat shock proteins in the presence and absence of HCV. With this purpose, human hepatoma Huh7.5 cells and Huh7.5 cells infected with the virus (HCV JFH-1) were subjected to RNA extraction and cDNA synthesis. The differential expression of 84 HSPs and chaperones was assessed by qPCR Array. The results demonstrate that five genes showed increased expression (over Log2 2), while five other presented reduced expression. To validate these results, the 10 differentially expressed genes were tested by real-time PCR in three different HCV cell culture models: subgenomic HCV replicon cells (SGR-JFH-1), JFH-1 infected cells (both genotype 2a) and subgenomic S52 cells (genotype 3). The HSPB8 gene showed increased expression in all of three tested models, consistent with qPCR Array results. Then we promoted the silencing of HSPB8 and observed an increase in viral replication. In contrast, when we increased an expression of HSPB8, HCV had a decrease in replication rate. The same procedure was adopted for the DNAJC5B, validated in the viral model genotype 3, and HCV showed replication pattern similar to that observed for the previous gene. These results suggest that HSPB8 may act as an intracellular factor against hepatitis C virus replication and DNAJC5B have the same function, but genotype 3 specific. We also evaluated direct interactions with HCV proteins and the results demonstrated a physical interaction between the HCV NS4B protein with HSPB8. These results can contribute for a better understanding of the mechanisms involved in HCV replication. / FAPESP: 2013/17253-9

Project title: Optimalization of the regime of marker's examination of clinically important infections in blood donors Project author: Daniela Dušková, M.D. Project supervisor: prof. Vladimír Tesař, M.D., Dr.Sc., MBA, FASN The aim of this project is to contribute to the discussion about introducing the methods of molecular biology into the routine blood donor testing in the transfusions departments in the Czech Republic. The theoretical part includes a brief history and some turning points in transfusion medicine. The next part within the theoretical section is dedicated to the problems of infectious diseases concerning transfusion and the general examination processes used during the selection of blood donors. The end of the theoretical part concentrates on existing possibilities of markers' examination of clinically important infections in blood donors, including the list of processes performed in the Czech Republic, the European Union and other countries. The practical part describes this study, ie. the routine screening test of blood donors using the CMIA method (a routine method) and using RT-Real Time PCR method (a molecular biology method) for detecting infectious markers (HCV, HBV, HIV). Within this part, the principle of both methods and the process of actual examinations are described in...

Le travail de recherche présenté dans ce mémoire de thèse a été centré sur une nouvelle famille de flavones aux propriétés antivirales. Mon travail de thèse avait pour premier objectif d'améliorer la synthèse de la ladanéine (tête de série) et permettre l'accès à d’autres analogues. Un développement méthodologique a permis de mettre au point une synthèse compatible avec les procédés industriels qui permette d'améliorer les rendements et de raccourcir significativement les délais d'obtention. De plus, aucune purification par colonne de silice n’est nécessaire. Une étude physico-chimique détaillée a ensuite été menée. Les propriétés acido-basiques de la série de composés ont d'abord été évaluées avant l'étude des propriétés électrochimiques. Ces données sont déterminantes pour une meilleure compréhension du mécanisme d'action de ces flavones. La complexation au Fe(III) a été également démontrée comme essentielle pour l’activité antivirale de ces composés. Les propriétés de complexation de ce cation ont donc été étudiées et ont apporté des informations importantes. Finalement, dans le but d’améliorer les propriétés pharmacocinétiques de ces agents virucides, des formulations originales avec le Mg(II), cation biocompatible, ont été élaborées et étudiées. / The research work presented in this manuscript was centered on a novel flavone series displaying potent antiviral activities toward enveloped viruses such as HCV. The first goal of my research work was to improve the synthesis of ladanein (the lead antiviral compound) and to allow an easy access to a broad range of analogues. A methodological approach allowed setting up a synthetic route compatible with industrial processes with high yields and significantly shortened preparation time. Furthermore, no silica gel column chromatography was needed throughout the synthetic route. A thorough physico-chemical study was then undertaken. The acido-basic properties of this homogenous series of compounds were first evaluated prior to the investigation of their electrochemical parameters. These data are essential for a deeper understanding of the mechanism of action of these polyphenolic compounds. Fe(III) was shown to be essential for the antiviral activity of these compounds and, hence, the Fe(III) complexation properties of the flavones have been studied and provided important information. Last but not least, in order to improve the pharmacokinetic properties of the flavones, original formulation approaches using the biocompatible Mg(II) cation were undertaken and thoroughly investigated.

L'hépatite C (VHC) est l'une des causes principales de maladies du foie dans le monde, qui représentent un risque élevé d'évoluer vers la cirrhose et le carcinome hépatocellulaire. Actuellement, le traitement standard de l’infection par le VHC est l'interféron pégylé-(peg-IFN) et la ribavirine. Bien que le taux de la réponse virale soutenue (RVS) au traitement se soit améliorée au cours de ces années, cette thérapie n'est pas efficace chez tous les patients. En outre, plusieurs effets secondaires toxiques, de complications et le coût élevé limitent la compliance du patient et l'efficacité du traitement. Il n'existe pas de modèle simple d'infection par le VHC et il est nécessaire de développer des modèles in vitro et in vivo utiles pour étudier la physiopathologie de l'infection par le VHC, y compris les événements précoces de l'infection aiguë (l'entrée du virus, des mécanismes immunologiques et génétiques prédictifs) ainsi que l'évaluation de la puissance des médicaments antiviraux contre le VHC. Nous rapportons ici, nos efforts visant à développer des modèles appropriés de l'infection par le VHC. Dans un premier temps, nous avons établi un modèle de petit animal pour étudier l'infection par le VHC. Tupaia est un petit animal, apparenté aux primates et peu couteux. Dans notre travail, nous avons étudié la susceptibilité du tupaia à l'infection par VHC. Douze tupaias adultes ont été inoculés avec le VHC provenant de sérum de patient et d'ARN du VHC (génotype 1a). Trois jeunes tupaias ont été artificiellement nourris pendant un mois et ensuite inoculés par le VHC provenant de sérum du patient. L'ARN du VHC, les anticorps anti-VHC et l’évolution des quasi-espèces du VHC ont été déterminées chez l'animal avant et après l'inoculation. L'infection transitoire et intermittente s'est produite chez deux des 3 jeunes tupaias et l’infection chronique par le VHC s’est produite chez quatre tupaias sur 12 tupaias adultes. Le tupaia devrait représenter un modèle utile pour l'étude de l’infection chronique par le VHC. Dans une deuxième étape, un système de culture in vitro d'hépatocytes primaires de Tupaia a été établi, dans lequel l'infection par le VHC ne pouvait être bloquée ni par le CD81 soluble ni par des anticorps dirigés contre le CD81. Pour comprendre ces résultats, nous avons cloné, séquencé la grande boucle extracellulaire (LEL) du CD81 chez le Tupaia et analysé l'interaction de la protéine d’enveloppe E2 du VHC avec la LEL du CD81 chez le Tupaia par un test « enzyme-linked immunosorbent assay » (EIA). Nous avons constaté que chez le Tupaia, la séquence d'acides aminés du LEL de CD81 qui se lie au VHC présentait en 6 résidus d'acides aminés différents par rapport à la séquence humaine et la capacité de LEL de CD81 à se lier à la proteine d’enveloppe E2 du VHC a également diminuée. La structure différente de CD81 chez l’homme et chez le tupaia pourrait expliquer l'altération de l'interaction entre CD81 et la proteine E2 du VHC. Ce résultat démontre un rôle important de LEL du CD81 pour l'entrée du VHC. Dans une troisième étape, nous avons développé un modèle ex vivo de culture de tranches de foie humain et leur infection par le VHC. Le développement de lignées cellulaires provenant d’hepatocarcinome, permissives à la réplication du VHC, a fourni d'importants nouveaux outils virologiques pour étudier les mécanismes de l'infection par le VHC, mais ce modèle expérimental reste relativement éloigné des conditions physiologiques et pathologiques. Nous rapportons ici le développement d'un nouveau modèle ex vivo utilisant la culture de tranches de foie humain adulte, démontrant, pour la première fois, la capacité d’isolats primaires ainsi que JFH -1, H77/C3, Con1/C3 (HCVcc), de répliquer et de produire de novo des particules virales infectieuses ayant un titre viral élevé… / Hepatitis C virus (HCV) is one of the major causes of liver disease all over the world which has a high risk to progress to cirrhosis and hepatocellular carcinoma. Currently, the licensed standard treatment of HCV infection is Pegylated-interferon (peg-IFN) and ribavirin. Although the sustained viral response (SVR) rate of treatment has improved during these years, this therapy is not effective in all patients. In addition, several toxic side effects, complication and high cost limit the patient compliance and the efficacy of the treatment. There is no easy model of HCV infection and it is necessary to develop useful in vitro and in vivo models to study the pathobiology of HCV infection, including early events of acute infection (viral entry, immunological mechanisms, and genetic predictors) as well as the evaluation of the potency of the HCV antiviral drugs. We report here in our efforts in developing suitable models of HCV infection. In a first step, we preliminary established a small animal model to study HCV infection. Tupaia is a small, closed related to primate and cost-effective animal. In our work, we investigated the susceptibly of tupaia to HCV infection. Twelve adult tupaias were inoculated with native HCV from patient serum and full-length HCV RNA (Genotype 1a). Three young tupaias were artificially breeded for a month and then inoculated by native HCV from patient serum. HCV RNA, anti-HCV and HCV quasi species evolution were determined in the animal before and after inoculation. Transient and intermittent infection occurred in two among 3 young tupaias and HCV chronic infection occurred in four among 12 adult tupaias. Tupaia should represent a useful model for study HCV chronic infection. In a second step, an in vitro culture system of primary tupaia hepatocytes has been established in which HCV infection could be blocked neither by the soluble CD81 nor by antibodies against CD81. To understand these results, we cloned, sequenced the large extracellular loop (LEL) of tupaia CD81 and analyzed the interaction of HCV E2 with the tupaia CD81 LEL by enzyme-linked immunosorbent assay (EIA). We found that in the tupaia the amino acids sequence of HCV CD81 LEL presented in 6 different amino acid residues compared with human CD81 LEL sequence and the CD81 LEL ability to bind to HCV E2 was also decreased. The different structure of CD81 between human and tupaia could explain the alteration of the interaction between HCV E2 and CD81. This result demonstrated an important role of CD81 LEL for HCV entry. In a third step, we developed an ex vivo model of human liver slices culture and their infection with HCV. The development of human cultured HCV-replication-permissive hepatocarcinoma cell lines has provided important new virological tools to study the mechanisms of HCV infection; however this experimental model remains distantly related to physiological and pathological conditions. Here, we report the development of a new ex vivo model using human adult liver slices culture, demonstrating, for the first time, the ability of primary isolates to undergo de novo viral replication with the production of high titer infectious virus, as well as JFH-1, H77/C3, Con1/C3 (HCVcc). This experimental model was validated by demonstrating the HCV neutralization or HCV inhibition, in a dose-dependent manner, either by CD81 or E2 specific antibodies or convalescent serum from a recovered HCV patient, or by anti-viral drugs. This new ex vivo model represents a powerful tool for studying the viral life cycle, dynamics of virus spread in the liver and also for evaluating the efficacy of the new antiviral drugs. In the last step, we evaluated the efficacy of the new antiviral drugs with our ex vivo model of human adult liver slices. HCV NS3/4A protease is essential for viral replication and has been one of the most important target for developing specific antiviral drug

Introduction : Le virus de l’hépatite C (VHC), est l’une des premières causes de pathologie hépatique dans le monde. Ce virus à ARN est responsable de l’hépatite C qui aboutit au développement de la cirrhose et du cancer du foie. Selon l’Organisation Mondiale de la Santé, le VHC infecte actuellement plus de 170 millions de personnes dans le monde, soit 3% de la population. L’hépatite C chronique connait toujours en Tunisie un taux de guérison faible pour le génotype 1 car le traitement standard actuellement disponible est la bithérapie interféron pégylé associé à la ribavirine. A l’heure actuelle, le développement de différentes molécules ciblant spécifiquement le VHC, appelées les antiviraux à action directe (AAD), apparait comme une potentielle révolution dans le traitement de l’infection par le VHC.Ces AAD comprennent les inhibiteurs de protéase (IP), les inhibiteurs nucléos(t)idiques (IN) et les inhibiteurs non-nucléosidiques (INN) de la polymérase NS5B ainsi que les inhibiteurs de la protéine NS5A. La quasi-espèce virale est formée d’un mélange complexe de variants viraux parmi lesquels se trouvent des variants associés à des degrés variables à la résistance aux AAD. Ces variants peuvent donc exister naturellement en absence de toute pression médicamenteuse et sont susceptibles d’avoir un impact sur la réponse aux différents traitements par AAD. Notre objectif était de déterminer la prévalence des variants associés à la résistance dans les souches tunisiennes circulantes en préambule à l’introduction deces molécules en Tunisie. Méthodes : L’amplification et le séquençage direct de la protéase NS3, de la polymérase NS5B ainsi que la région NS5A ont été effectuées chez 149 patients tunisiens naïfs de traitement et infectés par le VHC de génotype 1 (génotype 1b = 142 ; génotype 1a = 7). Résultats : Douze séquences NS3 (12/131 ; 9,2%) ont montré des mutations connes pour conférer une résistance aux IP. Une seule séquence (1/95 ; 1,1%) a montré la mutation V321I connue pour conférer une résistance aux IN-NS5B. Trente quatre séquences (34/95 ; 35,8%) ont montré des mutations connues pour diminuer la sensibilité des INN-NS5B. Une seule séquence de génotype 1a (1/7 ; 14,3%) et 17 séquences de génotype 1b (17/112 ; 16,2%) ont montré des mutations connues pour conférer une résistance au inhibiteurs de la protéine NS5A. Conclusions : Notre étude a permis de mettre en évidence la présence de substitutions conférant une diminution de la sensibilité aux AAD chez des patients tunisiens naïfs de tout traitement anti-VHC. Des études in situ seront nécessaires pour évaluer l’impact de ces mutations sur la réponse au traitement. / Introduction: Hepatitis C virus (HCV) is a major cause of liver disease worldwide. This RNA virus is responsible for hepatitis C, which leads to the development of cirrhosis and liver cancer. According to the World Health Organization, HCV infects more than 170 million people worldwide, about 3% of the population. Chronic hepatitis C still know in Tunisia low cure rates for genotype 1, because the currently standard treatment available is combination therapy of pegylated interferon plus ribavirin. At present, the development of different molecules that specifically target HCV, called direct-acting antivirals (DAA) appears as a potential revolution in the treatment of HCV infection. These DAA include protease inhibitors (PI), nucleos(t)ide (NI) and non-nucleoside inhibitors (NNI) for NS5B polymerase and NS5A inhibitors. The viral quasispecies is formed by a complex mixture of viral variants including variants associated with variable degrees of resistance to DAA. These variants may therefore exist naturally in absence of drug pressure and may affect response to different treatments by DAA. Our objective was to determine the prevalence of variants associated with resistance in circulating Tunisian strains preamble to the introduction of these molecules in Tunisia. Methods: Amplification and direct sequencing of NS3 protease, NS5B polymerase and NS5A region were performed in 149 Tunisian naïve patients infected with HCV genotype 1 (genotype 1b = 142; genotype 1a = 7) . Results: Twelve sequences NS3 (12/131; 9.2%) showed mutations known to confer resistance to PI. One sequence (1/95; 1.1%) showed the V321I mutation known to confer resistance to NS5B-IN. Thirty four sequences (34/95; 35.8%) showed mutations known to reduce the sensitivity of NS5B-INN. One genotype 1a sequence (1/7; 14.3%) and 17 genotype 1b sequences (17/112; 16.2%) showed mutations known to confer resistance to NS5A inhibitors.Conclusions: Our study highlighted the presence of substitutions conferring decreased susceptibility to DAA in naïve patients infected with HCV genotype 1. Field studies will be needed to evaluate the impact of these mutations on the treatment response.

The current treatment for hepatitis C virus (HCV) infection – combination therapy with pegylated interferon and ribavirin – elicits sustained responses in only ~50% of the patients treated. Greater cumulative exposure to ribavirin increases response to interferon-ribavirin combination therapy. A key limitation, however, is the toxic sideeffect of ribavirin, hemolytic anemia, which often necessitates a reduction of ribavirin dosage and compromises treatment response. Maximizing treatment response thus requires striking a balance between the antiviral and hemolytic activities of ribavirin. Current models of viral kinetics describe the enhancement of treatment response due to ribavirin. Ribavirin-induced anemia, however, remains poorly understood and precludes rational optimization of combination therapy. Here, we develop a new mathematical model of the population dynamics of erythrocytes that quantitatively describes ribavirin-induced anemia in HCV patients. Based on the assumption that ribavirin accumulation decreases erythrocyte lifespan in a dose-dependent manner, model predictions capture several independent experimental observations of the accumulation of ribavirin in erythrocytes and the resulting decline of hemoglobin in HCV patients undergoing combination therapy, estimate the reduced erythrocyte lifespan in patients and describe inter-patient variations in the severity of ribavirin-induced anemia. Further, model predictions estimate the threshold ribavirin exposure beyond which anemia becomes intolerable and suggest guidelines for the usage of growth hormones. A small fraction of the population (~30%) with polymorphisms in the ITPA gene shows protection from ribavirin-induced anemia. The optimum dosage of ribavirin that can be tolerated is then dependent on the ITPA polymorphisms. Coupled with a previous population pharmacokinetic study, our model yields a facile formula for estimating the optimum dosage given a patient’s weight, creatinine clearance, pretreatment hemoglobin levels, and ITPA polymorphism. The reduced lifespan we predict is in agreement with independent measurements from breath tests as well as estimates derived from in vitro studies of ATP depletion. The latter estimates also agree with the extent of ATP depletion due to ribavirin that we predict from a detailed analysis of the nucleoside metabolism in erythrocytes. Our model thus facilitates in conjunction with models of viral kinetics the rational identification of treatment protocols. Our formula for optimum dose presents an avenue for personalizing ribavirin dosage. By keeping anemia tolerable, the predicted optimal dosage may improve adherence, reduce the need for drug monitoring, and increase response rates.

Introduction International guidelines endorse the screening of patients for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Chlamydia trachomatis before assisted reproductive techniques (ART). At present no such guidelines exists in South Africa. At the Reproductive and Endocrine Unit (referred to as “the Unit”) of Steve Biko Academic Hospital, all patients with unknown HIV status are counselled and a blood sample is collected during the initial visit for automated laboratory based HIV screening. These HIV results are not available before semen samples are processed. Furthermore, patients are not screened for HBV, HCV and Chlamydia trachomatis. Couples attending the Unit are of a low to middle socio-economic status and experience financial constraints. Moreover, automated laboratory based assays are expensive to perform. Rapid testing is a cost effective and practical method from screening patients, with a 20–30 minute result turnover time. Until screening at the Unit is improved, the possible identification of semen characteristics that could indicate HIV infection would be a useful tool. Materials and Methods The following rapid point-of-care assays were evaluated: Determine® HIV-1/2 combo test (n=100), Determine® HBsAg test (n=100), DIAQUICK HCV kit (n=74), and the DIAQUICK Chlamydia trachomatis kit (n=30). For profiling, parameters from a basic semen analysis of HIV-positive males (n=60) were compared with HIV-negative males (n=60). Information pertaining to CD4 count, antiretroviral treatment and plasma viral load of HIV-positive males were analysed. Results From all patients included in the study, 8% tested positive for HIV. The risk of a female being HIV-positive was 3.73 times higher than for males. In the pilot study to explore rapid testing for HBV and HCV, 1% and 1.4% of patients tested positive respectively. When testing for Chlamydia trachomatis 31.3% of females, but no males tested positive. Comparing semen profiles, no significant differences were found between samples from HIV positive and negative males or between HIV positive males categorised by CD4 cell count (p>0.05). For the HIV-positive group with a detectable plasma HIV viral load (>40 copies/ml), a significant difference was observed in the semen viscosity (p=0.0460). Significant differences were noted in the sperm motility (immotile sperm p=0.0456, progressive sperm p=0.0192) of patients receiving antiretroviral (ARV) therapy. Discussion and Conclusion The use of rapid testing is an acceptable and feasible option for improving current screening protocols at the Unit. The absence of definite alterations in the semen characteristics of HIV-positive men further motivates the need for a simpler, point-of-care screening protocol. The prevalence of HBV was lower than that reported in the general population of South Africa and further investigation is needed. Although the sample size was small, HCV prevalence was similar to that of the general population. One third of females tested positive for Chlamydia trachomatis. The methodology used was possibly not appropriate for males. This study highlighted the need for guidelines that address the specialised needs of ART clinics in resource-limited and developing countries with a high HIV prevalence. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Obstetrics and Gynaecology / unrestricted

Project title: Optimalization of the regime of marker's examination of clinically important infections in blood donors Project author: Daniela Dušková, M.D. Project supervisor: prof. Vladimír Tesař, M.D., Dr.Sc., MBA, FASN The aim of this project is to contribute to the discussion about introducing the methods of molecular biology into the routine blood donor testing in the transfusions departments in the Czech Republic. The theoretical part includes a brief history and some turning points in transfusion medicine. The next part within the theoretical section is dedicated to the problems of infectious diseases concerning transfusion and the general examination processes used during the selection of blood donors. The end of the theoretical part concentrates on existing possibilities of markers' examination of clinically important infections in blood donors, including the list of processes performed in the Czech Republic, the European Union and other countries. The practical part describes this study, ie. the routine screening test of blood donors using the CMIA method (a routine method) and using RT-Real Time PCR method (a molecular biology method) for detecting infectious markers (HCV, HBV, HIV). Within this part, the principle of both methods and the process of actual examinations are described in...

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L’Organització Mundial de la Salut (OMS) estima que fins a un 3% de la població mundial ha estat infectada pel virus de l’hepatitis C i és la causa més important d’hepatitis crònica, cirrosi i de malaltia hepàtica terminal, que finalment acaba conduint a un transplantament de fetge. La relació entre la variabilitat en la seqüència del virus de l’hepatitis C i el desenvolupament de la malaltia hepàtica és de tipus multifactorial. La infecció crònica causa fibrosi hepàtica, fet que es veu accelerat per mecanismes desconeguts en el cas de pacients coinfectats amb VIH. La progressió de la malaltia produïda pel VHC en pacients coinfectats, està influenciada no només per factors demogràfics, epidemiològics o pels antecedents clínics dels pacients, si no també per diferències genètiques entre els diferents virus i els hostes.