Introdução: A infecção pelo VHC é uma epidemia de proporções globais, que se torna crônica em cerca de 85% dos indivíduos. Sobrecarga de ferro secundária a mutações HFE vem sendo proposta como fator agravante na evolução da hepatite crônica C. Objetivos: Avaliar se sobrecarga de ferro, mutações no gene HFE estão associadas a progressão da fibrose hepática e a carcinoma hepatocelular (CHC) em portadores crônicos VHC. Casuística e Métodos: De um banco de 2300 pacientes matriculados nos Ambulatórios de Hepatologia e de Transplante Hepático do HCFMUSP, selecionaram-se 320 portadores de hepatite crônica C. Os homens (55,6%) apresentaram 50,8 anos de idade em média e as mulheres 54,3. Obtiveram-se dados clínicos e laboratoriais da época da biópsia hepática, admissionais ou com pelo menos um ano de wash-out do tratamento antiviral. Considerou-se sobrecarga bioquímica níveis de ferro, saturação da transferrina e ferritina acima dos valores de referência. Sobrecarga de ferro tecidual foi identificada pela coloração de Perls graus 3-4. As mutações HFE C282Y e H63D foram pesquisadas pela técnica de PCR em tempo real, em DNA extraído de sangue total, após assinatura de TCLE aprovado pelo comitê de ética local. A fibrose hepática foi considerada avançada para graus 3-4 da classificação SBH/SBP e Metavir (n=167, 52,2%). CHC foi definido por biópsia ou por imagem típica por 2 métodos e AFP 400 mg/dL (n=45, 14,1%). Investigaram-se antecedentes de etilismo, diabetes mellitus, IMC e esteatose em biópsia hepática. A partir dos resultados de genotipagem HFE, constituiu-se um subgrupo caso-controle (n=182) com os portadores de mutações e pares de idade, sexo, etnia e IMC similares. Procederam-se análises de correlação bivariada e multivariada (IC=95%) nos grupos geral e caso-controle. Resultados: Quando se compararam casos com fibrose leve vs avançada, observaram-se níveis elevados de ferro em 17,7% vs 25,2% (p=0,019); saturação da transferrina em 24,3% vs 36,7% (p=0,001); ferritina em 25,8% vs 32,4% (p=0,040) e sobrecarga de ferro tecidual em 3,6% vs 1,4% (p=0,126). Quanto à mutação C282Y, observaram-se frequências alélicas de 0,9% vs 2,0% (p=0,110) e à H63D 5,0% vs 8,0% (p=0,033). No subgrupo casocontrole, as associações de C282Y e H63D com fibrose avançada ocorreram com significâncias de p=0,072 e 0,008, respectivamente. A análise multivariada, tendo fibrose como variável dependente nesse mesmo subgrupo, confirmou as associações com C282Y (OR=31,45; p=0,006) e H63D (OR=33,70; p=0,001). Neste mesmo subgrupo, a presença de pelo menos um alelo mutante esteve associada à ocorrência de CHC (p=0,015). Não se identificaram outros parâmetros associados a transformação carcinomatosa. Na análise multivariada, foram variáveis associadas a evolução da fibrose idade avançada (OR=2,36; p=0,000), etilismo (OR=2,18; p=0,007), saturação da transferrina (OR=2,11; p=0,010) e mutação H63D (OR=1,97; p=0,03). Discussão e Conclusões: Houve correlação de graus mais avançados de fibrose com níveis elevados de ferro, saturação da transferrina e ferritina; contudo, esses marcadores também podem estar igualmente elevados nos indivíduos com pouca fibrose. Ao contrário, em muitos casos, a sobrecarga bioquímica de ferro pode ser conseqüência da progressão da doença hepática, e não sua causa. As mutações H63D e C282Y ocorreram em associação com maiores graus de alteração arquitetural; mas, como não houve associação entre siderose tecidual e fibrose, é possível que seu papel na agressão hepática não ocorra diretamente por meio da sobrecarga de ferro. Os portadores de mutações HFE apresentam CHC com maior frequência que seus casos-controle. / Introduction: HCV infection is an epidemic of global proportions, which becomes chronic in about 85% of individuals. Iron overload due to HFE mutations has been proposed as aggravating factor in the evolution of chronic hepatitis C. Objectives: To assess whether iron overload, mutations in the HFE gene are associated with progression of liver fibrosis and hepatocellular carcinoma (HCC) in chronic HCV. Patients and Methods: From a database of 2300 patients enrolled in the outpatient clinics of Hepatology and Liver Transplantation, HCFMUSP, 320 patients with chronic hepatitis C were selected. Men (55.6%) were 50.8 years old on average and women 54.3. Admissional clinical and laboratory data at the time of liver biopsy were obtained; when patient had been previously treated with antiviral drugs, the wash-out period required was of at least one year. Biochemical iron overload was defined as iron, ferritin and transferrin saturation above the reference values. Tissue iron overload was identified by Perls staining of grades 3-4. The HFE C282Y and H63D mutations were searched by real-time PCR technique in DNA extracted from whole blood, after signing of FICT approved by the local ethics committee. The liver fibrosis was considered advanced for grades 3-4 in the classification SBH/SBP and METAVIR(n = 167, 52.2%). HCC was defined by biopsy or by typical image by 2 methods and AFP 400 mg / dL (n = 45, 14.1%). Personal history of alcoholism, diabetes mellitus, BMI and steatosis in liver biopsy were obtained. A casecontrol group was constituted based on the results of HFE genotyping (n = 182), subjects were paired by age, gender, ethnicity and BMI. Bivariate correlation and multivariate analysis (CI = 95%) groups in general and case-control were carried-out. Results: When comparing patients with mild vs advanced fibrosis, biochemical high levels of iron were detected in 17.7% vs 25.2% (p = 0.019), transferrin saturation in 24.3% vs 36.7% (p = 0.001), ferritin in 25.8% vs 32.4% (p = 0.040) and tissue iron overload in 3.6% vs 1.4% (p = 0.126). Regarding to HFE mutations, the allelic frequencies of C282Y were 0.9% vs 2.0% (p = 0.110); and of H63D, 5.0% vs 8.0% (p = 0.033). In casecontrol group, associations of C282Y and H63D with advanced fibrosis occurred with significance of p=0.072 and 0.008, respectively. Multivariate analysis with fibrosis as the dependent variable in that group, confirmed the associations with C282Y (OR = 31.45, p = 0.006) and H63D (OR = 33.70, p = 0.001). In this same subgroup, the presence of at least one mutant allele was associated with occurrence of HCC (p = 0.015). There were not any other parameters found in association with carcinomatous transformation. The multivariate analysis using fibrosis as dependent variable showed association with age (OR = 2.36, p = 0.000), alcoholism (OR = 2.18, p = 0.007), transferrin saturation (OR = 2.11, p = 0.010) and H63D mutation (OR = 1.97, p = 0.03). Discussion and Conclusions: Advanced degrees of fibrosis were correlated with high levels of iron, transferrin saturation and ferritin; however, these markers can also be elevated in individuals with slight fibrosis. In contrary, in many cases, the biochemistry of iron overload may be a consequence of progression of liver disease. C282Y and H63D mutations occurred in association with more advanced fibrosis. However, as there was not any correlation between tissue siderosis and fibrosis, it might be possible that liver injury occurs not by the iron overload pathway. Finally, carriers of HFE mutations were found with HCC more frequently than their casecontrol.

La polymérase NS5B du virus de l’hépatite C (VHC) porte une activité ARN polymérase ARN-dépendante essentielle pour la réplication de l'ARN génomique viral. Cette réplication implique la synthèse d'un intermédiaire de réplication de polarité négative. In vitro et probablement in vivo, la NS5B initie la synthèse d'ARN par un mécanisme de novo qui nécessite des interactions spécifiques entre la polymérase virale et des éléments des ARN viraux. Dans une première partie nous avons étudié le rôle du GTP et d’un domaine C-terminal nommé linker de la polymérase. Nos résultats démontrent que des concentrations élevées de GTP sont nécessaires pour la transition de l'initiation à l'élongation de la synthèse de l'ARN. Des mutations dans le linker à la position 556 ne modifient pas la concentration de GTP nécessaire pour la transition. Toutefois, l'initiation de la synthèse d'ARN est augmentée par la mutation S556K. Une analyse structurale menée en parallèle suggère une implication directe du linker dans l'initiation de novo de la synthèse de l'ARN. Dans les deuxièmes et troisièmes parties, nous avons étudié le rôle de motifs ARN dans la traduction et la synthèse de l’'ARN du VHC. Nous avons démontré que la tige boucle SL-E1 formée par la région entre les nt 177 et 222 de l'extrémité 3' de l’ARN (-) est importante pour la synthèse d'ARN in vitro par la NS5B recombinante et dans les cellules Huh7 exprimant le complexe de réplication (RC) du VHC. SL-E1 est impliquée dans l’initiation de la synthèse d’ARN, au moins in vitro. Nous avons également étudié le rôle des tiges boucles SLV et SLVI du gène core. Nos données n'ont pas montré de rôle évident de ces séquences ou de leur complément dans la synthèse de l'ARN in vitro par la NS5B recombinante et en culture cellulaire par le RC du VHC. Nous avons confirmé leur effet négatif sur la traduction IRES dépendante par interaction ARN-ARN longue distance entre SL-VI et le 5'UTR et démontré que le miR122 ne peut pas empêcher cet interaction. Par contre, la présence de SL-VI prévient l’inhibition de la traduction induite par l’interaction entre le domaine III de l’IRES et la tige boucle 5BSL3.2 en 3’ du génome. Ces résultats démontrent la complexité des interactions ARN/ARN et ARN/protéines dans la régulation de la réplication virale. / The hepatitis C virus (HCV) NS5B protein displays a RNA-dependent RNA polymerase activity essential for replication of the viral RNA genome. This replication involves the synthesis of a replication intermediate of negative polarity. In vitro and likely in vivo, the NS5B initiates RNA synthesis by a de novo mechanism which requires specific interactions between the polymerase and viral RNA elements. In the first part of results, we described a combined structural and functional analysis of HCV-NS5B to study the role of a C-terminal segment (termed linker) and of GTP in RNA synthesis. Our results demonstrated that high GTP concentrations are necessary for the transition from the initiation to the elongation of RNA synthesis, and that linker mutations at position S556 did not modify the GTP requirement of NS5B for this transition. However, the initiation of RNA synthesis was greatly enhanced by a S556K mutation. These results together with a structural analysis point to the direct involvement of the linker in the de novo initiation of RNA synthesis. In the second and third parts of results, we studied the role of RNA elements in RNA synthesis. We demonstrated that the SL-E1 stem–loop formed by nucleotides 177–222 from the 3’-end of the HCV (-) RNA is important for RNA synthesis both in vitro by the recombinant NS5B and in Huh7 cells by HCV replication complex (RC). We also showed that SL-E1 is involved in initiation of RNA synthesis, at least in vitro. Then we studied the role of other viral RNA elements in core coding sequences (SLV and SLVI stem loops) and the involvement of the microRNA miR122 in RNA translation and RNA synthesis. For SLV and SLVI, our data did not show any clear role of these core-coding sequences or of their complement in the (-) RNA in RNA synthesis both in vitro by the recombinant NS5B and in cell culture by HCV-RC. We confirmed their negative effect on HCV-IRES translation through long range RNA-RNA interaction between SL-VI sequences and the 5’UTR and demonstrated that miR122 cannot disrupted this interaction and switches the region to an open conformation. Conversely, our data indicated that the SL-VI domain can counteract the negative effect of the interaction between the domain III of IRES and the 5BSL3.2 stem loop localized at the 3’end of the genome. These results point to the complexity of RNA/RNA and RNA/proteins interactions in the HCV replication cycle.

Since the discovery in 1989 of hepatitis C virus (HCV) as the infectious agent responsible for the vast majority of post-transfusion non-A non-B hepatitis, blood transfusions are no longer a source for HCV transmission in Sweden. Anti-HCV testing was implemented for all blood donations in 1992. Since then intravenous drug use (IDU) has become the major route of transmission in the western world. Six genotypes and more than 80 subtypes of HCV have now been identified world-wide. These genotypes and subtypes are determined by genetic divergences between the HCV strains. Subtypes 1a, 1b, 2b, 2c, and 3a have global spread, while the other subtypes have a more limited geographical distribution. Little was known on the prevalence of HCV among blood donors and on which genotypes and subtypes of HCV were circulating in Sweden before the testing of all blood donations was implemented. The prevalence of anti-HCV was therefore investigated in sera sent to the Swedish Institute for Infectious Disease Control (SMI) from 412 patients; 241 were sampled between 1970 and 1991 before the universal screening in 1992, while 171 were sampled between 1992 and 2002. The samples derived from 193 (47%) blood donors, (104 sampled before, and 89 after 1992), and from seven other groups of patients. Two groups had suspected known routes of infection, intravenous drug use (IDU) 33 patients and hemodialysis, 16 patients, while it was unknown for the other patients. Anti-HCV was detected in 120 (29%) samples. The highest frequency was found among IDUs, (91%). Before general screening was implemented, 2.8% of the blood donors were positive for hepatitis C, whereas 28% of those sampled after 1992 were anti-HCV positive. Those latter samples were sent to SMI due to anti-HCV reactivity in a primary test at the blood centre. HCV RNA could be detected by PCR in 56 (47%) of the anti-HCV positive samples, the subtype could be determined by sequencing in 45 (80%) of those. The subtypes found were 1a in 31 %, 1b in 18%, 2b in 22%, and 3a in 27%. One sample was of subtype 2c. There was a tendency of increase of genotype 2 and a decrease in subtype 1a with time. 1a was found in 38% of the samples collected before 1992, while it was only found in 19% of the samples from 1992 or later. On the other hand genotype 2 was found in 17% sera sampled before 1992 and in 37% of the samples collected 1992 or later. It is not known if this genotype has recently been introduced into Sweden. Further analysis on larger series of samples is needed to confirm these preliminary results. / AcknowledgmentsI would like to express my gratitude to several people who have been supportive in different ways throughout this project.First of all, I want to thank my supervisor Helene Norder, for giving me the possibility to do my diploma thesis at the Department of Virology, Swedish Institute for Infectious Disease control (SMI) and for helping me during this study and for the many insightful conversations during the design and development stages of the application, and also for the many helpful comments and suggestions on the text of the thesis.I want to express my appreciation to my laboratory supervisor Regina Wallin, Camilla Jern and Josefine Ederth for helping me during the procedure for this study. Then, I want to thank my examiner Magnus Johansson from the Södertörns university collegefor his advice on writing this paper. Finally, I would like to thank my family and specially my mother Bahar Hamid for always supporting me during my whole life.Last, but not least, I would like to thank my friends Annika Andersson and Yourdons Yemane for being encouraging, understanding and always supportive.

Philosophiae Doctor - PhD / Hepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.

Le but du projet est l’étude des régulateurs de la voie de signalisation intracellulaire de la voie Pi(3)K-Akt au travers de l’analyse du suppresseur de tumeur PTEN (Phosphatase and tenson homolog) et de la sérine/thréonine kinase mTOR (Mamalian Target of Rapamycin). Cette étude à plusieurs objectifs :1. Modulation de la voie Akt par HCV sur des foies humains, foies de souris FL-N/35 au niveau basal dans un premier temps et lors d’un boost de la voie in vitro sur des cultures primaires d’hépatocytes murins.2. Etude de l’expression ainsi que des modifications post-traductionnelles des modulateurs de la voie PI(3)K dans un modèle murin exprimant (FL-N/35) ou non l’ORF complète du virus de l’hépatite C (VHC).3. Confirmer nos précédentes données avec l’invalidation de PTEN dans un modèle de souris KO pour PTEN.4. Etendre ses données au niveau moléculaire dans l’optique d’une étude mécanistique grâce à l’analyse in vivo, ex vivo et in vitro d’un modèle murin KO pour PTEN.5. Compléter cette étude par l’analyse des déterminants viraux impliqués dans la dérégulation de la signalisation intracellulaire grâce à l’étude de souris NS5A.6. Examiner l’impact de la dérégulation de la voie PI(3)K-Akt dans le développement des Carcinomes hépatocellulaires (CHCs) induit par le VHC. / The goal of myt thesis is to study regulators of intracellular signaling pathway of Pi route (3) K-Akt through the analysis of tumor suppressor PTEN (Phosphatase and tenson homolog) and serine / threonine kinase mTOR (Target of Rapamycin Mamalian). This study has several objectives:1. Modulation of the Akt pathway by HCV in human liver, mouse livers FL-N / 35 to the basal level in a first time and at a track boost in vitro on primary cultures of mouse hepatocytes.2. Study of the expression and post-translational modifications of modulators of PI path (3) K in a murine model expressing (FL-N / 35) or not the complete ORF of hepatitis C ( HCV).3. Confirming our previous data with the invalidation of PTEN in a knockout mouse model for PTEN.4. Extending its data at the molecular level with a view to a mechanistic study through analysis in vivo, ex vivo and in vitro of a knockout mouse model for PTEN.5. Complete this study by analyzing the viral determinants involved in the dysregulation of intracellular signaling through the NS5A mouse study.6. Examine the impact of deregulation of IP route (3) K-Akt in the development of hepatocellular carcinoma (CHCs) induced by HCV.

No description available.

The importance of interactions between transmembrane domains of integral membrane proteins has been well-established in a range of essential cellular functions. Most integral membrane proteins also possess regions that lie on the exterior of the membrane that may influence the ability of these transmembrane domains to interact. We sought to test this hypothesis by quantifying the energetics of transmembrane helix self-association in the absence and presence of an amphipathic helix that can bind to the membrane surface. The model chosen for this study was the major coat protein (MCP) of M13 bacteriophage, which has an N-terminal amphipathic helix linked to its single transmembrane segment via a flexible linker. Dimerization of both full-length MCP and a peptide containing only the transmembrane domain (MCPTM) was studied by solution NMR in SDS micelles. We found that there was an increase in the apparent dimerization affinity in the absence of the N-terminal helix. However, this increase in apparent affinity could be attributed to differences in detergent-binding properties of the two polypeptides in monomeric versus dimeric states when the empty micelle was considered to be a participant in the dimer dissociation. Preliminary results from the integral membrane protein, p7 of the hepatitis C virus are also presented in this thesis. It has been demonstrated that p7 enhances viral infectivity and accumulation, and that this function may require oligomerization in the membrane. While we encountered limitations due to challenges in the generation of sufficient quantities of pure p7 samples, we were able to perform circular dichroism spectroscopy under conditions that may favor different oligomeric states. These studies suggest that there is a change in the degree of helicity upon oligomerization, and suggest that SDS could be a suitable system to characterize the interactions of the p7 oligomer in the future.

Le diagnostic et le suivi des infections par le VIH, VHB et VHC restent un défi dans les pays à faibles ressources qui sont par ailleurs les plus touchés. Il est urgent de pouvoir disposer d'outils de biologie simples, fiables et peu chers pour le contrôle de ces infections virales. Le défi est immense d'un point de vue clinique et politique de santé. L'objectif de ce travail de recherche effectué dans le cadre de la présente thèse est de développer et valider des stratégies et de nouveaux outils de biologie adaptés au diagnostic et au suivi des hépatites virales et de l'infection VIH dans les pays à ressources limitées. Dans un premier temps, nous avons investigué sur les résultats de sérologie VIH discordants, car en biologie et en pratique clinique, il est important d'établir le statut sérologique réel des personnes dépistées avec des résultats clairs pour une prise de décision adéquate à l'échelle individuelle. Dans ce travail, nous avons trouvé que les résultats discordants par l'algorithme de dépistage des femmes enceintes au Burkina Faso, étaient dans 94% des cas de faux positifs dus au test Determine™ HIV-1/2 et 4% de faux négatifs liés au test Genie II™ HIV-1/HIV-2. En matière de santé publique, les femmes présentant ce type de résultat peuvent être considérées comme séronégatives dans les centres où des investigations supplémentaires ne sont pas possibles, surtout dans les pays comme le Burkina Faso où la prévalence de l'infection est basse avec une faible diversité génétique. Dans un second temps, nous avons focalisé nos travaux sur la faisabilité d'une stratégie de dépistage qui combine la détection des trois infections (VIH, VHB et VHC) sur une seule carte DBS. Dans cette étude pilote, nous avons démontré que le DBS collecté en parallèle au test rapide VIH dans un centre de dépistage volontaire permet d'une part de faire la confirmation du VIH par immunoblot, et d'autre part de compléter par le diagnostic des hépatites B et C par ELISA suivi de western blot et PCR pour la confirmation pour le VHC. Cette stratégie peut servir de modèle pour promouvoir et vulgariser le dépistage des hépatites virales B et C dans les pays à ressources limitées. Le DBS peut servir de contrôle et de confirmation du diagnostic du VIH, VHB et VHC. Par la suite, nous avons évalué la performance de deux tests de 4ème génération immunoluminométriques (Elecsys® HIV Combi PT assay, Roche Diagnostics et Liaison® XL Murex HIV Ab/Ag test, DiaSorin) sur des échantillons séchés sur papier filtre en comparaison au test de diagnostic rapide et aux échantillons de sérum frais prélevés chez les patients en primo-infection VIH. Ces travaux ont clairement montré que les deux tests de 4ème génération réalisés sur papier filtre offrent de bonnes performances dans le diagnostic de la primo-infection par rapport aux tests de diagnostic rapide sur sérum frais. Cette méthode peut être utilisée pour détecter précocement le VIH chez les personnes difficiles à atteindre et les populations vivant dans des zones périphériques des pays à ressources limitées. Enfin, nous avons mis au point une technique de PCR en temps réel de détection et de quantification de l'ADN du VHB. A cet effet, nous sommes partis de deux PCR « maison » ciblant deux régions génomiques différentes (gène X pour la qPCR 1 et gène S pour la qPCR 2) en comparaison à un test commercial de charge virale (Cobas AmpliPrep/Cobas TaqMan HBV Test, version 2.0, Roche Diagnostics). La qPCR 2 avec un seuil de détection à 91 UI/ml (vs 104 UI/ml pour la qPCR 1) a montré une meilleure performance dans la quantification de l'ADN du VHB. Cette qPCR 2 peu chère est aujourd'hui produite sous forme de kit par une start-up appelée OMUNIS. Ce kit en développement fera l'objet d'une évaluation multicentrique en France, en Afrique et en Asie du Sud-Est à travers un réseau de laboratoires sous la promotion de l'ANRS. / Diagnosis and management of hepatitis B, hepatitis C and HIV infections are a real challenge in middle and low-income countries. There is an urgent need for simple, reliable and inexpensive tools to control these infections in high prevalence sittings like Africa and Asia. The challenge is immense in clinical and public health policy hands. The main goal of this research work performed for our PhD is the development and validation of strategies and tools to diagnose and monitor HIV, HBV and HCV infections in resource-constrained countries. At a first step, we investigated the results of HIV discordant results, since it is important to establish the real HIV status of people tested with clear results for appropriate decision-making in biological and clinical practice. This work show that discordant results obtained in the algorithm of HIV screening among pregnant women in Burkina Faso, are false positive results in 94% of cases due to the Determine™ HIV-1/2 immunochromatographic test and false negative results in 4% of cases due to the Genie II ™ HIV-1 / HIV-2 test. In public health practice, women with this type of result can be considered as negative for HIV testing in centers where additional investigations are not possible, especially in countries like Burkina Faso with a low incidence and a low genetic diversity of HIV.In a second step, we focused our work on the feasibility of a screening strategy that detects HIV, HBV and HCV infections into a single card of DBS. In this pilot study, we demonstrated that DBS collected in parallel to HIV rapid testing in a voluntary counseling and testing center allows HIV confirmation using immunoblotting, and an additional testing by diagnosing HBV and HCV using ELISA followed by immunoblotting and PCR for HCV confirmation. This strategy can serve as a model to promote and scale-up the screening of HBV and HCV in resource-limited countries. DBS can be served as control and confirmation of HIV, HBV and HCV diagnosis. Furthermore, we evaluated the performance of two 4th generation chemiluminescent immunoassays (Elecsys HIV Combi PT assay, Roche Diagnostics and Liaison XL Murex HIV Ab/Ag test, DiaSorin) tested on filter paper samples in comparison to rapid diagnostic test and fresh serum samples from patients with acute HIV infection. These studies have clearly shown that the two 4th generation tests performed on filter paper offer good performance in terms of sensitivity for the diagnosis of HIV infection in its early phases compared with rapid diagnostic tests. This approach may be used in combination with HIV rapid tests in hard-to-reach individuals and populations living in remote areas of when an early HIV infection is suspected since rapid tests do not offer appropriate performance in this case.Finally, we developed a real-time PCR for HBV DNA detection and quantification. In this study, we evaluated two in-house PCR targeting two different regions of HBV genome (X gene for qPCR 1 and S gene for qPCR 2) in comparison with a commercial Roche HBV DNA test (Cobas AmpliPrep / Cobas TaqMan HBV Test, version 2.0, Roche Diagnostics) as a gold standard. The qPCR 2 with a low detection limit of 91 IU/ml (vs 104 IU/ml for 1 qPCR) showed a better performance in HBV DNA quantification. This inexpensive qPCR with best performance characteristics is producing by a start-up called OMUNIS. This kit will be evaluated in France, in Africa and in South and East Asia in a research study funded by ANRS (France REcherche Nord & sud Sida-hiv Hépatites).

Les tests de laboratoire basés sur des techniques de biologie moléculaire sont des outils inestimables pour le suivi des patients, en particulier dans le cadre de l’infectiologie. Dans ce contexte clinique, ils peuvent permettre d’établir un diagnostic, un pronostic ainsi que de déterminer la stratégie thérapeutique antivirale la plus adaptée. Les virus hautement variables tel que le virus de l’immunodéficience humaine (VIH), de l’hépatite B (VHB) et de l’hépatite C (VHC) sont présents sous forme de quasi-espèces au sein de leur environnement réplicatif. Lorsque des pressions de sélection s’exercent telle que l’administration d’un antiviral, une redistribution des variants majoritaires est fréquemment observée en variants mutés pour mieux s’adapter à cet environnement. Nos travaux ont permis, dans ce contexte, de valider l’impact clinique des mutations majoritaires mais aussi minoritaires grâce à la mise en œuvre de plusieurs techniques de séquençage (pyroséquençage, séquençage à haut débit et PCR en allèle spécifique). Nous avons également validé l’utilisation d’un logiciel simple, fiable et utilisable en routine par le clinicien pour l’interprétation clinique de la masse de données générées par le séquençage à haut débit. Enfin dans le contexte du test diagnostique, nous avons cliniquement validé sur une cohorte de patients infectés par le VHB, l’utilisation du papier buvard (Dried Blood Spot) comme support de prélèvement alternatif non invasif de diagnostic, en particulier pour les populations n’ayant pas accès aux structures classiques de soins et pour les pays en voie de développement. / Molecular biology based assays are invaluable tools for the patients follow-up. They can help to establish the prognosis, guide for the treatment decisions and assess the virological response to therapy. Highly variable viruses like Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C virus (HCV) which have a quasispecies distribution. Selection pressure on viral replicative environment such as an antiviral drug treatment, generally lead to a redistribution of the viral quasispecies with an increasing of the best adapted viral mutant. Our work allowed in this context to validate the clinical impact of majority but also minority mutations through the implementation of several sequencing techniques (pyrosequencing, high-throughput sequencing and allele-specific PCR). We also validated the use of a simple, reliable and routinely software solution by clinician for clinical interpretation of the mass of data generated by high-throughput sequencing. Finally, in the context of the diagnostic testing, we clinically validated in a cohort of patients infected with HBV, use the Dried Blood Spot technique as a supporting noninvasive diagnostic alternative sampling, especially for populations that have no access to conventional health structures and developing countries.

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