Orientador: Rejane Maria Tommasini Grotto / Banca: Dennis Armando Bertolini / Banca: Giovanni Faria Silva / Resumo: O vírus da Hepatite C (VHC) é um importante patógeno associado com doença hepática crônica sendo que alguns infectados podem desenvolver cirrose e carcinoma hepatocelular. O tratamento da hepatite C crônica visa a resposta virológica sustentada (RVS), definida como níveis de RNA viral indetectáveis no soro por seis meses depois do término do tratamento. Atualmente, a terapia padrão ouro é a combinação de interferon α peguilado e ribavirina, porém esse esquema terapêutico vem se mostrando eficaz em, apenas, 50% dos pacientes infectados com o genótipo 1, o mais prevalente no Brasil. Portanto, novas drogas mais eficazes e menos tóxicas estão sendo desenvolvidas para melhorar a assistência aos pacientes infectados pelo VHC, entre as quais merecem destaque os inibidores da serina protease NS3, a qual é uma enzima essencial para a replicação do VHC e assim um potencial alvo para novas terapias antivirais. Entretanto, a emergência de variantes resistentes é o maior obstáculo para o sucesso da terapêutica. Variantes resistentes já foram isoladas em pacientes tratados com os inibidores de protease e, estão associadas com a falência terapêutica. Porém o impacto dessas variantes resistentes em pacientes virgens de tratamento ainda não foi esclarecido e, esse tipo de informação pode avaliar o impacto dos inibidores de protease na terapia antiviral. O objetivo deste estudo foi avaliar a presença de mutações de resistência e polimorfismos genéticos na região genômica NS3 do VHC em 37 pacientes virgens de tratamento com inibidores de protease infectados com genótipo 1. RNA viral sérico foi utilizado como fonte para amplificação e seqüenciamento da região NS3 do VHC e, avaliar a presença de mutações de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Hepatitis C Virus (HCV) is an important pathogen associated with chronic hepatic disease and some infected patients can develop cirrhosis and hepatocellular carcinoma. The treatment of chronic hepatitis C aimed the sustained virological response (SVR), defined as having undetectable serum HCV RNA at the end of therapy for at least 6 months. Currently, the gold standard therapy is a combination of pegylated interferon-α and the ribavirin, however this treatment present efficacy in only 50% of patients infected with genotypes 1, the most prevalent in Brazil. Then, new drugs more effective and less toxic have been developed to improve the attendance of the HCV infected patients as the serine protease NS3 inhibitors, which is an enzyme essential to HCV replication and main target of new antiviral therapies. However, the emergence of drug resistant variants has been the major obstacle to therapeutic successful. Resistant variants have already been isolated in patients treated with protease inhibitors and, these resistant variants are associated with non response to treatment. But the impact of the resistant variants in naïve protease inhibitors patients is unclear yet and, this information can evaluate the impact of protease inhibitors in antiviral therapeutic. The goal of this study was evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV in 37 protease inhibitors-naive genotype 1 HCV infected patients. Serum viral RNA was used as source to amplification and sequencing of NS3 region of HCV and, evaluates the presence of resistance mutations and polymorphisms in this region. The results showed that only 07 (18.9%) samples presented resistant variants, the mutations... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:23:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-15Bitstream added on 2014-06-13T19:28:48Z : No. of bitstreams: 1 zeminian_lb_me_botfm.pdf: 506409 bytes, checksum: 08bea6131bbfbe54e0327803c5f6bb14 (MD5) / Ministério da Saúde / O vírus da Hepatite C (VHC) é um importante patógeno associado com doença hepática crônica sendo que alguns infectados podem desenvolver cirrose e carcinoma hepatocelular. O tratamento da hepatite C crônica visa a resposta virológica sustentada (RVS), definida como níveis de RNA viral indetectáveis no soro por seis meses depois do término do tratamento. Atualmente, a terapia padrão ouro é a combinação de interferon α peguilado e ribavirina, porém esse esquema terapêutico vem se mostrando eficaz em, apenas, 50% dos pacientes infectados com o genótipo 1, o mais prevalente no Brasil. Portanto, novas drogas mais eficazes e menos tóxicas estão sendo desenvolvidas para melhorar a assistência aos pacientes infectados pelo VHC, entre as quais merecem destaque os inibidores da serina protease NS3, a qual é uma enzima essencial para a replicação do VHC e assim um potencial alvo para novas terapias antivirais. Entretanto, a emergência de variantes resistentes é o maior obstáculo para o sucesso da terapêutica. Variantes resistentes já foram isoladas em pacientes tratados com os inibidores de protease e, estão associadas com a falência terapêutica. Porém o impacto dessas variantes resistentes em pacientes virgens de tratamento ainda não foi esclarecido e, esse tipo de informação pode avaliar o impacto dos inibidores de protease na terapia antiviral. O objetivo deste estudo foi avaliar a presença de mutações de resistência e polimorfismos genéticos na região genômica NS3 do VHC em 37 pacientes virgens de tratamento com inibidores de protease infectados com genótipo 1. RNA viral sérico foi utilizado como fonte para amplificação e seqüenciamento da região NS3 do VHC e, avaliar a presença de mutações de... / The Hepatitis C Virus (HCV) is an important pathogen associated with chronic hepatic disease and some infected patients can develop cirrhosis and hepatocellular carcinoma. The treatment of chronic hepatitis C aimed the sustained virological response (SVR), defined as having undetectable serum HCV RNA at the end of therapy for at least 6 months. Currently, the gold standard therapy is a combination of pegylated interferon-α and the ribavirin, however this treatment present efficacy in only 50% of patients infected with genotypes 1, the most prevalent in Brazil. Then, new drugs more effective and less toxic have been developed to improve the attendance of the HCV infected patients as the serine protease NS3 inhibitors, which is an enzyme essential to HCV replication and main target of new antiviral therapies. However, the emergence of drug resistant variants has been the major obstacle to therapeutic successful. Resistant variants have already been isolated in patients treated with protease inhibitors and, these resistant variants are associated with non response to treatment. But the impact of the resistant variants in naïve protease inhibitors patients is unclear yet and, this information can evaluate the impact of protease inhibitors in antiviral therapeutic. The goal of this study was evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV in 37 protease inhibitors-naive genotype 1 HCV infected patients. Serum viral RNA was used as source to amplification and sequencing of NS3 region of HCV and, evaluates the presence of resistance mutations and polymorphisms in this region. The results showed that only 07 (18.9%) samples presented resistant variants, the mutations... (Complete abstract click electronic access below)

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Submitted by FERNANDA CRISTINA WINCKLER null (fwinckler@fmb.unesp.br) on 2016-11-30T19:00:44Z No. of bitstreams: 1 DISSERTAÇÃO FINAL 30.11.16.pdf: 468887 bytes, checksum: 372ef2c27b73313e5608616205ed1d2e (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-02T16:28:26Z (GMT) No. of bitstreams: 1 winckler_fc_me_bot.pdf: 468887 bytes, checksum: 372ef2c27b73313e5608616205ed1d2e (MD5) / Made available in DSpace on 2016-12-02T16:28:26Z (GMT). No. of bitstreams: 1 winckler_fc_me_bot.pdf: 468887 bytes, checksum: 372ef2c27b73313e5608616205ed1d2e (MD5) Previous issue date: 2016-10-19 / A hepatite C é uma doença infecciosa que torna-se crônica em cerca de 85% dos infectados que poderão desenvolver cirrose e carcinoma hepato celular. O tratamento antiviral em muitos dos pacientes não é eficaz, principalmente quando estes portam o genótipo 1 e fibrose avançada, a resposta inflamatória também desempenha seu papel sobre a resposta virológica sustentada (RVS) durante o tratamento com Interferon Peguilado (PegIFN) associado a Ribavirina (RBV). Nesse estudo nosso objetivo principal foi avaliar a influência da resposta inflamatória através de células e citocinas/quimiocinas sobre a resposta virológica do paciente em tratamento antiviral com terapia tripla. Incluimos pacientes com RNA VHC+, nunca tratados (naive), portadores do genótipo 1, ambos os sexos e com fibrose avançada F3 (n=6); F4 (n=21) candidatos ao tratamento em regime triplo. Os pacientes tiveram suas amostras coletadas e analizadas nas semanas 0 e 12 do tratamento e os seguintes parâmetros foram analisados: IL-2, IL-4, IL-6, IL-8, IL-10, IL-17A, TNF-α, IFN-γ, RANTES, MCP-1, MIG, IP- 10, através de citometria de fluxo (método CBA). Foram incluídos 15 voluntários saudáveis (grupo controle) e 27 pacientes que foram separados em G1(RVS) e G2 (não RVS), a taxa de RVS foi de 63%. Os pacientes com hepatite C crônica tiveram os níveis circulantes de IP10, MCP-1, MIG, RANTES, IL-8 e IL-6 mais elevados quando comparados com voluntários saudáveis, quando comparados G1xG2 os níveis de RANTES (p=0,04) e IL-6 (p=0,02) foram associadas com a RVS na semana 0, seus níveis eram mais baixos em G1, na semana 12 os níveis de RANTES (p=0,04) e IL-8 (n=0,01) foram associados com a RVS, seus níveis são mais elevados em G2, a comparação entre as semanas 0 e 12 mostrou que em G1 os níveis de IL6 (p= 0,02) e MCP-1 (p=0,001) apresentam associação com o tratamento e em G2 os parâmetros associados ao tratamento foram RANTES (p=0,05) e MCP-1 (p=0,01). Os resultados sugerem que, a citocina IL-6 e a quimiocina RANTES estão associadas com a RVS na semana 0. Na semana 12, RANTES assim como IL-8 influenciam na RVS durante terapia antiviral em regime triplo. Quando comparado semana 0 e 12 em pacientes RVS, a citocina IL-6 está associada ao tratamento. Em pacientes não RVS, RANTES esta associada ao tratamento e MCP-1 está associada ao tratamento independente da resposta obtida. / Hepatitis C is an infectious disease which becomes chronic in about 85% of infected people who can develop cirrhosis and hepatocellular carcinoma. Antiviral therapy isn’t effective in many patients, especially when these patients are genotype 1 and have advanced fibrosis, the inflammatory response also plays a role on sustained virological response (SVR) during treatment with Pegylated (PegIFN) plus Ribavirin (RBV). The aim of this study was evaluate the influence of the inflammatory response by cells and cytocines/chemokines on the virologic response of the patients under antiviral treatment with triple therapy. We included patients with HCV RNA+, naive, genotype 1, both male and female and with advanced fibrosis F3 (n=6); F4 (n=21) for triple treatment regimen. Patients had their samples collected and analyzed at weeks 0 and 12 of treatment and the following parameters were analyzed: IL- 2, IL-4, IL-6, IL-8, IL-10, IL-17A, TNF-α, IFN-γ RANTES, MCP-1, MIG, IP-10 by flow cytometry (CBA method). Control group of 15 healthy volunteers and 27 patients, who were separated into GI (SVR) and G2 (not SRV), were included, the SVR rate was 63%. Patients with chronic hepatitis C had higher circulating levels of IP10, MCP-1, MIG, RANTES, IL-8 and IL-6 compared with healthy volunteers, when G1xG2 were compared, levels of RANTES (p=0,040 and IL-6 (n=0,02) were associated with a SVR at week 0 and its levels were lower in G1; at week 12, levels of RANTES (p=0,04) and IL-8 (p=0,01) were associated with a SVR and its levels were higher in G2. The comparison between weeks 0 and 12 showed that, in G1, the IL6 levels (p = 0.02) and MCP-1 (p = 0.001) were associated with the treatment and in G2, the parameters associated with the treatment were RANTES (p = 0.05) and MCP-1 (p = 0.01). The results suggest that the cytocine IL-6 and chemokine RANTES are associated with SVR at week 0. At week 12, RANTES as well as IL-8 influence in SVR during antiviral therapy in triple regimen. When weeks 0 and 12 in patients SVR are compared, the cytocine IL-6 is associated with treatment. In non-SVR patients, RANTES is associated with treatment and MCP-1 is associated with independent treatment of the patient's response.

Background: Immunological and virological responses to ART are important outcome indicators that are mostly used to evaluate the success of an ART program. A comparative performance between ART providers based on the two outcomes can be useful in optimising resources to underperforming providers and advising quality improvement plans. Aim: To compare immunological and virological responses of ART for adult HIV positive patients between providers in Tshwane District, Gauteng Province, South Africa. Methodology: This study was an analytical observational study that retrospectively compared patient treatment outcomes on immunological and virological responses between 16 Antiretroviral Therapy (ART) providers. The analysis compared baseline patients’ status on these two outcomes with their statuses after 6 and12 months on ART. Ordinary logistic regression was used to calculate Standardised Incidence Ratios (SIR), while multilevel model analysis was used to calculate specific provider random effects of poor immunological and virological responses. Results: After 6 months of treatment, the SIR of poor immunological outcome for all clinics under study, as predicted by the unadjusted logistic regression models was 0.29 (95% CI: 0.27-0.31), but varied from a low of 0.14 (95% CI: 0.00-0.40) to a high of 0.66 (95% CI: 0.13-1.20) between the clinics. Two clinics had a Standardised Incidence Ratio (SIR) of poor immunological response that was significantly below 1 (poor immunological rate below average), while three clinics had an SIR above 1 (poor immunological rate above average) under the unadjusted logistic models. After adjusting for the effects of gender, age, drug combination, religion and present virological status, no clinic had a SIR that was significantly below 1, but two clinics had a SIR that was significantly above 1. xi Under the logistic multilevel (MLLR) analysis, the unadjusted model flagged two clinics whose clinic specific effects were below zero (lower rate of poor immunological outcome below that of the total sample) and one clinic whose clinic specific effect was above zero (higher rate of poor immunological outcome below the total sample rate). The adjusted model showed that no clinic had residual effects that were significantly below or above zero. The confidence intervals for MLLR model were found not to be wider than those of the logistic regression (LR) models particularly for clinics with small sample sizes. A number of clinics changed the relative order of their SIR/random effects after case-mix adjustments under both the LR and MLLR modelling. For poor virological response, both the LRD and MLLR models indicated no clinic specific effects. The predicted poor virological response rate by the case-mix unadjusted LR model was 0.12 (95% CI 0.11 - 0.13). All clinics except one had SIRs that were not significantly different from 1. After adjusting for CD4 count and age, no clinic had an SIR that was significantly different from 1. Conclusions: Case-mix or patients baseline characteristics explained much of the variation in the Standardised Incidence Ratios (SIR) of poor immunological outcome after 6 months of patient treatment, while provider (clinic) specific effects explained much of the variation after 12 months of treatment. After 6 months of treatment, the results also showed that there were significant differences in the SIR between the clinics before case-mix adjustments, but the differences disappeared after case-mix adjustments. This shows that comparison of treatment outcomes between providers (clinics) can be misleading if no proper adjustment are made for confounding factors. Differences in the SIRs for poor virological outcome, after 6 months of patient treatment were no longer significant between clinics after taking account of CD4 count and age. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / School of Health Systems and Public Health / unrestricted

Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram clinicamente relevantes. Desde então, procuramos observar as diferenças entre melhor ou pior prognóstico de acordo com a imunogenética que mostrou diferenciação entre os grupos R e NR ao tratamento em relação ao prognóstico da resposta terapêutica. Quando as diferenças entre as sequências da NS5B e a resposta ao tratamento foram consideradas verificou-se que associada a mutação R254K, estava a C316N que poderia conduzir a uma não resposta à terapia no genótipo 1b. Os nossos dados também suportaram forte associação de IL-28B rs12979860, com elevada probabilidade de resposta à terapia de IFN + RBV. Nossos dados evidenciam a presença de pacientes virgens de tratamento que abrigam mutações de resistência previamente descritas na literatura. A análise dos fatores preditores de resposta virológica mostrou que a predição de boa resposta ou não ao tratamento e ainda da progressão da doença é dependente de uma importante interação entre a genética viral e a do hospedeiro. Fato este importante para que no momento de avaliação de diagnóstico e conduta terapêutica, o médico possa tomar medidas apropriadas para o tratamento de cada paciente individualmente independentemente do genótipo do HCV em questão. / It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid fragment of NS5B was examined in 69 Hepatitis C Virus drug naïve (HCV)-infected individuals in Rio de Janeiro, Brazil. In the second phase, the mutations revealed clinically relevant from the gene in question. Since then, we seek to observe the differences between better or worse prognosis according to immunogenetic showed that differentiation between the immunogenetics of the groups R and NR to treatment in relation to prognosis of therapeutic response. When the differences between the NS5B sequences at baseline and the treatment response were considered we found that R254K associated with C316N mutations could lead to a non-response to IFN-RBV therapy in genotype 1b. Our data also strong support the association of rs12979860 IL-28B polymorphism with high probability of response to IFN + RBV therapy. Our data highlight the presence of HCV genotypes from drug naïve patients harboring resistance mutations previously described in literature. The analysis of predictors virologic response demonstrated that the prediction of better or worse therapy response and further the disease progression is dependent of a significant interaction between viral and host genetics. This fact is important for diagnosis evaluation and clinical therapeutic, the medico can take appropriate measures to treat each individual patient irrespective of the genotype of HCV in question.

H θεραπεία της χρόνιας ηπατίτιδας C παραμένει πρόκληση ,ιδιαίτερα για κάποιους ασθενείς ,μια και ποικίλοι παράγοντες που αφορούν τόσο τον ιό όσο και τον ξενιστή σχετίζονται με χαμηλότερη ιολογική απάντηση στη θεραπεία .Ο γονότυπος τύπου 1 και το υψηλό ιϊκό φορτίο έναρξης είναι οι κύριοι ιϊκοί παράγοντες που σχετίζονται με χαμηλότερη ιολογική απάντηση στη θεραπεία ,ενώ για τον ασθενή οι παράγοντες που σχετίζονται με χαμηλότερη ιολογική απάντηση στη θεραπεία είναι η μη ανταπόκριση ή η υποτροπή σε προηγούμενη θεραπεία ,η παρουσία κίρρωσης ,η Αφροαμερικανική καταγωγή, η μεγαλύτερη των 40 ετών ηλικία, η μη συμμόρφωση στη θεραπεία και η παχυσαρκία. Τα οβάλ ηπατοκύτταρα είναι πρόδρομα ηπατικά κύτταρα που έχουν συσχετισθεί με την εξέλιξη της ηπατικής νόσου και την ανάπτυξη ηπατοκυτταρικού καρκινώματος σε πειραματικά μοντέλα .Πρόσφατα έχουμε αποδείξει ότι σε περιπτώσεις χρόνιας ηπατίτιδας τύπου Β ή C η παρουσία πρόδρομων ηπατικών κυττάρων σχετίζεται με τη σοβαρότητα της νόσου το βαθμό ίνωσης και το σχετιζόμενο κίνδυνο ανάπτυξης ηπατοκυτταρικού καρκινώματος Αυτή η μελέτη προσπάθησε να εντοπίσει τους παράγοντες που παίζουν καθοριστικό ρόλο στην μακροχρόνια ανταπόκριση στη θεραπεία ασθενών με χρόνια ηπατίτιδα C καθώς και τον ρόλο που μπορεί να έχει σε αυτή τη διαδικασία η ύπαρξη πρόγονων ηπατικών κυττάρων . Στόχος του έργου είναι η διερεύνηση ποικίλων κλινικών γενετικών εργαστηριακών και ιστολογικών παραμέτρων οι οποίες σχετίζονται με τη θεραπεία σε ασθενείς με χρόνια HCV λοίμωξη. Δευτερογενής στόχος της μελέτης είναι ο καθορισμός ομάδων υψηλού κινδύνου για την ανάπτυξη κίρρωσης και ηπατοκυτταρικού καρκίνου. ΣΧΕΔΙΑΣΜΟΣ 135 ενήλικες ασθενείς με θετική PCR και με βιοψία ήπατος που πιστοποιούσε χρόνια ηπατίτιδα εισήχθησαν στη μελέτη και τυχαιοποιήθηκαν να λάβουν είτε μονοθεραπεία με 3 MU ιντερφερόνης a2b τρεις φορές την εβδομάδα για 24 εβδομάδες ή συνδιασμό με ιντερφερόνη ή ή πεγκυλιωμένη ιντερφερόνη a2a (40 KD) μία φορά την εβδομάδα και προσθήκη 1000ή1200 mg ριμπαβιρίνης για 24 ή 48 εβδομάδες ανάλογα τον γονότυπο .Όλοι οι ασθενείς παρακολουθούνταν για την ασφάλεια την αντοχή και την αποτελεσματικότητα στο τέλος της εβδομάδας 2, 4, 8 και κάθε 4 εβδομάδες κατά τη διάρκεια της θεραπείας . Μετά την ολοκλήρωση της θεραπείας οι ασθενείς παρακολουθούνταν στις εβδομάδες 4, 12, 24 . Το πρώιμο τελικό σημείο ήταν η απώλεια του μετρήσιμου RNA του ιού της ηπατίτιδας C (HCV- RNA < 100c/ml) την εβδομάδα 24 μετά το τέλος της θεραπείας . Για να μελετήσουμε τη συσχέτιση των πρόγονων ηπατικών κυττάρων με την ανταπόκριση στη θεραπεία μελετήθηκαν 77 ηπατικές βιοψίες από ισάριθμους ασθενείς με χρόνια ηπατίτιδα C. Σαν μάρτυρες χρησιμοποιήθηκαν 10 φυσιολογικές ηπατικές βιοψίες .29 ασθενείς που ανταποκρίθηκαν στη θεραπεία και είχαν αρνητική PCR στο τέλος της προκαθορισμένης περιόδου αποτέλεσαν την ομάδα (Α) .29 ασθενείς που δεν ανταποκρίθηκαν στη θεραπεία και είχαν θετική PCR στο τέλος της προκαθορισμένης περιόδου αποτέλεσαν την ομάδα (Β) και τέλος 19 ασθενείς που υποτροπίασαν μετά αρχική ανταπόκριση στη θεραπεία αποτέλεσαν την ομάδα (Γ).Εξετάσθηκαν τομές παραφίνης πάχους 4 μm και καταμετρήθηκαν τα κύτταρα με μορφολογία οβάλ ηπατοκυττάρων δηλαδή AFPmRNA(+) και πρωτεΐνες CK19 (+) CK7(+) LCA(-) KAI CD34(-) Τα αποτελέσματα εκφράστηκαν σε ποσοστό επί τις %για τα κύτταρα με τα αντίστοιχα μορφολογικά χαρακτηριστικά και συσχετίστηκαν με τις αντίστοιχες κλινικές παραμέτρους. Ηπατικά προγονικά κύτταρα παρουσιάστηκαν και στις 87 βιοψίες αν και ήταν σημαντικά λιγότερα στις βιοψίες ελέγχου. Σύμφωνα με την έκφραση AFPmRNA η επί της % έκφραση ήταν :Ομάδα Β 53.4+ 1.3 > Ομάδα Γ 49+1.8 > Ομάδα Α 30.7 + 1.9 Οι ασθενείς που έλαβαν ιντερφερόνη ανταποκρίθηκαν καλύτερα στη θεραπεία αν ήταν < 40 είχαν γονότυπος 3,ιστορικό IV Χρήσης ουσιών αρνητική PCR-HCV (-) στην εβδομάδα 24 μετά θεραπεία &είχαν απουσία κίρρωσης ενώ είχαν μικρότερη πιθανότητα ανταπόκρισης αν είχαν ηλικία >40 έτη γονότυπο 1 ιστορικό μετάγγισης, αυξημένη ALT ή παρουσία κίρρωσης. Οι ασθενείς που έλαβαν θεραπεία συνδιασμού είχαν ευνοϊκή πρόγνωση αν εμφάνιζαν γονότυπο 2 ή 3 και μη ευνοϊκή αν είχαν γονότυπο 1,4 παρουσία κίρρωσης& αυξημένη γGt . Οι αυξημένες τρανσαμινάσες δεν αποτελούσαν προγνωστικό δείκτη. Στους ασθενείς που έλαβαν θεραπεία με πεγκυλιωμενη ιντερφερονη η ηλικία ή η απουσία κίρρωσης δεν αποτελούσε ανεξάρτητο προγνωστικό παράγοντα αντίθετα με την παραμονή αυξημένης ALT & γGt στον 6ο μήνα θεραπείας Ανεξάρτητοι προγνωστικοί παράγοντες για οποιαδήποτε θεραπευτικό σχήμα ήταν η Ηλικία (< 40 ετών )και η απουσία κίρρωσης .Οι ασθενείς με Φυσιολογικές τρανσαμινάσες αποτελούσαν το 40% των ασθενών μας στη δική μας μελέτη φάνηκε ότι ανταποκρίνονται καλύτερα στη θεραπεία και Σε μικρότερο ποσοστό εμφανίζουν κίρρωση ( 7% ) ενώ οι ασθενείς με κίρρωση Ανταποκρίνονται λιγότερο καλά σε όλες τις θεραπείες εμφανίζουν μεγαλύτερη διάρκεια λοίμωξης,λιπώδες ήπαρ σε μεγαλύτερο ποσοστό (64%) Έχουν αυξημένα ποσοστά ALT-γGt. Ενώ η παρουσία κίρρωσης είναι ανεξάρτητη από τον γονότυπο Τα ΠΗΚ υπάρχουν συχνά σε βιοψίες ήπατος ασθενών με ΧΗC και εκφράζουν AFPmRNA Υπάρχει σημαντική συσχέτιση με τη σοβαρότητα της νόσου και την ανταπόκριση στη θεραπεία Τα ΠΗΚ μπορεί να αποτελέσουν ανεξάρτητο προγνωστικό παράγοντα για την ανταπόκριση στη θεραπεία σε ασθενείς με χρόνια ηπατίτιδα C. / The treatment of chronic hepatitis C remains a challenge, particularly for certain patients as several virus related and patient related factors are associated with a lower virologic response to therapy. Hepatitis C virus genotype 1and a high baseline viral load are the major viral factors associated with a lower virologic response to therapy .Patient –related factors include previous relapse or non response to treatment, the presence of cirrhosis, the African America ethnicity, older age, contraindications to treatment and obesity. Oval hepatocytes (HK) are liver stem cells which are involved in the progress of liver disease and hepatocellular carcinoma development in experimental models. We have previously shown that in case of chronic hepatitis type B or C the presence of OH is related to the severity of the disease, the grade of fibrosis and the relative risk for HCG development. This study try to determine the factors effect the long term suppression of hepatitis C virus and also investigate the correlation of OH expression with treatment response in patients with chronic hepatitis C. Design 135 patients aged 18 years or more with positive HCV RNA and liver biopsy, were enrolled and randomly allocated to one of three regimens: 3mega units (MU) interferon a2btree times a week for 24 weeks, 3mega units (MU) interferon a2b tree times a week, plus 1000-12000 mg ribavirine per day for 24 weeks or 48 weeks or peg interferon alfa-2a (40KD) plus ribavirine 1000 or 1200 per day for 24 or 48 weeks because of different genotype. All patients were assessed for safety, tolerance and efficacy and the end of weeks 2, 4, 8and every 4 weeks during treatment. After treatment was completed patients were followed up on weeks 4, 12 and 24.The primary endpoint was loss of detectable HCV-RNA (serum HCV-RNA <100 copies/ml) at week 24 after treatment. The study comprised 77 liver biopsies obtained to an equal number of patients with chronic hepatitis C virus infection. To investigate the correlation of OH expression with treatment response ten normal liver biopsy were used as control. Twenty nine patients were assigned as responders (group A) 29 as non responders (group B) and 19 as releasers (group C). Paraffin sections (4μm thick) were subjected. To OH expretion . Cells with morphologic features of OH that were AFPmRNA or protein +/CK19+/CK7+ and LCA (-) /CD 34(-) were scored. Results were expressed as% of positive cells following morphometric analysis and correlated with the clinical parameters. Findings Sustained virological response at 24 weeks after treatment was found in 18( 22% ) of the 88 patient treated for 24 weeks with 3mega units (MU) interferon a2b three times a week ,35 ( 47%) of the 74 patient treated with the combination regiment, and 28 (60% ) of the 47 treated with peg interferon alfa-2a (40KD) plus ribavirine. Logistic recreation identified five independent factors significantly associated with response to treatment with interferon: genotype 2 or 3 , age forty years or less, minimal fibrosis stage, Ivd users , PCR negative at 6 months. Tree factors associated with the response to combination therapy: genotype 2 or 3, no cirrhosis, and high γgt the only factors significantly associated with response with the pegylated interferon treatment is high levels of γgt and alt to the sixth month of treatment. Oval hepatocyte expression was present in all 87 specimens, being significantly lower in controls compared with cases of chronic hepatitis C. According to the AFPmRNA expression, the grade for % OH expression was: (group B) non responders: 53.4+- 1.3> (Group C ) relapses :49+_1.8>(group A) responders30.7 +_1.9The difference was recorded as follows>(group A) vs. Bp<0.01, group A vs. C p<0.01, group B vs. C p<0.05. Conclusions This study demonstrates that OH are frequently present and expresses AFPmRNA in liver biopsies of patients with chronic hepatitis C. There is a significant association with the severity of disease and with response to treatment and may provide additional prognostic information and predict prognosis in cases of chronic hepatitis c. Age forty years or less and the presence of cirrhosis or no, they are independent factors significantly associated with response to any treatment. Patient with persistently normal or almost normal transaminase levels have higher virological response to therapy and less frequently cirrhosis Patient with cirrhosis exhibit lower response rates to therapy. The presents of cirrhosis is not related to genotype . Patients have frequently high levels of alt and γgt before and six months after therapy and have a worse virological response to any therapy.

Estima-se que a prevalência global da população mundial com hepatite C é de 3%. Pouco se sabe sobre a resposta ao tratamento com respeito à resistência viral. Algumas mutações no fragmento de 109 aminoácidos da NS5B são associadas com resistência ao interferon (IFN) e ribavirina (RBV). Estudos moleculares e clínicos identificaram fatores associados com o hospedeiro e vírus relacionados associada com a resposta ao tratamento, tal como o gene que codifica a IL-28B. Este estudo foi dividido em duas fases, cujos objetivos foram caracterizar a frequência de mutações que conferem resistência ao HCV e avaliar a relevância das mutações em pacientes Respondedores (R) ou Não Respondedores (NR) ao tratamento e caracterizar geneticamente as populações sobre polimorfismos genéticos nos SNPs da IL-28B em relação ao prognóstico da resposta ao tratamento. As amostras dos pacientes foram submetidas a testes de genotipagem e carga viral. As sequências geradas foram comparadas no BLAST e no banco de dados Los Alamos HCV. Realizamos o alinhamento das sequências homólogas e as mutações identificadas. Com base no genótipo e carga viral determinamos a classificação dos pacientes de acordo com a resposta à terapia. O DNA genômico foi isolado a partir de sangue periférico para a realização da tipagem de SNPs de IL-28B. A metodologia utilizada foi de PCR em tempo real utilizando sondas TaqMan SNP específico. A análise dos dados foi realizada utilizando GraphPad Prism com qui-quadrado, risco relativo (RR), Odds Ratio (OR) e intervalo de confiança de 95%, com um nível de significância de P <0,05. Foi encontrado na primeira fase deste estudo uma taxa significativa mutações associadas ao tratamento nas amostras estudadas. A prevalência de mutações associadas à resistência ao IFN e RBV bem como a novos medicamentos antivirais localizados no fragmento de 109 aminoácidos da NS5B foi examinado em 69 indivíduos infectados naïve no Rio de Janeiro, Brasil. Na segunda fase, as mutações foram clinicamente relevantes. Desde então, procuramos observar as diferenças entre melhor ou pior prognóstico de acordo com a imunogenética que mostrou diferenciação entre os grupos R e NR ao tratamento em relação ao prognóstico da resposta terapêutica. Quando as diferenças entre as sequências da NS5B e a resposta ao tratamento foram consideradas verificou-se que associada a mutação R254K, estava a C316N que poderia conduzir a uma não resposta à terapia no genótipo 1b. Os nossos dados também suportaram forte associação de IL-28B rs12979860, com elevada probabilidade de resposta à terapia de IFN + RBV. Nossos dados evidenciam a presença de pacientes virgens de tratamento que abrigam mutações de resistência previamente descritas na literatura. A análise dos fatores preditores de resposta virológica mostrou que a predição de boa resposta ou não ao tratamento e ainda da progressão da doença é dependente de uma importante interação entre a genética viral e a do hospedeiro. Fato este importante para que no momento de avaliação de diagnóstico e conduta terapêutica, o médico possa tomar medidas apropriadas para o tratamento de cada paciente individualmente independentemente do genótipo do HCV em questão. / It is estimated that the overall prevalence of the average world population with hepatitis C is 3%. Little is known about the treatment response with respect to viral resistance. Some mutations in the 109-aminoacid fragment of NS5B are associated to Interferon (IFN) and Ribavirin (RBV) resistance. Molecular and clinical studies have identified factors associated with the host and related viruses associated with response to treatment, as the gene encoding IL-28B. This study was divided into two phases whose objectives were to characterize the frequency of mutations conferring resistance to HCV viral evaluating the relevance of these in Responders (R) or Non-Responders (NR) patients to treatment and to characterize genetically the populations regarding genetic polymorphisms SNPs IL-28B in relation to prognosis of response to treatment for HCV. Patient samples were subjected to tests for genotyping and viral load. The sequences generated were compared in the BLAST and the Los Alamos database HCV. We conducted the alignment of homologous sequences and mutations identified. Based on virological parameters genotype and viral load determined the classification of patients according to response to therapy. Genomic DNA was isolated from peripheral blood for carrying out the typing of SNPs of IL-28B. The methodology used was real-time PCR using TaqMan probes specific SNPs. Data analysis was performed using GraphPad Prism with chi-square, relative risk (RR), Odds Ratio (OR) and confidence interval of 95% with a significance level of P <0.05. To study these biological parameters we associated the responsive patients, non-responders, the viral load, genotype, and IL-28B polymorphism to treatment outcome. We found in the first phase of this study a significant rate of treatment-associated mutations in the samples studied. The prevalence of mutations associated to resistance to interferon and ribavirin (IFN/RBV) as well new antiviral drugs located in the 109 aminoacid fragment of NS5B was examined in 69 Hepatitis C Virus drug naïve (HCV)-infected individuals in Rio de Janeiro, Brazil. In the second phase, the mutations revealed clinically relevant from the gene in question. Since then, we seek to observe the differences between better or worse prognosis according to immunogenetic showed that differentiation between the immunogenetics of the groups R and NR to treatment in relation to prognosis of therapeutic response. When the differences between the NS5B sequences at baseline and the treatment response were considered we found that R254K associated with C316N mutations could lead to a non-response to IFN-RBV therapy in genotype 1b. Our data also strong support the association of rs12979860 IL-28B polymorphism with high probability of response to IFN + RBV therapy. Our data highlight the presence of HCV genotypes from drug naïve patients harboring resistance mutations previously described in literature. The analysis of predictors virologic response demonstrated that the prediction of better or worse therapy response and further the disease progression is dependent of a significant interaction between viral and host genetics. This fact is important for diagnosis evaluation and clinical therapeutic, the medico can take appropriate measures to treat each individual patient irrespective of the genotype of HCV in question.

Le VIH-1 groupe O (VIH-1/O) est un groupe divergent de VIH-1, endémique au Cameroun, 140 cas ont été identifiés en France depuis 1990. L’objectif de ce travail était d’évaluer l’impact de la diversité du VIH-1/O sur les caractéristiques épidémiologiques, l’évolution naturelle de l’infection et sur la réponse immuno-virologique aux traitements.En France, le recueil de données de suivi uniques portant sur 101 patients ont permis de mettre en évidence que l’évolution naturelle des VIH-1/O était intermédiaire entre celle des VIH-1/M et celle des VIH-2. La résistance aux INNTI des patients VIH-1/O a été confirmée in vivo. La réponse immuno-virologique aux traitements a été comparée entre les VIH-1/O et les VIH-1/M suivis dans des cohortes en France et dans le cadre d’un essai clinique au Cameroun.Une des caractéristiques des VIH-1/O est une CVp significativement plus faible que celle des VIH-1/M avant l’initiation du traitement. Cela n’a pas d’impact sur la survenue de l’indétectabilité, qui est similaire dans les deux groupes après un an de traitement. La réponse immunologique des patients VIH-1/O est globalement proche de celle des patients VIH-1/M en France. Cependant, la comparaison entre deux populations appariés suivis au Cameroun a montré une réponse immuno-virologique plus faible des patients VIH-1/O, sans que cela ait d’impact clinique.La prise en charge des patients VIH-1/O suivant les recommandations mises en place pour les VIH-1/M est donc efficace si elle ne comporte pas d’INNTI. / HIV-1 group O (HIV-1/O) is genetically distinct from HIV-1/M and is endemic in Cameroon while in France 140 HIV-1/O were diagnoses since 1990. The aim of this work was to characterize HIV-1/O epidemiological characteristics, infection natural evolution and immuno-virological response to treatment.In France, based on a large series of 101 HIV-1/O patients, we demonstrated that the natural evolution of HIV-1/O was in between the evolution of HIV-2 and HIV-1/M, but closer to HIV-1/M. We also confirmed in vivo HIV-1/O natural resistance to INNTI treatment. Then, we compared HIV-1/O and HIV-1/M immune-virological response to ARV treatment. In France, we compared data from two cohorts and a clinical trial was performed in Cameroun. One of the HIV-1/O characteristic is the lower CVp before treatment compared to HIV-1/M in naïve patient. This difference had no impact on the virological responses to cART. The proportion of patients with an undetectable pVL was similar between the two groups one year after the beginning of the treatment. Immunological response was close between HIV-1/O and HIV-1/M in France. But when the two populations were compared in Cameroon, the immunological response to cART was lower for HIV-1/O patients than for HIV-1/M patients, although this diffence didn’t had clinical consequences.HIV-1/O medical care based on HIV-1/M guidelines is highly efficient if the treatment doesn’t include NNRTI treatment.

Introduction : Depuis 2015, l’OMS recommande la mise sous traitement antirétroviral systématique de tous les enfants infectés par le VIH pour réduire la mortalité précoce liée au VIH chez les nourrissons en l’absence de traitement. Cependant, malgré la disponibilité des tests de dépistage et des médicaments antirétroviraux, l’initiation précoce de ces traitements reste un défi majeur dans les pays à ressources limitées. L’étude ANRS 12140-PEDIACAM est mise en place pour évaluer la faisabilité, l’efficacité et la tolérance en routine du traitement précoce des enfants infectés par le VIH au Cameroun. Objectifs : Les objectifs de cette thèse visaient à étudier la mortalité et la réponse virologique à deux et quatre ans après l’initiation précoce d’un traitement antirétroviral chez les nourrissons infectés par le VIH, et à identifier les facteurs associés à l’obtention et au maintien d’un succès virologique.Méthodes : Les analyses ont porté sur les 190 enfants infectés par le VIH traités avant l’âge de 1 an (médiane=4 mois), inclus dans les trois sites cliniques du Cameroun participant à la cohorte prospective ANRS PEDIACAM initiée en 2007. La première étude a évalué les performances d’un critère basé sur le nombre de doses manquées de traitement rapporté dans un questionnaire d’observance pour dépister un échec virologique chez les nourrissons. La seconde étude portait sur l’estimation de la fréquence et des facteurs associés à l’obtention d’un succès virologique et à la mortalité à deux ans du traitement, utilisant un modèle de survie à risque compétitif. La troisième concernait l’évolution de la réponse virologique entre 2 et 4 ans du traitement selon le statut virologique obtenu à deux ans.Résultats : Les performances du questionnaire d’observance administré à l’accompagnant du nourrisson s’avèrent limitées, avec une valeur prédictive positive trop faible pour dépister un échec virologique en l’absence de charge virale disponible. La mortalité reste élevée à un an du traitement précoce (18,0% [IC95% : 13,0 - 24,0]). Elle est de 3,3% [IC95% : 0,4 - 6,2] entre 2 et 4 ans de traitement. La probabilité d’atteindre au moins un succès virologique avant 2 ans de traitement est de 80% environ, mais celle d’obtenir une suppression virologique maintenue sur au moins 6 mois n’est que de 67% au seuil de 1000 copies/mL, et de 60% au seuil de 400 copies/mL. A 4 ans du traitement initial, la proportion de charge virale contrôlée (<400 copies/mL) est de 75,2% [68,3-82,1]) chez les 144 enfants toujours vivants et suivis, mais pour 12% la charge virale n’a pas été mesurée. Le seul facteur associé significativement au succès virologique dans les 2 ans du traitement initial est la bonne observance rapportée par l’accompagnant. Et seuls un succès virologique obtenu à 2 ans et l’initiation plus récente du traitement antirétroviral sont associés à un charge virale contrôlée à 4 ans.Conclusion : Même si l’intérêt du traitement précoce des nourrissons infectés par le VIH est démontré, le succès virologique à moyen et long terme passe par des stratégies favorisant l’administration quotidienne soutenue des médicaments et une surveillance régulière de la réponse virologique. L’évaluation de l’observance par questionnaire présente une trop faible performance pour dépister précocement un échec virologique. Il est urgent de donner un accès large à la mesure de la charge virale en routine dans les pays à ressources limitées pour dépister rapidement les échecs virologiques chez les enfants recevant un traitement antirétroviral. / Introduction: Since 2015, the WHO recommends to start antiretroviral treatment promptly in all HIV-infected children in order to reduce HIV related mortality. Despite increasing availability of screening tests and antiretroviral drugs, early initiation of antiretroviral treatment (ART) remains challenging in resource-limited countries. The ANRS 12140-Pediacam study assesses feasibility, effectiveness and tolerability in routine practice of early treatment of HIV-infected children in Cameroon. Objectives: The objectives of this thesis are to study mortality and virologic response at 2 and 4 years of early initiation of ART in HIV-infected infants and identify factors associated with virologic success. Methods: The analysis concerned the 190 HIV-infected infants who have initiated ART no later than 1 year (median=4 months) and were enrolled in the 3 Cameroon clinical sites involved in the PEDIACAM prospective cohort study since 2007. The first study evaluated adherence criterium based on the number of missed doses as reported through an adherence questionnaire in oerder to detect virologic failure in infants. The second study concerned the evaluation of the frequency and the factors associated with virologic success and mortality at 2 years of ART initiation, using competing risk regression. The third study concerned the evolution of virologic response between 2 and 4 years of QRT initiation depending on virologic status achieved at 2 years of ART initiation. Results: The performances of adherence questionnaire administered to the infant's caregiver are limited; the positive predictive value is low for detecting virologic failure in the absence of viral load exam. The mortality is high at 1 year after early ART initiation (18.0% [95% CI: 13.0 – 24.0]). The mortality is 3.3% [95%CI: 0.4 – 6.2] between 2 and 4 years of ART initiation. The probability of achieving at least once virologic success within the first 2 years of ART is around 80.0% but the probability of maintaining virologic success for at least 6 months was 67% for threshold=1000 copies/mL and 60% for threshold=400 copies/mL. At 4 years of ART initiation, the proportion of virologic success (viral load<400 copies/mL) is 75.2% [68.3-82.1]) in the 144 children still alive among whom viral load exam was not performed. The only factor associated with virologic success at 2 years of ART initiation is good adherence as reported by the caregiver. Et seuls un succès virologique obtenu à 2 ans et l’initiation plus récente du traitement antirétroviral sont associés à un charge virale contrôlée à 4 ans.Conclusion: Although the interest of early ART in HIV-infected infants is demonstrated, the mid and long term virologic success pass through strategies enhancing supporting steady and daily administration of drugs and regular monitoring of virologic response. The steady evaluation of adherence as reported by questionnaire has a very low performance for early detecting virologic failure. It is urgent to widely get access to routine viral load exam in resource-limited countries for quickly detecting virologic failures in children receiving antiretroviral treatment.