A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues(R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA was used for relative quantification of viral protein in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity.

Patienten mit intravenösem Drogengebrauch (IVDU) sind in hochentwickelten Industrieländern die Hauptrisikogruppe für den Erwerb chronischer Hepatitis C-Virus (HCV)-Infektionen. Um die weitere Verbreitung des HCV zu verhindern, ist es essenziell, diesen Risikogruppen adäquaten Zugang zu diagnostischen und therapeutischen Optionen zu schaffen. Trotz vorhandener Evidenz zur effektiven und sicheren Therapie von HCV-infizierten Patienten mit IVDU bestehen nach wie vor von therapeutischer Seite Zurückhaltung und Sorge vor ungenügender Therapieadhärenz der Patienten. Dies führt leider noch oft dazu, dass diese wichtige HCV-Risikogruppe zu wenig untersucht und therapiert wird. In der Regel stehen urbane Ballungszentren im Blickpunkt der HCV-Forschung, da hier die entsprechenden Risikopopulationen stärker vertreten sind. In ländlichen Regionen mit weniger ausdifferenzierter medizinischer Infrastruktur ist der Zugang zur Diagnostik und Therapie der chronischen HCV-Infektion meist zusätzlich erschwert. Die Suchtklinik der Ilmkreiskliniken Arnstadt-Ilmenau gGmbH in Thüringen ist als Einrichtung der Grund- und Regelversorgung mit ihrem Einzugsgebiet über die Grenzen des Ilmkreises hinaus eine Anlaufstelle der stationären Suchtbehandlung. Hier wurden im Beobachtungszeitraum von 2000 bis 2014 weit mehr als 3000 Patienten mit einer Abhängigkeit von illegalen Drogen und Alkohol suchtmedizinisch behandelt. Schwerpunkt der vorliegenden Arbeit war es, die Ergebnisse der antiviralen Behandlung mit dem damaligen Standard bestehend aus pegyliertem Interferon und Ribavirin bei HCV-infizierten drogenabhängigen Patienten retrospektiv zu untersuchen. Es wurden alle Patienten, die mit einer Abhängigkeitserkrankung in der Suchtklinik Ilmenau behandelt wurden, auf positives Anti-HCV gescreent. Die Anti-HCV-Prävalenz in der Screeningkohorte betrug 15,5 %. In der Kohorte der Patienten mit intravenösem Drogengebrauch war die HCV-Prävalenz wesentlich höher: Knapp die Hälfte (44,4 %) aller Patienten mit IVDU (n = 475) hatte einmal Kontakt mit HCV. Diese regionale epidemiologische Beobachtung steht im Einklang mit Daten aus Multicenter-Studien aus den USA, Schottland und Westeuropa einschließlich Deutschland. Von allen 3197 während des stationären Aufenthaltes gescreenten Patienten der Suchtklinik Ilmenau hatten 354 Patienten eine persistierende chronische HCV-Infektion und fünf Patienten eine akute HCV-Infektion. Spontaneliminationen der chronischen HCV-Infektion waren bei 131 Patienten zu beobachten. Die Patientenkohorten mit chronischer HCV-Infektion und mit Spontanelimination wurden miteinander verglichen. Hochsignifikante Unterschiede zwischen beiden Kohorten waren für den Risikofaktor Haft zu beobachten: So fanden sich unter den Patienten mit Haftaufenthalten in der Eigenanamnese mehr persistierende chronische HCV-Infektionen und seltener Spontaneliminationen. Eine antivirale Peg-Interferon-basierte Therapie erhielten 103 der 354 chronisch HCV-infizierten Patienten (28,7%). Von diesen 103 Patienten waren 69 Patienten unter Opiatsubstitution. Wesentliche Kriterien für den Beginn einer antiviralen Therapie waren psychosoziale Stabilität, Fehlen potenzieller Störfaktoren (z. B. Arbeitslosigkeit, Haftantritt) und soziale Integration. Ein anhaltendes virologisches Ansprechen (SVR = Heilungsraten) 24 Wochen nach Therapieende erreichten 66 der 103 behandelten Patienten. Die SVR-Raten betrugen in Abhängigkeit vom HCV-Genotyp 64,1 %. Diese SVR-Raten sind vergleichbar mit den Ergebnissen aus zahlreichen Studien zur Therapie der chronischen Hepatitis bei Patienten ohne Suchterkrankung bzw. IVDU. 11 Patienten brachen die Therapie vorzeitig ab. Weitere 26 Patienten erreichten keine SVR. Bei drei Patienten kam es zum Therapieabbruch aufgrund von medikamentös bedingten Nebenwirkungen. Generell waren Depressionen, Haarausfall, Inappetenz neben Blutbildveränderungen wie Thrombopenie, Leukopenie oder Anämie zu diagnostizieren. Nur bei acht Patienten (7,8 %) kam es aufgrund von unzureichender Therapieadhärenz zum Therapieabbruch. Alle 66 erfolgreich therapierten Patienten konnten nach Erreichen einer SVR nach Therapieende über mindestens 12 Monate nachbeobachtet werden. Eine Reinfektion trat bei 25,8 % der erfolgreich therapierten Patienten auf. Ein Wechsel des HCV-Genotyps im Rahmen der Reinfektion wurde bei drei Patienten beobachtet. Nur in Ausnahmefällen wurde nach suchtmedizinischer Beratung und Reevaluation den Patienten erneut die PegInterferon-Ribavirin-Therapie angeboten. Zwei HCV-Reinfektionen wurden erneut konventionell erfolgreich therapiert. Die Arbeit zeigt, dass selbst mit einer Interferon-basierten Therapie bei ausgewählten Patienten mit IDVU in mehr als 60% der Fälle die chronische HCV-Infektion ausgeheilt werden kann. Die Rate der Rückfälle mit 25.8 % ist jedoch ein signifikantes Problem und zeigt, dass die Maßnahmen zur Reinfektionsprophylaxe noch nicht ausreichend umgesetzt werden. Trotz umfangreicher Aufklärungsarbeit durch Ärzte, Sozialzentren und Suchtberatungsstellen kann bei Patienten mit IVDU das Suchtpotential so hoch sein, dass trotz des Wissens um die Gefahr einer HCV-Infektion weiterhin intravenös konsumiert wird. Einen fortgesetzten intravenösen Drogenkonsum unter der antiviralen Therapie konnten wir bei 7.8 % der Patienten mit sehr hohem Suchtpotential feststellen. Bei diesen Patienten wurde die Therapie beendet. Für das Erreichen der WHO-Ziele einer 90 %-igen Reduktion der HCV-Neuinfektionen und einer 65 %-igen Reduktion der HCV-assoziierten Mortalität bis 2030 ist die Elimination der HCV-Infektion in bestimmten Risikogruppen mit der höchsten Inzidenz an Neuinfektionen von zentraler Relevanz. Diese als Mikroelimination bezeichnete Strategie wird leider noch nicht ausreichend in Deutschland umgesetzt, obwohl das Bundesministerium für Bildung und Forschung (BMBF) sich den Zielen der WHO in einer nationalen Strategie zur Eindämmung sexuell übertragbarer Erkrankungen ausgesprochen hat. Unsere Studie zeigt, dass auch in ländlichen Regionen mit Hilfe eines interdisziplinären Teams die HCV-Infektion bei Patienten mit Suchterkrankungen frühzeitig erkannt und erfolgreich behandelt werden kann. Durch den konsequenten Einsatz der heute zu Verfügung stehenden effektiven und sehr gut verträglichen Therapie mit direkt-wirkenden antiviralen Substanzen (DAAs) mit Heilungsraten von größer 95% bei Kurzzeittherapie über 8-12 Wochen könnte eine Elimination der HCV-Infektion bei diesen Hochrisikogruppen bereits in wenigen Jahren erreicht werden.:1 EINLEITUNG 6 1.1 PHYSIOLOGIE DER LEBER ALS ORGAN 6 1.2 HEPATITIDEN IM ÜBERBLICK 9 1.2.1 Definition, Klassifikation und Klinik der Hepatitiden 9 1.2.2 Hepatitis A 10 1.2.3 Hepatitis B 10 1.2.4 Hepatitis D 11 1.2.5 Hepatitis E 12 1.3 HEPATITIS C 13 1.3.1 Virologie 13 1.3.2 Epidemiologie 14 1.3.3 Prävalenz 14 1.3.4 Infektionswege, Risikofaktoren 14 1.3.5 Klinik der Hepatitis C 16 1.3.6 Klinische Symptome und natürlicher Verlauf 16 1.3.7 Überblick über die Therapie der chronischen HCV-Infektion 18 1.3.8 HCV-Koinfektion mit Hepatitis-B-Virus (HBV) 20 1.3.9 HCV-Koinfektion mit humanem Immunodefizienz-Virus (HIV) 21 1.3.10 Psychiatrische Komorbiditäten bei HCV-infizierten Patienten und ihre Relevanz für die Interferontherapie 21 1.4 SUCHTERKRANKUNGEN UND IHRE BEDEUTUNG FÜR HCV-INFIZIERTE PATIENTEN 23 2 ZIELE DER ARBEIT 28 3 PATIENTEN & METHODEN 30 3.1 ANGEBOT UND STRUKTUR DER KLINIK FÜR SUCHTMEDIZIN ILMENAU 30 3.2 THERAPIE DER CHRONISCHEN HCV-INFEKTION BEI DROGENABHÄNGIGEN PATIENTEN IN DER SUCHTKLINIK ILMENAU 34 3.3 MAßNAHMEN ZUR THERAPIEADHÄRENZ 37 3.4 EIN- UND AUSSCHLUSSKRITERIEN 38 3.5 PATIENTENKOHORTE 38 3.6 STATISTISCHE BERECHNUNGEN 43 4 ERGEBNISSE 44 4.1 PATIENTENKOLLEKTIV 44 4.1.1 Anti-HCV- und HCV-RNA-Seroprävalenz bei Patienten mit Suchterkrankung 44 4.1.2 Verlauf der Patienten mit positivem Anti-HCV-Status bei Erstdiagnose 46 4.1.3 Vergleich der persistierenden chronischen HCV-Infektionen mit Spontan-eliminationen 50 4.1.4 Patientenkohorte mit akuter HCV-Infektion 54 4.2 THERAPIE DER CHRONISCHEN HCV-INFEKTION 57 4.2.1 Vergleich der Patienten mit persistierender chronischer HCV-Infektion ohne Therapie mit den Patienten, die eine antivirale Therapie erhalten hatten (Therapiekohorte) 57 4.2.2 Verlauf der therapierten Patienten 65 4.2.3 Charakteristika von Patienten mit und ohne Erreichen der SVR 67 4.3 LANGZEITVERLAUF NACH ERFOLGREICHER PRIMÄRTHERAPIE 75 4.3.1 Reinfektionsraten 75 4.4 KASUISTIKEN 79 4.4.1 Therapieabbruch bei nonadhärenter opiatabhängiger Patientin mit fehlender Therapieadhärenz 79 4.4.2 Erfolgreiche Interferontherapie unter Suchtmittelsubstitution bei HCV-assoziierter Leberzirrhose 81 5 DISKUSSION 84 6 ZUSAMMENFASSUNG 95 7 LITERATUR- & QUELLENVERZEICHNIS 98 8 ANLAGEN 112 8.1 TABELLENVERZEICHNIS 112 8.2 ABBILDUNGSVERZEICHNIS 114 8.3 VERWENDETE FRAGEBÖGEN 119 8.4 EHRENWÖRTLICHE ERKLÄRUNG 121 8.5 LEBENSLAUF 122 8.6 DANKSAGUNG 124

Le traitement du VHC connaît une évolution rapide avec le développement d'antiviraux à action directe plus efficaces et mieux tolérés qui vont modifier les stratégies thérapeutiques, les facteurs prédictifs de réponse et les modalités de suivi des patients. Notre travail s'intéresse aux paramètres de suivi du traitement que sont les dosages pharmacologiques de ribavirine et le suivi des cinétiques virales lors d'une trithérapie. L'étude d'une cohorte prospective incluant 186 patients sous trithérapie par IP montre que 60% d'entre-eux présentent une SVR12 et que les facteurs prédictifs sont le génotype de l'IL28B et la réponse au précédent traitement. Une diminution de la filtration glomérulaire réversible est également observée. La mesure du taux résiduel de ribavirine permet de réduire les risques hématologiques chez des patients insuffisants rénaux, la réalisation de l'ASC témoigne d'une moins bonne exposition à la ribavirine chez des patients co-infectés par le VIH/VHC et la biodisponibilité de la ribavirine et la sévérité des anémies augmentent chez des patients traités par télaprévir. Au sein de la cohorte CUPIC, la négativation ou la diminution >50-70% de la charge virale initiale à S2 de trithérapie sont fortement prédictives de la SVR12. Cette mesure à S2 permet aussi de dépister les échappements viraux précoces. La place de la ribavirine est importante dans les associations thérapeutiques actuelles et futures. Sa surveillance pharmacologique peut avoir un intérêt au cours de futures multi-thérapies exposant à d'éventuelles interactions médicamenteuses / The rapid development of new direct antiviral agents (DAA) against HCV gives hope of more potent and well tolerated treatments. These new compounds will deeply modify therapeutic schedules, virological response prognostic factors and patients’ monitoring. The aim of our work was to define the relevance of ribavirin plasma concentration and viral kinetics monitoring during triple therapy. The study of a prospective cohort including 186 patients under triple therapy showed an SVR12 rate of 60%. Associated predictive factors were IL-28B genotype and previous treatment response. A reversible decrease of glomerular filtration rate was also observed. Ribavirin plasma concentration monitoring reduced hematological risks among patients with renal insufficiency. Early ribavirin plasma exposure showed an underexposure among HIV/HCV patients and ribavirin biodisponibility with severe anemia increased among telaprevir-treated patients. Within the CUPIC cohort, the initial viral load undetectability or decrease up to 50% or 70% at week 2 of triple therapy were predictive of SVR12. Moreover, this week 2 viral load assessment allowed the detection of early viral breakthrough. Ribavirin still plays a major role in current and future therapeutic strategies. Ribavirin monitoring could also be important during future multi-drug therapy that could be associated with drug interactions

Introdução: Informações de vida real relativas ao tratamento da hepatite C crônica com interferon peguilado (Peg-IFN) e ribavirina (RBV) servem para mensurar sua efetividade nos anos em que o seu uso foi amplamente difundido, além de auxiliar em tomada de decisões futuras. Objetivos: Avaliar a taxa de resposta virológica sustentada (RVS) nos pacientes com hepatite C crônica tratados com Peg-IFN e RBV, fora de protocolos de pesquisa. Determinar os fatores associados à obtenção de RVS, frequência e causas de interrupção precoce do tratamento e de redução ou interrupção temporária dos medicamentos. Métodos: Estudo observacional retrospectivo de uma coorte de pacientes de dois ambulatórios de um centro de referência brasileiro localizado em São Paulo/SP. Resultados: Dos 440 indivíduos analisados, 182 apresentaram RVS (prevalência: 41,4% [IC95%: 36,7 - 46,1]). A RVS ocorreu em 33,5% (104/310) dos pacientes com genótipo 1 e em 53,8% (7/13) e 60,5% (69/114) daqueles com genótipos 2 e 3, respectivamente. Após análise multivariada, a RVS esteve positiva e independentemente associada à presença dos genótipos 2 ou 3 (p < 0,001), ausência de esteatose (p = 0,025) e de tratamento prévio (p = 0,038). Os eventos adversos mais frequentemente relacionados à redução de dose ou suspensão temporária de Peg-IFN ou RBV foram anemia (15,6%) e plaquetopenia (3,9%). Dos eventos adversos que levaram 79 (18%) pacientes a interromper o tratamento precocemente, distúrbios psiquiátricos (15,1%) e anemia (13,9%) foram os mais frequentes. Conclusões: A taxa de RVS foi semelhante àquela obtida em outros estudos de vida real. A RVS esteve independentemente associada à: presença dos genótipos 2 ou 3, ausência de esteatose e ausência de tratamento prévio. As principais causas de redução de dose dos medicamentos foram anemia e plaquetopenia e de interrupção precoce do tratamento, desordens psiquiátricas, e citopenias / Introduction: The association of pegylated interferon (Peg-IFN) and ribavirin (RBV) was considered a first line treatment for chronic hepatitis C during the past decade. Routine clinical practice information and real-life treatment outcomes can guide future therapeutic strategies for this group of patients. Objectives: The main objective of our study was to determine the sustained virological response (SVR) rate under current clinical practice. The secondary objectives were: 1- to investigate the factors that before or during treatment could predict SVR 2- to identify the causes of treatment interruption. Method: This cross-sectional study enrolled hepatitis C patients treated with Peg-IFN and RBV in a tertiary outpatient clinic setting. Data were collected retrospectively on patients treated for hepatitis C. Demographics, treatment outcomes and potential predictors of outcome were recorded. Results: Among the 440 analyzed patients 182 achieved SVR (prevalence: 41.4% [95% CI: 36.7 to 46.1]). On an intention-to-treat basis, SVR rates were 33.5% (104/310), 53.8% (7/13) and 60.5% (69/114) in genotypes 1, 2 and 3 respectively. After multivariate analysis, SVR was independently associated with presence of genotypes 2 or 3 (p < 0.001), no hepatic steatosis (p=0.025) and absence of prior treatment (p = 0.038). Anemia (15.6%) and thrombocytopenia (3.9%) were the most frequent causes of treatment dose reduction. Among the adverse events that led 79 patients into treatment discontinuation, the most frequent were psychiatric complications (15.1%) and anemia (13.9%). Conclusion: In our cohort, the treatment success rate (SVR) was similar to that observed in other in real-life setting studies. The SVR was independently associated with: presence of genotypes 2 or 3, no hepatic steatosis and absence of prior treatment. Psychiatric disorders and anemia were the main causes of premature treatment discontinuation

Introdução: Informações de vida real relativas ao tratamento da hepatite C crônica com interferon peguilado (Peg-IFN) e ribavirina (RBV) servem para mensurar sua efetividade nos anos em que o seu uso foi amplamente difundido, além de auxiliar em tomada de decisões futuras. Objetivos: Avaliar a taxa de resposta virológica sustentada (RVS) nos pacientes com hepatite C crônica tratados com Peg-IFN e RBV, fora de protocolos de pesquisa. Determinar os fatores associados à obtenção de RVS, frequência e causas de interrupção precoce do tratamento e de redução ou interrupção temporária dos medicamentos. Métodos: Estudo observacional retrospectivo de uma coorte de pacientes de dois ambulatórios de um centro de referência brasileiro localizado em São Paulo/SP. Resultados: Dos 440 indivíduos analisados, 182 apresentaram RVS (prevalência: 41,4% [IC95%: 36,7 - 46,1]). A RVS ocorreu em 33,5% (104/310) dos pacientes com genótipo 1 e em 53,8% (7/13) e 60,5% (69/114) daqueles com genótipos 2 e 3, respectivamente. Após análise multivariada, a RVS esteve positiva e independentemente associada à presença dos genótipos 2 ou 3 (p < 0,001), ausência de esteatose (p = 0,025) e de tratamento prévio (p = 0,038). Os eventos adversos mais frequentemente relacionados à redução de dose ou suspensão temporária de Peg-IFN ou RBV foram anemia (15,6%) e plaquetopenia (3,9%). Dos eventos adversos que levaram 79 (18%) pacientes a interromper o tratamento precocemente, distúrbios psiquiátricos (15,1%) e anemia (13,9%) foram os mais frequentes. Conclusões: A taxa de RVS foi semelhante àquela obtida em outros estudos de vida real. A RVS esteve independentemente associada à: presença dos genótipos 2 ou 3, ausência de esteatose e ausência de tratamento prévio. As principais causas de redução de dose dos medicamentos foram anemia e plaquetopenia e de interrupção precoce do tratamento, desordens psiquiátricas, e citopenias / Introduction: The association of pegylated interferon (Peg-IFN) and ribavirin (RBV) was considered a first line treatment for chronic hepatitis C during the past decade. Routine clinical practice information and real-life treatment outcomes can guide future therapeutic strategies for this group of patients. Objectives: The main objective of our study was to determine the sustained virological response (SVR) rate under current clinical practice. The secondary objectives were: 1- to investigate the factors that before or during treatment could predict SVR 2- to identify the causes of treatment interruption. Method: This cross-sectional study enrolled hepatitis C patients treated with Peg-IFN and RBV in a tertiary outpatient clinic setting. Data were collected retrospectively on patients treated for hepatitis C. Demographics, treatment outcomes and potential predictors of outcome were recorded. Results: Among the 440 analyzed patients 182 achieved SVR (prevalence: 41.4% [95% CI: 36.7 to 46.1]). On an intention-to-treat basis, SVR rates were 33.5% (104/310), 53.8% (7/13) and 60.5% (69/114) in genotypes 1, 2 and 3 respectively. After multivariate analysis, SVR was independently associated with presence of genotypes 2 or 3 (p < 0.001), no hepatic steatosis (p=0.025) and absence of prior treatment (p = 0.038). Anemia (15.6%) and thrombocytopenia (3.9%) were the most frequent causes of treatment dose reduction. Among the adverse events that led 79 patients into treatment discontinuation, the most frequent were psychiatric complications (15.1%) and anemia (13.9%). Conclusion: In our cohort, the treatment success rate (SVR) was similar to that observed in other in real-life setting studies. The SVR was independently associated with: presence of genotypes 2 or 3, no hepatic steatosis and absence of prior treatment. Psychiatric disorders and anemia were the main causes of premature treatment discontinuation

Abbreviations ALT – alanine aminotransferase AST – aspartate aminotransferase BMI – body mass index CHC – chronic hepatitis C EBR – early biochemical response EHR – early histological response EIA – enzyme immunoassay EVR – early virological response HAI – hepatitis activity index HCV – hepatitis C virus HCV RNA - hepatitis C virus ribonucleic acid Helicobacter spp. – Helicobacter species H. pylory – Helicobacter pylori IFN – interferon α-2b PCR – polymerase chain reaction PEG IFN – peginterferon RBV – ribavirin SVR – sustained virological response SBR – Sustained biochemical response INTRODUCTION Chronic hepatitis due to hepatitis C virus (HCV) infection is a worldwide disease representing a serious public health problem. Chronic hepatitis C (CHC) infection affects nearly 170-200 million people worldwide. The prevalence of anti-HCV at this time in the general adult population of Lithuania is nearly 50 thousand (0.9%). Without effective treatment strategies, hepatitis C - related morbidity and mortality is expected to increase nearly 3-fold by the year 2015. Current hepatitis C therapies are aimed at achieving eradication of HCV infection as a means of delaying progression to end-stage liver disease and preventing the development of hepatocellular carcinoma. The treatment options include interferon (IFN), ribavirin (RBV) and peg interferon’s a-2a and a-2b (PEG IFN). IFN-based regiments for the treatment of CHC have become increasingly effective and are to eradicate virus... [to full text]

Hepatitis C is an infectious disease with an overall prevalence of 2.2% and Brazil 1.5%. Drug therapy consists of interferon α, peginterferon α and ribavirin. It is filled with aggressive treatment of adverse reactions, hence the importance of pharmacovigilance as an additional tool in monitoring the treatment and rational use of medicines. Thus, adverse reactions occurred in patients with chronic hepatitis C who were treated with peginterferon and ribavirin, were identified and quantified through a retrospective and observational study. The most prevalent reactions observed in 46 patients in the study, were: fatigue (84.8%), fever (82.6%), loss weight (80.4%), irritability (73.9%) and body pain (71.7%). Most reactions were classified as mild (95.1%), while like moderate, 4.5% and as serious, 0.4%. The adverse reactions caused the therapeutic management in 11 patients (23.9%) where it was, dose reduction for 7 patients (15.2%), temporary discontinuation of the treatment for 5 patients (10.9%) and permanent discontinuation for 3 patients (6.5%). Eleven potential drug interactions were identified in 9 patients (19.6 %), where the most frequent was among peginterferon α 2a and captopril (45.4%). Said that, it s noticed that the treatment for chronic hepatitis C is marked for many adverse reactions with variable severity, that may interfere on patient s quality of life or in compliance of the treatment and this may be exacerbated by potential drug interactions. Additionally we evaluated the pharmacovigilance system of the Hepatology service of the University Hospital of the Federal University of Sergipe of patients registered from January 2007 to July 2009. / A hepatite C é uma doença infecciosa, com prevalência global de 2,2% e no Brasil de 1,5%. A terapêutica medicamentosa é constituída pelo interferon α, peginterferon α e a ribavirina. É um tratamento agressivo repleto de reações adversas, daí a importância da farmacovigilância como ferramenta adicional no acompanhamento do tratamento e do uso racional dos medicamentos. Assim, Reações adversas ocorridas em pacientes com hepatite C crônica tratados com peginterferon e ribavirina foram identificadas, quantificadas e classificadas através de um estudo retrospectivo e observacional. As reações mais prevalentes observadas nos 46 pacientes do estudo foram: astenia (84,8%), febre (82,6%), perda de peso (80,4%), irritabilidade (73,9%) e dor no corpo (71,7%). A maior parte das reações foi classificada como leve (95,1%), enquanto que como moderada, 4,5% e como graves, 0,4%. As reações adversas acarretaram o remanejamento terapêutico de 9 pacientes (19,6%) nos quais houve, redução da dose para 7 (15,2%), interrupção temporária do tratamento para 5 (10,9%) e interrupção permanente para 3 pacientes (6,5%). Onze interações medicamentosas potenciais foram identificadas em 9 pacientes (19,6 %), nos quais a mais freqüente foi entre o peginterferon α 2a e o captopril (45,4%). Diante do exposto, observa-se que o tratamento para hepatite C crônica é marcado por várias reações adversas, de gravidade variável, que podem interferir na qualidade de vida do paciente ou no cumprimento do tratamento e que isto pode ser agravado pelas potenciais interações medicamentosas. Adicionalmente se avaliou o sistema de farmacovigilância do Ambulatório de Hepatologia do Hospital Universitário da Universidade Federal de Sergipe dos pacientes cadastrados de janeiro de 2007 a julho de 2009.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Chronic hepatitis C has been associated with non-organ-specific autoantibodies (NOSA) production. Despite of increasing number of researches about this subject, there is no agreement among the authors of which autoantibodies are produced during combinated therapy of interferon and ribavirin or the clinical relevance of NOSA in patientâs organism. Our aim was to evaluate the profile of NOSA in patients with chronic hepatitis C who attended to Walter CantÃdio Hospital (HUWC) and received combinated antiviral therapy (interferon-ribavirin). A total of 34 patients with hepatitis C were studied. Anti-nuclear antibody (ANA), anti-smooth muscle antibody (SMA), anti-liver/kidney microsomal antibody type 1 (LKM-1) and anti-mitochondrial antibody (AMA) were detected by indirect immunofluorescence. The presence of NOSA was related to clinical and epidemiological variables and to the outcome of antiviral combination therapy with interferon-alfa and ribavirin. Patients were classified as nonresponders, relapsers or long-term responders depending on the outcome of treatment. In our study, before therapy, 23 patients were NOSA positive (SMA was detected in 6 patients, SMA and AMA in 10 and SMA, AMA and ANA in 7). On the 24th week of treatment, 24 patientes were NOSA positive (SMA was detected in 4 patients, SMA and AMA in 10, ANA and SMA in 1, ANA and AMA in 1 and SMA, AMA and ANA in 8). NOSA behavior did not show significant variation during treatment. The overall rate of long-term response was 26,5% (9/34). Long-term response occurred in 17,4% (4/23) of NOSA positive patients and 45,5% (5/11) of NOSA negative patients. Positivity of autoantibodies was not associated with gender, age, viral genotype or aminotransferase levels. In conclusion, ANA was the only NOSA associated with treatment outcome. The absence of NOSA might indicate a significantly higher chance for viral clearance in response to combination therapy for chronic hepatitis C infection. / A hepatite crÃnica pelo vÃrus C tem sido associada à produÃÃo de autoanticorpos nÃo-ÃrgÃo especÃficos (NOSA). Apesar do aumento do nÃmero de pesquisas nessa Ãrea, ainda nÃo existe um consenso entre quais autoanticorpos tÃm seus nÃveis elevados devido ao tratamento combinado de interferon e ribavirina, nem sua influÃncia no desfecho do mesmo ou a relevÃncia clÃnica da presenÃa desses autoanticorpos no organismo do pacientes. O objetivo do presente estudo foi avaliar o perfil de NOSA em pacientes com hepatite C crÃnica atendidos no Hospital UniversitÃrio Walter CantÃdio (HUWC) e submetidos à terapia combinada de interferon-alfa e ribavirina. Para isso, um total de 34 pacientes com hepatite C foram estudados. Os anticorpos anti-nuclear (FAN), anti-mÃsculo liso (SMA), anti-microssomal de fÃgado e rim do tipo 1 (LKM-1) e anti-mitocÃndria (AMA) foram detectados atravÃs de imunofluorescÃncia indireta. A presenÃa de NOSA foi relacionada a variÃveis clÃnicas e epidemiolÃgicas e à resposta ao tratamento. Os pacientes foram classificados, em relaÃÃo à resposta ao tratamento, como nÃo respondedores, recidivantes ou respondedores (resposta virolÃgica sustentada). Em nosso estudo, 23 pacientes foram NOSA reagentes (SMA foi detectado em 6 pacientes, SMA e AMA em 10 e SMA, AMA e FAN em 7). Na 24 semana de tratamento, 24 pacientes foram NOSA reagentes (SMA foi detectado em 4 pacientes, SMA e AMA em 10, FAN e SMA em 1, FAN e AMA em 1 e SMA, AMA e FAN em 8). A variaÃÃo dos tÃtulos dos autoanticorpos durante o tratamento nÃo foi significativa. O percentual total de respondedores foi de 26,5% (9/34). A resposta virolÃgica sustentada foi obtida por 17,4% (4/23) dos pacientes NOSA reagentes e 45,5% (5/11) dos pacientes nÃo reagentes para NOSA. A presenÃa de autoanticorpos nÃo foi associada a gÃnero, idade, genÃtipo viral ou nÃveis de transaminases. Conclui-se que o FAN foi o Ãnico NOSA significativamente associado à resposta à terapia. A ausÃncia de NOSA indica uma tendÃncia à resposta virolÃgica sustentada no tratamento da hepatite C crÃnica.

Mon travail de thèse s'est focalisé sur la machinerie enzymatique impliquée dans la réplication du génome ARN du Syndrome Respiratoire Aigu Sévère-Coronavirus (SRAS-CoV). J'ai montré in vitro que l'activité ARN polymérase ARN-dépendante (RdRp) portée par nsp12 nécessite le complexe nsp7/nsp8, qui agit comme facteur de processivité. Grâce à ce complexe polymérase hautement actif, j'ai pu en suite étudier le mécanisme de "proofreading" (correction d'épreuve) associé aux coronavirus, pour lequel seulement des preuves indirectes avaient été assemblées. En effet, les coronavirus codent pour une activité exonucléase 3'-5' (nsp14-ExoN) qui lorsqu'elle est absente, entraine 14-fois plus d'erreurs de réplication en contexte cellulaire. In vitro, nous avons pu montrer que nsp14-ExoN est capable d'exciser l'ARN double brin ainsi qu'un nucléotide mésapparié en 3' de l'ARN en cours d'élongation. J'ai pu apporter pour la première fois une preuve directe de l'existence d'un système de réparation des erreurs au cours de la synthèse, mené par le complexe nsp7/nsp8/nsp12/nsp14. En effet, le complexe nsp7/nsp8/nsp12 ralentit jusqu'à 30-fois quand il rajoute une base mésappariée. Par sequençage, nous avons pu montrer la réparation de cette base mésappariée en presence de nsp14. Enfin, grâce à ce système in vitro nous avons une base pour comprendre l'inefficacité de la ribavirine sur des patients atteints du SRAS. En effet, la ribavirine, incorporée par le complexe polymérase, serait également excisée par nsp14, annihilant tout potentiel effet mutagenique. En conclusion, ce système va permettre de guider le développement d'antiviraux de type nucleoside analogues contre les coronavirus. / This work focused on the enzymatic machinery involved in Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) RNA replication and transcription. Firstly, I established a robust in vitro polymerase assay with the canonical SARS-CoV RNA-dependent RNA polymerase (RdRp) nsp12. I showed that nsp12, in order to engage processive RNA synthesis, needs two viral proteins, i.e. nsp7 and nsp8. This nsp7/nsp8 complex not only activates nsp12-RdRp, but also acts as a processivity factor. Thus, using this processive polymerase complex, I could investigate SARS-CoV proofreading for which only indirect evidences were reported. Indeed, coronaviruses encode for a 3'-5' exonuclease (nsp14-ExoN), putatively involved in a mechanism that proofreads coronavirus RNA during viral replication. We first showed in vitro that nsp14-ExoN, which is stimulated by nsp10, is able to excise specifically dsRNA as well as all primer/templates bearing a 3' mismatch on the primer. Moreover, we could confirm by sequencing that a RNA 3' mismatch was indeed corrected in vitro by the nsp7/nsp8/nsp12/nsp14 complex. We provide for the first time direct evidence that nsp14-ExoN, in coordination with the polymerase complex, is able to proofread RNA. Interestingly, using this in vitro system we found an element that could possibly explain the inefficacy of ribavirin therapeutic treatment on SARS-patients: ribavirin, which is incorporated by the SARS-CoV polymerase complex, would also be excised by nsp14. In conclusion, this system will drive future development of antivirals, particularly of the nucleoside analogue type, against coronaviruses.

Resumo: A composição de quasiespécies do vírus da Hepatite C (HCV) pode ter implicações importantes com relação à persistência viral e à resposta a terapia baseada em Interferon. A região NS5A completa foi analisada para avaliar se a composição de quasiespécies do HCV 1a/1b está relacionada à resposta ao tratamento combinado de interferon peguilado (PEGIFN) e ribavirina. Seiscentos e noventa seqüências correspondentes a região não estrutural 5A (NS5A) completa foram geradas a partir de amostras coletadas antes, durante a após a administração da terapia de pacientes respondedores, não respondedores e respondedores ao final do tratamento. Este estudo apresenta evidências de que a homogeneidade da composição de quasiespécies, e a baixa complexidade e diversidade da região NS5A em amostras préterapia estão associados à resposta virológica sustentada. Portanto, a alta diversidade e complexidade de quasiespécies podem fornecer ao vírus melhores oportunidades de evadir a terapia antiviral. Análises filogenéticas não demonstraram o agrupamento das seqüências de acordo os padrões específicos de resposta ao tratamento. Contudo, o agrupamento distinto de seqüências pré e pós-terapia foi observado, sugerindo que um processo adaptativo ocorreu durante o período analisado. Adicionalmente, a dinâmica evolutiva da composição de quasiespécies demonstrou estar sob pressão seletiva purificadora ou purificadora relaxada, o que é condizente com a população de quasiespécies diversificada no pré-terapia, seguida de um aumento em freqüência de quasiespécies predominantes nas amostras pós-tratamento, provavelmente devido a conferirem alguma vantagem ao vírus. Estes resultados sugerem que a diversidade de quasiespécies da região NS5A pode ser importante para o entendimento dos mecanismos de baixa resposta virológica sustentada em pacientes com Hepatite C crônica / Abstract: The quasispecies composition of Hepatitis C virus (HCV) could have important implications with regard to viral persistence and response to interferon-based therapy. The complete NS5A was analyzed to evaluate whether the composition of NS5A quasispecies of HCV 1a/1b is related to responsiveness to combined interferon pegylated (PEG-IFN) and ribavirin therapy. Six hundred and ninety full-length NS5A sequences were generated from samples collected before, during and after treatment from virological sustained responder, non-responder and the end-of-treatment responder patients. This study provides evidence that homogeneity of quasispecies composition, low diversity and less complexity of the NS5A region pre-therapy are associated with viral clearance. Therefore, higher diversity and complexity of quasispecies could offer the virus a better opportunity of evading anti-viral therapy. Phylogenetic relationships concerning complete NS5A sequences obtained from patients did not demonstrate clustering associated with specific response patterns. However, distinctive clustering of pre/post-therapy sequences was observed, suggesting that an evolutionary process occurred during the time course examined. In addition, the evolution of quasispecies over time was subjected to purifying or relaxed purifying selection. This could explain the initial diversified composition of quasispecies at baseline, followed by an increase in the frequency of a predominant quasispecies in 'after treatment' samples of non-responders and end-of-treatment responders, probably because it offers some advantage for the virus. These results suggest that quasispecies diversity of the NS5A region could be important for elucidating the mechanism underlying treatment failure in patients infected with chronic hepatitis C / Orientador: Paula Rahal / Coorientador: Isabel Maria Vicente Guedes Carvalho-Mello / Banca: Camila Malta Romano / Banca: Jonny Yokosawa / Banca: Maurício Lacerda Nogueira / Banca: Fátima Pereira de Souza / Doutor