Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-22T14:32:37Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Gabriela de Souza da Silva.pdf: 747981 bytes, checksum: c098f507cfc2f2c6e90b43f8f1a81fec (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-22T14:32:51Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Gabriela de Souza da Silva.pdf: 747981 bytes, checksum: c098f507cfc2f2c6e90b43f8f1a81fec (MD5) / Made available in DSpace on 2016-09-22T14:32:51Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Gabriela de Souza da Silva.pdf: 747981 bytes, checksum: c098f507cfc2f2c6e90b43f8f1a81fec (MD5) Previous issue date: 2016-04-27 / O fluconazol é um fármaco antifúngico de amplo espectro muito utilizado pela população. Contudo, são poucos os registros na literatura que imputem a segurança do uso do fluconazol e tampouco a dose mínima capaz de induzir lesões no material genético do paciente. Embora a Agência Nacional de Vigilância Sanitária (ANVISA) exija testes de genotoxicidade para admissão de novos medicamentos, os que têm seu registro expedido antes do ano de 2006 não trazem essa obrigatoriedade, incluindo-se aqui o fluconazol. Portanto, é de extrema importância estudos sobre a avaliação genotoxicológica de fármacos, principalmente os que são tão conhecidos e utilizados como o fluconazol. O presente estudo investigou os efeitos genotóxicos da cápsula e do princípio ativo do fluconazol através do teste cometa e do teste de proliferação celular e também os efeitos citotóxicos da cápsula e do princípio ativo do fluconazol através do teste de viabilidade celular. As concentrações testadas da cápsula e do princípio ativo do fluconazol foram 6, 12, 30, 60 e 120 μg/mL. Todos os ensaios foram realizados em triplicatas e o peróxido foi utilizado como controle positivo e tampão PBS 7,4 como controle negativo. Todas as concentrações testadas da cápsula do Fluconazol (6, 12, 30, 60 e 120 μg/mL) demonstraram ser capazes de interferir negativamente no processo de proliferação celular, diminuindo o número de células proliferadas em todas as concentrações testadas, quando comparadas ao controle negativo. Já no caso do princípio ativo do Fluconazol, apenas as três últimas concentrações (30, 60 e 120 μg/mL) interferiram negativamente no processo de proliferação celular. Na avaliação do índice de dano ao DNA (teste cometa), foi observado dano à estrutura de DNA nas concentrações 60 e 120 μg/mL da cápsula do Fluconazol. Já as concentrações testadas com o princípio ativo do Fluconazol não foi observado dano à estrutura de DNA. Foi verificado que a cápsula do Fluconazol possui atividade citotóxica sobre leucócitos humanos nas duas maiores concentrações testadas (60 e 120 μg/mL) de forma concentração dependente. Porém, o princípio ativo do Fluconazol não demonstrou ser citotóxico nas concentrações testadas (6 12, 30, 60 e 120 μg/mL). / Fluconazole is a broad-spectrum antifungal drug widely used by the population. However, there are few reports in the literature that imput the safety of the use of fluconazole and either the minimum dose capable of inducing lesions in the genetic material of the patient. Although the National Health Surveillance Agency (ANVISA) requires genotoxicity tests for admission of new medicines, which have their registration issued before 2006 do not bring this obligation, including here fluconazole. Therefore, it is extremely important genotoxicológica studies on the evaluation of drugs, especially such that are known and used as fluconazole. This study investigated the genotoxic effects of the capsule and the active principle of fluconazole by the comet test and the cell proliferation test and also the cytotoxic effects of the capsule and the active principle of fluconazole by cell viability test. The tested concentrations of the capsule and the active principle of fluconazole were 6, 12, 30, 60, and 120 mg/mL. All assays were performed in triplicate and peroxide was used as a positive control and PBS buffer as negative control 7.4. All tested concentrations of fluconazole capsule (6, 12, 30, 60 and 120 μg/mL) were capable of induce a negative interference in the cellular proliferation process, reducing the number of proliferating cells at all concentrations tested when compared to the negative control. In the case of the active ingredient, the fluconazole, only the last three concentrations (30, 60, and 120 μg/mL) were able to decrease the cellular proliferation process. In the assessment of DNA damage index (comet assay), there was observed damage of fluconazole capsule on the DNA structure at concentrations 60 and 120 μg/mL. On the other hand, the concentrations tested with the active ingredient of Fluconazole was not observed damage to the DNA structure. Fluconazole has been found that the capsule has a cytotoxic activity on human leukocytes at the two highest concentrations tested (60 and 120 μg/mL) as a concentration-dependent manner. However, the active ingredient of Fluconazole not induced any cytotoxic effect at the concentrations tested (6, 12, 30, 60, and 120 μg/mL).

Candida glabrata, geralmente comensal, surgiu como uma causa comum de infecções fúngicas graves com risco de morte. Dada a resistência crescente aos azóis, a orientação recente é utilizar as equinocandinas como a primeira escolha para o tratamento de infecções sistêmicas por C. glabrata. No entanto, esta é a primeira espécie de Candida que foi detectada com resistência significativa às equinocandinas. Esta levedura é capaz de colonizar tecidos do hospedeiro, bem como superfícies abióticas (catéteres, próteses) onde desenvolve um crescimento em multicamadas caracterizado como biofilme. A natureza da estrutura do biofilme e os atributos fisiológicos a ele conferidos resultam em uma resistência inerente a agentes antimicrobianos, impactando negativamente na saúde do paciente. Este estudo teve como objetivo a indução in vitro de resistência à anidulafungina em células planctônicas e sésseis de sete cepas sensíveis de C. glabrata, além da verificação do desenvolvimento de resistência cruzada com fluconazol. A indução de resistência foi realizada submetendo as cepas a concentrações sub-inibitórias do antifúngico. A determinação de concentração inibitória mínima através de microdiluição em caldo foi realizada previamente e posteriormente à indução de resistência e, também, para a verificação de resistência cruzada com fluconazol. O método de indução de resistência resultou em cepas fortemente resistentes à anidulafungina, com concentrações inibitórias mínimas variando de 1 a 2 μg/mL. Antes da indução da resistência, as formas planctônica e séssil das cepas eram todas sensíveis ou sensíveis dose-dependente ao fluconazol. Após a indução de resistência à anidulafungina esta sensibilidade ao fluconazol não foi mantida, tornando as cepas resistentes a este antifúngico. Clinicamente, esta resistência cruzada poderia implicar em falha terapêutica ao utilizar o fluconazol em pacientes previamente expostos a concentrações sub-inibitórias de anidulafungina por longos períodos. / Candida glabrata, usually commensal, has emerged as a common cause of serious life threatening fungal infections. Given the increasing resistance to azoles, the recent guidance is to use echinocandins as the first choice for the treatment of systemic infections by C. glabrata. However, C. glabrata is the first species of Candida that has been detected with significant resistance to echinocandins. This yeast is able to colonize host tissues as well as abiotic surfaces (catheters, prostheses) where it develops a multi-layer growth characterized as biofilm. The nature of the biofilm structure and the physiological attributes conferred on it, result in an inherent resistance to antimicrobial agents, negatively impacting the patient's health. This study aimed to induce in vitro resistance to anidulafungin in planktonic and sessile cells of seven sensitive C. glabrata strains, as well as to verify the development of cross-resistance with fluconazole. The induction of resistance was performed by subjecting the isolates to sub-inhibitory concentrations of the antifungal. The determination of minimum inhibitory concentration by broth microdilution was performed before and after induction of resistance and also for fluconazole cross-resistance verification. The resistance induction test resulted in strains strongly resistant to anidulafungin, with minimum inibitory concentrations ranging from 1 to 2 μg mL-1. Prior to induction of resistance, the planktonic and sessile forms of the strains were all sensitive or sensitive dose-dependent to fluconazole. However, after the induction of resistance to anidulafungin, this sensitivity to fluconazole was not maintained, making the strains resistant to this antifungal. Clinically, this cross-resistance could lead to therapeutic failure when using fluconazole in patients previously exposed to sub-inhibitory concentrations of anidulafungin for long periods.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 128-147).

Candida glabrata, geralmente comensal, surgiu como uma causa comum de infecções fúngicas graves com risco de morte. Dada a resistência crescente aos azóis, a orientação recente é utilizar as equinocandinas como a primeira escolha para o tratamento de infecções sistêmicas por C. glabrata. No entanto, esta é a primeira espécie de Candida que foi detectada com resistência significativa às equinocandinas. Esta levedura é capaz de colonizar tecidos do hospedeiro, bem como superfícies abióticas (catéteres, próteses) onde desenvolve um crescimento em multicamadas caracterizado como biofilme. A natureza da estrutura do biofilme e os atributos fisiológicos a ele conferidos resultam em uma resistência inerente a agentes antimicrobianos, impactando negativamente na saúde do paciente. Este estudo teve como objetivo a indução in vitro de resistência à anidulafungina em células planctônicas e sésseis de sete cepas sensíveis de C. glabrata, além da verificação do desenvolvimento de resistência cruzada com fluconazol. A indução de resistência foi realizada submetendo as cepas a concentrações sub-inibitórias do antifúngico. A determinação de concentração inibitória mínima através de microdiluição em caldo foi realizada previamente e posteriormente à indução de resistência e, também, para a verificação de resistência cruzada com fluconazol. O método de indução de resistência resultou em cepas fortemente resistentes à anidulafungina, com concentrações inibitórias mínimas variando de 1 a 2 μg/mL. Antes da indução da resistência, as formas planctônica e séssil das cepas eram todas sensíveis ou sensíveis dose-dependente ao fluconazol. Após a indução de resistência à anidulafungina esta sensibilidade ao fluconazol não foi mantida, tornando as cepas resistentes a este antifúngico. Clinicamente, esta resistência cruzada poderia implicar em falha terapêutica ao utilizar o fluconazol em pacientes previamente expostos a concentrações sub-inibitórias de anidulafungina por longos períodos. / Candida glabrata, usually commensal, has emerged as a common cause of serious life threatening fungal infections. Given the increasing resistance to azoles, the recent guidance is to use echinocandins as the first choice for the treatment of systemic infections by C. glabrata. However, C. glabrata is the first species of Candida that has been detected with significant resistance to echinocandins. This yeast is able to colonize host tissues as well as abiotic surfaces (catheters, prostheses) where it develops a multi-layer growth characterized as biofilm. The nature of the biofilm structure and the physiological attributes conferred on it, result in an inherent resistance to antimicrobial agents, negatively impacting the patient's health. This study aimed to induce in vitro resistance to anidulafungin in planktonic and sessile cells of seven sensitive C. glabrata strains, as well as to verify the development of cross-resistance with fluconazole. The induction of resistance was performed by subjecting the isolates to sub-inhibitory concentrations of the antifungal. The determination of minimum inhibitory concentration by broth microdilution was performed before and after induction of resistance and also for fluconazole cross-resistance verification. The resistance induction test resulted in strains strongly resistant to anidulafungin, with minimum inibitory concentrations ranging from 1 to 2 μg mL-1. Prior to induction of resistance, the planktonic and sessile forms of the strains were all sensitive or sensitive dose-dependent to fluconazole. However, after the induction of resistance to anidulafungin, this sensitivity to fluconazole was not maintained, making the strains resistant to this antifungal. Clinically, this cross-resistance could lead to therapeutic failure when using fluconazole in patients previously exposed to sub-inhibitory concentrations of anidulafungin for long periods.

Candida glabrata, geralmente comensal, surgiu como uma causa comum de infecções fúngicas graves com risco de morte. Dada a resistência crescente aos azóis, a orientação recente é utilizar as equinocandinas como a primeira escolha para o tratamento de infecções sistêmicas por C. glabrata. No entanto, esta é a primeira espécie de Candida que foi detectada com resistência significativa às equinocandinas. Esta levedura é capaz de colonizar tecidos do hospedeiro, bem como superfícies abióticas (catéteres, próteses) onde desenvolve um crescimento em multicamadas caracterizado como biofilme. A natureza da estrutura do biofilme e os atributos fisiológicos a ele conferidos resultam em uma resistência inerente a agentes antimicrobianos, impactando negativamente na saúde do paciente. Este estudo teve como objetivo a indução in vitro de resistência à anidulafungina em células planctônicas e sésseis de sete cepas sensíveis de C. glabrata, além da verificação do desenvolvimento de resistência cruzada com fluconazol. A indução de resistência foi realizada submetendo as cepas a concentrações sub-inibitórias do antifúngico. A determinação de concentração inibitória mínima através de microdiluição em caldo foi realizada previamente e posteriormente à indução de resistência e, também, para a verificação de resistência cruzada com fluconazol. O método de indução de resistência resultou em cepas fortemente resistentes à anidulafungina, com concentrações inibitórias mínimas variando de 1 a 2 μg/mL. Antes da indução da resistência, as formas planctônica e séssil das cepas eram todas sensíveis ou sensíveis dose-dependente ao fluconazol. Após a indução de resistência à anidulafungina esta sensibilidade ao fluconazol não foi mantida, tornando as cepas resistentes a este antifúngico. Clinicamente, esta resistência cruzada poderia implicar em falha terapêutica ao utilizar o fluconazol em pacientes previamente expostos a concentrações sub-inibitórias de anidulafungina por longos períodos. / Candida glabrata, usually commensal, has emerged as a common cause of serious life threatening fungal infections. Given the increasing resistance to azoles, the recent guidance is to use echinocandins as the first choice for the treatment of systemic infections by C. glabrata. However, C. glabrata is the first species of Candida that has been detected with significant resistance to echinocandins. This yeast is able to colonize host tissues as well as abiotic surfaces (catheters, prostheses) where it develops a multi-layer growth characterized as biofilm. The nature of the biofilm structure and the physiological attributes conferred on it, result in an inherent resistance to antimicrobial agents, negatively impacting the patient's health. This study aimed to induce in vitro resistance to anidulafungin in planktonic and sessile cells of seven sensitive C. glabrata strains, as well as to verify the development of cross-resistance with fluconazole. The induction of resistance was performed by subjecting the isolates to sub-inhibitory concentrations of the antifungal. The determination of minimum inhibitory concentration by broth microdilution was performed before and after induction of resistance and also for fluconazole cross-resistance verification. The resistance induction test resulted in strains strongly resistant to anidulafungin, with minimum inibitory concentrations ranging from 1 to 2 μg mL-1. Prior to induction of resistance, the planktonic and sessile forms of the strains were all sensitive or sensitive dose-dependent to fluconazole. However, after the induction of resistance to anidulafungin, this sensitivity to fluconazole was not maintained, making the strains resistant to this antifungal. Clinically, this cross-resistance could lead to therapeutic failure when using fluconazole in patients previously exposed to sub-inhibitory concentrations of anidulafungin for long periods.

Class of 2015 Abstract / Objectives: To assess the efficacy of fluconazole prophylaxis in the prevention of coccidioidomycosis in the post-heart transplant patient and to identify risk factors for coccidioidomycosis infection. Methods: Heart transplant patients with ICD-9 code V42.1 from October 2001 to October 2013, were selected and electronic medical records were retrospectively reviewed for coccidioidomycosis history, Coccidiodes serologies, reason for transplantation, immunosuppressive drug therapy regimens, rejection treatment course, fluconazole dose, and demographics. Negative Coccidiodes serology results post transplantation relative to negative Coccidiodes serology results prior to transplantation will be determined using a Chi Square test. Risk factors for disease contraction will be analyzed using multivariate logistic regression. Results: Between October 2001 and October 2013, 244 patients received a heart transplant at this institution. Fourteen (5.7%) heart transplant recipients with a negative Coccidiodes serology pre-transplantation had a positive Coccidiodes serology post-transplantation. Nine (64.2%) of those recipients received antifungal prophylaxis (p=0.16). Risk factors for developing a positive Coccidiodes serology included using tacrolimus (p=0.05) and non-ischemic cardiomyopathy (p=0.04). Conclusions: Antifungal prophylaxis does not reduce the risk of developing a positive Coccidiodes serology after heart transplantation. Risk factors for developing a positive Coccidiodes serology include the use of tacrolimus and having non-ischemic cardiomyopathy prior to transplant.

Class of 2016 Abstract / Objectives: The purpose of this study was to evaluate if fluconazole 200 mg is an acceptable alternative to the fluconazole 400 mg for fungal prophylaxis in allogenic hematopoietic stem cell patients. Lower fluconazole doses will decrease cost of therapy and may reduce adverse events associated with higher doses. Methods: This study was a retrospective chart review conducted at the Arizona Cancer Center. A total of 58 patients qualified for the study. Primary endpoints were number of days on fluconazole 200 mg and type and number of fungal infections that occurred within 1 year post transplant. Results: Out of the fifty-eight patients who qualified for the study, only eight patients had a breakthrough fungal infection while on 200 mg (13.7%) after one year. Three of those eight were identified as having systemic fungal infections (5.2%). Conclusions: Fluconazole 200 mg is a reasonable low-cost and low side effect alternative to fluconazole 400 mg for antifungal prophylaxis in allogenic hematopoietic stem cell patients.

Philosophiae Doctor - PhD / Background: Oropharyngeal candidiasis, caused by the fungus Candida, is the most common opportunistic infection affecting the quality of life of immunocompromised patients. Fluconazole is widely used as the first line of treatment for fungal infections. However, the inappropriate and misguided use of the drug has led to the evolvement of fluconazole-resistant Candida organisms. This arising resistance resulted in the urgent need for the development of new antimicrobial drugs. The aim of the present study was to investigate the antifungal action of K21, a novel antimicrobial quarternary ammonium compound, on fluconazole-resistant Candida species.

Objectives: Candida glabrata is the second most common cause of oral candidiasis, second only to C. albicans. Incidence of antifungal resistance has shown a steady increase for C. glabrata. Iron has shown to modulate C. albicans pathogenesis and affect drug-susceptibility. Here, we assess the effect of iron on the growth, antifungal-susceptibility, biofilms, and cell wall of C. glabrata.Methods: Growth, minimal inhibitory concentration (MIC), and biofilm experiments were conducted using 96-well polystyrene plates. Yeast Nitrogen Base medium was used for growth experiments. Cultures of C. glabrata and C. albicans were grown over two nights in respective media containing varying iron concentrations. Rosewell Park Memorial Institute medium was used for MIC and biofilm experiments. Serial dilution was performed to obtain desired concentrations of antifungal drugs. For all experiments, growth was assessed at OD600nm over 24 hours using BioTek Synergy Multi Mode Reader. Paraformaldehyde treated cells and specific stains were used for cell wall studies. Results: Growth of C. glabrata declined significantly below 5μM iron, while C. albicans continued to grow at decreasing iron concentrations, up to 0.5μM. MIC experiments revealed 1.562μM, 1.562μM, and 4μM, as the MIC for Deferasirox, Nystatin, and Fluconazole, respectively. Drug synergy experiments revealed a 128-fold reduction in the amount of Nystatin and Fluconazole needed, with the addition of 1/8th of Deferasirox concentration. The biofilm experiments were inconclusive and the cell wall studies showed decreased levels of mannan, chitin, and an increased β-glucan exposure in high iron conditions. Conclusion: C. glabrata is more sensitive to alterations in environmental iron when compared to C. albicans. Drug synergy experiments underscore the importance of Deferasirox in lowering the MICs of Nystatin or Fluconazole. This can allow use of classical antifungals at lower doses, thereby limiting their side effects. Cell wall studies discuss the effect of iron on the virulence of the C. glabrata. / Oral Biology

Le recours aux prescriptions sans ou hors AMM, dû principalement au manque d’essais cliniques, est très largement répandu en pédiatrie en particulier chez le nouveau-né. Les prescriptions hors AMM constituent un facteur de risque dans la survenue des effets indésirables médicamenteux (EIM). La pharmacovigilance est indispensable mais insuffisante afin d’évaluer le rapport bénéfice/risque des traitements en particulier lorsque les prescriptions hors AMM sont fréquentes. En combinant plusieurs approches pharmacoépidémiologiques, méta-analytiques et pharmacométriques, ce travail de thèse a pour objectifs de décrire les prescriptions hors AMM en néonatologie et explorer des approches qui permettront d’aboutir à mieux estimer le rapport bénéfice/risque des médicaments pour les enfants. Nous avons confirmé que le taux de prescription hors AMM en néonatologie est important (46%). Même en absence de soins de réanimation, plus de deux tiers des nouveaux-nés reçoivent au moins un médicament hors AMM pendant leur hospitalisation. En utilisant une approche méta-analytique explorant le rapport bénéfice risque du fluconazole en néonatologie en fonction du risque de base dans chaque étude (taux d’infection <10%), nous avons montré que la prophylaxie ciblée sur le risque de base serait plus bénéfique que la prophylaxie systématique des infections fongiques invasives. Nos travaux en pharmacovigilance pédiatrique, ont montré que les deux méthodes de détection actives (collaboration entre pharmaciens et cliniciens et le « trigger tool »), utilisant les dossiers patients informatisés, sont faisables et plus efficaces que la notification spontanée / Many drugs used to treat children in hospitals are either not licensed for use in children or are prescribed outside the terms of their product license (off-label prescribing) because of the lack of clinical trials in this population, and practical difficulties to involve children in clinical research. Drugs used within the specifications of the product license should therefore be less likely to cause ADRs compared to drugs that are either unlicensed or off-label for use in children. With different approaches such as pharmacoepidemiologic, meta-analytic and pharmacometrics, we sought to improve the estimate of the benefit risk ratio of medicines used for treating children. Our observational prospective study in a neonatal unit demonstrated that the prescriptions of unlicensed or off-label drugs in neonatal unit is common (46%). We also used meta-analytic approach in order to estimate the benefit risk ratio of the prophylactic use of fluconazole as a function of the baseline risk. Our results suggest that “systematic fluconazole prophylaxis for all VLBW in NICUs is not warranted by the available evidence and should be adapted to the baseline risk. We also showed that active drug monitoring by using electronic patient files by targeting review chart with a trigger tool in neonates and with close collaboration between the pharmacovigilance center, pharmacologists, and clinicians was necessary and feasible for improving the detection of ADRs in children