FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Nos Ãltimos anos, a melhoria dos mÃtodos de diagnÃstico micolÃgico e as doenÃas imunossupressoras causaram grande impacto na incidÃncia das micoses profundas e oportunistas em todo o mundo, fato que impulsionou a realizaÃÃo de estudos de prospecÃÃo por novas drogas antifÃngicas. A histoplasmose à uma micose sistÃmica, causada pelo fungo Histoplasma capsulatum var. capsulatum que, em pacientes hÃgidos, pode mimetizar a tuberculose quanto aos aspectos clÃnicos e radiolÃgicos. Alguns casos de histoplasmose refratÃria ao tratamento com drogas antifÃngicas convencionais vÃm sendo descritos. O objetivo deste trabalho foi determinar o efeito inibitÃrio, in vitro, das drogas antituberculose: isoniazida (INH), pirazinamida (PZA) e etambutol (EMB); antifÃngicas: anfotericina B (AMB), fluconazol (FLC), itraconazol (ITC) e voriconazol (VRZ) e de anÃlogos quÃmicos da isoniazida frente a cepas de H. capsulatum (n=30), assim como avaliar o emprego de diferentes meios de cultura para a realizaÃÃo dos testes de sensibilidade. Para isso, primeiramente, foram repicadas 18 cepas de H. capsulatum em Ãgar BHI e utilizadas na realizaÃÃo dos testes de sensibilidade frente aos agentes antituberculose citados e anÃlogos quÃmicos da isoniazida, isolados e em combinaÃÃo com os antifÃngicos FLC, ITC e VRZ por meio da tÃcnica de macrodiluiÃÃo em caldo. Cada uma das 12 cepas restantes foi repicada em Ãgar batata dextrose, Ãgar BHI, Ãgar malte a 2% e Ãgar lactrimel e, analisadas ao microscÃpio Ãptico quanto a presenÃa de macroconÃdios tuberculados, sendo quantificados de acordo com os parÃmetros: (0-10); (10-50); (>50) macroconÃdios/campo. As culturas foram empregadas na determinaÃÃo dos testes de sensibilidade frente aos agentes antifÃngicos AMB, FLC, ITC e VRZ, utilizando a tÃcnica de microdiluiÃÃo em caldo. As drogas antituberculose inibiram o crescimento das cepas in vitro com valores de CIM de 0,04 a 0,30 mg/mL para INH, 0,55 a 3,13 mg/mL para PZA e 1,56 a 6,25 mg/mL para EMB. No tocante Ãs drogas antifÃngicas, todas as cepas foram sensÃveis apresentando valores de CIM que variaram de 0,0625 a 0,25 Âg/mL para AMB; 15,62 a 62,5 Âg/mL para FLC; 0,0039 a 0,0312 Âg/mL para ITC e 0,00156 a 0,25 Âg/mL para VRZ. Quanto Ãs combinaÃÃes entre os fÃrmacos antituberculose e os derivados azÃlicos, todas foram capazes de inibir o crescimento in vitro das cepas de H. capsulatum, sendo detectado sinergismo nas nove combinaÃÃes. Os anÃlogos da isoniazida apresentaram valores de CIM 2, 4, 8 e 15 vezes superior a atividade da droga antituberculose padrÃo. A partir da anÃlise micromorfolÃgica do fungo repicados nos quatro meios de cultura foi identificado a menor quantificaÃÃo (0-10 macroconÃdios/campo) para Ãgar batata, Ãgar BHI, Ãgar malte e Ãgar lactrimel, perfazendo um total de 11, 10, 6 e 7 cepas, respectivamente. O meio de cultura Ãgar malte foi o mais adequado para produÃÃo de macroconÃdios (10-50) e (>50), norteando um total de 6 cepas, seguido do meio lactrimel, 5 cepas. Em relaÃÃo a determinaÃÃo da CIM e o meio de cultura utilizado para o procedimento, observou-se que quando o inÃculo era proveniente de cepas em Ãgar malte e Ãgar BHI foi possÃvel a visualizaÃÃo da CIM em 11 cepas. Enquanto repiques feitos em Ãgar batata e lactrimel nÃo foi possÃvel determinar os valores de CIM para 8 e 5 cepas, respectivamente. Os resultados deste estudo fornecem dados adicionais sobre o potencial antifÃngico das drogas antituberculose e suas interaÃÃes com os derivados azÃlicos. Entretanto, novos estudos se fazem necessÃrio, visando determinar os mecanismos de aÃÃo desses compostos no metabolismo celular dos fungos. / In the past years, the improvement of mycological diagnosis methods and immunosuppressive diseases have caused a great impact in the incidence of opportunistic and deep mycoses all around the world, which motivated the performance of new antifungal drugs prospective studies. Histoplasmosis is a systemic mycosis caused by the fungus Histoplasma capsulatum var. capsulatum, which may mimic tuberculosis, in healthy individuals, concerning clinical and radiological aspects.ome cases of histoplasmosis that are refractory to the treatment with conventional antifungal drugs have been described. The aim of this study was to determine the in vitro inhibitory effect of the antituberculosis drugs: isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB); antifungal drugs: amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC) and voriconazole (VRZ) and chemical analogs of isoniazid against strains of H. capsulatum (n=30), as well as to evaluate the use of different culture media for the performance of the susceptibility tests. For such, first, the antituberculosis agents INH, PZA and EMB and the analogs of isoniazid were tested isolatedely, and then, in association with the antifungal drugs FLC, ITC and VRZ, against 18 strains of H. capsulatum, previously grown onto BHI agar, through broth macrodilution technique. Each of the 12 remaining strains grown onto potato agar, BHI agar, 2% malt extract agar and lactritmel agar were microscopically analyzed, concerning the presence of tuberculate macroconidia, which were quantified as follows: 0-10, 10-50 and >50 macroconidia/field. Fungal cultures were used to determine the susceptibility of H. capsulatum to the antifungal agents AMB, FLC, ITC and VRZ, through broth microdilution methodology. The antituberculosis drugs inhibited the in vitro growth of the fungal strains, with MICs ranging from 0.04 to 0.30 mg/mL for INH; 0.55 to 3.13 mg/mL for PZA and 1.56 to 6.25 mg/mL for EMB. Concerning antifungal drugs, all the strains were susceptible, with MIC values ranging from 0.0625 to 0.25 Âg/mL for AMB; 15.62 to 62.5 Âg/mL for FLC; 0.0039 to 0.0312 Âg/mL for ITC, and 0.00156 to 0.25 Âg/mL for VRZ. When associating antituberculosis drugs with azole derivatives, all associations inhibited the in vitro growth of H. capsulatum strains, and synergy was observed for the nine combinations tested. Analogs of isoniazide presented MICs of 2, 4, 8 and 15-fold better than the standard antituberculosis drug. Basing on micromorphological analysis, the lowest quantification of macroconidia/field (0-10) was observed for 11, 10, 6 and 7 strains previously grown onto potato agar, BHI agar, malt agar and lactritmel agar, respectively. Malt agar was the most adequate medium for the production of macroconidia, 10-50 and >50/field, with a total of six strains; followed by lactritmel agar, with 5 strains. Concerning the relationship between MIC and culture medium used during the test, it was observed that inoculum from strains grown onto malt agar and BHI agar allowed the detection of the MIC for 11 strains. On the other hand, for those inocula grown onto potato agar and lactritmel agar, the MIC values were not detected for 8 and 5 strains, respectively. The results of this study provide additional data on the antifungal potential of antituberculosis drugs and their interactions with azole derivatives. However, new studies are necessary in order to determine the mechanism of action of these compounds on fungal cellular metabolism.

Orientador: Angélica Zaninelli Schreiber / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T18:50:03Z (GMT). No. of bitstreams: 1 Biancalana_FernandaSimasCorrea_D.pdf: 1602190 bytes, checksum: f9f6d1fcbc7dcd1bf6b7e3dc9f3c4a90 (MD5) Previous issue date: 2011 / Resumo: Nos últimos anos, os fungos demáceos têm sido intensamente reconhecidos como importantes patógenos, principalmente em pacientes imunocomprometidos, sendo reportadas como patogênicas mais de 100 espécies de 60 diferentes gêneros. O tratamento das infecções causadas por estes fungos é difícil, bastante demorado e algumas vezes sem sucesso, portanto, se fazem necessários mais estudos sobre a suscetibilidade destes patógenos a antifúngicos in vitro. Alguns autores têm demonstrado a excelente eficácia in vivo da terbinafina, em combinação com outros antifúngicos, para o tratamento destas infecções, porém são poucos os que avaliam seu efeito in vitro isoladamente, e menos ainda os relatos sobre associação desta com outros antifúngicos. A maioria dos trabalhos utiliza apenas o inóculo com conídios, que não é a principal morfologia do fungo encontrada no paciente. A realização de testes utilizando hifas poderia mimetizar as características do fungo no tecido infectado, demonstrando melhor o potencial terapêutico do antifúngico. Existem poucos relatos na literatura sobre avaliação dinâmica de crescimento de fungos, e nenhum deles realizado com fungos demáceos. Partindo deste princípio, foi investigada neste estudo a suscetibilidade in vitro de conídios e hifas fungos demáceos dos gêneros Alternaria, Bipolaris, Cladophialophora, Curvularia, Exophiala e Fonsecaea frente a anfotericina B, itraconazol e voriconazol e terbinafina, e esta última em combinação com os demais antifúngicos através da metodologia do tabuleiro de xadrez e avaliação dinâmica de crescimento. Quando avaliados isoladamente, o voriconazol foi o antifúngico mais ativo, seguido de itraconazol, terbinafina e anfotericina B, utilizando conídios. Quando foi feita a associação, os resultados indicaram 100% de sinergismo nas interações entre terbinafina e voriconazol, 96,5% de sinergismo entre anfotericina B e terbinafina e 75,9% de sinergismo entre terbinafina e itraconazol. Nenhum caso de antagonismo foi observado. Voriconazol e itraconazol demonstraram maior atividade frente a hifas dos isolados avaliados, seguidos de terbinafina e anfotericina B. Os isolados de C. bantiana e F. pedrosoi demonstraram altos valores de CIM frente a anfotericina B. Os valores de CIM obtidos para as hifas foram menores que os dos conídios, chegando a duas ou quatro diluições abaixo do valor da CIM. Os resultados demonstraram 100% de sinergismo entre a terbinafina e os azólicos ou a anfotericina quando utilizadas hifas no sistema BCT. Concluindo, os resultados obtidos neste trabalho sugerem que a forma de hifas dos isolados avaliados é mais suscetível que a de conídios frente aos antifúngicos avaliados tanto isoladamente, como em associação. / Abstract: In recent years, the dematiaceous fungi have been widely recognized as important pathogens, especially in immunocompromised patients, being reported as pathogenic, more than 100 species of 60 different genera. Treatment of infections caused by these fungi is difficult, time consuming and sometimes without success, therefore, more studies are needed about the susceptibility of the pathogens to antifungal agents in vitro. Some studies have demonstrated excellent in vivo efficacy of terbinafine in combination with other antifungals to treat these infections, but few ones have evaluated the in vitro effect of terbinafine alone, and even less are about association with other antifungal agents. Most used only the inoculum with conidia, which is not the essencial morphology of the fungus found in the patient. Testing susceptibility using hyphae could mimic the characteristics of the fungus in infected tissue, demonstrating more clearly the therapeutic potential of the antifungal agent. There are few reports in the literature on dynamic evaluation of mold growth, and none of them performed with dematiaceous fungi. With this assumption, was investigated in this study the in vitro susceptibility of terbinafine alone and in combination with amphotericin, itraconazole and voriconazole against conidia and hyphae of dematiaceous fungi belonging to genera: Alternaria, Bipolaris, Cladophialophora, Curvularia, Exophiala and Fonsecaea, using the checkerboard methodology and evaluation of dynamic growth. When evaluated alone, voriconazole was the most active antifungal agent, followed by itraconazole, terbinafine and amphotericin B, using the broth microdilution method with conidia. In the association, the results indicated 100% of synergism in interactions between voriconazole and terbinafine, 96.5% of synergism between amphotericin and terbinafine and 75.9% of synergism between terbinafine and itraconazole. No case of antagonism was observed. Voriconazole and itraconazole demonstrated the highest activity with the use of BCT with hyphae of isolates evaluated, followed by terbinafine and amphotericin B. Isolates of C. bantiana and F. pedrosoi showed higher MIC values compared to amphotericin B. MIC values obtained for hyphae were lower than those of conidia two or four dilutions below the MIC value. The results demonstrated 100% synergistic interactions between terbinafine and azoles or amphotericin when testing hyphae in BCT system. In conclusion, the present results suggest that the hyphae form of tested isolates are more susceptible than conidia to the antifungal agent evaluated alone or in combination. / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Orientador: Angelica Zaninelli Schreiber / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T15:37:56Z (GMT). No. of bitstreams: 1 Correa_FernandaSimas_M.pdf: 2038543 bytes, checksum: 170a6e791e2c64d599b88b8485092a96 (MD5) Previous issue date: 2007 / Resumo: Dermatófitos do gênero Microsporum acometem preferencialmente pele e pêlos, sendo M.canis e M.gypseum as espécies mais isoladas em nosso meio. Antifúngicos tópicos e sistêmicos são indicados para o tratamento destas dermatofitoses, que é considerado mais difícil quando comparado ao de outros gêneros de fungos queratinofílicos. Deste modo, a determinação da suscetibilidade aos antifúngicos in vitro destes microrganismos é de interesse para embasamento da terapêutica empírica, avaliação de falhas terapêuticas, e testes de novos antifúngicos. No entanto, os dermatófitos não foram incluídos no documento relacionado do CLSI (M38-A, 2002). Os estudos disponíveis até o momento, utilizando conídios dos fungos, apresentam resultados muito divergentes, devido a falta de padronização e o grande número de variáveis pré-analíticas envolvidas. Em adição, uma vez que a forma de hifas predomina no tecido infectado, há controvérsias sobre o fato de se a forma conidial seria adequada para os testes. Há poucos relatos na literatura sobre avaliação dinâmica de crescimento de fungos e a taxa de inibição destes por drogas antifúngicas, mas nenhum destes realizados com dermatófitos. Este estudo se propôs a determinar condições para realização do teste de suscetibilidade pelo método de microdiluição em caldo e padronizar a avaliação de crescimento dinâmico pelo sistema BCT® para seis cepas do gênero Microsporum, determinando respectivamente CIM e CFM e taxas de inibição de crescimento frente a ciclopirox olamina, terbinafina e griseofulvina. Os resultados obtidos apontaram: ágar batata como o melhor meio de cultura para produção de conídios, inóculo de 1x 103 células/ml, incubação de 7 dias a 28ºC e leitura final considerando 100% de inibição do crescimento para o teste de microdiluição em caldo. Os resultados de CIM variaram de 1,0 a 16,0 µg/ml para ciclopirox olamina, de 0,005 a 0,004 µg/ml para terbinafina e de 2,5 a 20,0 µg/ml para griseofulvina. No Sistema Automatizado BioCell-Tracer® os testes foram realizados com: concentração de PLL de 0,05%; inóculo ? 1x106 conídios/ml em 5?l; incubação a 30ºC por 72 a 96h; 30ºC como temperatura de realização do teste e tempo total de experimento de 3 horas e 30 minutos. Todas as cepas apresentaram taxa de inibição de crescimento acima de 85% na fase de exposição à droga frente aos antifúngicos avaliados, na concentração equivalente ao resultado de CIM obtido pela técnica de microdiluição em caldo e, em pelo menos, uma diluição abaixo / Abstract: Dermatophytes of the genus Microsporum preferentially invade skin and hair, being M.canis and M.gypseum the most prevalent species in our routine. Topics and systemic antifungal agents are indicated for the treatment of theses dermatophytoses, which is considered more difficult when compared to other genera of keratinofilic fungi. By the way, the determination of in vitro antifungal susceptibility of these organisms is interesting as a guide of empiric therapy, evaluation of therapeutic failures and to evaluate new antifungal agents. However, dermatophytes were not included in the CLSI document (M38-A, 2002). The studies available until now, using fungi conidia, present divergent results due to the lack of standartization and the large number of pre-analytical variables involved. In addiction, once the hyphae forms predominate in infected tissues, there are controversies about if the conidial form would be adequate for the tests. There are few articles in the literature about dynamical evaluation of fungi growth and their growth inhibition rate in contact with antifungal agents, but none with dermatophytes. This study proposed the determination of conditions for broth microdilution susceptibility test and the standardization of dynamical growth evaluation by the BCT® system for six strains of Microsporum sp, determining respectively MIC and MFC and the inhibition rate after application of ciclopirox olamine, terbinafine and griseofulvin. The results pointed: potato dextrose agar as the best culture medium for conidium production, inoculum concentration of 1x 103 conidia/ml, incubation for 7 days at 28ºC and test enpoint at 100% of growth inhibition for broth microdilution test. The MIC results ranged from 1,0 to16,0 µg/ml for ciclopirox olamine, 0,005 to 0,004 µg/ml for terbinafine and 2,5 to 20,0 µg/ml for griseofulvin. On the automatizated system BioCell-Tracer®, the tests were realized with: PLL concentration of 0,05%; inocula of ? 1x106 conídia/ml in 5?l; incubation at 30ºC for 72 to 96hand also 30ºC as the test temperature, 3 hours and 30 minutes as total time of experiment. All strains showed growth inhibition rate above 85% on drug exposure phase against the antifungal agents evaluated, in MIC obtained by both microdilution method and, at least, one dilution below / Mestrado / Patologia Clinica / Mestre em Ciências Médicas

Orientadores: Jose Francisco Hofling, Marta Cristina Teixeira Duarte / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-06T08:53:40Z (GMT). No. of bitstreams: 1 Furletti_VivianFernandes_M.pdf: 1126113 bytes, checksum: d4a24df57fea2a22c72726fc7f3e1ea6 (MD5) Previous issue date: 2006 / Resumo: A suscetibilidade a antifúngicos foi avaliada para espécies de Candida spp isoladas de três diferentes sítios da cavidade oral de 19 pacientes com doença periodontal, a saber: bolsa periodontal, mucosa bucal e sulco gengival. Um total de 140 amostras de leveduras foram isoladas e identificadas por métodos comumente utilizados em Micologia. Destas, 105 foram identificadas como C. albicans, 16 como C. tropicalis, 5 como C. parapsilosis, 4 como C. krusei, 3 como C. famata, 3 como C. norvegensis, 3 como C. dubliniensis e 1 como C. lusitaniae. As leveduras foram testadas frente aos antifúngicos fluconazol, itraconazol, cetoconazol e anfotericina B de acordo com a padronização do NCCLS. Dentre as amostras de C. albicans, 88% apresentaram susceptibilidade dependente da concentração (SDC) e 3,6% resistência à pelo menos um antifúngico azólico. Entre as outras espécies, 57% apresentaram SDC e 42,8% resistência a pelo menos um dos azólicos testados. Quanto a anfotericina B, 90% das C. albicans e 74,3% das não-albicans foram resistentes a este antifúngico. Não houve ocorrência de resistência para o fluconazol e apenas 3,6% das C. albicans e 40% das ¿não-albicans¿ foram SDC para este antifúngico. Os valores da Concentração Inibitória Mínima CIM50 e CIM90 dos antifúngicos azólicos para as amostras de C. albicans foram inferiores aos encontrados para as demais espécies. Para a anfotericina B, os valores de CIM50 e CIM90 não foram diferentes entre C. albicans e demais espécies. Apesar do baixo índice de resistência, foi observada elevada ocorrência de SDC para itraconazol e cetoconazol. Foi possível concluir que pacientes com doença periodontal apresentaram níveis de colonização por Candida spp relevantes, principalmente na mucosa bucal e bolsa periodontal, com uma importante ocorrência de SDC e resistência aos antifúngicos testados, o que está de acordo com dados recentes sobre a diminuição da sensibilidade a antifúngicos entre espécies de Candida / Abstract: The susceptibility to antifungal was evaluated for Candida spp isolated from three different sites of the oral cavity from 19 patients with periodontal illness, known as: periodontal pocket, oral mucosa and ridge gengival. A total of 140 yeasts were isolated and identified by classical methods in Micology. From this, 105 were identified as C. albicans, 16 as C. tropicales, 5 as C. parapsilosis, 4 as C. Krusei, 3 as C. famata, 3 as C.norvegensis, 3 as C.dubliensis and 1 as C. lusitaniae. The antifungal Fluconozole, Itraconazole, Ketoconazole and Anfotericin B were tested against the yeasts according to the NCCLS padronization. Among the samples of C. albicans, 88% showed susceptibility depending on the concentration (SCD) and 3,6% were resistant to at least one antifungal azoles studied. Among other species, 57% presented SDC and 42,8% showed resistance to at least one of the antifungal azoles tested. Regarding to Anfotericin B, 90% of the C. albicans isolates and 3% of the ¿non- albicans¿ showed resistance to this antifungal drug. There was no occurrence of resistance to the Fluconozale and only 3,6% of C. albicans and 40% of the ¿non-albicans¿ were SDC to this antifungal. The values of Minimum Inhibitory Concentration (MIC50 and MIC90) of the antifungal azoles to the samples of C. albicans were inferior to the ones found in the other species. For the Anfoterecin B, the values of MIC50 and MIC90 were not different among C. albicans and the other species. Besides the low percentage indice of resistance, it was observed high occurrence of SDC to Itraconazole and Cetoconazole. Patients with periondontal illness presented relevant levels of colonization by Candida spp, mainly at the oral mucosa and periodontal pocket showing important occurrence of SDC and resistance against the antifungals drugs tested according to the recent literature data about the decrease of sensibility to antifungals among Candida species / Mestrado / Microbiologia e Imunologia / Mestre em Biologia Buco-Dental

Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Several yeast species had previously been isolated from water systems in the North West Province, South Africa. Some of the identified species had, in other studies, been associated with superficial mucosal infections to life threatening diseases. Antifungal drugs are used to treat such yeast infections. However, due to prophylactic usage and continuous exposure some yeast species have developed resistance to some antifungal agents. The aim of this study was to determine the diversity and antifungal susceptibility of yeasts in selected rivers, Mooi River and Harts River in the North West Province, South Africa. Waters samples were collected from the rivers in summer and winter seasons. Physico-chemical parameters such as pH, temperature, total dissolved solids, chemical oxygen demand, nitrates and phosphates were measured to determine the water quality. Yeast colonies were enumerated at room temperature and 37°C using yeast-malt-extract agar (containing 100 ppm chloramphenicol). Pure isolates from 37°C were identified by biochemical tests and 26S rRNA gene sequencing. Yeast sequences of isolated yeasts were sent to Genbank. Phylogenetic tree was conducted to determine phylogenetic relationship between the yeast isolates. Disk diffusion antifungal susceptibility tests were conducted on the yeast species. Physico-chemical parameters of the water were within target water quality range for livestock farming but in most sampling sites out of range for irrigation use. pH, Nitrates, phosphates and chemical oxygen demand levels ranged from 7.40 to 8.64, 0 to 5.4 mg/L, 0 to 7.14 mg/L and 31 to 43 mg/L, respectively. Elevated levels of total dissolved solids were measured in all the sampling sites. Total yeast counts ranged between 320-4200 cfu/L and 27-2573 cfu/L for room temperature and 37˚C. All the yeast colonies isolated were non-pigmented. Diazonium Blue B tests determined the yeasts isolates as ascomycetes. Haemolysin and extracellular enzyme production tests were negative on all the isolates. Yeasts isolates were identified and belonged to the genera Arxiozyma, Candida, Clavispora, Cyberlindnera, Lecythophora, Pichia, Saccharomyces, and Wickerhamomyces. Saccharomyces cerevisiae and Candida glabrata were mostly isolated species. Furthermore, the results indicated that levels of yeast could be correlated to physico-chemical quality of water. A large number of isolates were resistant to azoles, especially fluconazole as well as other antifungal classes. Most of the Candida species were resistant to almost all the antifungals. Several of the isolated yeast species are opportunistic pathogens. They could cause infections in sensitive individuals during occasional direct contact especially immune compromised people. Resistance of these yeast species to antifungal agents is a major health concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A coccidioidomicose e a histoplasmose sÃo micoses sistÃmicas que acometem o homem e animais, causadas por espÃcies de fungos dimÃrficos, com Ãnfase para Coccidioides posadasii e Histoplasma capsulatum var. capsulatum, respectivamente. SÃo consideradas doenÃas profundas importantes, podendo culminar em diversas complicaÃÃes secundÃrias. Nos Ãltimos anos, a melhoria dos mÃtodos de diagnÃstico micolÃgico e o aumento da ocorrÃncia de doenÃas imunossupressoras causaram grande impacto na incidÃncia das micoses profundas e oportunistas no mundo. Apesar da existÃncia de terapias eficazes com antifÃngicos contra a coccidioidomicose e a histoplasmose, a busca por novas drogas para o tratamento destas doenÃas se faz necessÃria. Ciprofloxacina à uma droga antibacteriana clÃssica do grupo das fluoroquinolonas, que inibe a atividade catalÃtica da DNA girase e topoisomerase IV, essenciais na replicaÃÃo e transcriÃÃo do DNA bacteriano. Estudos verificaram que ciprofloxacina pode atuar na DNA girase dos fungos. Assim, o presente estudo visou avaliar o efeito inibitÃrio in vitro de ciprofloxacina (CIP) isolada e em combinaÃÃo com os antifÃngicos anfotericina B (AMB), itraconazol (ITC), voriconazol (VRC) e caspofungina (CAS) frente à C. posadasii e Histoplasma capsulatum var. capsulatum. Foram utilizados 16 cepas de C. posadasii na fase filamentosa, 16 cepas de H. capsulatum var. capsulatum na fase filamentosa e 9 cepas de H. capsulatum var. capsulatum na fase leveduriforme. O estudo foi conduzido em ensaio de macrodiluiÃÃo e microdiluiÃÃo em caldo, descritos nos documentos M-38A e M-27A2, padronizados pelo Clinical Laboratory Standards Institute (CLSI), sendo utilizados para C. posadasii e Histoplasma capsulatum var. capsulatum, respectivamente. A interaÃÃo das drogas foi analisada atravÃs do cÃlculo do Ãndice da ConcentraÃÃo InibitÃria FracionÃria (FICI), definido como a soma das relaÃÃes entre a concentraÃÃo inibitÃria mÃnima (CIM) de cada droga em combinaÃÃo e a CIM da mesma droga isolada, considerando os valores menores ou iguais a 0,5 indicativos de sinergismo. Com relaÃÃo Ãs cepas de C. posadasii, foram observadas interaÃÃes sinÃrgicas em todas as combinaÃÃes, com destaque para as associaÃÃes de CIP (3,125≤CIM≤12,5 ug mL-1) com ITC (0,0078≤CIM≤0,125 ug mL-1) (n=13/16), CIP (3,125≤CIM≤12,5 ug mL-1) com VRC (0,0078≤CIM≤0,0312 ug mL-1) (n=13/16) e CIP (3,125≤CIM≤12,5 ug mL-1) com CAS (2≤CIM≤8 ug mL-1) (n=14/16). Para as cepas de H. capsulatum na fase filamentosa, tambÃm foram observadas interaÃÃes sinÃrgicas em todas as combinaÃÃes, com destaque para as associaÃÃes de CIP (3,906≤CIM≤62,5 ug mL-1) com ITC (0,00006≤CIM≤0,0078 ug mL-1) (n=14/16) e CIP (31,25≤CIM≤125 ug mL-1) com VRC (0,0156≤CIM≤0,125 ug mL-1) (n=16/16). No tocante Ãs cepas de H. capsulatum na fase leveduriforme, foram observadas poucas interaÃÃes sinÃrgicas nas combinaÃÃes de drogas testadas. Nenhuma das associaÃÃes de drogas testadas apresentou antagonismo. Os dados obtidos apontam uma nova alternativa para o tratamento da coccidioidomicose e da histoplasmose, sendo necessÃrios novos estudos que visem investigar os mecanismos de aÃÃo dessas combinaÃÃes de drogas no metabolismo celular fÃngico, bem como o delineamento de experimentos in vivo para confirmar a significÃncia desses achados. / Coccidioidomycosis and histoplasmosis are systemic mycoses that occur in humans and other animals and are caused by the dimorphic fungi Coccidioides posadasii and Histoplasma capsulatum var. capsulatum, respectively. They are considered important deep mycoses that can lead to several secondary complications. In the past years, the improvement of the techniques applied in mycological diagnosis and the increase in the occurrence of immunocompromising diseases have caused a great impact in the incidence of deep and opportunistic mycoses in the world. In spite of the existence of effective antifungal therapy against coccidioidomycosis and histoplasmosis, the pursue of new drugs to treat theses diseases is necessary. Ciprofloxacin is a classic antibacterial drug that belongs to the group of fluoroquinolones, which inhibit the catalytic activity of DNA gyrase and topoisomerase IV, which are essential in bacterial DNA replication and transcription. Some studies have shown that ciprofloxacin can act on fungal DNA gyrase. Thus, the present study aimed at evaluating the in vitro inhibitory effect of ciprofloxacin (CIP), when associated with amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC) or caspofungina (CAS), on C. posadasii and H. capsulatum var. capsulatum. Sixteen strains of C. posadasii in the filamentous phase and 16 and 9 strains of H. capsulatum in the filamentous and yeast-like phase, respectively, were used. Broth macrodilution and microdilution assays were performed, as described in the documents M38-A and M27-A2, respectively, of the Clinical Laboratory Standards Institute (CLSI). Drug interaction was analyzed by calculating the fractional inhibitory concentration index (FICI), which is defined as the sum of the ratios between the minimal inhibitory concentration (MIC) of each combined drug and the MIC of the same drug isolatedly. Values of FICI smaller or equal to 0.5 indicate the occurrence of synergy. Concerning the isolates of C. posadasii, synergistic interactions were observed for all combinations, especially for the associations of CIP (3.125≤CIM≤12.5 ug mL-1) with ITC (0.0078≤CIM≤0.125 ug mL-1) (n=13/16), CIP (3.125≤CIM≤12.5 ug mL-1) with VRC (0.0078≤CIM≤0.0312 ug mL-1) (n=13/16) and CIP (3.125≤CIM≤12.5 ug mL-1) with CAS (2≤CIM≤8 ug mL-1) (n=14/16). For the isolates of H. capsulatum in the filamentous phase synergistic interactions were also observed for all combinations, with emphasis to the associations of CIP (3.906≤CIM≤62.5 ug mL-1) with ITC (0.00006≤CIM≤0.0078 ug mL-1) (n=14/16) and CIP (31.25≤CIM≤125 ug mL-1) with VRC (0.0156≤CIM≤0.125 ug mL-1) (n=16/16). For H. capsulatum in yeast-like phase, few synergistic interactions were observed for the tested drug combinations. None of the tested combinations presented antagonism. The obtained data may point at a new alternative for the treatment of coccidioidomycosis and histoplasmosis. Thus, it is necessary to investigate the mechanisms of action of these drug combinations on the fungal cellular metabolism and to perform in vivo experiments to confirm the relevance of these findings.

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Made available in DSpace on 2016-12-23T13:56:10Z (GMT). No. of bitstreams: 1 Dissertacao de Adrielle Hoffmann.pdf: 1434617 bytes, checksum: 427eb50baa714de01ab9deab183c0308 (MD5) Previous issue date: 2011-08-10 / As onicomicoses, infecções fúngicas de unhas, são causadas por fungos filamentosos dermatófitos e não-dermatófitos e leveduras e representam as micoses superficiais mais difíceis de serem diagnosticadas e tratadas. O tratamento é através do uso tópico e/ou oral com drogas antifúngicas e pode ainda envolver a remoção da unha. O uso de agentes tópicos concomitante à terapia sistêmica leva à melhores resultados clínicos e micológicos. A eficácia da associação medicamentosa pode estar associada à ação complementar entre as drogas, envolvendo diferentes níveis de penetração ungueal e, dependendo dos antifúngicos, de diferentes alvos de ação na célula fúngica. O objetivo do presente estudo consistiu em estabelecer a ocorrência de fungos filamentosos na etiologia das onicomicoses e a suscetibilidade in vitro destes às drogas terbinafina, ciclopirox olamina, cetoconazol e itraconazol, conforme o documento M38-A2 (2008) do CLSI. Foi tambem avaliada a combinação entre estas drogas antifúngicas através do cálculo do índice fracionário de concentração inibitória (IFCI). Nossos resultados demonstraram que prevalência das onicomicoses, no período de janeiro de 2009 a abril de 2011, em Vitória, ES, foi de 50% dentre as dermatomicoses. A maioria dos isolados (77%) foi obtido de pacientes do sexo feminino e o local de maior acometimento foram as unhas dos pés para ambos os sexos. As unhas das mãos foram mais acometidas por leveduras e as unhas dos pés, por fungos filamentosos. Em geral, os gêneros de fungos filamentosos mais predominantes na etiologia das onicomicoses foram Trichophyton spp (21,7%), Fusarium spp. (11,2%) e Scytalidium spp.(8,4%). Para fungos filamentosos, os testes de suscetibilidade in vitro mostraram que isolados de dermatófitos foram mais sensíveis que isolados do grupo não-dermatófitos. Entre os nãodermatófitos, Fusarium spp. foi menos inibido que Scytalidium spp., que por sua vez, foi menos inibido que o dermatófito Trichophyton spp. Dentre as combinações testadas não houve nenhum efeito antagônico e,, com exceção daquela entre itraconazol e cetoconazol, as demais apresentaram efeito sinérgico sobre algum isolado. A combinação entre drogas apresentou maiores índices de sinergismo para o gênero Scytalidium spp. O melhor resultado para este gênero foi também obtido pela combinação itraconazol e terbinafina / The onychomycosis, fungal nail infections, are caused by filamentous fungi dermatophytes and non-dermatophytes and yeasts and represent the most difficult superficial mycosis to be diagnosed and treated. Treatment involves use of topical and/or oral antifungal drugs, as well as removal of the nail. The use of topical agents concomitant to systemic therapy leads to better clinical and mycological results. The efficacy of combination therapy may be associated with a complementary action between the drugs, involving different levels of nail penetration and, depending on the antifungal, different targets of action in the fungal cell. The purpose of this study was to establish the occurrence of filamentous fungi in the etiology of onychomycosis and in vitro susceptibility to the drugs terbinafine, ciclopirox olamine, ketoconazole and itraconazole, according to document M38-A2 (2008) CLSI. It was also evaluated the combination among these antifungal drugs by calculating the fractional inhibitory concentration index (FICI). Our results showed that the prevalence of onychomycosis in the period from January 2009 to April 2011, in Vitoria, ES, Brazil, was 50% among the dermatomycosis cases. Most of the isolates (77%) were obtained from female patients and toenails were the local with greater involvement for both sexes. The fingernails were more affected by yeast and toenails, by filamentous fungi. In general, the genera of filamentous fungi more prevalent in the etiology of onychomycosis was Trichophyton spp (21.7%), Fusarium spp. (11.2%) and Scytalidium spp. (8.4%). For filamentous fungi, the in vitro susceptibility testing showed that dermatophytes were more susceptible than non-dermatophytes isolates. Among non-dermatophytes, Fusarium spp. was less inhibited than Scytalidium spp., which in turn, was less inhibited than the dermatophyte Trichophyton spp. Among the combinations tested, there was no antagonistic effect and,, with exception of ketoconazole and itraconazole, those ones showed synergistic effect for isolates. The best results were presented for combinations involving itraconazole and terbinafine. The drug combination with greater synergistic effect was observed for genus Scytalidium spp. being that the combination itraconazole and terbinafine presented also the best results for this genus

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Historicamente, as bactÃrias foram vistas como organismos que vivem isolados, no entanto, à claro, agora, que a grande maioria de bactÃrias existe em comunidades complexas, conhecidas como biofilmes. As bactÃrias em biofilmes se encontram aderidas a superfÃcies diversas, tanto abiÃticas como biÃticas (dente, osso, mucosa), compondo um ecossistema altamente estruturado, dinÃmico e organizado. Um exemplo de infecÃÃo que pode envolver a presenÃa formaÃÃo de biofilme à a otite - definida como inflamaÃÃo aguda ou crÃnica da orelha, podendo se estender desde o epitÃlio do conduto auditivo externo atà a orelha interna. Descrita como a doenÃa mais comum do canal auditivo externo em cÃes, possui uma etiologia multifatorial, que inclui fungos e bactÃrias, principalmente do gÃnero Staphylococcus. BactÃrias desse gÃnero sÃo comensais de pele e mucosas, mas podem atuar como patÃgenos oportunistas em condiÃÃes propÃcias e sÃo frequentemente associadas a uma ampla variedade de infecÃÃes em seres humanos e animais com vÃrios relatos de refratariedade aos tratamentos usuais. Em um biofilme, as bactÃrias podem ser dezenas de vezes mais resistentes aos antibiÃticos, quando comparadas Ãs mesmas bactÃrias em crescimento planctÃnico. O combate a infecÃÃes envolvendo bactÃrias em biofilme à um grande desafio da microbiologia mundial, que leva à busca de novas opÃÃes terapÃuticas. Com efeito, este estudo teve como objetivo avaliar o efeito inibitÃrio in vitro de seis substÃncias â trÃs agentes antimicrobianos clÃssicos, ciprofloxacina, cloranfenicol, gentamicina; e trÃs agentes antimicrobianos nÃo-clÃssicos timol, carvacrol, perÃxido de hidrogÃnio (H2O2) â contra cepas estafilocÃcicas de otite canina em crescimento planctÃnico e em biofilme. Foram avaliadas 54 cepas clÃnicas isoladas de secreÃÃo purulenta de cÃes com otite, divididas entre cinco espÃcies: S. intermedius, S. simulans, S. haemolyticus, S. epidermidis e S. lugdunensis. As 16 cepas classificadas como produtoras de biofilme (11 de S. intermedius e cinco de S. simulans), segundo resultado obtido pelo teste do Ãgar vermelho Congo e confirmaÃÃo por microscopia eletrÃnica de varredura (MEV), foram usadas em ensaios de microdiluiÃÃo em caldo para determinaÃÃo de concentraÃÃo inibitÃria mÃnima (CIM) e concentraÃÃo inibitÃria mÃnima em biofilme (CIMB). Com relaÃÃo Ãs cepas produtoras de biofilme, foram observados os seguintes resultados: os valores para ciprofloxacina foram 0,12 ≤CIM≤0,5 mcg/mL (mÃdia 0,28 mcg/mL) e 0,5≤CIMB≤8 mcg/mL (mÃdia 1,79 mcg/mL); para cloranfenicol 2≤CIM≤16 mcg/mL (mÃdia 7,41 mcg/mL) e 8≤CIMB≤32 mcg/mL (mÃdia 20,71 mcg/mL); para gentamicina 0,5≤CIM≤4 mcg/mL (mÃdia 2,09 mcg/mL) e 4≤CIMB≤64 mcg/mL (mÃdia 24,24 mcg/mL); para timol 32≤CIM≤512 mcg/mL (mÃdia 137,41 mcg/mL) e 256≤CIMB≤2048 mcg/mL (mÃdia 768 mcg/mL); carvacrol 32≤CIM≤512 mcg/mL (mÃdia 128 mcg/mL) e 256≤CIMB≤4096 mcg/mL (mÃdia 993,88 mcg/mL); e para H2O2 32≤CIM≤128 ppm (mÃdia 99,76 ppm) e 128≤CIMB≤4096 ppm (mÃdia 1874,82 ppm). Os dados obtidos apontam para o potencial terapÃutico dos seis antibiÃticos estudados no tratamento de infecÃÃes estafilocÃcicas associadas a biofilme, sendo necessÃrios novos estudos para investigar os mecanismos de aÃÃo dessas drogas sobre o biofilme, bem como o delineamento de experimentos in vivo para confirmar a significÃncia desses achados. / Historically, bacteria have been seen as isolated organisms; however, it is now clear that the vast majority of bacteria exist in complex communities known as biofilms. Bacteria in biofilms are adhered to various surfaces, both abiotic and biotic (teeth, bones, mucosa), often forming a highly dynamic ecosystem, which is structured and organized. An example of infection, which can involve the formation of biofilm is otitis - defined as an acute or chronic inflammation of the ear, which may extend from the external ear canal to the inner ear. Described as the most common disease of the external ear canal in dogs, it has a multifactorial etiology, including fungi and bacteria, especially of the genus Staphylococcus. Bacteria of this genus are commensal to skin and mucous membranes, but can act as opportunistic pathogens in favorable conditions and are often associated with a wide variety of infections in humans and animals with many reports of refractoriness to usual treatments. In biofilm, bacteria can be ten to a hundred times more resistant to antibiotics when compared to the same bacteria in planktonic growth. The struggle to fight infections involving bacterial biofilms is a major challenge of microbiology today, which, in turn, leads to the search for new therapeutic options. Thus, this study aimed to evaluate the in vitro inhibitory effect of six substances â three classic antimicrobial agents ciprofloxacin, chloramphenicol, gentamicin; and three non-classic antimicrobial agents thymol, carvacrol, hydrogen peroxide (H2O2) - against staphylococcal strains from canine otitis in planktonic growth and in biofilm. We analyzed 54 clinical strains isolated from purulent secretion of canine otitis, divided into five species: S. intermedius, S. simulans, S. haemolyticus, S.epidermidis and S. lugdunensis. The 16 strains classified as biofilm producers (11 S. intermedius and five S. simulans), according to result obtained by the Congo red agar test and confirmed by scanning electron microscopy (SEM), were used in broth microdilution assay for determination of minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC). With regards to the biofilm-producing strains, the following results were observed: the values for ciprofloxacin were 0,12 ≤MIC≤0,5 mcg/mL (mean 0,28 mcg/mL) and 0,5≤MBIC≤8 mcg/mL (mean 1,79 mcg/mL); for chloramphenicol 2≤MIC≤16 mcg/mL (mean 7,41 mcg/mL) and 8≤MBIC≤32 mcg/mL (mean 20,71 mcg/mL); for gentamicin 0,5≤MIC≤4 mcg/mL (mean 2,09 mcg/mL) and 4≤MBIC≤64 mcg/mL (mean 24,24 mcg/mL); for thymol 32≤MIC≤512 mcg/mL (mean 137,41 mcg/mL) and 256≤MBIC≤2048 mcg/mL (mean 768 mcg/mL); carvacrol 32≤MIC≤512 mcg/mL (mean 128 mcg/mL) and 256≤MBIC≤4096 mcg/mL (mean 993,88 mcg/mL); and for H2O2 for 32≤MIC≤128 mcg/mL (mean 99,76 ppm) and 128≤MBIC≤4096 ppm (mean 1874,82 ppm). The presented data indicates the therapeutic potential of the six antibiotics studied in the treatment of staphylococcal infections associated with biofilm, which warrants further studies to investigate the mechanisms of action of these drugs on biofilms and the design of in vivo experiments to confirm the significance of these findings.