A leishmaniose é uma doença negligenciada causada por protozoários parasitos do gênero Leishmania sp. e transmitida por vetores. Esta doença pode apresentar formas clínicas que variam de lesões cutâneas simples que podem desaparecer espontaneamente até a forma visceral, a qual acomete órgãos como baço, fígado, medula óssea e linfonodos, e pode levar a morte se não tratada corretamente. Além disso, de acordo com a Organização Mundial da Saúde as leishmanioses são endêmicas em 98 países. O tratamento ainda é feita principalmente com dois medicamentos, os antimoniais pentavalentes e anfotericina B, os quais causam uma série de efeitos secundários, além disso, relatos de resistência a esses fármacos têm sido publicados. Além disso, medicamentos alternativos aprovados para uso, como a miltefosina e pentamidina, também possuem sérios efeitos colaterais e nem sempre são efetivos para todas as espécies do parasito e/ou formas clínicas. Estes fatos demonstram que é necessário o desenvolvimento de fármacos alternativos para a quimioterapia das leishmanioses. Nesse sentido, o reposicionamento de fármacos se mostra como uma importante estratégia para o desenvolvimento de novas alternativas terapêuticas para a leishmaniose, pois fármacos já liberados para o uso em humanos podem ser reutilizados, diminuindo o tempo de desenvolvimento e custos empregados para o desenvolvimento de novos fármacos. Leishmania sp. possui como um dos principais lipídios o ergosterol, cuja rota metabólica é complexa e envolve uma série de enzimas, as quais, se corretamente bloqueadas poderão suprimir a produção do ergosterol e, portanto, a viabilidade de parasitos. Considerando estes aspectos, o presente estudo objetivou investigar a ação leishmanicida de fármacos que possuem ação inibitória em enzimas da via de esteróis como o fármaco anti-hiperlipidêmico rosuvastatina (inibidor da enzima 3-hidroxi-3-metil-glutaril-CoA redutase) e, os antifúngicos voriconazol, tiaconazol, fenticonazol (inibidores da enzima 14 alfa-metilesterol 14-demetilase), naftifina e tolnaftato (inibidores da enzima esqualeno epoxidase), e por fim a nistatina que age diretamente sobre ergosterol. O potencial contra formas promastigotas e amastigotas intracelulares de L. (L.) amazonensis, L. (L.) infantum e L. (V.) braziliensis foram avaliados, e então foram investigadas possíveis alterações ultraestruturais, fisiológicas em promastigotas de L. (L.) amazonensis (formação de poros de membrana, e alteração do potencial de membrana mitocondrial) ou nas células hospedeiras (produção de óxido nítrico, peróxido de hidrogênio, e mudanças no pH intracelular). O fenticonazol, tioconazol, nistatina e o tolnaftato foram capazes de eliminar as formas promastigotas e amastigotas intracelulares, sendo a nistatina, o tolnaftato e o fenticonazol os mais seletivos para amastigotas das três espécies de parasitos estudados. Por outro lado, rosuvastatina, voriconazol e a naftifina não apresentaram ação contra formas amastigotas intracelulares. Além disso, os fármacos com atividade leishmanicida alteraram morfologicamente e fisiologicamente a mitocôndria do parasito. Apesar de não haver estímulo da produção de H2O2 ou NO por parte dos fármacos analisados, o fenticonazol foi capaz de alcalinizar macrófagos hospedeiros infectados. Considerando que estes fármacos foram capazes de inibir o crescimento de multi-espécies do parasito no interior dos macrófagos, esses resultados sugerem que medicamentos antifúngicos podem ser interessantes alvos para reposicionamento da quimioterapia das leishmanioses tegumentar e visceral / Leishmaniosis is a neglected disease caused by parasitic protozoa belonging to the genus Leishmania sp. and transmitted by vectors. This disease presents different clinical forms ranging from single lesion, that can heal spontaneously, to the visceral form, that affects the spleen, liver, bone marrow and lymph nodes, and may lead to death if not properly treated. Additionally, according to the World Health Organization, leishmaniosis is endemic to 98 countries. The treatment is still carried out with two main drugs, antimonials and amphotericin B that induce side effects; in addition, reports about the emergence of parasite resistance have been published. Besides, alternative drugs such as miltefosine and pentamidine still have serious side effects and are not always effective to treat all Leishmania species and/or clinical form. In this regard, drug repurposing has been considered an important strategy to develop new therapeutic alternatives to leishmaniasis chemotherapy, since drugs already approved to human use may be reused, decreasing time and costs needed for the research of new drugs. Leishmania sp. possesses ergosterol as the main membrane lipid, and the metabolic pathway to produce this lipid is complex and involves different enzymes, which if correctly inhibited may suppress ergosterol production and, therefore, parasite viability. Considering these aspects, the aims of this study is to investigate the leishmanicidal potential of drugs who inhibit enzymes of the ergosterol pathway, such as the anti-hyperlipidemic drug rosuvastatin (inhibitor of the 3-hydroxy-3-methyl-glutaryl-CoA reductase), the antifungals voriconazole, tioconazole, fenticonazole, (inhibitors of the 14 Alpha-methylsterol 14-demethylase), naftifine and tolnaftate (inhibitors of squalene epoxidase), and nystatin, that acts directly on ergosterol. The anti-promastigote and anti-amastigote potentials of drugs against L. (L.) amazonensis, L. (L.) infantum and L. (V.) braziliensis were evaluated; ultrastructural and physiological alterations of L. (L.) amazonensis promastigotes (membrane pore formation and mitochondrion membrane potential alterations) and host macrophages (nitric oxide and hydrogen peroxide productions and changes to intracellular pH) were investigated. Fenticonazole, tioconazole, nystatin and tolnaftate were capable to eliminate promastigote and amastigotes, being the most selective drugs nystatin, tolnaftate and fenticonazole to all studied species amastigotes. Rosuvastatin, voriconazole and naftifine were unable to eliminate amastigote forms. Furthermore, the drugs that possess leishmanicidal effects affected parasite mitochondrion. Although these drugs did not trigger the production of H2O2 or NO, fenticonazole was able to alkalinize host infected macrophages. Considering these drugs were able to inhibit multiple species of the parasite growth in macrophages, these results suggest that antifungal drugs can be interesting targets to be repurposed in the cutaneous and visceral leishmaniasis

O complexo C. neoformans (no qual as espécies C. neoformans e C. gattii estão inseridas) é constituído por leveduras encapsuladas que causam infecção em humanos e animais, com distribuição mundial. Diante do reconhecimento da importância dessas leveduras, seja no meio ambiente ou na área médica, há um crescente interesse no desenvolvimento de pesquisas que possam levar a uma melhor compreensão de sua epidemiologia e estrutura. Cepas clínicas e ambientais pertencentes ao acervo de amostras do Departamento de Microbiologia do HCFMUSP e ao Laboratório de Leveduras Patogênicas do ICB-USP foram reidentificadas, estudadas quanto aos fatores relacionados à virulência (por pesquisa de exoenzimas), quanto ao perfil de suscetibilidade in vitro frente aos antifúngicos: anfotericina B, fluconazol e voriconazol pela utilização da técnica E-test®, e quanto aos aspectos epidemiológicos (estudo de prontuários dos pacientes). As cepas de origem clínica (60) e ambiental (42) foram reidentificadas e condiziam com o estabelecido pela literatura. Nota-se que quanto às cepas clínicas, 90% delas pertenciam à espécie C. neoformans e 10% à espécie C. gattii e das cepas ambientais, 95,2% eram C. neoformans e 4,8% eram C. gattii. Com relação à produção de exoenzimas, cepas de origem clínica (45%) apresentaram índice 2 (positiva) e cepas de origem ambiental (45,2%) apresentaram índice 3 (fortemente positiva) quanto à proteinase; quanto à fosfolipase, 71,7% das cepas de origem clínica apresentaram índice 2 e, 52,4% das cepas ambientais apresentaram índice 3. Todas as cepas foram sensíveis aos antifúngicos testados (com exceção de uma que era sensível de forma dose dependente ao fluconazol). Em estudo epidemiológico, a maioria dos pacientes acometidos de criptococose foi do sexo masculino (70%), com média de acometimento aos 47 anos; 25 pacientes (41,7%) foram ao óbito; a criptococose mais frequente (50%) foi a neurocriptococose; 50% dos pacientes tinham sorologia positiva para o HIV e 26,7% dos pacientes utilizaram anfotericina B associada ao fluconazol como tratamento. Ao final do trabalho chegamos a importantes conclusões: o meio CGB não se apresentou como método capaz de identificar eficazmente cepas C. gattii, sendo útil apenas como uma primeira triagem, devendo ser acoplado à técnica de PCR-RFLP; ensaios de produção da atividade fosfolipásica são extremamente relevantes, podendo-se afirmar que a fosfolipase pode ser a mais importante enzima na patogênese da criptococose (servindo como indicador das espécies do gênero Cryptococcus spp.); homens são mais acometidos de criptococose do que mulheres, sendo estas acometidas mais cedo. A leucemia de células linfoides granulares grandes (LGL), com expressão anômala de CD16+CD56+CD19+, encontrada em 1 paciente HIV negativo, infectado por C. gattii pode sugerir que a substituição de células imunocompetentes por células aberrantes com ineficiência funcional poderia ser a responsável pela imunossupressão do paciente. Esse fato sugere a importância de uma investigação detalhada em pacientes com meningite causada por Cryptococcus spp. com um sistema imune aparentemente competente / C. neoformans complex (the species C. neoformans and C. gattii are inserted) consists of encapsulated yeasts that cause infection in humans and animals, with worldwide distribution. With the recognition of the importance of these yeasts, either in the environment or in the medical field, there is a growing interest in developing research that may lead to a better understanding of its epidemiology and structure. Clinical and environmental strains of the collection of samples from the Department of Microbiology, HC-USP and the Laboratory of Pathogenic Yeasts of ICB- USP were reidentified, studied concerning the factors related to virulence (by exoenzymes research), as the susceptibility profile in vitro front of antifungal: amphotericin B, fluconazole and voriconazole by use of the E-test® technique, and as to the epidemiological (study of medical handbooks of patients). The strains of clinical (60) and environmental origin (42) were reidentified and established in the literature. We notice that as the clinical strains, 90% of them belonged to the species C. neoformans and (10%) to the species C. gattii and of theenvironmental strains, 95,2% were C. neoformans and 4,8 % were C. gattii. With respect to exoenzyme production, strains of clinical origin (4 %) had an index 2 and strains of environmental origin (45,2%) had an index 3, as the phospholipase, 71,7 % of the strains of clinical origin showed index 2 (positive) and 52,4 % of the environmental strains exhibited index 3 (strongly positive). All strains were susceptible to antifungal agents tested (except one that was sensitive dose dependente to fluconazole). In an epidemiological study, the majority of patients with cryptococcosis were male (70%), with age of 47 years, 25 patients (41,7%) died; the cryptococcosis more frequent (50%) was the neurocryptococcosis, 50 % of patients were seropositive for HIV and 26,7 % of patients received amphotericin B associated to fluconazole treatment. At the end of the study we got some important conclusions: the middle CGB is not presented as a method that effectively identify C. gattii strains , being useful only as a first triage, and should be coupled with PCR-RFLP; tests that measuring phospholipase activity are extremely relevant, we can affirm that the phospholipase may be the most important enzyme in the pathogenesis of cryptococcosis (serving as an indicator species of the genus Cryptococcus spp.), men are more affected of cryptococcosis than women, which are affected earlier as men with the age. The NK-cell leukemia found in 1 patient, HIV negative, infected by C. gattii and with a special expression of both their NK cells (CD19+CD16+CD56+ aberrant cells) gave origin to a lymphoid population anomalous CD56+CD19+, and this lymphoid tumor cells lineage may suggest that the substitution of immunocompetent cells by aberrant cells with a functional inefficiency could be responsible for the immunosuppression of the patient, and this suggests the importance of a detailed investigation in patients with meningitis caused by Cryptococcus spp., with an apparently competent immune system

INTRODUÇÃO: Embora a lista dos microrganismos envolvidos em fungemias esteja em expansão, as espécies de Candida permanecem como os principais agentes etiológicos, causando morbidade e mortalidade significativas. A introdução de novos antifúngicos na prática clínica tem reforçado a necessidade de estudos que monitorem mudanças na epidemiologia e no perfil de sensibilidade de Candida spp. a nível local e global. OBJETIVO: Avaliar a prevalência e incidência de candidemia no HC-FMUSP durante o ano de 2006 e determinar o perfil de suscetibilidade das espécies isoladas. MÉTODOS: Foi realizado um estudo retrospectivo no qual os pacientes identificados foram caracterizados de acordo com variáveis clinicas disponíveis nos sistemas laboratoriais. A concentração inibitória mínima (MIC) foi determinada pelo método de microdiluição Sensititre YeastOne Ò, considerado como referência, além do Etest Ò. As seguintes drogas foram avaliadas: anfotericina B (AMB), caspofungina (CAS), fluconazol (FLU), itraconazol (ITR), voriconazol (VOR) e posaconazol (POS). O método de disco difusão (DD) também foi utilizado para FLU e VOR. Os resultados obtidos pelas três metodologias foram comparados entre si. RESULTADOS: A prevalência de Candida em hemocultura foi de 3,5%. Foram identificados 136 casos de candidemia e a incidência global foi de 1,87 casos/1.000 admissões hospitalares e 0,27 casos por 1.000 pacientes dia. A mediana de idade foi de 40 anos e 58,1% dos pacientes eram homens. Neoplasia foi a doença de base mais frequente. Na distribuição das espécies, C. albicans foi a espécie mais isolada (52,2%), seguida por C. parapsilosis (22,1%), C. tropicalis (14,8%) e C. glabrata (6,6%). O perfil de sensibilidade foi determinado em 100 cepas de Candida. Destas, 100% mostraram-se suscetíveis a AMB e CAS; 98 % ao VOR; 91%, ao FLU e 66% ao ITR. Para o POS, MIC90 foi de 0,25 µg/mL. O percentual de concordância essencial (CE) e concordância categórica (CC) entre o teste de referência e o Etest Ò foi > 93%, exceto para o itraconazol (CE 80%, CC 70%). O percentual de CC para o fluconazol e voriconazol pelo DD versus Sensititre Ò e Etest Ò foi, respectivamente: FLU 94%, 95% e VOR 96%, 98%. CONCLUSÃO: A prevalência a incidência de candidemia encontrada e a distribuição das espécies concordaram com outros dados nacionais. Observou-se uma boa atividade in vitro da maioria das drogas para Candida spp., exceto itraconazol. Houve uma boa concordância essencial entre os métodos utilizados, exceto também para itraconazol. As discordâncias de categoria observadas entre os métodos resultaram em erros menores na maior parte dos casos / INTRODUCTION: Although the spectrum of fungi causing bloodstream infections continues to expand, Candida species remain responsible for the majority of cases, causing significant morbidity and mortality. The introduction of new antifungal agents in the clinical practice has increased the need for new studies for monitoring changes in the epidemiology and susceptibility profiles of candidemia at a local and global level. PURPOSE: To determine the prevalence and incidence of candidemia, species distribution and antifungal susceptibility patterns at large Brazilian tertiary public hospital during 2006. METHODS: Data were collected retrospectively. Patients were evaluated for selected variables. The minimal inhibitory concentrations (MIC) were determined using Sensititre YeastOne Ò, considered the reference method, and Etest Ò. The following drugs were tested: amphotericin B (AMB); caspofungin (CAS); and posaconazole (POS),fluconazole (FLU) and voriconazole (VOR). The disk diffusion method was also employed for FLU and VOR. MIC results obtained by Etest Ò, Sensititre YeastOne Ò and disk diffusion were compared. RESULTS: The prevalence of Candida spp. from blood cultures was 3,5%. One hundred and thirty-six cases of candidemia were identified. The overall incidence of candidemia was 1,87 cases per 1.000 admissions and 0,27 cases per 1.000 patient-days. 58.1% patients were male and the median age was 40 years. Cancer was the most frequent underlying disease. C. albicans was the most commonly identified species (52,2%), followed by C. parapsilosis (22,1%), C. tropicalis (14,8%) and C. glabrata (6,6%). The susceptibility profile of 100 Candida spp. isolates was: 100% to AMB B and CAS; 98 % to voriconazole; 91% to fluconazole; and 66% itraconazole. For posaconazole MIC90 was 0,25 µg/mL. The percentage of essential agreement (EA) and categorical agreement (CA) between the reference and test methods was > 93%, except for itraconazole (EA 80%, CA 70%). The (CA) between Sensititre YeastOne Ò versus disk diffusion and Etest Ò versus disk diffusion were respectively: FLU (94%) (95%) and VOR (96%) (98%). Minor errors accounted for the majority of all categorical errors. CONCLUSION: The prevalence and incidence of candidemia found in this study were in agreement with previous national reports. There was a good in vitro activity for most drugs tested against Candida spp. except for itraconazole. A good EA was demonstrated between methods except again for itraconazole. Minor errors accounted for the majority of all categorical errors for between methods.

Para que um produto de aplicação tópica apresente o efeito terapêutico é necessário que o fármaco seja liberado de seu veículo e atravesse a barreira do estrato córneo, atingindo seus sítios de ação nas camadas da pele nas quais o efeito é esperado. Os estudos de penetração cutânea in vitro, utilizando células de difusão de Franz modificadas, podem fornecer resultados úteis quanto à eficácia do produto. As dermatofitoses são micoses superficiais. São manifestações crônicas e resistentes ao tratamento, fato que se explica não só em decorrência da própria natureza destas afecções como também devido à baixa penetração de fármacos antimicóticos na pele. Dentre os antifúngicos disponíveis na terapêutica, ciclopirox olamina é uma substância que tem demonstrado elevada eficácia no tratamento de dermatofitoses. O presente trabalho teve como objetivo desenvolver formulações de uso tópico contendo 0,5% de ciclopirox olamina e avaliar a performance dessas formulações. Foi possível obter formulações estáveis fisicamente, contendo promotores de penetração cutânea. O sistema de amostragem automática utilizado para realização dos experimentos foi qualificado e apresentou-se em condições adequadas. / In order to present the pharmacological effect, a topical product must release the pharmaceutical active ingredient from its vehicle and this active must pass through the stratum corneum barrier, reaching the target layer of the skin, where its effect is desired. In vitro skin penetration studies, using modified diffusion Franz cells, can provide useful results regarding the product efficacy. Dermatophytosis is superficial mycosis resistant to treatment due to the disease nature and also to the low penetration profile of the antifungals into the skin. Among the antifungals available in the therapeutic arsenal, ciclopirox olamine has demonstrated a high efficacy in the treatment of dermatophytosis. The objectives of the present work were to develop topical formulations containing 0,5% of ciclopirox olamine and evaluate their performance. It was possible to obtain physically stable formulae, containing skin penetration enhancers. The automatic sampling system used in the experiments was qualified, showing adequate performance.

Para que um produto de aplicação tópica apresente o efeito terapêutico é necessário que o fármaco seja liberado de seu veículo e atravesse a barreira do estrato córneo, atingindo seus sítios de ação nas camadas da pele nas quais o efeito é esperado. Os estudos de penetração cutânea in vitro, utilizando células de difusão de Franz modificadas, podem fornecer resultados úteis quanto à eficácia do produto. As dermatofitoses são micoses superficiais. São manifestações crônicas e resistentes ao tratamento, fato que se explica não só em decorrência da própria natureza destas afecções como também devido à baixa penetração de fármacos antimicóticos na pele. Dentre os antifúngicos disponíveis na terapêutica, ciclopirox olamina é uma substância que tem demonstrado elevada eficácia no tratamento de dermatofitoses. O presente trabalho teve como objetivo desenvolver formulações de uso tópico contendo 0,5% de ciclopirox olamina e avaliar a performance dessas formulações. Foi possível obter formulações estáveis fisicamente, contendo promotores de penetração cutânea. O sistema de amostragem automática utilizado para realização dos experimentos foi qualificado e apresentou-se em condições adequadas. / In order to present the pharmacological effect, a topical product must release the pharmaceutical active ingredient from its vehicle and this active must pass through the stratum corneum barrier, reaching the target layer of the skin, where its effect is desired. In vitro skin penetration studies, using modified diffusion Franz cells, can provide useful results regarding the product efficacy. Dermatophytosis is superficial mycosis resistant to treatment due to the disease nature and also to the low penetration profile of the antifungals into the skin. Among the antifungals available in the therapeutic arsenal, ciclopirox olamine has demonstrated a high efficacy in the treatment of dermatophytosis. The objectives of the present work were to develop topical formulations containing 0,5% of ciclopirox olamine and evaluate their performance. It was possible to obtain physically stable formulae, containing skin penetration enhancers. The automatic sampling system used in the experiments was qualified, showing adequate performance.

Throughout the history of mankind, many infectious diseases have been treated with plant extracts. Venereal infections are one such group and are regarded as conditions that are highly responsive to traditional treatment. Aqueous, ethanol and ethyl acetate extracts of 13 plants used in South Africa for the treatment of venereal diseases were screened for in vitro antibacterial, antifungal, mutagenic and antimutagenic activities. Antibacterial activity was evaluated using the disc-diffusion and microdilution assays to determine the minimal inhibitory concentration (MIC) values of the extracts. The extracts were tested against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus, and the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Among the plants tested, Gunnera perpensa, Harpephyllum caffrum, Hypoxis latifolia and Ledebouria ovatifolia showed the best antibacterial activity. The aqueous rhizome extract of Gunnera perpensa displayed good activity against Gram-negative bacteria with an MIC value of 0.78 mg/ml, and against S. aureus (0.78 mg/ml). Aqueous and ethanol extracts of H. caffrum bark were active against both Gram-positive and Gram-negative bacteria. Hypoxis latifolia aqueous corm extracts exhibited very good MIC values against K. pneumoniae (0.78 mg/ml), E. coli and S. aureus (1.56 mg/ml). Ethanol and ethyl acetate bulb extracts of Ledebouria ovatifolia displayed good activity against Bacillus subtilis bacteria with MIC values of 0.78 mg/ml and 0.39 mg/ml respectively. Antifungal activity was evaluated using the microdilution bioassay. Good activity was shown by the ethanolic bark extracts of Bersama lucens and Harpephyllum caffrum against Candida albicans. Only in the case of Harpephyllum caffrum did aqueous extracts have activity against Candida albicans. In the Ames test, all plant extracts showed a negative genotoxic response except for ethanol and ethyl acetate bulb extracts of Cyrtanthus obliquus which induced mutations in TA98. Moderate antimutagenic activity was observed with the ethyl acetate extract of G. perpensa and the ethanolic extract of H. latifolia. High antibacterial and antifungal activity detected with Harpephyllum caffrum bark extracts resulted in an investigation on seasonal and geographical variation of this inhibitory activity. Seasonal variation in antibacterial and antifungal activities was investigated in order to determine the best collection time to ensure potential high medicinal activity in plant preparations. The highest inhibitory activity was detected with plant material collected in June and December 2003, with a decline in activity when collections were made in September 2004. The chemical profiles of TLC chromatograms were compared and little variation was found, particularly in the case of plant material obtained from the Botanic Garden of the University of KwaZulu-Natal and a 'Muthi' Shop in Pietermaritzburg. Identification of active compounds from G. perpensa and H. caffrum was not successful due to insufficient amounts of isolated fractions. / Thesis (Ph.D.)-University of KwaZulu-Natal, 2006.

Among all the major infectious human diseases, gastro-intestinal infections caused by microbial pathogens are a major cause of morbidity and infant death in developing countries, largely due to inadequate sewage disposal and contaminated water. Traditional health practitioners in South Africa play a crucial role in providing health care to the majority of the population. Many plants are locally used by South African traditional healers to treat microbial infections related to gastro-intestinal tracts. Ethnopharmacological and ethnobotanical studies using traditional knowledge as a selection strategy has given priority to certain plants for isolation and identification of plant novel bioactive compounds. Pharmacological and phytochemical studies of the investigated twelve medicinal plant species (from 10 families) extensively used as antimicrobials against gastro-intestinal infections was necessary to validate the use of the plants. Furthermore, to provide sufficient preliminary information for the isolation and identification of active compounds that are present in the investigated plants. Plant parts were sequentially extracted using petroleum ether (PE), dichloromethane (DCM) and 70% ethanol (EtOH). Cold water and boiled (decoction) extracts of the plant materials were prepared non- sequentially. Among the extracts, EtOH yielded the highest amount of plant substances. A total number of 85 extracts were evaluated for antibacterial activity, 80 for antifungal activity, 64 for anti-inflammatory activity, and 27 biologically active extracts were tested for genotoxicity. The microdilution method was used to determine the minimum inhibitory concentration values in the antibacterial assay against two Gram-negative bacteria (Escherichia coli ATCC 11775 and Klebsiella pneumoniae ATCC 13883) and two Gram-positive bacteria (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600). A modified microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values in the antifungal assay against Candida albicans. Cyclooxygenase assay was used to evaluate the anti-inflammatory activity of the extracts against cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes. The plant extracts were screened first at a concentration of 250 ƒÊg/ml per test sample, and then further screened at concentrations of 125 and 62.5 ƒÊg/ml for extracts that inhibited the COX-2 enzyme. The Ames test was used to test for genotoxicity in extracts that showed interesting pharmacological activities using Salmonella typhimurium strain TA98. Among the screened extracts, 25 extracts showed good antibacterial activity with MIC values . 1.0 mg/ml. Dichloromethane extracts exhibited the greatest antibacterial activity, and Gram-positive bacteria were most susceptible. The best antibacterial activity was exhibited by Becium obovatum leaf EtOH extracts with an MIC value of 0.074 mg/ml. A broad spectrum antibacterial activity was observed by leaf extracts of Cucumis hirsutus (PE), Haworthia limifolia (PE), Protea simplex (PE and DCM) and Dissotis princeps (EtOH) against both Gram-negative and Gram-positive bacteria. No interesting antibacterial activity was exhibited by water extracts with the exception of Dissotis princeps water extract with a good antibacterial activity against Gram-positive and Gram-negative bacteria. In the antifungal assay, 6 extracts showed interesting antifungal activity. Protea simplex leaf PE extract showed the best fungicidal activity with an MFC value of 0.014 mg/ml. The best overall antifungal activity was observed in plant EtOH extracts. Some extracts from Agapanthus campanulatus (leaves and roots), Dissotis princeps (leaves), Gladiolus dalenii (corms) and Protea simplex (leaves) showed good activity against Candida albicans. Twenty one extracts inhibited the COX-1 enzyme, while fifteen extracts inhibited the COX-2 enzyme at the lowest screening concentration of 62.5 ƒÊg/ml. The highest COX-1 inhibition at a concentration of 62.5 ƒÊg/ml was exhibited by Diospyros lycioides leaf PE extract (89.1%) while Agapanthus campanulatus root DCM extract showed the highest COX-2 inhibitory activity (83.7%) at the same concentration. In the Ames test, no genotoxicity was observed in any of the extracts, however more tests need to be done to confirm these results. Thin layer chromatograms of the organic solvent plant extracts were developed. The fingerprints of the plant extracts showed colours of bands at different Rf values when viewed under UV254 and UV366 suggesting that the investigated plant species contained different compounds in the extracts. In the quest to understand the source of the plants pharmacological activities, total phenolic compounds including condensed tannins, gallotannins and flavonoids were quantitatively investigated in terms of their amounts in the aqueous methanol extracts of the plants materials using spectrophotometric methods. Alkaloids and saponins were qualitatively determined. The amounts of total phenolics were determined by the Folin Ciocalteu assay, condensed tannins were determined by the butanol-HCl assay, while rhodanine and vanillin assays were used to determine the amounts of gallotannins and flavonoids respectively. Dragendorff reagent was used to detect alkaloids in the plant extracts on thin layer chromatographic plates, while the froth test was employed to detect saponins. Secondary metabolites varied with plant parts and species with Cyperus textilis (leaf) having the highest amounts of total phenolics, condensed tannins and flavonoids. The highest amount of gallotannins was detected in Protea simplex leaf extracts. All the investigated plant materials with the exception of Haworthia limifolia leaf, Protea simplex leaf, Antidesma venosum leaf and Dissotis princeps leaf tested positively to saponins. Alkaloids were detected in Haworthia limifolia leaf (PE and EtOH), Cucumis hirsutus leaf (EtOH), Becium obovatum root (DCM), Protea simplex root and bark (EtOH), Agapanthus campanulatus root (DCM) and leaf (EtOH), Cyperus textilis root (DCM), Vernonia natalensis leaf (PE), Antidesma venosum leaf (PE), Diospyros lycioides leaf (PE) and Dissotis princeps leaf (DCM) extracts. The results obtained from the investigation of the pharmacology and phytochemistry of the plant species used to treat microbial infections related to gastro-intestinal tracts, provide sufficient preliminary information to validate the use of some of the plants in traditional medicine. The information provided might be considered sufficient for further studies aimed at isolating and identifying the active compounds in the plant species, and evaluating possible synergism amongst the isolated compounds. / Thesis (M.Sc)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Orientador: Eduardo Luiz Bagagli / Resumo: Infecções fúngicas causadas por leveduras constituem um dos maiores problemas em pacientes hospitalizados em todo o mundo e têm se tornado uma importante causa de morbidade e mortalidade. Embora Candida albicans seja a espécie mais frequentemente isolada e sua forma de infecção ocorra geralmente por translocação endógena, tem-se observado um aumento das espécies não-albicans, com destaque para o complexo C. parapsilosis, cuja principal forma de infecção é exógena, provavelmente pelas mãos dos profissionais de saúde. Este trabalho propôs estudar os aspectos microbiológicos e ambientais das candidemias, com especial atenção para o complexo Candida parapsilosis, no hospital terciário HC/FMB/UNESP, Botucatu, utilizando-se de métodos moleculares, busca ativa dos agentes no ambiente hospitalar (ar, superfícies e mãos de profissionais de saúde), bem como determinar os fatores de virulência e susceptibilidade aos antifúngicos destas espécies e associação com o desfecho clínico. Os isolados clínicos (obtidos de hemoculturas, período 2007-2015) e ambientais (período 2014-2015) de C. parapsilosis sensu lato foram identificados pelo meio CHROMagar Candida, Vitek-2, sequenciamento do rDNA e perfis dos inteins VMA e ThrRS. Fatores de virulência (produção de proteinase, fosfolipase e biofilme) e perfis de susceptibilidade aos antifúngicos anfotericina B, fluconazol, voriconazol, caspofungina e micafungina foram estimados, e dados clínicos obtidos junto aos prontuários dos pacientes. Dentre ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fungal infections caused by yeasts are serious problems in hospitalized patients around the globe and have become a major cause of morbidity and mortality. Although Candida albicans is the most frequently isolated species and its infection usually occurs by endogenous translocation, an increase of non-albicans species has been observed, with emphasys for the C. parapsilosis complex, whose main route of infection is exogenous, probably by the hands of health professionals. This work proposes to study the microbiological and environmental aspects of candidemia, with special attention to the C. parapsilosis complex, in a public tertiary hospital HC/FMB/UNESP, in Botucatu, using the molecular methods, active search of agents in the hospital environment (air, surfaces and hands of health professionals), as well as to determine the virulence factors and susceptibility to the antifungals of these species and correlate with the patient clinical outcomes. The clinical isolates (obtained of the blood cultures, 2007-2015 period) and environmental (2014-2015 period) of the C. parapsilosis sensu lato were identified by CHROMagar Candida medium, Vitek-2, rDNA sequencing and VMA and ThrRS inteins profiles. Virulence factors (production of proteinase, phospholypase and biofilm) and profiles of susceptibility to antifungals amphotericin B, fluconazole, voriconazole, caspofungin and micafungin were estimated, and the clinical data were obtained from patient’s records. Among the clinical isolat... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2016-12-23T13:56:03Z (GMT). No. of bitstreams: 1 dissertacao-simonebrmaifrede.pdf: 1273260 bytes, checksum: d5439bcf28dc75da348359e5efa529ef (MD5) Previous issue date: 2009-03-06 / INTRODUÇÃO: Onicomicose é a infecção da unha causada por amplo espectro de espécies fúngicas, incluindo leveduras e fungos filamentosos dermatófitos e nãodermatófitos. Devido à variável suscetibilidade dos diversos agentes etiológicos às drogas antifúngicas, o diagnóstico laboratorial vem sendo considerado uma ferramenta importante para se estabelecer a etiologia e auxiliar na escolha do tratamento da onicomicose. Com base na elevada porcentagem de falha terapêutica no tratamento da onicomicose, tem-se evidenciado o interesse na padronização de testes de suscetibilidade in vitro de fungos filamentosos. OBJETIVOS: Estabelecer a freqüência das onicomicoses em relação a outras dermatomicoses; definir a etiologia das onicomicoses através do isolamento e identificação dos fungos; comparar o padrão de suscetibilidade entre fungos dermatófitos e não-dermatófitos às drogas fluconazol, cetoconazol, itraconazol, miconazol, ciclopirox, terbinafina e griseofulvina. MÉTODOS: As amostras clínicas foram colhidas através de raspagem e/ou fragmentação da unha e o exame microscópico direto foi realizado através do tratamento destas amostras com hidróxido de potássio (KOH) a 20% e tinta Parker. As culturas foram realizadas nos meios de ágar Sabouraud dextrose adicionado de 0,05 mg.mL-1 de cloranfenicol e ágar Mycose, incubados à temperatura ambiente e por um período de até 15 dias. A identificação dos fungos filamentosos foi baseada na observação de suas características macroscópicas e microscópicas e os testes de suscetibilidade in vitro a drogas antifúngicas foram baseados no Documento M38-A do CLSI. RESULTADOS: O diagnóstico laboratorial das dermatomicoses foi estabelecido em 69% dos 1.008 pacientes com lesões sugestivas de dermatomicoses encaminhados ao Laboratório de Diagnóstico Micológico do Depto. de Patologia / UFES, no período de 12/03/2004 a 14/08/2008. Onicomicose foi diagnosticada em 333 pacientes e os grupos de fungos mais isolados foram: leveduras 55,6%, fungos filamentosos não-dermatófitos 27,2% e dermatófitos 17,3%. Fungos dermatófitos foram mais inibidos in vitro que fungos não-dermatófitos. As drogas fluconazol e griseofulvina inibiram apenas fungos dermatófitos, enquanto a terbinafina foi a droga que mais inibiu os dois grupos de fungos e em baixas concentrações. A quantificação do inóculo por contagem em hemocitômetro e em placas de ágar Sabouraud revelou que o acerto do inóculo em espectrofotômetro pode ter uma boa correspondência se estabelecido em faixas de transmitância diferenciadas para os diversos tipos de fungos. CONCLUSÃO: É necessário estabelecer o diagnóstico laboratorial das onicomicoses, já que estas podem ser causadas por diversos agentes etiológicos e com diferentes suscetibilidades in vitro a várias drogas antifúngicas. / INTRODUCTION: Onychomycosis is the nail infection caused by a wide spectrum of fungi species, including yeasts, dermatophyte and nondermatophyte mould. Due to the variable susceptibility of the several etiologic agents to the antifungal drugs, the laboratorial diagnosis is being considered an important tool to establish the etiology and to help in the choice of the treatment of onychomycosis. Based on the high percentage of therapeutic flaw in the treatment of onychomycosis, there has been some evident interest in the standardization of the susceptibility tests in vitro of filamentous fungi. OBJECTIVES: To establish the frequency of the onychomycosis in relation to other dermatomycosis; to define the etiology of the onychomycosis by the isolation and identification of the fungi; to compare the pattern of susceptibility among dermatophytes and nondermatophytes mould to drugs such as fluconazole, cetoconazole, itraconazole, miconazole, ciclopirox, terbinafine and griseofulvine. METHODS: The clinical samples were collected by the scratching and/or fragmentation of the nail and the direct microscopic examination was made by the treatment of these samples with potassium hydroxide (KOH) at 20% and Parker ink. The cultures were made in dextrose agar Sabouraud with 0,05 mg.mL-1 of cloranphenicol and agar Mycosel, incubated to room temperature and for a period of up to 15 days. The identification of the filamentous fungi was based on the observation of its macroscopic and microscopic characteristics and the tests of susceptibility in vitro to the antifungal drugs were based on CLSI M38-A reference method. RESULTS: The laboratorial diagnosis of the dermatomycosis was established in 69% of the 1.008 patients with lesions that suggested dermatomycosis sent to the Laboratory of Mycologic Diagnosis from the Dept. of Pathology / UFES, in the period of 03/12/2004 to 08/14/2008. Onychomycosis was diagnosed in 333 patients and the groups of more isolated fungi were: yeasts 55,6%, nondermatophyte mould 27,2% and dermatophytes 17,3%. Dermatophytes were more inhibited in vitro than nondermatophytes ones. Drugs such as fluconazole and griseofulvine inhibited just dermatophytes fungi while terbinafine was the drug which most inhibited both groups of fungi and in low concentrations. The quantification of the inoculum for counting in haemocytometer and in plates of agar Sabouraud revealed that the correctness of the inoculum in espectrofotometer may have established a good correspondence in transmission bands differentiated for the various types of fungi. CONCLUSION: It is necessary to establish the laboratorial diagnosis of the onychomycosis, as these may be caused by several etiologic agents and with different susceptibilities in vitro to several antifungal drugs.

CNPQ / A produção e consumo de queijos finos especialmente queijo Azul e tipo Gorgonzola tem intensificado no Brasil. A natamicina é intencionalmente utilizada no processo industrial para este tipo de produto pelo seu potencial antifúngico. O uso da natamicina é permitido pela legislação brasileira na quantidade de até 5 mg/kg na casca, e não deve ser detectável a 2 mm de profundidade. No que se refere ao queijo tipo Gorgonzola são poucas as pesquisas direcionadas à determinação de natamicina, além de pouca fiscalização sobre seu uso e quase nenhuma análise sobre o perfil nutricional destes queijos. O objetivo deste estudo foi determinar a concentração, migração e declínio do teor de natamicina, assim como avaliar as características físicas de cor, físico-químicas e químicas além de avaliar o perfil de ácidos graxos do queijo Azul industrializado e tipo Gorgonzola comercializado na Região Sudoeste do Paraná e Oeste de Santa Catarina. Para isto, foram coletadas oito amostras de queijo tipo Gorgonzola de diferentes marcas em distintos municípios pertencentes a estas regiões, e na indústria foram coletadas amostras de queijo Azul ao longo da maturação. Na determinação de natamicina, foi utilizada espectrofotometria UV na terceira derivada na casca e internamente a cada 2 mm até a profundidade de 8 mm em queijo Azul e tipo Gorgonzola. Para a caracterização físico-química foram realizadas análises de umidade, cinzas, proteínas, lipídeos, carboidratos e valor calórico. Na avaliação química determinou-se o pH, atividade de água e concentração de sódio total. Para avaliação da cor utilizou-se o método instrumental CIELAB e o perfil de ácidos graxos foi determinado por cromatografia em fase gasosa. Do total de amostras de queijo avaliadas, os resultados da quantificação de natamicina mostraram que 50% das marcas apresentaram concentração na casca acima da quantidade preconizada pela legislação, enquanto que na massa do queijo 87,5% das marcas avaliadas ultrapassaram a concentração máxima de natamicina permitida. Em queijo Azul, houve declínio da natamicina ao longo da maturação, e a concentração de 1,5% do banho de imersão em natamicina foi ideal para que a partir dos 25 dias o queijo estivesse adequado para consumo. Por meio da caracterização físico-química de queijo tipo Gorgonzola foi possível observar que as marcas diferenciam-se sob a maioria dos aspectos, porém, ainda assim, todas tiveram valores baixos de umidade e altos percentuais de lipídeos e consequentemente elevado valor energético. Do perfil de ácidos graxos, foi possível constatar que majoritariamente, estes queijos são constituídos de ácidos graxos saturados (principalmente ácido Palmítico, ácido Esteárico e ácido Mirístico). Na maturação de queijo Azul houve redução da umidade, atividade de água, aumento do teor de sódio, cinzas e do pH. Na avaliação da cor observou-se que houve alterações com redução da luminosidade e aumento da predominância do verde sobre o amarelo. / The production and consumption of fine cheeses especially blue cheese and Gorgonzola type has stepped up in Brazil. Natamycin is intentionally used in the industrial process for this type of product for its antifungal potential. The use of natamycin is allowed by Brazilian legislation in the amount of up to 5 mg/kg in the bark, and should not be detectable at 2 mm depth. Regarding to the type cheese gorgonzola there are few researches directed to the determination of natamycin, besides the little supervision over its use and almost no analysis on the nutritional profile of these cheeses. The objective of this study was to determine the concentration, migration and decline of natamycin, as well as assess the physical characteristics of color, physical chemistry and chemistry in addition to assessing the fatty acids profile of blue cheese and Gorgonzola type sold in the southwest region of Paraná and west of Santa Catarina. For this, it was collected eight samples of cheese gorgonzola type of different brands in different municipalities belonging to these regions, and in industry were collected repetitions of blue cheese along the maturation. In the determination of natamycin, was used UV spectrophotometry in the third derived in the bark and internally to each 2 mm to a depth of 8 mm in blue cheese and Gorgonzola type. For the physico-chemical characterization were performed analyzes of moisture, ashes, proteins, lipids, carbohydrates and caloric value. In the chemical evaluation it was determined that the pH, water activity and concentration of sodium. For evaluation of the color we used the method CIELAB instrumental and the fatty acids profile was determined by gas chromatography. The total number of samples of cheese evaluated, the results of the quantification of natamycin showed that 50% of the brands showed, in the skin, concentration above of the quantity recommended by legislation, while the mass of the cheese 87.5% of registered evaluated exceeded the maximum concentration of natamycin allowed. In blue cheese, there was a decrease of natamycin along the maturation, and the concentration of 1.5% of the immersion bath in natamicyn was ideal for that from the 25 days the cheese was suitable for consumption. Through the physico-chemical characterization of cheese gorgonzola type it was possible to observe that the brands differ in most aspects, however, still, all had low levels of moisture and high percentages of lipids and consequently high energy value. From the fatty acids profile, it was established that mostly, these cheeses are made up of saturated fatty acids (mainly Palmitic acid, stearic acid and myristic acid). In the maturation of blue cheese, there was a reduction of moisture and the water activity, increased sodium content, ash and pH. In the evaluation of the color, it was observed that there have been changes with reduction of brightness and increased prevalence of green on the yellow.