Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: HIV-positive patients have a significantly weakened immune system which makes them highly susceptible for opportunistic infections, requiring additional treatment. Cryptococcal meningitis and oropharyngeal candidiasis are treated with oral fluconazole. A great potential for drug-drug interactions (DDIs) between fluconazole and antiretrovirals (ARVs), efavirenz, nevirapine, and lopinavir/ritonavir, exists due to interference in common metabolic pathways. The outcome may result in the development of adverse drug reactions or drug resistance and treatment failure. Aim: The primary aim of this thesis was to evaluate the effect of fluconazole on the pharmacokinetics of efavirenz, nevirapine and lopinavir/ritonavir in HIV-infected patients diagnosed with cryptococcal meningitis or oropharyngeal candidiasis. Methods: A prospective study was conducted in 80 HIV-positive, treatment experienced adults (≥18 years old) treated in three different outpatient clinics in the Western Cape region. Patients were subdivided according to ARV regimen and the use of fluconazole. A sparse sampling design was used and corresponding ARV serum concentrations were determined by established HPLC and GC methods. Fluconazole serum concentrations were determined by a newly developed HPLC method. Patient characteristics, concomitant medications, clinical test data and ARV serum concentrations were included in a NONMEM generated, onecompartment, open pharmacometric model with first order elimination to detect any drugdrug interactions between fluconazole and the studied ARVs. The secondary outcome was to establish which patient characteristics influence ARV pharmacokinetics. Results: From 80 outpatients, a total of 276 ARV serum samples (137 efavirenz, 67 nevirapine and 72 lopinavir) were collected for pharmacokinetic evaluation. Efavirenz clearance was correlated with race and concomitant use of rifampicin. No significant covariates were established in the nevirapine model. In the lopinavir model, concomitant use of clotrimazole and the antituberculosis combination isoniazid, pyrazinamide and rifampicin were identified as significant covariates. Discussion: No significant effects of fluconazole on the pharmacokinetics of any of the studied ARVs were observed. Varying efavirenz plasma concentrations in different ethnic populations may be due to differences in gene expression particularly CYP2B6. Coloured patients had significantly lower efavirenz serum concentrations (56.8% decrease in clearance), which has not been previously described in the South African context. Although gender was not a significant covariate in the nevirapine model, female patients tended to have higher nevirapine serum concentrations. TB treatment in all patients receiving lopinavir consisted of a combination of isoniazid, pyrazinamide and rifampicin, each with different effects on CYP isoenzymes. The exact contributing factor of each drug in the ultimate decrease in lopinavir clearance (46.4%) can therefore not be established. Conclusions: Given the limitations of the sample size in the present study, no statistical significant effect of fluconazole on the pharmacokinetics of the investigated ARVs could be demonstrated. A retrospective analysis of the data showed various co-factors that influence the pharmacokinetics of the investigated ARVs. This data needs to be confirmed in a prospective study as the identified covariates are appropriate in the management of HIVinfected patients in the South African context. / AFRIKAANSE OPSOMMING: Agtergrond: HIV-positief pasiënte het ‘n aansienlike verswakte immuunstelsel, wat hul hoogs vatbaar tot opportunistiese infeksies maak, en dus, addisionele behandeling benodig. Cryptococcal meningitis en orofaringeale kandidiase word met orale flukonasool behandel. As gevolg van middeling in algemene metaboliese paaie is daar ‘n groot moontlikheid van middel-middel interaksies tussen flukonasool en die antiretrovirale (ARV) middels, efavirenz, nevirapine, en lopinavir/ritonavir. Die uitkomste hiervan mag tot die ontwikkeling van nadelige middel-middel interaksies of middelweerstandigheid en mislukte behandeling lei. Doel: Die primêre doel van hierdie tesis was om die effek van flukonasool op die farmakokinetika van efavirenz, nevirapine en lopinavir/ritonavir in HIV geïnfekteerde pasiënte met gediagnoseerde cryptococcal meningitis en orofaringeale kandidiase te evalueer. Metodes: Die studie was met 80 HIV-positief, behandeling-ervare volwassenes (≥18 jaar) onderneem. Voorafgenoemde was in drie verskillende buitepasiëntklinieke in die Wes-Kaap behandel. Pasiënte was volgens ARV regimen en die gebruik van flukonasool, of dan nie, verder verdeel. ‘n Beperkte steekproef ontwerp was gebruik, en ooreenstemmende ARV serum konsentrasies is deurgevestigde HPLC en GC metodes vasgestel. Flukonasool serum konsentrasies was deur ‘n nuutontwikkelde HPLC metode vasgestel. Pasiëntkenmerke, gepaardgaande medikasie, kliniesetoets data en ARV serum konsentrasies was by ‘n NONMEM genereerde, een-kompartement, oop farmakometriese model met eerste orde eliminasie ingesluit om enige middel interaksies tussen flukonasool en die bestudeerde ARVs op te tel. Die sekondêre uitkomste was om vas te stel watter pasiënt kenmerke ARV farmakokinetika beïnvloed. Resultate:Uit 80 buitepasïente was ‘n totaal van 276 ARV serum monsters (137 efavirenz, 67 nevirapine en 72 lopinavir) vir farmakokinetiese evaluasie gekollekteer. Efavirenz opruiming was met ras gekorreleer asook gepaardgaande gebruik van rifampisien. Geen betekenisvolle ko-variante was in die nevirapine model vasgestel nie. In die lopinavir model het die gepaardgaande gebruik van clotrimazole en die anti-tuberkulose kombinasie isoniazied, pyrazinamied en rifampisien, lopinavir opruiming verminder. Bespreking: In hierdie studie is geen betekenisvolle effekte van flukanosool op die farmakokinetika van enige van die bestudeerde ARVs waargeneem nie. Afwisselende efavirenz plasma konsentrasies in verskillende etniese populasies mag aan verskille in geenuitdrukking, veral CYP2B6, toegeskryf word. Kleurling pasïente het betekenisvolle verlaagde efavirenz serum konsentrasies getoon (56.8% verlaging in opruiming). Hierdie bevinding is nog nooit voorheen in die Suid-Afrikaanse konteks beskryf nie. Alhoewel geslag nie ‘n beduidende ko-variant in die nevirapine model was nie, het vroulike pasïente geneig om hoer nevirapine serum konsentrasies te hê. TB behandeling, in alle pasïente wat lopinavir ontvang het, het uit die volgende kombinasie bestaan: isoniazied, pyrazinamied en rifampisien, elk met hul eie effekte op CYP isoensieme. Die presiese bydra van elke middel in die uiteindelike verlaging (46.4%) in lopinavir opruiming kan dus nie vasgestel word nie. Gevolgtrekking: Gegewe die beperkings van die steekproef in die huidige studie, kon geen statistiese beduidende effek van flukonazool op die farmakokinetika van die betrokke ARVs gedemonstreer word nie. ‘n Retrospektiewe analise van die data het gewys dat verskeidene ko-faktore die farmakokinetika van die betrokke ARVs beïnvloed. Hierdie data moet in ‘n prospektiewe studie bevestig word omdat die geidentifiseerde covariates die bestuur van MIV-positiewe pasiente in die Suid-Afrikaanse konteks te verbeter.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Kateřina Brokešová Supervisor: Ing. Martin Drastík, Ph. D. Thesis title: The use of core-shell columns for fluconazole determination A novel HPLC method for determination of fluconazole in dissolution test samples was developed and partly validated. A matrix formed by lactic and glycolic acid copolymer branched by different compounds was used as a drug carrier. Fluconazol was incorporated as the model drug. The concentration profile of fluconazole was studied by developed HPLC method during the dissolution test. A modern core-shell column Ascentis Express RP-Amide, 10 cm × 3.0 mm; 2.7 μm was employed. A mixture of acetate buffer pH 5.0:methanol (80:20) served as the mobile phase. The flow rate was 0.70 ml/min and the detection wavelength was 260 nm. The temperature of analysis was 50 řC. The retention time of fluconazole was 3.3 min and the whole analysis took just 4 min. Keywords: fluconazole, core-shell column, HPLC, PLGA

A ceratite fúngica é uma doença grave que pode levar à perda da visão. O tratamento consiste na aplicação de antifúngicos, no entanto a administração tópica não tem se mostrado efetiva devido aos mecanismos de defesa do olho que levam à baixa retenção da formulação no local de aplicação e à baixa permeação do fármaco através da córnea. Sendo assim, formulações mais adequadas e sistemas de liberação vêm sendo estudados na tentativa de melhorar a biodisponibilidade local de antifúngicos. No presente trabalho foram obtidos e caracterizados dois sistemas para a liberação ocular do fluconazol (FLU), um antifúngico de atividade reconhecida: um gel termorreversível in situ e micropartículas poliméricas. A permeação passiva e iontoforética do fármaco através da córnea foi estudada in vitro a partir dos sistemas desenvolvidos. O gel termorreversível in situ contendo 16% de poloxamer e 1,0% de quitosana apresentou temperatura de geleificação adequada, propriedades mucoadesivas, melhores parâmetros mecânicos (dureza, compressibilidade e adesividade) que ambos os polímeros separadamente e mostrou-se superior que micropartículas poliméricas, com fluxo de permeação passiva cerca de duas vezes maior. A maior quantidade de fármaco retido na córnea foi obtida após aplicação da iontoforese em solução aquosa do fármaco. Ainda assim, o fluxo iontoforético do FLU não foi significativamente diferente do fluxo passivo a partir do gel termorreversível. O desempenho in vivo desta formulação foi então avaliado em modelo animal. Estudos de permeação, utilizando a técnica de microdiálise para amostragem da câmara anterior, confirmaram a maior biodisponibilidade do fármaco. Por fim, exames de cintilografia em humanos confirmaram maior tempo de retenção na superfície ocular. Portanto, o gel termorreversível in situ obtido e caracterizado no presente trabalho e a iontoforese, representam sistemas promissores para a liberação ocular tópica do FLU. O primeiro possui características promotoras de permeação, prolongado tempo de retenção e facilidade de obtenção e administração, e o segundo é capaz de promover maior retenção do fármaco na córnea. / Fungal keratitis is a serious disease that can lead to loss of sight. The treatment consists in antifungal application; however topical administration has not shown to be effective due to defense mechanisms of the eye, which leads to low retention at the application site and low permeation of the drug trough the cornea. In this way, more suitable formulations and delivery systems have been studied in an attempt to increase local availability of antifungal agents. At the present work it was obtained and characterized two systems for the passive and iontophoretic ocular delivery of fluconazole (FLU), a well known antifungal agent: an in situ forming gel and polymeric microparticles. Passive and iontophoretic drug permeation across the cornea was studied in vitro from the developed systems. The in situ forming gel containing 16% of poloxamer and 1.0% of chitosan presented more adequate gelation temperature, mucoadhesive properties, improved mechanical parameters (strength, compressibility and adhesiveness) than both polymers separately and showed to be superior to the polymeric microparticles, with passive permeation flux almost twice higher. The greatest amount of drug retained in the cornea was achieved after iontophoresis application using an aqueous solution of the drug. Even though, the iontophoretic drug flux was not significantly different from the passive drug flux using the in situ forming gel. The in vivo performance of this formulation was then evaluated in an animal model. Permeation studies, using the microdialysis technique for the anterior chamber sampling, confirmed the increased drug availability. At the end, scintigraphy exams in humans confirmed the greater retention time at the ocular surface. Therefore, the in situ forming gel obtained and characterized at the present work and iontophoresis, represent promising systems for the topical ocular delivery of FLU. The first possess permeation enhancement properties, prolonged retention time and facility of obtention and administration, and the second is capable of promoting higher drug retention in the cornea.

A participação das espécies reativas de oxigênio (ERO) no mecanismo de ação dos antifúngicos azólicos, bem como a relação entre resistência aos antifúngicos e resposta ao estresse oxidativo, têm sido sugeridos. Entretanto, os dados ainda são inconclusivos e diferem entre os micro-organismos. Neste estudo estão apresentados os resultados da geração de ERO pelo fluconazol em isolados de C. glabrata sensíveis e resistentes a esse antifúngico e a resposta antioxidante da levedura. Nesses isolados, tratados e não tratados com fluconazol em concentração subinibitória, de acordo com sua concentração inibitória mínima (CIM), até fase de crescimento estacionário, foi avaliado se o fluconazol geraria ERO. Subsequentemente, foram analisadas as defesas antioxidantes glutationa peroxidase (GPx), superóxido dismutase (SOD), glutationa-S-transferase (GST), consumo de peróxido de hidrogênio e glutationa total, bem como possível dano oxidativo causado pelo fluconazol em lipídeos, proteínas e ácidos nucleicos e os níveis de nitritos e nitratos. Os resultados mostram que nos isolados de C. glabrata sensíveis e resistentes ao fluconazol, na presença do antifúngico, houve um aumento da geração ERO e maior atividade enzimática da GPx e SOD comparada a dos isolados não tratados com fluconazol, não havendo diferença estatística entre isolados sensíveis e resistentes nesses três parâmetros citados. Em relação à enzima GST, os isolados sensíveis mostraram maior atividade enzimática comparada aos resistentes, e quando as células sensíveis foram tratadas com fluconazol, a atividade da GST diminuiu. O fluconazol não induziu dano oxidativo em proteínas e em lipídeos, entretanto foi observado dano oxidativo ao DNA. Diante disso, sugere-se que o fluconazol gera ERO como parte do seu mecanismo antifúngico em C. glabrata em fase de crescimento estacionário, induzindo dano oxidativo no DNA. Como resposta, observa-se aumento na atividade enzimática da SOD e da GPx na levedura. O entendimento da resposta antioxidante de leveduras patogênicas tem importante interesse clínico, uma vez que o desenvolvimento racional de novas drogas antifúngicas requer conhecimento do metabolismo fúngico. / The participation of reactive oxygen species (ROS) in azoles antifungal mechanism of action has been suggested, as well as the relation between antifungal resistance and oxidative stress response. However, data are still inconclusive and differ between microorganisms. This study presents the results of ROS generation by fluconazole in fluconazole-susceptible and resistant C. glabrata strains and their antioxidant response. It was evaluated whether fluconazole generates ROS in those isolates treated and untreated with fluconazole at sub-inhibitory concentration according to their minimal inhibitory concentration (MIC). This treatment was conducted until stationary growth phase was reached. Subsequently, the antioxidant defenses glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione-S-transferase (GST), consumption of hydrogen peroxide and total glutathione, the possible oxidative damage in lipids, proteins and nucleic acids and the levels of nitrites and nitrates were analyzed. Results showed increased ROS generation in fluconazole-susceptible and resistant C. glabrata strains treated with fluconazole, and also higher GPx and SOD enzymatic activity, compared to untreated cells. No statistical difference of those three parameters was observed between susceptible and resistant strains. In relation to GST, susceptible strains demonstrated higher activity compared to the resistant ones, and when susceptible cells were treated with fluconazole the GST activity decreased compared to untreated. Fluconazole did not induce oxidative damage in proteins and in lipids, however oxidative DNA damage was observed. Therefore, it is suggested that fluconazole generates ROS as part of its antifungal mechanism in C. glabrata at stationary growth phase, inducing oxidative DNA damage. In response, there was increase in the enzymatic activity of SOD and GPx in yeast. The understanding of the pathogenic yeast antioxidant response has important clinical interest, since the rational development of new antifungal drugs requires knowledge about the fungal metabolism.

Introdução: Nas últimas décadas, as infeções fúngicas invasivas por leveduras tornou-se um importante problema de saúde pública, dado sua incidência crescente relacionada ao aumento da população suscetível. O reconhecimento destes patógenos em aspectos como, distribuicão ambiental e caraterísticas fenotípicas, são pilares essenciais para sua vigilância e controle. Objetivo: Descrever a frequência dos agentes de criptococose e outras leveduras com potencial patogênico e comparar essa frequência em excretas de aves silvestres em três municípios do estado de São Paulo, com vistas a melhor conhecimento da distribuição desses agentes no ambiente, além de determinar o perfil de suscetibilidade in vitro a antifúngicos de uso clínico. Método: No período de 2 anos, aves silvestres foram identificadas em áreas de circulação de 3 municípios de São Paulo (Praia Grande, Santos e Rio Claro) e submetidas à coleta de excretas para isolamento de leveduras com potencial patogênico. Análise microscópica e macroscópica para classificação presuntiva de gênero foram realizados em todas as colônias de leveduras obtidas das amostras de excretas. A suscetibilidade dos isolados de leveduras aos antífúngicos: fluconazol, voriconazol e anfotericina B foi determinada segundo método de referência europeu (AFST-EUCAST). Análise de dados: Foi utilizada a regressão de Poisson com a opção robusta para estimar razões de prevalência e identificar variáveis associadas com os principais isolados identificados, com opção de cluster para agrupar os isolados por excreta. Foi avaliado o nível de concordância entre os dois métodos de identificação (fenotípico e MALDI-TOF), utilizando o coeficiente Kappa. Adicionalmente, foi estimada a correlação entre os MIC´s dos fármacos estudados no total de espécies identificadas, utilizando o coeficiente de correlação de Spearman. Resultados: Das 294 excretas coletadas, 42,2 por cento continham leveduras, incluindo espécies de Candida 62 por cento , seguido por Rhodotorula 16,4 por cento , Cryptococcus 10,4 por cento , Trichosporon 6,6 por cento e Pichia 2,7 por cento . Muitas espécies, verificadas em alta frequência, tem forte potêncial de causar infecção invasiva, como: C. parapsilosis stricto sensu, C. tropicalis, Clavispora lusitaniae, C. krusei, C. orthopsilosis, C. glabrata, C. laurenti, C. albicans, C. metapsilosis, C. nivariensis e Meyerozyma guilliermondii. A resistência ao fluconazol, voriconazol e anfotericina B ocorreu nesses isolados, sendo documentada uma forte correlação entre a susceptibilidade, principalmente entre os azois (fluconazol e voriconazol), no entanto, a correlação mesmo sendo menor também foi significativa entre esses fármacos e a anfotericina. De 13 espécies de aves silvestres dispersoras de leveduras, as de maior frequência foram: Sula leucogaster 26,2 por cento , Turdus leucomelas 17 por cento , Larus dominicanus 15 por cento , Thalasseus maximus 11,2 por cento , Thalasseus acuflavidus 5,4 por cento , Tangara sayaca 4,4 por cento , Turdus amaurochalinus 3,7 por cento , Sterna hirundinacea e Pitangus sulphuratus 2,7 por cento . Os gêneros identificados apresentaram associações entre local, estação do ano e espécies de aves. Conclusões: Dentre as principais espécies de aves estudadas, 3 eram de hábitos migratórios (Thalasseus maximus, Thalasseus acuflavidus e Sterna hirundinacea) o que permite inferir dispersão interamericana de leveduras patogênicas. Diversas espécies resistentes a antifúngicos foram descritas, pela primeira vez, em excretas de aves silvestres conferindo a este estudo o valor de contribuir para o conhecimento da epidemiologia das infecções fúngicas por leveduras. / Background: In recent decades, invasive yeast fungal infections have become an important public health problem, due to their increasing incidence related to the increase in the susceptible population. The recognition of these pathogens in aspects such as environmental distribution and phenotypic characteristics are essential pillars for their surveillance and control. Objective: To describe the frequency of cryptococcosis agents and other yeasts with pathogenic potential and to compare this frequency in excreta of wild birds in three municipalities of the state of São Paulo, with a view to a better knowledge of the distribution of these agents in the environment, in addition to determining the profile of in vitro susceptibility to antifungals for clinical use. Method: During two years, wild birds were identified in circulation areas of three municipalities of São Paulo (Praia Grande, Santos and Rio Claro) and were screened for yeasts with pathogenic potential. Microscopic and macroscopic analysis for presumptive genus classification were performed in all yeast colonies obtained from excreta samples. Susceptibility of yeast isolates to antifungals: fluconazole, amphotericin B and voriconazole was determined according to the European reference method (AFST-EUCAST). Data analysis: Poisson regression was used with the robust option to estimate prevalence ratios and to identify variables associated with the main isolates identified, with option of cluster to group the isolates by excreta. The level of agreement between the two identification methods (phenotype and MALDI-TOF) was evaluated using the Kappa coefficient. Additionally, the correlation was estimated between MICs of the drugs studied in total of species identified, using Spearman\'s correlation coefficients. Results: Of the 294 excreta collected, half contained yeasts, including Candida species (62 per cent ), followed by Rhodotorula (16.4 per cent ), Cryptococcus (10.4 per cent ), Trichosporon (6.3 per cent ) and Pichia (2.7 per cent ). Many species, verified at high frequency, have a strong potential to cause invasive infection, such as: C. parapsilosis stricto sensu, C. tropicalis, Clavispora lusitaniae, C. krusei, C. orthopsilosis, C. glabrata, C. laurentii, C. albicans, C. metapsilosis, C. nivariensis and Meyerozyma guilliermondii. Resistance to fluconazole, voriconazole and amphotericin B occurred in these isolates and a strong correlation was reported between susceptibility, mainly between azole (fluconazole and voriconazole), however, the correlation, even though it was lower, was also significant between these drugs and amphotericin. From 13 species of wild birds dispersing yeasts, the ones with the highest frequency were: Sula leucogaster 26,2 per cent , Turdus leucomelas 17 per cent , Larus dominicanus 15 per cent , Thalasseus maximus 11,2 per cent , Thalasseus acuflavidus 5,4 per cent , Tangara sayaca 4,4 per cent , Turdus amaurochalinus 3,7 per cent , Sterna hirundinacea e Pitangus sulphuratus 2,7 per cent . The identified genera presented associations between site, season of the year and species of birds. Conclusion: Among the main species of birds studied, 3 were of migratory habits (Thalasseus maximus, Thalasseus acuflavidus and Sterna hirundinacea), which allows inferring the inter - American dispersion of pathogenic yeasts. Several species resistant to antifungal were described for the first time in excreta of wild birds, conferring to this study the value of contributing to the knowledge of the epidemiology of fungal infections by yeasts.

<p>&nbsp / </p> <p align="left">One of the most common HIV-associated opportunistic infections is candidiasis, caused by <i><font face="TimesNewRoman,Italic">Candida albicans </font></i><font lang="KO" face="TimesNewRoman">or other </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. The mechanisms of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">drug resistance in the African continent have also not been described. In this study, 255 </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">colonization mechanisms in HIV-infected African patients.</font></i></i></i></i></i></i></i></i></i></i></p>

<p>&nbsp / </p> <p align="left">One of the most common HIV-associated opportunistic infections is candidiasis, caused by <i><font face="TimesNewRoman,Italic">Candida albicans </font></i><font lang="KO" face="TimesNewRoman">or other </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. The mechanisms of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">drug resistance in the African continent have also not been described. In this study, 255 </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">colonization mechanisms in HIV-infected African patients.</font></i></i></i></i></i></i></i></i></i></i></p>

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (D.P.H.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Widespread and prolonged usage of azoles in recent years has led to the rapid development of drug resistance in Candida species. In Candida albicans, resistance to fluconazole causes cross-resistance to other antifungals and an increase in virulence, making treatment still more difficult because of the limited therapeutical options. Nonetheless, other species has emerged as significant pathogens of clinical importance. Fluconazole resistance has been clinically described in Candida dubliniensis isolates and it is easily induced by in vitro exposure to the drug, but little is known about its consequences. In the present study, two groups of C. dubliniensis isolates were evaluated and compared in some points with the closely related species, C. albicans. One group was composed by fluconazole-susceptible clinical isolates and the other was composed by fluconazole-resistant laboratory derivatives from the former, in order to examine the changes on phenotypic characteristics and antifungal susceptibility accompanying the development of resistance to fluconazole. Resistant derivatives showed minimal inhibitory concentrations equal or higher than 64 μg/mL and proved to keep most of phenotypic characteristics that were tested before resistance was induced. However, some strains were not found to produce pseudomycelia and chlamydospores. Against killer toxins, C. dubliniensis did not present biotypes enough to permit its differentiation from C. albicans. On the other hand, when proteinase activity was evaluated, C. albicans activity was significantly higher than C. dubliniensis . Resistant derivatives of C. dubliniensis showed proteinase activity similar to fluconazole-susceptible isolates, suggesting that fluconazole resistance may not necessarily result on an increase of virulence in this species. Partial atmosphere of CO2, fluconazole at subinhibitory concentrations, or combination of both conditions, as well as addition of antiretrovirals on induction culture medium did not influence on proteinase activity of C. albicans and C. dubliniensis isolates. Finally, fluconazole-resistant isolates showed cross-resistance with ketoconazole, itraconazole, ravuconazole and terbinafine. In addition, associations of amphotericin B or terbinafine with azoles resulted mainly on indifferent interactions. On fluconazole-susceptible isolates, the most positive interaction came from association of amphotericin B with voriconazole. When resistance was induced, the best activity was found when terbinafine was combined with itraconazole. / O uso prolongado e indiscriminado dos azólicos nos últimos anos permitiu um rápido desenvolvimento de resistência aos fármacos nas espécies de Candida. Em Candida albicans, a resistência ao fluconazol causa resistência cruzada a outros antifúngicos e aumento na virulência, tornando o tratamento ainda mais complicado por causa das opções terapêuticas limitadas. Ainda assim, outras espécies emergiram como patógenos significantes de importância clínica. A resistência ao fluconazol tem sido clinicamente descrita em isolados de Candida dubliniensis e é facilmente induzida pela exposição in vitro ao azólico, mas pouco se conhece sobre suas conseqüências. No presente estudo, dois grupos de isolados de C. dubliniensis foram avaliados e comparados em alguns aspectos com C. albicans. Um grupo era composto de isolados clínicos sensíveis ao fluconazol, e o outro, derivado do primeiro, era composto de isolados resistentes, com o intuito de analisar as alterações nas características fenotípicas e na suscetibilidade aos antifúngicos que acompanham o desenvolvimento de resistência ao fluconazol. Os derivados resistentes obtidos evidenciaram concentrações inibitórias mínimas maiores ou iguais a 64 μg/mL e mantiveram grande parte das características fenotípicas avaliadas anteriormente à indução da resistência. Entretanto, apenas o Ágar Suco de Tomate permitiu a identificação dessas estruturas em 100% dos isolados resistentes. Diante das toxinas killer , C. albicans e C. dubliniensis exibiram biotipos incapazes de permitir a diferenciação entre as espécies. Por outro lado, quando avaliada a atividade de proteinase, a de Candida albicans foi significativamente maior do que a de C. dubliniensis. Os derivados resistentes de C. dubliniensis evidenciaram atividade de proteinase semelhante a dos isolados sensíveis ao fluconazol, sugerindo que a resistência ao antifúngico pode não necessariamente acarretar aumento de virulência na espécie. Microaerofilia, fluconazol a concentrações subinibitórias, ou a combinação dessas duas condições, bem como a adição de anti-retrovirais ao meio de indução da enzima não exerceram influência sobre a atividade de proteinase em C. albicans e C. dubliniensis. C. dubliniensis resistentes ao fluconazol evidenciaram resistência cruzada com cetoconazol, itraconazol, ravuconazol e terbinafina. Em adição, as associações de anfotericina B ou terbinafina com azólicos resultaram principalmente em interações indiferentes. Em isolados sensíveis, a interação mais positiva resultou da associação de anfotericina B com voriconazol. Quando a resistência foi induzida, a melhor atividade foi encontrada quando a terbinafina foi combinada com itraconazol.