Submitted by Isaac Francisco de Souza Dias (isaac.souzadias@ufpe.br) on 2016-03-03T17:04:26Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao Carolina Maria da Silva.pdf: 976111 bytes, checksum: 727eabad7b0e248858d85fdda9638e23 (MD5) / Made available in DSpace on 2016-03-03T17:04:26Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertaçao Carolina Maria da Silva.pdf: 976111 bytes, checksum: 727eabad7b0e248858d85fdda9638e23 (MD5) Previous issue date: 2011-11-29 / CNPQ / Infecções fúngicas invasivas têm se tornado cada vez mais freqüentes em neonatos, principalmente devido ao aumento da sobrevivência de prematuros e a deficiência do sistema imune. Dessa forma, torna-se relevante o conhecimento dos fatores epidemiológicos e susceptibilidade aos antifúngicos, uma vez que permitem o melhor conhecimento dos fatores associados à doença e a resistência dos agentes etiológicos, além da concentração ideal do medicamento a ser administrado para inibir e /ou matar o agente causal da infecção. Nesse contexto, os objetivos deste estudo foram diagnosticar candidemia em neonatos, associando os fatores epidemiológicos predisponentes e o perfil de susceptibilidade às drogas antifúngicas dos agentes etiológicos. No período de março de 2010 a julho de 2011, foram feitas coletas das amostras clínicas em neonatos de Unidades de Terapia Intensiva Neonatal do Hospital Agamenon Magalhães e do Instituto de Medicina Integral Professor Fernando Figueira. O diagnóstico micológico foi realizado através do exame direto, cultura e identificação dos agentes etiológicos. Foram coletadas amostras de sangue de 301 pacientes e isoladas 30 culturas, sendo identificadas Candida albicans (11), C. parapsilosis (11), C. pelliculosa (5), C. glabrata (1), C. guilliermondii (1) e C. tropicalis (1). Dos 30 pacientes com hemoculturas positivas para fungos, 90% eram pré-termos, 60% do sexo masculino, 93,4% possuíam peso ao nascer inferior a 2,5kg e as condições clínicas mais associadas foram icterícia e síndrome do desconforto respiratório. A grande maioria dos pacientes fazia uso de dispositivo terapêutico invasivo, destacando-se nutrição parenteral (96,7%) e cateterismo umbilical (73,3%). Quanto à susceptibilidade antifúngica todos os isolados de levedura foram sensíveis a anfotericina B, porém foi observada resistência ao fluconazol e voriconazol, principalmente por C. albicans, e 7 dos 11 isolados de C. parapsilosis foram resistentes a anidulafungina. As infecções fúngicas invasivas são frequentes em neonatos, permanecendo as espécies de Candida como as mais isoladas. Pacientes prematuros de baixo peso e que fazem uso de dispositivos invasivos são os mais acometidos, o conhecimento destes dados aliados aos resultados de susceptibilidade antifúngica in vitro possibilitam a prevenção e o tratamento mais adequado destas infecções. / Invasive fungal infections have become increasingly frequent in neonates, due to the increased survival of premature and disability of the immune system. The knowledge of epidemiological factors of these infections, as well as testing susceptibility to antifungal agents is relevant in this group of patients, because they allow a better understanding of the factors associated with the disease, the evaluation of the occurrence of fungal resistance, and the optimal concentration of the drug to be administered to inhibit and / or kill the agent of infection. In this context, the objectives of our study were to detect candidemia in neonates, the epidemiological factors associated with these infections and determine the antifungal susceptibility profile of the isolates. The samples were collected in the Neonatal Intensive Care Units from Agamenon Magalhães Hospital and Institute of Integrative Medicine Fernando Figueira, according to the medical request, from March 2010 to July 2011. The samples were manipulated to perform the direct examination and culture and then purified and identified. Samples were collected from 301 patients and we had isolated yeasts in 30 samples of blood , they were identified as Candida albicans (11), C. parapsilosis (11), C. pelliculosa (5), C. glabrata (1), C. guilliermondii (1), C. tropicalis (1). Of the 30 patients with positive blood cultures for fungi, 90% were preterm, 60% male, 93.4% had birth weight below 2.5 kg and the more usual conditions associated were clinical jaundice and respiratory distress syndrome. The vast majority of patients used invasive therapeutic device, especially parenteral nutrition (96.7%) and umbilical catheterization (73.3%). The antifungal susceptibility showed that all isolates were sensible to amphotericin B but some were resistente to fluconazole and voriconazole, mainly species of C. albicans, and 7 of 11 isolates of C. parapsilosis were resistant to anidulafungin. Invasive fungal infections are common in neonates, remaining Candida species as the most isolated. Preterm infants with low birth weight and use of invasive devices are the most affected and this knowledge combined with the in vitro antifungal susceptibility results enables a better prevention and treatment of these infections.

Several Fungal species have the potential to cause a broad spectrum of diseases in humans, ranging from mild superficial to disseminated invasive infections that involve the bloodstream and vital organs. Invasive fungal infections are severe, life-threatening diseases that result in the deaths of 1.5 million patients each year. The most common fungal species responsible for the majority of invasive fungal infections include Candida, Cryptococcus, and Aspergillus. The current treatment options for invasive fungal infections are restricted to three classes of antifungals: Azoles, polyenes, and echinocandins. The emergence of new fungal species, especially C. auris, marked by high resistance profiles and increased mortality rates (30-60%), has further exacerbated the limitations in its therapeutic options. This emphasizes the urgent need for effective alternatives to combat these deadly pathogens. C. auris isolates exhibited high resistance capability especially against azole (fluconazole) and polyene (amphotericin B) antifungals. Here, we utilized the combinatorial strategy to screen ~3400 FDA-approved drugs and clinical compounds to identify hits that can enhance/restore the antifungal activity of azoles and amphotericin B against resistant C. auris. The HIV protease inhibitors (lopinavir and ritonavir) were identified as potent enhancers to the antifungal activity of azole drugs (fluconazole, voriconazole and itraconazole). We confirmed that lopinavir and ritonavir have the capability to interfere with fungal efflux pump machinery. The in vivo efficacy of the combination of azole antifungals and HIV protease inhibitors was also evaluated to discover the best combination of itraconazole, lopinavir and ritonavir. Three drugs (lansoprazole, rolapitant and idebenone) were identified to effectively enhance the antifungal effects of amphotericin B and overcome its resistance in C. auris. Furthermore, the synergistic interactions of these combinations were applied on other medically important Candida, Cryptococcus, and Aspergillus species. In a comprehensive mechanistic study, we discovered that lansoprazole interferes with an essential target in the fungal mitochondrial cytochrome system, cytochrome bc1. This interference induces oxidative stress in fungal cells and subsequently enhances the antifungal activity of amphotericin B. For rolapitant, a transcriptomic analysis along with ATP luminescence assays confirmed that rolapitant at sub-inhibitory concentrations significantly interferes with ATP production in C. auris. For idebenone, checkerboard assays confirmed the synergistic interactions between amphotericin B and idebenone against a diversity of medically important fungal species. This combination exhibited a rapid fungicidal activity within 4 hours. Additionally, the cytotoxicity of this combination was assessed in a cell line model of kidney cells. Based on the potent in vitro and in vivo synergistic relationships observed for the identified combinations, it can be concluded that our approach offers a new hope to restore the antifungal activity of the existing antifungal drugs, even against resistant fungal infections. Additionally, it provides valuable insights into identifying novel targets to overcome resistance in multidrug-resistant fungal pathogens. / Doctor of Philosophy / Fungi comprise a diverse group of organisms that interact with humans in many good and bad aspects. Candida auris, a recently identified fungus, poses a significant threat to patients with weak immune systems. Infections with C. auris can be associated with mortality rates of up to 60%. Notably, this fungus is characterized by its powerful spreading capability and displays extraordinary resistance to antifungal agents, rendering many existing antifungal drugs ineffective. As a result, there is an unmet need to find efficient treatments for such deadly fungal infections. In this study, several drugs were identified with the potential to restore the activity of traditional antifungal drugs. The study identified four promising drugs (lopinavir, lansoprazole, rolapitant, and idebenone) with the potential to enhance the activity of the antifungal drugs against C. auris. lopinavir showed great potential to enhance the activity of azole antifungals, including fluconazole, voriconazole, and itraconazole. Furthermore, three other drugs (lansoprazole, rolapitant, and idebenone) were identified for their potential to enhance the activity of amphotericin B, which is considered a last-line antifungal therapy. We clarified the mechanisms by which these drugs could restore the activity of antifungal agents. Finally, we confirmed the effectiveness of these combinations in animal models, providing valuable insights into their potential for clinical applications. In summary, our research has opened promising avenues to overcome resistance and develop new treatments for hard-to-treat fungal infections.

Invasive fungal infections are prevalent and often deadly in immunocompromised patients. There continues to be a pressing need for the development of novel antifungal compounds since there are currently only 13 compounds licensed for the treatment of invasive fungal infections and antibiotic-resistant strains have been emerging. CAY-1 is an antifungal steroidal saponin which was isolated from the fruit of the cayenne pepper plant in 0.1% yield. In Vitro studies of CAY-1 have shown it to be an effective antifungal agent against sixteen pathogenic fungal strains and it showed no cytotoxicity toward mammalian cells up to 100 ìg/mL. The development of a practical synthesis of CAY-1 will potentially allow for further exploration of its medicinal utility and provide the opportunity to synthesize derivatives of CAY-1 which could be investigated in structure-activity relationship studies. To this end, methods for the preparation of they CAY-1 aglycone and pentasaccharide moieties have been investigated. Through this work, several partially protected stereoisomers of the CAY-1 aglycone have been prepared which can be used for the synthesis of saponin derivatives of CAY-1 for structure-activity relationship studies. Definitive characterization of one of these isomers, 3á-hydroxy-(22S, 25R)-5á-spirostan-2â-yl acetate, was achieved by X-ray crystallography. Furthermore, a quantitative inversion of the C-3 stereochemical configuration of this compound was achieved via an acetate group migration of the corresponding mesylate. The possibility of competition between the acetate migration and substitution mechanisms with various nucleophiles was explored. The results, however, indicate that this inversion only occurs via the acetate migration. Additionally, the CAY-1 pentasaccharide synthesis poses two significant challenges. First, these results demonstrate that the central 2, 3-branched portion can be synthesized efficiently from a partially protected glucopyranosyl acceptor since the C-2 and C-3 alcohols differ in their reactivity in glycosylation reactions. The second challenge is the ƒÀ-(1¨4) linkage to the galactosyl acceptor which significantly increases the complexity of the synthesis as compared to literature reported syntheses of other branched oligosaccharides. Nonetheless, this ƒÀ-(1¨4) linkage was achieved using a disarmed trichloroacetimidate glucosyl donor.

Nas últimas décadas, tem sido relatado o aumento de casos de infecções fúngicas invasivas por agentes oportunistas, tais como Aspergillus spp., Fusarium spp. e fungos da ordem Mucorales, em pacientes hospitalizados, particularmente os imunocomprometidos. Como os sintomas são frequentemente inespecíficos, esses patógenos não são identificados inicialmente como agentes causais. Além disso, muitas vezes o diagnóstico só é estabelecido em estágios tardios da infecção, pois as metodologias diagnósticas de rotina, como cultura e microscopia, apresentam limitações caracterizadas, sobretudo, por baixa sensibilidade e especificidade. Os métodos moleculares, incluindo as amplificações de material genômico por PCR, em tecidos a fresco e parafinados, têm sido mais aplicados para a melhoria na detecção e identificação desses patógenos. A técnica de PCR em tempo real apresenta a vantagem de fornecer resultados com rapidez e elevada sensibilidade, além de minimizar os riscos de contaminação ambiental das amostras. Para aprimorar o conhecimento sobre a eficácia desta técnica na detecção e identificação dos patógenos, neste estudo foram utilizados camundongos da linhagem BALB/c para o desenvolvimento de modelos de infecção invasiva por Aspergillus fumigatus, Rhizopus arrhizus e Fusarium solani. Após inoculação dos fungos por via intravenosa, os órgãos (pulmão, fígado, rins, cérebro e coração) foram retirados para realização dos exames de cultura, histopatologia, PCR semi-nested e PCR em tempo real. As culturas foram realizadas em ágar Sabouraud dextrose e incubadas por 3 a 5 dias à temperatura ambiente. Para as análises histopatológicas, uma parte dos órgãos foi emblocada em parafina e cortes foram submetidos às colorações por hematoxilina-eosina e Gomori-Grocott. Para as análises moleculares, foram realizadas clonagens dos amplicons obtidos com os mesmos primers gênero-específicos (Aspergillus spp. e Fusarium spp.) e ordem-específicos (mucoráceos) empregados nas reações de PCR. Os clones foram empregados na técnica de PCR em tempo real para avaliação da sensibilidade analítica dos ensaios e como controles positivos. Os tecidos a fresco e parafinados foram submetidos a extrações de DNA, e as amostras foram avaliadas por PCR do tipo semi-nested, e por PCR em tempo real, empregando-se os primers gênero e ordem-específicos. Após 48-72 horas, observou-se crescimento fúngico nas culturas, porém a identificação macroscópica foi possível somente após 96 horas. Nos exames histopatológicos foram observadas presença de estruturas fúngicas e alterações na morfologia tecidual, confirmando a disseminação da infecção nos três modelos experimentais. Os dois métodos de PCR (convencional e tempo real) empregando os primers para Aspergillus e mucoráceos demonstraram 100% de especificidade. Além disso, os primers para mucoráceos amplificaram amostras de DNA dos gêneros Rhizopus, Mucor e Lichtheimia, confirmando serem ordem-específicos. Por outro lado, os testes moleculares com emprego de diversos primers de Fusarium apresentaram reatividade cruzada, principalmente com amostras de DNA de Aspergillus e Rhizopus. O limiar de detecção (LD) das PCR semi-nested para Aspergillus e Rhizopus foi de 50 fentogramas de DNA, enquanto na PCR em tempo real o LD foi de 20 fentogramas de DNA e 2x102 plasmídios/ul. Os dois métodos moleculares detectaram DNA de Aspergillus e Rhizopus, em 100% das amostras de tecido a fresco e parafinado, corroborando com os resultados de cultura e histopatologia. Os resultados do estudo demonstraram que a PCR em tempo real foi capaz de detectar e diferenciar Aspergillus e Rhizopus nos tecidos a fresco e parafinados, com 100% de especificidade e maior sensibilidade analítica quando comparada à PCR semi-nested. Entretanto, os resultados obtidos com diversos primers de Fusarium utilizados nos ensaios de PCR foram inconsistentes em relação à especificidade das amplificações. A PCR em tempo real constituiu um método rápido para diagnosticar, com acurácia, aspergilose e mucormicose em tecidos, podendo ser implementada como alternativa na rotina diagnóstica de laboratórios de patologia ou microbiologia. Por outro lado, a técnica apresentou baixa especificidade com os primers de Fusarium selecionados neste estudo. Outras sequências gênicas deste fungo deverão ser avaliadas na técnica molecular para se atingir resultados precisos / Increase of invasive fungal infections by opportunistic agents, such as Aspergillus sp., Fusarium sp. and fungi from the order Mucorales, in immunocompromised patients has been reported in the last decades. As the symptoms are often non-specific, diagnosis are only established in late stages of infection. Routine diagnostic methodologies, such as culture and direct microscopy, have limitations due to their low sensitivity and specificity. Molecular methods, including amplifications of nucleic acids by PCR in fresh and paraffined tissues, have been more frequently applied to improve the detection and identification of these pathogens. The real-time PCR (qPCR) technique has the advantage of providing fast results with high sensitivity, as well as minimizing the risks of environmental contamination of the samples. This study aimed to evaluate the effectiveness of this technique for detection and discrimination of the fungal pathogens in tissues taken from BALB/c mice intravenously inoculated with Aspergillus fumigatus, Rhizopus arrhizus and Fusarium solani. The organs (lung, liver, kidneys, brain and heart) were removed from animals and analysed by culture, histopathology, semi-nested PCR and qPCR. Recombinant plasmid clones containing small sequences of Aspergillus, Fusarium and mucoraceous were used to draw qPCR standard curves and to evaluate the analytical sensitivity of the assays. The fresh and paraffined tissues were submitted to DNA extractions, and samples were evaluated by semi-nested PCR and qPCR with genus-specific primers for Aspergillus and Fusarium, and order-specific primers for mucoraceous. Cultures were positive for all fresh tissue samples. Presence of typical fungal structures and alterations in tissue morphology were observed in the histopathological examinations, confirming the dissemination of infection in the three experimental models. Both PCR assays employing Aspergillus and mucoraceous primers demonstrated 100% specificity. On the other hand, molecular tests using at least six different Fusarium primers showed cross reactivity, mainly with Aspergillus and Rhizopus DNA samples. The limit of detection (LD) of the semi-nested PCR for Aspergillus and Rhizopus was 50 femtograms of DNA, while for the qPCR it was 20 femtograms of DNA, and 2x102 plasmids/?l. Both molecular methods detected Aspergillus and Rhizopus DNA in 100% of fresh and paraffined tissue samples, corroborating the results with cultures and histopathology. The results of the study demonstrated that qPCR assay was able to detect and differentiate Aspergillus and Rhizopus in fresh and paraffined tissues, with 100% of specificity and higher analytical sensitivity when compared to semi-nested PCR assay. However, the results obtained with several Fusarium primers in the PCR assays were inconsistent regarding specificity of the amplifications. Real-time PCR assay is a fast and accurate method to diagnose aspergillosis and mucormycosis in tissues, and could be implemented as an alternative tool for diagnostic routine in pathology or microbiology laboratories. On the other hand, the technique showed low specificity with the Fusarium primers selected in this study. Other gene sequences should be evaluated by PCR assays techniques to achieve accurate results

As infecções fúngicas invasivas (IFIs) são um problema de saúde pública e representam uma importante causa de morbidade e mortalidade. A Anfotericina B (AnfB) é a droga de escolha no tratamento das IFIs. Apesar da sua eficácia, a AnfB está associada com efeitos adversos como a nefrotoxicidade. Com o número elevado de pacientes suscetíveis às IFIs, estudos foram desenvolvidos e demonstraram que a nefrotoxicidade pode ser prevenida através do uso de AnfBs modificadas. Dessas modificações, a AnfB incorporada à uma emulsão lipídica (AnfB/EL) apresenta baixo custo e similar eficácia. Existe uma alta prevalência de deficiência de vitamina D (dVD) na população mundial. Sendo assim, a hipovitaminose D pode acelerar a progressão da doença renal e refletir em mau prognóstico em casos de nefrotoxicidade. Esse trabalho visa analisar se a deficiência da vitamina D pode induzir a nefrotoxicidade da AnfB/EL. Ratos Wistar foram divididos em quatro grupos: controle, animais que receberam dieta padrão por 34 dias; AnfB/EL, animais que receberam dieta padrão por 34 dias e injeção intraperitoneal de AnfB/EL (5mg/kg/dia) nos últimos 4 dias; dVD, animais que receberam dieta depletada em vitamina D por 34 dias; e dVD+AnfB/EL, animais que receberam dieta depletada em vitamina D por 34 dias e a injeção intraperitoneal de AnfB/EL (5mg/kg/dia) nos últimos 4 dias. Amostras de sangue, urina e tecido renal foram coletadas para a análise dos efeitos da droga sobre a função, hemodinâmica e morfologia renal. A associação de AnfB/EL com a hipovitaminose D levou a injúria renal, lesão tubular discreta, hiperfosfatúria, hipermagnesiúria, hipertensão e comprometimento do mecanismo de concentração urinária. Dessa forma, é essencial monitorar os níveis de vitamina D em pacientes tratados com AnfB/EL, uma vez que a deficiência dessa vitamina é um fator indutor de nefrotoxicidade associada ao uso da AnfB/EL / Invasive fungal infections (IFI) have become a worldwide serious health problem and represent a significant cause of morbidity and mortality. Amphotericin B (AmB) is the drug of choice for the treatment of IFI. Despite its efficacy, use of AmB has been associated with adverse reactions such as nephrotoxicity The increasing number of high-risk patients, who are more susceptible to IFI and are more likely to use AmB, has enabled the development of modified AmBs in order to reduce nephrotoxicity. Among these formulations, an extemporaneous lipid emulsion preparation of AmB (AmB/LE) is a lower cost alternative with similar benefits. Moreover, studies have shown a high prevalence of vitamin D deficiency (VDD) in immunocompromised individuals and patients hospitalized in intensive care units. Thus, vitamin D deficiency or insufficiency may hasten the progression of kidney disease and reflect on a worse prognosis in cases of acute kidney injury and drug-induced nephrotoxicity. In view of the high worldwide incidence of hypovitaminosis D, the aim of this study was to investigate whether vitamin D deficiency may induce AmB/LE-related nephrotoxicity. Wistar rats were divided into four groups: control, received a standard diet for 34 days; AmB/LE, received a standard diet for 34 days and AmB/LE (5mg/kg/day) intraperitoneally in the last 4 days; VDD, received a vitamin D-free diet for 34 days; and VDD+AmB/LE, received a vitamin D-free diet for 34 days and AmB/LE (5mg/kg/day) intraperitoneally in the last 4 days. Blood, urine and renal tissue samples were collected in order to evaluate the effects of the drug on renal function, hemodynamic and morphology. Association of AmB/LE and vitamin D deficiency led to impaired renal function, mild tubular injury, hypertension and urinary concentration defect. Hence, it is important to monitor vitamin D levels in AmB/LE treated patients, since vitamin D deficiency induces AmB/LE nephrotoxicity