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Host-fungal pathogen interactions: A study of Candida albicans and mammalian macrophage and epithelial cells at the transcriptional levelDelorey, Toni Marie 31 May 2019 (has links)
It is estimated that fungal infections kill greater than 1.6 million people annually, a number that is comparable to the number of deaths associated with tuberculosis. Candida species are the fourth leading cause of hospital-acquired blood infections and Candida albicans is the most common cause of these fungal blood infections (known as candidemia). Immunocompromised individuals, such as those who have HIV/AIDS, those undergoing chemotherapy treatments, or those on broad-spectrum antibiotics, are most likely to develop candidemia. Candidemia is associated with a 20-40% mortality rate. However, when patient treatment for candidemia is delayed for over 48 hours, associated mortality rates increase to 78%. Blood infections can disseminate Candida albicans throughout the body, eventually leading to infection in vital organs like the liver, kidney and brain. Optimal patient outcomes are achieved if antifungal therapy is given within 12 hours after a blood sample is obtained for culture and testing. However, current blood tests cannot reliably detect Candida this early and thus antifungals are not routinely given to patients in this time frame. Counterintuitively, it is believed that some fungi, like many bacteria, are non-harmful residents in small intestines of most adults and this hypothesis is supported by the fact that the most common fungal species in the human gut is Candida albicans. However, intestinal overgrowth of C. albicans is linked to Crohn's disease, and disease-causing forms of C. albicans can arise from commensal strains that once resided in the patient’s gastrointestinal tract. The specific molecular mechanisms by which C. albicans interacts with host immune cells versus intestinal cells, and those that trigger Candida pathogenicity remain unknown. Many strains of Candida albicans have developed resistance to azoles, the major class of drugs used to treat both superficial and systemic infections. In order to develop new treatments, we must better understand host-fungal pathogen biology to determine novel antifungal targets or therapeutics to fight fungal infections at the early stages of infection. In this work, we developed a novel tool that allows us to measure which genes are important to both the host and Candida albicans simultaneously, in specific infection states. We have applied this tool to measure gene expression in Candida albicans interacting with mammalian macrophages and small intestine epithelial cells– at both the population and single cell levels. When examining populations of sorted infection samples, we found that host immune cells both exposed to and infected with fungal cells exhibit similar expression patterns. In contrast, phagocytosed C. albicans exhibit unique expression patterns compared to those merely exposed to macrophages. We found that immune response genes in single, Candida infected macrophages exhibited bimodal expression patterns for some immune response genes. We also observed examples of expression bimodality in live Candida inside of single macrophages. Both Candida albicans and host small intestine epithelial cells demonstrate distinct patterns of expression when exposed to each other at the population level, compared to unexposed controls. However, the magnitude of these differences is dependent on the multiplicity of infection. Some expression programs overlapped with those observed in populations of Candida cells interacting with macrophages, with key differences. We also observed expression bimodality among epithelial cells infected with C. albicans. We believe the information obtained using this technique could be used when considering new antifungal or therapeutics targets; if uniform and high expression of particular in genes in Candida populations phagocytosed by macrophages or invading epithelial cells leads to high and early protein production, these proteins may be effective antifungal targets. Similarly, if some host immune response genes are not expressed in a population of Candida infected macrophages as uniformly and highly as expected, these genes or proteins could be target of a therapeutic for patients with Candida infections that are resistant to azoles.
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Oral candida in HIV positive women: influence of oral hygiene, clinical and social factors on the carriage rates and the influence of virulence of the organism on the development of clinical infectionOwotade, Foluso John January 2014 (has links)
Degree of Doctor of Philosophy in Medicine by research only
A thesis submitted to the Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, in fulfilment of the requirements for the
Degree of Doctor of Philosophy in Medicine.
Johannesburg, 2014 / Introduction
Patients with HIV infection frequently encounter oral candidiasis, caused by Candida species. However, factors responsible for Candida colonisation and development of oral candidiasis in these patients are controversial. This study investigated the effect of social and clinical factors on oral Candida colonisation in HIV positive women. In addition, virulence of these organisms during clinical infection, the role of non-albicans Candida and reinfections with C. albicans were investigated.
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The effect of Dodonaea viscosa var. Angustifolia (L.F.) on the ultrastructure of Candida albicans cell wall and biofilm formationNaicker, Serisha Devi January 2012 (has links)
Dissertation submitted to the Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, in fulfillment of the requirements for the
degree of Master of Science in Medicine.
Johannesburg, 2012 / Oral candidiasis is an infection prevalent in immunocompromised individuals. The main
causative agent is Candida albicans. Many antifungal agents are available and are effectively
used. However, due to the development of drug resistance, toxicity and poor solubility resulting
in poor absorption; medicinal plants have been investigated. Dodonaea viscosa var. angustifolia,
an indigenous South African plant has shown to have an antifungal effect including inhibition of
adherence of C. albicans to oral epithelial cells; which is the crucial first step of infection. This
study investigated the effect of the crude extract on the ultrastructure of C. albicans cell wall,
which might be responsible for the reduced adherence to oral epithelial cells. The effect of the
plant extract on C. albicans germ tube and biofilm formation was also studied since biofilm
structure allows for high resistance to antifungal agents and host defense mechanisms.
Crude plant extracts were prepared using dried leaves and acetone. Three C. albicans strains
were used throughout the study. Minimal fungicidal concentrations of plant extract were
determined using a microdilution technique. Three subinhibitory concentrations 3.125, 1.562 and
0.781 mg/ml were selected for further studies. The effect of these subinhibitory concentrations of
plant extract on the C. albicans cell wall structure, cell membrane, germ tube formation, biofilm
formation and cell wall proteins were studied using transmission electron microscopy, light
microscopy, scanning electron microscopy and SDS-PAGE respectively.
v
The subinhibitory concentrations of crude plant extract rendered C. albicans cell wall thinner and
at some places caused cell wall breakage and disruption. This effect increased with a decrease in
plant extract concentration. The cell membrane was also damaged by the plant extract showing
increased undulation. This effect was not concentration dependent. The subinhibitory
concentrations decreased C. albicans germ tube formation and the effect increased with an
increase in concentration. Biofilm formation was reduced by the plant extract and in addition,
hyphal formation by cells within the biofilm was also reduced. However, SDS-PAGE showed
that on a molecular level, the plant extract did not remove any specific adhesin proteins from the
cell wall.
The crude plant extract of D. viscosa var. angustifolia at high concentrations, kills C. albicans
and at low concentrations, renders the surviving cells avirulent. Therefore it has the potential to
be developed into an effective therapeutic agent to treat and prevent oral candidiasis. However,
further research is required to identify the mode of action of the extract, the specific chemicals
responsible for the effect, and the cytotoxicity.
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Ocorrência das espécies de leveduras isoladas de sangue e cateter de pacientes internados em Hospital Público Infantil de São Paulo (período 2007 a 2010). / Yeasts species isolated from blood and catheter from patients admitted into Children\'s Public Hospital of São Paulo (period 2007 to 2010).Oliveira, Vanessa Kummer Perinazzo de 14 December 2011 (has links)
Investigamos a ocorrência de espécies de leveduras isoladas em um hospital infantil. Pesquisamos a presença de C. dubliniensis; investigamos as espécies do complexo Candida parapsilosis e estudamos as espécies deste complexo quanto a produção de biofilme. Pesquisamos os fatores relacionados a virulência e o perfil de sensibilidade pelo método Etest®. Dentre os anos em estudo C. albicans foi a levedura mais isolada com 36.45% seguida de C. tropicalis 23.36%, C. parapsilosis sensu stricto 21.49%, Pichia anômala 5.61%, C. guilliermondii 4.67%, C. krusei 2.80%; C. orthopsilosis 1.87%, C. glabrata 1.87%, C. metapsilosis 0.94%, C. pararugosa 0.94%. C. dubliniensis não foi isolada nesta pesquisa. No estudo do complexo Candida parapsilosis, C. parapsilosis sensu estricto foi a espécie mais prevalente. Produziram biofilme apenas oito amostras de C. parapsilosis sensu estricto e uma amostra de C. orthopsilosis. As espécies não-albicans produziram mais proteinase e hialuronidase do que C. albicans. No estudo da fosfolipase e hemolisina, C. albicans foi a espécie mais produtora. Todas as amostras foram sensíveis para anfotericina B e para fluconazol quatro amostras foram resistentes. Duas amostras foram resistentes para caspofungina. / We investigated the occurrence of yeast species isolated in a public hospital. We researched the presence of C. dubliniensis and investigated the species within the complex Candida parapsilosis and studied this complex in relation to biofilm´s production. We also investigated the factors related to virulence and studied the sensibility profile by the \"Etest ®\" method. During the years of study, C. albicans was the most isolated yeast with 36.45% followed by C. tropicalis 23,36%, C. parapsilosis sensu stricto 21,49%, Pichia anomala 5,61%, C. guilliermondii 4.67%, C. krusei 2.80%, C. orthopsilosis 1.87%, C. glabrata 1.87%, C. metapsilosis 0.94%, C. pararugosa 0.94%. C. dubliniensis was not isolated in this study. In the study of Candida parapsilosis complex, C. parapsilosis sensu stricto was the most prevalent species. Only eight strains of C. parapsilosis sensu stricto and one strain of C. orthopsilosis produce biofilm. The non-albicans species showed greater activity of the proteinase and hyaluronidase than C. albicans. In the study of hemolysin and phospholipase, C. albicans was the most producer. All strains were susceptible to amphotericin B and to fluconazole four were resistant. For caspofungin, two strains were resistant.
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Análogos da 8-hidroxiquinolina como candidatos a agentes antimicóticos : estudo da atividade antifúngica, mecanismos de ação e parâmetros toxicológicos / Analogues of 8-hydroxyquinoline as candidate to antimycotic agents : study of antifungal activity, action mechanisms and toxicological parametersPippi, Bruna January 2018 (has links)
A incidência e gravidade de doenças fúngicas têm aumentado significativamente, apresentando elevados índices de morbidade e mortalidade. Além disso, a emergência de fungos resistentes aos fármacos disponíveis e a escassez de agentes antifúngicos seguros tem tornado ainda mais crítico o controle desses micro-organismos. Portanto, as atuais circunstâncias justificam a nossa pesquisa destinada a caracterizar a atividade anti-Candida e antidermatofítica, obter informações sobre o mecanismo de ação antifúngico e avaliar parâmetros toxicológicos de análogos de 8-hidroxiquinolina. Cinco derivados da 8-hidroxiquinolinas foram avaliados: clioquinol (composto 1), ácido 8-hidroxiquinolinil-5-sulfônico (composto 2), ácido 7-iodo-8-hidroxiquinolinil-5-sulfônico (composto 3), 8-hidroxiquinolinil-5-(N-4-clorofenil)sulfonamida (PH151) e 8-hidroxiquinolinil-5-(N-4-metoxifenil)sulfonamida (PH153). As 8-hidroxiquinolinas mostraram atividade antifúngica para todos os isolados de Candida e dermatófitos testados. Clioquinol 1 foi o composto que apresentou menores valores de CIM, seguido das sulfonamidas (PH151 e PH153) e ácidos sulfônicos (composto 2 e 3). Os compostos foram fungistáticos para C. albicans e fungicidas para dermatófitos (exceto PH151). A modelagem PK/PD mostrou EC50 variando entre 0,181 - 0,306 μg/mL para clioquinol 1, 0,873 - 1,329 μg/mL para PH151 e 7,105 - 8,865 μg/mL para PH153. Em relação aos mecanismos de ação envolvidos, todos os derivados mostraram efeito sobre a parede celular fúngica. Os ácidos sulfônicos 2 e 3 comprometeram, também, a integridade funcional da membrana plasmática; e clioquinol 1, PH151 e PH153 inibiram a formação de pseudo-hifas em C. albicans. Ainda, clioquinol 1 exibiu ação sobre biofilme de Candida formado em microplaca de poliestireno e em dispositivo intrauterino. Clioquinol 1, PH151 e PH153 mostraram ótimos efeitos antifúngicos via oral em D. melanogaster imunodeficientes infectadas com C. albicans. Os derivados da 8-hidroxiquinolina apresentaram baixa toxicidade em modelos alternativos como pele de orelha de porco, D. melanogaster e embriões de galinha. O composto 3 foi o único que demonstrou toxicidade nesses modelos, uma vez que foi letal para o embrião de galinha. Os estudos com embriões e larvas de zebrafish foram realizados apenas com os derivados PH151 e PH153 e mostraram mortalidade dependente da dose. A aparente toxicidade foi detectada nas fases embrio-larval, logo, não se pode inferir sobre potencial toxicidade em adultos; mas podem servir de alerta para uma possível embriotoxicidade. Em conjunto, os dados deste estudo apoiam o potencial do clioquinol 1, PH151 e PH153 para tratar candidíase sistêmica e dermatomicose. Por fim, estes compostos demonstram ser candidatos antimicóticos apropriados para avançar em estudos com roedores. / The incidence and severity of fungal diseases have significantly increased, presenting high morbidity and mortality rates. In addition, the emergence of drug-resistant fungi and the scarcity of safe antifungal agents have become the control of these microorganisms even more critical. Therefore, the current circumstances justify our research designed to characterize anti-Candida and antidermatophytic activity, gain insights into the antifungal action mechanism and to evaluate toxicological parameter of 8-hydroxyquinoline analogues. Five 8-hydroxyquinoline derivatives were evaluated: clioquinol (compound 1), 8-hydroxy-5-quinolinesulfonic acid (compound 2), 8-hydroxy-7-iodo-5-quinolinesulfonic acid (compound 3) 8-hydroxyquinoline-5-(N-4-chlorophenyl)sulfonamide (PH151) and 8-hydroxyquinoline-5-(N-4-methoxyphenyl)sulfonamide (PH153). The 8-hydroxyquinolines showed antifungal activity for all Candida and dermatophytes tested. Clioquinol 1 was the compound that presented lower MIC values, followed by sulfonamides (PH151 and PH153) and sulfonic acids (compounds 2 and 3). The compounds were fungistatic for C. albicans and fungicides for dermatophytes (except PH151). PK/PD modeling showed EC50 ranging from 0.181 - 0.306 μg/mL for clioquinol 1, 0.873 - 1.329 μg/mL for PH151 and 7.105 - 8.865 μg/mL for PH153. Regarding the mechanisms of action involved, all derivatives showed effect on the fungal cell wall. Sulphonic acids 2 and 3 also compromised the functional integrity of the cytoplasmic membrane; and clioquinol 1, PH151 and PH153 inhibited the formation of pseudohyphas in C. albicans. Further, clioquinol 1 exhibited action on Candida biofilm formed on polystyrene microplate and intrauterine device. Clioquinol 1, PH151 and PH153 showed excellent oral antifungal effects in immunodeficients D. melanogaster infected with C. albicans. The 8-hydroxyquinoline derivatives showed low toxicity in alternative models such as pig ear skin, D. melanogaster and chicken embryos. Compound 3 was the only one that demonstrated toxicity in these models, since it was lethal to the chicken embryo. Studies with zebrafish embryos and larvae were performed only with the PH151 and PH153 derivatives and showed dose-dependent mortality.The apparent toxicity was detected in the embryo-larval stages, therefore, it is not possible to infer about potencial toxicity in adults; but may be an alert for possible embryotoxicity.Taken together, data from this study support the potential of clioquinol 1, PH151 and PH153 to treat systemic candidiasis and dermatomycosis. Finally, these compounds demonstrate to be appropriate antimycotic candidates to advance in rodent models.
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Prospecção de novas moléculas, biopolímeros e suas associações com atividade antifúngica e antibiofilme sobre espécies patogênicas de Candida / Prospection of new molecules, biopolymers and their associations with antifungal and antibiofilme activity on Candida pathogenic speciesBergamo, Vanessa Zafaneli January 2018 (has links)
A incidencia de infecções por espécies patogênicas de Candida vem crescendo e Candida albicans é a espécie que continua sendo a mais prevalente nas candidíases. Entretanto, observa-se o aumento no número de infecções por espécies emergentes de Candida não-albicans (CNA). Neste trabalho é avaliada a prospecção de novas moléculas e biopolímeros com atividade antifúngica e antibiofilme sobre cepas de Candida. Formulações de enxaguantes bucais foram desenvolvidas a partir do sal imidazólico cloreto de 1-n-hexadecil-3-metilimidazólico (C16MImCl) e selenocianatos alílicos 1-3. As formulações de enxaguantes bucais contendo C16MImCl na ausencia ou presença de sorbato de potássio (F1, F2, respectivamente), apresentaram uma Concentração Inibitória Mínima (CIM) de 50 μg/mL e os selenocianatos alílicos 1-3 apresentaram atividade antibiofilme pelo método cristal violeta (150 μg/mL). Os resultados expressos em Unidades Formadoras de Colônia (UFC/mL) e corante (brometo de 3- (4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT) confirmaram que a formulação F2 exerceu um efeito antibiofilme similar ou mesmo maior do que F3 (presença de cloreto de cetilpiridinio - CP) e Colgate®. A formulação contendo selenocianatos alílicos 1 e 3, ambos na ausencia ou presença de sorbato de potassio, exibiram o mesmo nível de atividade antibiofilme para C. tropicalis (UFC/cm2). A técnica da Microscopia Eletrônica de Varredura (MEV) avaliou as formulações contendo C16MImCl e selenocianatos alílicos (1-3) sobre a resina acrílica e foram observadas ausência de arquitetura de biofilme e material de matriz extracelular. A técnica Hens Egg Test-Chorion Allantoic Membrane (HET-CAM) também foi empregada e os compostos em estudo não foram irritantes. Sugere-se que o C16MImCl e os selenocianatos alílicos (1-3) são potentes candidatos a agentes antifúngicos e antibiofilmes para o desenvolvimento de formulações de enxaguantes bucais. Alem disso, foram utilizados agentes antifúngicos comerciais, como anidulafungina (AND), Anfotericina (AMB), Cetoconazol (CTZ), Itraconazol (ITR), bem como C16MImCl e o CP com o intuito de avaliar a susceptibilidade de Candida spp na presença dos polímeros (PVOH, álcool polivinílico altamente amorfo e PDDA, cloreto de poli(dialildimetilamônio) em diferentes proporções de polímero/fármaco (1:1, 99:1 e 1:99). O PVOH não apresentou CIMs capazes de inibir o crescimento de todos os fungos testados. Quando o PVOH foi associado, verificou-se a eficácia de quatro dos seis fármacos testados (CTZ, ITR, C16MImCl e AND), mostrando também ampla atividade antifúngica contra Candida spp. Em alguns casos, em concentrações muito baixas quando comparadas aos fármacos puros. O PDDA em água apresentou atividade antifúngica. Seis antifúngicos (CTZ, ITR, CP, C16MImCl, AND e AMB) apresentaram seu efeito antifúngico potencializado quando associado ao polímero catiônico (PDDA). Verificou-se mudança no potencial zeta, tamanho de partícula e estrutura do fármaco/polímero indicando a possível formação de micelas que solubilizam os fármacos em água. Os resultados obtidos neste trabalho evidenciam fortes candidatos para a aplicação em várias áreas biomédicas, possibilitando o uso de uma série de fármacos antifúngicos de diferentes classes. / The incidence of infections by pathogenic species of Candida has been increasing and Candida albicans is the species that continues being the most prevalent in candidiasis. However, the number of other species of Candida non-albicans (CNA) is now higher. This work aims to evaluate the prospection of new molecules and biopolymers with antifungal activity and antibiofilm on strains of Candida. Mouthwashes formulations have been developed from the imidazolium salt 1-methyl-3-octylimidazole chloride (C16MImCl) and for allylic selenocyanates 1-3. The mouthwash formulations containing C16MImCl in the absence or presence of potassium sorbate (F1, F2, respectively) presented a Minimal Inhibitory Concentration (MIC) of 50 μg/mL and the allylic selenocyanates 1-3 presented antibiofilm activity by crystal violet method (150 μg/mL). Both the colony forming units (CFU/mL) and (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) results confirmed that the F2 formulation exerted a similar antibiofilm effect or even greater than F3 (presence of the cetylpyridinium chloride compound - CP) and Colgate®. The data obtained for formulations containing the allylic selenocyanates (1-3) indicated that formulations containing 1 and 3 (both in the absence or presence of potassium sorbate) exhibited the same level of antibiofilm activity for C. tropicalis (in CFU/cm2). The Scanning electron microscopy (SEM) technique evaluated the formulations containing C16MImCl and allylic selenocyanates (1-3) on the acrylic resin and showed no biofilm architecture and extracellular matrix material. The Hens Egg Test-Chorion Allantoic Membrane (HET-CAM) technique was employed and the compounds under study were not irritating. It is suggested that C16MImCl and allyl selenocyanates (1-3) are potent candidates for antifungal and antibiofilms agents for the development of mouthwashes formulations. In addition, commercial antifungal agents as Anidulafungin (AND), Anfotericin (AMB), Ketoconazole (CTZ), Itraconazole (ITR), as well as C16MImCl and CP were used in order to evaluate the susceptibility of Candida spp in the presence or absence of polymers (PVOH, highly amorphous polyvinyl alcohol and PDDA, poly diallyl dimethyl ammonium chloride)) in different polymer/drug ratios (1:1; 99:1 and 1:99). PVOH did not present MICs capable of inhibiting the growth of all fungi tested. When PVOH was associated the efficacy of four of the six drugs tested (CTZ, ITR, C16MImCl and AND) was demonstrated, showing broad antifungal activity against Candida spp. In some cases, in very low concentrations when compared to pure drugs. PDDA in water showed antifungal activity. Six antifungals (CTZ, ITR, CP, C16MImCl, AND and AMB) had their antifungal effect potentiated when associated with the cationic polymer (PDDA). Change in zeta potential, particle size and and structure of the drug/polymer indicating the possible formation of micelles that solubilize the drugs in water.The results obtained in this work show strong candidates for the application in several biomedical areas, allowing the use of a series of antifungal drugs of different classes.
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Tools to study the transition from fungal commensalism to systemic infectionSood, Prashant January 2019 (has links)
Candida albicans colonizes the gastrointestinal tract of up to 75 % healthy individuals. It usually cohabits the gut as an innocuous commensal. But in critically ill patients whose gut barrier, immune system and normal gut microbiota are compromised, C. albicans often transmigrates the gut barrier, transforms into an invasive pathogen and causes fatal systemic infections. The genetic transitions that drive this transformation in C. albicans have been a major focus of research and have led to the identification of key transcription factors that regulate this commensal-to-pathogen transition. However, the current challenge lies in identifying the downstream pathways and effectors that bring this transition into effect. This thesis addressed this challenge by developing 11 new bioinformatics tools, including 6 comprehensive databases, 4 novel software packages and 1 analysis framework. These databases included a comprehensive topological map of the mammalian gut biogeography, a C. albicans microarray database comprising of 3,091 publically available microarray transcript profiles, C. albicans RNA-seq gene expression and small variant databases extracted from 1,177 publically available RNA-seq samples, a C. albicans gene alias database comprising of 113,297 gene aliases representing the 6,735 open reading frames of C. albicans, and a C. albicans gene ontology slim comprising of 1,194 C. albicans-specific gene ontology terms. These databases were accompanied by a robust analysis framework which brought together these resources for quality control, batch correction and weighted gene co-expression network analysis. All these tools were finally employed in a pilot exploration of the C. albicans gut commensal-to-pathogen transition, which demonstrated the effectiveness of these bioinformatics resources. The analysis unveiled known regulators, uncharacterized gene networks, pathways and effectors potentially crucial for the C. albicans gut commensal-topathogen transition. These resources are a step towards a better understanding of this transition and can also be utilized for examining various other aspects of C. albicans biology.
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Population structure of Candida species : spatio-temporal distribution of strain types and association with ancient HomininsMoorhouse, Alexander James January 2016 (has links)
No description available.
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The role and regulation of Pxl1 in the hyphal steering of Candida albicansCruz e Almeida, Mariana January 2017 (has links)
No description available.
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Development of a novel electrochemical assay for the rapid detection of pathogenic fungi and identification of clinically relevant Candida speciesMuir, Alastair January 2010 (has links)
A number of fungal species, particularly Candida species, are opportunistic pathogens capable of causing disease in humans. These range from relatively mild infections in healthy individuals (e.g. thrush) to life threatening systemic infections and colonisation of major organs in immunocompromised individuals. Existing methods of identifying Candida species in clinical samples are time and resource intensive and are not always specific enough to differentiate between drug-susceptible and drug-resistant species. Atlas Genetics have developed a novel electrochemical assay for the detection of nucleic acid from target pathogens using PCR followed by hybridisation by labelled probes. The work describes the development of a suite of probes and optimisation of reaction conditions for the rapid detection of fungal pathogens using this assay. A suite of five species-specific probes was developed to detect the five most clinically relevant Candida species as well as a pan-fungal probe capable of detection of DNA from any fungal species. Additionally, since the assay was novel, the work investigated other aspects such as probe and primer design which led to the development of a quick bioinformatics approach for design of species-specific probes which is also described. The results demonstrated that species-specific detection of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei was possible, and that there was no cross reactivity exhibited by any probe with isolates from a test panel of other Candida species. The limit of detection of the assay was shown to be approximately one genome in 1ml of blood or 10 whole cells in 1ml of blood. The use of solid-state electrodes for detection provides an opportunity for miniaturisation of the assay into a robust, easily operated, portable system capable of rapid detection of pathogenic DNA in clinical samples either at point of care or in the microbiological laboratory.
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