Made available in DSpace on 2015-07-22T20:49:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-26 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A candidíase nosocomial é grande preocupação em hospitais terciários em todo o mundo. A infecção ocorre geralmente em pacientes com doenças neoplásicas e degenerativas e é considerada a quarta causa mais freqüente de infecções sangüíneas. O diagnóstico da candidemia ou candidíase hematogênica tem sido problemático porque os sinais e sintomas clínicos são inespecíficos, o que conduz a atrasos no diagnóstico e, conseqüentemente, na terapia antifúngica apropriada. Desenvolvemos a técnica de ELISA de inibição para a detecção do antígeno de 65 kDa em modelo experimental de candidemia e para o diagnóstico de pacientes em UTI com suspeita de candidemia. Anticorpo monoclonal anti-65 kDa foi produzido e testado para a detecção do antígeno comum de 65 kDa produzido por C. albicans, C. tropicalis, C. parapsilosis em modelos murinos candidemia. No modelo experimental o antígeno de 65 kDa foi detectado no soro do camundongos em concentrações variando de 0,012 a 3,25 μg / ml. Vinte pacientes com candidemia foram avaliados pelo teste de ELISA de inibição em soros seqüenciais. Dezesseis (80%) pacientes apresentavam o antígeno de 65 kDa em concentrações variando de 0,07 a 5,0 μg/ml. Os soros sequenciais de pacientes com candidemia apresentaram 3 diferentes padrões de antigenemia; 1 – clareamento total da antigenemia; 2-clareamento inicial e recidiva da antigenemia; e 3 – clareamento parcial da antigenemia. Nossos resultados indicam que a detecção da molécula de 65 kDa pode ser muito útil para o diagnóstico de candidemia por C. albicans, C. tropicalis e C. parapsilosis. Avaliamos também um possível papel biológico da molécula de 65 kDa. Os ensaios demonstraram que a proteína de 65 kDa tem ligantes de fibronectina de matriz extracelular, tendo um possível papel da adesão do fungo no hospedeiro. / Nosocomial candidiasis is a major concern in tertiary care hospitals worldwide. This infection generally occurs in patients with degenerative and neoplastic diseases and is considered the fourth most frequent cause of bloodstream infections. Diagnosis of candidemia or hematogenous candidiasis has been problematic because clinical signs and symptoms are nonspecific, leading to delays in diagnosis and, consequently, in appropriate antifungal therapy. We developed an inhibition ELISA for detection of a 65 kDa-antigen in an experimental model of candidemia and for diagnosis of patients in ICUs with suspected candidemia. An anti-65 kDa monoclonal antibody was tested for detection of the 65 kDa antigen produced by C. albicans, C. tropicalis, and C. parapsilosis in murine candidemia models. The 65 kDa-antigen was detected in sera at concentrations ranging from 0.012 to 3.25 μg/ml. A total of 20 human patients with candidemia were then evaluated with the inhibition ELISA using sequential sera. Sixteen (80%) patients had the 65 kDa-antigen in concentrations ranging from 0.07 to 5.0 μg/ml. Sequential sera from patients with candidemia presented 3 different patterns of antigenemia of the 65 kDa molecule; 1- total clearance of antigenemia; 2-initial clearance and relapse of antigenemia; and 3- partial clearance of antigenemia. Our results indicate detection of the 65 kDa protein may be a valuable tool in the diagnosis of candidemia by C. albicans, C. tropicalis, and C. parapsilosis. We also evaluated a possible biological role of the molecule of 65 kDa from C. albicans. The tests showed that the protein of 65 kDa is bound to fibronectin from extracellular matrix, and a possible role of the adhesion of the fungus in the host. / TEDE / BV UNIFESP: Teses e dissertações

Among fungal infections, those caused by Candida spp are the most recurrent. Candidaemias are the most emergent and amphotericin B has been the mainstay of therapy in spite of its therapeutic failures. Because of these limitations, susceptibility testing of fungi against antifungal agents has been evidencing a great importance in order to confirm susceptibility to the used antifungal agents. In this study, 144 Candida spp isolates from immunocompromised patients, that either evolved into death (Group 1) or survived to candidaemia episodes (Group 2), were assessed. Tests were made according to proceedings and culture media described on M27-A2 document currently provided by Clinical and Laboratory Standards Institute (CLSI). Media such as Yeast Nitrogen Base- dextrose (YNBd) and Antibiotic Medium were also tested, being used a higher number of Amphotericin B concentrations. Susceptibility values of strains from Group 1 were significantly higher than Group 2 when Antibiotic Medium 3 (AM3) and YNBd were used. The Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) media values for strains from patients that survived to candidaemia were, respectively, 0.5 μg/dL and 0.7 μg/dL; whereas MIC and MFC media values for strains from patients that evolved into death were, respectively, 0.6 μg/dL and 0.9 μg/dL. Significant differences were not observed between the two groups of strains when using RPMI 1640 medium. / Dentre as infecções fúngicas, aquelas provocadas por Candida spp são as mais recorrentes, sendo as candidemias a forma mais emergente, cujo tratamento mais utilizado tem sido à base de anfotericina B, apesar das falhas terapêuticas a esse poliênico. Devido a tais falhas, os testes de avaliação da suscetibilidade de fungos a agentes antifúngicos passam a ter grande importância para confirmar a suscetibilidade aos agentes antifúngicos usados. Neste estudo foram avaliadas 144 isolados de Candida spp, oriundas de pacientes imunodeprimidos que evoluíram ao óbito ou que sobreviveram aos episódios de candidemia. Os testes foram realizados de acordo com os procedimentos e meios de cultura descritos no documento M27-A2 do atual Clinical and Laboratory Standards Institute (CLSI). Testou-se também os meios Yeast Nitrogen Base dextrosado (YNBd) e Antibiotic Médium 3 (AM3) sendo utilizado um número maior de concentrações de anfotericina B. Os valores de suscetibilidade das cepas provenientes dos pacientes que evoluíram ao óbito foram significativamente maiores que os valores obtidos dos pacientes que sobreviveram a candidemia quando os meios utilizados foram o Antibiotic Médium 3 (AM3) e o YNBd, nos quais encontrou-se valores médios de Concentração Inibitória Mínima (CIM) igual a 0,5 μg/dL e valores de Concentração Fungicida Mínima (CFM) igual a 0,7 μg/dL para pacientes que sobreviveram a candidemia; valores médios de CIM igual a 0,6 μg/dL e de CFM igual a 0,9 μg/dL foram encontrados nas cepas dos pacientes que evoluiram ao óbito. Com o meio de cultura RPMI 1640 não foram observadas diferenças significativas nos valores de suscetibilidade entre os dois grupos de pacientes.

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Pereira.pdf: 2756512 bytes, checksum: 9892b08404437328cce673864effe453 (MD5) / Made available in DSpace on 2018-04-06T15:21:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação_Vivian do N. Pereira.pdf: 2756512 bytes, checksum: 9892b08404437328cce673864effe453 (MD5) Previous issue date: 2015-08-31 / Candidemia is an infection recognized as public health problem related to health assistance. Filling the knowledge gaps through epidemiological studies and diagnostic tools to respond emerging threats that may be involved in the disease process is among the actions to eliminate these infections. The objective of this research was to study the Candida yeasts isolated from blood cultures of patients admitted in 11 hospitals of Manaus in 2013, as well to study the clinical epidemiological aspects of candidemia, the antifungal profile of susceptibility and virulence factors. The detection of yeasts in the collected samples was performed by conventional methodology. The antifungal susceptibility was determined using E-Test® strips. The studied virulence factors were proteinase, phospholipase, hemolysin and biofilm formation. During the study period, there were 85 cases of infection in the blood of 81 patients with Candida spp. In relation to gender, 67.9% were male, the median age of adult patients was 53 years and between pediatric patients was 60 days. The main risk conditions were: previous use of antibiotics (98.6%), presence of central venous catheter (95.9%), ICU (84.9%), use of protectors of gastric mucosa (84.4 %) and one-third of patients are from natal care and 72.8% of patients had up to 1 year old. According to the clinical course of patients, 40.7% died. C. albicans was the most frequently isolated species (34.57%), followed by C. tropicalis (32.1%) and Candida parapsilosis (17.28%). A susceptibility test was performed against Caspofungin, Micafungin, and Voriconazole, where the resistance 12.1%, 4.3% and 4.3% was respectively found. The virulence factors related to production of enzymes was: Proteinase with 56.4% of the isolates being producers, Phospholipase 36.2% and 78.7% hemolysin with this capability being more expressive in the species C. albicans. The biofilm formation was present in 34% of the isolates whose C. tropicalis was the most significant in this factor. The importance of epidemiology studies and local phenotypic studies may contribute to improve decision-making process and propose more effective measures for control and prevention of these infections. / As Candidemias são Infecções Relacionadas à Assistência a Saúde que são reconhecidamente um problema de saúde pública, e dentre as várias ações como medidas de eliminação destas infecções, estão o preenchimento das lacunas de conhecimento para responder as ameaças emergentes, isto se dá por estudos epidemiológicos, de diagnóstico e dos fatores que podem estar envolvidos no processo de adoecimento. O objetivo deste trabalho foi estudar as leveduras do gênero Candida isoladas a partir de hemoculturas de pacientes internados em 11 Hospitais de Manaus no ano de 2013, bem como estudar os aspectos clínicos epidemiológicos das candidemias, o perfil de susceptibilidade antifúngica e fatores de virulência. A detecção das leveduras presentes nas amostras coletadas foi realizada por meio de metodologia convencional. A susceptibilidade antifúngica foi determinada utilizando fitas de Etest® seguindo as recomendações do fabricante. Os fatores de virulência estudados foram: proteinase, fosfolipase, hemolisinas e formação de biofilme. No período de estudo, foram registrados 85 casos de infecção na corrente sanguínea de 81 pacientes por Candida spp. Em relação ao gênero 67,9% eram do sexo masculino, a mediana das idades entre os pacientes adultos foi de 53 anos e entre os pediátricos de 60 dias. As principais condições de risco foram: o uso prévio de antibióticos (98,6%), presença de cateter venoso central (95,9%), internação em UTI/CTI (84,9%), uso de gastroprotetores (84,4%) e um terço dos pacientes são procedentes de maternidades, e 72,8% dos pacientes tinham até 1 ano de idade. Segundo a evolução clínica dos pacientes, 40,7% foram a óbito. C. albicans foi a espécie isolada com mais frequência (34,57%), seguido de C. tropicalis (32,1%), C. parapsilosis (17,28%). Foi realizado teste de susceptibilidade frente a Caspofungina, Micafungina e Voriconazol, onde a resistência encontrada foi de 12,1%, 4,3% e 4,3% respectivamente. Os fatores de virulência relacionados à produção de enzimas foram: Proteinase com 56,4% dos isolados sendo produtores, Fosfolipase 36,2% e Hemolisina 78,7 com esta capacidade; sendo mais expressiva nas espécies de C. albicans. A formação de biofilme estava presente em 34% dos isolados, sendo a C. tropicalis a mais expressiva neste fator. A importância de estudos de epidemiologia e estudos fenotípicos locais podem contribuir para a melhoria da tomada de decisão e propor medidas mais efetivas de controle e prevenção destas infecções.

INTRODUÇÃO: A frequência de Candida parapsilosistem apresentado considerável aumento em UTIs neonatais. Embora a taxa de resistência dessa espécie aos azólicos seja baixa, recentemente têm sido relatados surtos de candidemia por isolados resistentes. A capacidade de adesão e formação de biofilme por essa espécie confere maior potencial patogênico e resistência aos antifúngicos. Portanto, a vigilância epidemiológica, tanto da resistência aos antifúngicos como da virulência dos isolados, é fundamental para o controle e prevenção das infecções e surtoshospitalares. A técnica de MALDI-TOF MS pode ser uma ferramenta útil para realizar análises proteômicas das células planctônicas e sésseis de Candida parapsilosis,e identificar possíveis alvos terapêuticos ou biomarcadores, específicos do biofilme. MÉTODOS: Isolados clínicos do complexo Candida parapsilosis de dois hospitais universitários públicos brasileiros, foram submetidos à identificação por RAPD, RFLP e MALDI-TOF MS e aos testes de suscetibilidade aos antifúngicos. Ensaios de formação de biofilme foram realizados para quantificar a biomassa, a atividade metabólica e ainda, avaliar atividade in vitrodos antifúngicos contra as células sésseis dos isolados com alta formação de biofilme. A análise proteômica por MALDI-TOF MS das células planctônicas e sésseis dos isolados com alta formação de biofilme, foi realizada nas plataformas VITEK-MS(TM) e Microflex(TM). Isolados de Candida parapsilosis (sensu stricto) foram genotipados por PFGE e análise de microssatélites. Os genótipos foram correlacionados com dadosclínicos, para investigar a ocorrência de um surto em CTI adulto, e as sequências do gene ERG11dos isolados não suscetíveis aos azólicos (NSA) foram analisadas. RESULTADOS: Foram obtidos 38 isolados do complexo Candida parapsilosis, sendo Candida parapsilosis(sensu stricto) a espécie de maior frequência, superando 80% em ambos os hospitais, seguida de C. orthopsilosis e C. metapsilosis. Embora todos os isolados tenham sido suscetíveis à anfotericina B ( < 2 mg/L) e apresentado suscetibilidade intermediária à anidulafungina, caspofungina e micafungina ( > 0,002 mg/L), elevada frequência de não suscetibilidade (resistência ou suscetibilidade intermediária) ao fluconazol e voriconazol foi observada entre isolados de um dos hospitais. Alta formação de biofilme foi observada apenas entre os isolados da espécie Candida parapsilosis(sensu stricto). Por outro lado, a maioria dos isolados NSA, apresentou baixa formação de biofilme e baixa atividade metabólica. Apenas anfotericina B apresentou atividade contra os biofilmes de Candida parapsilosis. As duas plataformas de MALDI-TOF MS conseguiram diferenciar os perfis proteômicos das células planctônicas e sésseis dos isolados. A genotipagem de Candida parapsilosis(sensu stricto) revelou a persistência de isolados clonais NSA e a mutação A395T no gene ERG11foi identificada exclusivamente entre os isolados resistentes ao azólicos. O uso de corticosteroide foi associado, estatisticamente, com a ocorrência de isolados clonais NSA. CONCLUSÕES: Candida parapsilosis (sensu stricto) se mantém como a principal espécie do complexo em infecções sanguíneas. Isolados resistentes aos azólicos, com mutações no gene ERG11, ocorreram nos dois hospitais avaliados. A correlação dos genótipos com os dados clínicos evidenciou a ocorrência de um surto envolvendo isolados clonais NSA, com associação estatisticamente significativa, ao uso prévio de corticosteroides. Candida parapsilosis (sensu stricto) foi a única espécie que apresentou alta formação de biofilme, o qual demonstrou elevada resistência às equinocandinas. As duas plataformas de MALDI-TOF MS, diferenciaram os perfis proteômicos, das células planctônicas e sésseis de Candida parapsilosis, demonstrando o potencial emprego dessa tecnologia na identificação de possíveis alvos terapêuticos ou biomarcadores, específicos de biofilmes / INTRODUCTION: The frequency of Candida parapsilosis isolates has increased considerably in neonatal ICUs. Although resistance to azoles is usually low in this species, candidemia outbreaks by resistant isolates have been recently reported. Theability of adhesion and biofilm formation by this species confers higher pathogenic potential and resistance to antifungal agents. Therefore, establishment of profiles of antifungal susceptibility and virulence, besides the epidemiological surveillance ofC. parapsilosisisolates are essential for the control and prevention of nosocomial infections and outbreaks. The MALDI-TOF MS technique can be a useful tool to perform proteomic analyzes of the planktonic and sessile cells of Candida parapsilosis, identifying possible biofilm-specific therapeutic targets or biomarkers. METHODS: Candida parapsilosisclinical isolates from two Brazilian public university hospitals were identified by RAPD, RFLP and MALDI-TOF MS and submitted to antifungal susceptibility tests. Biofilm formation assays were carried out to quantify the biomass and metabolic activity, and to evaluate the in vitroactivity of antifungal drugs against the sessile cells of the isolates with high biofilm formation. Proteomic analysis of the planktonic and sessile cells of the isolates with high biofilm formation was performed in two MALDI-TOF MS platforms, VITEK-MS(TM) and Microflex(TM). Candida parapsilosis(sensu stricto) isolates were genotyped by PFGE and microsatellite analysis. The genotypes were correlated with clinical data to investigate the occurrence of an outbreak in the adult ICU andERG11gene sequences from non-susceptible to azoles (NSA) isolates were also analyzed. RESULTS: 38 clinical isolates of the Candida parapsilosiscomplex were obtained, with Candida parapsilosis(sensu stricto) being the most frequent species (exceeding 80% in both hospitals), followed by C. orthopsilosisand C. metapsilosis. Although all isolates were susceptible to amphotericin B ( < 2 mg/L) and showed intermediate susceptibility to anidulafungin, caspofungin e micafungin ( > 0,002 mg/L), high frequency of non-susceptibility (resistance or intermediate susceptibility) to fluconazole and voriconazole was observed among isolates from one of the hospitals. High biofilm formation was only observed among isolates of the Candida parapsilosis. (sensu stricto) species. On the other hand, most of the NSA isolates presented low biofilm formation and low metabolic activity. Only amphotericin B showed activity against Candida parapsilosisbiofilms. The two MALDI-TOF MS platforms were able to differentiate the proteomic profiles of planktonic and sessile cells of isolates. Candida parapsilosis(sensu stricto) genotyping revealed the persistence of clonal NSA isolates. The A395T mutation in the ERG11gene was identified exclusively among azole resistant isolates. The use of corticosteroid was statistically associated with the occurrence of clonal NSA isolates. CONCLUSIONS: Candida parapsilosis(sensu stricto) remains the main species of the complex in bloodstream infections. Azole-resistant isolates with mutations in the ERG11gene are emerging in the two hospitals evaluated. Additionally, the correlation between the genotypes and the clinical data showed the occurrence of an outbreak involving isolates resistant to azoles, with a statistically significant association with previous use of corticosteroids. Candida parapsilosis(sensu stricto) was the only species that presented high biofilm formation and resistance against echinocandins. The two MALDI-TOF MS platforms differentiated the proteomic profiles of the planktonic and sessile cells of Candida parapsilosis, demonstrating the potential use of this technology to identify possible biofilm-specific therapeutic targets or biomarkers

INTRODUÇÃO: A frequência de Candida parapsilosistem apresentado considerável aumento em UTIs neonatais. Embora a taxa de resistência dessa espécie aos azólicos seja baixa, recentemente têm sido relatados surtos de candidemia por isolados resistentes. A capacidade de adesão e formação de biofilme por essa espécie confere maior potencial patogênico e resistência aos antifúngicos. Portanto, a vigilância epidemiológica, tanto da resistência aos antifúngicos como da virulência dos isolados, é fundamental para o controle e prevenção das infecções e surtoshospitalares. A técnica de MALDI-TOF MS pode ser uma ferramenta útil para realizar análises proteômicas das células planctônicas e sésseis de Candida parapsilosis,e identificar possíveis alvos terapêuticos ou biomarcadores, específicos do biofilme. MÉTODOS: Isolados clínicos do complexo Candida parapsilosis de dois hospitais universitários públicos brasileiros, foram submetidos à identificação por RAPD, RFLP e MALDI-TOF MS e aos testes de suscetibilidade aos antifúngicos. Ensaios de formação de biofilme foram realizados para quantificar a biomassa, a atividade metabólica e ainda, avaliar atividade in vitrodos antifúngicos contra as células sésseis dos isolados com alta formação de biofilme. A análise proteômica por MALDI-TOF MS das células planctônicas e sésseis dos isolados com alta formação de biofilme, foi realizada nas plataformas VITEK-MS(TM) e Microflex(TM). Isolados de Candida parapsilosis (sensu stricto) foram genotipados por PFGE e análise de microssatélites. Os genótipos foram correlacionados com dadosclínicos, para investigar a ocorrência de um surto em CTI adulto, e as sequências do gene ERG11dos isolados não suscetíveis aos azólicos (NSA) foram analisadas. RESULTADOS: Foram obtidos 38 isolados do complexo Candida parapsilosis, sendo Candida parapsilosis(sensu stricto) a espécie de maior frequência, superando 80% em ambos os hospitais, seguida de C. orthopsilosis e C. metapsilosis. Embora todos os isolados tenham sido suscetíveis à anfotericina B ( < 2 mg/L) e apresentado suscetibilidade intermediária à anidulafungina, caspofungina e micafungina ( > 0,002 mg/L), elevada frequência de não suscetibilidade (resistência ou suscetibilidade intermediária) ao fluconazol e voriconazol foi observada entre isolados de um dos hospitais. Alta formação de biofilme foi observada apenas entre os isolados da espécie Candida parapsilosis(sensu stricto). Por outro lado, a maioria dos isolados NSA, apresentou baixa formação de biofilme e baixa atividade metabólica. Apenas anfotericina B apresentou atividade contra os biofilmes de Candida parapsilosis. As duas plataformas de MALDI-TOF MS conseguiram diferenciar os perfis proteômicos das células planctônicas e sésseis dos isolados. A genotipagem de Candida parapsilosis(sensu stricto) revelou a persistência de isolados clonais NSA e a mutação A395T no gene ERG11foi identificada exclusivamente entre os isolados resistentes ao azólicos. O uso de corticosteroide foi associado, estatisticamente, com a ocorrência de isolados clonais NSA. CONCLUSÕES: Candida parapsilosis (sensu stricto) se mantém como a principal espécie do complexo em infecções sanguíneas. Isolados resistentes aos azólicos, com mutações no gene ERG11, ocorreram nos dois hospitais avaliados. A correlação dos genótipos com os dados clínicos evidenciou a ocorrência de um surto envolvendo isolados clonais NSA, com associação estatisticamente significativa, ao uso prévio de corticosteroides. Candida parapsilosis (sensu stricto) foi a única espécie que apresentou alta formação de biofilme, o qual demonstrou elevada resistência às equinocandinas. As duas plataformas de MALDI-TOF MS, diferenciaram os perfis proteômicos, das células planctônicas e sésseis de Candida parapsilosis, demonstrando o potencial emprego dessa tecnologia na identificação de possíveis alvos terapêuticos ou biomarcadores, específicos de biofilmes / INTRODUCTION: The frequency of Candida parapsilosis isolates has increased considerably in neonatal ICUs. Although resistance to azoles is usually low in this species, candidemia outbreaks by resistant isolates have been recently reported. Theability of adhesion and biofilm formation by this species confers higher pathogenic potential and resistance to antifungal agents. Therefore, establishment of profiles of antifungal susceptibility and virulence, besides the epidemiological surveillance ofC. parapsilosisisolates are essential for the control and prevention of nosocomial infections and outbreaks. The MALDI-TOF MS technique can be a useful tool to perform proteomic analyzes of the planktonic and sessile cells of Candida parapsilosis, identifying possible biofilm-specific therapeutic targets or biomarkers. METHODS: Candida parapsilosisclinical isolates from two Brazilian public university hospitals were identified by RAPD, RFLP and MALDI-TOF MS and submitted to antifungal susceptibility tests. Biofilm formation assays were carried out to quantify the biomass and metabolic activity, and to evaluate the in vitroactivity of antifungal drugs against the sessile cells of the isolates with high biofilm formation. Proteomic analysis of the planktonic and sessile cells of the isolates with high biofilm formation was performed in two MALDI-TOF MS platforms, VITEK-MS(TM) and Microflex(TM). Candida parapsilosis(sensu stricto) isolates were genotyped by PFGE and microsatellite analysis. The genotypes were correlated with clinical data to investigate the occurrence of an outbreak in the adult ICU andERG11gene sequences from non-susceptible to azoles (NSA) isolates were also analyzed. RESULTS: 38 clinical isolates of the Candida parapsilosiscomplex were obtained, with Candida parapsilosis(sensu stricto) being the most frequent species (exceeding 80% in both hospitals), followed by C. orthopsilosisand C. metapsilosis. Although all isolates were susceptible to amphotericin B ( < 2 mg/L) and showed intermediate susceptibility to anidulafungin, caspofungin e micafungin ( > 0,002 mg/L), high frequency of non-susceptibility (resistance or intermediate susceptibility) to fluconazole and voriconazole was observed among isolates from one of the hospitals. High biofilm formation was only observed among isolates of the Candida parapsilosis. (sensu stricto) species. On the other hand, most of the NSA isolates presented low biofilm formation and low metabolic activity. Only amphotericin B showed activity against Candida parapsilosisbiofilms. The two MALDI-TOF MS platforms were able to differentiate the proteomic profiles of planktonic and sessile cells of isolates. Candida parapsilosis(sensu stricto) genotyping revealed the persistence of clonal NSA isolates. The A395T mutation in the ERG11gene was identified exclusively among azole resistant isolates. The use of corticosteroid was statistically associated with the occurrence of clonal NSA isolates. CONCLUSIONS: Candida parapsilosis(sensu stricto) remains the main species of the complex in bloodstream infections. Azole-resistant isolates with mutations in the ERG11gene are emerging in the two hospitals evaluated. Additionally, the correlation between the genotypes and the clinical data showed the occurrence of an outbreak involving isolates resistant to azoles, with a statistically significant association with previous use of corticosteroids. Candida parapsilosis(sensu stricto) was the only species that presented high biofilm formation and resistance against echinocandins. The two MALDI-TOF MS platforms differentiated the proteomic profiles of the planktonic and sessile cells of Candida parapsilosis, demonstrating the potential use of this technology to identify possible biofilm-specific therapeutic targets or biomarkers

Invasive Candida infection is an increasing cause of morbidity and mortality in the immunocompromised patient. Molecular diagnosis based on genomic amplification methods, such as real time PCR, has been reported as an alternative to conventional culture for early detection of invasive candidiasis. The template DNA extraction step has been the major limitation in most reported nucleic acid based assays, due to problems in breaking fungal cell walls and incomplete purification in PCR inhibitor substances. The aim of this study was to compare enzymatic cell wall disruption using recombinant lyticase with mechanical disruption using glass beads. The QIAamp tissue kit was compared with two automated DNA extraction robots, the BioRobot M48 and NucliSens easyMAG, to determine their sensitivity, reliability and duration for DNA release of C. albicans. Mechanical cell wall disruption shortened and facilitated the extraction procedure, but the quantity of released DNA was significantly lower than when enzymatic cell wall disruption was used. Use of robots did not significantly shorten the DNA extraction time, compared with manual DNA extraction. However the NucliSens easyMAG resulted in a higher yield of target DNA compared to the BioRobot M48 and the manual QIAamp tissue kit. / Invasiva svampinfektioner är ett stort problem hos patienter med dåligt immunförsvar. Förekomst av invasiva svampinfektioner har ökat under senare år och medför hög dödlighet. En svampinfektion som inte snabbt diagnostiseras och behandlas kan bli livshotande om patientens kondition är dålig. Candida albicans är den vanligaste orsaken till invasiva svampinfektioner. Med traditionell svampidentifiering kan det ta dagar till veckor att isolera och artbestämma svampen. En snabbare metod att detektera Candida är att använda sig av molekylärbiologiska metoder som påvisar svampens arvsmassa, DNA. Svampar har en cellvägg som är svår att bryta ner och därför är DNA extraktionssteget ett av de mest rapporterade problemen vid DNA svampdiagnostik. Syftet med denna studie var att jämföra enzymatisk och mekanisk cellväggsnedbrytning av C. albicans med hjälp av enzymet lyticase respektive glaskulor. Vi jämförde också en manuell metod med två automatiska robotar för att bestämma deras känslighet, tillförlitlighet och tidsåtgång för DNA-extraktion från C. albicans. De slutsatser som nåtts är att den enzymatiska cellväggsnedbrytningen var känsligare men betydligt mer tidskrävande än den mekaniska cellväggsnedbrytningen. Denna studie visade även att en av de automatiska systemen extraherade signifikant mer DNA än den manuella metoden.

<p><strong><br /></strong></p> / <p><strong>In Canada the incidence of yeast infections have increased over the past decade, which in turn has resulted in the increased mortality and morbidity rates among the immuno-compromised patients. Yeasts are ubiquitous in nature and constitute a healthy portion of human skin and gut flora. Factors such as the urban settings and food have been previous shown to influence the yeast flora people harbour. This makes us believe that to effectively tackle the rising yeast infections in Canada we need to not only conduct epidemiological yeast studies in clinical settings but should also understand the diversity and distribution of them in the urban environment. This thesis constitutes of an epidemiological fungemia study and an urban environmental yeast profiling study conducted in the city of Hamilton.</strong></p> <p><strong> </strong></p> <p><strong>In the first chapter of the thesis I discuss the results of the epidemiological candidemia study. We noticed that over the past decade the mean age of the population with candidemia in hospitals within Hamilton has increased by 10 years. DNA fingerprinting analysis suggested that 33% of the blood stream</strong><strong><em>Candida</em></strong><strong> </strong><strong>isolates from January 2005 to February 2009 belonged to 18 clusters, some of which were shared between wards and hospitals. we found that for each of the four species, strains isolated closer to each other temporally were overall genetically more similar to each other as well, which suggested that nosocomial sources likely caused repeated candidemia infections. The study is the first of its sort in Canada and the results of this chapter are expected to aid infection control practitioners in the Hamilton hospitals and make the stay of patients in hospitals safer.</strong></p> <p><strong> </strong></p> <p><strong>In the second chapter, we discuss the diversity and distribution of yeasts prevalent on trees in and around Hamilton. We identified a total of 88 environmental yeasts belonging to 20 species (based on ITS sequence data). The yeast populations were highly heterogeneous in both species and genotype composition. Among the 14 tree species sampled, yeasts were frequently found on cedar, cottonwood and basswood. Interestingly all the</strong><strong> <em>Candida parapsilosis</em> </strong><strong>strains were found from pine tree only. Some of the potential environmental factors shaping the distribution of yeast populations in Hamilton are discussed.</strong></p> / Master of Science (MSc)

Made available in DSpace on 2016-12-23T13:55:57Z (GMT). No. of bitstreams: 1 Descricao dos episodios de candidemia de origem nosocomial.pdf: 1812015 bytes, checksum: 9c5089ff58847dbbef9c102928ca12c2 (MD5) Previous issue date: 2006-09-04 / Este trabalho foi um estudo descritivo, que descreveu os episódios de candidemia nosocomial ocorridos no Hospital Universitário Cassiano Antônio de Moraes (HUCAM), entre 1.º de março de 2003 e 28 de fevereiro de 2006. Seu objetivo foi definir as taxas de prevalência e mortalidade desses espisódios, identificar as espécies envolvidas e relacionar a freqüência dos fatores de risco entre adultos e recém-nascidos (RNs), bem como entre os episódios de candidemia por Candida albicans (CA) e por Candida não-albicans (CNA). A metodologia Incluiu os casos de forma consecutiva, à medida que foi detectada hemocultura positiva para esses fungos. A coleta de dados dos pacientes foi realizada com base no prontuário médico, por meio de formulário padronizado e analisados utilizando o programa Social Package Statistical Science e englobando 45 casos de candidemia, sendo dezenove casos (42%) em neonatos. Foi observado que a incidência de candidemia nosocomial no HUCAM foi de 2,0/10.000 pacientes-dia, 1,9/1.000 altas e 16/10.000 admissões, sendo maior nos pacientes internados em unidades de terapia intensiva. As espécies não-albicans predominaram e Candida parapsilosis foi mais incidente entre os RNs; os grupos de pacientes susceptíveis mais envolvidos foram aqueles com internação em torno de três semanas, com síndrome séptica aguda e em uso de antibióticos; a freqüência dos fatores de risco foi similar entre os adultos e os RNs, assim como entre os episódios devidos a CA e os devidos a CNA; maior utilização de nutrição parenteral total foi encontrada entre os RNs e de diálise, entre os adultos (p < 0,05); a taxa de mortalidade global foi de 44% entre os adultos e de 58% entre os RNs; e a taxa de mortalidade atribuída foi de 16% entre os adultos e 26% entre os RNs. Foi concluído que a incidência de candidemia no HUCAM foi alta e associada com substancial mortalidade. / This work was a descriptive study, which described the episodes of nosocomial candidemias occurring at Hospital Universitário Cassiano Antônio de Moraes (HUCAM), between March 1st, 2003 and February 28th, 2006. Its aim was to define the rates of prevalence and mortality of these epidsodes, to identify the species involved and to relate the frequency of the risk factors among adults and newborn (NBs), as well as the epidsodes among candidemias caused by Candida albicans (CA) and Candida non-albicans (CNA). The methodology included the cases in a consecutive way, as positive hemoculture was detected for these fungi. The date collection of patients was based on the medical records, through a standard form and it was analyzed using the Social Package Statistical Science program, including 45 cases of candidemias, being nineteen cases (42%) in NBs. It was observed that the incidence of nosocomial candidemia at HUCAM was 2.0/10.000 patient-day, 1.9/1.000 discharges and 16/10.000 admissions, being greater in patients interned in intensive therapy unity. the CNA predominated and C. parapsilosis was more incidente among the NBs; the group of susceptible more involved were those interned for 3 weeks with acute septical syndrome and in use of antibiotics; the frequency of risk factor was similar among the episodes due to CA and the ones due to CAN; greater utilization of total parenteral nutrition was found in the NBs and the dialysis, among the adults (p< 0,005); the total mortality rate was 44% among adults and 58% among the NBs. And the attributed mortality rate was 16 in adults and 26% in NBs. It was concluded that incidence of candidemias at HUCAM was high and associated with substantial mortality.

No description available.

Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.