In the last two decades it has been seen a large increase in blood stream infections by Candida spp. The candidemias are associated with high morbidity and mortality, as well as the increase in hospital costs. The phenomenon of resistance highlights the importance of performing tests for susceptibility to antifungal agents. Such tests are improving constantly in an attempt to standardize their interpretations. This study evaluated the susceptibility of Candida spp. isolated from candidemias during a period of 15 years divided into three periods of five years in Santa Maria, RS, Brazil, interpreting minimum inhibitory concentrations according to the proposal of the CLSI and the new breakpoints recently proposed by Pfaller et al. Among the 525 isolates during the three periods, C. parapsilosis was the predominant species (49.14%), followed by C. albicans (34.47%), C. tropicalis (8.19%), C. glabrata (3.61%), C. lusitaniae (1.90%), C. guilliermondii (1.71%) and C.krusei (0.95%). During the study period and considering the two criteria, 136 non-susceptible isolates were found. According to CLSI, we identified 40 (7.61%) non-susceptible isolates, from wich 19 (3.61%) were resistant. According to Pfaller et al., 96 (18.28%) isolates presented themselves as nonsusceptible, being 11 (2.09%) resistant. All isolates were susceptible to amphotericin B and 3 presented themselves dose-dependent or intermediate suscpetible to flucytosine and caspofungin. Resistance was detected only among the azole antifungals: itraconazole (2.09%), fluconazole (1.14%) and voriconazole (0.38%). The isolation of species resistant to the antifungal agents used was not a prominent feature in this study. Detecting a higher number of non-susceptible species highlighted the relevance of evaluating of susceptibility tests by new breakpoints in order to find a more efficient therapy. / Nas últimas duas décadas observou-se um grande aumento nas infecções de corrente sanguínea por Candida spp. As candidemias estão associadas a altas taxas de morbidade e mortalidade, bem como ao incremento nos custos hospitalares. O fenômeno da resistência evidencia a importância da realização de testes de suscetibilidade aos agentes antifúngicos. Tais testes estão aprimorando-se constantemente na tentativa de uniformizar suas respectivas interpretações. O presente estudo avaliou a suscetibilidade de Candida spp. isoladas de candidemias por um período de 15 anos divididos em três períodos de cinco anos, em Santa Maria, RS, Brasil, interpretando as concentrações inibitórias mínimas de acordo com a proposta do CLSI e os novos breakpoints recentemente propostos por Pfaller et al. Entre os 525 isolados nos três períodos avaliados, C. parapsilosis foi a espécie predominante (49,14%), seguida por C. albicans (34,47%), C. tropicalis (8,19%), C. glabrata (3,61%), C. lusitaniae (1,90%), C. guilliermondii (1,71%), e C. krusei (0,95%). Durante o período estudado e considerando-se os dois critérios, foram detectados 136 isolados não sensíveis. Segundo o CLSI, foram identificados 40 (7,61%) isolados não sensíveis, sendo 19 (3,61%) resistentes. De acordo com Pfaller et al., 96 (18,28%) isolados apresentaram-se como não sensíveis, sendo 11 (2,09%) resistentes. Todos os isolados foram sensíveis a anfotericina B e 5 e 3 isolados apresentaram-se intermediários a flucitosina e caspofungina, respectivamente. Resistência foi detectada somente entre os antifúngicos azólicos: itraconazol (2,09%), fluconazol (1,14%), e voriconazol (0,38%). O isolamento de espécies resistentes aos agentes antifúngicos utilizados não foi uma característica marcante no presente estudo. Este estudo permitiu uma melhor visão da epidemiologia local. A detecção de um maior número de espécies não sensíveis ressaltou a relevância da avaliação dos testes suscetibilidade através dos novos breakpoints a fim de ser direcionada a uma terapia mais eficiente.

Candida species are ubiquitous, ranging from pure saprobes through endo-symbionts of animals, to pathogens in many animals including humans. Some of the pathogenic species are of medical importance, especially Candida albicans. However, the prevalence of other non-albicans Candida species as human pathogens has been increasing worldwide. The aim of this study was to use conventional phenotypic tests and molecular methods to isolate, identify and characterise 600 Candida isolates from three teaching hospitals in Ghana, namely Korle Bu, Komfo Anokye and Tamale from mid-January to April, 2012. The prevalence of these species in cases of genitourinary candidiasis and pelvic inflammatory disease was investigated. Preliminary identification and characterisation of Candida isolates using four conventional phenotypic tests showed that C. albicans was the most common species, which constituted 41% of the isolates whereas non-albicans Candida species constituted 59% of the total number of Candida isolates. In patients presenting with vulvovaginal candidiasis (VVC) for at least two or more times, chi-square analysis indicates that the frequency of Candida species isolated were not statistically different from patients presenting for the first time with VVC. Candida albicans was the most common species in vaginal swabs from patients presenting with vulvovaginal candidiasis (VVC) for the first time in each of the three locations, present in 53.4% of the total swabs. The other species that were present were C. glabrata (21.6%), C. parapsilosis (15.5%), C. tropicalis (4.7%) and C. krusei (4.7%). Similar Candida species distributions were found in swabs taken from patients presenting with suspected pelvic inflammatory disease (PID). Across the three locations, however, there was a significant difference in the frequency of C. albicans, which was present in 68 and 69.6% of patients from Komfo Anokye and Tamale, but only 26.7% of patients from Korle Bu. Urine samples were taken in two of the locations, Korle Bu and Tamale, from female patients presenting with candiduria. Statistical analysis indicated a significant difference in the frequency of Candida isolates in cases of candiduria between the two locations. In Korle Bu, C. glabrata was the most prominent species (37.8%) followed by C. albicans (22.4%), C. parapsilosis (21.7%), C. tropicalis (10.5%), C. krusei (7%) and C. lusitaniae (0.7%). In Tamale, the species distribution was C. albicans (60.9%), C. glabrata (21.7%), C. parapsilosis (13%) and C. krusei (4.3%). The data highlight the prevalence of species other than C. albicans in case of candidiasis in Ghana. Delineation of C. albicans strains using the 25S rDNA to investigate the genotypic variation among C. albicans isolates showed that genotype A constituted about 95% of the Ghanaian C. albicans isolates, genotypes B and C constituted 2.5% each respectively. The general-purpose genotype (GPG) which corresponds to clade 1 among C. albicans was also investigated to know the prevalence of clade 1 among the C. albicans isolates investigated. The presence or absence of general-purpose genotype (GPG) gene was used to categorise the 240 C. albicans to clade 1 or other clade. The test revealed that 64.2% had the GPG genotype which corresponds to clade 1 and the remaining 35.8% were of non-GPG genotype; thus belongs to other clades. The population structure of C. albicans from the three teaching hospitals indicates a mainly clonal and homogeneous population across the three sampling locations from Ghana. Molecular analyses of the transposable group 1 intron in the ITS1-5.8S-ITS2 region using universal primer pair ITS1 and ITS4 revealed the presence of two rare Candida species; Candida rugosa and Candida mesorugosa. To the best of my knowledge this is the first report of either of these in Africa. Antifungal susceptibility tests among Candida isolates recovered from patients presenting with clinically suspected or symptomatic candidal vaginitis for the first time and patients presenting with candidal vaginitis on two or more occasions revealed a high percentage of Fluconazole-resistant C. albicans. This study highlights the prevalence of species other than C. albicans in cases of candidiasis in Ghana. Furthermore, this study has also demonstrated that no single conventional phenotypic test has been highly efficient to delineate Candida species into their respective species type. Thus, development of an identification scheme, which can discriminate between Candida isolates both at species and strain levels, will have prognostic and therapeutic significance for effective patient management.

With the recent emergence of candidemia as a significant cause of mortality in our health care system, clinicians must identify methods to minimize the sequelae of infection of this type in patients already burdened with serious underlying conditions. While well established as a major cause of blood stream infection (BSI), candidemia has been shown to have some of the highest rates of inappropriate therapy when compared to infections from all other sources. Rates of inappropriate therapy may be even higher for some of the less common and antifungal resistant non-albicans candidemia. Identifying those patients at risk for the development of these types of infections will help improve clinical outcomes. Antifungal activity is dependent both on species and agent, describing the unique susceptibility patterns between Candida species can help identify the appropriate therapy. We performed a case-case-control study to identify clinical risk factors for the development of Candida glabrata candidemia compared to Candida albicans candidemia and an uninfected control using multivariate and logistic regression analysis. We observed that patients in the C. glabrata cohort were more likely to have gastrointestinal disorders and peripheral vascular disease than patients suffering from C. albicans BSIs. We also determined that when compared to the uninfected control group, patients with C. glabrata BSIs were more likely to have been prior colonized with C. glabrata, undergone dialysis, and have been catheterized with both arterial and urinary catheters. We concluded that patient exposure to unique clinical risk factors may be predictive of the development of future candidemia and may help distinguish between albicans versus non-albicans candidemia. We performed a drug susceptibility study using time-kill methods with the echinocandin antifungal agents on Candida parapsilosis and two newly identified species of Candida, C. orthopsilosis and C. metapsilosis. The echinocandins as a group displayed primarily fungistatic activity against the clinical isolates tested. However, we observed substantial variability in antifungal activity that varied by both the echinocandin used and Candida species analyzed. We concluded that this variability in activity that is both species and drug dependent should be considered when selecting the treatment of candidemia resulting from these non-albicans species.

Student Number: 0420652W - MSc(Med) Dissertation - School of Pathology - Faculty of Health Sciences / Candida species are opportunistic yeasts that cause infections in immunocompromised individuals such as HIV and cancer patients. Recent studies show that 5fluorouracil, a nucleoside analogue used for cancer treatment, increases Candida cell virulence. The aim of this study is to determine the effects of commonly used antiHIV nucleoside analogue drugs on the virulence of Candida albicans, the predominant species associated with oral candidiasis. Oral swabs were collected from antiretroviralnaïve HIVpositive individuals. C. albicans was characterised from 39 of these swabs using standard microbiological techniques and polymerase chain reaction. The effect of nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine, stavudine, didanosine and lamivudine, at predicted drug peak concentrations in patients, as well as half and double these concentrations on select virulence factors of C. albicans isolates were studied. In addition, antifungal susceptibility to amphotericin B was assessed. Not all 39 isolates were used in the assays because of delays in obtaining reagents from respective manufacturers. Results show no change in the adherence and biofilm formation of 29 isolates upon exposure to NRTIs. In contrast, a steady increase in the number of viable cells was observed upon exposure to double the peak concentration of lamivudine to 23 of the clinical isolates. All 31 isolates tested were susceptible to amphotericin B (MIC£1mg/ml). Although these results suggest that NRTIs may have little effect on the virulence of C. albicans it is postulated, that, in a dosedependent manner, cytidine analogues act similarly to 5FU by activating a signaltransduction pathway which stimulates proliferation.

Made available in DSpace on 2014-06-11T19:28:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-02-12Bitstream added on 2014-06-13T18:07:23Z : No. of bitstreams: 1 pires_jr_dr_arafo.pdf: 422001 bytes, checksum: 5fdba3ee936d59216f9c3b7bd4ad41a3 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi avaliar os efeitos do tratamento periodontal em pacientes diabéticos do tipo 2 sobre alterações de fluxo salivar, xerostomia, Candida spp. na saliva e candidose bucal clínica. Foram selecionados 20 pacientes diabéticos tipo 2 e 17 pacientes não-diabéticos de ambos os gêneros, com idade média entre 37 e 55 anos, portadores de doença periodontal crônica. Após exame clínico para avaliação das condições clínicas periodontais, presença de xerostomia e presença clínica de candidose bucal, amostras de saliva foram coletadas para mensuração do fluxo salivar (mL/min), contagem das colônias de Candida spp. (UFC/mL) e identificação das espécies. Esse procedimento foi realizado antes da terapia periodontal (baseline), que consistiu em raspagem e alisamento radicular, instrução de higiene bucal e motivação do paciente, aos 30 e 90 dias após a mesma. Os resultados demonstraram que os pacientes diabéticos apresentaram maior ocorrência de xerostomia e de candidose bucal, sendo estes colonizados por várias espécies de Candida spp. com predominância de C. albicans, sem no entanto, implicar no desenvolvimento da candidose clinicamente. A terapia periodontal, a motivação do paciente e a instrução de higiene bucal foram importantes para melhorar as condições clínicas periodontais e evitar a formação de reservatórios intrabucais de infecção. / In order to investigate the effect of periodontal therapy, in type 2 diabetes mellitus subjects about salivar flow rates, presence of xerostomia, presence of Candida species in the saliva and presence of buccal candidose, twenty type 2 diabetes mellitus subjects and seventeen no-diabetes mellitus subjects, with medium age among 33 to 55 years, presenting generalized chronic periodontal disease were selected. After initial clinical exam to analyze periodontal parameters, xerostomia and buccal candidose presence, saliva samples were assessed to quantify as millimeters of saliva generated per minute (mL/min), account yeasts by colony forming units per mL (CFU/mL) and identificated species. That procedure was accomplished before periodontal therapy (baseline) that it consisted in scaling and root planning, oral hygiene instructions and patient's motivation to the thirty and ninety days after these. Diabetics subjects showed to having hyposalivation in similar levels in no-diabetics groups and in all the analyzed periods, higher prevalence of xerostomia and higher occurrence of buccal candidose. Candida albicans were the prevalent yeast at all periods in both groups, but the presence of yeasts in saliva not implied in the development of buccal candidose. Concluded that the periodontal therapy, the patients' motivation and the buccal hygiene instructions improve periodontal conditions and avoid the formation of microbial reservoirs in mouth.

Orientador: Denise Madalena Palomari Spolidorio / Banca: Elaine Maria Sgavioli Massucato / Banca: Antonio Olavo Cardoso Jorge / Resumo: O objetivo deste estudo foi avaliar os efeitos do tratamento periodontal em pacientes diabéticos do tipo 2 sobre alterações de fluxo salivar, xerostomia, Candida spp. na saliva e candidose bucal clínica. Foram selecionados 20 pacientes diabéticos tipo 2 e 17 pacientes não-diabéticos de ambos os gêneros, com idade média entre 37 e 55 anos, portadores de doença periodontal crônica. Após exame clínico para avaliação das condições clínicas periodontais, presença de xerostomia e presença clínica de candidose bucal, amostras de saliva foram coletadas para mensuração do fluxo salivar (mL/min), contagem das colônias de Candida spp. (UFC/mL) e identificação das espécies. Esse procedimento foi realizado antes da terapia periodontal (baseline), que consistiu em raspagem e alisamento radicular, instrução de higiene bucal e motivação do paciente, aos 30 e 90 dias após a mesma. Os resultados demonstraram que os pacientes diabéticos apresentaram maior ocorrência de xerostomia e de candidose bucal, sendo estes colonizados por várias espécies de Candida spp. com predominância de C. albicans, sem no entanto, implicar no desenvolvimento da candidose clinicamente. A terapia periodontal, a motivação do paciente e a instrução de higiene bucal foram importantes para melhorar as condições clínicas periodontais e evitar a formação de reservatórios intrabucais de infecção. / Abstract: In order to investigate the effect of periodontal therapy, in type 2 diabetes mellitus subjects about salivar flow rates, presence of xerostomia, presence of Candida species in the saliva and presence of buccal candidose, twenty type 2 diabetes mellitus subjects and seventeen no-diabetes mellitus subjects, with medium age among 33 to 55 years, presenting generalized chronic periodontal disease were selected. After initial clinical exam to analyze periodontal parameters, xerostomia and buccal candidose presence, saliva samples were assessed to quantify as millimeters of saliva generated per minute (mL/min), account yeasts by colony forming units per mL (CFU/mL) and identificated species. That procedure was accomplished before periodontal therapy (baseline) that it consisted in scaling and root planning, oral hygiene instructions and patient's motivation to the thirty and ninety days after these. Diabetics subjects showed to having hyposalivation in similar levels in no-diabetics groups and in all the analyzed periods, higher prevalence of xerostomia and higher occurrence of buccal candidose. Candida albicans were the prevalent yeast at all periods in both groups, but the presence of yeasts in saliva not implied in the development of buccal candidose. Concluded that the periodontal therapy, the patients' motivation and the buccal hygiene instructions improve periodontal conditions and avoid the formation of microbial reservoirs in mouth. / Mestre