Accuracy in clinical examinations for the diagnosis of vulvovaginitis by CANDIDA SPP. and IN VITRO susceptibility to the main antifungals

Vulvovaginal candidiasis (VVC) is a common infection. This work aims to determine the positive predictive value (PPV) of the clinical diagnosis of VVC and to characterize Candida species isolated from the vaginal mucosa. This cross-sectional study was conducted from February 2016 to February 2017 at the Gynecology and Obstetrics Outpatient Clinic of the Hospital das Clínicas, in Goiânia, Goiás State, Brazil. The study included samples of vaginal secretion from 55 women who complained of vaginal discharge and itching as their main symptoms. The PPV of the clinical diagnosis of VVC was estimated in comparison to the laboratory culture method. The phenotypic methods and molecular tests were performed to identify Candida spp. In vitro susceptibility of Candida spp. isolates to fluconazole, itraconazole, clotrimazole, nystatin, and amphotericin B was determined using the broth microdilution assay. Yeast growth using the enzymes protease, phospholipase, and hemolysin was carried out in media containing respectively bovine albumin, egg yolk, and sheep erythrocytes. A PPV of 61.8% (34/55) was determined. Among the 55 vulvovaginal samples collected, we identified 36 isolates in which C. albicans was the most common species. High resistance to fluconazole and low minimal inhibitory concentration (MIC) values for clotrimazole, nystatin and amphotericin B were observed. All isolates were proteinase and hemolysin producers, while seven strains were phospholipase negative. The clinical diagnosis of VVC presented a moderate PPV, which meant that cultures had to be conducted in the laboratory to confirm infection. The high resistance to fluconazole and itraconazole indicated the importance of the in vitro susceptibility test.

Biofilm forming capability and antifungal susceptibility profile of Candida spp. from blood

Although Candida albicans remains the most frequent Candida species; however other species have emerged as important causes of candidiasis. In this work, we evaluated the in vitro susceptibility profile of C. albicans, C. parapsilosis, and C. tropicalis biofilms isolated from patients with candidemia to fluconazole, voriconazole, amphotericin B, and caspofungin. Differences between the biofilm ultrastructure of the three species were also determined. The isolates were phenotypically determined by growth on a ChromagarTM medium and assimilation profile on ID32C. The Scanning Electron Microscopy method (SEM) on biofilm was performed using polyurethane strips. For the in vitro susceptibility profile a microdilution in broth was used. Sessile cells were resistant to fluconazole, voriconazole and caspofungin. The resistance to amphotericin B was less pronounced and more variable between the tested isolates. In the SEM, slight differences in ultrastructural morphology for each species in biofilms were observed. Our results verified biofilm formation. Low susceptibility to the drugs in the three researched species confirmed the higher virulence of them.

Cell surface hydrophobicity and biofilm formation of candida yeast species in different culture media / Hidrofobicidade da superfície celular e formação de biofilme de Candida sp em diferentes meios de cultura

Cell surface adhesion is considered an essential step in the spread, infection and persistence of Candida yeasts in the host. Their ability to adhere on biotic and abiotic surfaces depends on several factors, including hydrophobicity. Once attached, these yeasts are capable of growing in biofilms, which are constituted of structured communities of encapsulated cells within an extracellular matrix, resistant to antifungal agents. In this context, this study aimed to analyze the cell surface hydrophobicity and specific biofilm formation of six Candida strains in different culture media: Sabouraud dextrose broth (SDB), artificial saliva (AS), Roswell Park Memorial Institute medium (RPMI 1640) and N-acetylglucosamine-yeast nitrogen base proline (NYP). Six yeasts of the genus Candida were studied: three C. albicans (Ca): Ca ATCC 10231 and the clinical isolates Ca34 and Ca05 and C. parapsilosis (Cp): Cp ATCC 22019 and the clinical isolates Cp120 and Cp38. Hydrophobicity was calculated as the percentage reduction in turbidity of the aqueous phase, due to the retention of the hydrophobic cells in hydrocarbon by the hydrocarbon-water biphasic assay following the MATH (Microbial Adhesion to Hydrocarbon) method. The biofilm formation index was calculated as the optical density obtained by the growth of the yeasts in the culture media in a polystyrene microtiter plate, subsequently stained with 1% violet crystal. The results showed that hydrophobicity varied according to the media and the yeasts studied, and two of these (Ca34 and Ca10231) presented significant variation between the media. A more hydrophobic character was observed in yeasts grown on RPMI-1640 medium, and those grown on Sabouraud dextrose broth appeared more hydrophilic. The specific biofilm formation index was more intense for RPMI 1640 than in other media, which was expected, due to its ability to induce the transition between yeast-hyphae morphology, which is one of the key factors involved in the adhesion of C. albicans on different surfaces. RPMI 1640 was the best medium for obtaining biofilm in vitro, due to its greater hydrophobicity, which can enhance cell adhesion to the polystyrene plate, and due to its nutrient content, necessary for complete cell growth and biofilm formation.

A adesão é considerada um passo essencial para a disseminação, infecção e persistência das leveduras do gênero Candida nos hospedeiros. A habilidade dessas leveduras em se aderir a superfícies bióticas e abióticas depende de fatores, incluindo a hidrofobicidade da superfície celular. Uma vez aderidas, estas leveduras são capazes de crescer como biofilmes, os quais são caracterizados como comunidades estruturadas de células encapsuladas dentro de uma matriz extracelular, resistentes a drogas antifúngicas. Neste contexto, objetivou-se analisar a hidrofobicidade da superfície celular e a formação específica de biofilme de Candida spp. em diferentes meios de cultura: caldo Sabouraud dextrose (CSD), saliva artificial (SA), Roswell Park Memorial Institute medium (RPMI 1640) e N-acetylglucosamineyeast nitrogen base-proline (NYP). Foram estudadas seis leveduras do gênero Candida, sendo três C. albicans (Ca): Ca ATCC 10231 e os isolados clínicos Ca34 e Ca05 e C. parapsilosis (Cp): Cp ATCC 22019 e os isolados clínicos Cp120 e Cp38. A hidrofobicidade foi calculada como a porcentagem da redução da turbidez da fase aquosa, devido a retenção das células hidrofóbicas no hidrocarboneto pelo método bifásico água-hidrocarboneto MATH (Teste de Adesão Microbiana a Hidrocarbonetos). O índice de formação de biofilme foi calculado com as densidades ópticas obtidas por meio do crescimento das leveduras nos meios de cultura em poços de placa de microtitulação de poliestireno e posteriormente corados com cristal violeta a 1%. Os resultados demonstraram que a hidrofobicidade variou de acordo com os meios e as leveduras estudadas, sendo que duas (Ca34 e Ca10231) apresentaram variação significativa entre os meios. Foi verificado um caráter mais hidrofóbico das células crescidas em RPMI 1640 e mais hidrofílico nas células crescidas em caldo Sabouraud dextrose. A formação específica de biofilme apresentou-se mais intensa em RPMI 1640 do que nos outros meios, o que já era esperado pela sua capacidade de induzir o processo de transição levedura-hifa, o qual é considerado um dos fatores cruciais envolvidos na adesão de leveduras. Com os resultados obtidos, infere-se que o RPMI 1640 é o melhor meio para obtenção de biofilme in vitro, pois as células foram mais hidrofóbicas, o que pode aumentar a adesão das

Invasive fungal infection in patients with hematologic disorders in a Brazilian tertiary care hospital

ABSTRACT INTRODUCTION: Invasive fungal infections (IFIs) are an important complication in immunocompromised individuals, particularly neutropenic patients with hematological malignancies. In this study, we aimed to verify the epidemiology and diagnosis of IFIs in patients with hematologic problems at a tertiary hospital in Goiânia-GO, Brazil. METHODS: Data from 117 patients, involving 19 cases of IFIs, were collected. The collected data included diagnosis methods, demographics, clinical characteristics, and in vitro susceptibility to different antifungal agents. Among the 19 cases, 12 were classified as proven IFI and 7 as probable invasive aspergillosis with detection of galactomannan in blood and presence of lung infiltrates in radiographic images. Logistic regression analysis showed that the proven and probable IFIs were associated with increased risk of death. Statistical analysis demonstrated that age, sex, and underlying disease were not independently associated with risk of death in IFI patients. RESULTS: Most bloodstream isolates of Candida spp. exhibited low minimum inhibitory concentrations (MICs) to all antifungal agents tested. Voriconazole and amphotericin had the lowest MICs for Aspergillus spp. and Fusarium spp., but Fusarium spp. showed the least susceptibility to all antifungals tested. Amphotericin B, fluconazole, and itraconazole were found to be inactive in vitro against Acremonium kiliense; but this fungus was sensitive to voriconazole. CONCLUSIONS: Considering the high number of IFI cases, with crude mortality rate of 6%, we could conclude that IFIs remain a common infection in patients with hematological malignancies and underdiagnosed ante mortem. Thus, IFIs should be monitored closely.

Effects of the essential oil of Thymus vulgaris L. Against Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast found in the environment, responsible for causing of meningoencephalitis in patients with a compromised immune system. In Brazil, cryptococcosis is the second cause of death among systemic mycoses. The limited efficacy of the available antifungal drugs used in its treatment has encouraged the search for therapeutic alternatives, such as medicinal plants. Thymus vulgaris, popularly known as thyme, is an aromatic plant whose essential oil (EO) possesses antifungal properties. The aim of this study was to assess the action of T. vulgaris EO on C. neoformans clinical isolates. This oil was analyzed by gas chromatography/mass spectrometry (GC/MS), which showed that its main components were thymol, ρ-cymene and linalool. Microdilution broth tests showed that this EO was effective against fungal isolates, with minimum inhibitory concentrations (MICs) ranging from 32 to 128 µg/mL. In vitro interaction tests between this oil and fluconazole (FCZ) showed no potentiation of the antifungal action of this drug. Its effect on mitochondrial metabolism of fungal cells was also evaluated and results demonstrated alterations on the mitochondrial enzyme activity of fungal cells only at concentrations >1,024 µg/mL. The results of the action of this EO on human erythrocytes indicated that it has low cytotoxic activity at MIC values. This investigation describes the antifungal action of T. vulgaris, showing its potential in the development of alternatives in the treatment of C. neoformans

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.

Pimenta pseudocaryophyllus inhibits virulence factors and promotes metabolic changes in Candida yeast

Introduction This is the first study to examine the in vitro susceptibility and the expression of virulence factors in Candida species in the presence of Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae), a Brazilian plant known as paucravo. Additionally, the mechanisms of action of the crude ethanol extract and the ethyl acetate and aqueous fractions of this plant were investigated. Methods The in vitro susceptibility of Candida was tested using the broth microdilution method, whereas an XTT reduction assay was used for biofilms. Adherence was determined by counting the number of yeast cells that adhered to 100 oral epithelial cells, and hyphal formation was verified in the hyphal induction medium M199. Flow cytometry with propidium iodide and FUN-1 was performed to assess the mechanism of action. Results The results revealed that the crude ethanol extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus inhibited the growth of Candida isolates at a minimal inhibitory concentration (MIC) ranging from 64 to 256µg/mL, whereas the 50% sessile minimal inhibitory concentration (SMIC50) ranged from 512 to >1,024µg/mL. Adherence and hyphal formation were significantly reduced in the presence of the crude ethanol extract and both fractions. Although cell membrane injury was detected, the predominant mechanism of action appeared to be the alteration of yeast metabolism, as demonstrated by flow cytometry. Conclusions Our results indicated that antifungal activity reduced the expression of virulence factors in yeast via the alteration of yeast metabolism, suggesting that the crude extract of P. pseudocaryophyllus and its fractions may contain novel antifungal agents. .

Clinical and microbiological features of cryptococcal meningitis

Introduction In this study, the clinical features, underlying diseases and clinical outcomes of patients with cryptococcosis were investigated. In addition, a molecular analysis of the Cryptococcus neoformans species complex isolated from these patients was performed. Methods A prospective study of 62 cases of patients with cryptococcal infection was conducted at the Hospital de Doenças Tropicais de Goiás Dr. Anuar Auad from 2009-2010. Cryptococcal meningitis cases were diagnosed by direct examination and cerebrospinal fluid (CSF) sample culture. The profiling of these patients was assessed. The CSF samples were submitted to India ink preparation and cultured on Sabouraud dextrose agar, and C. neoformans was identified by the production of urease, a positive phenoloxidase test and assimilation of carbohydrates. C. neoformans and C. gattii isolates were distinguished by growth on L-canavanine-glycine-bromothymol blue medium, and molecular analysis was conducted via PCR fingerprinting reactions using M13 and (GACA)4 primers. Results From the 62 patients with cryptococcosis, 71 isolates of CSF were obtained; 67 (94.4%) isolates were identified as C. neoformans var. grubii/VNI, and 4 (5.6%) were identified as C. gattii/VGII. Of these patients, 53 had an HIV diagnosis. The incidence of cryptococcosis was higher among patients 20-40 years of age, with 74.2% of the cases reported in males. Cryptococcus-related mortality was noted in 48.4% of the patients, and the symptoms were altered sensorium, headache, fever and stiff neck. Conclusions The high morbidity and mortality observed among patients with cryptococcosis demonstrate the importance of obtaining information regarding the epidemiological profile and clinical course of the disease in the State of Goiás, Brazil. .

Molecular analysis and dimorphism of azole-susceptible and resistant Candida albicans isolates / Análise molecular e dimorfismo de isolados de Candida albicans suscetíveis e resistentes aos azólicos

INTRODUCTION: Candida albicans is responsible for superficial or systemic infections known as candidiasis, which may be found in infected tissue as unicellular budding yeasts, hyphae, or pseudohyphae. In this study, the effects of both fluconazole and itraconazole antifungal agents on the hyphal formation and genotypic characterization of C. albicans isolates classified as either susceptible or resistant were investigated. METHODS: The hyphal production of five C. albicans isolates under the action of antifungal agents was investigated by culturing yeast on growth medium and on hyphal induction medium. The genotypic characterization was carried out for 13 isolates of C. albicans using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method. RESULTS: The dimorphism analysis showed that the hyphal formation was higher in resistant than in the susceptible isolates to both azoles. The RAPD-PCR method identified the formation of two different groups. In group A, four resistant and two susceptible isolates were clustered, and in group B, one resistant and six susceptible isolates were clustered. CONCLUSIONS: Considering that hyphal formation was higher in resistant isolates in the presence of azole drugs, we confirmed that the hyphal production is closely related to susceptibility to azoles. These drugs may affect the morphogenesis of C. albicans depending on their susceptibility to these drugs. In relation to RAPD-PCR, most resistant isolates classified in group A and susceptible isolates in group B demonstrated that this method presented a similar standard between the two groups, suggesting that by this technique, a strong correlation between genotypes and fluconazole-resistant samples may be found.

INTRODUÇÃO: Candida albicans é responsável por infecções superficiais ou sistêmicas conhecidas como candidíase, encontrada em tecidos infectados na forma de leveduras brotantes unicelulares, hifas ou pseudohifas. Neste estudo, os efeitos de agentes antifúngicos como o fluconazol e o itraconazol sobre a formação de hifas e caracterização genotípica de isolados de C. albicans suscetíveis ou resistentes foram investigados. MÉTODOS: A produção de hifas de cinco isolados de C. albicans, sob a ação de antifúngicos foi investigada pelo cultivo da levedura em meios de crescimento e de indução de hifas. A caracterização genotípica foi realizada para 13 isolados de C. albicans pelo método de RAPD-PCR. RESULTADOS: A análise do dimorfismo mostrou que a formação de hifas foi maior nos isolados resistentes do que nos suscetíveis aos antifúngicos. O método de RAPD-PCR identificou a formação de dois diferentes grupos. No grupo A, foram agrupados quatro isolados resistentes e dois suscetíveis e no grupo B um resistente e seis suscetíveis. CONCLUSÕES: Considerando que a formação hifal foi maior em isolados resistentes na presença de azólicos, concluimos que a produção hifal está muito relacionada a suscetibilidade a estes fámacos. Estes antifúngicos podem alterar a morfologia de C. albicans em dependência da sua suscetibilidade. No método de RAPD-PCR, o encontro da maioria dos isolados resistentes classificados como pertencentes ao grupo A e suscetíveis ao grupo B demonstrou que este método apresentou um padrão semelhante entre os dois grupos, sugerindo que por este método pode ser detectado uma estreita correlação entre genótipos e amostras resistentes ao fluconazol.

Identificação de espécies de candida em saliva de profissionais da saúde / Identification of candida species on the saliva of the hospital workers / La identificación de las especies de candida en salivas de profesionales de salud

As espécies de Candida são de ocorrência comum na cavidade bucal sendo que estas leveduras estão presentes em cerca de 50% de indivíduos saudáveis, podendo este porcentual variar dependendo da população estudada. Os processos infecciosos causados por Candida variam de quadros clínicos benignos, a quadros graves e fatais como as infecções invasivas e disseminadas. Neste estudo foi avaliada a colonização de leveduras do gênero Candida em 312 amostras de saliva de profissionais do Hospital das Clínicas da Universidade Federal de Goiás, na cidade de Goiânia, Goiás, Brasil, durante o período de julho a dezembro de 2007. As espécies de Candida isoladas da saliva foram identificadas através da produção de tubo germinativo e de clamidoconídios, assimilação de carboidratos e cultivo em meio CHROMagar® Candida. Os profissionais estudados mostraram colonização por leveduras do gênero Candida em 61,9% das amostras analisadas, sendo isolados C. albicans (58,5%), C. parapsilosis (24,4%), C. krusei (8,3%) e C. famata (3,6%). A divulgação destes resultados pode servir como um auxílio para a prevenção e controle de candidíase bucal nos trabalhadores da área de saúde que vivem em contato constante com indivíduos altamente suscetíveis a infecções.

Candida species are of common occurrence in the buccal cavity and these yeasts are present in about 50% of healthy individuals. This percentage can change depending on the studied population. The infectious processes caused by Candida vary of benign clinical pictures, to serious and fatal pictures as the invasive infections and disseminated. In this study it was evaluated the colonization of yeasts of the Candida genus on 312 saliva sample of the hospital workers from ?Hospital das Clínicas da Universidade Federal de Goiás? in the city of Goiania-Goiás/Brazil, during the period from July to December of 2007. Candida species were identified by production of germ-tube, chlamydospore formation, assimilation sugar tests and culture on CHROMagar® Candida. The hospital workers analyzed presented colonization of the saliva by yeasts of the Candida genus in 61.9% from samples studied, being isolated C. albicans (58.5%), C. parapsilosis (24.4%), C. krusei (8.3%) and C. famata (3.6%). The publication of these results can serve as an aid for the prevention and control of oral candidiasis in the workers of the area of health that live in contact with individuals susceptible to infections.

Especies de Candida son de ocurrencia común en la cavidad bucal y estas levaduras están presentes en cerca del 50% de individuos sanos. La frecuencia varía, dependiendo de las personas investigadas. Los procesos infecciosos causados por Candida varían de cuadros clínicos benignos, a los cuadros serios y fatales como las infecciones invasivas y diseminadas. En este estudio ha sido evaluado la colonización de las especies de Candida en la saliva de los obreros del Hospital das Clínicas de la Universidad Federal de Goiás en Goiânia, Brasil, entre julio y diciembre de 2007. El trabajo se llevó a cabo con 312 muestras de saliva. Las especies de Candida aisladas de la saliva fueron identificadas a través de la formación de tubo germinal y de clamidoconídio, asimilación de carbohidratos y cultivo en CHROMagar® Candida. En nuestro estudio, 61.9% de los trabajadores analizados del hospital presentaron colonización de la saliva por las levaduras del género Candida, estar aislado C. albicans (58.5%), C. parapsilosis (24,4%), C. krusei (8,3%) y C. famata (3,6%). La divulgación de estos resultados puede servir como una ayuda para la prevención y control de candidíase bucal en los trabajadores del área de salud que viven en contacto constante con individuos susceptibles a las infecciones.

Differences in exoenzyme production and adherence ability of Candida spp. isolates from catheter, blood and oral cavity / Diferenças na produção de exoenzimas e habilidade de aderência entre isolados de espécies de Candida provenientes do cateter, sangue e cavidade bucal

Phospholipase and proteinase production and the ability of adhesion to buccal epithelial cells (BEC) of 112 Candida isolates originated from oral cavity of HIV infected patients and from blood and catheter of intensive care unit patients were investigated. The proteinase production was detected by inoculation into bovine serum albumin (BSA) agar and the phospholipase activity was performed using egg yolk emulsion. A yeast suspension of each test strain was incubated with buccal epithelial cells and the number of adherence yeast to epithelial cells was counted. A percentage of 88.1 percent and 55.9 percent of Candida albicans and 69.8 percent and 37.7 percent of non-albicans Candida isolates produced proteinase and phospholipase, respectively. Non-albicans Candida isolated from catheter were more proteolytic than C. albicans isolates. Blood isolates were more proteolytic than catheter and oral cavity isolates while oral cavity isolates produced more phospholipase than those from blood and catheter. C. albicans isolates from oral cavity and from catheter were more adherent to BEC than non-albicans Candida isolates, but the adhesion was not different among the three sources analyzed. The results indicated differences in the production of phospholipase and proteinase and in the ability of adhesion to BEC among Candida spp. isolates from different sources. This study suggests that the pathogenicity of Candida can be correlated with the infected site.

A produção de proteinase e fosfolipase e habilidade de adesão à célula epitelial bucal de 112 isolados de Candida originadas da cavidade bucal de pacientes infectados pelo HIV e de sangue e cateter de pacientes hospitalizados foram investigados. A produção de proteinase foi detectada por inoculação em ágar soro albumina bovina e a atividade de fosfolipase foi realizada usando emulsão de gema de ovo. A suspensão de levedura de cada isolado foi incubada com célula epitelial e o número de leveduras aderidas a célula epitelial foi contada. Uma porcentagem de 88,1 e 55,9 por cento de C. albicans e 69,8 e 37,7 por cento de isolados de Candida não albicans produziram proteinase e fosfolipase, respectivamente. Candida não albicans obtidas do cateter foram mais proteolíticos que isolados de Candida albicans (p < 0,001). Isolados do sangue foram mais proteolíticos do que isolados do cateter e cavidade bucal, enquanto isolados da cavidade bucal produziram mais fosfolipase do que aqueles isolados do sangue e cateter. C. albicans isoladas da cavidade bucal e do cateter foram mais aderentes à célula epitelial bucal do que isolados de Candida não albicans, mas não houve diferença na adesão entre os três locais analisados. Os resultados indicaram diferenças na produção de fosfolipase e proteinase e na habilidade de adesão à célula epitelial bucal entre os isolados de Candida das diferentes fontes. Este estudo sugere que a patogenicidade de Candida spp pode estar correlacionada ao local infectado.

RAPD profile among Candida albicans isolates by using different primers / Perfil de amplificação randômica de DNA polimórfico (RAPD) de isolados de Candida albicans utilizando diferentes iniciadores

By using RAPD method, a total of 28 primers were screened for discrimination among Candidaalbicans isolates. Seven of these primers were selected because they presented reproducible DNAbanding patterns for all the strains of C. albicans. The same genetic profile was obtained by RAPD method using OPG 14 (5’-GGATGAGACC-3’) while the OPG 17 (5’- ACGACCGACA-3’) demonstrated capacity to differentiate the isolates of C. albicans in 4 genotypes. According to the results obtained, we concluded that by using these primers, the RAPD analysis may be useful in providing genotypic characteristics for C. albicans in epidemiological investigation.

Usando o método de amplificação randômica de DNA polimórfico (Randomly Amplified Polymorphic DNA analysis, RAPD), um total de 28 iniciadores (primers) foi estudado para discriminar entre os isolados de Candida albicans. Sete destes primers foram selecionados porque apresentaram padrões de banda de DNA reprodutíveis para todos os isolados de C. albicans. O mesmo perfil genético foi obtido usando OPG 14 (5-GGATGAGACC-3), enquanto o OPG 17 (5-ACGACCGACA-3) demonstrou capacidade de diferenciar os isolados em quatro genótipos. De acordo com os resultados obtidos, nós concluímos que usando estes primers, a análise de RAPD pode ser útil em prover características genotípicas em investigações epidemiológicas.

Comparison of in vitro activity of five antifungal agents against dermatophytes, using the agar dilution and broth microdilution methods / Comparação da atividade in vitro de cinco agentes antifúngicos para dermatófitos, usando os métodos de diluição em ágar e microdiluição em caldo

The purpose of this study was to compare the agar dilution and broth microdilution methods for determining the minimum inhibitory concentration (MIC) of fluconazole, itraconazole, ketoconazole, griseofulvin and terbinafine for 60 dermatophyte samples belonging to the species Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. The percentage agreement between the two methods, for all the isolates with < 2 dilutions that were tested was 91.6 percent for ketoconazole and griseofulvin, 88.3 percent for itraconazole, 81.6 percent for terbinafine and 73.3 percent for fluconazole. One hundred percent agreement was obtained for Trichophyton mentagrophytes isolates evaluated with ketoconazole and griseofulvin. Thus, until a reference method for testing the in vitro susceptibility of dermatophytes is standardized, the similarity of the results between the two methods means that the agar dilution method may be useful for susceptibility testing on these filamentous fungi.

O propósito do presente trabalho foi comparar os métodos de diluição em ágar e diluição em caldo para a determinação de concentração inibitória mínima de fluconazol, itraconazol, cetoconazol, griseofulvina e terbinafina para 60 amostras de dermatófitos pertencentes às espécies, Trichophyton rubrum, Trichophyton. mentagrophytes e Microsporum canis. A porcentagem de acordo entre os dois métodos para todos os isolados testados considerando-se valores < 2 diluições, foram de 91,6 por cento para cetoconazol e para griseofulvina, de 88,3 por cento para itraconazol, de 81,6 por cento para terbinafina e de 73,3 por cento para fluconazol. Uma concordância de 100 por cento foi obtido para isolados de Trichophyton mentagrophytes avaliados com cetoconazol e griseofulvina. Desta forma, até que um método de referência seja padronizado para testar a suscetibilidade in vitro para os dermatófitos, os resultados semelhantes encontrados para os dois métodos fazem com que o método de diluição em ágar possa ser útil no teste de suscetibilidade para estes fungos filamentosos.

Susceptibility testing of Candida albicans isolated from oropharyngeal mucosa of HIV+ patients to fluconazole, amphotericin B and Caspofungin: Killing kinetics of caspofungin and amphotericin B against fluconazole resistant and susceptible isolates / Teste de suscetibilidade de Candida albicans isoladas da mucosa orofaringeana de pacientes HIV+, ao fluconazol, anfotericcina B e caspofungina: Cinética de morte de caspofungina e anfotericina B contra isolados fluconazol resistentes e suscetíveis

A clear understanding of the pharmacodynamic properties of antifungal agents is important for the adequate treatment of fungal infections like candidiasis. For certain antifungal agents, the determination of Minimal Fungicidal Concentration (MFC) and time kill curve could be clinically more relevant than the determination of the Minimal Inhibitory Concentration (MIC). In this study, MIC and MFC to fluconazole, amphotericin B and caspofungin against C. albicans isolates and the killing patterns obtained with caspofungin and amphotericin B against susceptible and resistant strains to fluconazole were determined. The results of MICs showed that all C. albicans isolates were highly susceptible to amphotericin B, but two isolates were fluconazole resistant. The comparative analysis between MIC and MFC showed that MFC of fluconazole was fourfold higher than MIC in 41.9% of the C. albicans isolates. Same values of MFC and MIC of amphotericin B and caspofungin were found for 71% of the isolates. Correlation between time kill curves and MFC of amphotericin B and caspofungin against all 4 isolates tested was observed. The caspofungin killing effect was more evident at MFC in 6 hours of incubation than at MIC in this time, suggesting dependence of concentration. The similarity of results of time-kill curve and MFC values indicate that determination of MFC is an alternative for the detection of the fungicidal activity of these drugs.

Um claro entendimento das propriedades farmacodinâmicas dos agentes antifúngicos é de grande importância para o adequado tratamento das infecções fúngicas como a candidíase. Em alguns casos de escolha do agente antifúngico, a determinação da concentração fungicida minima (CFM) e a curva do tempo de morte podem ser mais clinicamente relevantes do que a concentração inibitória minima (CIM). Nesse estudo, foi avaliado a CIM e a CFM de fluconazol, anfotericina B e caspofungina em Candida albicans e ainda os padrões de morte obtidos com caspofungina e anfotericina B de isolados suscetíveis e resistentes ao fluconazol. Os resultados de CIM mostraram que todos os isolados de Candida albicans foram altamente suscetíveis à anfotericina B, entretanto dois isolados foram fluconazol resistentes. A análise comparativa de CIM e da CFM mostrou que o CFM de fluconazol foi quatro vezes superior à CIM para 41,9% dos isolados de Candida albicans. Valores iguais de CFM e CIM de anfotericina B e caspofungina foram encontrados para 71% dos isolados. Correlação entre a curva do tempo de morte e a CFM de anfotericina B e caspofungina contra quatro isolados testados foi observada. O efeito de morte de caspofungina foi mais evidente na CFM até 6 horas de incubação do que na CIM nesse mesmo tempo, sugerindo a dependência da concentração. A similaridade dos resultados da curva do tempo de morte e os valores de CFM indicam que a determinação da CFM é uma escolha alternativa na detecção da atividade fungicida destes agentes antifúngicos.

In vitro susceptibility testing of dermatophytes isolated in Goiania, Brazil, against five antifungal agents by broth microdilution method / Teste de suscetibilidade in vitro de dermatófitos isolados em Goiânia, Brasil, contra cinco agentes antifúngicos pelo método de microdiluição em caldo

The antifungal activities of fluconazole, itraconazole, ketoconazole, terbinafine and griseofulvin were tested by broth microdilution technique, against 60 dermatophytes isolated from nail or skin specimens from Goiania city patients, Brazil. In this study, the microtiter plates were incubated at 28 ºC allowing a reading of the minimal inhibitory concentration (MIC) after four days of incubation for Trichophyton mentagrophytes and five days for T. rubrum and Microsporum canis. Most of the dermatophytes had uniform patterns of susceptibility to the antifungal agents tested. Low MIC values as 0.03 µg/mL were found for 33.3 percent, 31.6 percent and 15 percent of isolates for itraconazole, ketoconazole and terbinafine, respectively.

Atividades antifúngicas de fluconazol, itraconazol, cetoconazol, terbinafina e griseofulvina foram testadas pelo método de microdiluição em caldo contra 60 isolados de dermatófitos. Os resultados mostraram que todos os isolados produziram crescimento claramente detectável a 28 ºC e a concentração inibitória mínima (CIM) foi determinada após quatro dias de incubação para Trichophyton mentagrophytes e cinco dias para T. rubrum e Microsporum canis. A maioria dos isolados teve um padrão uniforme de suscetibilidade para os agentes antifúngicos testados. Baixos valores de CIM como 0,03 µg/mL foram encontrados para 33,3 por cento, 31,6 por cento e 15 por cento dos isolados para itraconazol, cetoconazol e terbinafina, respectivamente.

Molecular epidemiological analysis of bloodstream isolates of candida albicans

Candidemia está geralmente relacionada à microbiota endógena, porém infecções exógenas originadas do staff ou do meio ambiente podem ocorrer. O método de DNA polimórfico amplificado aleatoriamente (RAPD) pode revelar variações específicas do isolado. Neste estudo, foi utilizada a análise de RAPD para avaliar a diversidade genética entre isolados de C. albicans, buscando-se encontrar similaridade entre os padrões de DNA dos isolados obtidos de amostras clínicas e ambientais da unidade de terapia intensiva (UTI) no Hospital das Clínicas da Universidade Federal de Goiás. Os primers denominados Cnd3 (5-CCAGATGCAC-3) e Cnd4 (5-ACGGTACACT-3) foram usados como primer na PCR. Perfis de RAPD do sangue e da urina, provenientes dos mesmos pacientes, mostraram-se idênticos em quase todas as amostras estudadas, exceto na de um paciente. O isolado da cama deste paciente tinha o mesmo genótipo da amostra obtida de seu sangue. Embora a maioria dos isolados de C. albicans provavelmente tivesse origem endógena, o encontro de isolados de pacientes com o mesmo perfil dos isolados do meio ambiente sugere que a candidemia pode resultar de uma fonte exógena.

Ocorrência de fungos filamentosos em acervo da Faculdade de Medicina da Universidade Federal de Goiás / Filamentous fungi in the archive of the Medicine Faculty of the Goias Federal University, Brazil

Microrganismos como fungos, bactérias e outros constituem freqüente ameaça para acervos de bibliotecas e arquivos, assim como para seus usuários, principalmente quando instalados em locais inadequados. A Faculdade de Medicina da Universidade Federal de Goiás abriga, em seu subsolo, uma sala na qual foi instalado um arquivo de livros de registros, classificadores, caixas de documentos e revistas guardados em estantes e armários. Amostras deste arquivo e do ar ambiente da sala foram coletadas para pesquisa de fungos. Foram isolados e identificados, neste estudo, Aspergillus spp, Penicillium spp, Mucor spp. e outros fungos filamentosos, que são amplamente distribuídos no meio ambiente, como solo e ar, e podem infectar o homem, causando doença quando encontram condições favoráveis ao seu desenvolvimento.

Fenotipagem de bactérias isoladas em hemoculturas de pacientes críticos / Phenotyping of bacteria isolated in blood cultures from critical patients

As infecções da corrente sangüínea representam uma grave complicação dos pacientes críticos, sendo a detecção de patógenos microbianos em hemoculturas um importante recurso diagnóstico. Esse estudo objetivou isolar e caracterizar bactérias do sangue de pacientes admitidos na unidade clínica de terapia intensiva de um hospital universitário, no período de abril/2003 a abril/2004. As bactérias isoladas foram identificadas por provas bioquímicas/enzimáticas e a detecção do perfil de suscetibilidade aos antimicrobianos pelo método de difusão em disco. Foram avaliadas 304 hemoculturas de 195 pacientes, sendo que 49 (16,1 por cento) apresentaram desenvolvimento microbiano. A espécie predominante foi Pseudomonas stutzeri (18,2 por cento). Os cocos Gram-positivos e as enterobactérias apresentaram maior resistência à ampicilina; vancomicina e linezolida foram os agentes mais ativos para cocos Gram-positivos e as carbapenemas para bastonetes Gram-negativos. Devido ao impacto das infecções da corrente sangüínea no contexto hospitalar, estudos adicionais são necessários para subsidiar medidas de prevenção e controle.

Bloodstream infections are a serious complication among critical patients. The detection of microbial pathogens in blood cultures is an important diagnostic tool. This study aimed to isolate and characterize bacteria from the blood of patients admitted to the clinical intensive care unit of a teaching hospital, between April 2003 and April 2004. The bacteria isolated were identified by biochemical/enzymatic tests and detection of the antimicrobial susceptibility profile using the disk diffusion method. A total of 304 blood cultures from 195 patients were evaluated. Forty-nine (16.1 percent) of the blood cultures presented microbe development. The predominant species was Pseudomonas stutzeri (18.2 percent). Gram-positive cocci and enterobacteria showed greater resistance to ampicillin; vancomycin and linezolid were the most active agents for Gram-positive cocci and carbapenems for Gram-negative rods. Because of the impact of bloodstream infection within the hospital context, additional studies are needed in order to give backing for prevention and control measures.

Identificação de espécies de Malassezia em pacientes com pitiríase versicolor em Goiânia-GO / Identification of Malassezia species in patients with pityriasis versicolor in Goiânia-GO

O objetivo deste estudo foi verificar a freqüência de pitiríase versicolor e identificar leveduras do gênero Malassezia, de pacientes encaminhados ao laboratório de Micologia da Universidade Federal de Goiás em Goiânia. Foram diagnosticados 95 casos de pitíriase versicolor e identificados quatro espécies de Malassezia: Malassezia furfur, Malassezia sympodialis, Malassezia globosa e Malassezia obtusa.

The objectives of this study were to investigate the frequency of pityriasis versicolor occurrence and to identify yeasts of the genus Malassezia in patients at the mycology laboratory of the Federal University of Goiás, in Goiânia, State of Goiás. Ninety-five cases of pityriasis versicolor were diagnosed, and four species of Malassezia were identified: Malassezia furfur, Malassezia sympodialis, Malassezia globosa and Malassezia obtusa.