Susceptibility to Oral Antiseptics and Virulence Factors Ex Vivo Associated with Candida spp. Isolated from Dental Prostheses.

PURPOSE: To isolate Candida spp. from dental prosthesis users' saliva and to evaluate the isolates for the presence of several virulence factors. This research also aimed to investigate the antifungal activity of 3 commercial mouthwashes/oral antiseptic formulations containing 0.12% chlorhexidine, 0.07% cetylpyridinium, or 0.075% cetylpyridinium against planktonic and sessile (biofilm mode) yeast cells. MATERIALS AND METHODS: Forty-three Candida yeasts were isolated from 32 of 70 selected patients, and the virulence factors of C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis species were investigated by polymerase chain reaction (PCR) and proteinase in plates. Minimum inhibitory concentration (MIC), and in vitro biofilm assay evaluated the antifungal activity of antiseptics. RESULTS: C. albicans, C. krusei, C. glabrata, C. tropicalis, and C. parapsilosis were detected in mono and mixed cultures. Only C. albicans displayed genes related to adhesion and proteinases (ALS2, ALS3, SAP1, and SAP3). The aspartate proteinase activity was found in 60.46% of isolates. The tested antiseptic formulations exhibited a MIC less than 1.25% toward yeasts in the planktonic mode. According to XTT ((2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay results, most Candida isolates and all mixed cultures formed biofilms within 24 hours. The evaluated antiseptic formulations were also active against biofilms. CONCLUSION: Most virulence factors investigated here (ALS2, ALS3, SAP1, and SAP3) occurred in the majority of the Candida spp. isolates, especially in C. albicans. The tested mouthwash formulations were effective against all the yeast isolates in both the planktonic and sessile growth modes. Developing alternative therapies that can avoid or control biofilm formation is necessary to prevent oral candidiasis and other Candida spp. infections.

FREQUENCY OF Candida SPECIES IN A TERTIARY CARE HOSPITAL IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL.

Infections by Candida species are a high-impact problem in public health due to their wide incidence in hospitalized patients. The goal of this study was to evaluate frequency, susceptibility to antifungals, and genetic polymorphism of Candida species isolated from clinical specimens of hospitalized patients. The Candida isolates included in this study were obtained from blood cultures, abdominal fluids, and central venous catheters (CVC) of hospitalized patients at the Clinical Hospital of the Federal University of Uberlândia during the period of July 2010 - June 2011. Susceptibility tests were conducted by the broth microdilution method. The RAPD-PCR tests used employed initiator oligonucleotides OPA09, OPB11, and OPE06. Of the 63 Candida isolates, 18 (28.5%) were C. albicans, 20 (31.7%) were C. parapsilosis complex species, 14 (22.2%) C. tropicalis, four (6.4%) C. glabrata, four (6.4%) C. krusei, two (3.3%) C. kefyr, and one (1.6%) C. lusitaniae. In vitro resistance to amphotericin B was observed in 12.7% of isolates. In vitro resistance to azoles was not detected, except for C. krusei. The two primers, OPA09 and OPB11, were able to distinguish different species. Isolates of C. albicans and C. parapsilosis complex species presented six and five clusters, respectively, with the OPA09 marker by RAPD-PCR, showing the genetic variability of the isolates of those species. It was concluded that members of the C. parapsilosis complex were the most frequent species found, and most isolates were susceptible to the antifungals amphotericin B, flucozanole, and itraconazole. High genetic polymorphisms were observed for isolates of C. albicans and C. parapsilosis complex species, mainly with the OPA09 marker.

FREQUENCY OF Candida SPECIES IN A TERTIARY CARE HOSPITAL IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL / Frequência de espécies de Candida em hospital terciário do Triângulo Mineiro, Minas Gerais, Brasil

Infections by Candida species are a high-impact problem in public health due to their wide incidence in hospitalized patients. The goal of this study was to evaluate frequency, susceptibility to antifungals, and genetic polymorphism of Candida species isolated from clinical specimens of hospitalized patients. The Candida isolates included in this study were obtained from blood cultures, abdominal fluids, and central venous catheters (CVC) of hospitalized patients at the Clinical Hospital of the Federal University of Uberlândia during the period of July 2010 - June 2011. Susceptibility tests were conducted by the broth microdilution method. The RAPD-PCR tests used employed initiator oligonucleotides OPA09, OPB11, and OPE06. Of the 63 Candida isolates, 18 (28.5%) were C. albicans, 20 (31.7%) were C. parapsilosis complex species, 14 (22.2%) C. tropicalis, four (6.4%) C. glabrata, four (6.4%) C. krusei, two (3.3%) C. kefyr, and one (1.6%) C. lusitaniae. In vitro resistance to amphotericin B was observed in 12.7% of isolates. In vitro resistance to azoles was not detected, except for C. krusei. The two primers, OPA09 and OPB11, were able to distinguish different species. Isolates of C. albicans and C. parapsilosis complex species presented six and five clusters, respectively, with the OPA09 marker by RAPD-PCR, showing the genetic variability of the isolates of those species. It was concluded that members of the C. parapsilosis complex were the most frequent species found, and most isolates were susceptible to the antifungals amphotericin B, flucozanole, and itraconazole. High genetic polymorphisms were observed for isolates of C. albicans and C. parapsilosis complex species, mainly with the OPA09 marker.

As infecções causadas por espécies de Candida são problema de grande impacto para a saúde pública, devido à alta incidência em pacientes hospitalizados e como causa de mortalidade. O presente estudo teve como objetivo avaliar a frequência de Candida spp. isoladas de pacientes hospitalizados, assim como a sensibilidade aos antifúngicos e o polimorfismo genético por RAPD-PCR. Os microrganismos incluíram isolados de hemocultura, líquido abdominal e ponta de cateter venoso central de pacientes internados no Hospital de Clínicas da Universidade Federal de Uberlândia, região do Triângulo Mineiro, Minas Gerais, Brasil, no período de julho de 2010-junho de 2011. Os testes de sensibilidade aos antifúngicos foram realizados por microdiluição em caldo e na análise por RAPD-PCR foram utilizados os oligonucleotídeos OPA09, OPB11, e OPE06. Dos 63 isolados, 18 (28,5%) foram C. albicans, 20 (31,7%) C. parapsilosis, 14 (22,2%) C. tropicalis, quatro (6,4%) C. glabrata, quatro (6,4%) C. krusei, dois (3,3%) C. kefyr, e um (1,6%) C. lusitaniae. Resistência in-vitro à anfotericina B foi observada em 12,7% dos isolados. Não foi observada resistência in-vitro aos azólicos, exceto para os isolados de C. krusei. Os oligonucleotídeos OPA09 e OPB11 possibilitaram distinguir diferentes espécies. Isolados de C. albicans apresentaram seis clusters e o complexo C. parapsilosis, cinco clusters, com o iniciador OPA09, por RAPD-PCR, mostrando a variabilidade genética daquelas espécies. Conclui-se que o complexo C. parapsilosis foi a espécie mais frequente, e a maioria dos isolados foi sensível in vitro aos antifúngicos testados. Alto polimorfismo genético foi observado para os isolados de C. albicans e complexo C. parapsilosis, principalmente com o oligonucleotídeo OPA09.

Sensibilidade de isolados de candida spp. a antifúngicos por disco-difusão em ágar e microdiluição em caldo / Antifungal susceptibility of isolates of candida spp. by disk diffusion and broth microdilution

Os testes de sensibilidade aos antifúngicos realizados pelo método de disco-difusão em ágar são práticos e bem conhecidos pelos profissionais do laboratório de microbiologia, entretanto apresentam particularidades que os diferem dos testes realizados para bactérias. O objetivo deste trabalho foi comparar as técnicas de disco-difusão em ágar e microdiluição em caldo na determinação da sensibilidade in vitro de isolados de Candida spp. a antifúngicos. Foram analisados 63 isolados clínicos de leveduras, que incluíram as espécies Candida parapsilosis complex (n = 20), Candida albicans (n = 18), Candida tropicalis (n = 14), Candida glabrata (n = 4), Candida krusei (n = 4), Candida kefyr (n = 2) e Candida lusitaniae (n = 1). As técnicas de disco-difusão em ágar e de microdiluição em caldo foram utilizadas para testar a sensibilidade em relação aos antifúngicos fluconazol, itraconazol e anfotericina B. A sensibilidade ao voriconazol foi determinada somente pela técnica de disco-difusão. Os halos ao redor dos discos de fluconazol variaram de 14 mm a 50 mm, e a CIM de 0,125 µg/mL a 32 µg/mL; para itraconazol, os halos variaram de 9 mm a 27 mm e a CIM de 0,03 µg/mL a 0,25 µg/mL; para anfotericina B, 9 mm a 21mm e 0,5 µg/mL a 2 µg/mL, respectivamente; para voriconazol, o diâmetro dos halos variaram de 19 mm a 50 mm. Para as três espécies, C. albicans, C. parapsilosis e C. tropicalis, a técnica de disco-difusão apresentou boa concordância com a microdiluição, especialmente em relação ao fluconazol, representando, assim, um recurso importante para os laboratórios reportarem os resultados dos testes de sensibilidade dos isolados dessas espécies ao fluconazol.

Antimicrobial susceptibility tests performed by disk diffusion method are practical and well known by professionals that work in the microbiology laboratory. The disk diffusion methodology used to verify the susceptibility of fungi to antifungal agents, however, has characteristics that differ from the tests for bacteria. The objective of this study was to evaluate the disk diffusion method to determine the in vitro susceptibility to antifungal agents of Candida species. We analyzed 63 clinical isolates of yeasts, which included Candida parapsilosis complex species (n = 20), Candida albicans (n = 18), Candida tropicalis (n = 14), Candida glabrata (n = 4), Candida krusei (n = 4), Candida kefyr (n = 2) and Candida lusitaniae (n = 1). The susceptibility tests to antifungal drugs was performed by disk diffusion methods and broth microdilution for antifungal fluconazole, itraconazole and amphotericin B. Voriconazole was used to test the susceptibility only by the disk diffusion method. The inhibition halos of growth around disks of fluconazole ranged from 14 mm to 50 mm and the MIC from 0.125 µg/mL to 32 µg/mL, for itraconazole, halos ranged from 9 mm to 27 mm and the MIC from 0.03 µg/mL to 0.25 µg/mL, for amphotericin B, 9 mm to 21 mm and 0.5 µg/mL to 2 µg/mL, respectively. The diameter of voriconazole disks varied from 19 mm to 50 mm. For the three species, C. albicans, C. parapsilosis and C. tropicalis, the disk diffusion method showed good agreement with the microdilution, especially to fluconazole, thus representing an important resource for medical laboratories reporting results of susceptibility testing of isolates of these species to fluconazole.

Comparison of primers for RAPD-PCR from environmental isolates of Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii complex

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e.g., CAV2 for C. albidus) may not to be applied to some species.

Comparison of primers for RAPD-PCR from environmental isolates of Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii complex.

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e.g., CAV2 for C. albidus) may not to be applied to some species.

Virulence attributes and genetic variability of oral Candida albicans and Candida tropicalis isolates.

The wide spectrum of candidiasis and its clinical importance encourage the research with the purpose of clarifying the mechanisms of pathogenicity and identification of virulence factors of Candida sp. Therefore, the aim of this study was to verify the adhesion capacity, protease activity and genotypic diversity of oral C. albicans and C. tropicalis isolates. The adhesion ability to the extracellular matrix glycoproteins laminin and fibronectin was evaluated using the ELISA technique. The research of proteases was carried out in agar plate containing bovine albumin and through a quantitative method in buffer solution containing haemoglobin. Intra and interspecies polymorphisms was verified through random amplified polymorphic DNA (RAPD) technique. All C. albicans and C. tropicalis isolates binded to immobilised laminin and fibronectin. Ca33 and Ct13 isolates had relative adhesion index significantly higher than the other isolates for both glycoproteins (P < 0.001). Protease activity was observed in all isolates of C. albicans using either the semi-quantitative or quantitative assay. The protease activity of C. tropicalis was better detected through the quantitative assay. The genotypic diversity by RAPD revealed a heterogeneous population in both species. Nevertheless, C. tropicalis presented higher genetic variability than C. albicans strains.

Effervescent tablets and ultrasonic devices against Candida and mutans streptococci in denture biofilm.

OBJECTIVE: To evaluate the antimicrobial action of effervescent tablets and ultrasound on Candida spp. and mutans streptococci from denture biofilm. BACKGROUND: It is not uncommon for edentulous patients to be elderly and find it difficult to brush their dentures. Hence, auxiliary methods are required for cleansing dentures as well as treating oral infections. MATERIALS AND METHODS: Seventy-seven complete denture wearers were randomly assigned into four groups: (A) Brushing with water (control); (B) Effervescent tablets; (C) Ultrasonic device (Ultrasonic Cleaner, model 2840 D); (D) Effervescent tablets and ultrasonic device. All groups brushed their dentures with a specific brush and water, three times a day, before applying their treatments. Denture biofilm was collected at baseline and after 21 days. The samples were collected by brushing the dentures with saline and the detached microbial cells were quantified by plating. Counts [log (CFU+1) ml(-1) ] of total aerobes, Candida spp. and mutans streptococci were compared by one-way anova or Kruskal-Wallis test (α = 0.05). RESULTS: No significant difference was found among the methods from C. albicans (p = 0.76), C. tropicalis (p = 0.94) and C. glabrata (p = 0.80). Lower counts were found for methods B and D when compared with the other methods against mutans streptococci (p < 0.001). Method B showed lower total aerobic counts than A, whereas C and D showed intermediate results (p = 0.011). CONCLUSION: The effervescent tablets significantly reduced mutans streptococci and total aerobes from denture biofilm. However, they was not as effective against C. albicans. Ultrasonic cleansing presented a discrete antimicrobial effect and was less effective than the tablets for complete denture disinfection.

Biotype stability of Candida albicans isolates after culture storage determined by randomly amplified polymorphic DNA and phenotypical methods.

Typing methods to evaluate isolates in relation to their phenotypical and molecular characteristics are essential in epidemiological studies. In this study, Candida albicans biotypes were determined before and after storage in order to verify their stability. Twenty C. albicans isolates were typed by Randomly Amplified Polymorphic DNA (RAPD), production of phospholipase and proteinase exoenzymes (enzymotyping) and morphotyping before and after 180 days of storage in Sabouraud dextrose agar (SDA) and sterilised distilled water. Before the storage, 19 RAPD patterns, two enzymotypes and eight morphotypes were identified. The fragment patterns obtained by RAPD, on the one hand, were not significantly altered after storage. On the other hand, the majority of the isolates changed their enzymotype and morphotype after storage. RAPD typing provided the better discriminatory index (DI) among isolates (DI = 0.995) and maintained the profile identified, thereby confirming its utility in epidemiological surveys. Based on the low reproducibility observed after storage in SDA and distilled water by morphotyping (DI = 0.853) and enzymotyping (DI = 0.521), the use of these techniques is not recommended on stored isolates.

Evaluation of the experimental inoculation of Cryptococcus albidus and Cryptococcus laurentii in normal mice: virulence factors and molecular profile before and after animal passage.

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37 degrees C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.

Candida albicans and Candida tropicalis in oral candidosis: quantitative analysis, exoenzyme activity, and antifungal drug sensitivity.

Candida albicans and C. tropicalis obtained from whole saliva of patients presenting signs of oral candidosis were assayed for quantification of colony forming units, exoenzyme activity (phospholipase and proteinase) and antifungal drug sensitivity (amphotericin B, fluconazole and itraconazole) by the reference method of the Clinical and Laboratory Standards Institute. The number of colony forming units per milliliter varied according to the Candida species involved and whether a single or mixed infection was present. Proteinase activity was observed in both C. albicans and C. tropicalis, but phospholipase activity was noted only in C. albicans. In vitro resistance to antifungals was verified in both species, but C. tropicalis appears to be more resistant to the tested antifungals than C. albicans.

Dermatophyte susceptibilities to antifungal azole agents tested in vitro by broth macro and microdilution methods.

The in vitro susceptibility of dermatophytes to the azole antifungals itraconazole, fluconazole and ketoconazole was evaluated by broth macro and microdilution methods, according to recommendations of the CLSI, with some adaptations. Twenty nail and skin clinical isolates, four of Trichophyton mentagrophytes and 16 of T. rubrum were selected for the tests. Itraconazole minimal inhibitory concentrations (MIC) varied from < 0.03 to 0.25 microg/mL in the macrodilution and from < 0.03 to 0.5 microg/mL in the microdilution methods; for fluconazole, MICs were in the ranges of 0.5 to 64 microg/mL and 0.125 to 16 microg/mL by the macro and microdilution methods, respectively, and from < 0.03 to 0.5 microg/mL by both methods for ketoconazole. Levels of agreement between the two methods (+/- one dilution) were 70% for itraconazole, 45% for fluconazole and 85% for ketoconazole. It is concluded that the strains selected were inhibited by relatively low concentrations of the antifungals tested and that the two methodologies are in good agreement especially for itraconazole and ketoconazole.

Dermatophyte susceptibilities to antifungal azole agents tested in vitro by broth macro and microdilution methods / Suscetibilidade in vitro de dermatófitos a azóis pelos métodos macro e microdiluição em caldo

The in vitro susceptibility of dermatophytes to the azole antifungals itraconazole, fluconazole and ketoconazole was evaluated by broth macro and microdilution methods, according to recommendations of the CLSI, with some adaptations. Twenty nail and skin clinical isolates, four of Trichophyton mentagrophytes and 16 of T. rubrum were selected for the tests. Itraconazole minimal inhibitory concentrations (MIC) varied from < 0.03 to 0.25 µg/mL in the macrodilution and from < 0.03 to 0.5 µg/mL in the microdilution methods; for fluconazole, MICs were in the ranges of 0.5 to 64 µg/mL and 0.125 to 16 µg/mL by the macro and microdilution methods, respectively, and from < 0.03 to 0.5 µg/mL by both methods for ketoconazole. Levels of agreement between the two methods (± one dilution) were 70 percent for itraconazole, 45 percent for fluconazole and 85 percent for ketoconazole. It is concluded that the strains selected were inhibited by relatively low concentrations of the antifungals tested and that the two methodologies are in good agreement especially for itraconazole and ketoconazole.

Foi avaliada a suscetibilidade in vitro de dermatófitos aos antifúngicos itraconazol, fluconazol e cetoconazol, pelos métodos macro e microdiluição em caldo, de acordo com as recomendações do CLSI, com algumas modificações. Foram estudados 20 isolados clínicos de lesões de unha e pele, sendo quatro Trichophyton mentagrophytes e 16 T. rubrum. A concentração inibitória mínima (CIM) para itraconazol variou de < 0,03 a 0,25 µg/mL pelo método da macrodiluição, e de < 0,03 a 0,5 µg/mL pela microdiluição em caldo; de 0,5 a 64 µg/mL e de 0,125 a 16 µg/mL para fluconazol, respectivamente, pela macro e microdiluição; e de < 0,03 a 0,5 µg/mL por ambos os métodos para cetoconazol. A concordância entre os dois métodos (considerando ± uma diluição) foi de 70 por cento para itraconazol, 45 por cento para fluconazol e 85 por cento para cetoconazol. Conclui-se que os isolados estudados foram inibidos por concentrações relativamente baixas dos antifúngicos testados, e os dois métodos apresentam boa concordância, especialmente para itraconazol e cetoconazol.

[Evaluation of melanin production by Cryptococcus species in four different culture media]. / Avaliação da produção de melanina por espécies de Cryptococcus em quatro diferentes meios de cultura.

The capacity of Cryptococcus spp to produce melanin in media containing phenol compounds is widely used for identifying these species in the laboratory. The aim of the present study was to compare the production of this pigment by Cryptococcus spp. in four culture media. Sixteen strains of Cryptococcus neoformans, 17 of Cryptococcus albidus, 13 of Cryptococcus laurentii and two of Cryptococcus uniguttulatus were tested in the following media: potato-carrot agar, Niger seed agar, sunflower seed agar and L-dopa agar. The melanin production was evaluated on the basis of colony pigmentation. Its production after five days of incubation was demonstrated by 93.8% of the strains of Cryptococcus neoformans in the media of potato-carrot agar, sunflower seed agar and L-dopa agar. From the isolates of Cryptococcus albidus, 29.4% produced the pigment in potato-carrot agar and L-dopa agar, 11.8% in Niger seed agar and 36% in sunflower seed agar. From Cryptococcus laurentii, 53.8% produced the pigment in potato-carrot agar and sunflower seed agar, 61.5% in L-dopa agar and 84.6% in Niger seed agar. Only one strain of Cryptococcus uniguttulatus presented slight production of the pigment, in potato-carrot agar.

Avaliação da produção de melanina por espécies de Cryptococcus em quatro diferentes meios de cultura / Evaluation of melanin production by Cryptococcus species in four different culture media

A capacidade de Cryptococcus spp produzir melanina em meios contendo compostos fenólicos é amplamente utilizada na identificação destas espécies no laboratório. O objetivo do presente trabalho foi comparar a produção desse pigmento em quatro meios de cultura por Cryptococcus sp. Foram testadas 16 cepas de Cryptococcus neoformans, 17 de Cryptococcus albidus, 13 de Cryptococcus laurentii, e 2 de Cryptococcus uniguttulatus nos meios: ágar batata e cenoura, ágar alpiste, ágar semente de girassol e ágar L-dopa. A produção de melanina foi avaliada com base na pigmentação das colônias, e demonstrada em 5 dias de incubação por 93,8 por cento das cepas de Cryptococcus neoformans nos meios ágar batata e cenoura, ágar semente de girassol e ágar L-dopa. Dos isolados de Cryptococcus albidus, 29,4 por cento produziram o pigmento em ágar batata e cenoura e L-dopa, 11,8 por cento em ágar alpiste, e 36 por cento em ágar girassol. De Cryptococcus laurentii, 53,8 por cento produziram em batata e cenoura e em semente de girassol, 61,5 por cento em L-dopa, 84,6 por cento em ágar alpiste. Somente uma cepa de Cryptococcus uniguttulatus produziu fracamente o pigmento em ágar batata e cenoura.

The capacity of Cryptococcus spp to produce melanin in media containing phenol compounds is widely used for identifying these species in the laboratory. The aim of the present study was to compare the production of this pigment by Cryptococcus spp. in four culture media. Sixteen strains of Cryptococcus neoformans, 17 of Cryptococcus albidus, 13 of Cryptococcus laurentii and two of Cryptococcus uniguttulatus were tested in the following media: potato-carrot agar, Niger seed agar, sunflower seed agar and L-dopa agar. The melanin production was evaluated on the basis of colony pigmentation. Its production after five days of incubation was demonstrated by 93.8 percent of the strains of Cryptococcus neoformans in the media of potato-carrot agar, sunflower seed agar and L-dopa agar. From the isolates of Cryptococcus albidus, 29.4 percent produced the pigment in potato-carrot agar and L-dopa agar, 11.8 percent in Niger seed agar and 36 percent in sunflower seed agar. From Cryptococcus laurentii, 53.8 percent produced the pigment in potato-carrot agar and sunflower seed agar, 61.5 percent in L-dopa agar and 84.6 percent in Niger seed agar. Only one strain of Cryptococcus uniguttulatus presented slight production of the pigment, in potato-carrot agar.

[Enzymatic activity, slime production and antifungal agent sensitivity of Candida sp]. / Atividade enzimática, produção de slime e sensibilidade a antifúngicos de Candida sp.

Abilith of Candida spp to secrete extracellular enzymes and slime has been associated as pathogenicity factors. Out of a total of 37 strains of Candida sp, 100% were proteinase producers, 83.8% were phospholipase producers, 64.9% were slime producers and 100% were sensitive to fluconazole and itraconazole. Seventeen typings (enzymes/slime) were found. This methodology presented a good discrimination rate (D=0.93) and could be used for phenotypic characterization of yeasts.

Atividade enzimática, produção de slime e sensibilidade a antifúngicos de Candida sp / Enzymatic activity, slime production and antifungal agent sensitivity of Candida sp

A habilidade de Candida spp secretar enzimas extracelulares e slime tem sido associada como fatores de patogenicidade. Do total de 37 cepas de Candida sp, 100 por cento foram produtoras de proteinase, 83,8 por cento fosfolipase, 64,9 por cento slime e 100 por cento sensíveis ao fluconazol e itraconazol. Foram encontradas 17 tipagens (enzima/slime). Esta metodologia apresentou um bom índice discriminatório (D=0,93) podendo ser utilizado na caracterização fenotípica das leveduras.

Abilith of Candida spp to secrete extracellular enzymes and slime has been associated as pathogenicity factors. Out of a total of 37 strains of Candida sp, 100 percent were proteinase producers, 83.8 percent were phospholipase producers, 64.9 percent were slime producers and 100 percent were sensitive to fluconazole and itraconazole. Seventeen typings (enzymes/slime) were found. This methodology presented a good discrimination rate (D = 0.93) and could be used for phenotypic characterization of yeasts.

[Occurrence of dermatophyte, in nails, feet and hands of university students]. / Ocorrência de dermatófitos em amostras de unhas, pés e mãos coletadas de estudantes universitários.

The objective of this paper was to evaluate the occurrence of dermatophytes, specifically in the nails, feet and hands of university students with and without lesions. Two hundred and eighty samples were collected; 31 (11.1%) were positive by direct examination, while only 20 (7.1%) showed dermatophyte growth in culture, as well as direct positive examination. Trichophyton rubrum was the most frequently isolated (80%) dermatophyte followed by T. mentagrophytes (20%). Considering the sites analyzed, dermatophyte occurrence was: 10.4% in toenails, 5% in foot skin, 2.5% in fingernails and 0.4% in hand skin.

In vitro susceptibility to antifungal agents of environmental Cryptococcus spp. isolated in the city of Ribeirão Preto, São Paulo, Brazil.

Infections by Cryptococcus strains other than C. neoformans have been detected in immunocompromised patients. Of these strains, three are considered human pathogens: C. albidus, C. laurenttii, and C. uniguttulatus. This study deals with the in vitro susceptibility of Cryptococcus to drugs such as amphotericin B, itraconazole, fluconazole, and 5-fluorocytosine. Environmental Cryptococcus isolates (50) distributed as follows: C. neoformans var. neoformans (16), C. albidus (17), C. laurentii (14), and C. uniguttulatus (3) were evaluated by the micro and macrodilution techniques, according to EUCAST and NCCLS recommendations, respectively. Considering both methodologies the respective minimal inhibitory concentrations (MIC) were 0.125 and 2 microg/ml for amphotericin B, 0.06 and 8 microg/ml for itraconazole, and 0.5 and more than 64 microg/ml for fluconazole and 5-fluorocytosine. Agreement percentages for the two methodologies were 100% for amphotericin B and fluconazole for all the strains tested. For itraconazole, the agreement percentage was 81.3% in the C. neoformans strain and 100% for all the others. All species had a agreement percentage of 94.1 to 100% when susceptibility to 5-fluorocytosine was tested. It is concluded that environmental isolates of C. neoformans var. neoformans, C. albidus, C. laurentii, and C. uniguttulatus may show high MICs against certain drugs, suggesting in vitro primary resistance to the antifungals tested.

Ocorrência de dermatófitos em amostras de unhas, pés e mãos coletadas de estudantes universitários / Occurrence of dermatophyte, in nails, feet and hands of universitary students

Este trabalho teve como objetivo, avaliar a presença de dermatófitos, especificamente em unhas, pés e mãos de estudantes universitários com e sem lesões sugestivas de dermatofitose. Foram coletadas 280 amostras dessas regiões, das quais 31 (11,1 por cento) apresentaram positividade apenas pelo exame direto, e 20 (7,1 por cento) tiveram, além do exame direto positivo, crescimento de dermatófito, mediante cultivo da amostra biológica. T. rubrum foi o dermatófito isolado com maior freqüência (80 por cento), seguido por T. mentagrophytes (20 por cento). Considerando os sítios analisados neste trabalho, a ocorrência de dermatófitos foi observada em 10,4 por cento nas unhas dos pés, 5 por cento nas escamas de pés, 2,5 por cento nas unhas das mãos e apenas 0,4 por cento nas escamas das mãos.