Trichophyton rubrum and Trichophyton interdigitale: genetic diversity among species and strains by random amplified polymorphic DNA method.

Onychomycosis is a common condition that represents up to 50% of all nail problems and 30% of all cases of dermatophytoses. Trichophyton rubrum and Trichophyton interdigitale are the most common agents involved in this condition. In cases of recurrent post-treatment onychomycosis, strain fingerprinting could reveal whether the original isolate is responsible, a new strain has been acquired or if multiple strains are involved. The aim of this study was to evaluate the efficacy of the RAPD method for species and strain differentiation of T. rubrum and T. interdigitale obtained from patients with subungeal distal-lateral onychomycosis. A set of 86 strains of onychomycosis causative dermatophytes were submitted to species differentiation and strain typing by RAPD method with two previously described primers. Both primers proved capable of strain differentiation when tested for each species. Nineteen molecular profiles were configured for T. rubrum isolates with primers 1 and 6. For T. mentagrophytes, ten molecular profiles were configured with primer 1 and twenty-one with primer 6. We found that T. interdigitale and T. rubrum species were grouped in different clusters when both primers were analyzed together. This study shows that these primers are valuable tools for strain differentiation with T. rubrum and T. intedigitale.

Identification and in vitro antifungal susceptibility testing of 200 clinical isolates of Candida spp. responsible for fingernail infections.

A total of 200 samples of Candida spp. that are responsible for fingernail infections were isolated in Belo Horizonte, MG, Brazil from April 2004 to May 2005. The samples were identified by routine microbiological techniques and had the following distribution: Candida parapsilosis (40.5%), C. albicans (31.5%), C. tropicalis (26%), and C. guilliermondii (2%). We performed in vitro susceptibility tests with ciclopiroxolamine, terbinafine, ketoconazole, itraconazole, and fluconazole using the CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antibiotic Susceptibility Testing) methodologies. The percentages of agreement between the two methodologies varied from 48 to 100% (the percentage increased to more than 60% for the majority of the samples). Percentages of agreement between the methodologies lower than 60% were seen with ketoconazole (57%) and itraconazole (48%) for samples of C. albicans and with fluconazole (54%) for samples of C. tropicalis. In general, we observed higher agreement between the values of the MICs obtained with both methodologies for ciclopiroxolamine and terbinafine for all tested species. With azoles, lower percentages of agreement between the methodologies were observed for samples C. albicans and C. tropicalis.

Successful treatment of a disseminated Sporothrix schenckii infection and in vitro analysis for antifungal susceptibility testing.

We report a case of a diabetic patient with localized osteoarticular sporotrichosis followed by widespread dissemination of the infection, which was successfully treated with amphotericin B. In addition, we evaluated the in vitro antifungal activity of 4 agents against both the mycelial and the yeast forms of the isolated fungus.

Evaluation of susceptibility of Trichophyton mentagrophytes and Trichophyton rubrum clinical isolates to antifungal drugs using a modified CLSI microdilution method (M38-A).

Onychomycosis is a common adult human mycosis, and dermatophytes of the Trichophyton genera are the most common causative agent. Many antimycotic agents are safe and highly effective for the treatment of dermatophytosis, and are available for clinical practice. Successful treatment depends on the ability of antifungal drugs to eradicate the fungal isolates. The aim of this work was to determine the MICs of four antifungal drugs (fluconazole, itraconazole, terbinafine and griseofulvin) recognized for ungual dermatophytosis treatment caused by Trichophyton species, especially Trichophyton mentagrophytes and Trichophyton rubrum. MICs were determined using a broth microdilution method in accordance with Clinical and Laboratory Standards Institute approved standard M38-A with some modifications, such as an incubation temperature of 28 degrees C, an incubation time of 7 days and inocula constituted of only microconidia. The results showed that the activities of terbinafine and itraconazole were significantly higher (MICs of <0.007-0.031 and 0.015-0.25 microg ml(-1), respectively) than other tested agents. All isolates had reduced susceptibility to fluconazole (1-64 microg ml(-1)). The MIC of griseofulvin varied among strains (MICs of 0.062-1 microg ml(-1)). The parameters adopted to perform susceptibility testing of T. rubrum and T. mentagrophytes to antifungal agents appeared to be suitable and reliable, and could contribute to the possible development of a standard protocol.

Molecular typing and antifungal susceptibility of Trichophyton rubrum isolates from patients with onychomycosis pre- and post-treatment.

Forty sequential isolates of Trichophyton rubrum were obtained from patients suffering from onychomycosis at two time points, before and after antifungal oral therapy. Strain differentiation by specific amplification of the two tandemly repeated elements (TRS-1 and TRS-2) of the ribosomal DNA of T. rubrum was performed. In addition, susceptibility tests were executed by the microdilution method with nine antifungal drugs: ketoconazole, itraconazole, fluconazole, miconazole, clotrimazole, isoconazole, griseofulvin, cyclopiroxolamine and terbinafine. The combination of TRS-1 with TRS-2 PCR amplification patterns configured 11 T. rubrum genotypes and the three most prevalent (genotypes 1-I, 5-I and 2-I) accounted for 67.5% of the isolates. Seven isolates (35%) obtained before antifungal oral therapy exhibited genotype 1-I compared to the 11 (55%) obtained after the treatment. Twelve patients exhibited different strains before and after the antifungal therapy. With respect to in vitro susceptibility testing, terbinafine was the most potent agent, followed by itraconazole, clotrimazole, isoconazole, miconazole, cyclopiroxolamine, ketoconazole, griseofulvin and fluconazole. Furthermore, an increase in the minimum inhibitory concentrations (MIC) were observed for most of the azole agents when testing isolates obtained post-treatment from four patients. This increase in MIC occurred concomitantly with the major occurrence of genotype 1-I for isolates obtained after oral therapy. These data attempt to consider the relevance of in vivo drug resistance for onychomycosis caused by T. rubrum.

In vitro susceptibility of isolates of Sporothrix schenckii to amphotericin B, itraconazole, and terbinafine: comparison of yeast and mycelial forms.

Forty-three clinical isolates of Sporothrix schenckii derived from humans and animals were evaluated in vitro for their susceptibility to amphotericin B, itraconazole, and terbinafine. MICs were determined by the method of micro dilution in liquid media, using protocols M27-A2 for the yeast form and M38-A for the mycelial form, both standardized by the Clinical Laboratory Standards Institute. In general, higher MICs were found for the mycelial form (intervals of up to two dilutions). In the case of amphotericin B, a significant difference in activity was observed, with higher values (p<0.05) found for the mycelial form. MICs for itraconazole and terbinafine were similar for both yeast and mycelial forms but slightly higher for mycelia. Although data presented here indicate different levels of susceptibility when both growth forms were compared, indicating an intrinsic difference between them, it is still difficult to draw a consensus as to which form correlates better with clinical findings. More studies are necessary to determine the criteria for in vitro tests that will lead to efficient therapeutic choices.

In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2% in-house rice flour to encourage sporulation, the addition of 2% glucose to the culture media (RPMI-1640), and an incubation temperature of 28 degrees C. In addition, the incubation period was 7d, the minimum inhibitory concentration (MIC) was defined as 80% growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC90 (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC50 values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.

Determination of susceptibility/resistance to antifungal drugs of Trichophyton mentagrophytes isolates by a macrodilution method.

Onychomycosis is a common adult human mycosis, and dermatophytes of the Trichophyton genera are the most frequently isolated microorganism. Globally, from 3% to 10% of the human population is attacked by ony cho mycosis, and many cases involve toenails. The aim of this work was to determine the minimal inhibitory concentrations (MICs) of antifungal drugs (fluconazole, ketoconazole, itraconazole, terbinafine, and griseofulvin) often used for the treatment of ungueal dermatophytosis caused by Trichophyton mentagrophytes. The MICs were determined by the broth medium macrodilution method. The results showed that activities of terbinafine and itraconazole were significantly higher (MIC <0.007-0.015 microg.mL -1 and MIC = 0.062-1.000 microg.mL -1, respectively). All isolates had reduced susceptibility to fluconazole (MIC = 16 to >64 microg.mL -1). The MICs of ketoconazole and griseofulvin varied among strains, ranging from 0.125 to 2.000 microg.mL -1 for ketoconazole and from 0.25 to 2.00 microg.mL -1 for griseofulvin. These MICs were higher than those of other studies cited, possibly because of differences in culture medium used in the other studies.

Antimicrobial susceptibility of microorganisms recovered from intraabdominal infections at Belo Horizonte, Brazil.

We evaluated antimicrobial susceptibility patterns of microorganisms isolated from intraabdominal infection of Brazilian patients, by agar dilution, agar diffusion, and E test. Among the strictly anaerobes, 57.7% were resistant to penicillin, 28.2% to clindamycin, and 9.9% to metronidazole. The majority of Escherichia coli and Staphylococcus were sensitive and resistant to almost all drugs, respectively. Half of Candida samples were resistant to itraconazole. Our data reinforce the importance of this kind of study to support rational antimicrobial therapy.

In vitro susceptibilities of isolates of Sporothrix schenckii to itraconazole and terbinafine.

Thirty isolates of the yeast form of Sporothrix schenckii were evaluated for in vitro susceptibility to itraconazole and terbinafine by the recommended NCCLS modified technique (M27-A2). The MICs of itraconazole obtained oscillated between 0.062 and 4.0 microg/ml, and those of terbinafine oscillated between 0.007 and 0.50 microg/ml; therefore, terbinafine showed greater in vitro activity.

Malassezia nana sp. nov., a novel lipid-dependent yeast species isolated from animals.

Five isolates of a novel species of the yeast genus Malassezia were isolated from animals in Japan and Brazil. Phylogenetic trees based on the D1/D2 domains of the large-subunit (26S) rDNA sequences and nucleotide sequences of the internal transcribed spacer 1 region showed that the isolates were conspecific and belonged to the genus Malassezia. They were related closely to Malassezia dermatis and Malassezia sympodialis, but were clearly distinct from these two species and the other six species of Malassezia that have been reported, indicating that they should be classified as a novel species, Malassezia nana sp. nov. Morphologically and physiologically, M. nana resembles M. dermatis and M. sympodialis, but can be distinguished from these species by its inability to use Cremophor EL (Sigma) as the sole lipid source and to hydrolyse aesculin. The type strain of M. nana is NUSV 1003(T) (=CBS 9557(T)=JCM 12085(T)).

Disseminated paracoccidioidomycosis: correlation between clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole.

The present study relates to a case of subacute multifocal paracoccidioidomycosis where the upper intestinal tract is involved. The involvement of the upper digestive tract is uncommon. The recommended therapeutic treatment plans and the difficulty in the treatment of paracoccidioidomycosis are discussed in association with susceptibility tests to antifungal drugs in vitro. This is the first report available in the literature showing, in parallel, clinical and in vitro resistance to ketoconazole and trimethoprim sulphamethoxazole, studied during the course of the disease.

Randomly amplified polymorphic DNA as a valuable tool for epidemiological studies of Paracoccidioides brasiliensis.

Randomly amplified polymorphic DNA (RAPD) has been successfully used to detect genetic variations among isolates of Paracoccidioides brasiliensis. However, the usefulness of this technique for assessing important parasitic properties is still unconfirmed. In the present work we further investigated the applicability of RAPD in revealing important intrinsic and extrinsic features of this fungus associated with geographical origin, time of isolation, source of clinical specimen, clinical forms of human disease and also in vitro and in vivo susceptibility to antimicrobial and antifungal drugs. The RAPD patterns allowed us to distinguish all of the analyzed strains, which included 26 clinical isolates, 2 animal isolates, and 1 environmental isolate of P. brasiliensis obtained from different geographic regions, confirming the strong discriminating power of this technique. A phenetic tree, build from the RAPD data, showed that although the two nonclinical Brazilian strains were set together the majority of the clinical Brazilian strains were randomly distributed through different sub-branches of a major cluster without any correlation to any of the parameters analyzed. A second major cluster, however, has grouped isolates from Mato Grosso and Roraima (Brazil) that not only were susceptible in vitro to trimethoprim-sulfamethoxazole but also produced a good in vivo response. These results open new vistas for epidemiological and clinical studies of P. brasiliensis.

Antifungal susceptibility of clinical and environmental isolates of Cryptococcus neoformans to four antifungal drugs determined by two techniques.

A total of 64 Cryptococcus neoformans strains, including clinical and environmental Brazilian isolates var. neoformans and var. gattii, were tested for susceptibility to amphotericin B, 5-flucytosine, fluconazole and itraconazole. The tests were performed according to the recommendations of National Committee of Clinical Laboratory Standards and the method of macrodilution in liquid medium of Shadomy et al. [Manual de Microbiologia Clínica, 4th ed. Buenos Aires: Editorial Medica Panamericana, 1987: 1229-38]. For most drugs there was a significant difference between the readings taken at 24 and 48 h with both methods. When the minimum inhibitory concentrations obtained by the two techniques were compared, significant differences were observed for amphotericin B and fluconazole. Overall, differences in drug susceptibility with respect to the origin of the isolates or the variety of the fungus were not observed. As an exception, the gattii variety exhibited a high resistance rate to amphotericin B when the technique of Shadomy et al. was applied, a fact possibly related to the greater difficulty for treatment of the disease caused by this fungal variety.

Identification of atypical strains of Malassezia spp. from cattle and dog.

Yeast species in the genus Malassezia are lipophilic with the exception of Malassezia pachydermatis. During a study of the occurrence of Malassezia species in the external ear of 964 cattle and 6 dogs in Minas Gerais, Brazil, six lipid-dependent isolates could not be identified to known species. Four isolates came from healthy cows, one from a cow with otitis, and one from a healthy dog. When tested with Tweens and Cremophor EL as single sources of lipids, the strains grew on all sources except Cremophor EL. None of the six strains hydrolyzed esculin, and all produced catalase. Pigment production from tryptophan was variable. Partial large subunit rRNA sequences were obtained for two isolates that remained viable in culture. The strain from the cow with otitis was identified as a lipid-dependent variant of M. pachydermatis, and the strain from the dog was an atypical variant of Malassezia furfur.

Characterization of Paracoccidioides brasiliensis atypical isolates by random amplified polymorphic DNA analysis.

Two atypical Paracoccidioides brasiliensis strains (yeast form at room temperature) have been isolated from chronically infected patients living in Brazil. Different random primers were used to characterize these isolates and compare them to typical strains. The RAPD patterns allowed the differentiation of all the selected isolates. Their genetic distance ranged from 5% to 80% of non-shared bands depending on the strains and the primer used. The RAPD data were used to build a Wagner phenogram, which showed two major branched with more than 56% of genetic distance separating them. No significant difference was observed between the atypical isolates and the others suggesting that specific genes are involved in the dimorphism phenomenon.

Prevalência de cândida na flora vaginal de mulheres atendidas num serviço de planejamento familiar / Prevalence of candida in the vagina of women attended at a family planning service

Objetivo: avaliar a prevalência de Candida sp. e a distribuiçäo de suas espécies na flora vaginal de mulheres numa clínica de planejamento familiar.Método: estudo transversal no qual se avaliaram prospectivamente 72 mulheres näo-grávidas, com ou sem queixas específicas, sendo coletadas amostras de secreçäo vaginal para cultura de leveduras, efetuada a mediçäo do pH vaginal e anotados dados de achados do exame ginecológico. Resultados: leveduras pertencentes ao gênero Candida foram encontradas em 18 casos (25 por cento). A C. albicans foi a espécie mais prevalente (77,8 por cento) a conseqüentemente 22,2 por cento foram näo-albicans. Entre as näo-albicans a espécie mais prevalente foi a C. glabrata (16,7 por cento) seguida pela C. parapsilosis (5,6 por cento). Prurido a ardor foram os únicos sintomas relacionados signficativamente com a presença de cândida. Foi observado que a C. glabrata, ao contrário da C. albicans, näo causa corrimento clinicamente verificável ao exame ginecológico. Näo foram verificadas associações de alguns fatores predisponentes (idade, escolaridade e uso de contraceptivos) com a presença ou näo de cândida. Conclusões: nossos resultados sugerem: a) uma alta prevalência de Candida sp. entre as mulheres (25 por cento); b) que as espécies näo?albicans desempenham um papel importante no meio vaginal; c) que prurido e ardor säo os sintomas mais comuns na presença de cândida e d) que a C. glabrata näo costuma causar corrimento vaginal ao exame ginecológico. Finalmente, é importante observar que nossos resultados säo consistentes com o que a literatura internacional tem mostrado nos últimos anos (AU)#S#as#HU

Case of mycotic keratis caused by Fusarium solani

O caso estudado refere-se a paciente de sexo masculino, 36 anos, feoderma, sem historia de doenca sistemica ou trauma ocular, apresentando ulcera corneana no olho esquerdo. Antes do estabelecimento do diagnostico especifico, o paciente foi tratado com antibiotico associado a corticosteroides durante um mes...

Biochemical analysis of the methylic antigen of Paracoccidioides brasiliensis

Yeast forms of five strains of Paracoccidioides brasiliensis (SN, 2, 18, 192 and JT-1) were cultured in a synthetic medium for obtaining methylic antigens. These antigens were lyophilized and studied for each strain, to determine their partial biochemical composition, through measurements of total lipid, protein and carbohydrate contents. Lipids of methylic antigens were purified and analysed for sterols, phospholipids, glycolipids, lipoproteins, and partial characterization of sterols. Significant differences were found among antigenic preparations derived from distinct P. brasiliensis strains, in relation to the quantitative determinations. On the other hand, sterol analysis revealed the presence of ergosterol, lanosterol and squalene in all samples. The diversity verified in the biochemical characteristics of antigens derived from different P. brasiliensis strains, confirm the need of using a pool of fungal samples in order to produce antigen preparations for serological procedures without hampering their sensitivity

Oxidative metabolism of Yeast cells of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis foi cultivado em meio de Fava Netto a 37-C. células na fase exponencial foram obtidas e homogeneizadas em tampäo fosfato 0,2M pH 7,2. Os experimentos de respirometria foram realizados a partir do extrato livre de células pelo método polarográfico. Entre os açúcares e alcoóis testados, manitol foi o substrato que exerceu maior estímulo sobre a respiraçäo endógena, enquanto glicose, xilose e trealose mostraram fraco efeito sobre a mesma. Glicerol, sacarose e maltose näo apresentaram nenhum efeito sobre a respiraçäo endógena. De todos os L aminoácidos testados glicina apresentou maior efeito estimulante sobre a respiraçäo endógena enquanto alanina mostrou o menor efeito. Entre os ácidos orgânicos, aspartato demonstrou o maior efeito estimulante sobre a respiraçäo endógena, enquanto oxalacetato e citrato exerceram fraco efeito sobre a mesma. Malato apresentou o menor efeito estimulante sobre a respiraçäo endógena