Padronização de multiplex PCR para detecção de dermatófitos em pelos e crostas de cães e gatos / Standardization of a PCR multiplex for the detection of dermatophytes in dogs and cats fur and crusts

Objetivou-se padronizar uma reação do tipo multiplex PCR (mPCR) para detectar Microsporum canis, Microsporum gypseum e o complexo Trichophyton mentagrophytes em amostras de pelos e/ou crostas de cães e gatos. 250 amostras de pelos e/ou crostas de cães e gatos foram analisadas por meio de exame direto e cultura, o DNA das mesmas foi extraído para mPCR. Primers foram desenhados e como controle positivo da reação utilizou-se o DNA extraído de colônias de M. canis (URM 6273), M. gypseum (URM 6921) e T. mentagrophytes (URM 6211), provenientes da Coleção de Culturas (Micoteca URM), Departamento de Micologia, Centro de Ciências Biológicas da Universidade Federal de Pernambuco (CCB/UFPE). Como controles negativos de reação, utilizou-se água destilada esterilizada e DNA extraído de Alternaria sp. para verificar a especificidade dos primers. Do total de amostras analisadas, 15 (6%) foram identificadas, em cultura, como dermatófitos, e destas, 10 foram M. canis, três M. gypseum e dois T. mentagrophytes (complexo). Destas 15 amostras positivas, 11 (73,3%) foram detectadas por meio da mPCR. Além destas, seis outras, negativas em cultura, foram identificadas como M. gypseum. Verificou-se uma boa concordância entre os resultados da cultura e mPCR (Kappa: 0,66). O protocolo padronizado neste estudo pode ser utilizado como um método de triagem, por apresentar uma sensibilidade maior que a da cultura, usado paralelamente aos exames de rotina, permitindo um diagnóstico em menor tempo.(AU)

The aim of this study was to standardize a multiplex PCR (mPCR) reaction to detect Microsporum canis, Microsporum gypseum and the Trichophyton mentagrophytes complex in dog and cat fur and/or crusts. 250 fur and/or crusts samples from dogs and cats were analyzed by direct examination and culture, DNA from them was extracted for mPCR. Primers were designed and the DNA extracted from colonies of M. canis (URM 6273), M. gypseum (URM 6921) and T. mentagrophytes (URM 6211) from the Collection of Cultures - URM Micoteca - Department of Mycology, Biological Sciences Center of the Federal University of Pernambuco (CCB / UFPE). As negative controls, sterile distilled water and DNA extracted from Alternaria sp., were used to verify the specificity of the primers. Of the total samples analyzed, 15 (6%) were identified in culture as dermatophytes, and of these, 10 were M. canis, three M. gypseum and two T. mentagrophytes (complex). Of these 15 positive samples, 11 (73.3%) were detected by mPCR. Besides these, six others, negative in culture, were identified as M. gypseum. There was good agreement between culture results and mPCR (Kappa: 0.66). The protocol standardized in this study can be used as a screening method, because it has a sensitivity greater than that of the culture, used in parallel to the routine exams, allowing a diagnosis in a shorter time.(AU)

Prof. Masao Ota.

Masao Ota was a Professor of Dermatology at Tokyo Imperial University. He is known to dermatologists around the world as the researcher who identified Nevus of Ota. He is also known for his research on Hansen's Disease. He was critical of the forced isolation policy and the sterilization law. He dreamt of developing chemotherapeutic measures and dedicated himself to cultivating Mycobacterium leprae. Among his accomplishments, those in the area of medical mycology are particularly remarkable. His discovery of Microsporum ferrugineum, his proposal for Trichophytia pompholyciformis, and his work on Ota-Langeron taxonomy based on the findings on fungus colonies are highly regarded and earned him the Ordre Royale de la Legion D'honneur. His accomplishments in the field of mycology are numerous; he has published a total of 39 research papers mostly in foreign languages. He was a leading world-class medical mycologist of his day. This review introduces some of his accomplishments and some episodes in his life.Furthermore, Masao Ota had a detailed knowledge of art and culture. Under the pseudonym of Kinoshita Mokutaro, he wrote poems, plays, and novels. He was also a painter. Particularly, his paintings in botany during his later years were published in the book "One Hundred Flower Sketches" after his death.Ota said, "The consequence of both science and art is global and humanitarian." He was one of the greatest men of culture in his time.

Toward a Novel Multilocus Phylogenetic Taxonomy for the Dermatophytes.

Type and reference strains of members of the onygenalean family Arthrodermataceae have been sequenced for rDNA ITS and partial LSU, the ribosomal 60S protein, and fragments of ß-tubulin and translation elongation factor 3. The resulting phylogenetic trees showed a large degree of correspondence, and topologies matched those of earlier published phylogenies demonstrating that the phylogenetic representation of dermatophytes and dermatophyte-like fungi has reached an acceptable level of stability. All trees showed Trichophyton to be polyphyletic. In the present paper, Trichophyton is restricted to mainly the derived clade, resulting in classification of nearly all anthropophilic dermatophytes in Trichophyton and Epidermophyton, along with some zoophilic species that regularly infect humans. Microsporum is restricted to some species around M. canis, while the geophilic species and zoophilic species that are more remote from the human sphere are divided over Arthroderma, Lophophyton and Nannizzia. A new genus Guarromyces is proposed for Keratinomyces ceretanicus. Thirteen new combinations are proposed; in an overview of all described species it is noted that the largest number of novelties was introduced during the decades 1920-1940, when morphological characters were used in addition to clinical features. Species are neo- or epi-typified where necessary, which was the case in Arthroderma curreyi, Epidermophyton floccosum, Lophophyton gallinae, Trichophyton equinum, T. mentagrophytes, T. quinckeanum, T. schoenleinii, T. soudanense, and T. verrucosum. In the newly proposed taxonomy, Trichophyton contains 16 species, Epidermophyton one species, Nannizzia 9 species, Microsporum 3 species, Lophophyton 1 species, Arthroderma 21 species and Ctenomyces 1 species, but more detailed studies remain needed to establish species borderlines. Each species now has a single valid name. Two new genera are introduced: Guarromyces and Paraphyton. The number of genera has increased, but species that are relevant to routine diagnostics now belong to smaller groups, which enhances their identification.

Chemical composition and antifungal activity of essential oil from Eucalyptus smithii against dermatophytes.

INTRODUCTION: In this study, we evaluated the chemical composition of a commercial sample of essential oil from Eucalyptus smithii R.T. Baker and its antifungal activity against Microsporum canis ATCC 32903, Microsporum gypseum ATCC 14683, Trichophyton mentagrophytes ATCC 9533, T. mentagrophytes ATCC 11480, T. mentagrophytes ATCC 11481, and Trichophyton rubrum CCT 5507. METHODS: Morphological changes in these fungi after treatment with the oil were determined by scanning electron microscopy (SEM). The antifungal activity of the oil was determined on the basis of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values. RESULTS: The compound 1,8-cineole was found to be the predominant component (72.2%) of the essential oil. The MIC values of the oil ranged from 62.5µg·mL-1 to >1,000µg·mL-1, and the MFC values of the oil ranged from 125µg·mL-1 to >1,000µg·mL-1. SEM analysis showed physical damage and morphological alterations in the fungi exposed to this oil. CONCLUSIONS: We demonstrated the potential of Eucalyptus smithii essential oil as a natural therapeutic agent for the treatment of dermatophytosis.

Chemical composition and antifungal activity of essential oil from Eucalyptus smithii against dermatophytes

ABSTRACT INTRODUCTION: In this study, we evaluated the chemical composition of a commercial sample of essential oil from Eucalyptus smithii R.T. Baker and its antifungal activity against Microsporum canis ATCC 32903, Microsporum gypseum ATCC 14683, Trichophyton mentagrophytes ATCC 9533, T. mentagrophytes ATCC 11480, T. mentagrophytes ATCC 11481, and Trichophyton rubrum CCT 5507. METHODS: Morphological changes in these fungi after treatment with the oil were determined by scanning electron microscopy (SEM). The antifungal activity of the oil was determined on the basis of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values. RESULTS: The compound 1,8-cineole was found to be the predominant component (72.2%) of the essential oil. The MIC values of the oil ranged from 62.5μg·mL−1 to >1,000μg·mL−1, and the MFC values of the oil ranged from 125μg·mL−1 to >1,000μg·mL−1. SEM analysis showed physical damage and morphological alterations in the fungi exposed to this oil. CONCLUSIONS: We demonstrated the potential of Eucalyptus smithii essential oil as a natural therapeutic agent for the treatment of dermatophytosis.

Effects of temperature variations and light exposure on the time to growth of dermatophytes using six different fungal culture media inoculated with laboratory strains and samples obtained from infected cats.

In this study, 5/6 commercially available fungal culture media were comparable with respect to first growth, first colour change, and first sporulation when inoculated with three strains of Microsporum canis, one strain of Microsporum gypseum, and one strain of Trichophyton species when incubated at either 25°C or 30°C. Five of six plates showed 100% growth at both temperatures. Five of six plates showed 100% growth when inoculated with naturally infective M canis hairs and spores. One commercial product packaged as a self-sealing incubation plate showed growth in only 65.4% of times and the plates were prone to desiccation. M canis inoculated plates were incubated under four different light exposures (24h of light, 24h of dark, 12h light/12h dark, and room lighting) and no differences in growth or sporulation were noted.

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.

Identification of Microsporum canis from dermatophytic pseudomycetoma in paraffin-embedded veterinary specimens using a common PCR protocol.

The effectiveness of a simple PCR protocol performed on paraffin-embedded tissues, obtained from histopathologically and culturally diagnosed cases of dermatophytic pseudomycetoma DPM was tested. The specimens were investigated using previously described primers (DH1L and DH1R) targeting the 18S rDNA gene and amplifying a 183-bp fragment. Microsporum canis was identified from all samples. The PCR protocol described in the present work demonstrated a 100% concordant result comparing the molecular characterisation with phenotypic characterisation of dermatophytes. Molecular biology could represent a valid identification tool in dermatophytic deep infections, when diagnosis cannot be achieved by cultural methods.

Micosis cutáneas y mucosas en niños: estudio retrospectivo de 314 casos

Se presentan 314 casos de micosis cutáneas en niños, que fueran diagnosticados en la Corporación para Investigaciones Biológicas (CIB), durante los años de 1987 a 1993. Se hace énfasis en la frecuencia de dermatofitosis, candidiasis y micosis superficiales. Las dermatofitosis fueron diagnosticadas en 210 pacientes (67 por ciento), con mayor frecuencia de tinea capitis y tinea corporis, 88 (42 por ciento) y 81 (38.5 por ciento) casos, respectivamente. La tinea pedis también ocupó lugar importante con 27 casos (13 por ciento). Entre los agentes etiológicos, Microsporum canis fue aislado de 94 pacientes con predominio de las lesiones de cuero cabelludo, 75 casos (80 por ciento). El M.gypseum ocupó el segundo lugar con 52 casos (25 por ciento) y fué además el principal causante de tinea corporis (20.7 por ciento) en este grupo de pacientes. La candidiasis se diagnosticó en 89 pacientes (28 por ciento); la localización más común fue la de la piel glabra (35 por ciento). Candida albicans fue la especie aislada con mayor frecuencia (50.5 por ciento), seguido por C.parapsilosis, 19 casos (21 por ciento). Entre las micosis superficiales, la pitiriasis versicolor se observó en 14 casos (4 por ciento), mientras que la piedra negra fue encontrada sólo en una oportunidad. Los resultados anotados permiten señalar la importancia de las micosis dérmicas en la población pediátrica

Origins of onychomycosis.

The invasion of human toenails by microorganisms is not always understood. Efficacious treatment, however, depends on establishing proper identification and understanding the improper diagnosis results in failed treatment. Most medical treatment presently is directed toward several fungal species. This article identifies these organisms by means of diagrams and transmission and scanning electron microscopy. The article also discusses microorganisms that are unresponsive to dermatophytic treatment and discusses how aspergillus, saprophytes, and yeasts can affect nails and mimic dermatophytic infections.

Identification and serotyping of Microsporum canis isolates by monoclonal antibodies.

Hybridoma cells were produced by fusing mouse myeloma cells with spleen cells from mice primed with an exoantigen of Microsporum canis. Three clones produced antibodies which were examined by the Western blot technique for their potential usefulness in the identification of M. canis isolates and differentiation of strains within the species. Based on reactions with immunological determinants, all of the M. canis isolates tested presented either species- or strain-specific domains. Monoclonal antibodies proved to be useful reagents for the identification of M. canis isolates and for the differentiation of strains within the species. A purified antigen depleted of common antigenic determinants was obtained in affinity chromatography by using monoclonal antibody.

Serological analysis of dermatophyte isolates with monoclonal antibodies produced against Microsporum canis.

Hybridoma cells produced by fusing myeloma cells with spleen cells from mice immunized with a soluble antigen of Microsporum canis yielded 30 antibody-producing clones. Six of these clones, propagated as ascites tumors in mice, showed two different types of monoclonal antibodies. The type 1 monoclonal antibody reacted with 17 heterologous and 10 homologous dermatophyte antigens. Type 2 monoclonal antibodies were unable to precipitate three antigens from different isolates of M. canis, thus suggesting the occurrence of different serotypes within the species.

Pyrolysis gas-liquid chromatography applied to a study of variation in Arthroderma tuberculatum.

Replicates of whole colonies of four species of closely related dermatophytes were analyzed by pyrolysis gas-liquid chromatography (PGLC). The four species included fifteen strains of Arthroderma tuberculatum, and two strains each of A. benhamiae, Nannizzia gypsea and N. incurvata. Individual peaks on different pyrograms were identified as homologous with the aid of internal markers by the superimposition of pyrograms. The peak height data extracted from the pyrograms of the fungal samples were analyzed to compute average similarities between pairs of pyograms. The average was calculated with each peak weighted equally, and log weighted for its information content. The results of the cluster analyses of proximities were generally similar. Most, but not all, replicates of each strain were similar enough to be clustered together. Some strains belonging to the same species were also similar enough to be grouped in one cluster. Other strains of a single species varied sufficiently to be put in separate clusters. The nearest neighbour to each OTU (pyrogram) was always a replicate of the same strain.