Homeopathy treatment of Malassezia Pachydermatis in vitro

A common clinical occurrence in dogs is otitis externa caused by excessive growth of yeasts Malassezia pachydermatis, which can become chronic after wrong treatments, in which microbial resistance can occur. Homeopathic remedies can be considered a successful alternative, selecting the medicine through the similitude principle. Herein, 50 µL of a 1:1000 dilution of Malassezia pachydermatis suspension at 0.5 McFarland scale was used to seed the yeast into Sabouraud dextrose agar plates using a Drigalski spreader to proceed with colony unit counting. Before being seeded, the yeast suspensions were treated with 1% of different homeopathic treatments previously selected from a pilot study, which means Sulphur 6cH, Dolichos pruriens 6cH, and Kali carbonicum 6cH, being water, and succussed water used as controls. For comparison, a set of Sabouraud dextrose agar plates containing 1% Tween 80 was seeded in parallel. The treatments were made blind and evaluated in triplicate. Contaminated cultures were withdrawn. The number of colonies per plate was assessed, and smears were made from the cultures to classify yeast growth according to cytomorphology on ImageJ®software. The preliminary results show no significant effect of all tested medicines compared to the controls. High data variability was also observed, mainly in those cultures whose medium was prepared with Twin 80. In conclusion, at this point of the study, no evidence of the effects of the studied medicines on Malassezia pachydermatis growth in vitro could be identified. The analysis of cytomorphology is still in course.

Genotyping of Malassezia pachydermatis disclosed genetic variation in isolates from dogs in Colombia / Genotipagem de Malassezia pachydermatis revelou variação genética em isolados de cães na Colômbia

Malassezia pachydermatis is a lipophilic and lipid-dependent yeast mostly isolated from animals' skin; hence, it is regarded as a zoophilic species causing otitis externa in dogs. Aspects associated with its epidemiology and pathogenicity is a matter of interest. This study aimed to conduct a molecular characterization of 43 isolates of M. pachydermatis obtained from dogs with otitis externa. For this purpose, the 5.8S internal transcribed spacer 2 (ITS2) and D1/D2 26S rRNA regions were amplified, sequenced and analyzed using restriction fragment length polymorphism (RFLP) with AluI, CfoI, and BstF5I endonucleases. Phylogenetic analyses revealed that these isolates grouped with the sequence types I, IV and V, previously proposed for M. pachydermatis. Interestingly, we found a new polymorphic RFLP pattern using BstF5I, these isolates were associated with the sequence types IV and V, nevertheless an association between polymorphic RFLP patterns, and fosfolipase activity or canine population data was not observed. These findings underline the genetic diversity of M. pachydermatis and provide new insights about the epidemiology of this species in the analyzed population.(AU)

Malassezia pachydermatis é uma levedura lipofílica e dependente de lipídios, principalmente da pele de animais. Sendo, por essa razão, considerada uma espécie zoofílica e causadora de otite externa em cães. Neste sentido, aspectos associados à sua epidemiologia e patogenicidade constituem um tema de interesse científico. O objetivo deste estudo foi realizar a caracterização molecular de 43 isolados de M. pachydermatis obtidos a partir de cães com otite externa. Para esta propósito, foram amplificadas, sequenciadas e analisadas com enzimas de restrição as regiões do gene 5.8S, do espaçador interno transcrito 2 (ITS2) e D1/D2 do 26S do rRNA pelo método RFLP, com as endonucleases AluI, CfOI e BstF5I. Análises filogenéticas revelaram que os isolados se agruparam com as sequências tipo I, IV e V de M. pachydermatis como já descrito anteriormente. De maneira interessante, se observou um novo RFLP polimórfico utilizando BstF5I. Os isolados que mostraram esse padrão foram associados com os padrões IV e V. No entanto, não foi observada associação entre padrões polimórficos de RFLP e atividade de fosfolipase ou dados da população canina. Estes resultados demonstram a diversidade genética de M. pachydermatis e fornecem novas perspectivas sobre a epidemiologia destas espécies na população analisada.(AU)

Dermatitis facial por Malassezia pachydermatis / Facial dermatitis caused by Malassezia pachydermatis

Se presenta un caso de dermatitis facial por Malassezia pachydermatis en un paciente de 34 años de edad, diagnosticado mediante clínica y estudio micológico. M. pachydermatis es una levadura que forma parte de la microbiota de mamíferos domésticos y animales salvajes, donde frecuentemente puede provocar dermatitis y otitis externa. En humanos, son escasas las infecciones por este agente, describiéndose desde infecciones superficiales hasta fungemias. En Chile se la ha encontrado como agente causal de pitiriasis versicolor y dermatitis seborreica principalmente en pacientes inmunocomprometidos.

We present a clinical case of facial dermatitis due to Malassezia pachydermatis in a patient of 34 years old, diagnosed by clinical and micology study. M. pachydermatis is a zoofilic yeast that is part of the microbiota of domestic mammals and wild animals, frequently causing dermatitis and external otitis. In humans, infections by this agent are very scarce, describing from superficial infections to fungemias. In Chile it has been found as causal agent of pitiriasis versicolor and seborrheic dermatitis mainly in inmunocompromised patients.

An update on microbiological causes of canine otitis externa in Campania Region, Italy

Objective: To update the recent knowledge of the microbiological causes of canine otitis externa in Campania Region (Italy) and the antibiotic susceptibility patterns of the iso-lated strains. Methods: A total of 122 dogs were examined by otoscopy, and auricular swab samples were collected from both ears in 74 dogs presenting clinical bilateral otitis and from single ears in 48 dogs displaying clinical unilateral otitis. Cytological examination, bacteriological analysis and antimicrobial susceptibility tests were performed. Results: Thirty-one out of 122 dogs were positive for yeast species (25.4%, 95% con-fidence interval (CI): 18.2%–34.2%) with a higher prevalence of Malassezia pachy-dermatis (21/31 isolates, 67.7%, CI: 48.5%–82.7%), and a total of 91 out of 122 dogs were positive for bacterial species (74.6%;CI:65.8%–81.8%) with a higher prevalence of Staphylococcus pseudintermedius (45/143 isolates, 31.5%, CI: 24.1%–39.8%). These results are the first description of Streptococcus agalactiae-associated otitis. The yeasts isolated showed high levels of susceptibility to all antifungal agents tested; on the con-trary all the isolated bacterial strains were highly resistant to at least four out of ten antimicrobial classes. Both Gram-positive and Gram-negative bacteria showed high resistance to amoxicillin/clavulanate and kanamycin hence they are not recommended as initial empirical therapy for the otitis treatment. Conclusions: This update illustrates an increase in antibiotic resistances providing an insight into the current knowledge of the therapeutic procedures followed on canine otitis externa in Italy. It also emphasizes the importance of considering the results of the microbiological and sensitivity tests to decide on an appropriate antibiotic therapy.

An update on microbiological causes of canine otitis externa in Campania Region, Italy

Objective: To update the recent knowledge of the microbiological causes of canine otitis externa in Campania Region (Italy) and the antibiotic susceptibility patterns of the isolated strains. Methods: A total of 122 dogs were examined by otoscopy, and auricular swab samples were collected from both ears in 74 dogs presenting clinical bilateral otitis and from single ears in 48 dogs displaying clinical unilateral otitis. Cytological examination, bacteriological analysis and antimicrobial susceptibility tests were performed. Results: Thirty-one out of 122 dogs were positive for yeast species (25.4%, 95% confidence interval (CI): 18.2%-34.2%) with a higher prevalence of Malassezia pachydermatis (21/31 isolates, 67.7%, CI: 48.5%-82.7%), and a total of 91 out of 122 dogs were positive for bacterial species (74.6%; CI: 65.8%-81.8%) with a higher prevalence of Staphylococcus pseudintermedius (45/143 isolates, 31.5%, CI: 24.1%-39.8%). These results are the first description of Streptococcus agalactiae-associated otitis. The yeasts isolated showed high levels of susceptibility to all antifungal agents tested; on the contrary all the isolated bacterial strains were highly resistant to at least four out of ten antimicrobial classes. Both Gram-positive and Gram-negative bacteria showed high resistance to amoxicillin/clavulanate and kanamycin hence they are not recommended as initial empirical therapy for the otitis treatment. Conclusions: This update illustrates an increase in antibiotic resistances providing an insight into the current knowledge of the therapeutic procedures followed on canine otitis externa in Italy. It also emphasizes the importance of considering the results of the microbiological and sensitivity tests to decide on an appropriate antibiotic therapy.

Malassezia pachydermatis en lesiones de piel en pacientes de la Quinta región de Chile / Malassezia pachydermatis on skin lesions in patients from the Fifth region of Chile

Introducción: M. pachydermatis es una levadura zoofílica que puede provocar pitiriasis versicolor y dermatitis seborreica. Objetivos: determinar la presencia de M. pachydermatis en pacientes con pitiriasis versicolor y dermatitis seborreica, además de evaluar el rendimiento del examen microscópico directo y cultivo, en estas infecciones. Materiales y Método: estudio descriptivo prospectivo realizado en el Hospital Carlos van Buren de Valparaíso, Chile, durante el período 2011-2013. Se seleccionaron pacientes con diagnóstico clínico de pitiriasis versicolor y dermatitis seborreica. La confirmación micológica fue realizada con examen microscópico directo y/o cultivo, además de pruebas especiales. Resultados: ingresaron al estudio 66 pacientes. El 60,6% fueron hombres, con un promedio de edad de 43 años. El 70% con VIH (+). El 90,9% presentó pitiriasis versicolor (principalmente hipopigmentadas en el tronco) y el 9% dermatitis seborreica. El examen microscópico directo fue positivo en el 95,4% de los casos y en el 62,12% de los cultivos. La especie más frecuente fue M. pachydermatis, seguida por M. furfur. Del total de pacientes: 50 se trataron por vía tópica, 15 con tópico más sistémico y 1 no fue tratado. Conclusiones: la mayoría de los pacientes ingresados a este estudio fueron hombres, VIH(+) y con lesiones de pitiriasis versicolor hipopigmentadas de tronco. La especie más frecuente fue M. pachydermatis. El tratamiento tópico fue el más indicado.

Introduction: M. pachydermatis is a zoophilic yeast that can cause pityriasis versicolor and seborrheic dermatitis. Objectives: to determine the presence of M. pachydermatis in patients with pityriasis versicolor and seborrheic dermatitis, in addition to evaluating the performance of direct microscopic examination and culture in these infections. Materials and Method: a prospective descriptive study conducted in the Carlos van Buren Hospital in Valparaíso, Chile, during the period 2011-2013. Patients with clinical diagnosis of pityriasis versicolor and seborrheic dermatitis were selected. Mycological confirmation was made with direct microscopic examination and / or culture, and special tests. Results: 66 patients entered the study. 60.6% were men, with an average age of 43 years. 70% with HIV (+). 90.9% had pityriasis versicolor (mostly hypopigmented in the trunk) and seborrheic dermatitis 9%. Direct microscopic examination was positive in 95.4% of cases and 62.12% of the cultures. The most frequent species was M. pachydermatis, followed by M. furfur. Of the patients: 50 were treated topically with 15 more systemic plus topical and 1 was untreated. Conclusions: most patients admitted to this study were men, HIV (+) and hypopigmented pityriasis versicolor lesions in the trunk. The most frequent species was M. pachydermatis. Topical treatment was the most appropriate.

Susceptibility variation of Malassezia pachydermatis to antifungal agents according to isolate source

Malassezia pachydermatisis associated with dermatomycoses and otomycosis in dogs and cats. This study compared the susceptibility of M. pachydermatis isolates from sick (G1) and healthy (G2) animals to azole and polyene antifungals using the M27-A3 protocol. Isolates from G1 animals were less sensitive to amphotericin B, nystatin, fluconazole, clotrimazole and miconazole.

Occurrence and identification of yeasts in dogs external ear canal with and without otitis / Ocurrencia y detección de levaduras en el conducto auditivo de los perros con y sin otitis

Objective. To analyze the presence of yeast in the external ear canal of 116 dogs with and without a diagnosis of otitis from veterinary clinic in the Chapecó city, Santa Catarina, Brazil, and to examine the secretion of the proteinase in isolates. Materials and methods. Were collected cerumen of conduct hearing of dogs of 16 different races 71% with pendular ear type, 5% of semi-pendular and 24% of the erect type. All dogs were previously evaluated by otoscopy and grouped in dogs with and without otitis. Results. Yeasts were isolated in 44 samples (approximately 36%), where Malassezia pachydermatis was identified in 95% of samples where were observed growth of yeasts. On 20 samples the proteinase enzyme showed strong activity in 31% isolates, were 21% of the dogs with otitis tested showed high proteolytic activity. Conclusions. We observed a variation of strains of M. pachydermatis-producing enzymes. The variation in production of these enzymes is probably more associated with different response to the action of the immune system of the animal in the tissue injury.

Objetivo. Se investigó la presencia de levaduras en el canal externo del oído de 116 perros de la clínica veterinaria en la ciudad de Chapecó, Santa Catarina,Brasil, en perros sanos y perros con otitis y se examinó la secreción de la proteinasa en las muestra aisladas. Materiales y métodos. Se recogieron cerumen del oído de perros de 16 razas diferentes, dónde 71% fue de oído de tipo pendular, 5% de semi-pendular y 24% del tipo erecto. Todos los perros fueron evaluados previamente por otoscopia y agrupados en perros con y sin otitis externa. Resultados. Las levaduras se aislaron en 44 muestras (aproximadamente 36%), donde Malassezia pachydermatis se identificó en el 95% de las muestras donde se observó el crecimiento de las levaduras. El 20 muestras la secreción de proteinasa mostró fuerte actividad en el 31% de los aislados y en 21% de los perros con otitis mostró alta actividad proteolítica. Conclusiones. Hemos observado una variación de cepas de M. pachydermatis productoras de enzimas. La variación en la producción de estas enzimas es probablemente más asociados con la respuesta diferente a la acción del sistema inmunológico del animal en la lesión tisular.

Pitiriasis versicolor por Malassezia pachydermatis: caso clínico / Pityriasis versicolor caused by Malassezia pachydermatis: clinical case

Se presenta un caso clínico de pitiriasis versicolor por M. pachydermatis en una paciente de 11 años de edad, diagnosticado mediante clínica, morfofisiología y biología molecular. Malassezia pachydermatis es parte de la microbiota común de diversos mamíferos domésticos y animales salvajes. En éstos se reconoce como agente causal de dermatitis y otitis externa. En humanos se han descrito escasas infecciones ya sea superficiales como invasoras, lo que puede atribuirse a la difícil transmisión y adaptación a partir de los animales domésticos. Se comentan los aspectos biológicos y epidemiológicos de esta infección zoonótica.

A clinical case of pityriasis versicolor caused by M. pachydermatis in an 11 year old patient which was diagnosed by means of clinic, morphophysiology and molecular biology is presented. Malassezia pachydermatis is part of regular microbiota in several domestic mammals as well as wild animals. It is recognized as the causing agent of dermatitis and external otitis in the latter. As to human beings, superficial infections have rarely been given an invasive character what can be explained by the difficult transmission and adaptation coming from domestic animals. Biological and epidemiological aspects of this zoonotic infection are described.

Molecular heterogeneity of Malassezia pachydermatis through RAPDPCR / Heterogeneidade molecular da Malassezia pachydermatis através de RAPD-PCR

Several methodologies in molecular biology have been used in theinvestigation of Malassezia pachydermatis and its differentiation into subtypes. Recentmolecular research of this species includes the use of samples isolated from canine otitisexterna and dermatitis, as well as from healthy animals, having in view an epidemiologicalstudy of the yeast. The aim of this study was to identify molecular differences in M.pachydermatis samples isolated from dogs with otitis externa. The M. pachydermatis strainswere analyzed by means of the Random Amplification Primer DNA - Polimerase ChainReaction (RAPD–PCR) for molecular heterogeneity research. DNA extraction was carriedout with phenol-chloroform and the RAPD technique using the AGAATCCGCC primer.A variation was observed in the number and arrangement of the bands among the 49studied isolates, grouped into nine patterns. Isolate groupings were not found to be relatedto animal breed, age or sex. It was concluded that M. pachydermatis has differences in itsmolecular profile, as shown by the molecular technique (RAPD – PCR), which allowsisolates to be classified into nine subtypes.

Várias metodologias em biologia molecular têm sido aplicadas para estudar aM. pachydermatis diferenciando-a em subgrupos. Recentemente utiliza-se a investigaçãomolecular desta espécie isolada de otite externa e dermatite, e também de isolados da mesmade animais hígidos, para um estudo epidemiológico da levedura. O objetivo deste trabalhofoi identificar diferenças moleculares entre isolados de M. pachydermatis obtidos de casos deotite externa canina. Para isto, amostras da levedura provenientes de cães com estaenfermidade foram estudadas através da técnica de Polimorfismo de DNA Amplificado aoAcaso - Reação da Polimerase em Cadeia (RAPD–PCR) para pesquisa de heterogeneidademolecular. A extração de DNA foi realizada no processo fenol-cloroformio e a técnica deRAPD foi estudada com o primer AGAATCCGCC. Pode-se observar com estametodologia, variação no número e posição das bandas entre os 49 isolados estudados,podendo-se agrupá-los em nove padrões. Os agrupamentos formados pelos isolados nãoapresentaram relação com raça, idade ou sexo do animal. Concluindo-se que a M.pachydermatis apresenta diferenças em seu perfil molecular, observado pela técnica molecular(RAPD-PCR) que permite classificar os isolados desta espécie em até nove subtipos.

CLSI broth microdilution method for testing susceptibility of Malasseziapachydermatis to thiabendazole / CLSI método de Microdiluição em Caldo para teste de suscetibilidade da Malassezia pachydermatis frente ao tiabendazol

Thiabendazole, classified as antiparasitic and also used as an antifungal drug, can be found as otological solution indicated for treatment of parasitic and fungal external otitis in small animals. Malassezia pachydermatis is a yeast recognized as a normal inhabitant on the skin and mucous membranes of dogs andcats. However, it is considered an opportunistic agent that causes external otitis and dermatitis in these animals. The aim of this study was to evaluate the in vitro effect of thiabendazole against 51 isolates of M. pachydermatis using the CLSI Broth Microdilution method that has been adapted for this yeast species (NCCLS, 2002). Based on this test, the Minimum Inhibitory Concentrations (MIC) of thiabendazol was calculated. Subsequently, the susceptibility of each isolate against this antifungal was determined. It wasobserved that the MIC of thiabendazole against M. pachydermatis ranged from 0.03 to > 4 μg/mL. A total of 13.7% of the isolates were found to be resistant, 47.1% were intermediate and 39.2% were sensitive to the drug. The rate of resistance of the yeasts against thiabendazole was similar to the results previously obtained with other antifungals, while the adapted broth microdilution technique used in this study proved to be efficient.

Tiabendazol, um fármaco classificado como antiparasitário e também usado como antifúngico, pode ser encontrado como solução otologica indicada no tratamento da otite externa parasitária e fungica em pequenos animais. Malassezia pachydermatis é uma levedura considerada habitante normalda pele e das mucosas de cães e gatos. Entretanto, considerase um agente do oportunista causador de otite externa edermatite nestes animais. A finalidade deste estudo foi avaliar o efeito in vitro do tiabendazol frente a 51 amostras de M. pachydermatis através do método CLSI de Microdiluição em Caldo adaptado para esta espécie de levedura (NCCLS, 2002). Baseado neste teste calculou-se as Concentrações InibitóriasMínimas (CIM) do tiabendazol e, subseqüentemente, foi detectada a suscetibilidade de cada amostra frente a este antifungico. Observou-se que a CIM do tiabendazol frente a M. pachydermatis variou de 0,03 a > 4 μg/mL. Estas amostras foram classificadas em resistente (13,7%), sensibilidade intermediária (47,1%) e sensível (39,2%). A resistência dalevedura frente ao tiabendazol mostrou similaridade com resultados anteriormente observados com outros antifúngicos e a adaptação da técnica de Microdiluição em Caldo utilizada mostrou-se eficiente.

Comparison of the broth microdilution technique and ETEST to ketoconazole front Malassezia Pachydermatis / Comparação da técnica de microdiluição em caldo e ETEST para o cetoconazol frente à Malassezia pachydermatis

Malassezia pachydermatis is recognized as a normal inhabitant and an opportunistic pathogen of the external ear canal and skin of dogs and cats. In special clinical conditions, and mainly in the cases of therapeutic failure related to external otitis and dermatitis complicated by this yeast, is recommended testing susceptibility to antifungal drugs. Different approaches of evaluating the susceptibility of yeasts faced to antifungals in laboratory exist, some of them are commercial approaches and others previously standardized by the CLSI (NCCLS,2002). The purpose of this study was to evaluate the susceptibility of 17 samples of M. pachydermatis from canine external otitis using two different in vitro antifungal susceptibility methods: the Etest®and the broth Microdilution Method (MD) with ketoconazole. The mean MIC observed between the 17 samples were 0.103mg/mL to ETEST and 0.0012mg/mL to MD ranging from 0.004 to 0.75mg/mL in ETEST and 0.0019 and 0.03mg/mL in MD using the same samples. By ETEST, two (11.8%) samples were resistant, eight(47.1%) susceptible and seven (41.1%) showed intermediate susceptibility. Through the MD it was observed four (23.5%) resistant samples, seven (41.2%) susceptible and six (35.3%) samples with intermediate susceptibility. Despite of the percentages being equivalent in each rank of susceptibility through the two techniques, the results do not correspond to the same sample. These results showed that there is an urgent need to standardize those values considered as parameters for growth inhibition of this yeast. Then a simple and efficient method could be used routinely in the laboratory practice.

Malassezia pachydermatis é reconhecida como um habitante normal e patógeno oportunista do meato acústico externo e da pele de cães e gatos. Em condições clínicas especiais e em casos de falha terapêutica relatada em otite externa e dermatite complicada por esta levedura, é recomendado o teste de suscetibilidade antifúngica. Existem diferentes métodos de avaliação da suscetibilidade da levedura frente a antifúngicos em laboratório, alguns métodos comerciais e outros previamente padronizados pelo CLSI (NCCLS, 2002). O objetivo deste estudo foi o de avaliar a suscetibilidade de 17 amostras de M.pachydermatis proveniente de otite externa canina por meio de duas técnicas in vitro de antifungigramas: o Etest® e a microdiluição em caldo (MC) com o cetoconazol. A media da Concentração Inibitória Mínima (CIM) observada entre as 17 amostras foram 0.103mg/mL para o ETEST e 0.0012mg/mL para a MC variando de 0.004 a0.75mg/mL no ETEST e entre 0.0019 e 0.03mg/mL na MC usando as mesmas amostras. Pelo ETEST, duas (11.8%) amostras foram resistentes, oito (47.1%) sensíveis e sete (41.1%) mostraram sensibilidade intermediária. Na MC foram observadas quatro (23.5%)amostras resistentes, sete (41.2%) sensíveis e seis (35.3%) amostras com sensibilidade intermediária. Apesar das porcentagens de sensibilidade serem semelhantes pelas duas técnicas, os resultados de CIM não correspondem na uma mesma amostra. Estes resultados mostraram que há uma urgente necessidade de padronização dos valores considerados como parâmetros para inibição do crescimento da levedura. Portanto, um método simples e eficiente deveria ser usado rotina na prática de laboratório.

Production of Antihypertensive Angiotensin I-Converting Enzyme Inhibitor from Malassezia pachydermatis G-14

To produce a novel antihypertensive angiotensin I-converting enzyme (ACE) inhibitor from yeast, a yeast isolate, designated G-14 showing the highest ACE inhibitory activity was obtained and identified as Malassezia pachydermatis based on morphological, biochemical and cultural characteristics. The maximal extracellular ACE inhibitor production was obtained from M. pachydermatis G-14 when the strain was cultured in YEPD medium containing 0.5% yeast extract, 3.0% peptone and 2.0% glucose at 30degrees C for 24 h and the final ACE inhibitory activity was 48.9% under the above condition.