Una experiencia de siete años en el laboratorio de Micología del Hospital de Pediatría Juan P. Garrahan / A seven-year experience in the Mycology Laboratory of Hospital de Pediatría Juan P. Garrahan

En noviembre del año 2015 nos incorporamos al Laboratorio de Micología del Servicio de Microbiología del Hospital Garrahan. En este breve resumen queremos compartir los avances logrados a través de nuestra experiencia durante siete años de trabajo profesional. Debido a los diagnósticos realizados y su complejidad, consideramos que el Hospital Garrahan, sus pacientes y la comunidad toda necesitan contar con un laboratorio de Micología que responda a sus necesidades. Creemos haber iniciado un camino que esperamos continúe y culmine con la creación de la Unidad de Micología (AU)

In November 2015 we joined the Mycology Laboratory of the Microbiology Service of the Hospital Garrahan. In this brief summary we want to share the advances achieved through our experience during seven years of professional work. Due to the diagnosis made and their complexity, we believe that the Hospital Garrahan, its patients and the entire community, need to have a Mycology laboratory that responds to their requirements. We believe we have started a path that we hope will continue and culminate with the creation of the Mycology Unit (AU)

[Performance evaluation of Rapid™ Yeast Plus (Remel) system from two different culture media]. / Evaluación del rendimiento del sistema Rapid™ Yeast Plus (Remel) a partir de 2 medios de cultivo diferentes.

Within the genus Candida, Candida albicans is the most commonly isolated species from clinical samples. Due to the emergence of other species which can show a higher index of antifungal resistance, a fast identification of these species is necessary. The aim of this work was to evaluate the performance of the RapID Yeast Plus system from two different subculture media formulations: Sabouraud dextrose agar adjusted by Emmons (the medium is indicated in the equipment insert) and Sabouraud glucose agar, which is the most frequently used in Buenos Aires City laboratories. One hundred and sixty-six clinical sample strains coming from different hospitals belonging to the Mycology Network of Buenos Aires City were studied. From the obtained results, we conclude that the conditions and culture medium indicated by the manufacturer should be followed.

[Evaluation of Vitek 2 for the identification of Candida yeasts]. / Evaluación del sistema Vitek 2 para la identificación de las principales especies de levaduras del género Candida.

The aim of this investigation was to evaluate the performance of Vitek 2 YST cards (bioMérieux, Inc., Hazelwood, MO, USA) for the identification of yeasts of the genus Candida. A total of 168 isolates were analyzed and the results were compared to those of the API 20 C AUX (24%) o API ID 32 C (76%) kits (bioMérieux, Marcy L'Etoile, France). Each isolate was grown in chromogenic agar and in corn meal agar (Oxoid, UK) to observe its micromorphology. C. albicans and C. dublininesis were identified by additional biochemical and molecular tests. The agreement observed was 98.3%. Only three isolates were incorrectly identified by Vitek 2: one strain of C .tropicalis and one strain of C. krusei were identified as C. parapsilosis by YST while one strain of C. krusei was identified with low discrimination. The average time for obtaining results was 18.25 h. Vitek 2 is a simple, safe and useful system for the identification of significant Candida species.

Evaluación del sistema Vitek 2 para la identificación de las principales especies de levaduras del género Candida. / [Evaluation of Vitek 2 for the identification of Candida yeasts].

The aim of this investigation was to evaluate the performance of Vitek 2 YST cards (bioMérieux, Inc., Hazelwood, MO, USA) for the identification of yeasts of the genus Candida. A total of 168 isolates were analyzed and the results were compared to those of the API 20 C AUX (24

) o API ID 32 C (76

) kits (bioMérieux, Marcy LEtoile, France). Each isolate was grown in chromogenic agar and in corn meal agar (Oxoid, UK) to observe its micromorphology. C. albicans and C. dublininesis were identified by additional biochemical and molecular tests. The agreement observed was 98.3

. Only three isolates were incorrectly identified by Vitek 2: one strain of C .tropicalis and one strain of C. krusei were identified as C. parapsilosis by YST while one strain of C. krusei was identified with low discrimination. The average time for obtaining results was 18.25 h. Vitek 2 is a simple, safe and useful system for the identification of significant Candida species.

Estudo da variabilidade genetica por RAPD, RFLP e sequenciamento por Microsatelites, antifungigrama ETEST, de isolados clinicos de Fonsecaea pedrosoi de pacientes com cromoblastomicose / Study of genetic variability by RAPD, RFLP and sequencing by microsatellites, eTest antifungal susceptibility of clinical isolates of Fonsecaea pedrosoi patients with chromoblastomycosis

A cromoblastomicose e uma infeccao subcutanea causada por fungos demacios. Essa micose ja foi diagnosticada em todo mundo, e frequente em paises de clima tropical, sendo o Brasil o primeiro pais em incidencia dessa doenca. Algumas especies de fungos demacios estao envolvidas na cromoblastomicose, no Brasil a mais comum e a Fonsecaea pedrosoi. Varios esquemas terapeuticos para o tratamento da cromoblastomicose ja foram utilizados, e ate o momento nao existe uma terapia padrao. A resistencia aos antifungicos tem sido diagnosticada grandemente, tano in vivo, como in vitro. Por causa dessa resistencia, os testes de susceptibilidade aos antifungicos precisam rapidamente ser adaptados a rotina laboratorial para uma melhor indicacao de antifungicos para os pacientes. O NCCLS desenvolveu o documento M38-A, antifungigrama acurado e preciso, mas trabalhosos, inviavel para a rotina, contudo a industria lancou a antifungigrama ETEST, bastante eficiente, pratico e rapido. E este sera nosso objeto de estudo, uma vez que o ETEST ainda nao foi testado frente aos fungos demacios. Os metodos moleculares serao aplicados de modo a estudar as variabilidades geneticas, caracter filogenetico e taxonomico, e tentaremos correlacionar as diferencas geneticas com o antifungigrama. Objetivando concluir que os metodos moleculares podem tornar-se uma ferramenta para ajudar, tanto na terapeutica como na correta identificacao do agente.

Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma reesei.

BACKGROUND: Methods for the extraction of DNA from filamentous fungi are frequently laborious and time consuming because most of the available protocols include maceration in liquid nitrogen after the mycelium has been grown in a liquid culture. This paper describes a new method to replace those steps, which involves the growth of the mycelium on cellophane disks overlaid on solid medium and the use of glass beads for cell wall disruption. RESULTS: Extractions carried out by this method provided approximately 2 microg of total DNA per cellophane disk for the filamentous fungus Trichoderma reesei. To assess the DNA's quality, we made a PCR (Polymerase Chain Reaction) amplification of a gene introduced by a transformation in this fungus's genome (hph gene), with successful results. We also confirmed the quality of the DNA by the use of Southern blotting to analyze the presence of the same gene, which was easily detected, resulting in a sharply defined and strong band. CONCLUSIONS: The use of this method enabled us to obtain pure DNA from Trichoderma reesei, dispensing with the laborious and time-consuming steps involved in most protocols. The DNA obtained was found to be suitable for PCR and Southern blot analyses. Another advantage of this method is the fact that several samples can be processed simultaneously, growing the fungus on multiple well cell culture plates. In addition, the absence of maceration also reduces sample handling, minimizing the risks of contamination, a particularly important factor in work involving PCR.

Phenol biodegradation using a repeated batch culture of Candida tropicalis in a multistage bubble column.

As in many other microorganisms, the growth rate of C. tropicalis is affected by phenol. Besides, when the yeast is aerobically cultivated in a medium containing phenol, using a bubble column, the yeast cell flotation phenomenon occurs, which makes the continuous operation of this type of reactor difficult. Therefore, a system of phenol degradation, which recycles the biomass separated by flotation, was devised in this work. In order to reduce the substrate toxicity observed at high phenol concentrations, the bubble column used in the biodegradation studies was fed in a semibatch mode. So, a semicontinuous system was implemented to treat effluents with relatively high concentrations (> 9,000 ppm) of phenol, by replacing periodically about 22% of the bioreactor operational volume. The phenol removal efficiencies obtained with this system were higher than 98.7%.

beta-Galactosidase production by Kluyveromyces marxianus cultured in shake flasks.

beta-Galactosidase production by K. marxianus was studied in shake flasks. Enzyme production was strongly influenced by culture conditions determining oxygen transfer rates. Working volume turned out to be the most significant parameter affecting enzyme yields: the higher the volume, the higher the specific activity of the enzyme. The use of dehydrated whey (as the source of lactose) resulted in a large increase in the specific enzyme activity as compared with the use of pure lactose. This effect is likely due to a lower dissolved oxygen concentration along the fermentation. In the range of kL a between 40 and 90 h-1, enzyme activity did not seem to depend on kL a, whereas a drastic drop in the final specific enzyme activity was observed for kL a values above 90 h-1.

Doses unicas semanais orais de fluconazol 150 mg no tratamento de tinea corporis/cruris e candidiase cutanea / Doses weekly only you pray of fluconazol 150 mg in the treatment of cutaneous tinea corporis/cruris and candidiasis

Noventa e cinco pacientes ambulatoriais com tinea corporis e/ou tinea cruris participaram de um estudo multicentrico nao comparativo aberto para investigar a seguranca e eficacia de 1-4 doses unicas semanais de fluconazol oral na dose de 150 mg. O trichophyton rabrum foi o organismo mais frequentemente isolado (67 de 86 pacientes avaliados micologicamente). Uma media de 2,6 doses de fluconazol foi administrada; pacientes infectados com Candida albicans ou Epidermophyton floccosum necessitaram, em media, de 2 doses enquanto foram necessarias 3-4 doses em pacientes infectados com outros organismos. A cura clinica foi obtida em 85 de 92 (92%) dos pacientes na ultima avaliacao depois do tratamento, tendo os sete pacientes restantes melhorado substancialmente. No seguimento a longo prazo, 28-30 dias apos a ultima dose, 80 de 91 (88%) pacientes foram considerados clinicamente curados, tres (3%) apresentaram melhora e oito (9%) tiveram insucesso terapeutico. Dentre os fracassos clinicos a longo prazo, houve um diagnostico de tinea corporis (3%) de taxa de insucesso) e sete diagnosticos de tinea cruris (12% de taxa de insucesso). Evidencias micologicas de infeccao ocorreram em apenas 1 de 86 pacientes seguidos ate o final do seguimento a longo prazo. Recidiva micologica ocorreu em nove (11%) dos pacientes do seguimeto a longo prazo; um paciente estava infectado pelo Trichophyton mentagrphytes e oito pacientes, pelo T. rubrum. Houve recidiva em 2 de 29 (7%) pacientes com tinea corporis e oito de 57 (14%) com tines cruris (um paciente que recidivou tinha tinea corporis e cruris). Nao se verificou correlacao entre o numero de doses recebidas e a resposta micologica ou as taxas de recidiva a longo prazo. O fluconazol foi bem tolerado; somente 5 de 95 pacientes tratados com fluconazol referiram efeitos adversos, um dos quais resultou em descontinuacao da terapia (urticaria moderada). A boa tolerancia comparada a dos outros antifugicos orais e a conveniencia de um esquema de dose unica semanal oral em comparacao aos tratamentos topicos e orais existentes tornam a dose unica oral semanal de fluconazol uma elternativa valiosa no tratamento da tinea corporis/cruris

Utilización de agar Mueller Hinton en micología: parte I / The evaluation of Mueller Hinton agar in micology: part I

Con el fin de probar la utilidad del Agar Mueller Hinton en micología médica, para futuros ensayos con drogas antifúngicas, se compara el desarrollo de Dermatofitos (M. canis, M. gypseum, T. rubrum, T. mentagrophytes y E. floccosum) y especies de Candida en este medio y en ASG 20% - 40%, YMA y DST. Se comprueba que el Agar Mueller Hinton es tan eficaz como el ASG para el crecimiento de estos hongos en los plazos recomendados para estudios de sensibilidad