XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:102-1

Oral / Poster

102-1	ATTEMPTS TO DETECT Paracoccidioides brasiliensis IN ENVIRONMENTAL AEROSOL SAMPLES USING CYCLONE SAMPLER AND EXTINCTION CULTURE TECHNIQUE
Authors:	Thales Domingos Arantes (UNESP - Universidade Estadual Paulista) ; Severino Assis da Graça Macoris (UNESP - Universidade Estadual Paulista) ; Eduardo Bagagli (UNESP - Universidade Estadual Paulista)

Abstract

Introduction: The exact location of Paracoccidioides brasiliensis in nature is still unknown; however, it is believed that the soil is its probable habitat, due to the high rates of infection in rural workers and also to the high frequency of isolation from armadillos belonging to the species Dasypus novencinctus and Cabassous centralis. The data on environmental isolation of P. brasiliensis is controversial because of the casual finds, with practically no repeatability. Objective: To detect and isolate P. brasiliensis in environmental aerosol samples, by using cyclonic air sampling based on capture and separation of aerosol particles according to their size in function of the volume and speed of the captured air. Material and Methods: The air was collected from three armadillo’s burrows where P. brasiliensis was previously detected in soil or isolated from armadillos, by using the cyclone sampler NIOSH Model BC 251, designed by CDC (Center for Disease Control and Prevention of Morgantown WV – USA. The sampling time ranged from 12-16 hours for each sample .The samples were evaluated by direct culture using the extinction culture technique in Mycosel Agar Medium at 35° C. For molecular detection the air samples were directly added to the PCR reagents mixture, without a previous DNA extraction, due to the low spore’s concentration. Nested-PCR of the ITS1/5.8S/ITS2 rRNA region was carried out by using the panfungal outer primers ITS4 and ITS5 and the specific inner primers PbITSE and PbITSR. In order to ensure the disruption of the spores the time and temperature of the initial denaturation step of the first PCR, were changed to 10 minutes and 98° C respectively. Partial Results: Although no positive culture for P. brasiliensis was so far obtained, the extinction culture technique provided a reduction of the excessive contaminant growth, avoiding the inhibition of slow growth fungi. Even so the detection of P. brasiliensis was positive for the molecular approach by obtaining a Nested-PCR band amplification (~387pb) with the specific inner primers PbITSE and PbITSR. We hope these new sampling and culture approaches help to clarify the habitat and ecological niche of this pathogen, with consequent adoption of more effective methods of preventing infections.

Keyword: Cyclone sampler, Extinction culture, P. brasiliensis

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