Isolamento and identification of Coccidioides immitis from soil samples related to outbreaks of coccidioidomycosis.
Coccidioidomycosis (CM) is a systemic mycosis that is endemic in the semi-arid Northeast of Brazil caused by the soil-inhabiting dimorphic fungus Coccidioides immitis non-CA [=C. posadasii]. In Brazil, the main epidemiological characteristic related to outbreaks of acute CM is the disturbance caused by armadillo hunters while digging them out of their infected burrows. This soil disturbing activity causes dust contaminated with arthroconidia of the fungus, which, once inhaled by the hunters, may provoke the infection or disease. The objectives of the present study were: (i) Isolate and identify agents of systemic mycoses by classical methodologies, detected in ambiental samples related to outbreaks of CM; (ii) Evaluate the efficiency of protocols for DNA extraction from ambiental soil samples using two distinct methodologies: a) liquid Nitrogen cryofracture and b) commercial extraction kit Fast DNA® SPIN® Kit For Soil Q-BIOgene; (iii) Develop specific primers for detection of C. immitis non-CA using bioinformatic tools; (iv) Evaluate and validate the specific primers for detection of C. immitis non-CA in DNA extracted from soil. Soil samples collected from two armadillo sites suspected to be contaminated by C. immitis non-CA were analyzed. Out of 10 soil samples collected in Elesbão Veloso, 3 (30%) were positive for C. immitis non-CA by mice inoculation, 2 (20%) were positive by direct PCR and 10 (100%) were positive as detected by the semi-nested PCR technique. Out of 23 soil samples collected in Caridade do Piauí, 3 (13,3%) were positive for C. immitis non-CA by mice inoculation, 6 (42,8%) out of 14 samples were positive by direct PCR and all (100%) of the 14 samples tested were positive as detected by the semi-nested PCR technique. This methodology represents a simple, sensitive and specific molecular technique to detect C. immitis non-CA in environmental soil samples and permits to determine the distribution of the ecotypes of this agent in the endemic areas. Moreover, it may be useful to identify clinical and environmental isolates suspected of C. immitis non-CA.
Advisor:Bodo Wanke; Marcia dos Santos Lazéra
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:genetica molecular e de microorganismos coccidioides coccidioidomicose coccidioidomycosis
Date of Publication:08/31/2006