New Restriction Fragment Length Polymorphism (RFLP) analysis markers for typing Aspergillus fumigatus strains.

Camile Pizeta Semighini, Steven Park1, David S. Perlin1, and Gustavo H

Author address: 

Faculdade de Ciencias Farmaceuticas de Ribeirão Preto, Universidade de São Paulo, Brazil and 1Public Health Research Institute, USA.

Abstract: 

Aspergillus fumigatus is an opportunistic fungus causing several respiratory diseases, such as allergic bronchopulmonary aspergillogis, aspergilloma and invasive aspergillosis. The later is presently a major cause of death amongst immunocompromised patients, associated with a high mortality rate (85%) even when appropriate treatment is used. The incidence of aspergillosis has increased significantly over the past two decades in parallel with the number of imunocompromised patients. Although many typing approaches have been proposed, an ideal epidemiological typing technique is not available that is applicable to a wide range of A. fumigatus isolates. In this study, we isolated and tested Restriction Fragment Length Polymorphism (RFLP) markers for A. fumigatus based on PCR-products amplified by the Random Amplified Polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A were amplified. The fragments Afd and Af5 were 85 % and 88 % identical at the DNA level to the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of the fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We have used both RAPD with the primer R108 and RFLP assays with Afut1, Afd, and Af4A as hybridization probes to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four cancer patients with recurrent aspergillosis following treatment with amphotericin B. Genetic relatedness was determined by using the Coefficient of Dice. The combination of these different methods was used to demonstrate that the isolates infecting the four patients were not identical. This approach should be valuable for molecular epidemiological investigations of Aspergillus infections, which should facilitate the development of preventive measures for patient management. Financial support: CNPq and FAPESP, Brazil.
2001

abstract No: 

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Full conference title: 

21st Fungal Genetics Conference
    • Fungal Genetics Conference 21st (2000)