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Journal of Clinical Microbiology, August 2005, p. 3869-3876, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.3869-3876.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

New Microsatellite Multiplex PCR for Candida albicans Strain Typing Reveals Microevolutionary Changes

Paula Sampaio,1 Leonor Gusmão,2 Alexandra Correia,1 Cíntia Alves,2 Acácio G. Rodrigues,2,4 Cidália Pina-Vaz,2,4 António Amorim,2,3 and Célia Pais1*

CBUM-Center of Biology, University of Minho, 4710-057 Braga, Portugal,1 IPATIMUP-Institut of Pathology and Molecular Immunology of Porto University, R. Roberto Frias, s/n 4200 Porto, Portugal,2 Faculty of Science, University of Porto, Porto, Portugal,3 Microbiology Department, Faculty of Medicine, University of Porto, Porto, Portugal4

Received 15 February 2005/ Returned for modification 28 March 2005/ Accepted 18 April 2005

Five new microsatellite loci were described and characterized for use as molecular markers for the identification and genetic differentiation of Candida albicans strains. Following the typing of 72 unrelated clinical isolates, the analysis revealed that they were all polymorphic, presenting from 5 to 30 alleles and 8 to 46 different genotypes. The discriminatory power obtained by combining the information generated by three microsatellites used in a multiplex PCR amplification strategy was 0.99, the highest ever reported. The multiplex PCR was later used to test a total of 114 C. albicans strains, including multiple isolates from the same patient collected from different body locations and along episodes of vulvovaginal infections. Three different scenarios for strain relatedness were identified: (i) different isolates that were revealed to be the same strain, (ii) isolates that were the same strain but that apparently underwent a process of microevolution, and (iii) isolates that corresponded to different strains. Analysis of the microevolutionary changes between isolates from recurrent infections indicated that the genotype alterations observed could be the result of events that lead to the loss of heterozygosity (LOH). In one case of recurrent infection, LOH was observed at the CAI locus, and this could have been related to exposure to fluconazole, since such strains were exposed to this antifungal during treatment. The analysis of microsatellites by a multiplex PCR strategy was found to be a highly efficient tool for the rapid and accurate differentiation of C. albicans strains and adequate for the identification of fine microevolutionary events that could be related to strain microevolution in response to environmental stress conditions.

* Corresponding author. Mailing address: Departamento de Biologia, Centro de Biologia da Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. Phone: (351)253604312. Fax: (351)253678980. E-mail: cpais{at}bio.uminho.pt.

Journal of Clinical Microbiology, August 2005, p. 3869-3876, Vol. 43, No. 8
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.8.3869-3876.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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