25S intron-restriction endonuclease genotyping of Candida albicans isolates

Abstract number: P1701

Karahan Z.C., Saran B., Agirbasli H., Tekeli A., Aksoy A.M.

Objectives: There isn't any universally accepted gold-standard genotyping method for differentiating Candida albicans isolates. The presence of transposable group I intron in the 25S rDNA gene makes C. albicans isolates divided into three genotypes by 25S intron analysis. Genotype-A isolates were previously shown to contain different subgroups which were determined by restriction endonuclease and sequence analysis. In this study, our aim was to establish a new genotyping method based on PCR amplification of 25S intron region followed by restriction endonuclease analysis with two different endonucleases.

Methods: 111 C. albicans strains obtained from different infection sites of 64 leukemic patients were genotyped. PCR amplification was performed by using primers designed to span the transposable group I intron region of the 25S rDNA gene. To obtain the genotypes, PCR products were digested by HaeIII and MspI restriction endonucleases. The resulting band patterns were determined by agarose gel electrophoresis. Groups were differentiated due to the differences in the patterns. Discriminatory power (DP) was calculated for each method.

Results: 25S intron analysis divided the isolates into three genotypes. Eightyone of the isolates were found to be genotype-A, 18 genotype-B, and 12 genotype-C (DP= 0.43). HaeIII restriction of genotype-A isolates gave 18 subgroups, while genotype B and genotype-C isolates clustered in single subgroups (DP = 0.89). MspI restriction of genotype A isolates gave 14 subgroups, while genotype-B and genotype-C isolates again clustered in single subgroups (DP = 0.80). When the results obtained by both enzyme digestions were combined, 33 subgroups were obtained for genotype-A isolates, while genotype-B and genotype-C isolates both gave single subgroups (DP = 0.92).

Conclusion: As the DP of 25S intron analysis is below the acceptable limits (<0.90), its use for investigating C. albicans strains epidemiologically would not be appropriate. By combining 25S intron genotyping with HaeIII and MspI restriction endonucelase analyses, the DP rises to 0.92 and it becomes suitable for epidemiological purposes. The method is easy to perform, cheap, and labour firendly.

Acknowledgement: This study was supported by a grant from the Biotechnology Institute of Ankara University (Project No: 2001K120240).