Evolutionary distances and identification of Candida species in clinical isolates by Randomly Amplified Polymorphic DNA (RAPD)

Fast and reliable identification of different species of the genus Candida is important to define adequate therapeutic decisions, because the differen t species have highly variable susceptibilities to antifungal drugs; azoles and amphothericin B. Accurate statistical records on case history and epid emiological studies also depend on effective identification. To address thi s problem we established a RAPD method that enabled direct identification o f five very common species of Candida. Initially, reference band patterns w ere established for C. albicans, C. tropicalis, C. parapsilosis, C. glabrat a and C. krusei. One of the primers, M2, showed remarkably conserved intra- specific patterns of approximately 10 bands each, ranging in size from 2.0 to 0.1 kb. These patterns were significantly different and species-specific . Few bands were conserved between different species of Candida, which was assumed to be consistent with their phylogenetic relatedness. In addition, band patterns were constant and reproducible and DNA isolated from single c olonies yielded sufficient DNA for identification. The reference band patte rns were then used, in blind experiments, to identify species of Candida in 50 randomly chosen samples, including clinical isolates and ATCC strains. RAPD results were 100% consistent with results obtained by conventional dia gnostic methods and were achieved in one day instead of several days taken by conventional methods. Because ideal identification methods should be con sistent with phylogeny and taxonomy we tested whether RAPD could be used to calculate genetic distances. Comparison of RAPD phylogenetic trees with 18 S rRNA trees showed significant differences in tree topologies which indica ted that RAPD data could not accurately measure the relative distances betw een different species. Also, computer simulations of RAPD random patterns w ere used to test whether the observed degree of RAPD band pattern similarit ies could occur at random. These simulations suggested that the level of in ter-specific band pattern similarities observed in our data could be obtain ed at random, while intraspecific pattern similarities could not. RAPD woul d be helpful to discriminate between isolates but not to quantitate the dif ferences. We suggest that the inaccurate estimate of genetic distances from RAPD is a general limitation of the technique and not a specific problem o f our identification method. Because of the repetitive character of the tar get sequences, genetic distances calculated from RAPD could be affected by paralogy, namely, recombination and duplication events not parallel with sp eciation events.