DNA pyrosequencing-based identification of pathogenic Candida species by using the internal transcribed spacer 2 region

Context. - The incidence of infections due to diverse Candida species is increasing, with correspondingly different antifungal susceptibility patterns. Routine yeast identification methods cause significant delays in appropriate patient management. Objective. - A DNA pyrosequencing strategy was evaluated for identification of pathogenic Candida species associated with human infections. Design. - Clinical (n = 51) and commercial (n = 9) Candida isolates were identified in a blinded, parallel study consisting of routine fungal cultures and biochemical analyses in comparison with DNA pyrosequencing. Results. - DNA pyrosequencing yielded species-level identification of all 60 Candida isolates, and sequencing interpretations agreed in all cases with results of biochemical and morphologic testing. Different Candida species were identified, such as C albicans, C dubliniensis, C glabrata, C guilliermondii, C krusei, C lusitaniae, C parapsilosis, and C tropicalis. Automated and manual approaches to DNA sequence interpretation, each coupled with the ldentifire identification software, demonstrated 100% agreement with respect to Candida species identification. Twenty-one isolates yielded intraspecies DNA sequence differences (90%-98% nucleic acid sequence identity) by automated interpretation. Sequence differences resulted from single-nucleotide polymorphisms or single-base additions/deletions, in addition to interpretative challenges in homopolymeric tracts. Conclusion. - DNA pyrosequencing coupled with automated DNA sequence alignment provides a practical approach for accurate and timely identification of Candida pathogens. Relatively rapid and facile genotypic studies by DNA pyrosequencing matched the effectiveness of extensive biochemical/morphologic studies for yeast identification.