title: Accurate identification of Candida parapsilosis (sensu lato) by use of mitochondrial DNA and real-time PCR.
abstract: Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the "gold standard" for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species.
authors.affiliation: Laboratório Especial de Micologia, Disciplina de Infectologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Ana Carolina R
authors.initials: AC
authors.lastname: Souza
authors.forename: Renata C
authors.initials: RC
authors.lastname: Ferreira
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Gonçalves
authors.forename: Guillermo
authors.initials: G
authors.lastname: Quindós
authors.forename: Elena
authors.initials: E
authors.lastname: Eraso
authors.forename: Fernando C
authors.initials: FC
authors.lastname: Bizerra
authors.forename: Marcelo R S
authors.initials: MR
authors.lastname: Briones
authors.forename: Arnaldo L
authors.initials: AL
authors.lastname: Colombo
mesh_terms: D002175:Candida / Q000145:classification* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D008828:Microbiological Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D009172:Mycology / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013020:South America; D013030:Spain; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D015345:Oligonucleotide Probes