Species identification and strain differentiation of clinical Candida isolates using the DiversiLab system of automated repetitive sequence-based PCR

Abstract

The DiversiLab system, which uses repetitive sequence-based PCR (rep-PCR) to genotype micro-organisms, was evaluated as a molecular typing tool for members of the genus Candida. Initially, 41 clinical Candida spp. (7 Candida krusei, 10 Candida parapsilosis, 7 Candida albicans, 10 Candida tropicalis and 7 Candida glabrata), previously identified at the species level by morphological and biochemical analysis, were analysed with the DiversiLab system. Species identification was confirmed by DNA sequence analysis of the contiguous internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). On the basis of an 80 % similarity threshold, rep-PCR consistently clustered like species and this set of isolates, along with five ATCC reference strains, was used to create a DNA fingerprint library with the DiversiLab software. Subsequently, an additional set of 115 clinical Candida isolates, identified biochemically as C. albicans (n594), C. glabrata (n58), C. parapsilosis (n55), C. tropicalis (n53), C. krusei (n53) and Candida lusitaniae (n52), isolated at a regional reference laboratory, were typed using DiversiLab. One hundred and six of these isolates clustered with members of the Candida library at .80 % similarity and thus could be assigned species identification, and initial calculations showed that identification via rep-PCR fingerprinting was 95 % concordant (101/106) with the biochemical/ morphological identification. However, ITS region sequencing of the five discrepant samples, as well as the nine isolates that were ,80 % similar to the database samples, showed that nine were misidentified with traditional biochemical/morphological methods. For the misidentified isolates, the sequence-based identification was in agreement with the DiversiLab clustering, yielding an actual correlation of .99 %. As traditional techniques can take several days to provide information about Candida at the genus/species level, genotyping with the DiversiLab system holds promise for more-rapid speciation of members of this genus. This system may also be useful for epidemiological studies such as source tracking that require Candida subspecies discrimination. INTRODUCTIONCandidiasis and candidaemia have emerged as common infections in debilitated hospitalized patients (Kauffman, 2006). Although Candida albicans remains the most important species causing these infections, non-albicans Candida spp. such as Candida glabrata have emerged as common pathogens, often accounting for more than 50 % of candidiasis cases (Clark & Hajjeh, 2002;Eggimann et al., 2003;Marchetti et al., 2004;Tortorano et al., 2004 Foulet et al., 2005;Lott et al., 2003;Sampaio et al., 2003; Stephan et al., 2002). Multilocus sequence typing is a highly discriminatory and stable method of unambiguous characterization of C. albicans (Bougnoux et al., 2002;Dodgson et al., 2003;Robles et al., 2004;Tavanti et al., 2003). However, the costs associated with this method of sequencing can be prohibitive and, along with the intensive data analysis requirements, may keep such technol...

Long-Term Rasamsonia argillacea Complex Species Colonization Revealed by PCR Amplification of Repetitive DNA Sequences in Cystic Fibrosis Patients

“…In the present study, a large set of clinical R. argillacea (sensu lato) isolates was investigated using the semiautomated DiversiLab system based on the analysis of repetitive DNA sequences. rep-PCR has been previously used successfully for species and/or strain differentiation in several fungal groups, including Candida and Aspergillus species, as well as dermatophytes and some dimorphic fungi (25,26,27,28,29). Recently, Matray et al (23) showed that this method also allows species identification within the Scedosporium apiospermum species complex, another group of sibling species encountered in patients with CF, as well as in CGD patients.…”

Microsatellite Genotyping Clarified Conspicuous Accumulation of Candida parapsilosis at a Cardiothoracic Surgery Intensive Care Unit

“…DiversiLab typing is a rapid and easy-to-perform method for initial screening during outbreak investigation for different bacterial species, as described by Fluit and colleagues for Acinetobater spp., Stenotrophomonas maltophilia, and the Enterobacter cloacae com- plex (9) and as recently described by Deplano and coworkers for Enterococcus faecium and Klebsiella pneumoniae (6). Regarding Candida species, Wise and colleagues did show that DiversiLab was able to discriminate between the medically important members of the genus Candida at the species level (24). However, when it comes to C. parapsilosis strain differentiation, our study could demonstrate that DiversiLab typing has limited discriminatory power and therefore needs to be replaced by a more discriminative method such as microsatellite genotyping to investigate outbreaks where C. parapsilosis is involved.…”

Molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii in China between 2007 and 2013 using multilocus sequence typing and the DiversiLab system

“…Automated repetitive PCR (DiversiLab system) [24] has been used in the strain identification and typing of some species of yeasts and molds [25][26][27][28][29][30]. The genotyping of C. neoformans and C. gattii using the DiversiLab system has not yet been reported.…”