Species identification and strain differentiation of clinical Candida isolates using the DiversiLab system of automated repetitive sequence-based PCR.
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 (n=94), C. glabrata (n=8), C. parapsilosis (n=5), C. tropicalis (n=3), C. krusei (n=3) and Candida lusitaniae (n=2), 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.
Wise
Mark G
MG
Bacterial Barcodes Inc., Athens, GA 30602, USA.
Healy
Mimi
M
Bacterial Barcodes Inc., Athens, GA 30602, USA.
Reece
Kristy
K
Bacterial Barcodes Inc., Athens, GA 30602, USA.
Smith
Rebecca
R
TriCore Reference Laboratories, Albuquerque, NM 87102, USA.
Walton
Dobbie
D
Bacterial Barcodes Inc., Athens, GA 30602, USA.
Dutch
Wendy
W
Bacterial Barcodes Inc., Athens, GA 30602, USA.
Renwick
Alex
A
Bacterial Barcodes Inc., Athens, GA 30602, USA.
Huong
Joe
J
Bacterial Barcodes Inc., Athens, GA 30602, USA.
Young
Steve
S
TriCore Reference Laboratories, Albuquerque, NM 87102, USA.
Tarrand
Jeffrey
J
Department of Laboratory Medicine, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
Kontoyiannis
Dimitrios P
DP
Department of Infectious Diseases, Infection Control and Employee Health, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
D001331:Automation; D002175:Candida / Q000145:classification* / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D002177:Candidiasis / Q000382:microbiology*; D016000:Cluster Analysis; D016172:DNA Fingerprinting; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D030541:Databases, Genetic; D015723:Gene Library; D006801:Humans; D016533:Mycological Typing Techniques / Q000379:methods*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012984:Software
D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D012340:RNA, Ribosomal, 5.8S