Open AccessSimple, Low-Cost Detection of Candida parapsilosis Complex Isolates and Molecular Fingerprinting of Candida orthopsilosis Strains in Kuwait by ITS Region Sequencing and Amplified Fragment Length Polymorphism Analysis
Author(s) -
Mohammad Asadzadeh,
Suhail Ahmad,
Ferry Hagen,
Jacques F. Meis,
Noura AlSweih,
Ziauddin Khan
Publication year - 2015
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0142880
Subject(s) - biology , candida parapsilosis , microbiology and biotechnology , internal transcribed spacer , amplicon , genetics , polymerase chain reaction , gene , ribosomal rna , antifungal
Candida parapsilosis has now emerged as the second or third most important cause of healthcare-associated Candida infections. Molecular studies have shown that phenotypically identified C . parapsilosis isolates represent a complex of three species, namely, C . parapsilosis , C . orthopsilosis and C . metapsilosis . Lodderomyces elongisporus is another species phenotypically closely related to the C . parapsilosis -complex. The aim of this study was to develop a simple, low cost multiplex (m) PCR assay for species-specific identification of C . parapsilosis complex isolates and to study genetic relatedness of C . orthopsilosis isolates in Kuwait. Species-specific amplicons from C . parapsilosis (171 bp), C . orthopsilosis (109 bp), C . metapsilosis (217 bp) and L . elongisporus (258 bp) were obtained in mPCR. Clinical isolates identified as C . parapsilosis (n = 380) by Vitek2 in Kuwait and an international collection of 27 C . parapsilosis complex and L . elongisporus isolates previously characterized by rDNA sequencing were analyzed to evaluate mPCR. Species-specific PCR and DNA sequencing of internal transcribed spacer (ITS) region of rDNA were performed to validate the results of mPCR. Fingerprinting of 19 clinical C . orthopsilosis isolates (including 4 isolates from a previous study) was performed by amplified fragment length polymorphism (AFLP) analysis. Phenotypically identified C . parapsilosis isolates (n = 380) were identified as C . parapsilosis sensu stricto (n = 361), C . orthopsilosis (n = 15), C . metapsilosis (n = 1) and L . elongisporus (n = 3) by mPCR. The mPCR also accurately detected all epidemiologically unrelated C . parapsilosis complex and L . elongisporus isolates. The 19 C . orthopsilosis isolates obtained from 16 patients were divided into 3 haplotypes based on ITS region sequence data. Seven distinct genotypes were identified among the 19 C . orthopsilosis isolates by AFLP including a dominant genotype (AFLP1) comprising 11 isolates recovered from 10 patients. A rapid, low-cost mPCR assay for detection and differentiation of C . parapsilosis , C . orthopsilosis , C . metapsilosis and L . elongisporus has been developed.