Simple, Low-Cost Detection of <i>Candida parapsilosis</i> Complex Isolates and Molecular Fingerprinting of <i>Candida orthopsilosis</i> Strains in Kuwait by ITS Region Sequencing and Amplified Fragment Length Polymorphism Analysis

<div><p><i>Candida parapsilosis</i> has now emerged as the second or third most important cause of healthcare-associated <i>Candida</i> infections. Molecular studies have shown that phenotypically identified <i>C</i>. <i>parapsilosis</i> isolates represent a complex of three species, namely, <i>C</i>. <i>parapsilosis</i>, <i>C</i>. <i>orthopsilosis</i> and <i>C</i>. <i>metapsilosis</i>. <i>Lodderomyces elongisporus</i> is another species phenotypically closely related to the <i>C</i>. <i>parapsilosis</i>-complex. The aim of this study was to develop a simple, low cost multiplex (m) PCR assay for species-specific identification of <i>C</i>. <i>parapsilosis</i> complex isolates and to study genetic relatedness of <i>C</i>. <i>orthopsilosis</i> isolates in Kuwait. Species-specific amplicons from <i>C</i>. <i>parapsilosis</i> (171 bp), <i>C</i>. <i>orthopsilosis</i> (109 bp), <i>C</i>. <i>metapsilosis</i> (217 bp) and <i>L</i>. <i>elongisporus</i> (258 bp) were obtained in mPCR. Clinical isolates identified as <i>C</i>. <i>parapsilosis</i> (n = 380) by Vitek2 in Kuwait and an international collection of 27 <i>C</i>. <i>parapsilosis</i> complex and <i>L</i>. <i>elongisporus</i> 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 <i>C</i>. <i>orthopsilosis</i> isolates (including 4 isolates from a previous study) was performed by amplified fragment length polymorphism (AFLP) analysis. Phenotypically identified <i>C</i>. <i>parapsilosis</i> isolates (n = 380) were identified as <i>C</i>. <i>parapsilosis</i> sensu stricto (n = 361), <i>C</i>. <i>orthopsilosis</i> (n = 15), <i>C</i>. <i>metapsilosis</i> (n = 1) and <i>L</i>. <i>elongisporus</i> (n = 3) by mPCR. The mPCR also accurately detected all epidemiologically unrelated <i>C</i>. <i>parapsilosis</i> complex and <i>L</i>. <i>elongisporus</i> isolates. The 19 <i>C</i>. <i>orthopsilosis</i> isolates obtained from 16 patients were divided into 3 haplotypes based on ITS region sequence data. Seven distinct genotypes were identified among the 19 <i>C</i>. <i>orthopsilosis</i> 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 <i>C</i>. <i>parapsilosis</i>, <i>C</i>. <i>orthopsilosis</i>, <i>C</i>. <i>metapsilosis</i> and <i>L</i>. <i>elongisporus</i> has been developed.</p>