Detection and Molecular Identification of Eight Candida Species in Clinical Samples by Simplex PCR

Abstract

Systemic candidiasis is a frequent opportunistic mycosis that can be life-threatening. Its main etiological agent is <i>Candida albicans</i>; however, the isolation of non-<i>albicans Candida</i> species has been increasing. Some of these species exhibit greater resistance to antifungals, so the rapid and specific identification of yeasts is crucial for a timely diagnosis and optimal treatment of patients. Multiple molecular assays have been developed, based mainly on polymerase chain reaction (PCR), showing high specificity and sensitivity to detect and identify <i>Candida</i> spp. Nevertheless, its application in diagnosis has been limited due to specialized infrastructure or methodological complexity. The objective of this study was to develop a PCR assay that detects and identifies some of the most common pathogenic <i>Candida</i> species and evaluate their diagnostic utility in blood samples and bronchial lavage. A pair of oligonucleotides was designed, CandF and CandR, based on sequence analysis of the 18S-ITS1-5.8S-ITS2-28S region of the rDNA of <i>Candida</i> spp., deposited in GenBank. The designed oligonucleotides identified <i>C. albicans</i>, <i>C. glabrata</i>, <i>C. tropicalis</i>, <i>C. parapsilosis</i>, <i>C. krusei</i>/<i>Pichia kudriazevii</i>, <i>C. guilliermondii</i>/<i>Meyerozyma guilliermondii</i>, <i>C. lusitaniae</i>/<i>Clavispora lusitaniae</i>, and <i>C. dubliniensis</i> using simplex PCR based on the amplicon size, showing a detection limit of 10 pg/μL of DNA or 10³ yeasts/mL. Based on cultures as the gold standard, it was determined that the sensitivity (73.9%), specificity (96.3%), and the positive (94.4%) and negative (81.2%) predictive values of the PCR assay with the designed oligonucleotides justify their reliable use in diagnosis.