dEarly diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the "psilosis" complex. During the last several decades, the impact and frequency of fungal infections have gained importance mainly due to an increasing number of immunocompromised patients (1, 2). Fungemia cases are being caused mainly by Candida species, which are the fourth most common microorganisms isolated from the blood samples (3, 4, 5). Sepsis due to Candida spp. is a very serious condition and has a higher mortality rate that for bacterial pathogens; reaching 54 to 64% in Candida-associated septic shock (6, 7). Moreover, the current changes in the epidemiology of invasive mycoses has highlighted a shift in the Candida species involved with a reduced proportion of Candida albicans and an increase in non-albicans species, which can show different susceptibility to various antifungal therapies (8,9,10).Early initiation of antifungal therapy is a critical step in the treatment of fungal infections. Therefore, quick, successful detection and identification of the etiological agents is crucial for early targeted therapy and favorable clinical patient outcome (11). Correct species identification is mostly based on phenotypic features and is usually time-consuming because a typical diagnostic workflow takes up to several days. Moreover, the phenotypic methods may lead to misidentification, particularly in the case of closely related species (12, 13). A significant improvement occurred when matrix-assisted laser desorption ionizationϪtime of flight mass spectroscopy (MALDI-TOF MS) was introduced as a routine laboratory procedure, enab...

Molecular techniques in fungal detection and identification represent an efficient complementary diagnostic tool which is increasingly used to overcome limitations of routinely used culture techniques. The aim of this study was to characterize Candida sp. representation in samples from urine, urinary catheter, and ureteral stent biofilm using ITS2 ribosomal DNA (rDNA) amplification followed by fluorescent capillary electrophoresis (f-ITS2-PCR-CE) and to compare the results with those obtained by culture. A total of 419 samples were analyzed, and 106 (25.2%) were found positive, out of which 17 (16%) were polyfungal. The positivity rate did not differ between samples from catheters and stents (23.6% versus 20.9%) or between catheter and stent corresponding urine samples (40.2% versus 30.2%). Ten different Candida species were detected, with Candida parapsilosis (31.4%), Candida albicans (26.5%), and Candida tropicalis (12.4%) predominating. f-ITS2-PCR-CE was evaluated as substantially less time-consuming and 8.3 times more sensitive than the routinely applied culture technique with 1 l of urine/sonicated fluid inoculated, detecting 67 (19.9%) versus 8 (2.4%) positive samples out of 337 initially analyzed samples. The culture sensitivity considerably improved to 1.7 times lower than that of f-ITS2-PCR-CE after the inoculation volume was increased to 100 l in the additional 82 samples. Moreover, the molecular technique, unlike routine cultivation, enabled precise pathogen composition determination in polymicrobial samples. In conclusion, the f-ITS2-PCR-CE method was shown to be a quick and efficient tool for culture-independent detection and identification of fungi in urinary tract-related samples, demonstrating a higher sensitivity than culture.