Evaluation of Fluorescent Capillary Electrophoresis for Rapid Identification of Candida Fungal Infections

Abstract

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...

Fluorescent Capillary Electrophoresis Is Superior to Culture in Detecting Candida Species from Samples of Urinary Catheters and Ureteral Stents with Mono- or Polyfungal Biofilm Growth

“…We optimized the method for ITS2 rDNA length polymorphism analysis using fluorescent capillary electrophoresis (f-ITS2-PCR-CE) and evaluated the technique's efficiency in our previous study (8), and we now proceeded to evaluate its applicability for urinary catheter, ureteral stent, and urine clinical samples. This technique enabled us to distinguish 26 out of 29 tested medically important Candida species.…”

“…Candida guilliermondii, Candida fermentati, and Candida carpophila were indistinguishable because of identical amplicon lengths. Amplicon length varied from 229 nucleotides (C. lipolytica) to 420 nucleotides (C. kefyr) (8).…”

“…Due to the final amplicon detection with capillary electrophoresis, a UNF1 primer 5= fluorescently labeled with a 6-carboxyfluorescein (6-FAM) dye was used. PCR was performed in a total volume of 20 l of HotStarTaq Master Mix (Qiagen, Germany) with 0.5 M of each primer and 2 mM final Mg 2ϩ concentration, using cycling conditions as described previously (8). A negative control of both DNA extraction (see above) and amplification (sterile DNA-free-water as a template) was used in each PCR run.…”

“…The fungi present in each sample were evaluated by comparing ITS2 lengths with our previously prepared internal laboratory database of the reference strain ITS2 lengths. Identification was considered successful if the analyzed sample length appeared within a 0.3-nucleotide (nt) interval of the respective reference strain's length (8).…”

“…In order to characterize polyfungal samples, several molecular techniques, employed mainly for environmental material testing in the field of molecular ecology, can be applied. These community profiling techniques include, for instance, denaturing/temperature gradient gel electrophoresis (DGGE/TGGE), singlestrand conformation polymorphism (SSCP), terminal restriction fragment length polymorphism (T-RFLP), cloning (7), or fluorescent capillary electrophoresis (CE) (8). The last method, based on utilizing the variability of the ITS2 ribosomal DNA (rDNA), has been proven to be superior to classical electrophoresis for analyzing DNA due to its higher separation efficiency, speed, and higher sensitivity and the fact that there is no need for sequencing (8,9).…”