Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

Abstract

Candida albicans is an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiating C. albicans from other Candida species are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel of C. albicans and various nontarget Candida spp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5-to 5.5-fold-higher discriminatory power for C. albicans than P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification of C. albicans in clinical and related applications, especially when combined with FCM.T he genus Candida includes several species that are pathogenic for humans, including Candida albicans, C. dubliniensis, C. krusei, C. glabrata, C. tropicalis, and others. Early detection and differentiation of pathogenic Candida species from patient samples are critical to patient outcomes. Candida albicans is the major pathogen in this genus, causing approximately one-half of all infections caused by Candida spp. (1, 2). Because Candida spp. differ in their patterns of resistance to common antifungals, differentiation of C. albicans (fluconazole responsive) from fluconazoleresistant species such as C. krusei, C. glabrata, and C. tropicalis is required for appropriate antimicrobial therapy (1-6). The close phenotypic similarity between C. dubliniensis and C. albicans further underlines the need for specific identification of C. albicans in clinical settings (7,8).Peptide nucleic acid (PNA) probes are synthetic DNA mimics with improved performance characteristics compared to DNA probes. Specifically, these characteristics include faster hybridization kinetics, enhanced single-mismatch discrimination, and improved penetration of structures such as the cell wall (9, 10). Fluorescence in situ hybridization (FISH) is a rapid method for whole-cell detection of specific microorganisms. The clinical utility of FISH for the detection of Candida spp. has been shown by using DNA-based probes targeting th...

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