Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

Abstract

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n ‫؍‬ 72), C. dubliniensis (n ‫؍‬ 58), C. glabrata (n ‫؍‬ 5), C. krusei (n ‫؍‬ 2), C. parapsilosis (n ‫؍‬ 4), and C. tropicalis (n ‫؍‬ 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.

Faster Identification of Pathogens in Positive Blood Cultures by Fluorescence In Situ Hybridization in Routine Practice

“…PNA probes have been used in several studies for the identification of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans in blood cultures. The sensitivities and specificities of these probes were all more than 98% (4,16,17,19,21). Although the sensitivity and specificity of individual probes (oligonucleotide and PNA) are very good, the usefulness of FISH as a diagnostic test depends on the probes included in the assay and is related to the prevalence of microorganisms in a specific setting.…”

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

“…PCR-based methods and other successful molecular diagnostic techniques, such as the peptide nucleic acid-fluorescent in situ hybridization method (1,39,47), evaluating the hybridization of specific fluorescent probes to RNA target sites, have been developed, but their implementation for the identification of medically important yeasts in the clinical laboratory has not yet been routinely established, possibly because they are not so easy to perform and require more or less sophisticated equipment.…”

Identification Of Candida Species From Blood Cultures With Fluorescent In Situ Hybridization (fish), Polymerase Chain Reaction-restriction Fragment Length Polymorphism (pcr-rflp) And Conventional Methods

“…[11,20,21] It has also been determined that PNA FISH test for C. albicans is more accurate than standard culture methods and results in significant cost saving. [31] Our results showed that FISH was compatible with PCR-RFLP and conventional methods, except multi-species candidemia.…”

“…in blood cultures are critical. [2,10,11] Current conventional methods for the identification of Candida spp. from positive blood cultures are based on subculture to appropriate fungal medium, including Sabouraud dextrose agar, or CHROMagar Candida medium, and isolated yeast colonies are identified by the phenotypic (germ tube formation, carbohydrate assimilation [e.g., API 20C, ID 32C], and chlamydospore formation on cornmeal agar).…”

“…It has been reported that sensitivity and specificity of this test are 100%. [11,20,21] There have been various probes to be used in FISH method such as rRNA-targeted oligonucleotide probes and peptide nucleic acid (PNA) probes. [22,23] Moreover, there has been commercial SeaFast sepsis kit, including yeast to be used for FISH.…”