Rapid Identification of Commonly EncounteredCandidaSpecies Directly from Blood Culture Bottles

Abstract

We report a rapid-cycle, real-time PCR method for identifying six Candida spp. directly from BACTEC blood culture bottles. Target sequences in the noncoding internal transcribed spacer regions of the rRNA operon were simultaneously amplified and interrogated with fluorescent probes to identify Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. lusitaniae; these account for 88% of the yeast species isolated from positive blood cultures in our laboratory. Any of the first four species can be identified in a single reaction using two fluorescent hybridization probe sets. The antifungal-resistant species C. krusei and C. lusitaniae are detected in a second reaction, also with two probe sets. The assay was validated with DNA extracted from BACTEC blood culture bottles positive for yeasts (n ‫؍‬ 62) and was 100% concordant with culture identification based on biochemical and morphological features of C. albicans (n ‫؍‬ 22), C. parapsilosis (n ‫؍‬ 10), C. tropicalis (n ‫؍‬ 1) C. glabrata (n ‫؍‬ 22), C. krusei (n ‫؍‬ 2), and C. lusitaniae (n ‫؍‬ 1). No cross-reactivity was observed in blood culture samples growing yeasts other than the above-mentioned species (n ‫؍‬ 4), in those growing bacteria (n ‫؍‬ 12), or in the absence of microbial growth. Our assay allows rapid (<2 h) and specific detection of the most common Candida spp. directly from positive blood cultures and may facilitate delivery of optimal antifungal therapy.

Simultaneous Detection and Identification of Candida , Aspergillus , and Cryptococcus Species by Reverse Line Blot Hybridization

“…Detection of oligonucleotide probe hybridization has been reported using microtitration plate-based enzyme immunoassay (5,17,30), Southern or slot blotting (4,6,23,28), and fluorogenic probes (8,19,24). We chose the RLB format, given the advantages of relative simplicity, low cost, ready availability of the materials and the methods, and ability to simultaneously analyze multiple specimens against multiple probes (7).…”

Rapid Identification of Candida Species and Other Clinically Important Yeast Species by Flow Cytometry

“…Rapid, reliable tests that identify a majority of species known to cause infections would greatly improve patient treatment. Many of the recently developed molecular-based assays are real-time assays that provide rapid results, but they are often limited in the number of species that can be detected in a single assay (18). Standard PCR-based assays are also limited in the number of species that can be identified from a single amplification.…”

Improved Identification of Yeast Species Directly from Positive Blood Culture Media by Combining Sepsityper Specimen Processing and Microflex Analysis with the Matrix-Assisted Laser Desorption Ionization Biotyper System

“…Current phenotypic methods for yeast identification, including colony morphology, germ tube test, urease activity, and the API 20C AUX strip (bioMeriuex, Durham, NC), take an additional 48 to 72 h to complete after the yeast-like fungal pathogens are observed in the blood culture media (33). Various molecular techniques, including real-time PCR, fluorescent in situ hybridization, and pyrosequencing, have been developed to speed the identification of blood-borne fungi (14,23,25), but they have not been implemented as routine techniques in the clinical microbiology laboratory.…”