Blastomyces dermatitidis and Histoplasma capsulatum are dimorphic fungi that often cause self-limited respiratory infections. However, they may also cause severe disseminated disease, depending on the level of the exposure to the organism and the host immune status. In addition, patients with infections caused by these fungi may have very similar clinical presentations. Although microbiologic culture is a standard method for detecting these pathogens, their recovery may require days to weeks, and the manipulation of cultures presents a significant safety hazard to laboratory personnel. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect and differentiate B. dermatitidis and H. capsulatum from culture isolates and directly from clinical specimens. Primers and fluorescence resonance energy transfer hybridization probes were designed to target the histidine kinase and glyceraldehyde-3-phosphate dehydrogenase genes of B. dermatitidis and H. capsulatum, respectively. The analytical sensitivity of the assay was determined to be 100 copies/l for both fungi. From culture isolates, the assay demonstrated 100% specificity and 100% sensitivity for B. dermatitidis and 100% specificity and 94% sensitivity for H. capsulatum. Detection directly from 797 clinical specimens demonstrated specificities and sensitivities of 99% and 86% for B. dermatitidis and 100% and 73% for H. capsulatum compared with the results for culture. This real-time PCR assay provides a rapid method for the detection of B. dermatitidis and H. capsulatum from culture isolates and directly from clinical specimens.

The Yeast Traffic Light PNA FISH kit (YTL) correctly identified Candida spp. in 207/216 (96%) positive blood cultures. Discordant results were seen with known cross-reacting species and cultures containing Candida lambica and Rhodotorula mucilaginosa. The YTL provides rapid, reliable identification of the five common Candida species found in blood cultures. Candida species are the leading cause of fungemia in both community-acquired and nosocomial infections (2). Two recent studies have shown that the use of PNA FISH probes to identify yeast in blood cultures decreases patient costs by directing appropriate antimicrobial therapy. Forrest et al. used the FDA-cleared Candida albicans PNA FISH kit (AdvanDx, Woburn, MA) for identification of C. albicans in 72 positive blood cultures (3). Rapid results allowed for a significant reduction in the use of caspofungin, with an overall cost savings of $1,729 per patient. In a similar study, Alexander and colleagues used a decision analytic model and showed an average savings of $1,837 per patient with the use of PNA FISH (1). Since the publication of these and other studies (6, 7, 9), additional probe kits have been cleared by the FDA, including the C. albicans/Candida glabrata PNA FISH kit and the Yeast Traffic Light PNA FISH kit (YTL).The performance of the C. albicans/C. glabrata PNA FISH kit has been previously reported (8). The YTL is a next-generation, three-probe system which is FDA cleared for the rapid identification of C. albicans/Candida parapsilosis, C. glabrata/Candida krusei and Candida tropicalis from positive blood cultures. Initially, the YTL was cleared using a 90-min probe hybridization step. Recently, a 30-min hybridization time was cleared, which further reduced the turnaround time to less than 2 h after the blood culture bottle signals as positive. In this study, we report the performance characteristics of the YTL kit using both the 90-and 30-min hybridization times. The reliability of the kit for the identification of the five claimed Candida species was evaluated, and the specificity of the probes was challenged using a wide variety of other yeast genera and species.Fifty-four blood cultures that were positive by Gram stain (44 yeast and 10 bacteria) were tested on the day of signal or were aliquoted and frozen at Ϫ20°C before testing as per the YTL product insert. In order to increase the number and diversity of yeasts tested, an additional 118 yeasts were inoculated into 5 ml of human red blood cells (Innovative Research, Navi, MI), and the simulated blood specimen was then placed into a Bactec MYCO/F lytic blood culture bottle (Becton Dickinson, Sparks, MD), incubated at 37°C, and tested with the YTL kit on the day that the bottle signaled positive. Subcultures of an additional 44 unusual yeast species grown on inhibitory mold agar (Becton Dickinson) were also tested for potential cross-reactivity of nontargeted yeast. The reference method for identification of the yeast used in the study was Sanger sequencing of the D2 large-subunit (LSU) RNA g...