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Vol. 31, No. 4
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1993, p. 845-850
0095-1137/93/040845-06$02.00/0 Copyright © 1993, American Society for Microbiology
Evaluation of Commercially Available Acridinium Ester-Labeled Chemiluminescent DNA Probes for Culture Identification of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum LESLIE STOCKMAN,' KATHLEEN A. CLARK,2 JOHN M. HUNT,1 AND GLENN D. ROBERTS'* Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905,1 and Gen-Probe, Inc., San Diego, California 921212 Received 31 July 1992/Accepted 28 December 1992
Four commercially available acridinium ester-labeled DNA probes directed against rRNA were evaluated for their ability to identify Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and Cryptococcus neoformans in culture. rRNA was extracted by sonication of 1- to 2-mm2 portions of cultures of fungi in two chaotropic reagents with glass beads. Following a heat inactivation step, the extracts were hybridized in solution with probes specific for each pathogen. The acridinium ester reporter moiety of nonhybridized probe was selectively hydrolyzed, and chemiluminescence of specific DNA:RNA hybrids was quantitated in relative light units with a luminometer. A positive identification required a relative light unit value of 250,000. Sensitivity and specificity of the probes were determined by probing cultures of the respective pathogenic fungi (target) and nontarget fungi. Both mycelial and yeast forms of the dimorphic fungi (B. dermatitidis and H. capsulatum) were tested. For B. dermatitidis, sensitivity and specificity were 87.8 and 100%, respectively (74 target and 219 nontarget fungi tested). For C. immitis, sensitivity and specificity were 99.2 and 100%, respectively (122 target and 164 nontarget fungi tested). For H. capsulatum, sensitivity and specificity were 100 and 100o, respectively (86 target and 154 nontarget fungi tested). For C. neoformans, sensitivity and specificity were 97 and 100o, respectively (100 target and 230 nontarget fungi tested). For B. dermatitidis, C. immitis, and C. neoformans, repeat testing increased the respective sensitivities to 97.3, 100, and 100%. The high sensitivities and specificities of the probes, the relatively short time (less than 1 h) required to perform the assay, and the availability of standardized reagent kits make the acridinium ester-labeled DNA probes well suited to laboratories in need of a rapid method to identify these fungal pathogens. Further, use of the probes to identify pathogenic fungi as soon as colonies appear on primary recovery media significantly shortens the time to reporting. reference isolates of fungi in the Mayo Clinic culture collection which were identified by conventional methods (5). Cultures varied in age at the time of testing, but all were less than 30 days old. Fungi were grown at 30°C on a variety of selective and nonselective media commonly used in a clinical microbiology laboratory, and the dimorphic fungi were converted to their yeast form at 35°C. Spherules of C. immitis were produced in Converse culture medium (2). Media included brain heart infusion agar containing 10% sheep blood, 16 ,ug of chloramphenicol per ml, and 5 ,ug of gentamicin per ml, with and without 0.5 mg of cycloheximide per ml; inhibitory mold agar; cottonseed agar; lactrimel agar; Middlebrook 7H10 agar; mycobiotic agar; Sabouraud dextrose agar (Emmon's modification); yeast extract agar; and yeast nitrogen base agar (5, 20). Methods for sample preparation and hybridization provided by the manufacturer were followed. Briefly, rRNA was extracted by sonication of 1- to 2-mm2 samples of fungal cultures in chaotropic reagents with glass beads for 15 min at 25°C. Following a heat inactivation step at 95°C for 15 min, the RNA extracts were hybridized for 15 min at 60°C in solution with probes specific for B. dermatitidis, C. immitis, H. capsulatum, or C. neoformans. The acridinium ester reporter moiety of nonhybridized probe was hydrolyzed in
Rapid molecular diagnostic methods for the detection and/or identification of prokaryotic and eukaryotic microorganisms based on unique rRNA sequences are being developed in rapidly increasing numbers (4, 6). Such probe-based methods are especially important for the dimorphic fungal pathogens, which often grow slowly and require an extended time for identification (10, 11). This report presents an evaluation of four nucleic acid probes designed by GenProbe, Inc. (San Diego, Calif.) for the culture identification of Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and Cryptococcus neoformans. (A portion of the information in this report was presented at the 90th Annual and 91st General Meetings of the American Society for Microbiology [17, 18].) MATERIALS AND METHODS
Commercially available AccuProbe culture identification reagent kits (Gen-Probe) designed for identification of four
fungal pathogens, B. dermatitidis, C immitis, H. capsulatum, and C. neoformans, were evaluated against target as well as nontarget organisms. These comprised clinical and *
Corresponding author. 845
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STOCKMAN ET AL. TABLE 1. Summary of AccuProbe results used for culture identification Result for probe:
Characteristic
C immitis
No. of target organisms (>50,000 RLU) No. of target organisms (
