Evaluation of novel broad-range real-time PCR assay for rapid detection of human pathogenic fungi in various clinical specimens

Abstract

In the present study, a novel broad-range real-time PCR was developed for the rapid detection of human pathogenic fungi. The assay targets a part of the 28S large-subunit ribosomal RNA (rDNA) gene. We investigated its application for the most important human pathogenic fungal genera, including Aspergillus, Candida, Cryptococcus, Mucor, Penicillium, Pichia, Microsporum, Trichophyton, and Scopulariopsis. Species were identified in PCR-positive reactions by direct DNA sequencing. A noncompetitive internal control was applied to prevent false-negative results due to PCR inhibition. The minimum detection limit for the PCR was determined to be one 28S rDNA copy per PCR, and the 95% detection limit was calculated to 15 copies per PCR. To assess the clinical applicability of the PCR method, intensive-care patients with artificial respiration and patients with infective endocarditis were investigated. For this purpose, 76 tracheal secretion samples and 70 heart valve tissues were analyzed in parallel by real-time PCR and cultivation. No discrepancies in results were observed between PCR analysis and cultivation methods. Furthermore, the application of the PCR method was investigated for other clinical specimens, including cervical swabs, nail and horny skin scrapings, and serum, blood, and urine samples. The combination of a broad-range real-time PCR and direct sequencing facilitates rapid screening for fungal infection in various clinical specimens.

FIG. 1.

Amplification plots for various specimens inoculated with 10⁶ CFU/ml of C. albicans. Nucleic acids were extracted from urine (thin solid lines), serum (dotted lines), EDTA-anticoagulated blood (dashed lines), and TS (dashed-dotted lines) samples inoculated with 10⁴ to 10⁶ CFU/ml of C. albicans (ATCC 10231) as described in Materials and Methods. Nucleic acids were analyzed in triplicate by 28S rDNA real-time PCR. Amplification plots for specimens inoculated with 10⁶ CFU/ml are shown, and the C_T for 28S rDNA (upper panel) and the IC (lower panel) are arranged tabularly. Thick solid lines indicate negative controls (noninoculated specimen).