Rapid identification of fungal pathogens: Molecular approaches for a new millennium

The increasing prevalence of invasive fungal disease in immunocompromised patients is confounded by inadequate methods for pathogen detection. Molecular methods have been developed to address this problem because conventional methods for pathogen identification often lack sensitivity, specificity and speed, and some infectious organisms are difficult to culture. Nucleic acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional methods by taking advantage of genus- and species-specific DNA sequence variations. Numerous methods have been employed that exploit both large- and small-scale genomic differences. Yet, polymerase chain reaction (PCR)-based amplification and analysis of DNA sequence polymorphisms in the internal transcribed spacer 2 region of conserved ribosomal (r)RNA genes is currently the most reliable approach for fungal identification. When combined with fluorescence-based oligonucleotide detection systems, such as TaqMan and Molecular Beacons, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination. These probe systems offer rapid turn around time (< 2 h) and are suitable for high throughput, automated, multiplex-type operations that are critical for clinical diagnostic laboratories.