Fungal Molecular Diagnostics

Fungal PCR Assays at Mayo Clinic

February 2011

Now that we have discussed the utility of nucleic acid hybridization probes, DNA sequencing, and PNA FISH probes for assisting in identification of fungi growing in culture, let’s switch gears and discuss molecular methods available for the direct detection of fungi in clinical specimens without the need for culture. Currently this is a very, very short list of methods consisting of a handful of laboratory-developed PCR assays that target specific fungi. In our laboratory, we have 2 of these real-time PCR assays that have been used routinely for several years. The first assay provides for the direct detection of Pneumocystis jiroveci from respiratory specimens such as bronchoalveolar lavage fluid and respiratory tissues. The second assay allows for the direct detection of Coccidioides immitis and Coccidioides posadasii from respiratory specimens, body fluids, and tissues. Within the next few months, we will begin offering a third real-time PCR assay that provides a single tube assay for detection and differentiation of Histoplasma capsulatum and Blastomyces dermatitidis from respiratory specimens, body fluids, and tissue.

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Jump to section:

• Introduction
• Objectives
• Traditional Methods of Fungal Diagnostics
• Traditional Methods
• Potential Advantages of Fungal Molecular Diagnostics
• Currently Available Molecular Diagnostics for Fungi
• Nucleic Acid Hybridization Probes³
• Nucleic Acid Hybridization Probes
• Nucleic Acid Hybridization Probes for Fungi
• Nucleic Acid Hybridization Probes
• DNA Sequencing for Fungi
• Ribosomal Structure: Fungi
• Traditional Sequencing Workflow
• Sequence Analysis
• Current Applications of D2 Fungal Sequencing at the Mayo Clinic
• Rapid, Direct Identification of Yeast from Blood Cultures
• PNA-FISH Basics
• PNA FISH for Candida spp—Direct Detection from Blood
• Yeast Identification Timeline from Positive Blood Bottle
• Potential Advantages of Direct Detection
• Fungal PCR Assays at Mayo Clinic
• Real-time PCR Workflow
• Pneumocystis PCR
• Pneumocystis LC-PCR Melt Curves
• Concordance of the Pneumocystis LightCycler PCR Assay and Calcofluor White Stain for BAL Fluid
• Coccidioides species Real-time PCR
• Melt-curve Analysis of Coccidioides spp Grown in Culture
• Utility of Coccidioides PCR Testing in the Clinical Setting⁴
• Meningeal Coccidioidomycosis Diagnosed by Real-Time PCR Analysis of Cerebrospinal Fluid⁵
• Histoplasma capsulatum/Blastomyces dermatitidis Real-Time PCR
• What lies in the future for fungal molecular diagnostics?
• Mass Spectrometric Identification Of Fungi
• Summary
• References
• Questions?
• Disclosure