Fungal Molecular Diagnostics

Current Applications of D2 Fungal Sequencing at the Mayo Clinic

February 2011

Not every yeast or mold identification requires DNA sequencing. Many identifications are made quite easily and cost-effectively using traditional methods. Here at Mayo, we selectively utilize D2 DNA sequencing as a tool when traditional methods aren't providing a clear answer or when traditional methods require inordinate amounts of time. For example, it has been very helpful for some yeast that are difficult to identify using phenotypic methods such as the API20C assimilation test. Further, we have had great success using it to rapidly rule-out pathogens when fungi are persistently nonsporulating, as often occurs in the summer months. D2 sequencing has been very helpful for several cases of difficult to identify molds. And finally, D2 sequencing is extremely useful to confirm questionable nucleic acid hybridization probe results in instances where one may be considering a false-positive probe due to a cross-reacting organism. Where necessary, dermatophyte identification can be achieved an average of 9 days faster using sequencing as opposed to phenotypic methods.

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Current Applications of D2 Fungal Sequencing

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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