Fungal Molecular Diagnostics

Traditional Methods of Fungal Diagnostics

February 2011

Before we begin discussing the use of molecular diagnostics, let us recall that until very recently, the methods used by mycologists for the identification of fungi in the laboratory have been largely unchanged for decades. Once received in the laboratory, a patient specimen will typically be plated onto several different types of media and these media will be incubated at a constant temperature for up to 4 weeks. Laboratory staff will periodically examine the plates for growth and when growth is noted, the staff will try to identify the fungus using both macroscopic and microscopic morphologies. Often, subculture of the fungus to other types of media, followed by additional incubation time may be required in order to induce sporulation or the development of characteristic structures that can be used in making a visual identification. If identification cannot be made based on morphology alone, there are a few biochemical tests that can sometimes be helpful in identification, most often for yeast.

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Traditional Methods of Fungal Diagnostics

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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