Fungal Molecular Diagnostics

Histoplasma capsulatum/Blastomyces dermatitidis Real-Time PCR

February 2011

The last PCR assay that I will discuss briefly is a new one that will be introduced this year. That assay allows for the detection and identification of Histoplasma capsulatum and Blastomyces dermatitidis in a single tube either from culture isolates or directly from patient respiratory or tissue specimens. We recently submitted a manuscript describing the assay for peer-review evaluation. The use of distinctly labeled fluorophores on the FRET probes allows for the detection and differentiation of Histoplasma capsulatum and Blastomyces dermatitidis from a single tube using different detection wavelengths. We are very excited about this assay since we will be able to rapidly detect and distinguish between these 2 dimorphic pathogens that have overlapping areas of endemicity and that often can have overlapping clinical pictures.

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• 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