Fungal Molecular Diagnostics

Mass Spectrometric Identification Of Fungi

February 2011

One technology that is rapidly entering the clinical diagnostic arena today is mass spectrometry. Once the purview of chemists, mass spectrometry is rapidly finding in-roads into microbiology in the areas of bacteria and even fungal identification. In a recent publication by Stevenson et al at the National Institutes of Health, matrix-assisted, laser desorption ionization coupled with time of flight detection (or MALDI-TOF) mass spectrometry was utilized to identify 23 species of yeast from 6 different genera. Why would you want to throw a "big gun" like MALDI-TOF at yeast identification? Well, for one thing, MALDI-TOF uses very few reagents and consumables so it's a very "green" technology that can be done quite cheaply in terms of reagent and FTE costs. Equipment cost is also reasonable. It's rapid to perform, requiring only a few minutes from isolate to answer and it's technically simple for the technologist. Further, it appears to have wide applicability as a number of groups have reported success with bacterial identification using this approach.

We recently evaluated the ability of mass spectrometry to rapidly identify yeast in our laboratory and concur with the findings of Stephenson et al. We anticipate that MALDI-TOF will allow us to replace several slower phenotypic tests that we currently use for yeast identification at a comparable overall cost. What else does the future hold? It's exciting to think about the possibilities offered by technologies such as next generation sequencing but, at this time, the technology is too expensive and lacks the data analysis tools necessary to make it routinely applicable in clinical microbiology. Time will tell if this or other molecular methods continue to make in-roads into the highly traditional field of diagnostic mycology.

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Mass Spectrometric Identification Of Fungi

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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