Fungal Molecular Diagnostics

Coccidioides species Real-time PCR

February 2011

Another PCR assay that has been very useful in our laboratory is the Coccidioides species real-time PCR assay, which can detect both Coccidioides immitis and Coccidioides posadasii in respiratory specimens and tissue. This assay was developed to decrease the time required for detection the organism relative to the time required for growth of the organism in culture, which can be 10 or more days. The PCR assay is rapid compared with culture, is more sensitive and specific than serology, and has the additional advantage of providing improved safety for laboratory workers since they spend less time manipulating growing cultures of this highly infectious mold. We utilize the Coccidioides PCR assay in several ways. It is used for the rapid identification of Coccidioides from organisms grown in culture. This provides us with another method of identification should the nucleic acid hybridization probe ever experience availability problems, plus it obviates the need for the lab to keep a growing culture of Coccidioides in the laboratory as a positive probe control. Continuous culture of Coccidioides is a problem for laboratories because not only is it a safety hazard for lab personnel, but it is a regulatory headache since Coccidioides species are listed as Select Agents by the Centers for Disease Control and Prevention Select Agent program. Finally, the PCR assay has been extremely useful for the direct detection of Coccidioides species from patient respiratory, body fluid, and tissue specimens where it can provide a rapid turnaround time without the need to wait for a culture to grow.

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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