Development and validation of a quantitative real-time PCR assay for the early diagnosis of coccidioidomycosis.

Abstract

A new real-time polymerase chain reaction (RT-PCR) assay based on a Coccidioides genus-specific molecular beacon probe was developed for the detection of coccidioidomycosis and validated with tissues from animal models and clinical samples. The assay showed high analytic reproducibility (r(2) > 0.99) and specificity for cultured strains (100%); the lower limit of detection was 1 fg of genomic DNA/μL of reaction. Fungal burdens in the organs of mice infected with Coccidioides posadasii strain Silveira were more accurately quantified by RT-PCR compared to colony-forming unit for all tissues. The RT-PCR assay was positive for 97.7% of spleen and 100% of liver or lung. Progression of infection in all organs was similar by both methods (P > 0.05). The sensitivity of the assay also was 100% for paraffin-embedded samples and samples from patients with positive cultures. Our RT-PCR assay is effective for the diagnosis and monitoring of Coccidioides infection, and its use also avoids the biohazard and time delay of identifying cultures in the clinical setting.

• Authors: Sara Gago, María José Buitrago, Karl V Clemons, Manuel Cuenca-Estrella, Laurence F Mirels, David A Stevens
• Journal: Diagnostic microbiology and infectious disease (Diagn Microbiol Infect Dis) Vol. 79 Issue 2 Pg. 214-21 (Jun 2014) ISSN: 1879-0070 [Electronic] United States
• PMID: 24657173 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Validation Study)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Topics

• Animals
• Coccidioides (genetics, isolation & purification)
• Coccidioidomycosis (diagnosis)
• Colony Count, Microbial (methods)
• Early Diagnosis
• Female
• Humans
• Mice
• Molecular Diagnostic Techniques (methods)
• Real-Time Polymerase Chain Reaction (methods)
• Reproducibility of Results
• Sensitivity and Specificity