title: 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.affiliation: Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Gago
authors.affiliation: Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: María José
authors.initials: MJ
authors.lastname: Buitrago
authors.affiliation: California Institute for Medical Research, San Jose, CA, USA; Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA, USA; Department of Medicine, Stanford University, Stanford, CA, USA. Electronic address: Clemons@cimr.org.
authors.forename: Karl V
authors.initials: KV
authors.lastname: Clemons
authors.affiliation: Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Cuenca-Estrella
authors.affiliation: California Institute for Medical Research, San Jose, CA, USA; Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA, USA.
authors.forename: Laurence F
authors.initials: LF
authors.lastname: Mirels
authors.affiliation: California Institute for Medical Research, San Jose, CA, USA; Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA, USA; Department of Medicine, Stanford University, Stanford, CA, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Stevens
mesh_terms: D000818:Animals; D003045:Coccidioides / Q000235:genetics / Q000302:isolation & purification*; D003047:Coccidioidomycosis / Q000175:diagnosis*; D015169:Colony Count, Microbial / Q000379:methods; D042241:Early Diagnosis; D005260:Female; D006801:Humans; D051379:Mice; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
keywords: Coccidioidomycosis; Diagnosis; RT-PCR