title: Development and validation of a quantitative PCR assay for diagnosis of scedosporiosis.
abstract: Scedosporium apiospermum and Scedosporium prolificans are fungal pathogens that can cause severe human infections, including disseminated mycosis in immunocompromised patients. Two real-time PCR (RT-PCR) assays for the diagnosis of these species were developed and validated for the classification of clinical strains and for the detection of DNA in clinical samples by use of a murine model of invasive infection. A total of 14 clinical strains and 141 samples, including blood, serum, and lung samples from infected CD1 mice, were analyzed. Each RT-PCR methodology used a species-specific molecular beacon probe targeting a highly conserved region of the fungal ribosomal DNA gene. Results showed 100% specificity and a detection limit of 10 fg of DNA for both assays. The sensitivities for the S. prolificans-specific PCR assay were 100% for cultured clinical strains, 95.5% for lung tissues, 85% for serum, and 83.3% for blood. For S. apiospermum, the sensitivities were 100% for clinical strains and 97.2%, 81.8%, and 54.5% for lung tissues, serum, and blood, respectively. Both techniques can be useful for clinical diagnosis, and further studies are warranted.
authors.affiliation: Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Spain.
authors.forename: Maria V
authors.initials: MV
authors.lastname: Castelli
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Buitrago
authors.forename: Leticia
authors.initials: L
authors.lastname: Bernal-Martinez
authors.forename: Alicia
authors.initials: A
authors.lastname: Gomez-Lopez
authors.forename: Juan L
authors.initials: JL
authors.lastname: Rodriguez-Tudela
authors.forename: Manuel
authors.initials: M
authors.lastname: Cuenca-Estrella
mesh_terms: D000818:Animals; D001769:Blood / Q000382:microbiology; D004271:DNA, Fungal / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D008168:Lung / Q000382:microbiology; D008297:Male; D051379:Mice; D009181:Mycoses / Q000175:diagnosis*; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D021681:Scedosporium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D016019:Survival Analysis
chemical_list: D004271:DNA, Fungal; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes