title: New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections.
abstract: The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFI diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real-time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time. In this work, a new panfungal real-time PCR assay using the combination of an intercalating dye and sequence-specific probes was developed. After DNA amplification, a melting curve analysis was also performed. The technique was standardized by using 11 different fungal species and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed by collecting those melting curves obtained from fungal species included in the standardization assay. Results showed high reproducibility (coefficient of variation [CV] < 5%; r > 0.95) and specificity (100%). The overall sensitivity of the technique was 83.3%, with the group of fungi involved in the infection detected in 77.8% of the positive samples with IFIs covered by molecular beacon probes. Moreover, sequencing was avoided in 67.8% of these "probe-positive" results, enabling report of a positive result in 24 h. This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.
authors.affiliation: Mycology Department, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Clara
authors.initials: C
authors.lastname: Valero
authors.affiliation: Mycology Department, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: de la Cruz-Villar
authors.affiliation: Mycology Department, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Óscar
authors.initials: Ó
authors.lastname: Zaragoza
authors.affiliation: Mycology Department, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain buitrago@isciii.es.
authors.forename: María José
authors.initials: MJ
authors.lastname: Buitrago
mesh_terms: D004271:DNA, Fungal / Q000235:genetics*; D005658:Fungi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D000072742:Invasive Fungal Infections / Q000175:diagnosis* / Q000382:microbiology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D004271:DNA, Fungal