title: Detection of fungal DNA by real-time polymerase chain reaction: evaluation of 2 methodologies in experimental pulmonary aspergillosis.
abstract: The capabilities of 2 quantitative polymerase chain reaction (PCR) assays for detecting pulmonary aspergillosis were analyzed. Both methodologies were real-time (RT) based and were compared with quantitative cultures and galactomannan (GM) antigen detection in a rabbit model of invasive aspergillosis. A total of 106 samples including blood, serum, lung, and brain from 3 controls and 9 infected New Zealand rabbits were analyzed. The RT-PCR methodologies were an Aspergillus fumigatus-specific assay using fluorescent resonance energy transfer technology targeting a highly conserved region of the fungal 18S rRNA gene and a panfungal assay to amplify the internal transcribed spacer regions 1 and 2 from fungal rRNA gene complex, employing SYBRGreen fluorescent dye as a detector. The specificity for both PCR base assays, culture, and GM determination was 100%. The sensitivity of the specific PCR assay was 88.9% in lung samples, 66.6% in serum, 55.5% in blood, and 33.3 in brain specimens. The panfungal assay had a sensitivity of 33.3% in lung and serum samples, being brain and blood specimens invariably negative. Otherwise, 100% of the lungs resulted positive for culture, and all serum samples showed a GM index above 1.0 after 2 days of infection. The specific RT-PCR assay is a reliable technique to detect A. fumigatus DNA in vivo comparable to cultures and GM determination. The panfungal RT-PCR assay exhibited low sensitivity to diagnose invasive aspergillosis in rabbits advising against its clinical introduction.
authors.affiliation: Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Gomez-Lopez
authors.forename: Mayte T
authors.initials: MT
authors.lastname: Martin-Gomez
authors.forename: Pilar
authors.initials: P
authors.lastname: Martin-Davila
authors.forename: Pedro
authors.initials: P
authors.lastname: Lopez-Onrubia
authors.forename: Joan
authors.initials: J
authors.lastname: Gavalda
authors.forename: Jesus
authors.initials: J
authors.lastname: Fortun
authors.forename: Albert
authors.initials: A
authors.lastname: Pahissa
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; D001228:Aspergillosis / Q000175:diagnosis*; D001232:Aspergillus fumigatus / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D008172:Lung Diseases, Fungal / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012331:RNA, Fungal / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D011817:Rabbits; D012680:Sensitivity and Specificity
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D012331:RNA, Fungal; D012337:RNA, Ribosomal, 18S