title: Detection and measurement of fungal burden in a guinea pig model of invasive pulmonary aspergillosis by novel quantitative nested real-time PCR compared with galactomannan and (1,3)-β-D-glucan detection.
abstract: We developed and assessed the diagnostic value of a novel quantitative nested real-time (QNRT) PCR assay targeting the internal transcribed spacer region of ribosomal DNA (rDNA) in a guinea pig model of invasive pulmonary aspergillosis. Groups of 5 immunosuppressed animals that were infected using an aerosol chamber with Aspergillus fumigatus conidia were humanely terminated 1 h postinoculation and at days 3, 5, 7, and 11 postchallenge, and lung tissue, bronchoalveolar lavage (BAL) fluid, whole blood, and serum samples were collected. The QNRT PCR results obtained with the serum and BAL fluid were compared to those achieved with galactomannan and (1→3)-β-d-glucan assays. High fungal burden levels were detected by QNRT PCR in both lung tissue and BAL fluid in all infected animals at each time point, and the sensitivity of each assay in BAL fluid was 100% by day 3 and remained so through the remainder of the study. The sensitivity of detection of fungi in whole blood and serum samples was significantly lower, and some samples remained negative by all three assays despite the advanced stage of the infection. From these data, we can conclude that this novel QNRT PCR method was highly sensitive for the detection of A. fumigatus from different types of samples in this model. In addition, BAL fluid samples appeared to be the most suitable for the early diagnosis of invasive pulmonary aspergillosis. When testing serum, the use of a combination of available assays may increase the possibility of early detection of this opportunistic mycosis.
authors.affiliation: Department of Internal Medicine—Hematooncology, Faculty of Medicine of Masaryk University and University Hospital Brno, Brno, Czech Republica. mlengerova@fnbrno.cz
authors.forename: Martina
authors.initials: M
authors.lastname: Lengerova
authors.forename: Iva
authors.initials: I
authors.lastname: Kocmanova
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Racil
authors.forename: Kristyna
authors.initials: K
authors.lastname: Hrncirova
authors.forename: Sarka
authors.initials: S
authors.lastname: Pospisilova
authors.forename: Jiri
authors.initials: J
authors.lastname: Mayer
authors.forename: Laura K
authors.initials: LK
authors.lastname: Najvar
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Wiederhold
authors.forename: William R
authors.initials: WR
authors.lastname: Kirkpatrick
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Patterson
mesh_terms: D000818:Animals; D001232:Aspergillus fumigatus / Q000302:isolation & purification*; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D015169:Colony Count, Microbial / Q000379:methods; D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D004195:Disease Models, Animal; D005690:Galactose / Q000031:analogs & derivatives; D006168:Guinea Pigs; D055744:Invasive Pulmonary Aspergillosis / Q000382:microbiology* / Q000473:pathology; D008168:Lung / Q000382:microbiology; D008297:Male; D008351:Mannans / Q000032:analysis*; D009172:Mycology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011509:Proteoglycans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D047071:beta-Glucans / Q000032:analysis*
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D008351:Mannans; D011509:Proteoglycans; D047071:beta-Glucans; C012990:galactomannan; C010770:polysaccharide-K; D005690:Galactose