title: RT-qPCR detection of Aspergillus fumigatus RNA in vitro and in a murine model of invasive aspergillosis utilizing the PAXgene® and Tempus™ RNA stabilization systems.
abstract: Molecular diagnosis of invasive aspergillosis (IA) is a potentially life-saving tool in the care of at-risk individuals. To date, the development of PCR-based diagnostic tests has been hampered by the lack of standardization in the methods for such critical activities. In this study, we used both spiked volunteer blood samples and a murine model of IA to test the utility of the PAXgene and Tempus systems for stabilization and isolation of fungal RNA from blood as part of an evaluation of a new diagnostic strategy. In spiking experiments, RNA isolation followed by RT-qPCR that targeted the 18S gene was compared to a standard DNA isolation and qPCR assay that targeted the ITS ribosomal region. We demonstrated that both PAXgene and Tempus RNA stabilization and extraction systems followed by RT-qPCR had similar performance in detecting fungal RNA in blood samples from Aspergillus fumigates-infected mice. In spiked samples, the Tempus system performed better than the PAXgene system as it detected 100% of all samples spiked with 10 or 20 germinated Aspergillus conidia/ml blood sample as compared to the PAXgene system which detected 33% and 56% of the samples spiked with 10 or 20 conidia/ml, respectively. The stabilization of fungal nucleic acids in blood samples and its efficient isolation by a commercial method is an important step in the development of standardized molecular diagnostic tools that are needed to improve the outcomes for individuals with IA.
authors.affiliation: Department of Clinical Microbiology, Sir Patrick Dun Research Laboratory, Trinity College Dublin, St James's Hospital, Dublin, Ireland. o.morton@uws.edu.au
authors.forename: Charles Oliver
authors.initials: CO
authors.lastname: Morton
authors.forename: Antonella
authors.initials: A
authors.lastname: de Luca
authors.forename: Luigina
authors.initials: L
authors.lastname: Romani
authors.forename: Thomas Richard
authors.initials: TR
authors.lastname: Rogers
mesh_terms: D000818:Animals; D001228:Aspergillosis / Q000175:diagnosis* / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D004271:DNA, Fungal / Q000097:blood / Q000235:genetics; D005800:Genes, Fungal; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D020871:RNA Stability; D012331:RNA, Fungal / Q000097:blood* / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000032:analysis* / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013172:Spores, Fungal / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D012331:RNA, Fungal; D012337:RNA, Ribosomal, 18S; D011933:Reagent Kits, Diagnostic