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

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.