Rapid detection of common pathogenic Aspergillus species by a novel real-time PCR approach

Invasive aspergillosis is a major cause of morbidity and mortality in immunocompromised and critically ill patients. Standard culture based methods for the diagnosis of Aspergillus infections have limited sensitivity and specificity and are time consuming. The recent availability of novel molecular based diagnostic techniques offers the potential of rapid, highly sensitive and specific pathogen detection. In this study, we aimed to develop a diagnostic assay to detect simultaneously common pathogenic Aspergillus species including Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus in whole blood specimen. A real-time PCR/probe set assay was designed to amplify the 18sRNA genomic region of Aspergillus. In addition to a previously probe set validated for the detection of A. fumigatus ('Asp.fum'), two other probe sets ('Asp.ter'; 'Asp.nig') were synthesised with 100% sequence homology to A. terreus or A. niger. Specificity testing demonstrated amplification of Aspergillus DNA, but not DNA from a panel of other common pathogenic fungi and bacteria and/or human DNA. Sensitivity testing in serial dilution assays revealed a lower detection limit of 1-5 CFU ml(-1) whole blood. However, no single probe set was most sensitive for all Aspergillus species tested and a combination of all three probe sets was necessary to achieve maximal overall assay sensitivity. Furthermore, inclusion of a subsequent probe melting temperature (Tm) analysis demonstrated species-specific Tm-patterns, useful to differentiate simultaneously between the different Aspergillus species detected. We describe a highly specific and sensitive real-time PCR approach to detect and differentiate the most common pathogenic Aspergillus species in a rapid 'same day' assay.