Rapid detection and identification of Aspergillus from lower respiratory tract specimens by use of a combined probe-high-resolution melting analysis.

ABSTRACT

Diagnosis of invasive aspergillosis (IA) requires increasingly rapid molecular methods that enable sensitive detection and discrimination between species. We designed and evaluated a real-time PCR-based method that combined melting temperature (T(m)) calling analysis of a specific probe with high-resolution melting analysis of the full amplicon. The test correctly identified 78 isolates of Aspergillus section Fumigati and non-Fumigati sections of Aspergillus with a limit of detection of 10(2) conidia/ml (10(2) fg/ml). No cross-reactivity with other fungi was found. The assay was further validated on lower respiratory tract specimens containing Aspergillus or not. It successfully identified Aspergillus to section level in 56 of 59 specimens. With culture as the gold standard, our assay shows 100% sensitivity and specificity and constitutes an efficient alternative for identification of Aspergillus in lower respiratory tract samples.