Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing

Guidelines on diagnosis and management of Aspergillus disease recommend a multi-test approach including CT scan, culture, fungal biomarker tests, microscopy and fungal PCR. First-line treatment of confirmed invasive aspergillosis (IA) consists of drugs in the azole family; however, the emergence of azole-resistant isolates has negatively impacted the management of IA. Failure to detect azole-resistance dramatically increases mortality rates of azole-treated patients. Despite drug susceptibility tests not being routinely performed currently, we suggest including resistance testing whilst diagnosing Aspergillus disease. Multiple tools, including DNA sequencing, are available to screen for drug-resistant Aspergillus in clinical samples. This is particularly beneficial as a large proportion of IA samples are culture negative, consequently impeding susceptibility testing through conventional methods. Pyrosequencing is a promising in-house DNA sequencing method that can rapidly screen for genetic hotspots associated with antifungal resistance. Pyrosequencing outperforms other susceptibility testing methods due to its fast turnaround time, accurate detection of polymorphisms within critical genes, including simultaneous detection of wild type and mutated sequences, and most importantly it is not limited to specific genes nor fungal species. Here we review current diagnostic methods and highlight the potential of pyrosequencing to aid in a diagnosis complete with a resistance profile to improve clinical outcomes.