Development and Validation of LAMP Primer Sets for Rapid and Correct Identification of Aspergillus fumigatus Carrying the cyp51A TR46 Azole Resistance Gene

Infections due to triazole-resistant Aspergillus fumigatus are increasingly reported worldwide and are associated with treatment failure and mortality. The principal class of azole-resistant isolates is characterized by the presence of tandem repeats of 34 bp or 46 bp (TR34 or TR46) within the promoter region of the cyp51A gene. Loop-mediated isothermal amplification (LAMP) is a widely >used nucleic acid amplification system with high rapidity and specificity. In this paper, we report a new LAMP assay method to detect the 46 bp tandem repeat insertion in the cyp51A gene promoter region named TR46-LAMP assay, based on the use of newly designed specific LAMP primer sets. TR46 is a high-prevalence allele that is associated with the occurrence of multi-triazole resistance of A. fumigatus in patients as well as isolates from the environment. This newly designed TR46-LAMP assay was validated as a useful method for specific detection of azole-resistant A. fumigatus isolates bearing TR462 as well as TR463 in the cyp51A gene promoter region. It could also differentiate azole-resistant isolates of TR46 tandem repeats from those with TR34 tandem repeats in cyp51A genes. These results showed this TR46-LAMP method is specific, rapid, and also provides crucial insights to enable the development of novel antifungal therapeutic strategies against severe fungal infections due to A. fumigatus with TR46 tandem repeats.