Development and validation of LAMP primer sets for rapid identification of Aspergillus fumigatus carrying the cyp51A TR46 azole resistance gene

Plinio Trabasso,Daisuke Hagiwara,Akira Watanabe,Yuzuru Mikami,Tetsuhiro Matsuzawa,Maria Luiza Moretti,Teppei Arai

doi:10.1038/s41598-021-96651-7

Abstract

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 tandem repeats of 34 bp or 46 bp within the promoter region of the cyp51A gene. Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid amplification system that is fast and specific. Here we describe a LAMP assay method to detect the 46 bp tandem repeat insertion in the cyp51A gene promoter region based on novel LAMP primer sets. It also differentiated strains with TR46 tandem repeats from those with TR34 tandem repeats. These results showed this TR46-LAMP method is specific, rapid, and provides crucial insights to develop novel antifungal therapeutic strategies against severe fungal infections due to A. fumigatus with TR46 tandem repeats.

Highlights

Infections due to triazole-resistant Aspergillus fumigatus are increasingly reported worldwide and are associated with treatment failure and mortality
There are several reports of fatal invasive aspergillosis caused by A. fumigatus carrying the TR46 in acute myeloid leukemia (AML) patients and hematopoietic stem cell transplant recipients[12]
We report a novel Loop-mediated isothermal amplification (LAMP) assay method that selectively detects triazole resistant A. fumigatus strains due to the presence of double TR46(TR462) or triple TR46(TR463)in the cyp51A promoter region

Summary

Introduction

Infections due to triazole-resistant Aspergillus fumigatus are increasingly reported worldwide and are associated with treatment failure and mortality. We describe a LAMP assay method to detect the 46 bp tandem repeat insertion in the cyp51A gene promoter region based on novel LAMP primer sets It differentiated strains with TR46 tandem repeats from those with TR34 tandem repeats. Resistance to azole antifungals can be due, in general, to two major genetic mechanisms; point mutation(s) in the cyp51A open reading frame with or without tandem repeat (TR) of 34 or 46 base pair (bp) in the promoter region of the gene ( TR34 or TR46) and overexpression of oligonucleotide sequence in the cyp51A gene[5]. A rapid and specific method to identify the presence of TR would contribute to faster therapeutic decision-making[14]

Methods

Results

Conclusion