P001 Characteristics and dynamics of azole-resistant Aspergillus fumigatus variants emerging over a 28-year period in the Netherlands
Authors: Song Yinggai , Buil Jochem B , Melchers Willem J G , Zoll Jan , Verweij Paul E 
Published: September 2022

Abstract

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM

Background
Aspergillus fumigatus, a globally distributed opportunistic pathogen, is the main cause of invasive aspergillosis, especially in immunocompromised patients with high mortality. The emergence of azole-resistant A. fumigatus isolates has been a significant concern worldwide and an important clinical problem.

Objectives
We aim to determine the presence of variants in a large collection of clinical A. fumigatus isolates from the Netherlands, if the number of variants increased over time and if the presence of additional short nucleotide polymorphisms (SNPs) or tandem repeats (TR) variations impacted on the triazole phenotype.

Methods
The Radboud University Medical Center has collected 11 813 clinical A. fumigatus isolates since 1994. The collection includes isolates cultured from patients admitted to our own center, isolates sent from other hospitals for identification and in vitro susceptibility testing, and isolates sent from five university medical centers and five teaching hospitals that contribute to the national Aspergillus resistance surveillance. The genotypes were detected by Cyp51A Sanger sequencing. All isolates were subjected to in vitro susceptibility testing using the EUCAST microdilution reference method. Minimal inhibitory concentrations (MICs) were determined for itraconazole, voriconazole, posaconazole, in all isolates and for isavuconazole in isolates cultured in 2015 and thereafter.

Results
In total, 1826 A. fumigatus isolates harbored azole-resistant mutations in the Cyp51A-gene with 92 genotypes. Tandem Repeat-associated resistance genotypes accounted for 55.43% of the variants and were involved in 1728 isolates (94.63%). TR34/L98H and TR46/Y121F/T289A resistance mutations remained dominant, and increasingly additional SNPs in the Cyp51A-gene or changes to the gene promoter were observed. The G448S mutation was relatively common and present in various genetic backgrounds. This SNP was most often found in isolates harboring the TR46 resistance mechanism (8 variants) and was also observed in two variants in the TR34 genetic background. TR34 and TR46 resistance mutations are associated with 1170 (64.07%) isolates that exhibited a pan-azole resistance phenotype, 547 (29.96%) a multi-azole resistance phenotype, and 75 (4.11%) resistance to a single azole. TR34/L98H confers high itraconazole resistance, while T289A confers high voriconazole resistance in the TR46 background. Isolates with a G448S point mutation show high MICs for both voriconazole and itraconazole. The TR34/L98H/T289A/G448S isolate showed low itraconazole MICs but high voriconazole resistance, and mutations in the promoter region, TR34/C-86 G/L98H, and (T-66 G)/TR34/L98H variants, showed increased voriconazole and isavuconazole MIC compared with the parent phenotype. TR46/Y121F/M172I/T289A/G448S variant was observed with an increased itraconazole (GM MIC 16 mg/L, 1→16 mg/l) and decreased voriconazole (GM MIC 18.664 mg/l, 4→16 mg/l) compared with the parent MIC of TR46/Y121F/T289A, while TR92/Y121F/M172I/T289A/G448S and TR46/Y121F/ T289A/G448S variants showed the consistent MIC distribution with parent genotype. The variants with more combination mutations showed pan-azole resistance with increased MIC distribution.

Conclusion
Our survey showed a significant increase in resistance genotypes in clinical A. fumigatus over a period of 28 years. Azoles resistance phenotypes vary from resistant variants in clinical isolates; it is an implication for clinical A. fumigatus infection treatment options and antifungal stewardship.