Azole-Resistant Aspergillus fumigatus Among Danish Cystic Fibrosis Patients: Increasing Prevalence and Dominance of TR34/L98H

Abstract

Azole-resistant (azole-R) Aspergillus is an increasing challenge worldwide. Patients with cystic fibrosis (CF) are at risk of Aspergillus colonization and disease due to a favorable lung environment for microorganisms. We performed a nationwide study in 2018 of azole-non-susceptible Aspergillus in CF patients and compared with data from two prior studies. All airway samples with mold isolates from patients monitored at the two CF centers in Denmark (RH, Jan-Sept and AUH, Jan-Jun) were included. Classical species identification (morphology and thermo-tolerance) was performed and MALDI-TOF/β-tubulin sequencing was performed if needed. Susceptibility was determined using EUCAST E.Def 10.1, and E.Def 9.3.2. cyp51A sequencing and STRAf genotyping were performed for azole-non-susceptible isolates and relevant sequential isolates. In total, 340 mold isolates from 159 CF patients were obtained. The most frequent species were Aspergillus fumigatus (266/340, 78.2%) and Aspergillus terreus (26/340, 7.6%). Azole-R A. fumigatus was cultured from 7.3% (10/137) of patients, including 9.5% (9/95) of patients at RH and 2.4% at AUH (1/42), respectively. In a 10-year perspective, azole-non-susceptibility increased numerically among patients at RH (10.5% in 2018 vs 4.5% in 2007-2009). Cyp51A resistance mechanisms were found in nine azole-R A. fumigatus from eight CF patients. Five were of environmental origin (TR 34 /L98H), three were human medicine-driven (two M220K and one M220R), and one was novel (TR 34 3 /L98H) and found in a patient who also harbored a TR 34 /L98H isolate. STRAf genotyping identified 27 unique genotypes among 45 isolates and ≥2 genotypes in 8 of 12 patients. This included one patient carrying two unique TR 34 /L98H isolates, a rare phenomenon. Genotyping of sequential TR 34 3 /L98H and TR 34 /L98H isolates from the same patient showed only minor differences in 1/9 markers. Finally, azole-R A. terreus

Azole Resistance in Aspergillus fumigatus: A Five-Year Follow Up Experience in a Tertiary Hospital With a Special Focus on Cystic Fibrosis

“…In Denmark, Risum et al. recently reported the occurrence of AR Af strains in 7.3% (10/137) CF patients ( Risum et al., 2020 ).…”

“…A global rate of resistance of A. fumigatus is however a metric with little significance, especially if we do not take into account variability between patients and drugs. During CF, particularly-high rates of AR Af have been reported ( Stevens et al., 2003 ; Mortensen et al., 2011 ; Burgel et al., 2012 ; Morio et al., 2012 ; Meis et al., 2016 ; Prigitano et al., 2017 ; Risum et al., 2020 ; Wiederhold and Verweij, 2020 ) while azoles are recommended in the guidelines for the management of allergic bronchopulmonary aspergillosis (ABPA) ( Stevens et al., 2003 ).…”

The Medical Triazole Voriconazole Can Select for Tandem Repeat Variations in Azole-Resistant Aspergillus Fumigatus Harboring TR34/L98H Via Asexual Reproduction

“…Our laboratory observations support the recent report of recovery of genetically closely related TR 34 3 /L98H and TR 34 /L98H isolates from a cystic fibrosis patient. The patient had been treated with VOR and POS (MC Arendrup, personal communication), which could have selected TR 34 3 /L98H harboring A. fumigatus strains in the lung [ 9 ]. However, the TR 34 /L98H patient isolate was recovered after the isolates harboring TR 34 3 /L98H and no ancestral isolate was available [ 9 ], therefore it is unclear whether the TR 34 3 -variant evolved in the host under VOR and POS selection from a TR 34 or vice versa.…”

“…Recently a TR 34 3 /L98H variant was recovered from a cystic fibrosis patient who also harbored a TR 34 /L98H isolate. Microsatellite typing showed that the TR 34 3 /L98H and TR 34 /L98H shared eight of nine markers, which supports possible in vivo acquisition of an additional TR [ 9 ]. Apparently not the resistance selection route, but other factors such as chemical characteristics of the azole, the reproduction mode the fungus goes through and azole stress duration might determine which resistance mutation is selected for [ 10 ].…”

“…TR-mediated resistance is highly dominant, especially TR 34 , accounting for up to 70% of resistance mutations in surveillance studies [ 11 ]. Furthermore, over time increased variation in TRs is observed with both increasing TR length, i.e., TR 34 , TR 46 , TR 53 , and TR 120 , as well as increasing numbers of TR-duplications, i.e., TR 46 , TR 46 3 and TR 46 4 [ 5 , 12 , 13 ] and TR 34 and TR 34 3 [ 9 ].The repeated promoter sequence in the variants TR 46 , TR 53 , TR 120 TR 46 3 , and TR 46 4 all contain the repeated sequence of TR 34 . It has been shown that the repeat sequence in TR 34 is bound by both the sterol regulatory element binding protein SrbA, and the CCAAT binding complex (CBC).…”