Azole-Resistant Aspergillus fumigatus Harboring the TR34/L98H Mutation: First Report in Portugal in Environmental Samples

Abstract

Introduction: The frequency in detection of azole-resistant Aspergillus fumigatus isolates has increased since 2010. In Portugal, the section Fumigati is one of the most frequent, and resistant strains to have been found in clinical and environmental contexts. Although several cryptic species within the Fumigati section show intrinsic resistance to azoles, one factor driving (acquired) resistance is selective pressure deriving from the extensive use of azoles. This is particularly problematic in occupational environments where high fungal loads are expected, and where there is an increased risk of human exposure and infection, with impact on treatment success and disease outcome. The mechanisms of resistance are diverse, but mainly associated with mutations in the cyp51A gene. Despite TR34/L98H being the most frequent mutation described, it has only been detected in clinical specimens in Portugal. Methods: We analyzed 99 A. fumigatus isolates from indoor environments (healthcare facilities, spas, one dairy and one waste sorting unit) collected from January 2018 to February 2019 in different regions of Portugal. Isolates were screened for resistance to itraconazole, voriconazole and posaconazole by culture, and resistance was confirmed by broth microdilution. Sequencing of the cyp51A gene and its promoter was performed to detect mutations associated with resistance. Results: Overall, 8.1% of isolates were able to grow in the presence of at least one azole, and 3% (isolated from the air in a dairy and from filtering respiratory protective devices in a waste sorting industry) were pan-azole-resistant, bearing the TR34/L98H mutation. Conclusion: For the first time in Portugal, we report environmental isolates bearing the TR34/L98H mutation, isolated from occupational environments. Environmental surveillance of the emergence of azole-resistant A. fumigatus sensu stricto strains is needed, to ensure proper and timely implementation of control policies that may have a positive impact on public and occupational health.

Aspergillus Section Fumigati in Firefighter Headquarters

“…The identification of the Aspergillus section Fumigati through passive and active sampling has already been reported [4,[34][35][36][37][38]. However, other studies developed in Portuguese occupational environments presented a lower prevalence of Aspergillus sp.…”

Cytotoxicity of Aspergillus Section Fumigati Isolated from Health Care Environments

“…Unfortunately, the section Fumigati was classified based on macroscopic and microscopic characteristics and because of that it was impossible to identify the species among the section. However, in previous studies held by the same team also with environmental isolates from different indoor environments, results revealed a good correlation between phenotypic and molecular identification [19,64]. Further studies should comprehend molecular identification of Aspergillus isolates and cytotoxicity analyses.…”

“…Malt extract agar (MEA) supplemented with chloramphenicol (0.05%), and dichloranglycerol agar (DG18) were used to increase selectivity for fungal growth. Sabouraud dextrose agar (SDA) and SDA supplemented with 4 mg/L itraconazole (ITR), 1 mg/L voriconazole (VOR) or 0.5 mg/L posaconazole (POS) were used for screening of azole resistance (adapted from EUCAST 2018) and following the procedures already reported [5,18,19]. The reference strain A. fumigatus ATCC 204,305 was used as a negative control and a pan-azoleresistant strain was used as a positive control (both kindly provided by Reference Unit for Parasitic and Fungal Infections, Department of Infectious Diseases of the National Institute of Health, from Dr. Ricardo Jorge).…”

“…Of note, 17 Aspergillus section Fumigati isolates (88.2% from environmental sampling) were able to grow in at least one azole (4 mg/L ITR), including 7 isolates from environmental samples (mostly air impaction) that were able to grow in two different azoles (4 mg/L ITR, and 1 mg/L VOR), of which 4 were able to grow in the three tested azoles (4 mg/L ITR, 1 mg/L VOR, and 0.5 mg/L POS) [5,19,27,28]. Regarding nasal samples, two Aspergillus section Fumigati isolates were able to grow in 4 mg/L ITR, one of which was from PHCC staff, and the other from controls.…”

“…The induction of immunosuppression and inflammation by exposure to fungi and bioaerosols is also described [13,14]. An additional concern regarding A. fumigatus sensu stricto is the emergence of acquired azole-resistance in clinical practice and in the environment [10,[15][16][17][18][19].…”