Novel qPCR demonstrates azole-resistant TR34/L98H and TR46/Y121F/T289A Aspergillus fumigatus, in air sporesamplings around Danish agricultural fields

Rasmus Krøger Hare *1, Thies Marten Heick 2, Lise Jørgensen 2, Maiken C. Arendrup 1;3;4

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1 Statens Serum Institut, Mycology Unit, Copenhagen, Denmark; 2 Aarhus University, Flakkebjerg Agricultural Sciences, Slagelse, Denmark; 3 Copenhagen University Hospital, Rigshospitalet, Clinical Microbiology Laboratory, Copenhagen, Denmark; 4 Copenhagen University, Department of Clinical Medicine, Copenhagen, Denmark


Background: Azole-resistant (AR) Aspergillus fumigatus is increasing worldwide and arise through medical and environmental azole use. Environmentally driven AR today accounts for the majority of AR cases in Denmark. We developed a novel Taqman qPCR for detection of the two dominating environmental AR mechanisms (TR34/L98H and TR46/Y121F/T289A) and applied the assay on spore-trap samples from air.

Materials/methods: Primers and probes were designed specifically for the promotor of cyp51A in A. fumigatus based on in-silico analysis covering >800 cyp51A Genbank sequences including >200 sequences from 44 Aspergillus non-fumigatus species. Three probes was included specific for A. fumigatus, TR34 and since 2018 also TR46. Sensitivity was evaluated by 10-fold dilution series of normalized DNA concentrations and by CFU experiments with spore suspensions subjected to bead-beating and Nuclisens easyMag (biomérieux, Denmark) DNA extraction. Environmental air-sampling was performed using Burkhard spore collectors, with tape-strips corresponding to 24-hour samplings. Three sampling-periods were included, 2012-2014 (N=512), 2016 (N=645) and 2018 (N=439), collected across Denmark. DNA was extracted from tape as previously described (Duvivier et al. 2013).

Results: Based on DNA quantification and CFU experiments, the limit of detection (LOD) was estimated to 50 fg (<20 copies) and 50-200 CFU/mL, respectively with no differences between the three probes (A. fumigatus, TR34 and TR46). A. fumigatus cyp51A was detected in 47 (9%), 87 (13.5%) and 122 (28%) samples in 2012-2014, 2016 and 2018, respectively. Increased detection rate may be correlated to improved DNA processing and continuous optimisation of the PCR assay. Two samples with TR34 were discovered in 2016, four TR34 in 2018 but none in 2012-2014 (Table). 

Conclusions: Environmental AR A. fumigatus was detected in 2016 and 2018 despite low content of A. fumigatus on tape samples. This is probably underrepresented as the AR spores constitute a minority among total A. fumigatus spores and thus potentially often below LOD. Although, this assay (single-copy gene target) displays lower sensitivity than diagnostic Aspergillus PCRs (multi-copy gene target) and lack extensive specificity testing, the assay offers a novel method for direct detection of TR34 and TR46 in clinical samples and for screening of environmentally driven azole resistance in complex specimens.

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European Congress of Clinical Microbiology and Infectious Diseases 2020
    • ECCMID 30th (2020)