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. 2020 Mar 13;63(5):430–436. doi: 10.1111/myc.13064

First azole‐resistant Aspergillus fumigatus isolates with the environmental TR46/Y121F/T289A mutation in Iran

Fatemeh Ahangarkani 1,2, Ynze Puts 2, Mojtaba Nabili 3, Sadegh Khodavaisy 4, Maryam Moazeni 5,6, Zahra Salehi 7, Melika Laal Kargar 7, Hamid Badali 5,6,8, Jacques F Meis 2,9,✉
PMCID: PMC7217147  PMID: 32056319

Summary

Background

Azole resistance in Aspergillus fumigatus is an emerging problem and reported from all continents. As triazole antifungals are the mainstay of therapy in the management of invasive aspergillosis, azole‐resistant A fumigatus has become a major medical concern and with complicated clinical management.

Objective

Screening of environmental presence of azole‐resistant A fumigatus in Iran.

Methods

Compost from Northern Iran, collected between 2017 and 2018, was screened for the presence of azole‐resistant A fumigatus with azole‐containing agar. Phenotypic MICs were obtained from selected, molecularly confirmed isolates. cyp51A gene sequencing and genotyping of azole‐resistant isolates were done.

Results

Among 300 compost samples, three A fumigatus isolates had high voriconazole MICs (≥16 mg/L) and harboured the TR46/Y121F/T289A mutation in the cyp51A gene. Microsatellite typing of these isolates showed that two strains had the same allele across all nine examined microsatellite loci and were genotypically related to Indian azole‐resistant strains. The other isolate had a different genotype.

Conclusion

This is the first report of A fumigatus with TR46/Y121F/T289A mutation from the region. Monitoring and surveillance of antifungal susceptibility of clinical A fumigatus is warranted in Iran and elsewhere in the region.

Keywords: Aspergillus fumigatus, azole resistance, compost, TR34/L98H, TR46/Y121F/T289A

1. INTRODUCTION

Invasive aspergillosis due to azole‐resistant A fumigatus has become a major medical concern associated with high mortality in immunocompromised individuals.1, 2, 3 Azole resistance in A fumigatus emerges due to long‐term treatment with azole antifungals, but also extensive exposure of the fungus to azole compounds in the environment is a major driver of resistance selection.4, 5, 6, 7 Resistance to azole drugs in A fumigatus is mainly linked to multiple amino acid substitutions in the cyp51A gene. The most described cyp51A‐mediated resistance mechanism is a 34‐basepair (bp) sequence tandem repeat (TR34) in the promoter region of cyp51A gene combination with the L98H substitution followed by a double substitution combined with a 46 bp tandem duplication in the cyp51A promoter (TR46/Y121F/T289A).8, 9 These mutations lead to high‐level resistance to triazole antifungals of A fumigatus isolates from both azole‐naive and azole‐treated patients.10 Furthermore, several institutions report a rise in resistant strains found in environmental niches such as soil samples, paddy fields, aerial samples of hospitals and compost.11, 12, 13, 14, 15, 16, 17, 18, 19 Compost (decaying plant waste material) is an important source of A fumigatus. It is known that high concentrations of azole‐resistant A fumigatus spores are released during incomplete composting processes, especially when azole residues from agricultural waste are present.11, 13, 14, 15, 20, 21, 22, 23 Azole‐resistant A fumigatus with the TR34/L98H mutation in the environment has been reported earlier in Iran.24 Here, we report for the first time the presence of resistance due to the TR46/Y121F/T289A mutation.

2. MATERIAL AND METHODS

2.1. Sample collection and identification of azole resistent A fumigatus isolates

Three hundred compost samples from different regions of Iran (Mazandaran province (n = 200) and Tehran province (n = 100), located about 300 km apart, were collected during 2017‐2018. To recover A fumigatus strains, 1 cm2 compost was dissolved in 5 mL sterile saline solution containing Tween 40 (0.05%), vortexed, and allowed to settle. Briefly, according to a previously described protocol, for primary screening of azole‐resistant A fumigatus strains from the supernatant, 100 μL was plated on a Sabouraud dextrose agar plate (SDA; Difco), supplemented with 4 and 1 mg/L itraconazole and voriconazole, respectively, and incubated at 45°C for 72 hours in the dark.23 Identification of Aspergillus section Fumigati was performed based on both macroscopic and microscopic characteristics. Subsequently, molecular identification with DNA sequencing of the partial b‐tubulin gene, using TUB2a (5‐ TGACCCAGCAGATGTT‐3) and TUB2b (5‐GTTGTTGGGAATCCACTC‐3), was done.25

2.2. In vitro antifungal susceptibility testing

To detect azole‐resistant A fumigatus, colonies that grew on screening media supplemented with itraconazole and voriconazole and confirmed by DNA sequencing as A fumigatus were selected and evaluated with microbroth dilution minimum inhibitory concentration (MIC) testing using the Clinical and Laboratory Standards Institute document for filamentous fungi.26 Paecilomyces variotii (ATCC 22319) and Candida parapsilosis (ATCC 22019) were used as quality controls.

2.3. Detection of cyp51A gene mutations

Isolates with reduced susceptibility to voriconazole (MIC >2 µg/mL) were tested with a mixed‐format real‐time PCR assay as described before.27 Isolates with TR46/Y121F/T289A mutations were further confirmed by sequencing the cyp51A gene as described previously.28

2.4. Microsatellite typing

Genotyping of A fumigatus isolates was performed with a panel of nine short tandem repeats (STRs) loci (namely STRAf 2A, 2B, 2C, 3A, 3B, 3C, 4A, 4B and 4C), as previously described.29

Genetic relatedness between the environmental Iranian azole‐resistant TR46/Y121F/T289A isolates was compared with already barcoded clinical and environmental isolates from different countries (The Netherlands, India, Tanzania, France, Colombia, Ireland, China and Germany) using BioNumerics software version 7.6.1.(Applied Maths NV).

3. RESULTS

A total of 63 samples from both Mazandaran (n = 44) and Tehran province (n = 19) yielded A fumigatus on SDA supplemented with itraconazole and voriconazole. A total of 57 A fumigatus isolates had high MICs level of itraconazole (>8 mg/L) (55/57) or voriconazole (>2 mg/L) (51/57) by in vitro antifungal susceptibility testing. Exploring the mechanisms of resistance by mixed‐format real‐time PCR and sequencing of cyp51A and its promoter in these isolates showed that resistant isolates had different resistance mechanisms including forty‐four with TR34/L98H and three isolates with TR46/Y121F/T289A (1 isolate with TR46/Y121F/M172I/T289A/G448S and two isolates with TR46/Y121F/T289A mutations in the cyp51 region). All three isolates with TR46/Y121F/T289A mutation in cyp51A and high voriconazole MIC (≥16 mg/L) were isolated from Mazandaran province. Microsatellite analysis revealed two different genotypes among isolates harbouring TR46/Y121F/T289A. Two strains had the same alleles across all nine examined microsatellite loci and the other differed by seven loci (2C, 3A, 3B, 3C, 4A, 4B and 4C). For comparison of the genotype of these isolates with other isolates from different countries, a minimum‐spanning tree was constructed based on nine STRAf loci of different azole‐resistant isolates.29 Two Iranian A fumigatus TR46/Y121F/T289A isolates were related to resistant isolates originating from India while the third was genetically different from other strains (Figure 1).

Figure 1.

Figure 1

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Minimum‐spanning tree showing the genetic relationship of A fumigatus genotypes harbouring TR46/Y121F/T289A mutation in cyp51A allele. Genotypic relationship of three Iranian isolates is illustrated by arrows

4. DISCUSSION

Azole resistance in A fumigatus due to TR34/L98H and more recent TR46/Y121F/T289A mutations in cyp51A has been described from wide geographical areas among both clinical and environmental isolates.30, 31 This is linked to the widespread use of azole fungicides in agriculture rather than the clinical use of antifungal drugs.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 19, 20, 21 The fungicide‐driven mutation TR46/Y121F/T289A has been first detected in the Netherlands in 2009.32 Since then, several reports have demonstrated that, particularly this mutation, corresponded to voriconazole resistance in A fumigatus in European, African, American and Asian countries. We report the presence of TR46/Y121F/T289A now also from Iran, where until now only TR34/L98H had been isolated.24, 33, 34, 35 This mutation, conferring voriconazole resistance, has been reported from both environmental (China,17, 21 Taiwan,13 United Kingdom,11 Colombia,12, 14 France,16, 36 Germany,37 India,19 the Netherlands32 and Tanzania18) and clinical sources (Spain,38, 39 United Kingdom,40 France,41 Portugal,42, 43 Argentina,44 Taiwan,45 Germany,46, 47, 48, 49 China,50, 51 Japan,52 United States,53, 54 Denmark,10 Belgium 7, 55, 56, 57 and the Netherlands7, 32, 58, 59, 60) (Table 1). All previous studies conducted in Iran for monitoring the mechanism of resistance among azole‐resistant A fumigatus in both clinical and environmental samples showed that the TR34/L98H mutation was reported with increasing frequency from 3.3% in 2013 to 6.6% in 2016.24, 34 Microsatellite typing of the current isolates showed that two A fumigatus isolates with TR46/Y121F/T289A isolates were related to resistant isolates from India in 2014. It is noteworthy that the first isolates with TR34/L98H in Iran in 2013 were also genotypically related to some resistant clinical and environmental TR34/L98H isolates from India and the Netherlands.24 It is anticipated that strains with the TR46/Y121F/T289A or TR34/L98H mutation will spread rapidly to other geographical regions especially due to the use of azole‐based agricultural fungicides which is a significant factor in the increase of multiple‐triazole‐resistant A fumigatus.4 In our study, most of the azole‐resistant A fumigatus isolated from Iranian compost were from Mazandaran province located in Northern Iran where agricultural activity and subsequently the usage of fungicides are generally higher than in other regions of Iran. Compost is a main ecological niche for A fumigatus, and azole residues have been reported to be present in commercial compost.20 Snelders et al61 showed that 86% of triazole‐resistant A fumigatus isolates recovered from environmental sources such as compost had the same resistance mechanism as found in clinical isolates. Compost is used widely in gardens and indoor plants in Iran, and has a key role in the exposure of azole‐resistant A fumigatus to susceptible hosts. Although CLSI and EUCAST have published standardised antifungal susceptibility testing methods to determine the in vitro efficacy of antifungal drugs, most clinical microbiology laboratories in Iran do not routinely perform antifungal susceptibility testing of Aspergillus isolates as recommended by ESCMID guidelines.62 Therefore, the true prevalence of resistance and mechanism of resistance in clinical A fumigatus isolates in Iran is unknown. Reports of clinical isolates harbouring this mutation are mainly limited to developed countries in Europe, East Asia and the USA, maybe because of the lack of routine antifungal susceptibility testing in less developed countries (Table 1). With this background in mind, the emergence of a new azole resistance mechanism in A fumigatus isolated from environmental sources in Iran is concerning as this mechanism has been associated with therapeutic failure with voriconazole, the first‐line treatment for invasive aspergillosis. Nevertheless, correlations between in vitro susceptibility results and clinical outcomes are not really estimated for A fumigatus. Compost used for planting surroundings of immunocompromised patient's houses could be a source of invasive aspergillosis due to azole‐resistant A fumigatus. 21, 22 Our study reinforces the importance of surveillance studies to monitor antifungal susceptibility of clinical isolates in Iran.

Table 1.

Reports of azole resistant Aspergillus fumigatus isolates carrying TR46/Y121F/T289A mutation

Number Country Year of publishing Number of environmental isolates Number of clinical isolates
Present report Iran 2020 3 –
1 China 202021 6 –
2 Taiwan 201913 3 –
3 Spain 201938 – 1
4 Belgium ‐ Netherlands 20197 – 12
5 United kingdom 201911 6 –
6 France 201941 – 2
7 Colombia 201914 8 –
8 Netherlands 201858 – 5
9 France 201816 9 –
10 Portugal 201842 – 1
11 Argentina 201844 – 1
12 Taiwan 201845 – 3
13 Portugal 201843 – 1
14 Germany 201846 – 1
15 Germany 201747 – 1
16 Belgium 201755 – 4
17 Colombia 201712 17 –
18 United kingdom 201740 – 1
19 France 201736 21 1
20 China 201617 2 –
21 Netherlands 201659 – 4
22 Japan 201652 – 1
23 China 201650 – 1
24 United States 201653 – 2
25 United States 201554 – 1
26 China 201551 – 1
27 Spain 201539 – 1
28 Germany 201537 6 –
29 France 201541 – 2
30 Germany 201548 – 2
31 Netherlands 201560 – 1
32 Belgium 201456 – 1
33 Denmark 201410 – 1
34 Germany 201449 – 1
35 Tanzania 201418 4 –
36 India 201419 6 –
37 Netherlands 201332 14 21
38 Belgium 201257 – 1
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CONFLICT OF INTEREST

JFM received grants from Pulmozyme and F2G. He has been a consultant to Scynexis and received speaker's fees from United Medical, TEVA and Gilead Sciences. The other authors report no conflicts of interest.

AUTHORS CONTRIBUTIONS

FA and HB conceived this study. MN, SK, MM, ZS and MLK were responsible for collection of the isolates and data acquisition. FA and YP did the molecular analysis. FA and JFM wrote the concept paper and all authors critically reviewed the manuscript prior to submission.

ETHICS APPROVAL

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as the research in this article related to micro‐organisms.

ACKNOWLEDGMENTS

The authors are grateful to Masume Farhadi, Miad Banaye Gholrizi and Fatemeh Barimani for their supportive help during our sampling. We thank Iman Haghani for excellent technical assistance and help with antifungal susceptibility testing. FA was the recipient of an ESCMID observership grant to visit ESCMID observership centre 58, CWZ.

Ahangarkani F, Puts Y, Nabili M, et al. First azole‐resistant Aspergillus fumigatus isolates with the environmental TR46/Y121F/T289A mutation in Iran. Mycoses. 2020;63:430–436. 10.1111/myc.13064

Funding information

This study was supported by National Institutes for Medical Research Development (NIMAD), Grant/Award Number: 982677; Mazandaran University of Medical Sciences, Sari, Iran, Grant/Award Number: 1352.

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