Next Article in Journal
Mucosal Immunity to Gut Fungi in Health and Inflammatory Bowel Disease
Previous Article in Journal
A New Benzaldehyde Derivative Exhibits Antiaflatoxigenic Activity against Aspergillus flavus
Previous Article in Special Issue
Potential Implication of Azole Persistence in the Treatment Failure of Two Haematological Patients Infected with Aspergillus fumigatus
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
JoF
Volume 9
Issue 11
10.3390/jof9111104
jof-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genomic Epidemiology Identifies Azole Resistance Due to TR34/L98H in European Aspergillus fumigatus Causing COVID-19-Associated Pulmonary Aspergillosis

by
Benjamin C. Simmons
1,2,*,
Johanna Rhodes
1,3,
Thomas R. Rogers
4,
Paul E. Verweij
3,5,6,
Alireza Abdolrasouli
7,8,
Silke Schelenz
9,10,
Samuel J. Hemmings
1,
Alida Fe Talento
4,11,12,
Auveen Griffin
13,
Mary Mansfield
4,
David Sheehan
4,
Thijs Bosch
6 and
Matthew C. Fisher
1
1
Medical Research Council Centre for Global Infectious Disease Analysis, Imperial College London, London W2 1PG, UK
2
UK Health Security Agency, London EP14 4PU, UK
3
Department of Medical Microbiology, Radboudumc Center for Infectious Diseases (RCI), Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
4
Department of Clinical Microbiology, St. James’ Hospital Campus, Trinity College Dublin, D08 NHY1 Dublin, Ireland
5
Radboudumc-CWZ Center of Expertise for Mycology, Radboudumc Center for Infectious Diseases (RCI), Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
6
Center for Infectious Disease Research, Diagnostics and Laboratory Surveillance, National for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands
7
Department of Infectious Diseases, Imperial College London, London W2 1NY, UK
8
Department of Infectious Diseases, King’s College Hospital, London SE5 9RS, UK
9
Infection Sciences, King’s College Hospital, London SE5 9RS, UK
10
School of Immunology & Microbial Sciences, King’s College London, London WC2R 2LS, UK
11
Department of Microbiology, Our Lady of Lourdes Hospital, A92 VW28 Drogheda, Ireland
12
Department of Microbiology, Royal College of Surgeons, D02 YN77 Dublin, Ireland
13
Department of Microbiology, St. James’ Hospital, D08 NHY1 Dublin, Ireland
 
add Show full affiliation list
 
remove Hide full affiliation list
*
Author to whom correspondence should be addressed.
J. Fungi 2023, 9(11), 1104; https://doi.org/10.3390/jof9111104
Submission received: 30 September 2023 / Revised: 31 October 2023 / Accepted: 3 November 2023 / Published: 13 November 2023
(This article belongs to the Special Issue Azole Resistance in Aspergillus spp.)
Browse Figures
Review Reports Versions Notes

Abstract

:
Aspergillus fumigatus has been found to coinfect patients with severe SARS-CoV-2 virus infection, leading to COVID-19-associated pulmonary aspergillosis (CAPA). The CAPA all-cause mortality rate is approximately 50% and may be complicated by azole resistance. Genomic epidemiology can help shed light on the genetics of A. fumigatus causing CAPA, including the prevalence of resistance-associated alleles. We present a population genomic analysis of 21 CAPA isolates from four European countries with these isolates compared against 240 non-CAPA A. fumigatus isolates from a wider population. Bioinformatic analysis and antifungal susceptibility testing were performed to quantify resistance and identify possible genetically encoded azole-resistant mechanisms. The phylogenetic analysis of the 21 CAPA isolates showed that they were representative of the wider A. fumigatus population with no obvious clustering. The prevalence of phenotypic azole resistance in CAPA was 14.3% (n = 3/21); all three CAPA isolates contained a known resistance-associated cyp51A polymorphism. The relatively high prevalence of azole resistance alleles that we document poses a probable threat to treatment success rates, warranting the enhanced surveillance of A. fumigatus genotypes in these patients. Furthermore, potential changes to antifungal first-line treatment guidelines may be needed to improve patient outcomes when CAPA is suspected.

1. Introduction

Aspergillus fumigatus is the predominant causative agent of invasive aspergillosis (IA) [1,2], with an estimated annual prevalence of > 250,000 cases globally and a mortality rate of 30 to 95% [3,4]. Normally, only those who are immunocompromised or immunosuppressed are highly susceptible to developing IA [2]; however, there is a widening group of patients who are at risk of IA, including those with severe influenza or COVID-19.
In 2019–2020, the novel virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing COVID-19 disease, spread across the globe. Opportunistic pathogens have been widely reported as causing secondary infections in COVID-19 patients with lung damage and a notable proportion of these are fungal coinfections (12.6%) [5]. A large proportion of fungal coinfections in COVID-19 cases are caused by Aspergillus species, including A. fumigatus, giving rise to COVID-19-associated pulmonary aspergillosis (CAPA) [6,7]. The European Confederation for Medical Mycology (ECMM) and the International Society for Human and Animal Mycology (ISHAM) developed a consensus definition for CAPA at the end of 2020, stating that CAPA is a form of IA that is “in temporal proximity to a preceding SARS-CoV-2 infection” [7]. The global prevalence of CAPA is reported to be between 3.8 to 40%, with 15.1% of ICU-admitted COVID-19 patients fulfilling the ECMM definition of CAPA [6,7,8,9,10]. Furthermore, CAPA is characterised by low survival, with mortality ranging from 44 to 75% [10,11,12]. The MYCOVID cohort study showed that COVID-19 patients who received intensive care treatment and positive sputum cultures for A. fumigatus but could not be classified as CAPA had higher mortality (45.8%) than those who had negative A. fumigatus cultures (32.1%) [10]. However, this was lower than the group who had CAPA (61.8%) [10]. Therefore, so-called colonisation may not be as harmless as previously thought.
In recent years, A. fumigatus resistance to the azole antifungal drug class has emerged, with its prevalence rapidly increasing, and recently has been declared a public health issue [13,14]. In the environment, population genomic studies have determined that depending on the country, between 2.2 to 20% of isolates are azole-resistant, and this proportion is as high as 95.2% in Vietnam [15,16,17,18,19,20]. A cause for concern is the increase in the proportion of azole-resistant A. fumigatus (ARAf) identified in patients, as this is associated with treatment failure, increased mortality rates, and a doubling of health care costs [13,14]. In CAPA, the prevalence of azole resistance has not yet been established, as the sample size of the studies has been too small. Two CAPA genomic epidemiology studies have reported no resistant isolates [21,22] while one German study reported 22.2% (n = 6/27) [23] as resistant. Using the Clinical and Laboratory Standards Institute (CLSI) method, a second German study (n = 4) did not find any resistant isolates [24]. Finally, a Portuguese study reported a high prevalence of ARAf of 45.5% (n = 5/11 [25].
Genomic epidemiological methods have played a key role in delineating the genetic basis of ARAf [13,26,27]. The primary locus, with a high-frequency of non-synonymous SNPs (nsSNPs), known to be involved in azole resistance is the cyp51A gene encoding the 14α-sterol demethylase enzyme [13,26]. Common amino acid substitution hotspots are G54, L98H, G138, M220, and G448 (Table A1). Point mutations occur in isolates cultured from patients who have had long-term exposure to azole therapy [26]. Additionally, several tandem repeats (TRs) in the gene promoter region of cyp51A lead to the overexpression of the gene and are commonly associated with point mutations in the cyp51A gene (Table A1). The genotypes of A. fumigatus that contain TR-mediated resistance are more common in environmental isolates and isolates obtained from azole-naïve patients. However, there is increasing awareness that resistance mechanisms in ARAf are complex and involve multiple genes (Table A1) [16,26,27,28,29,30,31,32]. In studies that identified ARAf in CAPA isolates, Kirchoff et al. [23] discovered only one CAPA isolate that contained TR34/L98H and five polymorphisms of ARAf via a non-cyp51A mechanism.
Whilst the development of a clinical definition for CAPA has aided clinicians in its diagnosis and ensuring treatment is commenced in a timely manner to improve patient outcomes, important questions remain as to the genetic characteristics and identity of A. fumigatus causing CAPA and the prevalence of antifungal resistance [13,21,22,23,24,33]. In A. fumigatus, genomic epidemiological methods have begun to unravel the genetics of environmental and clinical antifungal resistance and the potential mechanisms of dispersion [1,13,16,32]. To date, there are only five genomic epidemiologic studies of CAPA using multiple methods [21,22,23,24,25]. The results of these analyses were inconclusive on the genetic and epidemiological relatedness of A. fumigatus causing CAPA. The current study is the largest transnational genomic epidemiological investigation to date of CAPA isolates to determine where the genotypes of CAPA isolates group in the wider A. fumigatus population. Secondly, this study aimed to identify the mechanisms and determine the frequency of azole-resistant polymorphisms in CAPA isolates.

2. Materials and Methods

2.1. CAPA Definition

The definition of CAPA was based on the 2020 ECMM/ISHAM consensus criteria [7]. In summary, patients must have a positive SARS-CoV-2 polymerase chain reaction (PCR), require intensive care for COVID-19 and have signs of invasive pulmonary aspergillosis (IPA) infection. Ideally, IPA is confirmed through the histopathological or direct microscopic detection of fungal hyphae obtained by lung biopsy, therefore, showing signs of tissue invasion and/or damage [7,34]. However, samples from bronchoalveolar lavage, bronchial aspirate, and tracheal aspirate were used as alternatives, as lung biopsies could rarely be performed in CAPA patients [7]. A positive SARS-CoV-2 PCR test must occur between two weeks prior to hospital admission and up to 96 h after ICU admission. CAPA may be further categorised depending on the sensitivity of the diagnostic method used. That is, CAPA can be either proven, probable, or possible [7,11]. A fourth category was added in cases where A. fumigatus was isolated from the sputum of a patient with COVID-19 receiving intensive care, but there were no clear signs of invasive disease [10]. Furthermore, there was no follow-up bronchoscopy to determine if there was an invasive disease. Therefore, for the first analysis, the patients were categorised as non-CAPA and only colonised with A. fumigatus (‘colonising’). In the second analysis, the colonising isolates were categorised as clinical non-CAPA isolates.

2.2. Fungal Isolates

Twenty-one CAPA isolates from four European countries between 2020–2021 were included (Table 1). In this study, all CAPA isolates were recovered as per the ‘standard of care’ based on the ECMM criteria [7]. Four CAPA isolates (CAPA-A–D) were from four separate cases that originated from two Cologne hospitals in Germany [24,35]. Six CAPA isolates were from London, UK (C422–C425, C611, C612). A further two isolates originated from two hospitals in the Netherlands (C403 and C408). A further nine isolates were from two hospitals in Dublin, Ireland (C434–C441, C444). Isolate C444 has previously been described in a case report [36].
In this study, the genetic relatedness of CAPA isolates was compared to (1) A. fumigatus isolates from non-CAPA IA cases, and isolates colonising non-CAPA patients; and (2) non-CAPA clinical and environmental isolates from the wider A. fumigatus population. The first analysis involved comparing CAPA isolates to two other groups. The first group comprised twelve A. fumigatus isolates from eleven patients with different types of IA (C120, C137–C140, C143, C307, C323, C360, C372, C376, C442). This included four isolates from a patient with necrotising aspergillosis, one trauma patient, two allergic bronchopulmonary aspergillosis (ABPA) patients, two with ABPA and asthma, and one with chronic pulmonary aspergillosis (CPA), and two with IPA (Table A2). The selection criteria for these IA isolates were based upon the European Organization for Research and Treatment of Cancer and the Mycoses Study Group definition for invasive disease [34], and were from the UK and Ireland. The second group comprised eight ‘colonising’ isolates: seven from three Dutch Hospitals (C402, C404–C407, C409, C410) and one from a patient in Ireland (C443) [32,37,38]. The second analysis involved comparing 21 CAPA, 167 non-CAPA clinical, and 73 environmental A. fumigatus isolates. In summary, 218 UK and Irish A. fumigatus isolates that had been analysed by Rhodes et al. [32] and 23 additional A. fumigatus isolates (C307, C323, C360, C372, C376, C402, C404–C407, C409, C410, C426–C433, C442, C443) were used (Table A2). All 261 isolates can be found in the Microreact project [37] at https://microreact.org/project/mPbPTWS2jTvvdNGmuDVyia-capa (accessed on 4 November 2023).

Clinical Characterisation of the CAPA Isolates

Applying the ECMM/ISHAM criteria, sixteen isolates were from probable CAPA cases (C422–C425, C434–C441, C444, C611, CAPA-C, and CAPA-D) and five were from possible CAPA cases (C403, C408, C612, CAPA-A, and CAPA-B).

2.3. Antifungal Susceptibility Testing

Susceptibility to azole antifungal agents was carried out on all 21 of the CAPA isolates within this study. Isolates were first screened using the low-cost tebuconazole screening test (Tebucheck) to determine which isolates were resistant and then VIPcheckTM was used to give more information on which medical azoles the isolates were resistant to [39]. Fifteen of the CAPA isolates were prepared as per the protocol set out in Brackin et al. [39]. Briefly, isolates were inoculated in 25 cm3 tissue culture flasks containing Sabouraud dextrose agar and culture for a minimum of 2 days at 45 °C. Conidia were collected in sterilised 0.05% (v/v) Tween-80 (Calbiochem®, Sigma-Aldrich, Gillingham, UK using filtration through sterilised glass-wool. A final standardised spore suspension was achieved with a cell density reading at 600 nm between 0.09 and 0.12 using sterilised 0.05% (v/v) Tween-80 with a standard spectrophotometer [39]. Twenty microlitres of final standardised spore suspensions were inoculated into Tebucheck multi-well and VIPcheckTM (Mediaproducts BV, Groningen, NL) plates. The Tebucheck multi-well plate consists of four wells comprising of drug-free (growth control well), 6 mg/L, 8 mg/L, and 16 mg/L of tebuconazole [39]. All Tebucheck multi-well plates were made in-house as per the protocol set out in Brackin et al. [39]. The VIPcheckTM multi-well plate consists of wells with 4 mg/L itraconazole, 2 mg/L voriconazole, 0.5 mg/L posaconazole, and a drug-free growth control well [40]. Due to a lower concentration of spore suspension used, cultured plates were left at 45 °C to ensure there was complete growth in the control wells (48 h). Each strain was compared to a non-CAPA clinical isolate C154, which was EUCAST susceptible and verified by both Tebucheck and VIPcheckTM.
The scoring of Tebucheck plates is as follows: a well that had no growth (0 to <10% of the well) was scored 0; if there was partial growth (10 to 50% growth) the well was scored 0.5; and if there was full growth (50 to 100%) then the well was scored 1 [39]. Any isolates with a score greater than 1 were considered resistant to tebuconazole [39].
The scoring of VIPcheckTM can be found in Buil et al. [40]. Briefly, after 24 h, if there was uninhibited growth in any well which contained an azole, then the well would be scored 2 and the isolate would be resistant to that azole. If there was minimal growth, then this would be scored 1 and the isolate would therefore be partly resistant. If there was no growth in the azole-containing wells, then the well would be scored a 0 and the isolate would be considered susceptible to the azole.
Antifungal susceptibility data from previously published research was included in this study. The isolates C1–C200, E9–E206, and U1-3 [32]; C307, C323, C360, C372, and C376 [38]; and C444 [36] susceptibility testing was performed as per the protocol set out in Rhodes et al. [32], based upon the recent EUCAST methodology [41]. The Dutch isolates (C403–C410) had susceptibility testing performed using EUCAST methodology [41]. Finally, azole-resistant minimum inhibitory concentrations (MICs) were measured on two Irish isolates (C438 and C441) and the previously published CAPA-A to D [24], using CLSI broth dilution technique M38-A2 by the Mycology Reference Laboratory Bristol UK [42]. A caveat of the CLSI method is that it has different breakpoint values compared to the EUCAST method [41,42].

2.4. Genomic DNA Preparation and Whole Genome Sequencing

Whole-genome libraries of the A. fumigatus isolates were prepared and sequenced by different research teams and at different times; thus, different platforms were used. Extraction of genomic DNA from the Irish and UK CAPA isolates (n = 15) and some additional Irish non-CAPA isolates (clinical, n = 2, and environmental, n = 8) was carried out at Imperial College London. Briefly, gDNA was extracted using the MasterPureTM Complete DNA and RNA Purification Kit (Lucigen, Middleton, USA), including an additional bead-beating step using a FastPrep-24TM. Extracted gDNA was purified using a DNeasy Blood and Tissue Kit (Qiagen, Germany), with the concentration measured using a Qubit fluorometer and dsDNA Broad Range Assay kit (Invitrogen, ThermoFisher Scientific, Karnataka, India). DNA purity was assessed using NanoDropTM spectrophotometry. Purified gDNA was stored at −20 °C prior to the construction of gDNA libraries, normalisation, and indexing (Earlham Institute, Norwich, UK). Libraries were run on a NovaSeq 6000 SP v1.5 flow cell (Illumina, Cambridge, UK) to generate 150 bp paired-end reads.
CAPA (C403, C408) and colonising (clinical non-CAPA) (C402, C404–C407, C409, C410) isolates from the Netherlands (n = 9) were sequenced using NextSeq 550 sequencer (Illumina), and 218 UK and Irish isolates and the five IA isolates (C307, C323, C360, C372, C376) were all sequenced using a HiSeq2500 (Illumina) sequencer [32]. All these isolates generated 50-150bp paired-end reads. The four CAPA isolates obtained from Germany were sequenced from single-stranded circular DNA (ssCir DNA) using the MGISEQ2000 [24,43].

2.5. Bioinformatics Analysis

Raw Illumina whole-genome sequence (WGS) reads were quality checked using FastQC v0.11.9 (Brabham institute) and subsequently aligned to the Af293 reference genome using the Burrow-Wheeler Aligner alignment tool with maximal exact methods algorithm [44,45]. The quality of the alignment was improved and converted to sorted BAM format using sequence alignment/map (SAM) tools v1.15. To minimise inaccurate identification of single-nucleotide polymorphisms (SNPs) due to PCR duplication error, duplicated reads were marked using Picard v2.18.7. SNPs were identified using ‘Haplotype Caller’ from the Genome Analysis Toolkit v4.0. To ensure high confidence calls, SNPs had to achieve at least 1 parameter, QD < 2.0, fisher strand > 60.0, mapping quality < 40.0, mapping quality rank sum test < −12.5, read positive rank sum test < −8.0, and SOR > 4.0. The above expressions have been rigorously tested and benchmarked [32]. SNPs were mapped to genes using vcf-annotator v0.5 (Broad Institute).

2.6. A. fumigatus and MAT Identification

Previously, 223 UK and Irish A. fumigatus isolates have been confirmed to be members of the A. fumigatus species complex using molecular methods [32]. In this study, the CAPA isolates were confirmed as A. fumigatus for congruency with Af293 using the nucleotide sequence of the calmodulin (CaM) gene from Af293, utilising the basic local alignment searching tool (BLAST, v2.13.0) from the National Centre of Biotechnology Information (NCBI) [46,47]. A CAPA isolate was identified as A. fumigatus if the top BLAST hit had a percentage identification of >99% [48].
The mating type of all 261 A. fumigatus isolates was identified using BLAST v2.13.0 (NCBI), using sequences from Pyrzak et al. [49].

2.7. Phylogenetic and Spatial Analyses

Whole-genome SNP data were converted to the presence/absence of an SNP with respect to the reference. SNPs identified as low confidence during variant filtration were assigned as missing. In the first analysis, as there were fewer than 50 taxa, maximum likelihood (ML) phylogenies were constructed using the gamma model (GTRGAMMA) of rate heterogeneity and rapid bootstraps over 1000 replicates in RAxML v8.2.9 (Stematakis, 2006 RAxML-VI-HPC) [50]. In the second analysis, with more than 50 taxa, maximum likelihood (ML) phylogenies were constructed using the CAT approximation (GTRCAT) of rate heterogeneity and rapid bootstraps over 1000 replicates in RAxML v8.2.9 (Stematakis, 2006 RAxML-VI-HPC) [50]. Phylogenies were annotated and visualised in ggtree and ggtreeExtra in R (v4.2.2).
Genetic similarity and population allocation were investigated via principal component analysis (PCA) and discriminant analysis of principal components (DAPC) based on whole genome SNP data in R (v4.2.2). DAPC was performed using adegenet [51]. To determine the number of principal components (PCs) to retain, the a-score method in the adegenet package was used. As the loadings of the PCs themselves are generally uninformative, to gain insight into how each isolate contributes to the cluster’s composition, compoplots (adegenet) were generated.

2.8. Statistical Analyses

Descriptive analysis of data was conducted in R v4.2.2 and Microsoft Excel. Chi (χ2)-test was performed to address the association between CAPA and azole-drug resistance. Significant value was considered for p-values < 0.05. Yates’s continuity correction was used to calculate χ2 in cases when values for a variable were less than 20.

3. Results

3.1. WGS of 21 CAPA Isolates

The reference-guided methods were used to analyse the genetic diversity of twenty-one CAPA isolates from four European countries (Table 1). All the isolates were confirmed to be A. fumigatus, sharing 100% similarity with the calmodulin (CaM) gene of the Af293 genome. All the sequenced genomes mapped > 96.2% (mean 97.8%) to the reference genome Af293 (Table 1), with a mean coverage of 151× (128–167×) for the German and Dutch CAPA isolates, and 42.1× (27.6–56.2×) for the UK and Irish isolates, reflecting the different sequencing methods and platforms used. The normalised whole-genome depth of coverage confirmed an absence of aneuploidy events in the CAPA isolates. However, deletions and duplications were observed; all the isolates observed had an approximately 260 kilo-base pairs (Kbp) deletion in Chromosome IV (Figure A1). This phenomenon has been observed in previous WGS studies, and the region of the rRNA repeat cluster may be included in this deletion [44,52]. Deletions were also found in Chromosome I (region of > 200 Kb), VII (region of > 300 kb), and VIII (approx. 60 Kb region), in 7 (24.1%), 14 (48.3%), and 8 (27.5%) isolates, respectively (Figure A1). These regions have not been identified as having any known importance in the context of azole resistance. Multiple peaks were observed in all the chromosomes from each isolate, which relate to the presence of copy number variations (CNVs).
An even proportion of mating idiomorphs in the CAPA isolates was observed, with 52% (n = 11/21) containing the MAT1-1 gene and 48% (n = 10/21) containing the MAT1-2 gene. This is consistent with what is expected in the wild, and is suggestive of a sexually recombining population [49,53]. Combining the CAPA isolates with the UK and Ireland A. fumigatus isolates, there was a bias towards the MAT1-2 idiomorph (60.9%), which was statistically significant (χ2-test, p-value = 0.014, degrees of freedom, d.f. = 2). This means that the population may be more likely to reproduce asexually [32,49,53]. Although, there was no significant difference between source types (Table A3).
On average, the CAPA isolates differed from each other by 26,706 SNPs. There was only one CAPA isolate pair (C439–C440) that was highly similar (< 2671 SNPs); however, these were obtained from different patients. When the CAPA isolates were compared to the IA and colonised isolates, they differed by 30,986 SNPs.

3.2. Azole Resistance within CAPA Isolates Primarily Centred on Known Polymorphisms within cyp51A

Three CAPA (C438, C441, C444) isolates obtained from different locations in Ireland contained azole-resistant polymorphisms (TR34/L98H) (Table 2, Table A4, and Table A5). No known drug-resistant polymorphisms were identified in the Dutch, UK, or German CAPA isolates.
Susceptibility testing was performed to confirm the isolates containing azole-resistant polymorphisms have raised MICs to azole drugs. Of the CAPA isolates, three had scores of greater than 1 in VIPcheckTM and/or Tebucheck (C438, C441 and C444) (Table 2). Thus, three of the 21 CAPA isolates were ARAf. The remaining CAPA isolates scored 1 in VIPcheckTM and/or Tebucheck, and, therefore, are susceptible phenotypically. These results were verified independently using EUCAST and the CLSI broth microdilution methods (Table A4 and Table A5) [24,32,36].

3.3. Phylogenetic and Spatial Analysis Shows CAPA Isolates Are Highly Related to Non-CAPA Clinical and Environmental A. fumigatus

To test the phylogenetic relatedness of A. fumigatus cultured from CAPA patients with the existing collection of A. fumigatus from IA and colonised patients, the phylogenetic analysis of twenty-one CAPA, twelve non-CAPA IA isolates, and eight non-CAPA colonising isolates from four European countries was conducted (Table A2). This analysis revealed two broadly divergent clades, which are referred to as clade A and clade B, as previously reported (Figure 1 and Figure A2) [32,54]. Of the CAPA isolates, the majority (n = 15, 71.4%) were in clade B, and the remainder were in clade A (n = 6, 28.6%) (Figure 1 and Table A3). All CAPA ARAf isolates (C438, C441, C444) were situated in clade A. All the Dutch CAPA isolates were from clade B, whereas only 55.6% of the Irish CAPA isolates were from clade B. The UK and German CAPA isolates had 84.3% and 75% of isolates in clade B, respectively. Of the IA isolates (C120, C137–C140, C143, C307, C323, C360, C372, C376, C442), eight (66.6%) were in clade A and four (33.3%) were in clade B. The IA cohort contained ten ARAf isolates, which all contained cyp51A polymorphisms. The eight IA isolates in clade A were ARAf and two (C120 and C372) of the four IA isolates in clade B were ARAf isolates (Figure 1 and Appendix Table A4 and Table A5). Finally, seven of the eight colonising isolates comprised clade B and all the isolates where not ARAf.
Multivariate methods were used to identify and describe the genetically related clusters. The principal component analysis (four principal components, PCs) identified that the CAPA isolates come from the same population as the IA isolates (Figure 2A). Interestingly, the colonising isolates formed a subsection of the CAPA isolates (Figure 2A). The discriminant analysis of principal components (DAPC) and composition plot (five PCs) confirmed this observation (Figure 2B,C). All the isolate genotypes contained a mixture of CAPA, IA, and coloniser, with twenty-one of the isolates containing >20% CAPA genome (Figure 2B,C). Despite this, the isolates labelled as IA or coloniser are not genetically distinct from the CAPA isolates.
To gain a more in-depth understanding of the genetic relatedness of the CAPA isolates and whether they represent a distinct genetic identity or are drawn from the wider population of A. fumigatus, the phylogenetic relationship of the CAPA isolates with the wider A. fumigatus population was investigated. A phylogenetic analysis was conducted on 240 clinical and environmental isolates and twenty-one CAPA isolates (Figure A4). Again, two broadly divergent clades were observed: clades A and B (Figure 3, Figure A3 and Figure A4).
In total, 137 isolates (52.5%) lay within clade A and 124 isolates (47.5%) were from clade B. As seen in the first analysis, the CAPA isolates were largely associated with clade B (n = 15, 71.4%) (Figure 3). The proportions of each isolate (CAPA, non-CAPA clinical, and environmental) source type were associated with different clades, which were significantly different (χ2-test, p < 0.001; d.f. = 2) (Table A3). Additionally, the isolates containing azole-resistant polymorphisms were significantly associated with clade A (χ2-test, p < 0.001; d.f. = 1).
A multivariate analysis of the underlying genomic structure of the isolates confirmed that the genomes of the CAPA isolates could not be distinguished from the non-CAPA clinical and the environmental A. fumigatus isolates originating from the UK and Ireland (Figure 4).

4. Discussion

The COVID-19 pandemic has created a large and growing global cohort of patients at risk of developing A. fumigatus coinfections. Additionally, CAPA has higher mortality than COVID-19 itself (16 to 25% excess mortality rate) [7]. This, in part, has been due to the challenges of determining whether COVID-19 patients have IA [7,10]. Therefore, delays in commencing prompt antifungal treatment may have led to higher mortality rates [55]. Furthermore, WGS could address these issues by providing information on potential common sources, the genetic relatedness of the CAPA isolates with the wider A. fumigatus population, and the presence of ARAf, informing clinicians about the most effective way to treat CAPA patients [1,13]. In this study, WGS was carried out on twenty-one CAPA isolates from four European countries to explore the genomic epidemiology of A. fumigatus causing CAPA.
The present genomic analysis of CAPA isolates yielded three major findings. First, the CAPA isolates comprised a diverse range of A. fumigatus genotypes. Secondly, the CAPA genomes are an even mix of A. fumigatus genotypes found in previous studies in either the clinical environment or the environment. Finally, our study provides evidence of ARAf in at-risk patient cohorts. However, an important caveat is that the only ARAf found were from one country, likely due to the overall small sample size of A. fumigatus studied.
To gain insight into CAPA’s genetic and epidemiological relatedness, twenty-one CAPA isolates were compared to the different types of A. fumigatus samples including clinical non-CAPA and environmental isolates. This analysis provided evidence that the genotypes of the A. fumigatus isolates from the CAPA patients are genetically diverse, with two broadly divergent clades: A and B. A proportion of the CAPA isolates clustered in clade B (n = 15, 71.4%). In the sample of the IA isolates, 64% were in clade A. The phenomenon of the two-clade structure of A. fumigatus species was first identified in a large global genetic epidemiology study of over 4000 A. fumigatus isolates and has since been replicated in different A. fumigatus populations [32,54,56].
Even though the genotypes of the CAPA isolates in this study were biased towards clade B, the isolates were genetically diverse. The genetic relatedness between the CAPA isolates is on average 26,706 SNPs. The genetic relatedness between the isolates was higher when compared across the twelve IA and eight colonising isolates (average 30,986 SNPs) and the wider A. fumigatus population (25,448 SNPs), comparable to previous studies [32,56]. Only one pair of isolates was highly related (<2671 SNPs; C439–C440) and originated from the same country, but were from different patients, suggesting the patients were exposed to the same environmental source. However, no epidemiological information was available to investigate this possibility.
Secondly, using hypothesis-free population genetic methods, we identified that the CAPA isolates were a mixture of A. fumigatus genotypes. The genotypes from the 21 CAPA isolates showed genetic overlapping with the A. fumigatus genotypes isolated from the patients with IA, and those obtained from other patients and the environment. In the five previous genomic epidemiological studies of CAPA, the genotypes from the CAPA isolates were only compared with non-CAPA clinical isolates or COVID-19 patients who were colonised with A. fumigatus and had no active IA [21,22,23,24,25]. The results of Steenwyk et al. suggest that overall, the CAPA genotypes are drawn from the wider clinical population of A. fumigatus isolates, despite observing some genetic clustering [24]. This has been replicated in a larger transnational genomic study [21]. A larger multicentre study using microsatellite methods to genotype isolates identified that the genotypes obtained were genetically distinct from one another [23]. This finding was replicated in one Spanish and one Portuguese study using tandem repeats within the exons of surface proteins and erg coding genes (TRESPERG), and microsatellite methods, respectively, to compare isolates obtained from CAPA patients and the environment [22,25]. The authors identified that the CAPA isolates were from a diverse genetic pool [22,23,25]. However, compared to WGS, TRESPERG and microsatellite methods are limited in their power to provide an in-depth analysis of the genetic structure and diversity within a population [57,58], due to only utilising a small proportion of the whole genome [58]. Therefore, this study is the first to conduct an in-depth analysis to delineate the genetic diversity of A. fumigatus in CAPA cases. Furthermore, to show that the genotypes of A. fumigatus obtained from patients with CAPA are a genetically diverse mixture of clinical and environmental A. fumigatus genotypes, WGS is best placed to investigate [27,57].
COVID-19 patients who develop CAPA may have acquired A. fumigatus isolates from the environment. This is supported by the evidence that the genotypes of the CAPA isolates in this study are a mixture of genomes representative of both clinical and environmental A. fumigatus isolates. Previously, studies have identified that clinical non-CAPA A. fumigatus isolates are genetically similar to those sourced from the environment [22,25,32,56]. Additionally, ARAf has been isolated from several azole-naïve IA and CAPA cases [36,59,60] suggesting their resistance was pre-acquired from environmental (community and hospital) inocula. It has been hypothesised that patients who develop IA are first colonised with A. fumigatus isolates from airborne conidia in their environment. Thus, CAPA patients may acquire and become colonised with A. fumigatus on exposure to airborne A. fumigatus conidia in either the community or from the hospital environment. These conidia, under favourable conditions (immunosuppression from SARs-CoV-2 and drugs used to treat COVID-19, such as dexamethasone and tazoluzimab), develop hyphae and subsequently invade the surrounding tissue, leading to the onset of CAPA. If the patient is being exposed or treated with triazoles, it is at this stage that resistant inocula will remain in the patient’s airway.
The present study used bioinformatic tools and drug susceptibility tests to identify three ARAf out of 21 CAPA isolates containing the predominant cyp51A polymorphism TR34/L98H and that were phenotypically resistant to at least one azole. Furthermore, all three were pan-azole-resistant [36]. No further CAPA isolates had known azole-resistant polymorphisms and were susceptible to azoles phenotypically. A limitation of the current study is the screening tests to infer if an isolate is potentially susceptible or resistant. Thus, a robust and standardised approach (e.g., EUCAST broth microdilution method) is required to give the accurate and valid phenotyping of the isolates [26,39,41]. In a large Spanish tertiary hospital of 28 CAPA patients, the ARAf prevalence was zero [22]. This is despite ARAf being previously isolated from patients throughout Spain [61]. Similarly, a recently published small transnational study (n = 11) and a small German study (n = 4) did not identify any ARAf in the sample of CAPA isolates [21,24]. In a German multicentre study, the prevalence of ARAf isolates was found to be 22.2% (n = 6/27) [23]. Only one of the six CAPA isolates was identified to have known cyp51A polymorphisms conferring resistance [23]. In the Netherlands, a screening programme of CAPA patients identified one patient in a cohort of twenty-two to have an ARAf strain (4.5%) [62].
In the wider patient population, the prevalence of ARAf has been increasing in the last 30 years from 0.43% to 2.2% in London [63]. Different countries have recently reported varying levels: Germany at 3.5%, Denmark at 6.1%, Spain at 7.4%, Netherlands at 11.7%, and Japan at 12.7% [17,20,61,64,65]. In the UK, the prevalence of ARAf in the air was recently found to range from 3 to 9%, depending on the season [16,19]. Thus, the consideration of ARAf surveillance in CAPA and wider patient populations who may be exposed to Aspergillus conidia in the hospital air could be recommended owing to this near-ubiquitous exposure. However, this is not a cause for changing patient treatments.
There are some limitations to this study. During the COVID-19 pandemic, there were challenges in diagnosing CAPA. Therefore, relatively few patients were diagnosed with CAPA, so the sample size of this study was small. The small sample size of the CAPA isolates prevents the drawing of strong conclusions. Thus, future studies should include larger sample sizes. Secondly, it would have been useful to have clinical indicators and outcomes for the CAPA isolates to analyse any associations between the genotype and clinical phenotypes. This would help with understanding CAPA and improve patient outcomes. Finally, ARAf in CAPA was only identified in isolates from one country, owing to a small sample size. Thus, conclusions about the ARAf prevalence should be taken with caution. Ensuing studies with larger sample sizes from multiple countries across the globe would be better suited to determine the overall prevalence of ARAf in CAPA.

5. Conclusions

The present study is the largest transnational epidemiological analysis of the genomic relationship between CAPA isolates and the wider A. fumigatus population. We demonstrate that the genomes of the CAPA isolates comprised a diverse mixture of genotypes from the wider A. fumigatus population. The CAPA genomes are similar to those found in other clinical and environmental A. fumigatus, and are not composed of a unique sub-population. Finally, ARAf is identified in this at-risk group of patients.
Future directions should focus on developing user-friendly and easily accessible surveillance methodologies for assaying A. fumigatus for both clinical and environmental settings. Surveillance programmes should include methods of monitoring antifungal resistance, owing to the observed prevalence of resistance to frontline clinical azoles (e.g., itraconazole, voriconazole, isavuconazole, and posaconazole). Furthermore, exposure assessments could be used to aid clinical decisions on the most effective course of antifungal treatment. Finally, a review of local antifungal treatment guidelines in response to the increasing prevalence of ARAf needs to be further considered.

Author Contributions

Conceptualization, B.C.S., J.R. and M.C.F.; methodology, B.C.S., J.R. and M.C.F.; software, B.C.S. and J.R.; validation, B.C.S. and J.R.; formal analysis, B.C.S.; investigation, B.C.S.; resources, B.C.S., J.R. and M.C.F.; data curation, J.R., A.A., S.S., T.R.R., A.F.T., M.M., D.S., S.J.H., P.E.V. and T.B.; writing—original draft preparation, B.C.S.; writing—review and editing, B.C.S., J.R., M.C.F., T.R.R., P.E.V., A.A., A.F.T., A.G., M.M. and S.S.; visualization, B.C.S.; supervision, J.R., M.C.F., T.R.R., A.A. and P.E.V.; project administration, B.C.S.; funding acquisition, M.C.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by NERC (nos. NE/P001165/1 and NE/P000916/1), the UK Medical Research Council (MRC) (no. MR/R015600/1), and Wellcome Trust (no. 219551/Z/19/Z). M.C.F. is supported by the CIFAR Fungal Kingdoms Program.

Institutional Review Board Statement

All clinical isolates were collected and analysed as part of the ‘standard of care’ and all isolates were anonymised. The investigation of cases from which these isolates (C1–C200, E9–E206, U1–U3, four German CAPA isolates, and C444) originated had ethics approval and the data are publicly available [24,32,36]. The patients whose isolates were C435–C441, C443 were identified as having CAPA as part of an audit/service improvement that had prior approval of the the Research and Innovation Office, and of the Clinical Service Director, St. James’s Hospital, Dublin, Ireland.

Informed Consent Statement

Not applicable.

Data Availability Statement

All raw reads have been submitted to the European Nucleotide Archive (ENA) under Project Accession no. PRJEB60964. Two hundred and twenty-one raw reads of clinical non-CAPA and environmental A. fumigatus isolates have also been deposited under project accession no. PRJEB27135. The raw short reads of four German CAPA isolates have previously been deposited to the NCBI’s GenBank database under BioProject accession no. PRJNA673120.

Conflicts of Interest

M.C.F. and J.R. have received honoraria from Gilead Sciences for presentations. T.R. has received a research grant from Pfizer Healthcare Ireland outside of this work. A.F.T. has received a research grant from Gilead Sciences outside of this work. S.S. has received honoraria from Gilead Sciences and Pfizer for presentations.

Appendix

Table A1. Polymorphisms reported in the literature that are associated with azole resistance in A. fumigatus, were used to search for azole resistance in the CAPA isolates in this study.
Table A1. Polymorphisms reported in the literature that are associated with azole resistance in A. fumigatus, were used to search for azole resistance in the CAPA isolates in this study.
GenePolymorphismReference
Afu4g06890—cyp51ATR34 [32]
TR34/L98H [15,16,17,23,30,32,56,64,65,66,67,68,69,70,71,72,73]
TR34/L98H/S297T/F495I [15,18]
TR34/L98H/T289A/I364V/G448S [16,32]
TR46/Y121F/T289A [16,17,30,32,64,66,68,69,74]
TR46/L98H/T289A/I364V/G448S [16]
(TR46)2/Y121F/M172V/T289A/G448S [16]
TR53 [16]
TR120 [16]
F46Y/M172V/N248T/D255E/E427K [15,65,75,76]
F46Y/M172V/E427K [23]
G138C [77,78]
Y121F [18]
Y431C [75,77,78]
G434C [77,78]
G432S [18]
M220L [17,75]
M220T [30,79]
M220R [18,80]
M220I [18,31,79]
M220K [18,67]
M220V [81]
N248K [15,67,72,82]
A284T [30,75]
H285Y [75,77]
G54E [30,32,67,75]
G54R [18,30,31,32,72,75,80]
G54W [18,30,31,32,65,81]
G54V [30,80]
G448S [18,30,81]
F219V [30]
F219I [30,65,75]
F219L [18]
F219C [81]
P216H [30]
P216S [18]
P216L [32,65,75,77,80,81]
P216A [31]
H147Y [77]
I242V [16,82]
I266N [31]
(TR130)3/D430G [16]
Afu3g09850—MshAE812G [28]
A45T
P329T
E467N
Afu6g05300—hapEP88L [83]
Afu7g01960—Unknown transcription factor167* [84]
Afu2g03700—hmg1L273F [81]
Afu2g03700—hmg1S305P/M220I [81]
Afu4g06890—cyp51A
Afu2g03700—hmg1A640V/A9T [31]
Afu4g06890—cyp51A
Afu2g03700—hmg1H237Y/N157S [31]
Afu4g03630—erg6
Afu2g03700—hmg1S269C/G54R [31]
Afu4g06890 –cyp51A
Afu4g03630—erg6W320G [83]
Afu4g04820—erg25W218 *
Afu4g11240—aarAF481S
Afu5g07960—C2H2Y347 *
Afu1g17440—ABC drug transporterY1149N [32]
Afu1g17440—ABC drug transporterOver expression [85]
Afu2g14250—NctALoss-of-function mutations [29]
Afu3g02340—NctBLoss-of-function mutations
Afu2g16260—microtubule bundle proteinUnknown [27]
Afu4g04680—FGGY-family kinaseUnknown
Afu4g04960—uncharacterized ORFUnknown
Polymorphisms with * refer to a premature stop codon.
Table A2. Characteristics of additional clinical and environmental isolates of A. fumigatus used in this study to provide context for CAPA isolates, and details of WGS alignments.
Table A2. Characteristics of additional clinical and environmental isolates of A. fumigatus used in this study to provide context for CAPA isolates, and details of WGS alignments.
Isolate IDCity of OriginSourceNumber of Aligned Reads (Millions)Percentage of Reference Genome Covered (%)Mean Depth of Coverage (X)
C1LeedsClinical54.697.1189.3
C2LeedsClinical50.896.0175.4
C3LeedsClinical45.596.4157.3
C4LeedsClinical45.798.2158.8
C5LeedsClinical46.699.6167.8
C6LeedsClinical52.697.4185.2
C34DublinClinical6.697.134.6
C35DublinClinical6.197.531.6
C36DublinClinical6.597.333.6
C37DublinClinical6.698.534.5
C38DublinClinical6.698.634.7
C39DublinClinical7.197.237.1
C40DublinClinical7.197.737.1
C41DublinClinical6.998.036.4
C42DublinClinical7.39838.3
C43DublinClinical7.797.840.4
C44DublinClinical6.99734.7
C45DublinClinical6.79734.5
C46DublinClinical6.197.631.8
C47DublinClinical1.999.633.1
C48DublinClinical6.595.333.8
C49DublinClinical7.196.336.5
C50DublinClinical7.196.436.8
C51DublinClinical7.697.840.1
C52DublinClinical8.498.144.1
C53DublinClinical7.596.838.5
C54DublinClinical7.598.239
C55DublinClinical7.097.136.2
C56DublinClinical6.697.234.1
C57DublinClinical8.097.141.3
C58DublinClinical7.397.137.7
C59DublinClinical7.697.739.5
C60DublinClinical6.19732
C61DublinClinical7.397.538.3
C62DublinClinical6.597.334.3
C63DublinClinical6.997.536.3
C64DublinClinical7.096.836.7
C65DublinClinical7.196.837.1
C66DublinClinical7.197.337
C67DublinClinical7.397.2538.4
C68DublinClinical6.69734.3
C69DublinClinical6.397.332.9
C70DublinClinical7.097.436.1
C71DublinClinical6.496.933.3
C72DublinClinical6.597.632.6
C73DublinClinical7.497.739
C74DublinClinical7.398.138.3
C75DublinClinical7.797.640.3
C76DublinClinical8.597.644.2
C77DublinClinical8.797.245
C78DublinClinical6.697.134.4
C79DublinClinical8.297.842.6
C80DublinClinical6.897.435.1
C81EdinburghClinical5.997.629.6
C82BrightonClinical6.397.532
C85LondonClinical5.897.229.2
C86EdinburghClinical5.797.729.1
C87EdinburghClinical5.697.528.6
C88LondonClinical6.097.330.1
C89LondonClinical6.59733
C90CambridgeClinical6.897.134
C91LiverpoolClinical6.197.430.4
C92CardiffClinical6.397.731.8
C93BristolClinical6.697.333.1
C94LondonClinical6.597.832.9
C95LondonClinical6.297.131.4
C96EdinburghClinical6.697.733.2
C97LondonClinical6.29731.2
C99CardiffClinical6.198.431.1
C100BirminghamClinical6.398.432.3
C101CambridgeClinical6.297.331.3
C102LeicesterClinical6.397.631.9
C103LondonClinical6.398.532.5
C104LondonClinical6.597.632.8
C105LondonClinical6.097.530.6
C106LondonClinical5.896.329.6
C107LondonClinical5.697.928.5
C108LondonClinical5.896.829.2
C109LondonClinical5.897.629.7
C110LondonClinical5.595.227.8
C111LondonClinical5.697.728.5
C112LondonClinical5.897.829.5
C113LondonClinical6.097.730.6
C114LondonClinical6.597.632.9
C115LondonClinical5.997.830
C116LondonClinical6.197.730.6
C117LondonClinical6.497.732.5
C118LondonClinical5.895.329.2
C119LondonClinical5.995.329.8
C120LondonIA (ABPA)6.39731.7
C121LondonClinical6.397.532.2
C122LondonClinical6.595.732.8
C123LondonClinical6.497.732.3
C124LondonClinical6.596.932.5
C125LondonClinical6.497.632.4
C126LondonClinical6.397.631.7
C127LondonClinical6.297.031.7
C128LeedsClinical6.996.834.8
C129LeedsClinical7.296.635.9
C130LeedsClinical7.396.536.6
C131LeedsClinical6.996.424.6
C132LeedsClinical7.296.335.9
C133LeedsClinical6.896.233.9
C134LeedsClinical7.096.734.8
C135LeedsClinical6.996.734.6
C136LeedsClinical7.096.635.1
C137LondonIA (NAf) 7.097.935.7
C138LondonIA (NAf)6.497.932.5
C139LondonIA (NAf)6.398.232.3
C140LondonIA (NAf)6.298.231.9
C141LondonClinical6.396.831.9
C142LondonClinical6.397.131.9
C143LondonIA (Trauma)6.396.731.8
C144LondonClinical6.698.433.8
C145LondonClinical6.697.934.2
C146LondonClinical6.496.832.4
C147LondonClinical6.498.233.2
C148LondonClinical6.697.932.8
C149LondonClinical6.596.633.3
C150LondonClinical6.597.732.6
C151LondonClinical6.497.232.7
C152LondonClinical6.497.236.8
C153LondonClinical7.396.537.3
C154LondonClinical7.497.437.5
C155LondonClinical7.398.439.7
C156LondonClinical7.798.435.6
C157LondonClinical7.197.235
C158LondonClinical7.097.739
C159LondonClinical7.698.136
C160LondonClinical7.297.238.2
C161LondonClinical7.598.029.2
C162LondonClinical5.895.633.6
C163LondonClinical6.697.74
C164LondonClinical7.298.236
C165LondonClinical6.896.634.3
C166LondonClinical6.894.734.5
C167LondonClinical6.897.234.2
C168LondonClinical7.098.136
C169LondonClinical6.797.434.3
C170LondonClinical7.198.2436.7
C171LondonClinical6.697.833.2
C172LondonClinical6.596.933.2
C173LondonClinical6.896.634.1
C191DublinClinical9.197.046.5
C192DublinClinical9.997.249.9
C193DublinClinical8.997.645.2
C194DublinEnvironmental8.997.245.5
C195DublinEnvironmental9.396.247
C196DublinClinical10.396.151.7
C197DublinEnvironmental8.796.344.1
C198DublinClinical9.196.045.8
C199DublinClinical8.497.742.3
C200DublinClinical9.296.846.7
C307UKIA (IPA)5.797.321.6
C323UKIA (ABPA and asthma)6.197.323.5
C360UKIA (ABPA)2.597.78.9
C372UKIA (CPA)7.198.229.5
C376UKIA (ABPA and asthma)6.998.027.8
C402NetherlandsCOVID—Colonising31.397.5139.1
C404NetherlandsCOVID—Colonising49.498.3 216.4
C405NetherlandsCOVID—Colonising26.198.3 117.0
C406NetherlandsCOVID—Colonising28.397.1 124.2
C407NetherlandsCOVID—Colonising37.798.6 166.3
C409NetherlandsCOVID—Colonising33.197.9 147.3
C410NetherlandsCOVID—Colonising29.998.3132.0
C426IrelandEnvironmental10.797.743.0
C427IrelandEnvironmental11.497.644.9
C428IrelandEnvironmental10.597.841.6
C429IrelandEnvironmental9.797.338.1
C430IrelandEnvironmental11.797.244.8
C431IrelandEnvironmental11.697.844.7
C432IrelandEnvironmental9.197.737.1
C433IrelandEnvironmental1.097.84.5
C434IrelandClinical10.696.641.9
C442IrelandIA (IPA)11.997.845.6
C443IrelandCOVID—Colonising9.797.539.2
U1DublinClinical6.297.932.3
U2DublinClinical7.497.6338.5
U3DublinClinical7.597.139.2
E9WalesEnvironmental6.197.631.8
E10WalesEnvironmental7.297.838
E11WalesEnvironmental6.597.732.7
E12WalesEnvironmental6.296.632.2
E13WalesEnvironmental6.797.434.7
E14WalesEnvironmental7.398.038.4
E15WalesEnvironmental6.496.933.5
E16WalesEnvironmental6.897.535.5
E17WalesEnvironmental7.097.136.5
E18WalesEnvironmental7.497.038.6
E19WalesEnvironmental6.997.535.9
E20WalesEnvironmental7.297.037
E21WalesEnvironmental7.695.639.6
E22WalesEnvironmental7.497.837.1
E23WalesEnvironmental6.896.435.1
E24WalesEnvironmental7.597.239
E25WalesEnvironmental6.299.633.3
E26WalesEnvironmental6.496.333.4
E27WalesEnvironmental7.097.736.5
E28WalesEnvironmental6.997.735.6
E29WalesEnvironmental0.899.746.5
E30WalesEnvironmental7.497.438.5
E31WalesEnvironmental6.197.732
E32WalesEnvironmental7.098.637
E33WalesEnvironmental5.398.226.6
E34WalesEnvironmental6.298.232.6
E35WalesEnvironmental5.797.630.2
E36WalesEnvironmental5.897.330.4
E37WalesEnvironmental5.997.030.5
E38WalesEnvironmental6.497.633.2
E39DublinEnvironmental8.197.242.5
E40DublinEnvironmental8.196.741.6
E41DublinEnvironmental7.897.140.6
E42DublinEnvironmental7.197.536.7
E43DublinEnvironmental7.296.637.1
E44DublinEnvironmental8.098.139.4
E45DublinEnvironmental7.496.137.9
E46DublinEnvironmental7.397.937.7
E48DublinEnvironmental6.897.835.6
E49DublinEnvironmental7.397.538.1
E50DublinEnvironmental6.697.234.5
E52DublinEnvironmental7.397.238
E53DublinEnvironmental7.697.839.2
E81DublinEnvironmental6.698.231.5
E82DublinEnvironmental7.098.233.1
E83DublinEnvironmental8.198.338.1
E84DublinEnvironmental7.898.036.8
E107NottinghamEnvironmental9.798.044.1
E133NottinghamEnvironmental9.498.243.3
E136YorkshireEnvironmental8.196.137.1
E190DidcotEnvironmental8.596.543.3
E191LondonEnvironmental9.997.350.7
E192LondonEnvironmental9.097.345.5
E193LondonEnvironmental9.197.946.9
E194LondonEnvironmental8.997.445.4
E195LondonEnvironmental8.696.643.9
E201AberdeenEnvironmental1.699.748.9
E202AberdeenEnvironmental10.097.850.5
E203AberdeenEnvironmental10.097.150.8
E204AberdeenEnvironmental10.297.952.3
E205AberdeenEnvironmental9.694.549.9
E206AberdeenEnvironmental9.397.547.1
ABPA, allergic bronchopulmonary aspergillosis. CPA, chronic pulmonary aspergillosis. IA, invasive aspergillosis. IPA, invasive pulmonary aspergillosis. NAf, necrotising A. fumigatus.
Table A3. Relative frequencies of clade assignment, mating type idiomorph, and cyp51A polymorphisms for CAPA, clinical, and environmental A. fumigatus isolates in this study.
Table A3. Relative frequencies of clade assignment, mating type idiomorph, and cyp51A polymorphisms for CAPA, clinical, and environmental A. fumigatus isolates in this study.
Number of Isolate Types (%)χ2-test p-Value
CAPA (n = 21)Clinical (n = 167)Environmental (n = 73)
Clade
A6 (28.6)75 (44.9)56 (76.7)0.002 *
B15 (71.4)92 (55.1)17 (23.3)0.001 *
MAT idiomorph
MAT1-111 (52.4)73 (43.7)18 (24.7)0.056
MAT1-210 (47.6)94 (56.3)55 (75.3)0.158
Cyp51A genotype
TR34/L98H3 (14.3)47 (28.1)42 (57.5)<0.001 *
TR34001 (1.4)0.041 *
TR34/L98H/T289A/I364V/G448S04 (2.4)00.091
TR46/Y121F/T289A00 7 (9.6)<0.001 *
P216L02 (1.2)00.093
G54W09 (5.4)00.030 *
G54E01 (0.6)00.041 *
G54R03 (1.8)1 (1.4)0.295
WT18 (85.7)101 (60. 5)22 (30.1)0.002 *
Resistance to >= 1 azole
Yes3 (14.3)76 (45.5)43 (58.9)0.026 *
No18 (85.7)88 (52.7)19 (26.0)<0.001 *
Test not performed03 (1.8)11 (15.1)<0.001 *
(p-value *, is significant if the value p < 0.05).
Table A4. Azole drug susceptibility of CAPA, clinical, and environmental A. fumigatus isolates grown in minimal media and the associated candidate polymorphisms.
Table A4. Azole drug susceptibility of CAPA, clinical, and environmental A. fumigatus isolates grown in minimal media and the associated candidate polymorphisms.
Isolate IDITR MIC (mg/L)VOR MIC
(mg/L)		POS MIC
(mg/L)		TRGene of A. fumigatus Amino Acid Substitution
Afu4g06890Afu1g17440AFu7g01960Afu4g03630
C438*>1642TR34L98HE581A/Y1149NWTWT
C441*840.5TR34L98HE581A/Y1149NWTWT
C444>3221TR34L98HE581A/Y1149NI149V/
P160S		WT
C720.120.038WTWTG166AI149V/
P160S		A281V
C9912NDWTWTG166AI149V/
P160S		A281V
C10081NDWTWTN1073HI149V/
P160S		WT
C109>160.250.125WTWTG166AI149V/
P160S		WT
C110>1621WTWTG166AI149V/
P160S		WT
C111820.5WTWTG166AI149V/
P160S		A281V
C118>1622WTWTG166AI149V/
P160S		WT
C119>1622WTWTG166AI149V/
P160S		WT
C121>1621WTWTG166AI149V/
P160S		A281V
C13020.1250.03WTWTH222R/R645CI149V/
P160S		WT
C144221WTWTE581A/Y1149NI149V/
P160S		WT
C146210.125WTWTV1439LI149V/
P160S		WT
C14720.50.125WTWTG166AI149V/
P160S		WT
C15020.250.03WTWTG166AI149V/
P160S		WT
C310.250.06WTWTWTA87SWT
C410.50.25WTWTWTA87SWT
C50.50.1250.06WTWTWTP160SWT
C60.50.50.06WTWTE581A/S715G/Y1149NWTWT
C36<0.030.060.03WTWTN1073HI149V/P160SWT
C370.060.060.06WTWTT743S/Y1149NWTWT
C390.250.1250.06WTWTN1073HI149V/P160SA281V
C580.120.120.015WTWTG166AI149V/P160SA281V
C590.060.060.03WTWTG166AI149V/P160SA281V
C730.030.120.015WTWTG166AI149V/P160SA281V
C740.030.120.015WTWTG166AI149V/P160SWT
C750.030.120.015WTWTG166AI149V/P160SWT
C760.250.250.12WTWTG166AI149V/P160SWT
C770.250.250.06WTWTG166AI149V/P160SWT
C95ND0.25NDWTWTG166A/V1439LI149V/P160SWT
C14810.250.06WTWTN1073HI149V/P160SA281V
C1540.060.060.03WTWTG166AI149V/P160SWT
C1580.060.060.03WTWTG166AI149V/P160SWT
C1610.060.060.03WTWTG166AI149V/P160SA281V
C1690.060.060.03WTWTG166AI149V/P160SA281V
C3600.060.30.015WTWTG166A/N1073HI149V/P160SA281V
C376<0.015<0.015<0.015WTWTG166AI149V/P160SWT
C402NDNDNDWTWTN1073I149V/P160SWT
C4030.250.250.063WTWTR645C/H222RWTWT
C4040.250.50.063WTWTN1073P160SWT
C4050.250.250.063WTWTG166AP160SWT
C4060.50.50.125WTWTG166AI149V/P160SWT
C4070.250.50.063WTWTN1073I149V/P160SWT
C4080.1250.50.031WTWTN1073I149V/P160SWT
C4090.50.250.125WTWTN1073I149V/P160SWT
C4100.50.50.063WTWTN1073I149V/P160SWT
C442NDNDNDTR34L98HE581A/Y1149NWTWT
EUCAST clinical breakpoint MICs: ITR, itraconazole (>1 mg/L); VOR, voriconazole (>1 mg/L); POS, posaconazole (>0.25 mg/L) [41]. TEB, tebuconazole (breakpoint >4 mg/L). ND, test not performed. EUCAST broth microdilution method was used to test C1-C200, E9-E206, and U1-U3 by Rhodes et al. [32]; C444 by Mohamed et al. [36]; and C307, C323, C360, C372, and C376 by Armstrong-James [38]. C438* and C441* were tested using the Clinical and Laboratory Standards Institute (CLSI) method [42]. The epidemiological cut-off values for the CLSI method: ITR, itraconazole (>1 mg/L); VOR, voriconazole (>1 mg/L); POS, posaconazole (>1 mg/L) [42].
Table A5. Azole drug susceptibility of A. fumigatus isolates used in study grown in minimal media and the associated polymorphisms.
Table A5. Azole drug susceptibility of A. fumigatus isolates used in study grown in minimal media and the associated polymorphisms.
Isolate
ID		ITR MIC (mg/L)VOR MIC (mg/L)POS MIC
(mg/L)		TEB ScoreResistance
Marker		Clade MembershipMating Type
C1>1610.53TR34/L98HAMAT1-1
C2>1610.52TR34/L98HAMAT1-1
C310.250.06NDWTBMAT1-2
C410.50.251WTAMAT1-1
C50.50.1250.06NDWTBMAT1-2
C60.50.50.061WTAMAT1-1
C34>160.50.51TR34/L98HAMAT1-2
C35>1610.5NDTR34/L98HAMAT1-2
C36<0.030.060.03NDWTBMAT1-1
C370.060.060.06NDWTAMAT1-1
C380.060.250.061WTAMAT1-1
C390.250.1250.06NDWTBMAT1-1
C400.1250.250.06NDWTAMAT1-1
C410.060.250.06NDWTBMAT1-2
C420.060.250.03NDWTBMAT1-1
C430.250.250.25NDWTBMAT1-1
C440.250.250.12NDWTBMAT1-2
C450.030.120.015NDWTBMAT1-2
C460.030.120.015NDWTBMAT1-2
C470.030.120.015NDWTBMAT1-2
C480.030.120.015NDWTBMAT1-2
C490.030.0150.015NDWTBMAT1-2
C500.030.0150.015NDWTBMAT1-1
C510.120.250.06NDWTBMAT1-1
C520.250.120.06NDWTBMAT1-1
C530.060.250.03NDWTBMAT1-1
C54<0.0150.120.03NDWTBMAT1-2
C550.250.120.06NDWTBMAT1-2
C560.060.250.03NDWTBMAT1-2
C570.060.060.03NDWTBMAT1-2
C580.120.120.015NDWTBMAT1-1
C590.060.060.03NDWTBMAT1-1
C600.030.120.015NDWTAMAT1-1
C610.250.120.06NDWTAMAT1-1
C620.120.120.03NDWTAMAT1-1
C630.030.250.015NDWTAMAT1-1
C640.030.250.015NDWTAMAT1-1
C650.030.120.015NDWTAMAT1-1
C660.250.250.12NDWTAMAT1-1
C670.030.120.015NDWTAMAT1-1
C680.030.120.015NDWTAMAT1-1
C690.250.250.06NDTR34/L98HAMAT1-2
C7010.50.25NDTR34/L98HAMAT1-2
C71110.25NDTR34/L98HAMAT1-2
C720.120.038NDWTBMAT1-1
C730.030.120.015NDWTBMAT1-1
C740.030.120.015NDWTBMAT1-2
C750.030.120.015NDWTBMAT1-2
C760.250.250.12NDWTAMAT1-1
C770.250.250.06NDWTBMAT1-1
C78421NDTR34/L98HAMAT1-2
C791620.5NDTR34/L98HAMAT1-2
C80440.5NDTR34/L98HAMAT1-2
C81220.5NDTR34/L98HAMAT1-1
C82164NDNDTR34/L98HAMAT1-2
C8512NDNDTR34/L98HAMAT1-2
C86>160.5NDNDTR34/L98HAMAT1-1
C87>164ND1TR34/L98HAMAT1-2
C8824ND1TR34/L98HAMAT1-2
C8914ND3TR34/L98HAMAT1-2
C90161ND4TR34/L98HAMAT1-2
C91>161NDNDTR34/L98HAMAT1-1
C92>161NDNDTR34/L98HAMAT1-2
C93162NDNDTR34/L98HAMAT1-1
C94162ND4TR34/L98HAMAT1-1
C95ND0.25NDNDWTBMAT1-2
C96>162NDNDTR34/L98HAMAT1-1
C97410.25NDTR34/L98HAMAT1-1
C9912NDNDWTBMAT1-2
C10081NDNDWTBMAT1-2
C101161NDNDTR34/L98HAMAT1-2
C102>160.125NDNDG54RBMAT1-1
C103>160.250.5NDG54EAMAT1-1
C104>162NDNDTR34/L98HAMAT1-2
C105>1620.25NDTR34/L98HAMAT1-2
C106>160.1254NDG54WBMAT1-1
C107>16224TR34/L98HAMAT1-2
C108>160.032NDG54WBMAT1-1
C109>160.250.125NDWTBMAT1-2
C110>1621NDWTBMAT1-2
C111820.51WTBMAT1-2
C112>160.1258NDG54WBMAT1-2
C113>160.12516NDG54WBMAT1-2
C114>160.1258NDG54WBMAT1-2
C115>1622NDTR34/L98HAMAT1-2
C1161621NDTR34/L98HAMAT1-1
C117>160.12516NDG54WBMAT1-2
C118>16223WTBMAT1-2
C119>16223WTBMAT1-2
C120>160.252NDP216LBMAT1-1
C121>16211WTBMAT1-2
C122>160.12516NDG54WBMAT1-1
C1231610.25NDL98HAMAT1-1
C124>160.060.25NDG54WAMAT1-1
C1251620.5NDTR34/L98HAMAT1-2
C126>1620.25NDTR34/L98HAMAT1-2
C127>160.1250.5NDP216LBMAT1-2
C1280.50.1250.03NDWTBMAT1-2
C12910.1250.03NDWTBMAT1-2
C13020.1250.03NDWTBMAT1-2
C131>160.250.125NDTR34/L98HAMAT1-1
C132160.250.125NDTR34/L98HAMAT1-1
C133>160.250.125NDTR34/L98HAMAT1-1
C1340.50.1250.03NDWTBMAT1-2
C13510.1250.03NDWTBMAT1-2
C13610.1250.03NDWTBMAT1-2
C13716>1644TR34/L98H/T289A/
I364V/G448S		AMAT1-2
C13816>1644TR34/L98H/T289A/
I364V/G448S		AMAT1-2
C13916>164NDTR34/L98H/T289A/
I364V/G448S		AMAT1-2
C14016>1644TR34/L98H/T289A/
I364V/G448S		AMAT1-2
C141>1620.54TR34/L98HAMAT1-2
C142>1620.53TR34/L98HAMAT1-2
C14340.50.1254TR34/L98HAMAT1-1
C144221NDWTAMAT1-1
C145>161>16NDG54WAMAT1-1
C146210.1253WTBMAT1-2
C14720.50.125NDWTBMAT1-1
C14810.250.06NDWTBMAT1-2
C1490.060.060.03NDWTBMAT1-1
C15020.250.03NDWTBMAT1-1
C1510.060.060.0151WTBMAT1-2
C1520.060.060.015NDWTBMAT1-2
C153160.50.25NDTR34/L98HAMAT1-2
C1540.060.060.031WTBMAT1-1
C1550.060.060.015NDWTBMAT1-1
C1560.060.060.03NDWTBMAT1-1
C1570.060.060.03NDWTBMAT1-2
C1580.060.060.03NDWTBMAT1-1
C1590.060.1250.03NDWTAMAT1-1
C1600.060.060.03NDWTBMAT1-1
C1610.060.060.03NDWTBMAT1-1
C1620.060.060.034WTBMAT1-2
C1630.060.1250.06NDWTBMAT1-1
C1640.1250.1250.03NDWTBMAT1-2
C1650.1250.060.031WTBMAT1-2
C1660.060.060.03NDWTBMAT1-2
C1670.060.060.03NDWTBMAT1-1
C1680.060.060.03NDWTAMAT1-1
C1690.060.060.03NDWTBMAT1-2
C1700.1250.060.03NDWTAMAT1-1
C171>160.060.25NDG54RBMAT1-2
C1720.060.030.03NDWTBMAT1-1
C1730.060.060.0153WTBMAT1-2
C191420.5NDTR34/L98HAMAT1-2
C192220.5NDTR34/L98HAMAT1-1
C1930.520.5NDTR34/L98HAMAT1-2
C194NDNDNDNDTR34/L98HAMAT1-1
C195NDNDNDNDTR46/Y121F/T289AAMAT1-2
C1963220.5NDTR34/L98HAMAT1-2
C197NDNDNDNDTR46/Y121F/T289AAMAT1-2
C198NDNDNDNDWTBMAT1-2
C1990.750.1250.25NDWTAMAT1-2
C2000.030.120.25NDWTBMAT1-2
C307NDNDND2TR34/L98HAMAT1-1
C323>40.250.06NDTR34/L98HAMAT1-2
C3600.060.30.0151WTBMAT1-2
C372NDNDNDNDG54WBMAT1-2
C376<0.015<0.015<0.0151WTBMAT1-2
C402NDNDNDNDWTBMAT1-1
C4030.250.250.063NDWTBMAT1-2
C4040.250.50.063NDWTBMAT1-2
C4050.250.250.063NDWTBMAT1-2
C4060.50.50.125NDWTBMAT1-2
C4070.250.50.063NDWTBMAT1-2
C4080.1250.50.031NDWTBMAT1-1
C4090.50.250.125NDWTBMAT1-1
C4100.50.50.063NDWTBMAT1-1
C422NDNDND1WTBMAT1-2
C423NDNDND1WTBMAT1-1
C424NDNDND1WTBMAT1-2
C425NDNDND1WTAMAT1-1
C426NDNDND1WTBMAT1-1
C427NDNDND1WTAMAT1-2
C428NDNDND1WTBMAT1-2
C429NDNDND1WTAMAT1-1
C430NDNDND1WTAMAT1-1
C431NDNDND1WTBMAT1-1
C432NDNDND1WTBMAT1-2
C433NDNDND3WTAMAT1-2
C434NDNDND1WTAMAT1-1
C435NDNDND1WTBMAT1-2
C436NDNDND1WTBMAT1-1
C437NDNDND1WTBMAT1-1
C438*>16423TR34/L98HAMAT1-1
C439NDNDND1WTBMAT1-2
C440NDNDND1WTBMAT1-2
C441*840.54TR34/L98HAMAT1-1
C442NDNDND3TR34/L98HAMAT1-1
C443NDNDND1WTAMAT1-1
C444>32213TR34/L98HAMAT1-1
C611NDNDND1WTBMAT1-2
C612NDNDND1WTBMAT1-2
CAPA-A*0.50.51NDWTAMAT1-2
CAPA-B*0.50.51NDWTBMAT1-2
CAPA-C*0.50.51NDWTBMAT1-1
CAPA-D*0.50.51NDWTBMAT1-1
E9>1620.123TR34/L98HAMAT1-2
E10>1620.25NDTR34/L98HAMAT1-2
E11>1620.12NDTR34/L98HAMAT1-2
E12>168<0.54TR34/L98HBMAT1-2
E13>1620.123TR34/L98HAMAT1-2
E14>1620.25NDTR34/L98HAMAT1-2
E15>1640.25NDTR34/L98HAMAT1-2
E16>1620.12NDTR34/L98HAMAT1-2
E17>1680.5NDTR34/L98HAMAT1-2
E18>1620.25NDTR34/L98HAMAT1-2
E19>1640.25NDTR34/L98HAMAT1-2
E20>1620.12NDTR34/L98HAMAT1-2
E21>16>80.5NDTR34/L98HAMAT1-2
E22>168<0.5NDTR34/L98HAMAT1-2
E23>1620.12NDTR34/L98HAMAT1-2
E24>1620.12NDTR34/L98HAMAT1-2
E25>1620.12NDTR34/L98HAMAT1-2
E26>1610.25NDTR34/L98HAMAT1-2
E27>162<0.25NDTR34/L98HAMAT1-2
E28>1620.0253TR34/L98HAMAT1-2
E29>1620.123TR34/L98HAMAT1-2
E30>1620.12NDTR34/L98HAMAT1-2
E31>1620.12NDTR34/L98HAMAT1-2
E32>1640.25NDTR34/L98HAMAT1-2
E33>160.251NDG54RAMAT1-2
E34>1610.25NDTR34/L98HAMAT1-2
E35>1640.25NDTR34/L98HAMAT1-2
E36>1640.5NDTR34/L98HAMAT1-2
E37>1620.25NDTR34/L98HAMAT1-2
E38>1620.12NDTR34/L98HAMAT1-2
E39NDNDND3TR34/L98HAMAT1-2
E40NDNDND4TR46/Y121F/T289AAMAT1-2
E41NDNDNDNDTR34/L98HAMAT1-2
E42NDNDND4TR34/L98HAMAT1-2
E43NDNDND4TR34/L98HAMAT1-1
E44NDNDNDNDTR46/Y121F/T289ABMAT1-1
E45NDNDND4TR34/L98HAMAT1-2
E46NDNDND4TR46/Y121F/T289AAMAT1-2
E48NDNDNDNDTR46/Y121F/T289ABMAT1-1
E49NDNDNDNDTR34/L98HAMAT1-2
E50NDNDNDNDTR46/Y121F/T289AAMAT1-2
E52NDNDNDNDWTBMAT1-1
E53NDNDNDNDWTBMAT1-2
E810.060.060.06NDWTAMAT1-1
E820.1250.060.061WTAMAT1-2
E830.060.060.03NDWTBMAT1-2
E840.1250.060.061WTAMAT1-1
E1070.1250.1250.06NDWTAMAT1-2
E1330.1250.1250.03NDWTBMAT1-2
E1360.1250.1250.06NDWTBMAT1-2
E190>160.50.25NDTR34/L98HAMAT1-1
E191>160.50.25NDTR34/L98HAMAT1-2
E1920.060.060.03NDWTBMAT1-2
E1930.1250.060.03NDWTBMAT1-1
E1940.250.060.03NDWTBMAT1-2
E1950.250.060.03NDWTBMAT1-1
E20140.5ND4TR34/L98HAMAT1-1
E202162NDNDTR34AMAT1-1
E20340.25NDNDTR34/L98HAMAT1-1
E20410.5NDNDWTBMAT1-2
E20540.5NDNDTR34/L98HAMAT1-2
E20610.5NDNDTR34/L98HAMAT1-1
U10.030.250.015NDWTBMAT1-2
U20.030.120.015NDWTBMAT1-2
U30.060.250.03NDWTBMAT1-1
EUCAST clinical breakpoint MICs: ITR, itraconazole (>1 mg/L); VOR, voriconazole (>1 mg/L); POS, posaconazole (>0.25 mg/L) [41]. TEB, tebuconazole (breakpoint > 4 mg/L). ND, test not performed. EUCAST broth microdilution method was used to test C1-C200, E9-E206, and U1-U3 by Rhodes et al. [32]; C444 by Mohamed et al. [36]; and C307, C323, C360, C372, and C376 by Armstrong-James [38]. C438*, C441*, and CAPA A*–D* were tested using the Clinical and Laboratory Standards Institute (CLSI) method [42]. The epidemiological cut-off values for the CLSI method: ITR, itraconazole (>1 mg/L); VOR, voriconazole (>1 mg/L); POS, posaconazole (>1 mg/L) [42].
Jof 09 01104 g0a1
Figure A1. Normalised depth of coverage by locus indicative of duplications and deletion events. (a) C440 Chromosome 1. The deleted region is also found in C408 and C439–C441. (b) C440 chromosome 4. Two regions, a 500 kbp deletion identified in all isolates, and a 1300 kbp deletion around the centromere of Chromosome 4. (c) C440 Chromosome 7. Two deletions in Chromosome 7, relating to the centromere and a region of 1600 kbp found in CAPA-A, CAPA-B, C403, 408, C436 and C439-C441, 444. (d) C444 Chromosome 8. Small deletion of 1400 kbp found in Chromosome 8 in isolates CAPA-B, CAPA-C, C424, C436, C441, 444.
Figure A1. Normalised depth of coverage by locus indicative of duplications and deletion events. (a) C440 Chromosome 1. The deleted region is also found in C408 and C439–C441. (b) C440 chromosome 4. Two regions, a 500 kbp deletion identified in all isolates, and a 1300 kbp deletion around the centromere of Chromosome 4. (c) C440 Chromosome 7. Two deletions in Chromosome 7, relating to the centromere and a region of 1600 kbp found in CAPA-A, CAPA-B, C403, 408, C436 and C439-C441, 444. (d) C444 Chromosome 8. Small deletion of 1400 kbp found in Chromosome 8 in isolates CAPA-B, CAPA-C, C424, C436, C441, 444.
Jof 09 01104 g0a1
Jof 09 01104 g0a2
Figure A2. Phylogenetic tree of CAPA isolates, A. fumigatus IA control isolates from London, UK, and isolates from colonising COVID-19 patients (Netherlands and Ireland). Rooted ML phylogenetic tree with bootstraps support over 1000 replicates performed on WGS SNP data. Branch length represents the average number of SNPs. * 0.04 = 3242 SNPs.
Figure A2. Phylogenetic tree of CAPA isolates, A. fumigatus IA control isolates from London, UK, and isolates from colonising COVID-19 patients (Netherlands and Ireland). Rooted ML phylogenetic tree with bootstraps support over 1000 replicates performed on WGS SNP data. Branch length represents the average number of SNPs. * 0.04 = 3242 SNPs.
Jof 09 01104 g0a2
Jof 09 01104 g0a3
Figure A3. Phylogenetic tree of CAPA and A. fumigatus clinical and environmental isolates from Ireland and the UK. Rooted ML phylogenetic tree, showing: right track 1, if the isolate contains cyp51A polymorphism; middle track 2, the clade the isolate is located; and left track 3, the source of the isolate (CAPA, clinical, or environmental). Branch length represents the average number of SNPs. * 0.1 = 33,839 SNPs.
Figure A3. Phylogenetic tree of CAPA and A. fumigatus clinical and environmental isolates from Ireland and the UK. Rooted ML phylogenetic tree, showing: right track 1, if the isolate contains cyp51A polymorphism; middle track 2, the clade the isolate is located; and left track 3, the source of the isolate (CAPA, clinical, or environmental). Branch length represents the average number of SNPs. * 0.1 = 33,839 SNPs.
Jof 09 01104 g0a3
Jof 09 01104 g0a4
Figure A4. Phylogenetic tree of CAPA and A. fumigatus clinical and environmental isolates from Ireland and the UK. Rooted ML phylogenetic tree with bootstraps support over 1000 replicates performed on WGS SNP data. Branch length represents the average number of SNPs. * 0.1 = 33,839 SNPs.
Figure A4. Phylogenetic tree of CAPA and A. fumigatus clinical and environmental isolates from Ireland and the UK. Rooted ML phylogenetic tree with bootstraps support over 1000 replicates performed on WGS SNP data. Branch length represents the average number of SNPs. * 0.1 = 33,839 SNPs.
Jof 09 01104 g0a4

References

  1. Brackin, A.; Hemmings, S.; Fisher, M.; Rhodes, J. Fungal Genomics in Respiratory Medicine: What, How and When? Mycopathologia 2021, 186, 589–608. [Google Scholar] [CrossRef] [PubMed]
  2. Ullmann, A.; Aguado, J.; Arikan-Akdagli, S.; Denning, D.; Groll, A.; Lagrou, K.; Lass-Flörl, C.; Munoz, R.L.P.; Verweij, P.; Warris, A.; et al. Diagnosis and management of Aspergillus diseases: Executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin. Microbiol. Infect. 2018, 24, e1–e38. [Google Scholar] [CrossRef] [PubMed]
  3. Brown, G.; Denning, D.; Gow, N.; Levitz, S.; Neta, M.; White, T. Hidden Killers: Human Fungal Infections. Sci. Transl. Med. 2012, 4, 165rv113. [Google Scholar] [CrossRef] [PubMed]
  4. Bongomin, F.; Gago, S.; Oladele, R.; Denning, D. Global and Multi-National Prevalence of Fungal Diseases—Estimate Precision. J. Fungi 2017, 3, 57. [Google Scholar] [CrossRef] [PubMed]
  5. Pakzad, R.; Malekifar, P.; Shateri, Z.; Zandi, M.; Rezayat, S.; Soleymani, M.; Karimi, M.; Ahmadi, S.; Shahbahrami, R.; Pakzad, I.; et al. Worldwide prevalence of microbial agents’ coinfection among COVID-19 patients: A comprehensive updated systematic review and meta-analysis. J. Clin. Lab. Anal. 2022, 36, e24151. [Google Scholar] [CrossRef]
  6. Baddley, J.; Thompson, G.; Chen, S.; White, P.; Johnson, M.; Nguyen, M.; Schwartz, I.; Spec, A.; Ostrosky-Zeichner, L.; Jackson, B.; et al. Coronavirus Disease 2019-Associated Invasive Fungal Infection. Open Forum Infect. Dis. 2021, 16, ofab510. [Google Scholar] [CrossRef]
  7. Koehler, P.; Bassetti, M.; Chakrabarti, A.; Chen, S.; Colombo, A.; Hoenigl, M.; Klimko, N.; Lass-Florl, C.; Oladale, R.; Vinh, D.; et al. Defining and managing COVID-19-associated pulmonary aspergillosis: The 2020 ECMM/ISHAM consensus criteria for research and clinical guidance. Lancet Infect. Dis. 2021, 21, e149–e162. [Google Scholar] [CrossRef]
  8. Casalini, G.; Giacomelli, A.; Ridolfo, A.; Gervasoni, C. Invasive Fungal Infections Complicating COVID-19: A Narrative Review. J. Fungi 2021, 7, 921. [Google Scholar] [CrossRef]
  9. Feys, S.; Almyroudi, M.; Braspenning, R.; Lagrou, K.; Spriet, I.; Dimopoulos, G.; Wauters, J. A Visual and Comprehensive Review on COVID-19-Associated Pulmonary Aspergillosis (CAPA). J. Fungi 2021, 7, 1067. [Google Scholar] [CrossRef]
  10. Gangneux, J.-P.; Dannaoui, E.; Fekkar, A.; Luyt, C.-E.; Botterel, F.; Prost, N.D.; Tadié, J.-M.; Reizine, F.; Houzé, S.; Timsit, J.-F.; et al. Fungal infections in mechanically ventilated patients with COVID-19 during the first wave: The French multicentre MYCOVID study. Lancet Respir. Med. 2022, 10, 180–190. [Google Scholar] [CrossRef]
  11. Verweij, P.; Brüggemann, R.; Azoulay, E.; Bassetti, M.; Blot, S.; Buil, J.; Calandra, T.; Chiller, T.; Clancy, C.; Crnely, O.; et al. Taskforce report on the diagnosis and clinical management of COVID-19 associated pulmonary aspergillosis. Intensive Care Med. 2021, 47, 819–834. [Google Scholar] [CrossRef] [PubMed]
  12. Dellière, S.; Dudoignon, E.; Voicu, S.; Collet, M.; Fodil, S.; Plaud, B.; Chousterman, B.; Bretagne, S.; Azoulay, E.; Mebazaa, A.; et al. Combination of Mycological Criteria: A Better Surrogate to Identify COVID-19-Associated Pulmonary Aspergillosis Patients and Evaluate Prognosis? J. Clin. Microbiol. 2022, 60, e0216921. [Google Scholar] [CrossRef] [PubMed]
  13. Fisher, M.; Alastruey-Izquierdo, A.; Berman, J.; Bicanic, T.; Bignell, E.; Bowyer, P.; Bromley, M.; Bruggemann, R.; Garber, G.; Cornely, O.; et al. Tackling the emerging threat of antifungal resistance to human health. Nat. Rev. Microbiol. 2022, 20, 557–571. [Google Scholar] [CrossRef] [PubMed]
  14. World Health Organization. First Meeting of the WHO Antifungal Expert Group on Identifying Priority Fungal Pathogens: Meeting Report; World Health Organization: Geneva, Switzerland, 2020. [Google Scholar]
  15. Duong, T.; Le, T.; Tran, K.; Nguyen, P.; Nguyen, H.; Nguyen, T.; Nguyen, H.; Nguyen, B.; Fisher, M.; Rhodes, J.; et al. Azole-resistant Aspergillus fumigatus is highly prevalent in the environment of Vietnam, with marked variability by land use type. Environ. Microbiol. 2021, 23, 7632–7642. [Google Scholar] [CrossRef] [PubMed]
  16. Shelton, J.; Collins, R.; Uzzelll, C.; Alghamdi, A.; Dyer, P.; Singer, A.; Fisher, M. Citizen Science Surveillance of Triazole-Resistant Aspergillus fumigatus in United Kingdom Residential Garden Soils. Appl. Environ. Microbiol. 2022, 88, e02061-02021. [Google Scholar] [CrossRef]
  17. Fischer, J.; Koningsbruggen-Reitschel, S.v.; Rietschel, E.; Vehreschild, M.; Wisplinghoff, H.; Krönke, M.; Hamprecht, A. Prevalence and molecular characterization of azole resistance in Aspergillus spp. isolates from German cystic fibrosis patients. J. Antimicrob. Chemother. 2014, 69, 1533–1536. [Google Scholar] [CrossRef]
  18. Risum, M.; Hare, R.; Gertsen, J.; Kristensen, L.; Rosenvinge, F.; Sulim, S.; Abou-Chakra, N.; Bangsborg, J.; Røder, B.; Marmolin, E.; et al. Azole resistance in Aspergillus fumigatus. The first 2-year’s Data from the Danish National Surveillance Study, 2018–2020. Mycoses 2022, 65, 419–428. [Google Scholar] [CrossRef]
  19. Shelton, J. Environmental Drivers of Azole-Resistance in Aspergillus fumigatus and Its Relevance to Human Health; Imperial College London: London, UK, 2021. [Google Scholar]
  20. Tsuchido, Y.; Tanaka, M.; Nakano, S.; Yamamoto, N.; Matsumura, Y.; Nagao, M. Prospective multicenter surveillance of clinically isolated Aspergillus species revealed azole-resistant Aspergillus fumigatus isolates with TR34/L98H mutation in the Kyoto and Shiga regions of Japan. Med. Mycol. 2019, 57, 997–1003. [Google Scholar] [CrossRef]
  21. Mead, M.; Castro, P.d.; Steenwyk, J.; Gangneux, J.; Hoenigl, M.; Prattes, J.; Rautemaa-Richardson, R.; Guegan, H.; Moore, C.; Lass-Flörl, C.; et al. COVID-19 Associated Pulmonary Aspergillosis isolates are genomically diverse but similar to each other in their responses to infection-relevant stresses. Microbiol. Spectr. 2023, 11, e05128-22. [Google Scholar] [CrossRef]
  22. Rasilla, T.d.l.; González-Jiménez, I.; Fernández-Arroyo, A.; Roldán, A.; Carretero-Ares, J.; García-Clemente, M.; Telenti-Asensio, M.; García-Prieto, E.; Martínez-Suarez, M.; Vázquez-Valdés, F.; et al. COVID-19 Associated Pulmonary Aspergillosis (CAPA): Hospital or Home Environment as a Source of Life-Threatening Aspergillus fumigatus Infection? J. Fungi 2022, 8, 316. [Google Scholar] [CrossRef]
  23. Kirchoff, L.; Braun, L.; Schmidt, D.; Dittmer, S.; Dedy, J.; Herbstreit, F.; Buer, J.; Rath, P.; Steinmann, J.; Verhasselt, H. COVID-19-associated pulmonary aspergillosis in ICU patients in a German reference centre: Phenotypic and molecular characterisation of Aspergillus fumigatus isolates. Mycoses 2022, 65, 458–465. [Google Scholar] [CrossRef] [PubMed]
  24. Steenwyk, J.; Mead, M.; Castro, P.d.; Valero, C.; Damasio, A.; Santos, R.d.; Labella, A.; Li, Y.; Knowles, S.; Raja, H.; et al. Genomic and Phenotypic Analysis of COVID-19-Associated Pulmonary Aspergillosis Isolates of Aspergillus Fumigatus. Microbiol. Spectr. 2021, 9, e0001021. [Google Scholar] [CrossRef] [PubMed]
  25. Morais, S.; Toscano, C.; Simões, H.; Carpinterio, D.; Viegas, C.; Veríssimo, C.; Sabino, R. Comparison of Multi-locus Genotypes Detected in Aspergillus fumigatus Isolated from COVID Associated Pulmonary Aspergillosis (CAPA) and from Other Clinical and Environmental Sources. J. Fungi 2023, 9, 298. [Google Scholar] [CrossRef] [PubMed]
  26. Rogers, T.; Verweij, P.; Castanheira, M.; Dannaoui, E.; White, P.; Arendrup, M. Molecular mechanisms of acquired antifungal drug resistance in principal fungal pathogens and EUCAST guidance for their laboratory detection and clinical implications. J. Antimicrob. Chemother. 2022, 77, 2053–2753. [Google Scholar] [CrossRef] [PubMed]
  27. Barber, A.; Sae-Ong, T.; Kang, K.; Seelbinder, B.; Li, J.; Walther, G.; Panagiotou, g.; Kurzai, O. Aspergillus fumigatus pan-genome analysis identifies genetic variants associated with human infection. Nat. Microbiol. 2021, 6, 1526–1536. [Google Scholar] [CrossRef]
  28. Reis, T.d.; Silva, L.; Castro, A.d.; Carmo, R.d.; Marini, M.; Silveria, J.d.; Ferreira, B.; Rodrigues, F.; Lind, A.; Rokas, A.; et al. The Aspergillus fumigatus Mismatch Repair MSH2 Homolog Is Important for Virulence and Azole Resistance. Mol. Sphere 2019, 4, e00416–e00419. [Google Scholar] [CrossRef]
  29. Furukawa, T.; Rhijn, N.v.; Fraczek, M.; Gsaller, F.; Davis, E.; Carr, P.; Gago, S.; Fortune-Grant, R.; Rahman, S.; Gilsenan, J.; et al. The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus. Nat. Commun. 2020, 11, 427. [Google Scholar] [CrossRef]
  30. Novak-Frazer, L.; Anees-Hill, S.; Hassan, D.; Masania, R.; Moore, C.; Richardson, M.; Denning, D.; Rautemaa-Richardson, R. Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens. J. Antimicrob. Chemother. 2020, 75, 3501–3509. [Google Scholar] [CrossRef]
  31. Takazono, T.; Ito, Y.; Tashiro, M.; Nakano, Y.; Hirayama, T.; Hosogaya, N.; Saijo, T.; Yamamoto, K.; Imamura, Y.; Miyazaki, T.; et al. Transition of triazole-resistant Aspergillus fumigatus isolates in a Japanese tertiary hospital and subsequent genetic analysis. J. Infect. Chemother. 2021, 27, 537–539. [Google Scholar] [CrossRef]
  32. Rhodes, J.; Abdolrasouli, A.; Dunne, K.; Sewell, T.; Zhang, Y.; Ballard, E.; Brackin, A.; Rhijn, N.v.; Chown, N.; Tsitsopoulou, A.; et al. Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment. Nat. Microbiol. 2022, 7, 663–674. [Google Scholar] [CrossRef]
  33. Armstrong-James, D.; Youngs, J.; Bicanic, T.; Abdolrasouli, A.; Denning, D.; Johnson, E.; Mehra, V.; Pagliuca, T.; Patel, B.; Rhodes, J.; et al. Confronting and mitigating the risk of COVID-19 Associated Pulmonary Aspergillosis (CAPA). Eur. Respir. J. 2020, 56, 2002554. [Google Scholar] [CrossRef]
  34. Donnelly, J.; Chen, S.; Kauffman, C.; Steinbach, W.; Baddley, J.; Verweij, P.; Clancy, C.; Wingard, J.; Lockhart, S.; Groll, A.; et al. Revision and Update of the Consensus Definitions of Invasive Fungal Disease from the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin. Infect. Dis. 2020, 71, 1367–1376. [Google Scholar] [CrossRef] [PubMed]
  35. Koehler, P.; Cornely, O.; Böttiger, B.; Dusse, F.; Eichenauer, D.; Fuchs, F.; Hallek, M.; Jung, N.; Klein, F.; Persigehl, T.; et al. COVID-19 associated pulmonary aspergillosis. Mycoses 2020, 63, 528–534. [Google Scholar] [CrossRef] [PubMed]
  36. Mohamed, A.; Hassan, T.; Trzos-Grzybowska, M.; Thomas, J.; Quinn, A.; O’Sullivan, M.; Griffin, A.; Rogers, T.; Talento, A. Multi-triazole-resistant Aspergillus fumigatus and SARS-CoV-2 co-infection: A lethal combination. Med. Mycol. Case Rep. 2021, 31, 11–14. [Google Scholar] [CrossRef] [PubMed]
  37. Argimón, S.; Abudahab, K.; Goater, R.; Fedosejev, A.; Bhai, J.; Glasner, C.; Feil, E.; Holden, M.; Yeats, C.; Grundmann, H.; et al. Microreact: Visualizing and sharing data for genomic epidemiology and phylogeography. Microbiol. Genom. 2016, 2, e000093. [Google Scholar] [CrossRef]
  38. Armstrong-James, D. (Department of Infectious Diseases, Imperial College London, London SW7 2AZ, UK); Scourfiled, A. (Department of Infectious Diseases, Imperial College London, London SW7 2AZ, UK); Abdolrasouli, A. (Department of Infectious Diseases, Imperial College London, London SW7 2AZ, UK). Azole drug susceptibility of clinical A. fumigatus isolates grown in minimal media using EUCAST method (C307, C323, C360, C372, and C376), 2018. Unpublished work.
  39. Brackin, A.; Shelton, J.; Abdolrasouli, A.; Fisher, M.; Sewell, T. A low-cost tebuconazole-based screening test for azole-resistant aspergillus fumigatus. Curr. Protoc. Microbiol. 2020, 58, e112. [Google Scholar] [CrossRef]
  40. Buil, J.B.; Lee, H.A.L.v.d.; Rijs, A.J.M.M.; Zoll, J.; Hovestadt, J.A.M.F.; Melchers, W.J.G.; Verweij, P.E. Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck. Antimicrob. Agents Chemother. 2017, 61, e01250-17. [Google Scholar] [CrossRef]
  41. Guinea, J. Updated EUCAST Clinical Breakpoints against Aspergillus, Implications for the Clinical Microbiology Laboratory. J. Fungi 2020, 6, 343. [Google Scholar] [CrossRef]
  42. Espinel-Ingroff, A.; Diekema, D.; Fothergill, A.; Johnson, E.; Pelaez, T.; Pfaller, M.; Rinaldi, M.; Canton, E.; Turnidge, J. Wild-Type MIC Distributions and Epidemiological Cutoff Values for the Triazoles and Six Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document). J. Clin. Microbiol. 2010, 48, 3251–3257. [Google Scholar] [CrossRef]
  43. Korostin, D.; Kulemin, N.; Naumov, V.; Belova, V.; Kwon, D.; Gorbachev, A. Comparative analysis of novel MGISEQ-2000 sequencing platform vs. Illumina HiSeq 2500 for whole-genome sequencing. PLoS ONE 2020, 15, e0230301. [Google Scholar] [CrossRef]
  44. Nierman, W.; Pain, A.; Anderson, M.; Wortman, J.; Kim, H.; Arroyo, J.; Berriman, M.; Abe, K.; Archer, D.; Bermejo, C.; et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 2005, 438, 1151–1156. [Google Scholar] [CrossRef] [PubMed]
  45. Heng, L.; Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef]
  46. Samson, R.; Visagie, C.; Houbraken, J.; Hong, S.; Hubka, V.; Klaasen, C.; Perrone, G.; Seifert, K.; Susca, A.; Tanney, J.; et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud. Mycol. 2014, 78, 141–173. [Google Scholar] [CrossRef] [PubMed]
  47. FungiDB. Afu4g10050 Calmodulin. 2015. Available online: https://fungidb.org/fungidb/app/record/gene/Afu4g10050 (accessed on 14 July 2022).
  48. Garnica, S.; Schön, M.; Abarenkov, K.; Riess, K.; Liimatainen, K.; Niskanen, T.; Dima, B.; Frøslev, T.; Jeppesen, T.; Peintner, U.; et al. Determining threshold values for barcoding fungi: Lessons from Cortinarius (Basidiomycota), a highly diverse and widespread ectomycorrhizal genus. FEMS Microbiol. Ecol. 2016, 92, fiw045. [Google Scholar] [CrossRef]
  49. Pyrzak, W.; Miller, K.; Miller, B. Mating Type Protein Mat1-2 from Asexual Aspergillus fumigatus Drives Sexual Reproduction in Fertile Aspergillus nidulans. Eukaryot. Cell 2008, 7, 1029–1040. [Google Scholar] [CrossRef]
  50. Stamatakis, A. RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 2006, 22, 2688–2690. [Google Scholar] [CrossRef]
  51. Jombart, T.; Collins, C. A tutorial for Discriminant Analysis of Principal Components (DAPC) Using Adegenet 2.0.0. Available online: https://adegenet.r-forge.r-project.org/files/tutorial-dapc.pdf (accessed on 14 July 2022).
  52. Garcia-Rubio, R.; Monzon, S.; Alcazar-Fuoli, L.; Cuesta, I.; Mellado, E. Genome-Wide Comparative Analysis of Aspergillus fumigatus Strains: The Reference Genome as a Matter of Concern. Genes 2018, 9, 363. [Google Scholar] [CrossRef]
  53. Paoletti, M.; Rydholm, C.; Schwier, E.; Anderson, M.; Szakacs, G.; Lutzoni, F.; Debeaupuis, J.; Latge, J.; Denning, D.; Dyer, P. Evidence for Sexuality in the Opportunistic Fungal Pathogen Aspergillus fumigatus. Curr. Biol. 2005, 15, 1242–1248. [Google Scholar] [CrossRef]
  54. Sewell, T.; Zhu, J.; Rhodes, J.; Hagen, G.; Meis, J.; Fisher, M.; Jombart, T. Nonrandom distribution of azole resistance across the global population of Aspergillus fumigatus. Mbio Am. Soc. Microbiol. 2019, 10, e00392–e00419. [Google Scholar] [CrossRef]
  55. Boyd, S.; Loh, K.; Lynch, J.; Alrashed, D.; Muzzammil, S.; Marsh, H.; Masoud, M.; Ihsan, S.; Matin-Loeches, I. Elevated Rates of Ventilator-Associated Pneumonia and COVID-19 Associated Pulmonary Aspergillosis in Critically Ill Patients with SARS-CoV2 Infection in the Second Wave: A Retrospective Chart Review. Antibiotics 2022, 11, 632. [Google Scholar] [CrossRef]
  56. Abdolrasouli, A.; Rhodes, J.; Beale, M.; Hagen, F.; Rogers, T.; Chowdhary, A.; Meis, J.; Armstrong-James, D.; Fisher, M. Genomic Context of Azole Resistance Mutations in Aspergillus fumigatus Determined Using Whole-Genome Sequencing. mBio 2015, 6, e00536-15. [Google Scholar] [CrossRef] [PubMed]
  57. Bruske, E.; Ottoe, T.; Frank, M. Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance. Malar. J. 2018, 17, 376. [Google Scholar] [CrossRef] [PubMed]
  58. Garcia-Rubio, R.; Escribano, P.; Gomez, A.; Guinea, J.; Mellado, E. Comparison of Two Highly Discriminatory Typing Methods to Analyze Aspergillus fumigatus Azole Resistance. Front. Microbiol. 2018, 9, 1626. [Google Scholar] [CrossRef] [PubMed]
  59. Meijer, E.; Dofferhoff, A.; Hoiting, O.; Buil, J.; Meis, J. Azole-Resistant COVID-19-Associated Pulmonary Aspergillosis in an Immunocompetent Host: A Case Report. J. Fungi 2020, 6, 79. [Google Scholar] [CrossRef] [PubMed]
  60. Gonzalez-Jimenez, I.; Lucio, J.; Menéndez-Fraga, M.D.; Mellado, E.; Peláez, T. Hospital Environment as a Source of Azole-Resistant Aspergillus fumigatus Strains with TR34/L98H and G448S Cyp51A Mutations. J. Fungi 2021, 7, 22. [Google Scholar] [CrossRef]
  61. Escribano, P.; Rodríguez-Sánchez, B.; Díaz-García, J.; Martín-Gómez, M.; Ibáñez-Martínez, E.; Rodríguez-Mayo, M.; Pelâez, T.; Pedrosa, E.G.-G.d.l.; Tejero-García, R.; Marimón, J.; et al. Azole resistance survey on clinical Aspergillus fumigatus isolates in Spain. Clin. Microbiol. Infect. 2021, 27, 1170.E1171–1170.E1177. [Google Scholar] [CrossRef]
  62. Grootveld, R.v.; Paasen, J.v.; Boer, M.d.; Claas, E.; Kuijper, E.; Beek, M.v.d.; Group, L.-C.-R. Systematic screening for COVID-19-associated invasive aspergillosis in ICU patients by culture and PCR on tracheal aspirate. Myoces 2021, 64, 641–650. [Google Scholar] [CrossRef]
  63. Abdolrasouli, A.; Petrou, M.; Park, H.; Rhodes, J.; Rawson, T.; Moore, L.; Donaldson, H.; Holmes, A.; Fisher, M.; Armstrong-James, D. Surveillance for Azole-Resistant Aspergillus fumigatus in a Centralized Diagnostic Mycology Service, London, United Kingdom, 1998–2017. Front. Microbiol. 2018, 9, 2234. [Google Scholar] [CrossRef]
  64. Lestrade, P.; Bentvelsen, R.; Schauwvlieghe, A.; Schalekamp, S.; Velden, W.v.d.; Kuiper, E.; Paassen, J.v.; Hoven, B.v.d.; Lee, H.v.d.; Melchers, W.; et al. Voriconazole Resistance and Mortality in Invasive Aspergillosis: A Multicenter Retrospective Cohort Study. Clin. Infect. Dis. 2019, 68, 1463–1471. [Google Scholar] [CrossRef]
  65. Linden, J.v.d.; Snelders, E.; Kampinga, G.A.; Rijnders, B.; Mattsson, E.; Debets-Ossenkopp, Y.; Kuijper, E.; Tiel, F.; Melchers, W.; Verweij, P. Clinical Implications of Azole Resistance in Aspergillus fumigatus, the Netherlands, 2007–2009. Emerg. Infect. Dis. 2011, 17, 1846–1854. [Google Scholar] [CrossRef]
  66. Burks, C.; Darby, A.; Londoño, L.; Momany, M.; Brewer, M. Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance. PLoS Pathog. 2021, 17, e1009711. [Google Scholar] [CrossRef] [PubMed]
  67. Bustamante, B.; Illescas, L.; Posadas, A.; Campos, P. Azole resistance among clinical isolates of Aspergillus fumigatus in Lima-Peru. Med. Mycol. 2020, 58, 54–60. [Google Scholar] [CrossRef] [PubMed]
  68. Sewell, T. Elevated Prevalence of Azole-Resistant Aspergillus fumigatus in Urban versus Rural Environments in the United Kingdom. Antimicrob. Agents Chemother. 2019, 63, e00548-19. [Google Scholar] [CrossRef] [PubMed]
  69. Chowdhary, A.; Sharma, C.; Boom, M.v.d.; Yntema, J.; Hagen, F.; Verweij, P.; Meis, J. Multi-azole-resistant Aspergillus fumigatus in the environment in Tanzania. J. Antimicrob. Chemother. 2014, 69, 2979–2983. [Google Scholar] [CrossRef]
  70. Chowdhary, A.; Kathuria, S.; Randhawa, H.; Gaur, S.; Klaassen, C.; Meis, J. Isolation of multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR34/L98H mutations in the cyp51A gene in India. J. Antimicrob. Chemother. 2012, 67, 362–366. [Google Scholar] [CrossRef]
  71. Chang, H.; Ashu, E.; Sharma, C.; Kathuria, S.; Chowdhary, A.; Xu, J. Diversity and origins of Indian multi-triazole resistant strains of Aspergillus fumigatus. Mycoses 2016, 59, 450–466. [Google Scholar] [CrossRef]
  72. Talbot, J.; Subedi, S.; Halliday, C.; Hibbs, D.; Lopez-Ruiz, F.; Harper, L.; Park, R.; Cuddy, W.; Biswas, C.; Cooley, L.; et al. Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates. J. Antimicrob. Chemother. 2018, 73, 2347–2351. [Google Scholar] [CrossRef]
  73. Wu, C.; Wang, H.; Lee, J.; Lo, H.; Dai, C.; Chou, P.; Ko, W.; Chen, Y. Azole-resistant Aspergillus fumigatus isolates carrying TR34/L98H mutations in Taiwan. Mycoses 2015, 58, 544–549. [Google Scholar] [CrossRef]
  74. Vermeulen, E.; Maertens, J.; Schoemans, H.; Lagrou, K. Azole-resistant Aspergillus fumigatus due to TR46/Y121F/T289A mutation emerging in Belgium, July 2012. Euro Surveill. 2012, 17, 20326. [Google Scholar] [CrossRef]
  75. Buil, J.; Hare, R.; Zwaan, B.; Arendrup, M.; Melchers, W.; Verweij, P. The fading boundaries between patient and environmental routes of triazole resistance selection in Aspergillus fumigatus. PLoS Pathog. 2019, 15, e1007858. [Google Scholar] [CrossRef]
  76. Majima, H.; Arai, T.; Kusuya, Y.; Takahashi, H.; Watanabe, A.; Miyazaki, Y.; Kamei, K. Genetic differences between Japan and other countries in cyp51A polymorphisms of Aspergillus fumigatus. Mycoses 2021, 64, 1354–1365. [Google Scholar] [CrossRef] [PubMed]
  77. Zea, S.; Toyotome, T. Azole-resistant Aspergillus fumigatus as an emerging worldwide pathogen. Microbiol. Immunol. 2021, 66, 135–144. [Google Scholar] [CrossRef]
  78. Albarrg, A.; Anderson, M.; Howard, S.; Robson, G.; Warn, P.; Sanglard, D.; Denning, D. Interrogation of Related Clinical Pan-Azole-Resistant Aspergillus fumigatus Strains: G138C, Y431C, and G434C Single Nucleotide Polymorphisms in cyp51A, Upregulation of cyp51A, and Integration and Activation of Transposon Atf1 in the cyp51A Promoter. Antimicrob. Agents Chemother. 2011, 55, 5113–5121. [Google Scholar] [CrossRef]
  79. Howard, S.; Pasqualotto, A.; Anderson, M.; Leatherbarrow, H.; Albarrag, A.; Harrison, E.; Gregson, L.; Bowyer, P.; Denning, D. Major variations in Aspergillus fumigatus arising within aspergillomas in chronic pulmonary aspergillosis. Mycoses 2012, 56, 434–441. [Google Scholar] [CrossRef]
  80. Ballard, E.; Melchers, W.; Zoll, J.; Brown, A.; Verweij, P.; Warris, A. In-host microevolution of Aspergillus fumigatus: A phenotypic and genotypic analysis. Fungal Genet. Biol. 2018, 113, 1–13. [Google Scholar] [CrossRef] [PubMed]
  81. Takeda, K.; Suzuki, J.; Watanabe, A.; Narumoto, O.; Kawashima, M.; Fukami, T.; Tamura, A.; Nagai, H.; Matsui, H.; Kamei, K. High detection rate of azole-resistant Aspergillus fumigatus after treatment with azole antifungal drugs among patients with chronic pulmonary aspergillosis in a single hospital setting with low azole resistance. Med. Mycol. 2020, 59, 327–334. [Google Scholar] [CrossRef] [PubMed]
  82. Shalhoub, S.; Luong, M.; Howard, S.; Richardson, S.; Singer, L.; Chaparro, C.; Keshavjee, S.; Akinlolu, Y.; Rotstein, C.; Mazzulli, T.; et al. Rate of cyp51A mutation in Aspergillus fumigatus among lung transplant recipients with targeted prophylaxis. J. Antimicrob. Chemother. 2015, 70, 1064–1067. [Google Scholar] [CrossRef]
  83. Camps, S.; Dutilh, B.; Arendrup, M.; Rijs, A.; Snelders, E.; Huynen, M.; Verweij, P.; Melchers, W. Discovery of a hapE Mutation That Causes Azole Resistance in Aspergillus fumigatus through Whole Genome Sequencing and Sexual Crossing. PLoS ONE 2012, 7, e50034. [Google Scholar] [CrossRef]
  84. Ballard, E.; Weber, J.; Melchers, W.; Tammireddy, S.; Whitfield, P.; Brakhage, A.; Brown, A.; Verweij, P.; Warris, A. Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism. Fungal Genet. Biol. 2019, 130, 98–106. [Google Scholar] [CrossRef]
  85. Rybak, J. Challenging the Paradigm of Clinical Triazole Resistance in Aspergillus fumigatus; The University of Tennessee Health Science Center: Memphis, TN, USA, 2019. [Google Scholar] [CrossRef]
Jof 09 01104 g001
Figure 1. Phylogenetic tree of isolates from CAPA, IA, and colonising patients. Unrooted maximum likelihood (ML) phylogenetic tree over 1000 replicates performed on WGS SNP data, showing the following: inner track 1, the source of the isolate (CAPA, colonising or IA); middle track 2, if the isolate contains cyp51A polymorphism; and outer track 3, the clade in which the isolate is located.
Figure 1. Phylogenetic tree of isolates from CAPA, IA, and colonising patients. Unrooted maximum likelihood (ML) phylogenetic tree over 1000 replicates performed on WGS SNP data, showing the following: inner track 1, the source of the isolate (CAPA, colonising or IA); middle track 2, if the isolate contains cyp51A polymorphism; and outer track 3, the clade in which the isolate is located.
Jof 09 01104 g001
Jof 09 01104 g002aJof 09 01104 g002b
Figure 2. Multivariate analysis of isolates from CAPA, IA, and colonising patients. (A) Scatterplot of the principal component analysis (PCA) A. fumigatus genotypes using the first four principal components (PCs) illustrating the genetic identity of CAPA and IA control isolates. (B) Density plot of the discriminant PCA (DAPC), broadly identifying two distinct clusters, CAPA and control. (C) Composition plot highlights that all the isolates’ genotype is composed of genetic material identified as CAPA, IA, or colonising. Four isolates contain 70% control compared to CAPA, whereas there are 21 isolates the genome membership of which is mostly CAPA (>80%).
Figure 2. Multivariate analysis of isolates from CAPA, IA, and colonising patients. (A) Scatterplot of the principal component analysis (PCA) A. fumigatus genotypes using the first four principal components (PCs) illustrating the genetic identity of CAPA and IA control isolates. (B) Density plot of the discriminant PCA (DAPC), broadly identifying two distinct clusters, CAPA and control. (C) Composition plot highlights that all the isolates’ genotype is composed of genetic material identified as CAPA, IA, or colonising. Four isolates contain 70% control compared to CAPA, whereas there are 21 isolates the genome membership of which is mostly CAPA (>80%).
Jof 09 01104 g002aJof 09 01104 g002b
Jof 09 01104 g003
Figure 3. Phylogenetic tree of CAPA and A. fumigatus clinical and environmental isolates from Ireland and the UK. Unrooted ML phylogenetic tree with over 1000 replicates performed on WGS SNP data, showing the following: outer track 1, if the isolate contains cyp51A polymorphism; middle track 2, the clade in which the isolate is located; and inner track 3, the source of the isolate (CAPA, clinical, or environmental).
Figure 3. Phylogenetic tree of CAPA and A. fumigatus clinical and environmental isolates from Ireland and the UK. Unrooted ML phylogenetic tree with over 1000 replicates performed on WGS SNP data, showing the following: outer track 1, if the isolate contains cyp51A polymorphism; middle track 2, the clade in which the isolate is located; and inner track 3, the source of the isolate (CAPA, clinical, or environmental).
Jof 09 01104 g003
Jof 09 01104 g004aJof 09 01104 g004b
Figure 4. Multivariate analysis of CAPA and A. fumigatus clinical and environmental isolates from Ireland and the UK. Multivariate analysis, (A) scatterplot of the PCA A. fumigatus genotypes using the first five PCs illustrating the genetic identity of CAPA isolates, and clinical and environmental isolates from Ireland and the UK. (B) Scatterplot of the DPCA identifying 3 types form separate clusters, with CAPA and clinical with some overlap. (C) A composition plot comparing the genetic composition of each isolate. The plot highlights that each isolate is a mixture of genotypes identified as CAPA, environment, and clinical non-CAPA.
Figure 4. Multivariate analysis of CAPA and A. fumigatus clinical and environmental isolates from Ireland and the UK. Multivariate analysis, (A) scatterplot of the PCA A. fumigatus genotypes using the first five PCs illustrating the genetic identity of CAPA isolates, and clinical and environmental isolates from Ireland and the UK. (B) Scatterplot of the DPCA identifying 3 types form separate clusters, with CAPA and clinical with some overlap. (C) A composition plot comparing the genetic composition of each isolate. The plot highlights that each isolate is a mixture of genotypes identified as CAPA, environment, and clinical non-CAPA.
Jof 09 01104 g004aJof 09 01104 g004b
Table 1. Characteristics of CAPA isolates of A. fumigatus used in this study and details of alignments.
Table 1. Characteristics of CAPA isolates of A. fumigatus used in this study and details of alignments.
Isolate IDCountrySample TypeNo. of Aligned Reads (Millions)Mean Depth of Coverage (x)Percentage of Reference
Genome
Covered (%)
CAPA-AGermanyCAPA—Possible35.8146.798.4
CAPA-BGermanyCAPA—Probable40.1166.798.5
CAPA-CGermanyCAPA—Probable37.5157.498.1
CAPA-DGermanyCAPA—Possible34.2147.597.3
C403NetherlandsCAPA—Possible37.2165.098.6
C408NetherlandsCAPA—Possible28.5127.798.3
C422UKCAPA—Probable11.845.596.2
C423UKCAPA—Probable15.656.296.5
C424UKCAPA—Probable11.245.198.6
C425UKCAPA—Probable11.043.097.2
C434IrelandCAPA—Probable10.641.996.6
C435IrelandCAPA—Probable10.341.497.7
C436IrelandCAPA—Probable9.839.398.2
C437IrelandCAPA—Probable9.638.997.4
C438IrelandCAPA—Probable10.542.697.7
C439IrelandCAPA—Probable11.445.198.3
C440IrelandCAPA—Probable7.827.697.9
C441IrelandCAPA—Probable9.236.397.9
C443IrelandCAPA—Probable9.739.297.5
C444IrelandCAPA—Probable11.043.297.9
C611UKCAPA—Probable11.043.797.6
C612UKCAPA—Possible11.044.097.5
Table 2. Azole drug susceptibility of CAPA isolates grown in minimal media and the associated candidate polymorphisms.
Table 2. Azole drug susceptibility of CAPA isolates grown in minimal media and the associated candidate polymorphisms.
Isolate
ID		VIPcheckTM ScoreTebucheck
Score		Resistance
Marker
ITRVORPOS
C1540001WT
C403 *NDNDNDNDWT
C408 *NDNDNDNDWT
C422NDNDND1WT
C423NDNDND1WT
C424NDNDND1WT
C425NDNDND1WT
C4340001WT
C435NDNDND1WT
C4360001WT
C4370001WT
C4381113TR34/L98H
C439NDNDND1WT
C4400001WT
C4411104TR34/L98H
C444NDNDND3TR34/L98H
C6110001WT
C612NDNDND1WT
CAPA-A *NDNDNDNDWT
CAPA-B *NDNDNDNDWT
CAPA-C *NDNDNDNDWT
CAPA-D *NDNDNDNDWT
C154 was used as a control, as it had previously been shown to be EUCAST susceptible [32] and in this study, this confirmed by Tebucheck and VIPcheckTM. VIPcheckTM score is 2 for uninhibited growth, 1 for minimal growth in well, and 0 for no growth. The wells contain either ITR 4mg/L, POS 0.5 mg/L, or VOR 2mg/L [40]. Tebucheck score of 1 = fully susceptible to tebuconazole (0mg/L); 2 = resistant, growth in 6 mg/L of tebuconazole; 3 = resistant, growth in 8 mg/L of tebuconazole; 4 = resistant, growth in 16 mg/L of tebuconazole [39]. A score of 0.5 was given if there was partial growth (10 to 50%) in a well [39]. * 6 Isolates did not have VIPcheckTM or Tebucheck results. C403 and C408 had azole drug susceptibility using EUCAST broth microdilution method and CAPA A–D using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method (Table A5).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Simmons, B.C.; Rhodes, J.; Rogers, T.R.; Verweij, P.E.; Abdolrasouli, A.; Schelenz, S.; Hemmings, S.J.; Talento, A.F.; Griffin, A.; Mansfield, M.; et al. Genomic Epidemiology Identifies Azole Resistance Due to TR34/L98H in European Aspergillus fumigatus Causing COVID-19-Associated Pulmonary Aspergillosis. J. Fungi 2023, 9, 1104. https://doi.org/10.3390/jof9111104

AMA Style

Simmons BC, Rhodes J, Rogers TR, Verweij PE, Abdolrasouli A, Schelenz S, Hemmings SJ, Talento AF, Griffin A, Mansfield M, et al. Genomic Epidemiology Identifies Azole Resistance Due to TR34/L98H in European Aspergillus fumigatus Causing COVID-19-Associated Pulmonary Aspergillosis. Journal of Fungi. 2023; 9(11):1104. https://doi.org/10.3390/jof9111104

Chicago/Turabian Style

Simmons, Benjamin C., Johanna Rhodes, Thomas R. Rogers, Paul E. Verweij, Alireza Abdolrasouli, Silke Schelenz, Samuel J. Hemmings, Alida Fe Talento, Auveen Griffin, Mary Mansfield, and et al. 2023. "Genomic Epidemiology Identifies Azole Resistance Due to TR34/L98H in European Aspergillus fumigatus Causing COVID-19-Associated Pulmonary Aspergillosis" Journal of Fungi 9, no. 11: 1104. https://doi.org/10.3390/jof9111104

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop