High prevalence of triazole resistance in clinical Aspergillus fumigatus isolates in a specialist cardio-thoracic centre

Alireza Abdolrasouli1,2, Andrew Scourfield2,3, Johanna L. Rhodes4, Anand Shah5, J. Stuart Elborn5, Matthew C. Fisher4, Silke Schelenz6, Darius Armstrong-James2

Affiliations

1 Diagnostic Mycology Service, Department of Medical Microbiology, North West London Pathology, Imperial College Healthcare National Health Service Trust, London, UK

2 Fungal Pathogens Laboratory, National Heart and Lung Institute, Imperial College London, UK

3Department of Clinical Pharmacology, Guys and St Thomas’ Foundation NHS Trust, London, UK

4MRC Centre for Global Infectious Disease Analysis, Imperial College London, UK

5Department of Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, UK

6Department of Medical Microbiology, Royal Brompton and Harefield NHS Foundation Trust, London, UK

Article Word Count: 2,057

Abstract Word Count: 266

Key words: Aspergillus fumigatus, azole resistance, cyp51A, antifungal drugs

Corresponding author: Alireza Abdolrasouli

Diagnostic Mycology Service, Department of Medical Microbiology, North West London Pathology, Imperial College Healthcare National Health Service Trust, 4th Floor East Wing Charing Cross Hospital, Fulham Palace Road, London W6 8RF, United Kingdom

E-mail: a.abdolrasouli@imperial.ac.ukTel: +44 (0)20 3311 7830

ABSTRACT

Objectives: To evaluate the prevalence of triazole-resistant Aspergillus fumigatus and common molecular cyp51A polymorphisms amongst clinical isolates in a specialised cardio-thoracic centre in London, United Kingdom.

Methods: All A. fumigatus isolates independent of clinical significance were prospectively analysed from April 2014 to March 2016. Isolates were screened with a 4-well VIPcheck™ plate to assess triazole susceptibility. Resistance was confirmed with standard microbroth dilution method according to EUCAST reference guidelines. Triazole-resistant A. fumigatus isolates were subjected to a mixed-format RT-PCR assay (AsperGenius®) to detect common cyp51A alterations.

Results: We identified 167 unique clinical A. fumigatus isolates from 135 patients. Resistance to at least one azole antifungal drug was confirmed in 22/167 (13.2%) of isolates from 18/135 (13.3%) patients, including 12/74 (16.2%) patients with cystic fibrosis (CF). Sputum was the most common clinical sample from which azole-resistant A. fumigatus was isolated. The highest detection rate of azole resistant A. fumigatus was among the 11-20 year age group. All triazole-resistant isolates (n = 22) were resistant to itraconazole, 15 showed cross-resistance to posaconazole and 10 demonstrated resistance to voriconazole. No pan-azole resistant A. fumigatus was identified. TR34/L98H was identified in 6/22 (27.3%) of azole resistant isolates and detectable in 5/12 (42%) of patients with CF.

Conclusion: In our specialised cardio-thoracic centre the prevalence of triazole resistant A. fumigatus in respiratory samples is alarmingly high (13.2%). The majority of azole resistant isolates were from patients with CF. We found a higher prevalence of the environmentally driven mutation TR34/L98H in our A. fumigatus isolates than published UK data from other specialist respiratory centres which may reflect differing patient populations managed at these institutions.

INTRODUCTION

Aspergillus fumigatus is a ubiquitous ascomycete mould that can cause a wide spectrum of clinical syndromes. The pathological effects of A. fumigatus depend largely on the interplay between the pathogen and host immune response ranging from asymptomatic colonisation to life-threatening infection. Invasive aspergillosis (IA), the most severe form of A. fumigatus infection, predominantly affects immunocompromised patients while immune hyperactivity can lead to allergic bronchopulmonary aspergillosis (ABPA) and fungal sensitization in severe asthma (SAFS). In those with structural lung disease A. fumigatus can cause chronic pulmonary aspergillosis (CPA) and aspergilloma. More recently, Aspergillus bronchitis has been described predominantly affecting people with cystic fibrosis (CF), bronchiectasis, lung transplant recipients and those receiving mechanical ventilation in intensive care units [1].

Triazoles are the most widely used antifungal agents in prophylaxis and treatment of Aspergillus-related infections [2] but in those with allergic disease corticosteroids are often preferred [3]. The Infectious Diseases Society of America (IDSA) guideline recommends voriconazole the potent, broad-spectrum, triazole antifungal as the first line agent for the primary treatment of IA [2]. Over the last decade there has been increasing reports of A. fumigatus resistant to triazoles with subsequent treatment failure in some patients causing a major clinical concern [4, 5]. The emergence and worldwide occurrence of azole-resistant A. fumigatus (ARAf) isolates has raised the realistic possibility that, in future, mould-active azoles may cease to be effective.

In A. fumigatus the cyp51A gene encodes lanosterol 14-α demethylase, a cytochrome P450 enzyme required for the biosynthesis of ergosterol an essential component of the fungal cell membrane. This enzyme is the target of triazole drugs and a key site of development of azole resistance [6]. The environmentally-occurring TR34/L98H allele is a prevalent molecular mechanism of resistance to triazoles in A. fumigatus consisting of a tandem repeat (TR) of 34 bases in the promoter region of the cyp51A gene and substitution of leucine-to-histidine at codon 98 [7]. This mutation is found worldwide in the environment but also in clinical isolates of A. fumigatus [8, 9]. Another cyp51A-mediated resistance alteration TR46/Y121F/T289A has more recentlly been described in A. fumigatus and leads to high-level voriconazole resistance [10]. Furthermore, numerous mutations in cyp51A hot spots have been reported that confer resistance to triazoles in vitro and evolve during prolonged azole treatment in patients with chronic forms of aspergillosis [6].

The true prevalence of ARAf is largely unknown due to limited sampling across populations and geographical distribution, and is complicated by the use of differing detection methods. Based on prevalence surveys across the world rates of azole resistance in the clinic range from 0.6 to 27% [11]. In the UK, Public Health England have published data from the National Mycology Reference Laboratory (MRL) showing a recent increase in the number of A. fumigatus isolates with reduced susceptibility to itraconazole from 1.4% in 2012 to 8.5% in 2016 when using the clinical and laboratory standards institute (CLSI) method [12]. Similarly, voriconazole and posaconaozle demonstrated reduced susceptibility of 4.7% and 6.9% respectively. In 2015, 6.5% of A. fumigatus isolates referred to MRL showed reduced susceptibility to itraconazole in contrast to 15% reported from the Mycology Reference Centre in Manchester using European Committee on Antimicrobial Susceptibility Testing (EUCAST) method [12]. This regional variation in prevalence of azole resistance in clinical A. fumigatus isolates is likely related to different patient populations treated in different centres. Unpublished data from a recent survey in a centralised mycology laboratory in West London revealed low prevalence of azole resistance (2.2%) in 356 isolates over a 2-year period (2015-2017) (A Abdolrasouli, Diagnostic Mycology Service, North West London Pathology).

A review of studies across Europe investigating ARAf in CF have shown an average prevalence of 4.2% from a sample of 664 patients. In this series TR34/L98H was found to be the most common resistance mechanism in 60.7% of 17 ARAf isolates [13]. In the UK, TR34/L98H was detected in 6% (2/34) ARAf culture positive isolates from patients attending the National Aspergillosis Centre in Manchester [14].

In this study we aim to define the prevalence of azole-resistant A. fumigatus in unselected isolates of A. fumigatus that are obtained from patients at Royal Brompton and Harefield NHS Trust (RBHT) that are isolated using routine diagnostic workflows. Currently, approximately 600 adult and 340 paediatric patients are actively followed with CF in this centre.

MATERIALS AND METHODS

Fungal isolates: A. fumigatus isolates were prospectively collected from April 2014 to March 2016 independent of clinical significance. All isolates were cultured from routine clinical samples submitted to the diagnostic microbiology laboratory at the RBHT. Local standard operative procedures were followed to culture clinical samples and to identify fungal isolates. Identification of isolates was based on their colonial characteristics and microscopic features. All fungal isolates identified as A. fumigatus during the study period were sub-cultured and saved on Sabouraud-dextrose agar slopes (Oxoid, Basingstoke, UK). These isolates were later transferred to the Fungal Pathogens Laboratory, based at the National Heart and Lung Institute, Imperial College London for further investigation.

Azole resistance screen and confirmation: All A. fumigatus isolates were screened for azole susceptibility using a 4-well VIPcheck™ plate (Balis Laboratorium, Boven-Leeuwen, Netherlands) as previously described [15, 16]. In addition, any azole resistant isolate was confirmed with a standard microbroth dilution method according to EUCAST reference guideline [17] for seven antifungal agents and also an itraconazole E-test. Identification of isolates with azole resistant phenotype (i.e. growth on azole-containing wells in VIPcheck™ plate, raised minimum inhibitory concentration (MIC) to any triazole agents on microbroth dilution testing and raised MIC to itraconazole using an E-test) was confirmed by growth at 45°C to exclude most cryptic species that may exhibit intrinsic resistance to triazole antifungal agents.

Mechanism of resistance: In order to investigate the common cyp51A-dependant mechanisms of resistance all A. fumigatus isolates with azole-resistant phenotype were characterised for resistance mechanisms using AsperGenius® RT-PCR assay (PathoNostics, Maastricht, Netherlands). This quantitative PCR reaction targets the single copy cyp51A gene of A. fumigatus and detects the TR34, L98H, Y121F and T289A mutations in order to differentiate wild-type (WT) from mutant A. fumigatus strains via melting curve analysis. Fungal genomic DNA samples were prepared as described previously [18]. For the this PCR, the positive control from the assay was used as a standard for the melting peaks and was tested simultaneously to determine whether the melting peak of the amplicon represented WT or cyp51A mutations.

RESULTS

From April 2016 to March 2016, 167 unselected clinical isolates of A. fumigatus were prospectively analysed from 135 patients, median age 37 years (IQR 24 to 52 years). Resistance to triazoles was confirmed in 22/167 (13.1%) isolates of A. fumigatus from 18/135 (13.3%) patients, 12 female and 6 male, median age 30 year (IQR 21 to 46 years). A summary of methodology and results is depicted in Figure 1.

Sputum was the most common clinical sample for ARAf to be detected in 19/22 isolates, with one case each for lung cavity tissue, sternal wound and tracheal aspirate. The vast majority of A. fumigatus isolates originated from patients with respiratory diseases (150/167, 89.8%) (Figure 2a). We also sampled A. fumigatus isolates from eight lung transplant recipients, however no azole resistance was seen in this group. Cystic fibrosis was the commonest diagnosis that was associated with ARAf accounting for 16/22 (73%) of isolates from 12 patients giving a prevalence of ARAf among unselected CF isolates of 16.2% (12/74). There were four ARAf isolated from non-CF respiratory patients (interstitial lung disease, ABPA and two with bronchiectasis, one of whom had an aspergilloma) and two from cardiac patients; one isolate recovered from a sternal wound sample collected post aortic valve replacement and second cultured from a tracheal aspirate of a mechanically ventilated patient on intensive care with a left ventricular assist device. The largest numbers of A. fumigatus isolates were from patients in the age group 21-50 years although the highest rate of ARAf was amongst the 11 to 20 year age group (Figure 2b).

All ARAf isolates (n = 22) were resistant to itraconazole (MIC >2mg/L) from which 15 isolates showed cross-resistance to posaconazole (MIC >0.5 mg/L) and 10 isolates demonstrated reduced susceptibility to voriconazole (MIC >2mg/L) (Figure 2c). There were no pan-azole resistant A. fumigatus identified. All of the resistant isolates showed wild-type (WT) MICs for amphotericin B and echinocandin agents.

TR34/L98H was found in 6/22 (27%) of ARAf isolates from RBHT (Table 1). In CF patients with ARAf, the TR34/L98H mutation was the most commonly occurring mechanism of resistance in 42% (5/12) of tested isolates. The additional isolate with TR34/L98H was from a patient in the intensive care unit. No TR46/Y121F/T289A was detected among azole-resistant isolates. 16/22 (73%) of azole-resistant A. fumigatus isolate tested with AsperGenius® showed WT cyp51A genotype.

DISCUSSION

This prospective culture-based surveillance of unselected clinical isolates of A. fumigatus from our specialist cardio-thoracic centre in North West London revealed a prevalence of 13.1 % ARAf after screening with VIPcheck™ and confirmatory MIC testing. The prevalence of ARAf in unselected isolates from CF patients at RBHT was 16.2%; this is markedly higher than the prevalence of 4.2% reported in a review of studies from Portugal, Germany, Denmark, Italy and two from France [13]. Higher prevalence of ARAf has been reported in CF depending on the patient group and location. Two subsequent separate French studies reported higher prevalence of A. fumigatus with reduced susceptibility to itraconazole in CF cohorts; Morio et al. reported an 8% prevalence of itraconazole resistance among isolates obtained from CF patients admitted to the Nantes University Hospital [19], and in a more recent study, Guegan and colleagues found a prevalence of 12.2% ARAf amongst isolates from CF patients followed up at Rennes University Hospital [20]. Similarly, a recent study conducted in a Belgian University Hospital found 16% azole resistance among CF patients using a combination of culture-based methods and molecular tools [21].

The environmental resistance mutation TR34/L98H was identified in 42% (5/12) patients with CF and ARAf in our centre. This mutation had been identified in more than 90% ARAf isolates from Netherlands [22, 23] and is also found in a high proportion of patients with CF [24-26]. However, in these studies prior azole exposure had occured in most patients (83-100%) suggesting other mechanisms of resistance may contribute [25-27]. In the National Aspergillosis Centre, UK, TR34/L98H did not appear to be a predominant mechanism of resistance being identified only in 6% of culture-positive ARAf isolates [14]. Notably, a study of ABPA and CPA patients from this centre where Aspergillus DNA was detected using PCR in culture-negative sputum showed 55.2% prevalence of TR34/L98H [28], though this alarmingly high prevalence has not been reproduced in any other study so far.

The absence of TR46/Y121F/T289A mutation among our triazole-resistant isolates is consistent with our MIC data as this allele is associated with high-level of voriconazole resistance which was not seen in our cohort (Table 1). Furthermore, 16/22 (73%) of azole-resistant A. fumigatus isolate in this study tested with AsperGenius® showed WT cyp51A genotype. Although the molecular mechanism of azole resistance among these six isolates remains unknown, point mutations in cyp51A gene might be responsible for raised MICs to triazole antimycotic agents in a proportion of these isolates. Non-cyp51A mediated mechanisms like efflux pumps might also play a role and requires further investigation using genomics tools.

Previous studies have identified an association between ARAf infection and adverse outcomes (ref). However, these studies predominantly described more invasive forms of aspergillosis in haemato-oncological or intensive care settings [29]. In contrast, CF patients can be transiently or chronically colonised with A. fumigatus [18], some of these developing Aspergillus sensitisation or ABPA which are associated with a decline in lung function and increased airflow obstruction [30]. Although the treatment using antifungals of CF patients with isolation of Aspergillus species from respiratory specimens remain controversial, a systematic review by Moreira et al. showed that antifungal treatment for ABPA in CF patients demonstrated potential benefits in terms of clinical outcomes [31]. In most of the included studies, antifungal therapy was oral triazole-based. Therfore, if high prevalence of resistance to triazole agents occurs, then this may pose a clinical challenge in management of these patients.

Further clinical characterisation of CF patients in our cohort with ARAf is warranted to investigate potential contributory factors such as previous azole exposure, therapeutic levels of triazole drugs and adverse effects on morbidity and mortality. A longitudinal enhanced surveillance programme is urgently needed to examine larger number of isolates including sequential isolates from individual patients over time, and from cohorts of patients with different underlying clinical disorders. Molecular typing of serial isolates using whole genome sequencing will provide information on common genotypes of A. fumigatus present in CF airways and will aid in our understanding of the molecular mechanisms and epidemiological sources of antifungal resistant A. fumigatus.

Acknowledgments

The authors would like to thank the microbiology staff at the Royal Brompton and Harefield NHS Foundation Trust for assistance in fungal isolate collection.

Funding

No specific funding has been received for this study.

Transparency declaration

A. A. has been paid for talks and received travel support from Gilead. The authors of this manuscript have no conflicts of interest to disclose.

Author contributions

AA, JLR, MCF and DAJ contributed to the conception and design of the research. AA performed all experiments. All authors contributed to the analysis and interpretation of data and the drafting and revising of this manuscript.

FIGURE 1

(a) Summary of screening approach and confirmatory testing for detection of azole resistance in clinical A. fumigatus isolates, (b) the VIPcheck™ consists of a 4-wells plate, which contains RPMI agar supplemented with three medical azoles itraconazole (4 mg/L), voriconazole (1 mg/L) and posaconazole (0.5 mg/L), in wells 1 to 3 respectively and a azole-free growth control (well No. 4). After 48 hours incubation at 37°C, an azole-resistant A. fumigatus showed growth in wells 1 and 4 (left panel) while an azole-susceptible isolate only grew in well No.4 (right panel), (c) itraconazole E-test on RPMI agar confirmed raised MIC in an azole-resistant strain (left). In contrast, azole-susceptible isolate (right panel) exhibited wile-type (i. e. sensitive) MIC.

FIGURE 2

(a) Breakdown of A. fumigatus isolates by clinical classification of patients, (b) Age range of patients harbouring azole-resistant A. fumigatus compared to azole-susceptible, (c) distribution of the itraconazole (black bars), voriconazole (grey bars) and posaconazoe (white bars) MICs using EUCAST standard microbroth dilution method for 22 clinical azole-resistant isolates of A. fumigatus. HSTC; hematopoietic stem cell transplantation, GVHD; graft versus host disease, ECMO; extracorporeal membrane oxygenation, ITU; intensive care unit, ILD; interstitial lung disease, COPD; chronic obstructive pulmonary disease, ABPA; allergic bronchopulmonary aspergillosis, LRTI; lower respiratory tract infection, LTR; lung transplant recipient, CF; cystic fibrosis.

Table 1

Characteristics of triazole-resistant A. fumigatus isolates (n = 22) in this study. TA; tracheal aspirate, ILD; interstitial lung disease, CF; cystic fibrosis, ABPA; allergic bronchopulmonary aspergillosis, MIC; minimum inhibitory concentration, MEC; minimum effective concentration, ITC; itraconazole, VRC; voriconazole, PCZ; posaconazole, AMB; amphotericin B, MIF; micafungin, ANF; anidulafungin, CAS; caspofungin, WT; wild-type.

Patient No

Sample type

Patient category

MIC or MEC (mg/L)

cyp51A alteration

ITC

VRC

PCZ

AMB

MIF

ANF

CAS

RBH-1

Sputum

ILD, aspergillosis

>32

0.125

4

0.125

<0.015

<0.015

0.25

WT

RBH-2

Sputum

CF

>32

2

2

0.25

0.015

0.015

0.25

TR34/L98H

RBH-3

Sternum

Heart valve replacement

>32

0.03

2

0.25

<0.015

<0.015

0.06

WT

RBH-4

Sputum

CF

>32

0.25

0.125

0.25

<0.015

<0.015

0.06

WT

RBH-5

Sputum

CF

>32

2

1

0.5

<0.015

<0.015

0.125

WT

RBH-6

Sputum

CF

8

2

0.5

0.25

0.015

0.015

0.06

WT

RBH-7

Sputum

CF

>32

0.125

8

0.25

0.0.15

0.015

0.25

WT

RBH-7

Sputum

CF

>32

0.125

16

0.25

0.015

0.015

0.25

WT

RBH-7

Sputum

CF

>32

0.125

8

0.25

0.015

0.015

0.125

WT

RBH-7

Sputum

CF

>32

0.125

16

0.25

0.015

0.015

0.25

WT

RBH-8

TA

Left ventricular assist device

>32

2

2

0.25

0.015

0.015

0.25

TR34/L98H

RBH-9

Sputum

CF

16

2

1

0.5

0.015

0.015

0.125

TR34/L98H

RBH-10

Sputum

CF

>32

2

2

0.5

<0.015

<0.015

0.25

WT

RBH-10

Sputum

CF

>32

2

2

0.5

<0.015

<0.015

0.125

WT

RBH-11

Sputum

ABPA

>32

0.25

2

0.5

0.015

0.015

0.125

WT

RBH-12

Sputum

CF

>32

2

1

0.5

0.015

0.015

0.06

WT

RBH-13

Sputum

Bronchiectasis

>32

0.125

16

0.25

0.015

0.015

0.125

WT

RBH-14

Sputum

CF

16

1

0.25

0.25

0.002

0.002

0.03

TR34/L98H

RBH-15

Lung

Bronchiectasis, aspergilloma

>32

0.06

0.25

0.125

0.002

0.002

0.03

WT

RBH-16

Sputum

CF

16

2

0.5

0.25

0.002

0.002

0.03

TR34/L98H

RBH-17

Sputum

CF

>32

2

0.25

0.25

0.002

0.002

0.03

TR34/L98H

RBH-18

Sputum

CF

>32

0.125

0.5

0.5

0.002

0.002

0.03

WT

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