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: [email protected]: +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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