Abstract

Objectives : Several studies, especially in Europe, have recently reported the emerging phenomenon of azole resistance in Aspergillus fumigatus , but very few data are available in France. Our study aimed to determine the resistance prevalence in A. fumigatus isolates recovered from clinical samples over a 1-year period in two university hospital centers.

Methods : All A. fumigatus isolates were screened for azole resistance using RPMI agar plates supplemented with itraconazole and voriconazole. Resistance was then confirmed by the EUCAST method. A part of the beta-tubulin gene was amplified for resistant isolates to confirm the A. fumigatus species, and the Cyp51A gene and its promoter were afterward sequenced to detect mutations potentially responsible for this resistance.

Results : One hundred sixty-five A. fumigatus isolates were recovered from 134 patients. Three isolates recovered from three patients were found resistant with MICs of >8 mg/l, 4 mg/l, and 1 mg/l for itraconazole, voriconazole, and posaconazole, respectively. The TR 34 /L98H mutation, previously and largely described in other countries, was detected in the three isolates.

Conclusion : Our study demonstrated the occurrence of azole resistance among unselected A. fumigatus clinical isolates, with an overall prevalence of 1.8%.

Introduction

Aspergillus fumigatus resistance to azole drugs seems to be an emerging problem in many countries worldwide [ 1 ]. In Europe, the prevalence of this resistance is variable depending on the centers [ 2 ], but several studies have showed high prevalence, particularly in The Netherlands [ 3 ] and in the United Kingdom [ 4 ]. In France, although resistance has been detected many years ago [ 5 , 6 ], only a very few studies have been conducted so far [ 7–9 ], and the overall prevalence remains largely unknown.

Azole-resistance in A. fumigatus is mainly related to mutations in the Cyp51A gene coding for the 14-alpha-demethylase, the target enzyme of azole drugs. Many mutations have been described and may originate from selective pressure either in patients receiving long-term azole therapy or in the environment due to fungicides used in agriculture. Alternate mechanisms of resistance, not related to Cyp51A mutations, have also been described [ 10 ].

The aim of this prospective study was to estimate the prevalence of azole resistance in A. fumigatus isolates recovered from patients over a 1-year period (2012) at two centers. The Cyp51A mutations potentially involved in azole resistance were also investigated.

Materials and methods

Isolates

All isolates of A. fumigatus recovered over a period of 1 year (from January 2012 to December 2012) in clinical samples of patients from two centers (Hôpital Henri Mondor (HMN) and centre hospitalier intercommunal Créteil (CHIC)) were prospectively identified and stored for subsequent analysis. HMN and CHIC are tertiary hospitals with 1300 and 509 beds, respectively. Both immunocompromised patients (hematological malignancies, solid organ transplantations), and immunocompetent patients with chronic respiratory diseases including cystic fibrosis are hospitalized in these centers. For patients with resistant isolates, clinical data including underlying disease and previous exposure to azole drugs were retrieved. During the study period, 1737 pulmonary samples, including sputum (40%), bronchial fluid (33%), and BAL (27%), were collected and processed for mycology. From these, 165 (9.5%) samples recovered from 134 patients were positive in culture for A. fumigatus . Among these patients, 52% were immunocompetent, 40% were immunocompromised, and for 8% the underlying condition was unknown.

Table 1.

Characteristics of patients with azole-resistant isolates of Aspergillus fumigatus .

Patient Age Underlying Type of Sample Date of  MIC a (μg/ml)  Previous azole 
(years) disease infection  isolation   treatment b 
      ITZ VRZ PSZ  
58 Lung cancer Aspergilloma Bronchial fluid 06/12/2012 >8 VRZ 
62 COPD Colonization Bronchial fluid 11/04/2012 >8 no 
82 COPD ABPA sputum 11/10/2012 >8 no 
Patient Age Underlying Type of Sample Date of  MIC a (μg/ml)  Previous azole 
(years) disease infection  isolation   treatment b 
      ITZ VRZ PSZ  
58 Lung cancer Aspergilloma Bronchial fluid 06/12/2012 >8 VRZ 
62 COPD Colonization Bronchial fluid 11/04/2012 >8 no 
82 COPD ABPA sputum 11/10/2012 >8 no 

a MIC, Minimum Inhibitory Concentration as determined by EUCAST.

b During the 3-months period before recovering of the A. fumigatus isolate.COPD: Chronic Obstructive Pulmonary Disease; ABPA: Allergic Broncho-Pulmonary Aspergillosis; ITZ: Itraconazole; VRZ: Voriconazole; PSZ: Posaconazole.

Screening for resistance

Isolates (one per sample) were revived from storage and subcultured on Malt-extract agar slants. In vitro susceptibility to azoles was screened by subculturing each isolate on RPMI agar plates supplemented with itraconazole (Sigma-Aldrich, Saint-Quentin Fallavier France) at 4 mg/l and voriconazole (Sigma-Aldrich) at 1 mg/l as previously described [ 11 ].

In vitro antifungal susceptibility testing

In vitro resistance detected by the screening tests was confirmed by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) microdilution broth reference method [ 12 ]. EUCAST clinical breakpoints [ 13 ] were used to categorize the isolates. Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were included as quality controls. Minimum inhibitory concentrations (MICs) were determined visually after 48h of incubation at 35°C with a complete inhibition endpoint.

Molecular identification

For resistant strains, a molecular identification was performed to ensure that isolates were A. fumigatus sensu stricto . Isolates were cultured on Malt-extract agar slants for 3 days at 37°C. Aspergillus conidia were suspended in ATL tissue lysis buffer (Roche Diagnostics, Meylan, France) and mechanically disrupted in a MagNa Lyser instrument (Roche). DNA extraction was performed using the QIAmp DNA Mini Kit (Qiagen, Courtaboeuf, France) according to manufacturer's instructions. A part of the beta-tubulin gene was polymerase chain reaction (PCR) amplified [ 9 ] and sequenced on an Applied Biosystems ABI PRISM 3130XL genetic analyzer.

Cyp51A sequencing

For each azole resistant isolate, the whole Cyp51A gene and its promoter were PCR amplified [ 9 ] and sequenced as described above. Sequences were compared with that of a wild type isolate (GenBank acc. number AF338659) using SeqScape software v2.5 (Applied Biosystems).

Results

Three isolates from three patients were found to be itraconazole- and voriconazole-resistant based on the screening with azole-containing agar plates. The partial beta-tubulin sequence confirmed that all three isolates belonged to A. fumigatus species sensu stricto . The azole resistance was confirmed by the EUCAST reference microdilution technique with MICs of >8 mg/l, 4 mg/l, and 1 mg/l for itraconazole, voriconazole, and posaconazole, respectively (see Table 1 ). The same MIC values were obtained for the three isolates.

In the present study the prevalence of azole resistance was 1.8% (3/165) among the tested isolates and 2.2% (3/134) of the patients enrolled. Sequencing of the Cyp51A and deduced protein sequence demonstrated a TR 34 /L98H mutation for the three resistant isolates.

Case 1: A 58 years old patient previously treated by a combination of surgery, chemotherapy and radiation therapy for a lung cancer in 2008 was admitted in 2012. An excavated lesion of the left apex led to the diagnosis of aspergilloma. The patient was subsequently treated with voriconazole 400 mg/day for a period of 5 months with initial resolution of symptoms. A bronchial fluid grew an A. fumigatus 6 weeks after the end of the medical treatment. A radical surgery was performed in January 2013. The outcome was favorable.

Case 2: A 62 year-old former smoker patient with chronic obstructive pulmonary disease (COPD) was admitted in April 2012 in the context of bacterial pulmonary nodules. The patient was successfully treated with amoxicillin - clavulanic acid. Favorable clinical outcome on antibacterial treatment and negativity of specific Aspergillus antibodies suggested a colonization process by A. fumigatus . The nodules were stable, 1 year later. The patient never received azole treatment.

Case 3: An 82 year-old COPD patient was diagnosed in 2011 with allergic broncho-pulmonary aspergillosis (ABPA). He received itraconazole at 200 mg/d between May and September 2011. The patient was admitted in October 2012 for an exacerbation of ABPA. He was then successfully treated by a combination of antibiotics and corticosteroids. The azole-resistant A. fumigatus was isolated from sputum in October 2012.

Discussion

In the present study the prevalence of azole-resistance was 1.8% in unselected population of patients. Screening of unselected isolates of A. fumigatus has seldom been performed in France [ 2 , 5 ]. In one of these studies a similar prevalence of 2.6% was found [ 5 ].

The other few studies conducted in France have targeted a specific population such as cystic fibrosis (CF) or haematological patients. In CF patients, a relatively high prevalence of resistance (4.6 to 8%) has been reported [ 7 , 8 ]. Indeed, these patients are more likely to harbour resistant isolates due to previous azole exposure. In contrast a low resistance rate of 1.1% has been found in haematological patients [ 9 ].

Azole resistance in A. fumigatus has been reported in several countries in Europe such as The Netherlands [ 14 ], United Kingdom [ 15 ], Germany [ 16 ], Sweeden [ 17 ], and Denmark [ 18 ]. More recently, resistant isolates have been detected outside of Europe in the United States, China, India, and Iran [ 1 ].

In our study, the three resistant isolates harboured the TR 34 /L98H mutation, which has been largely described in other countries [ 1 ] and in France [ 7 , 8 ]. This mutation seems to be acquired in the environment and is not related to selective pressure due to previous azole treatment. This is probably the case of patient #2 and #3 who did not receive azole treatment less than 3 months before the recovery of the resistant isolate.

In conclusion, our results provide new data about azole resistance in France and confirmed the occurrence of azole resistance with a prevalence around 2% over a 12-month period. These results highlight the need to perform antifungal susceptibility testing in A. fumigatus isolates obtained in microbiology laboratories. Larger studies are needed to better estimate the actual prevalence in different geographical areas in France and in different populations of patients and to investigate the environment for the detection of resistant isolates.

Funding

This work was supported by a grant from MSD.

Declaration of interest

ED has received grants from Gilead, Ferrer, and Biorad, payment for lectures from Gilead, MSD, and Schering, and has been consultant for Astellas and Innothera.

FB has received payment for lectures from MSD.

All other authors have no conflict to declare.

References

1.
Vermeulen
E
Lagrou
K
Verweij
PE
Azole resistance in Aspergillus fumigatus : a growing public health concern
Curr Opin Infect Dis
 
2013
26
493
500
2.
Van der Linden
JWM
Arendrup
MC
Verweij
PE
SCARE network
Prospective international surveillance of azole resistance in Aspergillus fumigatus : SCARE-Network
Paper presented at: Abstract of the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC)
 
2011
Chicago, IL
Abstract M-490. American Society for Microbiology, Washington, DC, USA
3.
Snelders
E
van der Lee
HA
Kuijpers
J
et al
Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism
PLoS Med
 
2008
5
e219
4.
Bueid
A
Howard
SJ
Moore
CB
et al
Azole antifungal resistance in Aspergillus fumigatus : 2008 and 2009
J Antimicrob Chemother
 
2010
65
2116
2118
5.
Dannaoui
E
Persat
F
Monier
MF
et al
In-vitro susceptibility of Aspergillus spp. isolates to amphotericin B and itraconazole
J Antimicrob Chemother
 
1999
44
553
555
6.
Dannaoui
E
Borel
E
Monier
MF
et al
Acquired itraconazole resistance in Aspergillus fumigatus
J Antimicrob Chemother
 
2001
47
333
340
7.
Morio
F
Aubin
GG
Danner-Boucher
I
et al
High prevalence of triazole resistance in Aspergillus fumigatus , especially mediated by TR/L98H, in a French cohort of patients with cystic fibrosis
J Antimicrob Chemother
 
2012
67
1870
1873
8.
Burgel
PR
Baixench
MT
Amsellem
M
et al
High prevalence of azole-resistant Aspergillus fumigatus in adults with cystic fibrosis exposed to itraconazole
Antimicrob Agents Chemother
 
2012
56
869
874
9.
Alanio
A
Sitterle
E
Liance
M
et al
Low prevalence of resistance to azoles in Aspergillus fumigatus in a French cohort of patients treated for haematological malignancies
J Antimicrob Chemother
 
2011
66
371
374
10.
Camps
SM
Dutilh
BE
Arendrup
MC
et al
Discovery of a HapE mutation that causes azole resistance in Aspergillus fumigatus through whole genome sequencing and sexual crossing
PLoS One
 
2012
7
e50034
11.
van der Linden
JW
Camps
SM
Kampinga
GA
et al
Aspergillosis due to voriconazole highly resistant Aspergillus fumigatus and recovery of genetically related resistant isolates from domiciles
Clin Infect Dis
 
2013
57
513
520
12.
Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST), Rodriguez-Tudela JL, Donnelly JP et al. EUCAST Technical Note on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia-forming moulds
Clin Microbiol Infect
 
2008
14
982
984
13.
Verweij
PE
Howard
SJ
Melchers
WJ
et al
Azole-resistance in Aspergillus : proposed nomenclature and breakpoints
Drug Resist Updat
 
2009
12
141
147
14.
van der Linden
JW
Snelders
E
Kampinga
GA
et al
Clinical Implications of Azole Resistance in Aspergillus fumigatus , the Netherlands, 2007–2009
Emerg Infect Dis
 
2011
17
1846
1854
15.
Howard
SJ
Cerar
D
Anderson
MJ
et al
Frequency and evolution of Azole resistance in Aspergillus fumigatus associated with treatment failure
Emerg Infect Dis
 
2009
15
1068
1076
16.
Bader
O
Weig
M
Reichard
U
et al
cyp51A-Based mechanisms of Aspergillus fumigatus azole drug resistance present in clinical samples from Germany
Antimicrob Agents Chemother
 
2013
57
3513
3517
17.
Chryssanthou
E
In vitro susceptibility of respiratory isolates of Aspergillus species to itraconazole and amphotericin B. acquired resistance to itraconazole
Scand J Infect Dis
 
1997
29
509
512
18.
Mortensen
KL
Jensen
RH
Johansen
HK
et al
Aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: a laboratory-based study with focus on Aspergillus fumigatus azole resistance
J Clin Microbiol
 
2011
49
2243
2251