AAC Accepted Manuscript Posted Online 18 May 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00233-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Nationwide surveillance of azole resistance in Aspergillus disease
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Edith Vermeulen1, Johan Maertens1,2, Annelies De Bel3, Eric Nulens4, Jerina Boelens5, Ignace
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Surmont6, Anna Mertens7, An Boel8, Katrien Lagrou1,9*
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*Corresponding author: Katrien Lagrou, National Reference Center for Mycosis, University Hospitals Leuven,
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Herestraat 49, 3000 Leuven, Belgium;
[email protected]; phone +32 16 347 098, fax +32 16 347 931
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Department of Microbiology and Immunology, Catholic University Leuven, Leuven, Belgium
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Department of Haematology, University Hospitals Leuven, Leuven, Belgium
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Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel
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(VUB), Brussels, Belgium
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Department of Microbiology, AZ Sint-Jan Brugge-Oostende AV, Bruges, Belgium
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Department of Microbiology, Ghent University Hospital, Ghent, Belgium
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Department of Microbiology, Heilig Hart Ziekenhuis Roeselare-Menen, Roeselare, Belgium
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Department of Microbiology, ZNA Hospitals site Middelheim, Antwerp, Belgium
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Department of Clinical Microbiology, Onze-Lieve-Vrouwziekenhuis, Aalst, Belgium
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National Reference Center for Mycosis, Department of Laboratory Medicine, University Hospitals Leuven, Leuven,
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Belgium
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Running title: Azole resistance in aspergillosis
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ABSTRACT (209 words)
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Aspergillus disease affects a broad patient population, from patients with asthma to
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immunocompromised patients. Azole resistance is increasingly reported in both clinical and
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environmental Aspergillus strains. The prevalence and clinical impact of azole resistance in
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different patient populations is currently unclear.
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This one-year prospective multicenter cohort study aimed to provide detailed epidemiological
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data on Aspergillus resistance among patients suffering from Aspergillus disease in Belgium.
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Isolates were prospectively collected in 18 hospitals (April 2011 to April 2012) for
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susceptibility testing. Clinical and treatment data were collected with a questionnaire.
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Outcome was evaluated to one year after the patient’s inclusion.
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220 Aspergillus isolates were included from 182 patients. The underlying conditions included
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invasive aspergillosis (n= 122 patients), allergic bronchopulmonary aspergillosis (APBA)
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(n=39 patients), chronic pulmonary aspergillosis (n=10 patients), Aspergillus bronchitis (n=7
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patients) and aspergilloma (n=5 patients). Azole resistance prevalence was 5.5% (95% CI
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2.8–10.2%), namely 7.0% (4/57; 95% CI 2.3–17.2%) in patients suffering from APBA,
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bronchitis, aspergilloma or chronic aspergillosis and 4.6% in patients with invasive
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aspergillosis (5/108; 95%CI 1.7–10.7%). The 6-week survival in invasive aspergillosis was
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52.5%; whilst susceptibility testing revealed azole resistance in only 2/58 of the deceased
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patients. The clinical impact of A. fumigatus’ resistance was limited in our patient population
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suffering from Aspergillus' diseases.
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MANUSCRIPT (2269 words)
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INTRODUCTION
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Aspergilli are saprophytic molds, which are ubiquitous in our living environment.
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Aspergillosis represents a spectrum of disease affecting millions of people worldwide,
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ranging from life-threatening angio-invasive infections in immunocompromised patients to
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chronic infections and allergic syndromes. Aspergillus fumigatus is the primary human
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pathogenic species. Per current international guidelines, voriconazole is the antifungal drug of
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choice in invasive aspergillosis (IA) and itraconazole mainly in the chronic and allergic
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disease entities (1). However, azole resistance can affect patients via two routes (2).
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Resistance can develop gradually during the course of antifungal therapy (3). Secondly, azole-
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naïve patients, such as most IA patients, may suffer from azole resistant disease when they
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become infected with a strain which gained resistance properties externally (4). In the latter
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only a few predominant resistance mechanisms occur, namely the CYP51A alterations
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TR34/L98H and TR46/Y121F/T289A (4,5). Such resistant strains are selected in the
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environment, by the agricultural use of azole fungicides (2). Since 2007, reports of A.
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fumigatus azole resistance emerged from different countries and patient populations. The
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prevalence of “environmental resistance” ranges from 0 to 6.5%, with the highest percentage
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reported in the Netherlands (2-13). The ongoing spread of environmental resistance in and
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outside Europe is of clinical concern. Whether azole resistance is an independent risk factor
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for clinical failure remains uncertain. A threshold rate of resistance prevalence that would trigger a
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change in the empiric therapeutic regimen is yet to be determined (14). In real-life registries, the
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first-line use of voriconazole has been consistently and independently associated with
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improved response and decreased IA-attributable mortality when compared to other mold-
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active agents (15-18). Detailed epidemiological findings are essential to the debate at hand.
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The public health risk associated with the environmental triazole use has recently been 3
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assessed by the European Centre for Disease Control, but remains inconclusive since
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resistance development is poorly understood and its patient and economic impact needs
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further investigation (19).
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This one-year prospective multicenter cohort study aimed to investigate the prevalence of
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azole resistance in Belgium, among patients suffering from Aspergillus disease and its clinical
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impact.
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METHODS
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Clinical surveillance
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Inclusion criteria
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Aspergillus spp. cultured from clinical samples were prospectively collected in 18 Belgian
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hospitals caring for intensive care, hematology and/or transplant patients from April 2011 to
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April 2012. Only isolates from patients with documented Aspergillus disease were included.
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The participating microbiologists evaluated the clinical relevance of positive cultures by
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revising the medical files and the patients’ laboratory, radiology and pathology data, in
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consultation with the treating physician. IA cases were classified as “proven” or “probable”
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disease following the revised European Organisation for Research and Treatment of
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Cancer/Mycosis Study Group (EORTC/MSG) definitions (20). In addition, patients with a
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high clinical suspicion of IA, with no other apparent cause of disease and no response to
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broad-spectrum antibiotics, for which mold-active antifungal therapy was initiated, were
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included despite not fulfilling the EORTC/MSG criteria (referred to as “non-EORTC/MSG
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IA” in the remainder of the text). The principal investigators assessed whether the latter cases
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fulfilled the criteria for “putative” IA, as postulated by Blot (21). Of note, the Blot criteria are
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host and clinical criteria designed to judge Aspergillus cultured from BAL or endotrachial
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aspirate only. In this study, we also judged patients with sputum cultures as eligible for
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"putative" IA, if galactomannan (GM, Aspergillus antigen) positivity in blood (index ≥ 0.5) or 4
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broncho-alveolar lavage fluid (BAL) (index ≥ 0.8) supported the sputum culture as
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microbiological evidence.
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Patients with non-EORTC/MSG IA and chronic obstructive pulmonary disease (COPD) were
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also assessed according to the criteria designed by Bulpa (22).
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Allergic bronchopulmonary aspergillosis (ABPA) was defined following the minimal
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diagnostic criteria from the Cystic Fibrosis Foundation Consensus conference (23). Isolates
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from patients with acute ABPA, ABPA exacerbation and remission were included. Isolates
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from patients with chronic necrotizing (CNPA), cavitary (CCPA) or fibrosing (CFPA)
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pulmonary aspergillosis, single sinus or lung aspergilloma or Aspergillus bronchitis were
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identified based on clinical, radiologic and serologic findings (total IgE, specific IgE, specific
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IgG) (24,25). Supplemental table S1 summarizes all diagnostic criteria. The susceptibility
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result of maximum one isolate of the same species/patient/month was included, unless their
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susceptibility differed by interpretation for at least one azole.
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Susceptibility testing
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All Aspergillus isolates cultured in the participating centers were sent to the National
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Reference Center for Mycosis for analysis. Broth microdilution susceptibility testing was
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performed with the pure substances of itraconazole (Janssen Pharmaceutica), voriconazole
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(Pfizer) and posaconazole (Merck), following the Clinical Laboratory Standards Institute
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M38-A2 protocol. All Aspergillus colonies that grew were touched to prepare the inoculum
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(0.5 McFarland). The following interpretative criteria, based on epidemiological cut-off
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values (ECV), were applied for A. fumigatus: itraconazole or voriconazole minimal inhibitory
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concentration (MIC) ≤1 mg/L was considered susceptible, 2 mg/L intermediate and >2 mg/L
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resistant; posaconazole MIC ≤0.25 mg/L susceptible, 0.5 mg/L intermediate and >0.5 mg/L
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resistant (26). Susceptibility of isolates not belonging to A. fumigatus complex was judged by
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conformance with their respective ECV (26).
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Genotyping
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All Aspergillus isolates with non-wildtype susceptibility (an intermediate or resistant MIC to
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≥ one tested azole) were identified with beta-tubulin sequencing (27). Non-wild type A.
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fumigatus sensu stricto isolates were also submitted to CYP51A sequencing, as published
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(10), but with an adaptation to primer AF766F (5’TTCGGATCGGACGTGGTGT3’) due to
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observed interference of the CYP51A-mutation Y121F with the original primer.
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Statistical analysis
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Clinical signs and symptoms, treatment regimens, underlying disease and azole pre-exposure
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data were collected from the participating centers using a questionnaire. Outcome data were
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collected by revision of the medical files up to one year after the patient’s inclusion. This
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study was approved by the ethical committee University Hospitals Leuven, with reference
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S53024 (Belgian national reference B322201110423).
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All statistical analyses were performed with GraphPad Software, Inc. Confidence intervals
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(95% CI) for a proportion were calculated with the modified Wald method. The level of
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significance (p) was calculated with Fisher’s exact test (proportions), t-test for independent
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samples (age) or Mann-Withney test (GM medians). Two‐tailed p-values