Nationwide surveillance of azole resistance in Aspergillus disease

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May 18, 2015 - Vandecandelaere Patricia (Regionaal Ziekenhuis Jan Yperman, Ieper); .... Alanio A, Sitterlé E, Liance M, Farrugia C, Foulet F, Botterel F, .... Baxter CG, Dunn G, Jones AM, Webb K, Gore R, Richardson MD, Denning DW. 2013 ...
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