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.
2
Nationwide surveillance of azole resistance in Aspergillus disease
3
Edith Vermeulen1, Johan Maertens1,2, Annelies De Bel3, Eric Nulens4, Jerina Boelens5, Ignace
4
Surmont6, Anna Mertens7, An Boel8, Katrien Lagrou1,9*
5
*Corresponding author: Katrien Lagrou, National Reference Center for Mycosis, University Hospitals Leuven,
6
Herestraat 49, 3000 Leuven, Belgium;
[email protected]; phone +32 16 347 098, fax +32 16 347 931
7
1
Department of Microbiology and Immunology, Catholic University Leuven, Leuven, Belgium
8
2
Department of Haematology, University Hospitals Leuven, Leuven, Belgium
9
3
Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel
1
10
(VUB), Brussels, Belgium
11
4
Department of Microbiology, AZ Sint-Jan Brugge-Oostende AV, Bruges, Belgium
12
5
Department of Microbiology, Ghent University Hospital, Ghent, Belgium
13
6
Department of Microbiology, Heilig Hart Ziekenhuis Roeselare-Menen, Roeselare, Belgium
14
7
Department of Microbiology, ZNA Hospitals site Middelheim, Antwerp, Belgium
15
8
Department of Clinical Microbiology, Onze-Lieve-Vrouwziekenhuis, Aalst, Belgium
16
9
National Reference Center for Mycosis, Department of Laboratory Medicine, University Hospitals Leuven, Leuven,
17
Belgium
18 19
Running title: Azole resistance in aspergillosis
20 21 22 23 24 1
25
ABSTRACT (209 words)
26
Aspergillus disease affects a broad patient population, from patients with asthma to
27
immunocompromised patients. Azole resistance is increasingly reported in both clinical and
28
environmental Aspergillus strains. The prevalence and clinical impact of azole resistance in
29
different patient populations is currently unclear.
30
This one-year prospective multicenter cohort study aimed to provide detailed epidemiological
31
data on Aspergillus resistance among patients suffering from Aspergillus disease in Belgium.
32
Isolates were prospectively collected in 18 hospitals (April 2011 to April 2012) for
33
susceptibility testing. Clinical and treatment data were collected with a questionnaire.
34
Outcome was evaluated to one year after the patient’s inclusion.
35
220 Aspergillus isolates were included from 182 patients. The underlying conditions included
36
invasive aspergillosis (n= 122 patients), allergic bronchopulmonary aspergillosis (APBA)
37
(n=39 patients), chronic pulmonary aspergillosis (n=10 patients), Aspergillus bronchitis (n=7
38
patients) and aspergilloma (n=5 patients). Azole resistance prevalence was 5.5% (95% CI
39
2.8–10.2%), namely 7.0% (4/57; 95% CI 2.3–17.2%) in patients suffering from APBA,
40
bronchitis, aspergilloma or chronic aspergillosis and 4.6% in patients with invasive
41
aspergillosis (5/108; 95%CI 1.7–10.7%). The 6-week survival in invasive aspergillosis was
42
52.5%; whilst susceptibility testing revealed azole resistance in only 2/58 of the deceased
43
patients. The clinical impact of A. fumigatus’ resistance was limited in our patient population
44
suffering from Aspergillus' diseases.
45 46 47 48 2
49
MANUSCRIPT (2269 words)
50
INTRODUCTION
51
Aspergilli are saprophytic molds, which are ubiquitous in our living environment.
52
Aspergillosis represents a spectrum of disease affecting millions of people worldwide,
53
ranging from life-threatening angio-invasive infections in immunocompromised patients to
54
chronic infections and allergic syndromes. Aspergillus fumigatus is the primary human
55
pathogenic species. Per current international guidelines, voriconazole is the antifungal drug of
56
choice in invasive aspergillosis (IA) and itraconazole mainly in the chronic and allergic
57
disease entities (1). However, azole resistance can affect patients via two routes (2).
58
Resistance can develop gradually during the course of antifungal therapy (3). Secondly, azole-
59
naïve patients, such as most IA patients, may suffer from azole resistant disease when they
60
become infected with a strain which gained resistance properties externally (4). In the latter
61
only a few predominant resistance mechanisms occur, namely the CYP51A alterations
62
TR34/L98H and TR46/Y121F/T289A (4,5). Such resistant strains are selected in the
63
environment, by the agricultural use of azole fungicides (2). Since 2007, reports of A.
64
fumigatus azole resistance emerged from different countries and patient populations. The
65
prevalence of “environmental resistance” ranges from 0 to 6.5%, with the highest percentage
66
reported in the Netherlands (2-13). The ongoing spread of environmental resistance in and
67
outside Europe is of clinical concern. Whether azole resistance is an independent risk factor
68
for clinical failure remains uncertain. A threshold rate of resistance prevalence that would trigger a
69
change in the empiric therapeutic regimen is yet to be determined (14). In real-life registries, the
70
first-line use of voriconazole has been consistently and independently associated with
71
improved response and decreased IA-attributable mortality when compared to other mold-
72
active agents (15-18). Detailed epidemiological findings are essential to the debate at hand.
73
The public health risk associated with the environmental triazole use has recently been 3
74
assessed by the European Centre for Disease Control, but remains inconclusive since
75
resistance development is poorly understood and its patient and economic impact needs
76
further investigation (19).
77
This one-year prospective multicenter cohort study aimed to investigate the prevalence of
78
azole resistance in Belgium, among patients suffering from Aspergillus disease and its clinical
79
impact.
80
METHODS
81
Clinical surveillance
82
Inclusion criteria
83
Aspergillus spp. cultured from clinical samples were prospectively collected in 18 Belgian
84
hospitals caring for intensive care, hematology and/or transplant patients from April 2011 to
85
April 2012. Only isolates from patients with documented Aspergillus disease were included.
86
The participating microbiologists evaluated the clinical relevance of positive cultures by
87
revising the medical files and the patients’ laboratory, radiology and pathology data, in
88
consultation with the treating physician. IA cases were classified as “proven” or “probable”
89
disease following the revised European Organisation for Research and Treatment of
90
Cancer/Mycosis Study Group (EORTC/MSG) definitions (20). In addition, patients with a
91
high clinical suspicion of IA, with no other apparent cause of disease and no response to
92
broad-spectrum antibiotics, for which mold-active antifungal therapy was initiated, were
93
included despite not fulfilling the EORTC/MSG criteria (referred to as “non-EORTC/MSG
94
IA” in the remainder of the text). The principal investigators assessed whether the latter cases
95
fulfilled the criteria for “putative” IA, as postulated by Blot (21). Of note, the Blot criteria are
96
host and clinical criteria designed to judge Aspergillus cultured from BAL or endotrachial
97
aspirate only. In this study, we also judged patients with sputum cultures as eligible for
98
"putative" IA, if galactomannan (GM, Aspergillus antigen) positivity in blood (index ≥ 0.5) or 4
99
broncho-alveolar lavage fluid (BAL) (index ≥ 0.8) supported the sputum culture as
100
microbiological evidence.
101
Patients with non-EORTC/MSG IA and chronic obstructive pulmonary disease (COPD) were
102
also assessed according to the criteria designed by Bulpa (22).
103
Allergic bronchopulmonary aspergillosis (ABPA) was defined following the minimal
104
diagnostic criteria from the Cystic Fibrosis Foundation Consensus conference (23). Isolates
105
from patients with acute ABPA, ABPA exacerbation and remission were included. Isolates
106
from patients with chronic necrotizing (CNPA), cavitary (CCPA) or fibrosing (CFPA)
107
pulmonary aspergillosis, single sinus or lung aspergilloma or Aspergillus bronchitis were
108
identified based on clinical, radiologic and serologic findings (total IgE, specific IgE, specific
109
IgG) (24,25). Supplemental table S1 summarizes all diagnostic criteria. The susceptibility
110
result of maximum one isolate of the same species/patient/month was included, unless their
111
susceptibility differed by interpretation for at least one azole.
112 113
Susceptibility testing
114
All Aspergillus isolates cultured in the participating centers were sent to the National
115
Reference Center for Mycosis for analysis. Broth microdilution susceptibility testing was
116
performed with the pure substances of itraconazole (Janssen Pharmaceutica), voriconazole
117
(Pfizer) and posaconazole (Merck), following the Clinical Laboratory Standards Institute
118
M38-A2 protocol. All Aspergillus colonies that grew were touched to prepare the inoculum
119
(0.5 McFarland). The following interpretative criteria, based on epidemiological cut-off
120
values (ECV), were applied for A. fumigatus: itraconazole or voriconazole minimal inhibitory
121
concentration (MIC) ≤1 mg/L was considered susceptible, 2 mg/L intermediate and >2 mg/L
122
resistant; posaconazole MIC ≤0.25 mg/L susceptible, 0.5 mg/L intermediate and >0.5 mg/L
5
123
resistant (26). Susceptibility of isolates not belonging to A. fumigatus complex was judged by
124
conformance with their respective ECV (26).
125 126
Genotyping
127
All Aspergillus isolates with non-wildtype susceptibility (an intermediate or resistant MIC to
128
≥ one tested azole) were identified with beta-tubulin sequencing (27). Non-wild type A.
129
fumigatus sensu stricto isolates were also submitted to CYP51A sequencing, as published
130
(10), but with an adaptation to primer AF766F (5’TTCGGATCGGACGTGGTGT3’) due to
131
observed interference of the CYP51A-mutation Y121F with the original primer.
132 133
Statistical analysis
134
Clinical signs and symptoms, treatment regimens, underlying disease and azole pre-exposure
135
data were collected from the participating centers using a questionnaire. Outcome data were
136
collected by revision of the medical files up to one year after the patient’s inclusion. This
137
study was approved by the ethical committee University Hospitals Leuven, with reference
138
S53024 (Belgian national reference B322201110423).
139
All statistical analyses were performed with GraphPad Software, Inc. Confidence intervals
140
(95% CI) for a proportion were calculated with the modified Wald method. The level of
141
significance (p) was calculated with Fisher’s exact test (proportions), t-test for independent
142
samples (age) or Mann-Withney test (GM medians). Two‐tailed p-values
