Abstract Objectives Aspergillus fumigatus is the most prevalent filamentous fungus in the respiratory tract of patients with cystic fibrosis (CF). The aim of this prospective multicentre study was to investigate the prevalence of azole-resistant A. fumigatus (ARAF) in respiratory secretions from CF patients across Germany and to characterize ARAF isolates by phenotypic and molecular methods. Methods Twelve tertiary care centres from Germany participated in the study. In total, 2888 A. fumigatus isolates from 961 CF patients were screened for ARAF by using azole-containing agar plates. Antifungal susceptibility testing of isolates was performed by broth microdilution according to EUCAST guidelines. Analysis of mutations mediating resistance was performed using PCR and sequencing of the cyp51A gene. Furthermore, genotyping by microsatellite PCR was performed. Results Of a total of 2888 A. fumigatus isolates, 101 isolates from 51 CF patients were found to be azole resistant (prevalence per patient 5.3%). The Essen centre had the highest prevalence (9.1%) followed by Munich (7.8%), Münster (6.0%) and Hannover (5.2%). Most ARAF isolates (n = 89) carried the TR34/L98H mutation followed by eight G54E/R, one TR46/Y121F/T289A and one F219S mutation. In two isolates no mutation was found. Genotyping results showed no major clustering. Forty-five percent of CF patients with ARAF had previously received azole therapy. Conclusions This is the first multicentre study analysing the prevalence of ARAF isolates in German CF patients. Because of a resistance rate of up to 9%, susceptibility testing of A. fumigatus isolates from CF patients receiving antifungal treatment should be part of standard diagnostic work-up. Introduction Cystic fibrosis (CF) is the most common lethal autosomal hereditary disorder in Caucasians with an incidence of approximately 1 in 2500 live births.1 The CF lung is highly vulnerable to respiratory colonization/infections from bacterial and fungal organisms resulting in recurrent and chronic inflammation and tissue remodelling. Aspergillus fumigatus is the most prevalent filamentous fungus in the respiratory tract of patients with CF.2,3 Recent data from Germany showed that around 30% of CF patients are colonized with A. fumigatus.3 Allergic bronchopulmonary aspergillosis (ABPA) and Aspergillus bronchitis are the most frequent Aspergillus-associated complications, occurring in 2% to 25% of CF subjects.4 ABPA is associated with airway damage and decline in lung function.4 Several studies around the world5,6 have reported on the emergence of resistance in A. fumigatus to azole compounds with the first isolates in Germany being detected in 2012.7,8 Pulmonary colonization with azole-resistant A. fumigatus (ARAF) in CF patients is of medical concern. When antifungal therapy is necessary, treatment options are limited and currently no oral drug is available.9,10 In addition, knowledge of the local epidemiology of ARAF, particularly for CF patients colonized with A. fumigatus listed for lung transplantation (LTX), might be of importance. CF patients undergoing LTX are at high risk of developing Aspergillus-associated infections9 and azole-based prophylaxis/treatment in LTX patients is recommended during and after transplant surgery for an extended period according to recent guidelines.11 Previous single-centre studies from European countries reported ARAF prevalence in CF patients ranging from 0% to 8%.12–17 Data from Germany are scarce because most clinical laboratories do not routinely perform antifungal susceptibility testing of A. fumigatus. Here, we conducted the first prospective, multicentre study to elucidate the prevalence of ARAF in German CF patients. All ARAF isolates underwent phenotypic and molecular characterization, including microsatellite genotyping to explore their molecular epidemiology. Materials and methods Collection of A. fumigatus isolates Twelve German university hospitals with dedicated CF departments participated in the study and collected A. fumigatus isolates which were grown from respiratory specimens taken from CF patients from January 2012 to April 2016. Centres were allowed to collect several isolates from one patient. The following centres participated, in alphabetical order (number of isolates): Aachen (41), Cologne (162), Dresden (32), Düsseldorf (7), Essen (1011), Frankfurt (137), Hannover (1144), Heidelberg (40), Homburg/Saarbrücken (5), Munich (75), Münster (218) and Ulm (16). All isolates were further investigated in the central laboratory of the Institute of Medical Microbiology, University Hospital Essen, Essen, Germany. Screening for ARAF and species identification All aspergilli were plated onto a Sabouraud dextrose agar containing 4 mg/L itraconazole to screen for azole resistance. In the case of growth, species identification was performed by characteristic micro- and macromorphological criteria and β-tubulin sequencing as previously described.18 Susceptibility testing All A. fumigatus isolates that grew on the screening agar were further tested using broth microdilution according to the EUCAST 9.3 standard.19 Susceptibility was assessed for itraconazole, voriconazole, posaconazole (Sigma–Aldrich Life Sciences, Taufkirchen, Germany) and isavuconazole (Basilea Pharmaceutica, Basel, Switzerland). A. fumigatus ATCC 204305 was included in the testing as a quality control strain. DNA isolation All isolates with at least one elevated MIC for any azole were further analysed for underlying mutations. From Sabouraud agar inoculated with the isolate, three 5 mm2 agar blocks were punched out and lysed using MagNA Lyser (Roche). For DNA extraction and purification, the Maxwell 16 instrument was used with the Maxwell 16 LEV Total RNA Purification Kit (Promega, Mannheim, Germany). Determination of mutations in cyp51A To detect the three most common mutations all isolates were tested with a commercially available multiplex PCR (AsperGenius®, PathoNostics, Maastricht, The Netherlands). Next, the cyp51A gene was sequenced from those isolates that were negative in the AsperGenius assay, as described.20 Sequences were then analysed using the FunResDB-A database, a web resource for genotypic susceptibility testing of A. fumigatus,21 and matched with the non-mutated cyp51A sequence. Amino acid substitutions were compared with published mutations and concomitant cross-resistance to azoles. Microsatellite PCR Microsatellite PCR was performed on all ARAF isolates as described previously by de Valk et al.22 PCR products were analysed by capillary electrophoresis using an ABI 3130 sequence analyser (Applied Biosystems). As size standard, GeneScan 1200 LIZ Dye Standard (Applied Biosystems) was used. The fragment length of all nine microsatellite markers was analysed. Unweighted pair group analysis was used to calculate distance between isolates. Geneious 8.1.4 software was used to assign peak maxima and bin data for specific fragment lengths of microsatellite loci. Fragment length tables were exported to Microsoft Excel 2013 and PHYLOViZ 2.0/PHYLOViZ online software for further analysis. Patient characteristics Patient data concerning sex, age, Aspergillus-associated disease (e.g. ABPA, Aspergillus bronchitis), LTX status and previous azole administration were collected. Clinical data were available from 781/961 CF patients (81.3%) including 49/51 patients with an ARAF isolate. The study was reviewed and approved by the Ethics Committee of the Medical Faculty of the University of Duisburg-Essen (17-7668-BO). Statistical analysis Statistical analysis was performed with Excel 2013 and GraphPad Prism 6.0. The two-sided t-test was used for analysis of significance concerning sex and age distribution of ARAF and non-ARAF CF patients. Results Twelve centres across Germany participated in the study. In total, 2888 A. fumigatus isolates from 961 CF patients (mean age, 29.5 years; male, 49.6%) were analysed for azole resistance. Overall, 101 A. fumigatus isolates resistant to at least one azole compound were found. Azole resistance was detected in 7/12 (58%) participating centres. The overall prevalence of azole resistance calculated per patient was 5.3% (Figure 1). The resistance rate varied between the centres (Figure 1). The Essen centre in West Germany had the highest prevalence (9.1%), followed by Munich in Southern Germany (7.8%), then Münster and Hannover (6.0% and 5.2%) in West and Northern Germany, respectively. The antifungal susceptibility testing results (MIC50, MIC90 and MIC range) for the triazole agents itraconazole, voriconazole, posaconazole and isavuconazole against the 101 ARAF isolates are shown in Table 1 and Figure 2. Table 1. MIC50, MIC90 and MIC range for ARAF isolates (n = 101) Itraconazole Voriconazole Posaconazole Isavuconazole MIC50 (mg/L) >8 8 1 16 MIC90 (mg/L) >8 16 2 >16 MIC range (mg/L) 2 to >8 0.5 to >16 0.5 to >8 0.5 to >16 Itraconazole Voriconazole Posaconazole Isavuconazole MIC50 (mg/L) >8 8 1 16 MIC90 (mg/L) >8 16 2 >16 MIC range (mg/L) 2 to >8 0.5 to >16 0.5 to >8 0.5 to >16 Table 1. MIC50, MIC90 and MIC range for ARAF isolates (n = 101) Itraconazole Voriconazole Posaconazole Isavuconazole MIC50 (mg/L) >8 8 1 16 MIC90 (mg/L) >8 16 2 >16 MIC range (mg/L) 2 to >8 0.5 to >16 0.5 to >8 0.5 to >16 Itraconazole Voriconazole Posaconazole Isavuconazole MIC50 (mg/L) >8 8 1 16 MIC90 (mg/L) >8 16 2 >16 MIC range (mg/L) 2 to >8 0.5 to >16 0.5 to >8 0.5 to >16 Figure 1. View largeDownload slide Prevalence of ARAF in patients with CF in 12 German CF centres. 2888 isolates from 961 CF patients were tested. The information shown is: centre name; prevalence of ARAF in CF patients; number of CF patients with ARAF/total number of CF patients. Figure 1. View largeDownload slide Prevalence of ARAF in patients with CF in 12 German CF centres. 2888 isolates from 961 CF patients were tested. The information shown is: centre name; prevalence of ARAF in CF patients; number of CF patients with ARAF/total number of CF patients. Figure 2. View largeDownload slide Distribution histograms of MICs of itraconazole, voriconazole, posaconazole and isavuconazole for ARAF isolates (n = 101). Figure 2. View largeDownload slide Distribution histograms of MICs of itraconazole, voriconazole, posaconazole and isavuconazole for ARAF isolates (n = 101). A single mutation was found in 90 ARAF isolates by multiplex PCR, with the TR34/L98H alteration being the most prevalent (n = 89; 88%). One isolate harboured a TR46/Y121F/T289A mutation. Molecular characterization of the remaining 10 ARAF isolates demonstrated that mutations in the cyp51A gene were present in all ARAF isolates except two. Seven isolates harboured a G54E substitution, one a G54R substitution, one an F219S mutation and two isolates had a WT cyp51A gene. The eight ARAF isolates with the G54E (n = 7) or G54R (n = 1) mutations were detected from the same CF patient. Microsatellite genotyping outcomes are depicted in a minimum spanning tree (Figure 3). Most of the detected genotypes showed a polyclonal distribution (Figure 3). In two cases the same ARAF genotype from two different patients (2133/H344 and H616/H333) was found and in one case a cluster from two different patients (M1 and M2/M3) was found, suggesting transmission or acquisition from the same source. CF patients with chronic ARAF colonization showed stable genotypes over time in most cases. Figure 3. View largeDownload slide Microsatellite typing cluster analysis of ARAF isolates via a minimum spanning tree, using the goeBURST algorithm (n = 101). Circle colour indicates the centre of specimen isolation. Circle size indicates the prevalence of the exact genotype found. A bicoloured circle shows isolates of the same genotype, but two different centres (2133, Essen/H344, Hannover) and patients. Red numbers show calculated genetic distance. In Hannover (H616/H333) and Munich (M1/M2/3) the same genotypes were found in different patients. Figure 3. View largeDownload slide Microsatellite typing cluster analysis of ARAF isolates via a minimum spanning tree, using the goeBURST algorithm (n = 101). Circle colour indicates the centre of specimen isolation. Circle size indicates the prevalence of the exact genotype found. A bicoloured circle shows isolates of the same genotype, but two different centres (2133, Essen/H344, Hannover) and patients. Red numbers show calculated genetic distance. In Hannover (H616/H333) and Munich (M1/M2/3) the same genotypes were found in different patients. The age and sex of the CF patients with and without ARAF showed no statistically significant differences. The mean age of non-ARAF patients was 30.3 years and for the ARAF cohort was 31.1 years. The sex distribution among the non-ARAF population showed 49.9% male patients and 49.0% male patients in the ARAF cohort. Fourteen percent of the ARAF patients were listed for LTX and 12% had already had a transplant. In total, 45% of patients had previously received an azole treatment (mainly itraconazole and voriconazole) before ARAF detection. Twenty-two percent of CF patients with ARAF suffered from ABPA. Discussion Azole-resistant invasive aspergillosis (IA) is associated with a high mortality in patients with haematological malignancy or other critical illness.23,24 Knowledge of the epidemiology, characteristics, genotypes and resistance patterns of ARAF isolates in CF patients is scarce. Until now, five single-centre studies and one dual-centre study, all from European countries (Denmark, France, Germany, Italy and Portugal),12–17 have been conducted to determine the burden of ARAF in CF patients, analysing a total of 2654 A. fumigatus isolates from 664 patients. However, these studies differed in their design concerning screening, susceptibility testing and MIC interpretation strategies. Thus, direct comparison with our data is difficult.8 In addition, prevalence data from CF patients undergoing LTX were not reported. IA is also a frequent and severe complication in CF patients undergoing LTX, in particular those who are chronically colonized with A. fumigatus.9 Among our CF patients with ARAF, 26% were listed for LTX or had already been transplanted, thereby being predisposed for azole-resistant IA in the post-transplant period. So far, only one study from Canada has investigated the occurrence of ARAF in LTX recipients with voriconazole prophylaxis.25 Interestingly, no azole-resistant isolate was found in that cohort. In the absence of any randomized clinical data on the best treatment strategies, only expert opinions on ARAF treatment are available at the moment.26 A combination of voriconazole with an echinocandin or the use of liposomal amphotericin B was favoured by most participants of an expert panel if the susceptibility is unknown and the environmental resistance rates are ≥10%. In cases of confirmed IA with an ARAF isolate, liposomal amphotericin B was recommended. Liposomal amphotericin B and echinocandins can only be administered intravenously at present. Thus, in the case of azole resistance, oral administration of non-azole antifungals is not possible, which limits outpatient care of CF patients.10 In the present multicentre study including 2888 A. fumigatus isolates from 961 CF patients a prevalence of 5.3% (51/961 patients) ARAF was determined, which is comparable to data from France (4.6%; 6/131)12 and Denmark (4.5%; 6/133),11 but higher than in the only previously available study from Germany (3.4%, 4/119). In line with other studies including clinical isolates from CF, the most frequently detected underlying mutation was TR34/L98H. Furthermore, the rate of ARAF-colonized CF patients that had previously been exposed to azoles (45%) is comparable with summarized data from the six published studies (67%) indicating a combination of environmental acquisition of azole-resistant isolates and in vivo selection through azole therapy.9,10 This assumption is in line with recent data from Italy where ARAF isolates from CF patients and the environment showed identical genotypes.17 In addition, none of the 123 CF patients from Verona received an antifungal drug and no ARAF isolate was found, whereas in Milan 23% of CF patients had azole exposure within 6 months prior to ARAF isolation and the prevalence was found to be 8.2%.17 It was shown that ARAF isolates found in the environment in Germany (12%) exhibit a west–east distribution peaking in the middle of Germany.27 Our data on ARAF distribution in German CF patients are partly in agreement with this observation. However, it has to be kept in mind that the three centres with the highest prevalence (Essen, Munich and Hannover) are specialized for LTX and thus likely have a higher number of severely ill patients compared with the other centres. In contrast, it has also been hypothesized that the German west–east distribution can be explained by the geographical proximity to the Netherlands where ARAF is present in high numbers.28 However, ARAF isolates from German HSCT patients showed no phylogenetic similarity to selected clinical and environmental isolates from the Netherlands.24 To our knowledge, this study is the largest multicentre study on ARAF in the CF population. However, it has several limitations that have to be mentioned. Firstly, the number of collected isolates differed considerably between different centres. The ARAF prevalence in two centres which contributed two-thirds of isolates was 5.2% and 9.1%. Thus, they had a high influence on the overall resistance rate. In addition, centre-dependent effects such as the use of azoles, the number of LTX patients, different diagnostic strategies etc. likely had an influence on the overall prevalence of azole resistance. Furthermore, we screened for azole resistance with an itraconazole-containing agar only. It has been reported that some mutations, e.g. TR46/Y121F/T289A, only lead to increased posaconazole and voriconazole MICs, but do not impact itraconazole MICs. Therefore, the detection of some ARAF isolates could have been missed by our approach. Furthermore, we were not able to provide detailed clinical data from our whole patient cohort concerning lung function, co-colonization with bacteria, comorbidities, treatment courses etc. to perform a multivariate analysis for evaluating risk factors or clinical implications for CF patients with ARAF compared with non-ARAF or to perform a subgroup analysis for the LTX cohort. In summary, this large multicentre study showed that 5.3% of German CF patients harbour an ARAF isolate. Thus, susceptibility testing of A. fumigatus isolates from CF patients should be done routinely in all patients receiving azole-based antifungal treatment. Acknowledgements We thank all members of the Institute of Medical Microbiology, University Hospital Essen, for helpful suggestions and discussions. Funding This work was supported by Mukoviszidose e.V. (German CF Foundation; grant number 1502). Transparency declarations A. Hamprecht has received honoraria for lectures and advisory boards from Gilead and Astellas. F. S. has received honoraria for advisory boards from Gilead and Proteasis. J. F. M. has received grants from Astellas, Basilea, F2G and Merck, has been a consultant to Astellas, Basilea, Scynexis and Merck, and has received speaker’s fees from Astellas, Merck, United Medical, TEVA and Gilead. J. S. has received honoraria for lectures and advisory boards from Gilead Sciences and TEVA. All other authors: none to declare. References 1 Ratjen F , Doring G. Cystic fibrosis . Lancet 2004 ; 361 : 681 – 9 . Google Scholar CrossRef Search ADS 2 Pihet MJ , Carrere B , Cimon D et al. Occurrence and relevance of filamentous fungi in respiratory secretions of patients with cystic fibrosis–a review . Med Mycol 2009 ; 47 : 387 – 97 . Google Scholar CrossRef Search ADS PubMed 3 Ziesing S , Suerbaum S , Sedlacek L. Fungal epidemiology and diversity in cystic fibrosis patients over a 5-year period in a national reference center . Med Mycol 2016 ; 54 : 781 – 6 . Google Scholar CrossRef Search ADS PubMed 4 Maturu VN , Agarwal R. Prevalence of Aspergillus sensitization and allergic bronchopulmonary aspergillosis in cystic fibrosis: systematic review and meta-analysis . Clin Exp Allergy 2015 ; 45 : 1765 – 78 . Google Scholar CrossRef Search ADS PubMed 5 Camps SM , van der Linden JW , Li Y et al. 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Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Apr 19, 2018
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