Azole preexposure affects the Aspergillus fumigatus population in patients

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1 AAC Accepts, published online ahead of print on 18 June 2012 Antimicrob. Agents Chemother. doi: /aac Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 AAC R3 Azole preexposure affects the Aspergillus fumigatus population in patients Alexandre Alanio, 1,2 Odile Cabaret, 2 Emilie Sitterlé, 2 Jean-Marc Costa, 2 Sylvain Brisse, 3 Catherine Cordonnier, 4 Stéphane Bretagne 1,2* 1 Institut Pasteur, Unité de mycologie moléculaire, Paris, France. 2 Laboratoire de Parasitologie-Mycologie, Groupe hospitalier Albert Chenevier-Henri Mondor, Assistance Publique-Hôpitaux de Paris (APHP), Créteil, France. 3 Institut Pasteur, Genotyping of Pathogens and Public Health, Paris, France 4 Département d Hématologie, Groupe hospitalier Albert Chenevier-Henri Mondor, APHP and, and University Paris-Est-Créteil, Créteil, France. * Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, Hôpital Henri Mondor-APHP, Créteil, France. Phone: Fax: bretagne@univ-paris12.fr Keywords: Aspergillus fumigatus; invasive aspergillosis; cyp51a gene; SNP; itraconazole; voriconazole; posaconazole; preexposure; azole susceptibility; genotyping; clonal complex Total number of words: 977 words Abstract : 74 words Running title: Azole pre-exposure and A. fumigatus isolates 1

2 Abstract The relationship between azole preexposure in 86 patients and the genotype, azole susceptibility and cyp51a polymorphisms of 110 corresponding Aspergillus fumigatus isolates was explored. Isolates carrying serial polymorphisms (F46Y-M172V±N248T±D255E±E427K) had higher itraconazole MICs (p=0.04), although <2µg/ml using the EUCAST methodology, were associated with two genetic clusters (p<0.001), and with voriconazole pre-exposure in patients (p=0.016). Voriconazole pre-exposure influences the distribution of A. fumigatus isolates with selection of isolates carrying cyp51a polymorphisms and higher itraconazole MICs. Downloaded from on July 24, 2018 by guest 2

3 Azole-resistance in environmental and clinical Aspergillus fumigatus isolates is becoming a major preoccupation since emerging azole-resistance was described in the Netherlands (26) and the UK (9, 18). The prevalence of azole-resistant isolates was found to be 5.3 % in The Netherlands in 2011 (28) and 5.8% in the ARTEMIS global surveillance study (22). The major mechanism involved in azole resistance is modification of the azole target, Cyp51A protein (14- alpha-demethylase), with several mutations in cyp51a gene responsible for various resistant phenotypes (19). Numerous polymorphisms have also been described in azole-sensitive isolates (14, 19). We took advantage of our single-center, hospital-based cohort study of consecutives A. fumigatus isolates prospectively collected from patients of the Hematology Department of our hospital between 2006 and 2009 (3) to study the impact of azole preexposure in patients on the isolates recovered by analyzing cyp51a gene polymorphism, in-vitro azole susceptibility, and the distribution of genotypes based on microsatellite markers. We analyzed 110 isolates from 86 patients after exclusion of isolates with mixed genotypes (n=4) and with identical genotypes in a given patient (n=4), to rule out the eventuality to test several times the same isolate. For each isolate, the whole cyp51a gene and promoter were sequenced as previously described (3) and genotyping performed using four previously described microsatellite loci (7). MIC susceptibility testing using Etest (ABbioMérieux) revealed that one isolate from an azolenaïve patient was itraconazole-resistant (MIC=16µg/ml) and had its own genotype (3). The 109 remaining isolates were considered as azole-sensitive (itraconazole and voriconazole MICs<2µg/ml and posaconazole MIC<0.25µg/ml) (30). Unique single nucleotide polymorphisms (SNPs) were observed in 3 isolates: t173a, t1167a (N248K) and g1207t (D262Y). Several [8-12] synonymous and non-synonymous SNPs were observed in 13 isolates (thereafter ssnps isolates for serial SNPs isolates) recovered from 9 patients and classified into 4 groups based on their cyp51a sequences (Table 1). After random selection, 10 of these ssnps isolates were compared 3

4 to 10 isolates with the wild-type (WT) cyp51a sequence (GenBank: AY048754) using EUCAST methodology for itraconazole, voriconazole and posaconazole sensitivity. The itraconazole MICs were significantly higher although <2µg/ml and with less than 2 fold-dilution differences in ssnps isolates (Table 1) than in WT isolates (Wilcoxon rank-sum test, p=0.04, sum of ranks= vs. 78, U=23.00), with no significant difference for the other azoles. Genotyping of the 110 isolates revealed ninety-five different genotypes. Fourty-nine geneticallydifferent isolates were recovered from 50 patients. Genetically identical isolates (n=25) were collected from 23 patients (2-5 patients per genotype) who had or had not received azole therapy. Thirteen patients had iterative pulmonary isolates (n=36 ; range : 2-7), some collected before or after azole therapy. For all of these 13 patients, the genotypes of the subsequent isolates were genetically different compared to that of the first isolates as already reported in pulmonary samples of hematological patients (4, 6, 12, 29). The genetic variability of ssnps isolates compared with WT isolates was studied using minimum spanning tree method (MST, BioNumerics software v6.5) to group genotypes into genetic clusters (GCs) with the most stringent definition of GCs, i.e. tolerating only an allele difference in one marker, as already reported (8, 13, 16, 20, 23). Principal component analysis (PCA) and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering analysis was also performed [MeV v4.6.1 software (24)]. These three analyses highlighted that ssnps isolates belonged to distinct cloud (PCA) or clusters (MLS, UPGMA tree) compared to WT isolates (data not shown). Among ssnps isolates, two distinct genetic clusters (GC1, n=6 ; GC2, n=3) and 3 genetically-unrelated isolates were identifyed (Table 1). The ssnps isolates were significantly associated with GC1 and GC2 genetic clusters compared with WT isolates (Fischer exact test, p<0.0001, OR=225 [ ,392] and p=0.005, OR= 29 [ ], respectively). 4

5 To investigate whether azole preexposure affects A. fumigatus population in patients, we analyzed azole preexposure at the time of recovery for each isolate. Twenty-six (24%) isolates were recovered under voriconazole therapy from 14 patients treated for invasive aspergillosis [from 10 days to >2 years]. Voriconazole preexposure was significantly associated with ssnps isolates (Fischer exact test, p=0.016, OR=4.3 [ ]). Since different genotypes were recovered iteratively in 13 patients, some before and some after azole therapy, the patient-based analysis did not show any significant association between patients with ssnps and voriconazole preexposure (p=0.159, OR=3.0 [ ]). Along with the association between voriconazole preexposure and ssnps isolates, we found higher itraconazole EUCAST MICs in ssnps isolates compared with WT sequence isolates. Although this slight difference in MICs could be dismissed as non-significant since the MICs remained below the accepted threshold (2µg/ml) for resistant isolates (30), this finding is consistent with a better tolerance of these isolates to azole drugs. This could explain why these ssnps isolates are less likely to acquire high-level azole-resistance than WT isolates (15) despite that they have been described with (19) or without (14, 19) hotspot mutations responsible for high-level azole-resistance. Our findings are consistent with Escribano et al. who reported MIC at 2µg/ml for 3 out of 4 in such ssnps isolates, which belonged to distinct GCs compared to WT isolates (14). It is unlikely that such serial polymorphisms appear during medical azole therapy in different individuals, whereas the more plausible hypothesis is the environmental pressure exerted by the massive use of 14α-demethylase inhibitors in agriculture (1, 2, 21, 25). This could explain the clonal expansion observed for ssnps isolates in distinct genetic clusters, as reported for azole-resistant TR/L98H isolates (10, 22, 26). Since azole-resistance can be associated with lower virulence in mice (5), there is a need to study the virulence of these ssnps isolates. 5

6 Invasive aspergillosis has considerably changed during the past years with numerous modifications in its management, especially the prescription of azole as first-line therapy (17) or as prophylaxis (11, 27). Since voriconazole might select A. fumigatus isolates with specific Cyp51A polymorphisms associated with a slightly better in-vitro tolerance to itraconazole, our findings suggest that this phenomenon warrants continual surveillance. Acknowledgments This work has been partly presented at the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy, September 2011, Chicago IL, USA (Poster M-293). We thank Jean-Michel Thiberge and Laure Diancourt (Plate-forme de Génotypage des pathogènes et Santé Publique PF8, Institut Pasteur) for their help in building the minimum spanning trees, and Damien Hoinard and Dorothée Raoux-Barbot (Centre National de Référence Mycologie et Antifongiques, Institut Pasteur) for their help in performing EUCAST assays. 6

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12 1 2 Table 1: Substitutions in Cyp51A protein, EUCAST MICs, and genotype of the A. fumigatus isolates with serial single nucleotide polymorphisms in cyp51a sequence (ssnps isolates) recovered from 9 patients. 3 c ssnps MICs (µg/ml, range) n patient Microsatellite markers Genetic isolates Substitution(s) in Cyp51A protein a ITC VRC PSC Gt b A B C D clusters d (n=13) 8 F46Y, M172V, E427K GC GC GC GC GC GC GC S e 1 F46Y, M172V, N248T, E427K S e 3 F46Y, M172V, N248T, D255E, E427K GC GC GC2 1 F46Y, M172V, N248T, D255E S e 4 Gt: genotype ; GC: genetic cluster ; S: Singleton 5 1

13 a The substitutions t18c, c283t, t489a (F46Y), c560t, c619t, g690a (M172V), a937g and a1497g SNPs of cyp51a gene were common to all isolates, while a1166c (N248T), c1188g (D255E), g1702 (E427K), and t1785c were inconstantly found. b The numbers correspond to the genotype of the isolates and are arbitrary. c The figures correspond to the length in base pairs of the PCR products of the four microsatellite markers (A-D) and are function of the number of repeats of the microsatellite at each locus. d The genetic clusters (GC) were determined for all isolates using minimum spanning tree, principal component analysis, and UPGMA clustering. GC1 and GC2 were genetically distinct from WT isolates using these three analyses. e The isolates that had more than one allelic mismatch with the other isolates were considered as singletons (S). Downloaded from on July 24, 2018 by guest 2