Is Azole Resistance in Aspergillus fumigatus a Problem in Spain?

Transcription

1 AAC Accepts, published online ahead of print on 29 April 2013 Antimicrob. Agents Chemother. doi: /aac Copyright 2013, American Society for Microbiology. All Rights Reserved Is Azole Resistance in Aspergillus fumigatus a Problem in Spain? Pilar Escribano Teresa Peláez Patricia Muñoz Emilio Bouza Jesús Guinea* 1, 2, 3 1, 2, 3, 4 1, 2, 3, 4 1, 2, 3, 4 1, 2, 3, 4 1 Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Marañón, Universidad Complutense de Madrid, Madrid, Spain. 2 Instituto de Investigación Sanitaria del Hospital Gregorio Marañón. 3 CIBER de Enfermedades Respiratorias (CIBERES CD06/06/0058), Palma de Mallorca, Spain. 4 Medicine Department, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain. Running title: Azole resistance in Aspergillus fumigatus complex. Key words: Aspergillus fumigatus, Fumigati, Azole resistance, Aspergillus lentulus, Aspergillus novofumigatus, Aspergillus viridinutans, cyp51a gene. Abstract word count: 243 Text word count: 2365 Address correspondence to: Jesús Guinea Servicio de Microbiología Clínica y Enfermedades Infecciosas-VIH Hospital General Universitario Gregorio Marañón C/ Dr. Esquerdo, Madrid, Spain Phone: Fax: jguineaortega@yahoo.es This study was presented in part at the 23 rd European Congress on Clinical Microbiology and Infectious Diseases (ECCMID), Berlin, Germany, 2013 (P-996). 1

2 ABSTRACT Aspergillus fumigatus complex comprises A. fumigatus and other morphologically indistinguishable cryptic species. We retrospectively studied 362 A. fumigatus complex isolates (n=353 samples) from 150 patients with proven/probable invasive aspergillosisor aspergilloma (n=23/121/6) admitted to the hospital from 1999 to Isolates were identified using the β-tubulin gene, and only 1 isolate per species found in each sample was selected. Antifungal susceptibility to azoles was determined using the CLSI M38-A2 procedure. Isolates were considered resistant if they showed an MIC above the breakpoints for itraconazole, voriconazole, or posaconazole (>2/>2/>0.5 µg/ml). Most of the samples yielded only 1 species (A. fumigatus [n=335], A. novofumigatus [n=4], A. lentulus [n=3], A. viridinutans [n=1], and Neosartorya udagawae [n=1]). The remaining samples yielded a combination of 2 different species. Most of the patients were infected by a single species (A. fumigatus [n=143] or A. lentulus [n=2]). The remaining 5 patients were co-infected with multiple A. fumigatus complex species, although A. fumigatus was always involved; 4 of the 5 patients were diagnosed from 2009 onward. Cryptic species were less susceptible than A. fumigatus. The frequency of resistance among A. fumigatus complex/a. fumigatus to itraconazole, voriconazole, and posaconazole was 2.5%/0.3%, 3.1%/0.3%, and 4.2%/1.8%, respectively, in the per isolate analysis and 1.3%/0.7%, 2.6%/0.7%, and 6%/4% in the per patient analysis. Only 1 of the 6 A. fumigatus isolates in which the cyp51a gene was sequenced had a mutation at position G448. The proportion of patients infected by azole-resistant A. fumigatus isolates was low. 2

3 61 INTRODUCTION Invasive aspergillosis (IA), an opportunistic infection that affects patients with different degrees of immunosuppression (1-7), is usually treated with voriconazole (8). Patients at high risk of IA receive antifungal prophylaxis with posaconazole and itraconazole (9). These agents show potent in vitro activity against clinical isolates of the so-called Aspergillus fumigatus complex (10-13). The A. fumigatus complex includes several morphologically indistinguishable species, such as A. fumigatus sensu stricto (hereafter referred to as A. fumigatus), and the cryptic species A. lentulus, A. novofumigatus, A. viridinutans, and Neosartorya udagawae (2, 6, 14). Cryptic species show reduced susceptibility to azoles (15). Resistance in A. fumigatus isolates is conferred mainly by specific point mutations in the cyp51a gene (16-22). Recent reports from different European countries including the Netherlands, the United Kingdom, and France have alerted to an increase in the frequency of A. fumigatus isolates showing phenotypic resistance to itraconazole, voriconazole, and posaconazole (23-28). However, in most studies, only 1 colony per patient was studied and cryptic species were excluded (24-27, 29, 30). The selection of a single colony per sample would lead us to underestimate azole resistance or the presence of cryptic species if they are present in a low proportion. No recent data are available on the susceptibility to azoles of A. fumigatus complex isolates collected in Spain. We studied the antifungal susceptibility of a large collection of A. fumigatus complex isolates from 150 patients with proven or probable IA or aspergilloma admitted to a large tertiary hospital in Madrid, Spain. 3

4 85 MATERIALS AND METHODS Hospital description and study population. This study was carried out at a large teaching hospital serving a population of approximately 715,000 inhabitants in the city of Madrid. The institution cares for all types of patients at risk of acquiring aspergillosis, including solid organ and bone marrow transplant recipients and patients with hematological malignancies, HIV infection, and chronic obstructive pulmonary disease (COPD). We selected 150 patients with diseases caused by A. fumigatus complex admitted to the hospital from 1999 to Six had aspergilloma and 144 had IA (proven, n=23; probable, n=121) according to the revised criteria of the European Organization for Research and Treatment of Cancer (31, 32). Patients with COPD fulfilled Bulpa s criteria (31, 32). The clinical manifestations of IA were lower respiratory tract infection (n=136), sinusitis (n= 3), wound infection (n=8), central nervous system infection (n=8), and other conditions (n=3). The main underlying conditions of the patients were hematological cancer (12.8%), solid cancer (14.7%), cirrhosis (9.3%), COPD (50%), neutropenia (9.3%), and immunosuppression in solid organ recipients (13.3%). Samples and isolates. We studied 353 samples from the above-mentioned 150 patients (2.3 samples per patient), in whom A. fumigatus complex was isolated. Samples were taken from the lower respiratory tract (n=306), biopsy specimens (n=16), wounds (n=23), and other sources (n=8) before being cultured on both bacterial and mycological media. In 91 of the 150 patients, 2 or more samples were studied, and the mean number of days between the first and the last sample was All colonies resembling A. fumigatus complex that grew on 4

5 the culture plates were subcultured and further studied independently, yielding 837 isolates (2.3 samples per patient, 2.4 isolates per sample, and 5.6 isolates per patient). The 837 isolates were identified by amplifying and sequencing the β-tubulin gene (33). Genomic DNA was extracted from conidia suspensions with the DNeasy Tissue Kit (QIAGEN GmbH, Hilden, Germany) and initially treated with lyticase (Sigma-Aldrich Corporation, St Louis, Missouri, USA) for 2 hours at 37ºC. PCR amplifications were carried out using the βtub1/2 primers (34). We used the same conditions and temperature profiles for both independent amplification regions. Briefly, amplifications were performed in 50 µl of reaction solution containing 50 mm KCl, 15 mm Tris-ClH ph 8.8, 1.5 mm MgCl 2, 0.2 mm of each dntp, 0.5 µm for each pair of primers, 25 ng genomic DNA, and 2.5 units of AmpliTaqGold DNA Polymerase LD (Applied Biosystems). The temperature profiles chosen for the amplifications were as follows: an initial step of 5 minutes at 94 C, 35 cycles of 30 seconds at 94 C, 45 seconds at 50 C, 2 minutes at 72 C, and a final step of 5 minutes at 72 C. PCR reactions were carried out in a T- Gradient thermocycler (BIOMETRA, Germany). PCR products were purified using an Illustra GFX PCR DNA and gel band purification kit (Amersham Bioscience UK, Ltd., Little Chalfont, United Kingdom). Double-stranded DNA sequencing of the PCR products was performed using the Big Dye Terminator v3.1 sequencing kit and capillary electrophoresis with a 3130xl analyzer (Applied Biosystems, Inc.). A homology search of all the sequenced amplicons was performed through NCBI BLAST to determine whether the sequences matched A. fumigatus or the cryptic species. The sequences of the different A. fumigatus complex species were obtained from BLAST and used as controls. 5

6 We selected 362 of the 837 isolates according to the following criteria: (i) only 1 colony per species found was selected in samples with 2 different species; (ii) the isolate showing the highest MIC was chosen in cases of multiple isolates from the same species. From 1999 to 2005, only 1 colony per sample was stored, and isolates were collected retrospectively. From 2006 onward, all visible colonies grown in the media were stored, and isolates were collected prospectively. Antifungal susceptibility and sequencing of the A. fumigatus cyp51a gene. Antifungal susceptibility to itraconazole, voriconazole, and posaconazole was determined using the CLSI M38-A2 procedure (35). The final concentration of the antifungal agents tested ranged from μg/ml to 8 μg/ml. A. fumigatus complex isolates were considered resistant if they showed an MIC above the breakpoints recently proposed by Verweij et al. (36) (>2 μg/ml for itraconazole and voriconazole and >0.5 μg/ml for posaconazole). In the absence of specific breakpoints for the cryptic species, we applied the same breakpoints chosen for A. fumigatus. We calculated the rate of azole resistance for A. fumigatus complex and for A. fumigatus; the rate of resistance was shown per isolate and per patient. The percentage of isolates showing an MIC above the epidemiological cutoff values (ECVs) proposed by Pfaller et al (37) and Espinel- Ingroff et al (38) was calculated (>1 μg/ml for itraconazole and voriconazole and >0.5 μg/ml for posaconazole). We obtained the cyp51a gene sequence (39) of A. fumigatus isolates that were considered resistant or that showed an MIC above the ECVs. 159 RESULTS 6

7 Distribution of A. fumigatus complex species found in the samples. Most of the samples yielded only 1 species (A. fumigatus [n=335], A. novofumigatus [n=4], A. lentulus [n=3], A. viridinutans [n=1], and N. udagawae [n=1]). In the remaining samples, a combination of 2 different species was present (A. fumigatus + A. lentulus [n=6], A. fumigatus + A. novofumigatus [n=1], A. fumigatus + N. udagawae [n=1], A. novofumigatus + A. viridinutans [n=1]). A. fumigatus was found in 97% of the samples. Species of A. fumigatus complex causing aspergillosis. Of the 150 patients, 143 were infected exclusively by A. fumigatus, 2 were infected by A. lentulus, and the remaining 5 patients were co-infected by A. fumigatus plus other cryptic species (Table 1). The 7 patients infected by cryptic species had heterogeneous underlying predisposing conditions, mainly solid or hematological cancer, and were mostly diagnosed from 2009 onward (Figure 1). Males accounted for 43%, and all had invasive pulmonary aspergillosis. Despite frequent therapy with combined antifungal agents, the mortality rate was invariably high (86%). The prognosis was poor in patients receiving azoles or amphotericin B. Serum galactomannan was studied in 5 patients; the result was positive in 4. Isolation of cryptic species proved to be a surrogate marker of poor prognosis in the patients infected. Antifungal susceptibility and cyp51a gene sequencing. The overall rate of azole-resistance of A. fumigatus complex/a. fumigatus to 1 or more azoles was 4.2%/1.8%. The frequencies of resistance of A. fumigatus complex/a. fumigatus were as follows: itraconazole, 2.5%/0.3%; voriconazole, 3.1%/0.3%; and 7

8 posaconazole, 4.2%/1.8% (Figure 2). The percentage of isolates showing an MIC above the ECVs for itraconazole, voriconazole, and posaconazole, respectively, was 4.2%, 5%, and 4.2%. Fifteen A. fumigatus complex isolates were resistant to posaconazole (A. novofumigatus, n=6; A. lentulus, n=3; and A. fumigatus, n=6). The cyp51a gene was sequenced in the 6 isolates showing resistance to 1 or more azoles, and a point mutation leading to the G448S amino acid substitution was found in 1 isolate. The remaining 5 isolates showed a wild-type cyp51a gene sequence, and only posaconazole showed an MIC above the breakpoints (1 µg/ml). Isolates of A. fumigatus were more susceptible to azoles than isolates of cryptic species (Table 2). Among the cryptic species, N. udagawae was the most susceptible (Table 1, patients 2 and 7). In patients with multiple isolates from the same species, no differences in antifungal susceptibility were found between the isolates (data not shown). The percentage of patients infected by itraconazole, voriconazole, and posaconazole-resistant A. fumigatus complex/a. fumigatus isolates was 1.3%/0.7%, 2.6%/0.7%, and 6%/4%, respectively. The percentage of patients infected by an isolate resistant to 1 or more azoles was 6.7%. DISCUSSION The spectrum of patients at risk of IA has expanded, with the result that azoles are increasingly used for the treatment and prevention of IA. In some countries, the increase in the number of azole-resistant A. fumigatus isolates has restricted management of patients with this devastating disease. 8

9 In the Netherlands, the percentage of patients infected by azole-resistant Aspergillus has been growing since 2000 and now stands at 12% (24, 29). Most were infected by isolates showing the typical Dutch mutation in the cyp51a gene, which consists of an amino acid substitution in L98H and the insertion of a 34-bp tandem repeat in the promoter of the cyp51a gene (19). The use of azoles in agriculture has been proposed as the source of these resistant isolates (40). In the UK, Howard et al (26) illustrated the development of secondary resistance in up to 28% of A. fumigatus isolates from a series of patients previously treated with azoles (mostly itraconazole) (26). As expected, isolates from these patients showed several mutations in the cyp51a gene. Azole resistance has also been reported albeit sporadically in France (41), India (30), Japan (42), China (43), Denmark (44), Sweden (45), Norway (46), and Germany (47). Our results showed that 6.7% of patients were infected by resistant A. fumigatus complex isolates. When only A. fumigatus was considered, azole resistance was found in 6 patients (4%); the isolates from 5 patients were considered resistant only to posaconazole, and the MIC (1 µg/ml) was only a 1- fold dilution above the breakpoint. Furthermore, the cyp51a gene sequence was wild-type, and most of the patients responded clinically to azoles. A potential explanation could be that we studied antifungal susceptibility using the CLSI methodology, but that the breakpoints were proposed for the EUCAST method. Further evaluation of the breakpoints proposed for posaconazole is required. The isolates from the remaining patient showed a mutation in the cyp51a gene, thus demonstrating the presence of resistance. The patient had been living in another city and was transferred to our hospital to be treated for IA in 2011 (48), 9

10 suggesting that resistance to azoles in A. fumigatus isolates collected in our hospital is minimal (0.7% of patients infected by this species) None of our isolates showed the typical Dutch mutation that is emerging in other areas. The low rate of azole resistance detected could be explained by the kind of patients included, who were mostly treated for acute forms of IA. However, a high prevalence of A. fumigatus isolates carrying the L98 mutation has been reported in patients with chronic conditions, such as cystic fibrosis (28, 49, 50). Another possible explanation is that resistant isolates in the environment were infrequent: other authors were unable to detect azole-resistant A. fumigatus isolates in the environment of Madrid (10, 51). We began exhaustive collection of isolates in 2006, when all available colonies resembling A. fumigatus complex were isolated and identified using morphological and molecular procedures. However, we remained unable to detect azoleresistant A. fumigatus isolates that would have been missed if a single colony had been selected. In contrast, we were able to simultaneously isolate A. fumigatus together with other cryptic species in 9 samples. The number of patients infected by cryptic species has been growing since 2009, possibly owing to the large number of isolates collected during this period. Nevertheless, patients infected by cryptic species, either alone or together with A. fumigatus, had a poor prognosis despite antifungal treatment. We did not use azole-containing plates to screen resistant isolates, a procedure commonly used in other studies (24, 29). Unfortunately, the antifungal susceptibility of the isolates before contact with itraconazole was not reported in those studies. It should be noted that the azole MICs for isolates grown on plates containing itraconazole are higher than the MICs for the same isolates grown on 10

11 itraconazole-free plates (52). The MICs of the isolates reported here were not affected by previous exposure to azoles Our study is subject to a series of limitations. We only analyzed cultivable isolates. A recent report showed a high percentage of azole resistance in noncultivable A. fumigatus in lower respiratory tract samples after DNA amplification (53). As we only included isolates from a single hospital, our findings might not reflect the situation of other areas in Spain. The number of cryptic species isolates found in our study is low; however, the increase in the number of cases of IA caused by these isolates warrants further attention. As this is not a populationbased study, the precise prevalence of azole-resistance is unknown. Collection of isolates was not homogeneous during the study period only 1 colony per sample was collected from 1999 to 2006 and the presence of cryptic species during this period may have been underestimated. However, the emergence of cryptic species was more evident from 2009 onwards, although the study of multiple colonies per sample began in 2006 and 2 cases of IA were diagnosed before We conclude that the rate of azole resistance is very low in A. fumigatus strains isolated from patients admitted to our hospital. The number of cryptic species isolated was also low, but has been increasing since It is important to study several isolates per patient in order to reveal the presence of cases of coinfection by cryptic species ACKNOWLEDGMENTS 282 We would like to thank Thomas O Boyle for editing and proofreading the article. 11

12 283 This study does not present any conflicts of interest for its authors This study was partially financed by grant ref. CP09/00055 from the Fondo de Investigación Sanitaria (FIS, Instituto de Salud Carlos III) and Gilead Sciences. Jesús Guinea (MS09/00055) and Pilar Escribano (CD09/00230) are supported by the FIS. We are grateful to Ainhoa Simón Zárate, who holds a grant from the FIS (Línea Instrumental Secuenciación), for her participation in the sequencing analysis. The 3130xl Genetic Analyzer was partially financed by grants from the FIS (IF , IF ). Downloaded from on October 21, 2018 by guest 12

13 TABLES AND FIGURES TABLE 1. Summary of the 7 patients infected by cryptic species of Aspergillus fumigatus complex. The species involved and the antifungal susceptibility of the isolates are shown. Patient code Underlying conditions Antifungal Year of Outcome ITC VRC POS Species found Treatment diagnosis 2 1 Corticosteroids AMB Poor A. lentulus Hematological cancer + COPD VRC + CAS Poor A. fumigatus N. udagawae COPD VRC Poor A. lentulus Cirrhosis + solid cancer VRC + AMB Poor A. fumigatus A. lentulus A. fumigatus 7 5 Hematological cancer MYC followed by VRC A. lentulus Poor followed by AMB + CAS A. novofumigatus A. viridinutans 5 6 Solid cancer VRC followed by CAS A. fumigatus Favorable followed by POS A. lentulus A. fumigatus 6 7 Solid cancer AMB + MYC + VRC Poor A. lentulus N. udagawae ICU, intensive care unit; COPD, chronic obstructive pulmonary disease; GM, galactomannan; ITC, itraconazole; VRC, voriconazole; POS, posaconazole; AMB, amphotericin B; COPD, chronic obstructive pulmonary disease; CAS, caspofungin; MYC, micafungin. 2

14 TABLE 2. Antifungal susceptibility of the 362 A. fumigatus complex isolates studied to itraconazole, voriconazole, and posaconazole. Antifungal susceptibility is also shown for A. fumigatus and the cryptic species (A. lentulus, A. novofumigatus, A. viridinutans, and N. udagawae) No. of isolates MIC 90 (range) Itraconazole Voriconazole Posaconazole A. fumigatus complex ( ) 1 ( ) 0.5 (0.06-1) A. fumigatus (0.25-4) 1 ( ) 0.5 (0.06-1) Cryptic species (1-16) 8 (1-16) 1 (0.25-1) Downloaded from on October 21, 2018 by guest 2

15 FIGURE 1. Number of patients with invasive aspergillosis during the study period. Patients were grouped as infected by A. fumigatus sensu stricto (AfumSS) isolates and infected by cryptic species isolates Patients with cryptic species isolates Patients with AfumSS- isolates

16 FIGURE 2. MIC distribution of itraconazole, voriconazole, and posaconazole for the 362 A. fumigatus complex isolates. Resistant isolates were defined as those with an MIC of voriconazole or itraconazole > 2 µg/ml or an MIC of posaconazole > 0.5 µg/ml No. of isolates ITC VRC POS MICs Downloaded from on October 21, 2018 by guest 4

17 REFERENCES 1. Kontoyiannis DP, Marr KA, Park BJ, Alexander BD, Anaissie EJ, Walsh TJ, Ito J, Andes DR, Baddley JW, Brown JM, Brumble LM, Freifeld AG, Hadley S, Herwaldt LA, Kauffman CA, Knapp K, Lyon GM, Morrison VA, Papanicolaou G, Patterson TF, Perl TM, Schuster MG, Walker R, Wannemuehler KA, Wingard JR, Chiller TM, Pappas PG Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, : overview of the Transplant- Associated Infection Surveillance Network (TRANSNET) Database. Clin Infect Dis 50: Neofytos D, Horn D, Anaissie E, Steinbach W, Olyaei A, Fishman J, Pfaller M, Chang C, Webster K, Marr K Epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of Multicenter Prospective Antifungal Therapy (PATH) Alliance registry. Clin Infect Dis 48: Neofytos D, Fishman JA, Horn D, Anaissie E, Chang CH, Olyaei A, Pfaller M, Steinbach WJ, Webster KM, Marr KA Epidemiology and outcome of invasive fungal infections in solid organ transplant recipients. Transpl Infect Dis 12: Denning DW Aspergillus and aspergillosis-progress on many fronts. Med Mycol 44 Suppl:1. 5. Pagano L, Caira M, Candoni A, Offidani M, Martino B, Specchia G, Pastore D, Stanzani M, Cattaneo C, Fanci R, Caramatti C, Rossini F, Luppi M, Potenza L, Ferrara F, Mitra ME, Fadda RM, Invernizzi R, Aloisi T, Picardi M, Bonini A, Vacca A, Chierichini A, Melillo L, de Waure C, Fianchi L, Riva M, Leone G, Aversa F, Nosari A Invasive aspergillosis in patients with acute myeloid leukemia: SEIFEM-2008 registry study. Haematologica 95(4):

18 Guinea J, Torres-Narbona M, Gijón P, Muñoz P, Pozo F, Peláez T, de Miguel J, Bouza E Pulmonary aspergillosis in patients with chronic obstructive pulmonary disease: incidence, risk factors, and outcome. Clin Microbiol Infect 16: Singh N, Husain S Invasive aspergillosis in solid organ transplant recipients. Am J Transplant 9 Suppl 4:S Herbrecht R, Denning DW, Patterson TF, Bennett JE, Greene RE, Oestmann JW, Kern WV, Marr KA, Ribaud P, Lortholary O, Sylvester R, Rubin RH, Wingard JR, Stark P, Durand C, Caillot D, Thiel E, Chandrasekar PH, Hodges MR, Schlamm HT, Troke PF, de Pauw B Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 347: Cornely OA, Maertens J, Winston DJ, Perfect J, Ullmann AJ, Walsh TJ, Helfgott D, Holowiecki J, Stockelberg D, Goh YT, Petrini M, Hardalo C, Suresh R, Angulo-Gonzalez D Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia. N Engl J Med 356: Guinea J, Peláez T, Alcalá L, Ruiz-Serrano MJ, Bouza E Antifungal susceptibility of 596 Aspergillus fumigatus strains isolated from outdoor air, hospital air, and clinical samples: analysis by site of isolation. Antimicrob Agents Chemother 49: Sabatelli F, Patel R, Mann PA, Mendrick CA, Norris CC, Hare R, Loebenberg D, Black TA, McNicholas PM In vitro activities of posaconazole, fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically important molds and yeasts. Antimicrob Agents Chemother 50: Cuenca-Estrella M, Gómez-López A, Mellado E, Buitrago MJ, Monzón A, Rodríguez-Tudela JL Head-to-head comparison of the activities of currently available antifungal agents against 3,378 Spanish clinical isolates of yeasts and filamentous fungi. Antimicrob Agents Chemother 50:

19 Peláez T, Álvarez-Pérez S, Mellado E, Serrano D, Valerio M, Blanco JL, García ME, Muñoz P, Cuenca-Estrella M, Bouza E Invasive aspergillosis caused by cryptic Aspergillus species: a report of two consecutive episodes in a patient with leukemia. J Med Microbiol 62(Pt 3): Pappas PG, Alexander BD, Andes DR, Hadley S, Kauffman CA, Freifeld A, Anaissie EJ, Brumble LM, Herwaldt L, Ito J, Kontoyiannis DP, Lyon GM, Marr KA, Morrison VA, Park BJ, Patterson TF, Perl TM, Oster RA, Schuster MG, Walker R, Walsh TJ, Wannemuehler KA, Chiller TM Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET). Clin Infect Dis 50: Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot Cell 4: Díaz-Guerra TM, Mellado E, Cuenca-Estrella M, Rodríguez-Tudela JL A point mutation in the 14alpha-sterol demethylase gene cyp51a contributes to itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother 47: Mellado E, García-Effrón G, Alcázar-Fuoli L, Cuenca-Estrella M, Rodríguez-Tudela JL Substitutions at methionine 220 in the 14alpha-sterol demethylase (Cyp51A) of Aspergillus fumigatus are responsible for resistance in vitro to azole antifungal drugs. Antimicrob Agents Chemother 48: Howard SJ, Webster I, Moore CB, Gardiner RE, Park S, Perlin DS, Denning DW Multi-azole resistance in Aspergillus fumigatus. Int J Antimicrob Agents 28: Mellado E, García-Effrón G, Alcázar-Fuoli L, Melchers WJ, Verweij PE, Cuenca-Estrella M, Rodríguez-Tudela JL A New Aspergillus fumigatus Resistance Mechanism Conferring In Vitro Cross-Resistance to 7

20 Azole Antifungals Involves a Combination of cyp51a Alterations. Antimicrob Agents Chemother 51: Camps SM, van der Linden JW, Li Y, Kuijper EJ, van Dissel JT, Verweij PE, Melchers WJ Rapid Induction of Multiple Resistance Mechanisms in Aspergillus fumigatus during Azole Therapy: a Case Study and Review of the Literature. Antimicrob Agents Chemother 56: Bellete B, Raberin H, Morel J, Flori P, Hafid J, Manhsung RT Acquired resistance to voriconazole and itraconazole in a patient with pulmonary aspergilloma. Med Mycol 48: Kuipers S, Bruggemann RJ, de Sevaux RG, Heesakkers JP, Melchers WJ, Mouton JW, Verweij PE Failure of posaconazole therapy in a renal transplant patient with invasive aspergillosis due to Aspergillus fumigatus with attenuated susceptibility to posaconazole. Antimicrob Agents Chemother 55: Verweij PE, Mellado E, Melchers WJ Multiple-triazole-resistant aspergillosis. N Engl J Med 356: Snelders E, van der Lee HA, Kuijpers J, Rijs AJ, Varga J, Samson RA, Mellado E, Donders AR, Melchers WJ, Verweij PE Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med 5:e Snelders E, Huis In 't Veld RA, Rijs AJ, Kema GH, Melchers WJ, Verweij PE Possible environmental origin of resistance of Aspergillus fumigatus to medical triazoles. Appl Environ Microbiol 75: Howard SJ, Cerar D, Anderson MJ, Albarrag A, Fisher MC, Pasqualotto AC, Laverdiere M, Arendrup MC, Perlin DS, Denning DW Frequency and evolution of Azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis 15:

21 Bueid A, Howard SJ, Moore CB, Richardson MD, Harrison E, Bowyer P, Denning DW Azole antifungal resistance in Aspergillus fumigatus: 2008 and J Antimicrob Chemother 65: Morio F, Aubin GG, Danner-Boucher I, Haloun A, Sacchetto E, Garcia- Hermoso D, Bretagne S, Miegeville M, Le Pape P High prevalence of triazole resistance in Aspergillus fumigatus, especially mediated by TR/L98H, in a French cohort of patients with cystic fibrosis. J Antimicrob Chemother 67(8): van der Linden JW, Snelders E, Kampinga GA, Rijnders BJ, Mattsson E, Debets-Ossenkopp YJ, Kuijper EJ, Van Tiel FH, Melchers WJ, Verweij PE Clinical implications of azole resistance in Aspergillus fumigatus, The Netherlands, Emerg Infect Dis 17: Chowdhary A, Kathuria S, Randhawa HS, Gaur SN, Klaassen CH, Meis JF Isolation of multiple-triazole-resistant Aspergillus fumigatus strains carrying the TR/L98H mutations in the cyp51a gene in India. J Antimicrob Chemother 67: De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, Pappas PG, Maertens J, Lortholary O, Kauffman CA, Denning DW, Patterson TF, Maschmeyer G, Bille J, Dismukes WE, Herbrecht R, Hope WW, Kibbler CC, Kullberg BJ, Marr KA, Munoz P, Odds FC, Perfect JR, Restrepo A, Ruhnke M, Segal BH, Sobel JD, Sorrell TC, Viscoli C, Wingard JR, Zaoutis T, Bennett JE Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis 46: Bulpa P, Dive A, Sibille Y Invasive pulmonary aspergillosis in patients with chronic obstructive pulmonary disease. Eur Respir J 30:

22 Balajee SA, Borman AM, Brandt ME, Cano J, Cuenca-Estrella M, Dannaoui E, Guarro J, Haase G, Kibbler CC, Meyer W, O'Donnell K, Petti CA, Rodríguez-Tudela JL, Sutton D, Velegraki A, Wickes BL Sequence-based identification of Aspergillus, Fusarium, and Mucorales species in the clinical mycology laboratory: where are we and where should we go from here? J Clin Microbiol 47: Staab JF, Balajee SA, Marr KA Aspergillus section Fumigati typing by PCR-restriction fragment polymorphism. J Clin Microbiol 47: Clinical and Laboratory Standards Institute Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi. Approved Standard CLSI Document M38-A2. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania. 36. Verweij PE, Howard SJ, Melchers WJ, Denning DW Azoleresistance in Aspergillus: proposed nomenclature and breakpoints. Drug Resist Updat 12: Pfaller MA, Diekema DJ, Ghannoum MA, Rex JH, Alexander BD, Andes D, Brown SD, Chaturvedi V, Espinel-Ingroff A, Fowler CL, Johnson EM, Knapp CC, Motyl MR, Ostrosky-Zeichner L, Sheehan DJ, Walsh TJ Wild-type MIC distribution and epidemiological cutoff values for Aspergillus fumigatus and three triazoles as determined by the Clinical and Laboratory Standards Institute broth microdilution methods. J Clin Microbiol 47: Espinel-Ingroff A, Diekema DJ, Fothergill A, Johnson E, Peláez T, Pfaller MA, Rinaldi MG, Cantón E, Turnidge J Wild-Type MIC Distributions and Epidemiological Cutoff Values for the Triazoles and Six Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 document). J Clin Microbiol 48(9): Escribano P, Recio S, Peláez T, Bouza E, Guinea J Aspergillus fumigatus strains with mutations in the cyp51a gene do not always show 10

23 phenotypic resistance to itraconazole, voriconazole, or posaconazole. Antimicrob Agents Chemother 55: Verweij PE, Snelders E, Kema GH, Mellado E, Melchers WJ Azole resistance in Aspergillus fumigatus: a side-effect of environmental fungicide use? Lancet Infect Dis 9: Alanio A, Sitterle E, Liance M, Farrugia C, Foulet F, Botterel F, Hicheri Y, Cordonnier C, Costa JM, Bretagne S Low prevalence of resistance to azoles in Aspergillus fumigatus in a French cohort of patients treated for haematological malignancies. J Antimicrob Chemother 66: Tashiro M, Izumikawa K, Minematsu A, Hirano K, Iwanaga N, Ide S, Mihara T, Hosogaya N, Takazono T, Morinaga Y, Nakamura S, Kurihara S, Imamura Y, Miyazaki T, Nishino T, Tsukamoto M, Kakeya H, Yamamoto Y, Yanagihara K, Yasuoka A, Tashiro T, Kohno S Antifungal Susceptibilities of Aspergillus fumigatus Clinical Isolates Obtained in Nagasaki, Japan. Antimicrob Agents Chemother 56: Lockhart SR, Frade JP, Etienne KA, Pfaller MA, Diekema DJ, Balajee SA Azole resistance in Aspergillus fumigatus isolates from the ARTEMIS global surveillance study is primarily due to the TR/L98H mutation in the cyp51a gene. Antimicrob Agents Chemother 55: Mortensen KL, Johansen HK, Fuursted K, Knudsen JD, Gahrn-Hansen B, Jensen RH, Howard SJ, Arendrup MC A prospective survey of Aspergillus spp. in respiratory tract samples: prevalence, clinical impact and antifungal susceptibility. Eur J Clin Microbiol Infect Dis 30: Chryssanthou E In vitro susceptibility of respiratory isolates of Aspergillus species to itraconazole and amphotericin B. acquired resistance to itraconazole. Scand J Infect Dis 29: Warris A, Klaassen CH, Meis JF, De Ruiter MT, De Valk HA, Abrahamsen TG, Gaustad P, Verweij PE Molecular epidemiology of Aspergillus fumigatus isolates recovered from water, air, and patients shows two clusters of genetically distinct strains. J Clin Microbiol 41:

24 Rath PM, Buchheidt D, Spiess B, Arfanis E, Buer J, Steinmann J First Reported Case of Azole-Resistant Aspergillus fumigatus Due to the TR/L98H Mutation in Germany. Antimicrob Agents Chemother 56: Peláez T, Gijón P, Bunsow E, Bouza E, Sánchez-Yebra W, Valerio M, Gama B, Cuenca-Estrella M, Mellado E Resistance to voriconazole due to a G448S substitution in Aspergillus fumigatus in a patient with cerebral aspergillosis. J Clin Microbiol 50: Mortensen KL, Jensen RH, Johansen HK, Skov M, Pressler T, Howard SJ, Leatherbarrow H, Mellado E, Arendrup MC Aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: a laboratory-based study with focus on Aspergillus fumigatus azole resistance. J Clin Microbiol 49: Burgel PR, Baixench MT, Amsellem M, Audureau E, Chapron J, Kanaan R, Honore I, Dupouy-Camet J, Dusser D, Klaassen CH, Meis JF, Hubert D, Paugam A High Prevalence of Azole-Resistant Aspergillus fumigatus in Adults with Cystic Fibrosis Exposed to Itraconazole. Antimicrob Agents Chemother 56: Mortensen KL, Mellado E, Lass-Florl C, Rodríguez-Tudela JL, Johansen HK, Arendrup MC Environmental study of azole-resistant Aspergillus fumigatus and other aspergilli in Austria, Denmark, and Spain. Antimicrob Agents Chemother 54: Escribano P, Recio S, Peláez T, González-Rivera M, Bouza E, Guinea J In Vitro Acquisition of Secondary Azole Resistance in Aspergillus fumigatus Isolates after Prolonged Exposure to Itraconazole: Presence of Heteroresistant Populations. Antimicrob Agents Chemother 56: Denning DW, Park S, Lass-Florl C, Fraczek MG, Kirwan M, Gore R, Smith J, Bueid A, Moore CB, Bowyer P, Perlin DS High-frequency triazole resistance found In nonculturable Aspergillus fumigatus from lungs of patients with chronic fungal disease. Clin Infect Dis 52: