Manuel Cuenca Estrella S20 Fungicides in Agriculture – the only Source of Fungal Resistance? Current rates of resistance, impact on clinical use and prescribing of drugs Manuel Cuenca Estrella May, 2015

Conflict of interest disclosure In the past 5 years, M.C.E. has received grant support from Astellas Pharma, bioMerieux, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Schering Plough, Soria Melguizo SA, Ferrer International He has been an advisor/consultant to the Panamerican Health Organization, Astellas Pharma, Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough. He has been paid for talks on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, Astellas Pharma and Schering Plough.

Mold susceptibility profile Species Antifungal ANF FC FLC ITC VRC POS EQUIN A. fumigatus S R Aspergillus flavus S-I Aspergillus terreus I-R Aspergillus niger S-I-R** Fusarium spp. Scedosporium spp. Mucorales

Epidemiology of moulds Hematological patients SOT Neofytos CID’09 Pagano CID’07 Marr CID’02 Husain CID’03 Aspergillus 80% 94.5% 77.3% 69.8% Zygomycetes 9.7% 1.1% 8.6% 5.6% Fusarium 2.2% 3.2% 9.2% 3.7% Scedosporium --- 2.9% Other 1.78% 15% Let’s start with epidemiology. In all works collected the most frequent genera causing IMIs is Aspergillus. Then, there are geographical differences. In some places, Zygomycetes are the second most frequent family and in other the genera Fusarium. Besides, Scedosporium is another genera involved in IMIS but in some places a miscellaneous number of species cause significant number of cases.

Results from Spain FILPOP_2012 Population-based survey Molds isolated from deep samples (including respiratory ones) Two months (spring and fall) 30 Spanish hospitals Molecular ID and AST

AAC 2013; 57:3380 10% resistance rate to AmP, VRC, ITC. Mucorales, Scedosporium and Fusarium

Epidemiology of resistance in A. fumigatus To know the epidemiology of moulds is tough because many reasons. As most of them are opportunistic infections in immunocompromised patients a system for classified them as proven or probable has been decided. However, only those cases with a culture positive from a sterile sample are valid for epidemiology purposes and those cases are a minority due to the low rate of sterile samples that can be obtained from patients and also the high rate of false negative results of culture procedures. Besides, identification to level species of moulds are not possible by means of microscopy examination of sterile material or indirect tests. Finally, blood cultures, the gold standard for yeasts epidemiology are not useful for moulds.

Relevant Mycoses. Global burden According to LIFE and GAFFI (www Relevant Mycoses. Global burden According to LIFE and GAFFI (www.life-worldwide.org)

No patients with a +ve culture % patients with azole R isolates Now the second situation with A. fumigatus that is the mergence of secondary resistance to azole drugs. In the chart the evolution of resistance to azole in the Netherlands is shown. Since 2000 an increase in the detection of resistant isolates have been detected while the number of isolates analyzed is not very different 9

This finding has been also found in other settings as in UK This finding has been also found in other settings as in UK. In 2007, 17% of isolates received in the Regional Mycology Laboratory of Manchester were resistant to azole drugs but the most worrisome finding was the increase of isolates showing cross resistance to all azole drugs available 10

World Surveys on A. fumigatus complex resistance. Rates of resistance according to ECOFFs Journals and Congresses Country Itraconazole Voriconazole Posaconazole Spain Cuenca-Estrella et al 8% 4.5% 4.4% USA Diekema et al 7% 1% France Bretagne et al 2% The Netherlands Snelders et al 12.8% 10% United Kingdom Howard et al 17%

Resistance…? Associated to long-term antifungal use Associated to fungicides in the environment. Public Health problem (ECDC consultation) Sibling/cryptic species To know the epidemiology of moulds is tough because many reasons. As most of them are opportunistic infections in immunocompromised patients a system for classified them as proven or probable has been decided. However, only those cases with a culture positive from a sterile sample are valid for epidemiology purposes and those cases are a minority due to the low rate of sterile samples that can be obtained from patients and also the high rate of false negative results of culture procedures. Besides, identification to level species of moulds are not possible by means of microscopy examination of sterile material or indirect tests. Finally, blood cultures, the gold standard for yeasts epidemiology are not useful for moulds.

Epidemiology of aspergillosis is tough because………….. EORTC criteria: Probable aspergillosis (50-80% in last clinical trials) Proven aspergillosis (50-75%) by microscopic examination only. No ID of species Low performance of cultures Laboratory contaminants To know the epidemiology of moulds is tough because many reasons. As most of them are opportunistic infections in immunocompromised patients a system for classified them as proven or probable has been decided. However, only those cases with a culture positive from a sterile sample are valid for epidemiology purposes and those cases are a minority due to the low rate of sterile samples that can be obtained from patients and also the high rate of false negative results of culture procedures. Besides, identification to level species of moulds are not possible by means of microscopy examination of sterile material or indirect tests. Finally, blood cultures, the gold standard for yeasts epidemiology are not useful for moulds.

Haematologica. 2012;97(7):963-5.

Aguado et al. Clin Infect Dis. 2014;Oct 21. pii: ciu833

PCRAGA STUDY P=0.027 Aguado et al. Clin Infect Dis. 2014;Oct 21. pii: ciu833

ABC Azole Strain with low MIC           Cyp51A Cyp51B

Mutations in Cyp51A ABC Strain with high MIC Azole Cyp51A Cyp51B

UP regulation of Cyp51A ABC Strain with high MIC Azole Cyp51A Cyp51B

34-bp sequence in the promoter Gen Cyp51A in a resistant strain Duplication of 34-bp sequence in the promoter Cyp51A L98H TR-R-ITC x 8 237

No patients with a +ve culture % patients with azole R isolates Now the second situation with A. fumigatus that is the mergence of secondary resistance to azole drugs. In the chart the evolution of resistance to azole in the Netherlands is shown. Since 2000 an increase in the detection of resistant isolates have been detected while the number of isolates analyzed is not very different 23

TR34/L98H TR46/Y121F/T289A

New mechanisms of resistance in Aspergillus The azole target cyp51A is a hotspot for mutations that confer phenotypic resistance, but in an increasing number of resistant isolates the underlying mechanism remains unknown. Increase of expression and mutations related to efflux pumps Mutation in the CCAAT-binding transcription factor complex subunit HapE. A P88L substitution in HapE. Camps et al Plos One 2012 Arendrup et al, Denning et al, Mellado et al, Verweij et al

Figure 3 3D Representation of three aligned structures of CYP51 with the ligands in their active site, constructed by using the Yasara software. Snelders Plos One 2012

Description of A. fumigatus resistant to triazoles (both tandem repeats). Clinical isolates Environmental isolates Both Assessed but not detected (resistance in vitro or point mutations described)

Description of A. fumigatus resistant to triazoles (both tandem repeats). Kidd et al. Mycoses 2015 Liu et al. AAC 2015 Steinmann et al. JAC 2015 Van Ingen et al. JAC 2015 Pham et al. EID 2014 Astvad et al. AAC 2014 Ahmad et al. Environ Res 2014 Prigitano et al. Euro Surveill 2014 Chowdary et al. JAC 2014 Rocchi et al. JCM 2014 Faria-Ramos et al. BMC Mic 2014 Ziołkowska et al. Poult Sci 2014 Kikuchi et al. JIC 2014 Seyedmousavi et al. EID 2013 Chowdary et al. PloS One 2012 Pelaez et al. JCM 2012 Vermeulen et al. Eur Surveill 2012 Rath et al. AAC 2012 Morio et al. JAC 2012 Howard et al. EID 2009 Verweij et al. NEJM 2007

Other issues in resistances in Aspergillus To know the epidemiology of moulds is tough because many reasons. As most of them are opportunistic infections in immunocompromised patients a system for classified them as proven or probable has been decided. However, only those cases with a culture positive from a sterile sample are valid for epidemiology purposes and those cases are a minority due to the low rate of sterile samples that can be obtained from patients and also the high rate of false negative results of culture procedures. Besides, identification to level species of moulds are not possible by means of microscopy examination of sterile material or indirect tests. Finally, blood cultures, the gold standard for yeasts epidemiology are not useful for moulds.

Aspergillus cryptic species FILPOP 11% cryptic species 15% cryptic species Balajee et al, JCM 2009 Alastruey-Izquierdo et al. AAC 2013 Alastruey-Izquierdo et al. ANYAS 2012

Results from Spain. FILPOP. Population-based survey. 325 isolates Aspergillus spp. (sensu stricto) N % TOTAL 277 85% (4 cases per 100,000 pop) A. fumigatus 156 48% A. flavus 26 8% A. terreus A. tubingensis (section Nigri) 22 6.8% A. niger 21 6.5% A. nidulans 8 2.5%

Cryptic Species n AMB ICZ VCZ PCZ CPF MCF ANF A. lentulus 26 3 2.3 3.4 0.23 1.6 0.1 N. hiratsukae 9 1.7 0.9 1.1 0.16 0.11 0.03 N. pseudofischerii 6 0.25 4 2.51 0.22 0.86 A. fumigatiaffiinis 4.8 5 3.1 0.4 0,03 N. udagawae 2 0.6 0.3 A. viridinutans 0,7 16 0,25 5,66 0,06 0,09 A. tubingensis 22 0.42 0.76 0.09 0.05 A. calidoustus 19 8.6 6.2 6.8 0.5 0.04 A. insuetus 0.7 11.3 8 2.8 5.6 1.4 A. keveii 1 A. alliaceus 30 19.25 0.2 12.15 3.8 1.9 By Alastruey-Izquierdo

20% 47% A. terreus Other species Dissemination 63% 32% AmB response CNS 31% --- Skin 29% AmB response 20% 47%

Clinical relevance To know the epidemiology of moulds is tough because many reasons. As most of them are opportunistic infections in immunocompromised patients a system for classified them as proven or probable has been decided. However, only those cases with a culture positive from a sterile sample are valid for epidemiology purposes and those cases are a minority due to the low rate of sterile samples that can be obtained from patients and also the high rate of false negative results of culture procedures. Besides, identification to level species of moulds are not possible by means of microscopy examination of sterile material or indirect tests. Finally, blood cultures, the gold standard for yeasts epidemiology are not useful for moulds.

Not enough data but.. Strengthen epidemiological surveillance of isolates in all EU members Investigation of the environmental origin Implement molecular methods in culture negative specimens

8% PREVALENCE in patients with cystic fibrosis

30% resistance in haematological cases and 88% mortality

38% of 952 clinical strains were resistant to triazoles Of these, 225 (26.5%) contained the TR34/L98H mutation, 98 (10.5%) contained the TR46/Y121F/T289A mutation

Conclusions The antifungal resistance in molds is relevant already if prevalence of Mucorales, Scedosporium and Fusarium is high (empirical and first-line treatment) The triazole resistance can be high also in A. fumigatus in some geographical areas and patients populations (cystic fibrosis, other chronic respiratory diseases and some haematological patients) Other Aspergillus species can be resistant such as cryptic species High mortality, some recommendations in new guidelines are compulsory Surveillance; Environmental studies (media supplemented with ITC) Direct molecular detection

PCR in tissues. Proven IFI

Sequencing and analysis

The AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from ICU (resistant and susceptible to triazoles)