Manuel Cuenca Estrella May, 2015

1. 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

2. Conflict of interest disclosure • In the past 5 years, M.C.E. has received grant support from AstellasPharma, bioMerieux, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Schering Plough, SoriaMelguizo SA, FerrerInternational • He has been an advisor/consultant to the Panamerican Health Organization, AstellasPharma, 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, AstellasPharma and Schering Plough.

3. Mold susceptibility profile

4. Epidemiology of moulds

5. 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

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

7. Epidemiology of resistance in A. fumigatus

8. Relevant Mycoses. Global burdenAccording to LIFE and GAFFI (www.life-worldwide.org)

9. No patients with a +ve culture % patients with azole R isolates

11. World Surveys on A. fumigatus complex resistance. Rates of resistance according to ECOFFs Journals and Congresses

12. Resistance…? • Associated to long-term antifungal use • Associated to fungicides in the environment. Public Health problem (ECDC consultation) • Sibling/cryptic species

13. 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

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

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

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

18. Azole ABC Strain with low MIC Cyp51A Cyp51B

19. Azole Mutations in Cyp51A Strain with high MIC ABC Cyp51A Cyp51B

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

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

23. No patients with a +ve culture % patients with azole R isolates

24. TR34/L98H TR46/Y121F/T289A

25. 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

26. 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

27. Description of A. fumigatus resistant to triazoles (both tandem repeats).

28. 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

29. Other issues in resistances in Aspergillus

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

31. Results from Spain. FILPOP. Population-based survey. 325 isolates

32. Cryptic Species By Alastruey-Izquierdo

36. Clinical relevance

37. 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

38. 8% PREVALENCE in patients with cystic fibrosis

39. 30% resistance in haematological cases and 88% mortality

40. 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

41. 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

42. PCR in tissues. Proven IFI

44. Sequencing and analysis

45. 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)