1. 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

2. 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.

3. 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

4. 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.

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
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.

8. Relevant Mycoses. Global burden According to LIFE and GAFFI (www
Relevant Mycoses. Global burden According to LIFE and GAFFI (

9. 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

10. 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

11. 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%

12. 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.

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
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.

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. ABC
Azole
Strain with low MIC
         
Cyp51A Cyp51B

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

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

22. 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

23. 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

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).
Clinical isolates
Environmental isolates
Both
Assessed but not detected (resistance in vitro or point mutations described)

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
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.

30. 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

31. 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%

32. 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

35. 20% 47% A. terreus Other species Dissemination 63% 32% AmB response
CNS
31%
---
Skin
29%
AmB response
20%
47%

36. 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.

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)