1. Aspergillus Resistance
Don Sheppard M.D.
Associate Professor, Dept of Microbiology and Immunology
Director, Division of Infectious Diseases
McGill University 1

2. Conflict of Interest Statement
Dr Sheppard has received research support or been a consultant for:
Pfizer Canada
Astellas Canada,
Merck Canada 2

3. Azole resistance in Aspergillus
Many questions!
Should I be concerned?
How do I look for it?
How do I treat it?
Can I still use prophylaxis?

4. Types of resistance Intrinsic Acquired Primary Secondary
Species whose MIC distibution is elevated
Acquired
Primary
Environmental acquisition of resistance in a normally sensitive strain
Linked to environmental fungicide use in Europe
Secondary
Emergence during therapy
Most commonly in chronic pulmonary aspergillosis

5. Intrinsic resistance
Elevated azole MICs are observed in many non-fumigatus species
Usti group
A. calidoustus – MIC>8 all azoles
Often resistant to other antifungals also
Nigiri group
Some show elevated MICs
Poor correlation with species identity
Seyedmousavi & Verweij, In Handbook of Antimicrobial Resistance 2015

6. Acquired Primary (Environmentally acquired)
First described in Netherlands
Genotype clustering suggests clonal origin
Combined mutations in promotor and coding sequence of CYP51A
TR34/L98H best studied
VCZ >2, PCZ 0.5, ITRA >16
Now reported in Europe, India and China
TR46/Y121F/T289A
Newest mutation
Spreading rapidly in Europe; Environmental strains in India
VCZ MIC >16, PCZ , ITRA = 4-16
Verweijj et al. Lancet ID, 2009:59(3);789-95
Van der Linden et al, CID 2013:57;

7. Acquired Secondary (during therapy)
Most common reported in chronic infections
Incidence also increasing – up to 20% of patients in Manchester, UK
Heterogenous genotypes and mechanisms
CYP51a mutations in less than half of patients
Isolated PCZ/ITRA resistance common
G54R, P216L and G448S
Preserved susceptibility to VCZ
M220
Variable VCZ susceptibility
Howard et al. EID, 2009:15(7);
Bueid et al., JAC 2010:65; 2116–2118

8. Epidemiology of resistance
20% in 2009
Seyedmousavi & Verweij, In Handbook of Antimicrobial Resistance 2015

9. Challenges in diagnosis
IA now commonly diagnosed by GM or other molecular testing
Culture is insensitive (~30% for BAL)
MIC testing not available in many centres
When available, turn around time is long
Molecular based detection of resistant isolates is not standardized and rarely available
Forced to rely on local epidemiology and analysis of clinical failures

10. Management

11. Clinical data
High failure rate in treatment of successful strains makes clinical correlation challenging
Multiple reports of clinical failure on therapy with voriconazole
Resistance associated with increased mortality in several studies when initially treated with voriconazole
Majority of patients succesfully treated to date received L-AMB

12. Clinical data
High failure rate in treatment of successful strains makes clinical correlation challenging
Multiple reports of clinical failure on therapy with voriconazole
Resistance associated with increased mortality in several studies when initially treated with voriconazole
Majority of patients succesfully treated to date received L-AMB

13. Voriconazole use for Treatment
ƒAUC in HSCT patients: ~ 15-20
For resistant strains (MIC = 2): ƒAUC/MIC = 7-10
Activity < 35% of expected
Mavridou et al, AAC 2010:54(11):

14. Posaconazole: Treatment
Multiple studies have examined Pk/Pd of posaconazole in mouse models
Different endpoints – fungal burden vs survival
Target endpoints identified AUC/MIC: 167 or 100
Lewis et al, AAC 2014:58(11):
Howard et al, JID, 2011;203:1324–32

15. Predicted Posaconazole Exposure-Response: low MIC
Tablet
AUC suspension
~ 15μg/ml
AUC tablet ~ 35μg/ml
Formulation
AUC24h
MIC
AUC/MIC
Probability of
> target 167
> target 100
Suspension 15
0.015
1000
99%
Tablet 35
2333
Lewis et al, AAC 2014:58(11):
Howard et al, JID, 2011;203:1324–32

16. Predicted Posaconazole Exposure-Response: Sensitive, high MIC
Tablet
AUC suspension
~ 15μg/ml
AUC tablet ~ 35μg/ml
Formulation
AUC24h
MIC
AUC/MIC
Probability of
> target 167
> target 100
Suspension 15
0.125
120
15%
64%
Tablet 35
280
70%
95%
Lewis et al, AAC 2014:58(11):
Howard et al, JID, 2011;203:1324–32

17. Predicted Posaconazole Exposure-Response: Resistant
Tablet
AUC suspension
~ 15μg/ml
AUC tablet ~ 35μg/ml
Formulation
AUC24h
MIC
AUC/MIC
Probability of
> target 167
> target 100
Suspension 15
0.5 30 0%
Tablet 35 70
10%
24%
Lewis et al, AAC 2014:58(11):
Howard et al, JID, 2011;203:1324–32

18. What about prophylaxis?
Studies suggest cellular levels may be more important to mediate protection against infection
Tissue levels are significantly higher than serum levels
Should we be using the same targets?
Can these be achieved with tablet formulations?

19. In vitro model of the human alveolus
Simulates two compartments:
alveolar airspace
pulmonary capillary
Emphasize the space, epi layer, endo layer, blood Air chamber blood chamber and cell bilayer. !st component is this alveolar bilayer the second is need dynamic system(Need flow)
To study the relationship among the pathogenesis of early IA, the kinetics of diagnostic markers and the outcome of antifungal therapy
Model provides a strategy by which relationships among pathogenesis and antifungal drug theapy for IA may be further understood.
Population model and monte carlo simulation to further explore the clinical implications of the experimental results.
Hope (2009) Med. Mycology S291-S298

20. Dynamic Model of Antifungal Prophylaxis
Infect alveolar space with conidia
Antifungal
We used a flow by system to pump in drug at a controlled rate. We were able to control drug concentrations by pumping drug through the system. The alveolar cell layer is exposed to the drug containing media.
Pump
Sample media for antifungal and galactomannan concentrations

21. Dynamic in vitro model of posaconazole prophylaxis
Model of human pharmacokinetics/pharmacodynamics
Challenge
In our model we are able to mimic human-like PK associated with a single dose. We achieved Peak concentrations approaching 0.5 mg/L and trough concentrations/clearance of 0 between 24 and 48 hours. We then challenged these cells at different points along this PK curve to assess whether they were resistant to infection.
Only observe growth in system that did not contain any drug. As far as this data goes, 96 hours after free-drug levels are gone the cellular concentration of drug remains active. Suggests that free-drug levels may not determine success in prophylaxis
Make it a point to explain GM relation to fungal growth
Administration of a single 200 µg dose of posaconazole resulted in peak serum levels varying between µg/ml after 8-12 hours, with serum levels becoming undetectable after 48 hours
Peak concentration of 0.45 mg/L is protective

22. Dynamic in vitro model of posaconazole prophylaxis against an TRL98H isolate
Challenge
Peak concentration is protective, although sustained levels of 0.45μg/ml are required

23. Posaconazole prophylaxis PK/PD in mice
No loss of activity noted with TRL98H strains, only with strains MIC>16
Seyedmousavi et al. AAC 2015:59(3)

24. Prophylaxis and Treatment Pk Targets Differ
Outline – epidemiology of resistance – intrinsic strains, environmental/primary vs secondary resistance
Focus on TR strains – range of MICs
Diagnosis of resistance
Importance of culture for sensitivity and epidemiology –
Molecular tests?
How to treat – vori – treatment modelling
PK of IV/Po
Posa – prophyl modelling of targets
PK of new formulations
Swich to L-AMB
Treatment targets higher than those for prophylaxis
Seyedmousavi et al. AAC 2015:59(3)

25. Posaconazole as prophylaxis for resistant A. fumigatus
Protection from high dose conidial exposure
(~1x107/ mouse)
MIC =0.5
Massive challenge model – no differences in survival between 0.5 and WT
Seyedmousavi et al. AAC 2015:59(3)

26. L-AMB treatment of azole resistant strains
VCZ MIC
0.25
0.5 4 16
Seyedmousavi et al. AAC 2013:57(4)

27. When to switch empiric therapy: How effective is L-AmB ?
12 wk Survival
VCZ %
AmB %
L-AMB?
Bart-Jan Kullberg, 2013

28. Overall Conclusions and Perspectives
Resistance is emerging
VCZ not drug of choice
Posa prophylaxis effective at least to MIC 0.5
Posa role in treatment less clear – higher targets
L-AMB is probably best for treatment
When to switch empiric therapy?
Local epidemiology
Difference in efficacy between VCZ and L-AMB?

29. Thank you !