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

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

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?

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

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

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 0.25-2, ITRA = 4-16 Verweijj et al. Lancet ID, 2009:59(3);789-95 Van der Linden et al, CID 2013:57; 513-20

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);1068-76 Bueid et al., JAC 2010:65; 2116–2118

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

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

Management

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

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

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):4758-64

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):6767-72 Howard et al, JID, 2011;203:1324–32

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):6767-72 Howard et al, JID, 2011;203:1324–32

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):6767-72 Howard et al, JID, 2011;203:1324–32

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):6767-72 Howard et al, JID, 2011;203:1324–32

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?

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

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

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 0.2 - 0.7 µg/ml after 8-12 hours, with serum levels becoming undetectable after 48 hours Peak concentration of 0.45 mg/L is protective

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

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)1487-94

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)1487-94

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)1487-94

L-AMB treatment of azole resistant strains VCZ MIC 0.25 0.5 4 16 Seyedmousavi et al. AAC 2013:57(4)1866-71

When to switch empiric therapy: How effective is L-AmB ? 12 wk Survival VCZ - 70.8% AmB - 57.9% L-AMB? Bart-Jan Kullberg, 2013

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?

Thank you !