1. Azole resistance in Aspergillus – is it a problem? Dr Susan J Howard The University of Manchester & Regional Mycology Laboratory Manchester

2. Agenda Frequency of acquired azole resistance in the clinical setting Cross-resistance between the triazole agents Clinical risk factors How resistant infections occur Issues associated with detection of resistance

3. Acquired azole resistance Azoles extensively used to treat aspergillosis Standardised methodology (CLSI & EUCAST) Predominantly in A. fumigatus Primarily itraconazole data First resistant case late 1980s but most post-millennium Frequency ~2% cases aspergillosis Denning et al, AAC. 1997;41:1364-8

4. Breakpoints Verweij PE et al, DRU. 2009;12:141-7

5. Clinical azole resistance reported

6. Verweij PE et al, DRU. 2009;12:141-7

7. Number of patients overall 5% Significant increase since 2004 (Fishers exact test P<0.0001) Significant increase since 2004 (Fishers exact test P<0.0001)

8. Manchester as a centre Specialist service for the management of aspergillosis 2009 National Aspergillosis Centre www.nationalaspergillosiscentre.org.uk Susceptibility testing is routinely conducted may explain high frequency of itra resistance but does not explain the change in frequency why?

9. Azole cross-resistance Itra resistance = 100% Posa resistance = 74% Vori resistance = 65% Amb resistance = 0% Howard SJ et al. EID. 2009;15:1068-76

10. Number of patients

11. Clinical data Clinical data were available for 14 patients 2 invasive aspergillosis (IA) 9 chronic pulmonary aspergillosis (CPA) 2 allergic bronchopulmonary aspergillosis (ABPA) 1 Aspergillus bronchitis Highest frequency in those with aspergillomas 13 had prior azole exposure (1 – 30 months) 6 had low drug exposures 8 patients failed therapy and 5 failed to improve (1 not treated) Howard SJ et al, EID. 2009;15:1068-76. Howard SJ et al, CMI. Epub 2009

12. Case 64 M COPD, bronchiectasis, Mycobacterium avium pulmonary infection Chronic pulmonary aspergillosis 2003 Azole susceptible A. fumigatus Itra therapy Low itra drug exposure (rifabutin) Ambisome twice for 2wk - some clinical improvement 4 mo itra resistant isolate (G54R) 4 mo later, another itra res isolate (G54E) Increased precipitins titre, radiological progression

14. Case Oct 2004 vori, 500 > 400 mg daily Good levels (0.72-1.66mg/L) Radiological and serological improvement

15. Case Oct 2004 vori, 500 > 400 mg daily Good levels (0.72-1.66mg/L) Radiological and serological improvement 20 mo isolate vori resistant (G448S), posa MIC 1mg/L keep checking MICs! Sept 2006 posa therapy 800mg daily Good levels (1.18-1.9mg/L) Slow continued improvement ?same/different genetic type microsatellite typing

16. Howard SJ et al, EID. 2009;15:1068-76. unrelated strains

17. Howard SJ et al, EID. 2009;15:1068-76.

18. Snelders et al, PLoS Medicine. 2008;5:e219

19. cyp51A mutations intron start codon stop codon Regulatory sequences Intron Exons

20. cyp51A mutations 394 297 495 440 491 22 432 242 448 138 5498220 intron start codon stop codon

21. cyp51A mutations 394 297 495 440 491 22 432 242 448 138 5498220 hot-spots intron start codon stop codon

22. Nijmegen 98 220 297495 Manchester 216 147431 138448 434 5498 220 427 94%3% 12%6%9% Snelders et al, PLoS Medicine. 2008;5:e219 Howard SJ et al, EID. 2009;15:1068-76

23. Snelders et al, PLoS Medicine. 2008;5:e219 Environmental sampling Poster 103!

24. Evolution and environmental acquisition

25. What about when cultures are negative? Cultures frequently falsely negative in all forms of aspergillosis Cyp51A mutation detected by real-time PCR Prospective study on sputum samples Samples split for culture and PCR 30 samples PCR positive (Ct <38) and culture negative analysed for the most common mutations; G54, L98, G138, M220, TR All assays were done blinded to treatment and any mycology data Balashov et al, JCM. 2005, Trama et al, JCM 2005, Garcia-Effron et al, JCM 2008

26. Preliminary study findings G54 – 0/30 G138 – 0/25 M220 – 4/25 (16%) L98 – 23/25 (92%) TR – 19/30 (63%) TR+L98 – 15/25 TR and L98 alterations both found in isolation TR+L98H+M220 – 2/25 Overall 17/30 (57%) have evidence of a cyp51A mutation known to be associated with resistance Park, Perlin, Denning; unpublished preliminary data

27. Preliminary study findings Of 17 patients with resistance: 6/8 had ABPA/SAFS 10/20 had CPA 1/2 had bronchiectasis (controls) 3 were taking itraconazole (2 clearly failing Rx) 3 were taking voriconazole (1 clearly failed Rx) 5 were taking posaconazole (3 responders, 2 primary Rx) 4 had received no azole therapy 2 unknown currently 6 had known azole resistant infection Pros and cons Park, Perlin, Denning; unpublished preliminary data

28. Harrison E et al, ICAAC. 2009;M-1720 cyp51A mutation identified no cyp51A mutation

29. Conclusions Significant clinical import Environmental acquisition and emergence in situ, as a result of azole exposure Currently low frequency but increasing Risk of cross-resistance is high Routine susceptibility testing now required (real-time PCR may be useful if culture -ve)