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

Azole resistance in Aspergillus – is it a problem? Dr Susan J Howard The University of Manchester & Regional Mycology Laboratory Manchester. Agenda. Frequency of acquired azole resistance in the clinical setting Cross-resistance between the triazole agents Clinical risk factors

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

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  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)

  8. Manchester as a centre → Specialist service for the management of aspergillosis2009National 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

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

  14. 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 • Sept 2006 posa therapy 800mg daily • Good levels (1.18-1.9mg/L) • Slow continued improvement keep checking MICs! • ?same/different genetic type → microsatellite typing

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

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

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

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

  19. stop codon start codon intron 54 98 220 22 138 242 394 495 297 432 491 440 448 cyp51A mutations

  20. stop codon start codon intron 54 98 220 22 138 242 394 495 297 432 491 440 448 cyp51A mutations “hot-spots”

  21. Nijmegen 297 495 98 220 94% 3% 12% 6% 9% Manchester 138 216 427 448 147 431 54 98 434 220 Snelders et al, PLoS Medicine. 2008;5:e219 Howard SJ et al, EID. 2009;15:1068-76

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

  23. Evolution and environmental acquisition

  24. 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

  25. 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

  26. 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

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

  28. 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)

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