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Azole resistance in Aspergillus
– is it a problem?
Dr Susan J HowardThe University of Manchester &
Regional Mycology Laboratory Manchester
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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
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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
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Breakpoints
Verweij PE et al, DRU. 2009;12:141-7
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Clinical azole resistance reported
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Verweij PE et al, DRU. 2009;12:141-7
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Nu
mb
er o
f p
atie
nts
overall5%
Significant increase since 2004
(Fishers exact test P<0.0001)
Significant increase since 2004
(Fishers exact test P<0.0001)
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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?
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Azole cross-resistance
Itra resistance = 100%
Posa resistance = 74%
Vori resistance = 65%
Amb resistance = 0%
Howard SJ et al. EID. 2009;15:1068-76
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Nu
mb
er o
f p
atie
nts
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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
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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
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Case
• Oct 2004 vori, 500 > 400 mg daily• Good levels (0.72-1.66mg/L)• Radiological and serological improvement
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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
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Howard SJ et al, EID. 2009;15:1068-76.
unrelated strains
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Howard SJ et al, EID. 2009;15:1068-76.
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Snelders et al, PLoS Medicine. 2008;5:e219
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cyp51A mutations
intronstart
codon
stop codon
Regulatory sequences
Intron
Exons
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cyp51A mutations
394
297
495
440491
22
432
242
448
138
54 98 220
intronstart
codon
stop codon
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cyp51A mutations
394
297
495
440491
22
432
242
448
138
54 98 220
“hot-spots”
intronstart
codon
stop codon
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Nijmegen
98220
297 495
Manchester216
147 431
138 448
43454 98
220
427
94% 3%
12% 6% 9%
Snelders et al, PLoS Medicine. 2008;5:e219 Howard SJ et al, EID. 2009;15:1068-76
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Snelders et al, PLoS Medicine. 2008;5:e219
Environmental sampling
Poster 103!
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Evolution and
environmental
acquisition
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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
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Preliminary study findings
• G54 – 0/30G138 – 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 isolationTR+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
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Preliminary study findings
• Of 17 patients with resistance:6/8 had ABPA/SAFS10/20 had CPA1/2 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 therapy2 unknown currently
• 6 had known azole resistant infection
• Pros and cons
Park, Perlin, Denning; unpublished preliminary data
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Harrison E et al, ICAAC. 2009;M-1720
cyp51A genotype in azole resistant isolates
1992
-200
620
0720
080
5
10
15cyp51A WTcyp51A SNP
Year
Res
ista
nt
iso
late
s
cyp51A mutation identified
no cyp51A mutation
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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)