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20th Anniversary Meeting of the Fungal Research Trust
Aspergillus resistance – it's on the increase Dr Sue Howard
June 2011London, UK
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Aspergillus resistance
– it's on the increase
Dr Susan J Howard
The University of ManchesterManchester Academic Health Science Centre
NIHR Translational Research Facility in Respiratory Medicine University Hospital of South Manchester NHS Foundation Trust
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Aspergillus fumigatus
Aspergillus terreus
Aspergillus flavus
Aspergillus niger
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Other Aspergillus species
Klick MA. Identification of common Aspergillus species. CBS.
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Intrinsic resistance in Aspergillus
flavus - ++ ++ ++ + ++ - ++
fumigatus - ++ ++ ++ + ++ +/- ++
FLU ITR VOR POS RAV AMB 5FC CANDINS
niger - ++ ++ ++ + ++ - ++
terreus - ++ ++ ++ + - - ++
Acquired resistance development
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Acquired resistance
• Mostly azole resistance in A. fumigatus reported
1) most common species
2) primary therapy (itra, vori, posa)
• Standardised methodology
• First resistant case late 1980s
but most >2000
Denning et al, 1997. AAC 41:1364-8
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Agenda
• How common are resistant infections?
• What are the clinical risk factors?
• How does resistance occur?
• Is cross-resistance a clinical problem?
• How can we detect resistance?
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Agenda
• How common are resistant infections?
• What are the clinical risk factors?
• How does resistance occur?
• Is cross-resistance a clinical problem?
• How can we detect resistance?
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Clinical azole resistance reported
Frequency ~2% (0-15%)
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overall10%
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 frequencywhy?
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-20
-15
-10
-5
0
5
10
15
20
1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007
Year
Pe
rce
nta
ge
re
sis
tan
ce
Manchester
Nijmegen
Denning AAC 1997;41:1364-8 Verweij DRU 2009;12:141-7
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Agenda
• How common are resistant infections?
• What are the clinical risk factors?
• How does resistance occur?
• Is cross-resistance a clinical problem?
• How can we detect resistance?
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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 EID 2009;15:1068-76 Howard CMI 2010;16:683-8
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Agenda
• How common are resistant infections?
• What are the clinical risk factors?
• How does resistance occur?
• Is cross-resistance a clinical problem?
• How can we detect resistance?
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Resistance mechanism
Azole drug
ergosterol biosynthesisergosterol biosynthesis
Lanosterol ErgosterolE
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intronstart
codon
stop codon
The cyp51A gene
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54 98 220
“hot-spots”
intronstart
codon
stop codon
The cyp51A gene
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Snelders PLoS M 2009;5:e219
Holland
98220
297 495
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Rodriguez-Tudela AAC 2008;52:2468-72
Holland
98220
297 495
Spain
9854220
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Holland
98220
297 495
Spain
9854220
Manchester216
147 431
138 448
43454 98
220
Bueid JAC 2010; 65:2116-8 Howard EID 2009;15:1068-76
28% 19%53%
94% 3%
14% 6% 11%
284219
495
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• Striking variety of cyp51A mutations
• Including previously reported mutations
(including the hot-spots)
• Some novel (147, 216, 431 & 434) – as
yet uncharacterised
• Of 7 patients with multiple resistant isolates, 4 revealed different mutations over time
Manchester findings
Howard EID 2009;15:1068-76
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Patient 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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Patient case
• Oct 2004 vori, 500 > 400 mg daily• Good levels (0.72-1.66mg/L)• Radiological and serological improvement
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Patient case
• Oct 2004 vori, 500 > 400 mg daily• Good levels (0.72-1.66mg/L)• Radiological and serological improvement
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Patient 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 EID 2009;15:1068-76
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Evolution in the lung!
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Environmental sampling
Snelders PLoS M 2008;5:e219
?agricultural azole use
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Harrison E 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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Resistance mechanism
Azole drug
ergosterol biosynthesisergosterol biosynthesis
Lanosterol ErgosterolE
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Resistance mechanism
Azole drug
ergosterol biosynthesisergosterol biosynthesis
Lanosterol ErgosterolE
EE
EE
E
E
EE
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Al-Barrag unpublished
Rel
ati
ve
exp
ress
ion
of
CA
P51
A t
o B
eta
tub
uli
n
WT
WT
codon 138
(GGC→TGC)
codon 138
(GGC→TGC)
codon 431
(TAC→TGC)
codon 434
(GGC→TGC)
11/04 11/04 06/05 06/05
CYP51A overexpression with target mutations
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Resistance mechanism
Azole drug
ergosterol biosynthesisergosterol biosynthesis
Lanosterol ErgosterolE
AzoleAzole
Azole
Azole
Azole
Azole
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cyp51A
0
2
4
6
8
10
12
14
16
18
20
Re
lati
ve
ex
pre
ss
ion cyp51B
0
2
4
6
8
10
Rela
tive e
xp
ressio
n
AfuMDR1
0
2
4
6
8
10
Rela
tive e
xp
ressio
n
AfuMDR2
0
2
4
6
8
10
Rela
tive e
xp
ressio
n
AfuMDR3
0
2
4
6
8
10
Rela
tive e
xp
ressio
n
AfuMDR4
0
2
4
6
8
10
Rela
tive e
xp
ressio
n
atr-F
0
2
4
6
8
10
Rela
tive e
xp
ressio
n
expression of efflux transporters
Al-Barrag unpublished
Limited evidence in Aspergillus
currently
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Other as yet
un-identified
mechanisms??
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Agenda
• How common are resistant infections?
• What are the clinical risk factors?
• How does resistance occur?
• Is cross-resistance a clinical problem?
• How can we detect resistance?
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Azole cross-resistance
Itra resistance = almost all
Posa resistance = 74%
Vori resistance = 65%
Amb resistance = 0%
Howard EID 2009;15:1068-76
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0
10
20
30
40
50
60
70
80
90
100
1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
Year
Num
ber
of p
atie
nt c
ases
Multi-azole resistant
Itraconazole & posaconazole resistant
Voriconazole resistant
Itraconazole resistant
Fully susceptible
0% 0%
7%
3%
0%
5%
5%
5%
7%
17%
0%
14%
20%
Bueid JAC 2010; 65:2116-8
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Agenda
• How common are resistant infections?
• What are the clinical risk factors?
• How does resistance occur?
• Is cross-resistance a clinical problem?
• How can we detect resistance?
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Detection options
• MICs slow
• Cultures frequently falsely negative in all forms of aspergillosis
• Direct cyp51A mutation from primary specimenby real-time PCR most common mutations = G54, L98, M220, TR
• 55.1% cyp51A mutations (culture –ve)
• Pro’s and con’s (other/no mutations & cost vs. -ve cultures & speed)
Denning CID 2011; 52:1123-9
Need to do MICs
still!
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Conclusions
• Resistance is clinically significant
• Evidence of both environmental acquisition and emergence of resistance in the lung
• Increasing frequency
• Risk of cross-resistance is high/limited options
• Need to monitor susceptibility routinely
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Thank you