20th Anniversary Meeting of the Fungal Research Trust
Aspergillus resistance – it's on the increase Dr Sue Howard
June 2011London, UK
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
Aspergillus fumigatus
Aspergillus terreus
Aspergillus flavus
Aspergillus niger
Other Aspergillus species
Klick MA. Identification of common Aspergillus species. CBS.
Intrinsic resistance in Aspergillus
flavus - ++ ++ ++ + ++ - ++
fumigatus - ++ ++ ++ + ++ +/- ++
FLU ITR VOR POS RAV AMB 5FC CANDINS
niger - ++ ++ ++ + ++ - ++
terreus - ++ ++ ++ + - - ++
Acquired resistance development
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
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?
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?
Clinical azole resistance reported
Frequency ~2% (0-15%)
overall10%
Significant increase since 2004
(Fishers exact test P<0.0001)
Significant increase since 2004
(Fishers exact test P<0.0001)
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?
-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
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?
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
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?
Resistance mechanism
Azole drug
ergosterol biosynthesisergosterol biosynthesis
Lanosterol ErgosterolE
intronstart
codon
stop codon
The cyp51A gene
54 98 220
“hot-spots”
intronstart
codon
stop codon
The cyp51A gene
Snelders PLoS M 2009;5:e219
Holland
98220
297 495
Rodriguez-Tudela AAC 2008;52:2468-72
Holland
98220
297 495
Spain
9854220
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
• 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
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
Patient case
• Oct 2004 vori, 500 > 400 mg daily• Good levels (0.72-1.66mg/L)• Radiological and serological improvement
Patient case
• Oct 2004 vori, 500 > 400 mg daily• Good levels (0.72-1.66mg/L)• Radiological and serological improvement
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
Howard EID 2009;15:1068-76
Evolution in the lung!
Environmental sampling
Snelders PLoS M 2008;5:e219
?agricultural azole use
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
Resistance mechanism
Azole drug
ergosterol biosynthesisergosterol biosynthesis
Lanosterol ErgosterolE
Resistance mechanism
Azole drug
ergosterol biosynthesisergosterol biosynthesis
Lanosterol ErgosterolE
EE
EE
E
E
EE
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
Resistance mechanism
Azole drug
ergosterol biosynthesisergosterol biosynthesis
Lanosterol ErgosterolE
AzoleAzole
Azole
Azole
Azole
Azole
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
Other as yet
un-identified
mechanisms??
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?
Azole cross-resistance
Itra resistance = almost all
Posa resistance = 74%
Vori resistance = 65%
Amb resistance = 0%
Howard EID 2009;15:1068-76
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
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?
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!
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
Thank you