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