Azole resistance in Aspergillus

– is it a problem?

Dr Susan J HowardThe 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

• How resistant infections occur

• Issues associated with detection of resistance

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

Breakpoints

Verweij PE et al, DRU. 2009;12:141-7

Clinical azole resistance reported

Verweij PE et al, DRU. 2009;12:141-7

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)

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?

Azole cross-resistance

Itra resistance = 100%

Posa resistance = 74%

Vori resistance = 65%

Amb resistance = 0%

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

Nu

mb

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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

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

Case

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

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

unrelated strains

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

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

cyp51A mutations

intronstart

codon

stop codon

Regulatory sequences

Intron

Exons

cyp51A mutations

394

297

495

440491

22

432

242

448

138

54 98 220

intronstart

codon

stop codon

cyp51A mutations

394

297

495

440491

22

432

242

448

138

54 98 220

“hot-spots”

intronstart

codon

stop codon

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

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

Environmental sampling

Poster 103!

Evolution and

environmental

acquisition

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

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

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

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

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)