Azole resistance in Aspergillus

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• Azole resistance in Aspergillus
• is it a problem?
• Dr Susan J Howard
• The 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. 1997411364-8
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Breakpoints
Verweij PE et al, DRU. 200912141-7
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Clinical azole resistance reported
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Verweij PE et al, DRU. 200912141-7
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overall 5
Significant increase since 2004 (Fishers exact
test Plt0.0001)
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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. 2009151068-76
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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. 2009151068-76. 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 gt 400 mg daily
• Good levels (0.72-1.66mg/L)
• Radiological and serological improvement

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Case

• Oct 2004 vori, 500 gt 400 mg daily
• Good levels (0.72-1.66mg/L)
• Radiological and serological improvement
• 20 mo isolate vori resistant (G448S), posa MIC
  1mg/L

• Sept 2006 posa therapy 800mg daily
• Good levels (1.18-1.9mg/L)
• Slow continued improvement

keep checking MICs!

• ?same/different genetic type ? microsatellite
  typing

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unrelated strains
Howard SJ et al, EID. 2009151068-76.
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Howard SJ et al, EID. 2009151068-76.
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Snelders et al, PLoS Medicine. 20085e219
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cyp51A mutations
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cyp51A mutations
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cyp51A mutations

• hot-spots

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Snelders et al, PLoS Medicine. 20085e219
Howard SJ et al, EID. 2009151068-76
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Environmental sampling
Snelders et al, PLoS Medicine. 20085e219
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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 lt38) 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 0/30
• G138 0/25
• M220 4/25 (16)
• L98 23/25 (92)
• TR 19/30 (63)
• TRL98 15/25
• TR and L98 alterations both found in isolation
• TRL98HM220 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/SAFS
• 10/20 had CPA
• 1/2 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 therapy
• 2 unknown currently
• 6 had known azole resistant infection
• Pros and cons

Park, Perlin, Denning unpublished preliminary
data
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cyp51A mutation identified no cyp51A mutation
Harrison E et al, ICAAC. 2009M-1720
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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)