Lass-Flörl CorneliaInnsbruck Medical University

Divison of Hygiene and Medical Microbiology

Is azole resistance increasing amongst Aspergillus species?

Innsbruck Medical University

Faculty disclosure

• Invited speaker: Pfizer, Gilead, MSD, Schering-Plough

• Consultant: Pfizer, Gilead, Schering-Plough• Research Grants: Pfizer, Gilead, Schering-Plough

Concentration that inhibits the growth of fungi = Minimum inhibitory concentration

(expressed as µg/ml)

Several methods can be used to define the MIC MIC reading = depends on the method used

(EUCAST, CLSI, E-test,…)

To determine the susceptibility of fungi to antifungal agents

AmB

5FC

FLU

ITRA

VORI

CASPO

Increasing Concentrations

candida

MICs defined via E-test

Growth of fungus

MIC= zone of inhibition

Susceptibility testing: The big gaps

– MICs help, but hard to standardize

– Correlations appear possible based on individual isolates. Broad correlations based on multiple isolates are still lacking

– Understanding this helps a lot when trying to correlate outcome with MIC

- Some patients get better despite MICs

- Some patients just don’t get better despite MICs

– No rule when it correlates/not correlates

Rex, 2005

Aspergillus terreus Amphotericin B Intrinsic

Candida glabrata Azoles Intrinsic and acquired

Candida krusei Azoles Intrinsic

Candida lusitaniae Amphotericin B Intrinsic and acquired

Histoplasma capsulatum Fluconazole Acquired

Has Antifungal Susceptibility Testing Come of Age?Pfaller, Rex 2002

Facing In Vitro/In Vivo Correlation with Fungi

In vitro Azole-Resistance Differences within the world!

• 1997: first published case of ITC-resistant isolates of A. fumigatus (UK)

• 2000: 4.2% with higher MICs to ITC (>8 µg/ml) in a surveillance study (UK)

• 1945-1998: 0.3% in the Netherlands• 2000: 2.5%; 2002: 4.9%; 2006: 6.6% (NL)• 2006: 2% in Spain• 2007: 0% in Austria

Lass-Flörl, 2009

In vivo resistance

• 2002 first cases of what appears to be multiple-azole resistant in A. fumigatus

• In vitro and in vivo correlations• Treatment: polyene or candine or azoles! • Reports derive from UK and The Netherlands

Denning 1997, Verweij, 1998, Moore 2001, Howard, 2006

Multiple-triazole-resistant aspergillosis.

Verweij PE, Mellado E, Melchers WJ

N Engl J Med. 2007 356 (14):1481-3.

Clinical Feature Primary resistance + Secondary resistance

++

In vivo-in vitro correlation

++

Risk factors Long term azole treatment: ++ Long term azole prophylaxis: ++

Cross-resistance ++ Multi-drug azole resistance

++

Outcome Lethal: ++ Plus signs (+) indicate the severity Lass-Flörl, 2009

Frequency doese increase in some centres!

• Resistance of A. fumigatus clinical isolates to triazoles (ITC, VOR, POS) has been reported with increasing frequency,

• although it is generally considered an uncommon phenomenon.

14-α-sterol demethylase

Azoles: Voriconazole, Posaconazole, Itraconazole

Ergosterol

Lanosterol

Azole Resistance Mechanisms

Altered drug uptake Increased drug efflux

1. Changes in drug import/export

cyp 51A

cyp 51A

Mutations in cyp51A

cyp 51Acyp 51A

Overexpression of drug target

2. Alterations in sterol biosynthesis

AZOLE RESISTANT ASPERGILLUS FUMIGATUS

Amino acid changes Resistant drugs M220I, M220V, M220K, M220T

ITC-resistance and reduced activity to POS, VOR, RAV

G54R, G54E, G54W, G54V

ITC and POS

L98H Multidrug-azole resistance (ITC, POS, VOR, ravuconazole)

G138C ITC, VRC and long-term treatment

Mellado (2004,2005, 2007), Verweij 2007, Snelders 2009

Defintions: Azole- Resistance

1. Azole:a single drug resistance (ITC and VOR > 4µg/ml, POS > 2µg/ml)

2. Multi-azole resistance: resistant against at least two or more drugs

3. Panazole: resistant against all azoles tested

Denning, Verweij, 2009

Cross resistance

• Cross-resistance between azole drugs appears to exist in vivo and in vitro and depends on specific mutations in Cyp51A

• Between ITC and POS

• Not so evident between ITC and VOR

Oakley 2000, Moore 2000

Conclusion

• Increase in some centres

• In vivo and in vitro

• Associated with prolonged azole treatment

• Molecular mechanisms are well knwon