4 To determine the susceptibility of fungi to antifungal agents 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,…)
6 MICs defined via E-test Growth of fungusMIC= zone of inhibition
7 Susceptibility testing: The big gaps MICs help, but hard to standardizeCorrelations appear possible based on individual isolates. Broad correlations based on multiple isolates are still lackingUnderstanding 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 MICsNo rule when it correlates/not correlatesRex, 2005
8 Facing In Vitro/In Vivo Correlation with Fungi Aspergillus terreusAmphotericin BIntrinsicCandida glabrataAzolesIntrinsic and acquiredCandida kruseiCandida lusitaniaeHistoplasma capsulatumFluconazoleAcquiredHas Antifungal Susceptibility Testing Come of Age?Pfaller, Rex 2002
10 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): 0.3% in the Netherlands2000: 2.5%; 2002: 4.9%; 2006: 6.6% (NL)2006: 2% in Spain2007: 0% in AustriaLass-Flörl, 2009
11 In vivo resistance2002 first cases of what appears to be multiple-azole resistant in A. fumigatusIn vitro and in vivo correlationsTreatment: polyene or candine or azoles!Reports derive from UK and The NetherlandsDenning 1997, Verweij, 1998, Moore 2001, Howard, 2006
12 Multiple-triazole-resistant aspergillosis. Verweij PE, Mellado E, Melchers WJN Engl J Med (14):
14 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.
16 Azole Resistance Mechanisms Altered drug uptakeIncreased drug efflux1. Changes in drug import/exportcyp 51Acyp 51A2. Alterations in sterol biosynthesiscyp 51Acyp 51AOverexpression ofdrug targetMutations incyp51A
19 Defintions: Azole- Resistance Azole:a single drug resistance (ITC and VOR > 4µg/ml, POS > 2µg/ml)Multi-azole resistance: resistant against at least two or more drugsPanazole: resistant against all azoles testedDenning, Verweij, 2009
20 Cross resistanceCross-resistance between azole drugs appears to exist in vivo and in vitro and depends on specific mutations in Cyp51ABetween ITC and POSNot so evident between ITC and VOROakley 2000, Moore 2000
21 Conclusion Increase in some centres In vivo and in vitro Associated with prolonged azole treatmentMolecular mechanisms are well knwon
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