Background: Acquired resistance has been sporadically detected for almost any drug-bug combination. Infections due to such isolates are mostly seen after long-term exposure to antifungal compounds. An important exception, however, is the emergence of azole resistance in environmental and clinical isolates of A. fumigatus in the Netherlands. Here 6% of the isolates are azole cross resistant, 94% of which share the same resistance mechanism and infections involving such isolates have been diagnosed in azole naí¯ve patients. So far echinocandin resistance has only been demonstrated in patients following echinocandin exposure and apparently involves all three compounds. Susceptibility test methods: The reference methodologies CLSI and EUCAST susceptibility testing are both methods based on broth dilution. The methods differ in medium composition (glucose concentration), inoculum size and shape of micro titre wells (flat or round) but they are more alike than different. Whilst the endpoint reading is straight forward for azoles and amphotericin B due to the growth versus no growth pattern of inhibition, the endpoint determination for the echinocandins is more difficult to determine due to significant trailing growth. Thus for the echinocandins an MEC (minimum effective concentration) is defined as the concentration leading to aberrant hyphal growth. Recent data suggest that echinocandins resistance is not always reliably detected using this method indicating that further work is necessary in order to improve the in vitro susceptibility testing format for these compounds. Etest diffusion testing is an attractive approach as this testing principle is well known in most laboratories of clinical microbiology and due to its simplicity. For the echinocandins again however significant trailing in the inhibition zone makes the endpoint reading difficult. Recently, the use of screening agars containing fixed azole concentrations has been used and inter-laboratory testing demonstrated these to be well performing and attractive for initial screening for azole resistance. Breakpoint development process: Breakpoints for Aspergillus spp. have not yet been established. An important and mandatory step in the process is the establishment of wild type (WT) distributions for each species and antifungal compound. Based upon these epidemiological cut off values (ECOFFs) can be determined as the upper limit for isolates with no resistance mechanisms. Recent work has established ECOFFs for EUCAST and CLSI. Until clinical breakpoints are established these may serve as susceptibility breakpoints defining isolates without acquired resistance mechanisms. Molecular testing: Underlying molecular mechanisms have been described for acquired resistance to azoles and echinocandins. For the azoles the primary mechanism is mutation in the target cyp51A gene. Mutational hotspots confirmed to cause resistance have been characterised in the gene at codons 54, 98, 138, and 220 and additionally, mutations at codons 46, 147, 172, 216, 248, 255, 427, 431, 434 and 448 have been demonstrated in clinical isolates with azole resistance. However, clinical isolates with azole resistance but no mutations in the cyp51A gene have recently been reported. For the echinocandins acquired resistance in a clinical isolate was recently associated with increased expression level of the FSK gene. In laboratory manipulated strains mutations in the ECM33 gene (afuEcm33) encoding cell wall proteins or mutations in the FKS1 gene encoding a subunit of the target enzyme have been demonstrated. In conclusion susceptibility testing of Aspergillus is becoming more and more important not only from an academic and epidemiological point of view but for selecting appropriate treatment in individual patient cases. Molecular testing is a robust and reliable way of detecting characterised resistance mechanisms, however, resistant isolates with yet uncharacterised resistance mechanisms are continuously reported making in vitro susceptibility testing necessary as well.
Full conference title:
4th Advances Against Aspergillosis
- AAA 4th (2010)