Wild-Type MIC Distributions and Epidemiological Cutoff Values for the Triazoles and Six Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document)

Abstract

Among the filamentous (mold) fungal infections, those caused by Aspergillus fumigatus and other Aspergillus spp. are the most common; these infections are associated with high morbidity and mortality, especially in the immunocompromised host (3,7,15,33). The triazoles itraconazole, voriconazole, and posaconazole have a broad spectrum of in vitro activity against molds and are important therapeutic agents for the systemic treatment and prevention of severe mold infections, including aspergillosis (33). Although acquired azole resistance in Aspergillus spp. has been documented since the late 1990s (8), it was uncommon. However, these reports have increased in the last few years, especially in Europe (4, 13, 24, 25, 31). The azoles act by blocking the pathway of ergosterol biosynthesis, specifically the enzymes 14-␣-sterol demethylases A and B. These cytochrome enzymes are encoded by cyp51 (A and B) genes in A. fumigatus and other Aspergillus spp. (19). More importantly, multiazole resistance or cross-resistance has been associated with point mutations in the cyp51A gene in A. fumigatus by substitution of the glycine at position 54 and methionine at position 220 by different amino acids (4, 9, 12), but substitution of L98H and 2 copies of 34 bp in the cyp51A promoter and other mutations also have been identified (13,16,20,25,31). The TR/L98H point mutation has been responsible for an increased level of cyp51A expression (25,29,31), and several mutations have been associated with patient failure on triazole treatment (13, 29, 31); however, other host and drug factors cannot be ignored (e.g., azole bioavailability).The Clinical and Laboratory Standards Institute (CLSI) has developed a reference broth microdilution method for antifungal susceptibility testing of molds (CLSI M38-A2 document) (5). The availability of reference methodologies has enabled the recognition of the complexity of cross-resistance among triazoles (9,18,20,22,24,25) and the proposal for epidemiologic cutoff values (ECVs) for A. fumigatus and itraconazole, posaconazole, and voriconazole by both CLSI (22) and the European Committee of Antibiotic Susceptibility Testing (AFST-EUCAST) (24) methodologies. Clinical breakpoints are not available for mold testing by the CLSI methodology versus any antifungal agent. However, breakpoints based on MIC distributions, pharmacokinetic and pharmacodynamic (PK/PD) parameters, animal data, and clinical experience have been proposed for the EUCAST reference method for A. fumigatus and the three triazoles (30). In the absence of clinical breakpoints, ECVs could help to characterize the susceptibility of Aspergillus isolates to itraconazole, posaconazole, and voriconazole and to monitor the emergence of strains with mutations in the cyp51A gene and/or reduced antifungal triazole

In Vitro Activities of Isavuconazole and Comparator Antifungal Agents Tested against a Global Collection of Opportunistic Yeasts and Molds

“…The MIC distributions for isavuconazole, the other mold-active triazoles, and the echinocandins for five species of Aspergillus are shown in Table 3. In general, the MIC distributions for agents in both classes conformed to the WT distributions described previously (28,40). Isavuconazole MIC results were Յ2 g/ml for all species except A. niger SC, for which MIC values of Ն4 g/ml were determined for 27% of the isolates tested, similar to findings reported by Guinea and colleagues (7).…”

“…Second, we used molecular methods to confirm the identification of the less common species of Candida, as well as the non-Candida yeasts and all of the filamentous fungi. Finally, we applied the newly revised clinical breakpoints (CBPs) for the echinocandins, fluconazole, and voriconazole to determine the resistance profiles of various Candida species (27) and used the epidemiological cutoff values (ECVs) for these agents, as well as itraconazole and posaconazole, to detect emerging resistance among less common species of Candida (27) and among Aspergillus isolates (28).…”

High prevalence of clinical and environmental triazole-resistant Aspergillus fumigatus in Iran: is it a challenging issue?

“…Also, MECs were defined microscopically as the lowest concentrations of drugs leading to the growth of small, rounded and compact hyphal forms, compared with long, unbranched hyphal clusters in the controls (CLSI, 2008). Considering the breakpoints proposed by Verweij et al (2009a) for itraconazole, voriconazole (susceptible: < 2 mg l À1 ; intermediate: 2 mg l À1 ; resistant: > 2 mg l À1 ) and posaconazole (susceptible: < 0.5 mg l À1 ; intermediate: 0.5 mg l À1 ; resistant: > 0.5 mg l À1 ), itraconazole (1 mg l À1 ), voriconazole (1 mg l À1 ) and posaconazole (0.5 mg l À1 ) with MICs above the proposed epidemiological cut-off values against A. fumigatus isolates were selected for further analysis (Espinel-Ingroff et al, 2010;Rodriguez-Tudela et al, 2008). PCR amplification started with an initial denaturation at 95 C for 1 min, followed by 35 cycles of denaturation at 94 C (60 s), 60 C (30 s) and 72 C (60 s) and a final 10 min extension at 72 C. PCR products were run on 2 % agarose gel (Ahmad et al, 2014).…”

Echinocandin and Triazole Antifungal Susceptibility Profiles for Clinical Opportunistic Yeast and Mold Isolates Collected from 2010 to 2011: Application of New CLSI Clinical Breakpoints and Epidemiological Cutoff Values for Characterization of Geographic and Temporal Trends of Antifungal Resistance

“…The recent surveillance focus has been on how best to detect the emergence of resistance within a population of a given fungal species (2,7,8,(12)(13)(14)36). The development of epidemiological cutoff values (ECVs) has aided in the detection of non-WT strains in given populations, allowing a more focused approach to the molecular definition of strains presenting less-susceptible profiles (6,8,11,13,33,37).…”