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    • br Methods for antifungal testing of mucorales species The

    2024-03-28


    Methods for antifungal testing of mucorales species The Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methods currently in place for in vitro antifungal susceptibility testing of filamentous fungi [8], [9] have been validated and are recommended for testing Mucorales isolates [5]. Whereas the inoculum size, incubation temperature and reading endpoint are the same as for other filamentous fungi (e.g. Aspergillus spp.), the incubation time is shorter (16–24 h for Mucorales compared with 48 h for Aspergillus spp.) due to the rapid growth exhibited by members of Mucorales. Increased minimum inhibitory concentrations (MICs) may be recorded when the incubation time is extended to 48 h [13], [14]. For example, when testing Mucorales D609 sale with the EUCAST method, MICs have been found to be two dilutions higher for azoles and one dilution higher for AmB at 48 h compared with 24 h [13]. Similarly, in tests of an azole by the CLSI method, 56% of the strains examined showed an MIC increase of at least two dilution steps between 24 h and 48 h [14]. This is an important point to consider for further standardisation of antifungal susceptibility methods. Currently, both the CLSI and EUCAST methods include a recommended incubation time of 24 h. Nevertheless, the results obtained using these two protocols have been known to differ [13], [15]. In a recent study comparing the EUCAST and CLSI methods for testing of Mucorales species, the geometric mean MIC of AmB was demonstrated to be more than three-fold higher with the former compared with the latter, with an essential agreement of 66.1%. In the same study, the geometric mean MICs of posaconazole and isavuconazole were also found to be increased when using the EUCAST method compared with the CLSI procedure, albeit with higher levels of essential agreement (87% for posaconazole and 98.3% for isavuconazole) [15]. This implies that determination of clinical breakpoints should be not only species-specific but also method-specific. Commercial tests such as Etest have also been evaluated for antifungal susceptibility testing of Mucorales in several publications. Overall agreement between Etest and the CLSI or EUCAST reference methods for AmB and posaconazole varies from ca. 70% to ca. 100%, sometimes according to the species tested [16], [17], [18], [19]. Moreover, depending on the authors and the drugs involved, Etest was either considered a good alternative or was not recommended. In addition to in vitro techniques, animal models of mucormycosis have been used to evaluate the efficacy of antifungal agents, both as monotherapies and combination treatments [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35].
    In vitro susceptibility profiles and in vivo efficacy of antifungal drugs
    How to overcome resistance: from in vitro interaction studies to combination therapy Because the efficacy of monotherapies for the treatment of patients with mucormycosis is suboptimal, combining antifungal drugs may represent a promising therapeutic strategy [45]. The main rationale behind combination therapy is that treatment efficacy may be improved by the synergistic interaction of two or three drugs with distinct targets of action. Theoretically, this approach could also prevent the emergence of resistance, although resistance acquired during therapy has not yet been reported for Mucorales isolates. Combination therapies have been tested in vitro and in animal models, and some clinical data are also available. In vitro evaluations of double-drug combinations including diverse molecules as partners (AmB, azoles, echinocandins, flucytosine and terbinafine, as well as non-antifungal drugs) have revealed interactions ranging from indifferent to synergistic, but antagonism is generally not observed. In recent studies, combinations of AmB with either posaconazole, isavuconazole or anidulafungin, of posaconazole with anidulafungin, and of isavuconazole with micafungin have proven to be largely indifferent in their effects on various Mucorales species [4], [18]. In contrast, synergistic effects of various antifungal combinations have been noted in vitro against 33–94% of M. irregularis isolates [46].