Elsevier

Drug Resistance Updates

Volume 12, Issue 6, December 2009, Pages 141-147
Drug Resistance Updates

Azole-resistance in Aspergillus: Proposed nomenclature and breakpoints

https://doi.org/10.1016/j.drup.2009.09.002Get rights and content

Abstract

Reports of itraconazole resistance in Aspergillus fumigatus have been more frequent since the millennium. Identifying azole resistance is critically method dependent; nevertheless reproducible methods, reflective of in vivo outcome, are now in routine use. Some isolates also have elevated MICs to posaconazole and voriconazole. Multiple mechanisms of resistance are now known to be responsible, with differing degrees of azole cross-resistance, including mutations in the Cyp51A gene at G54, L98 + TR, G138, M220, G448. Establishing breakpoints for Aspergillus is probably impossible with clinical data alone for multiple reasons yet there is an urgent need to do so. We propose the following breakpoints for A. fumigatus complex using the proposed EUCAST susceptibility testing methodology: for itraconazole and voriconazole, <2 mg/L (susceptible), 2 mg/L (intermediate) and >2 mg/L (resistant); for posaconazole, <0.25, 0.5 and >0.5 mg/L respectively. We recognize that additional work will be needed to confirm these proposed breakpoints, including in vivo and clinical correlative responses. We also propose nomenclature for genotypic resistance, in the event an isolate is not cultured, typified by ITZgR, VCZgI, POSgR (G54W) indicating that the isolate has a G54W substitution with a corresponding phenotype of resistance to itraconazole and posaconazole and intermediate susceptibility to voriconazole.

Introduction

Aspergillus species cause a wide range of diseases including allergic syndromes, chronic pulmonary and nasal sinus aspergillosis and acute and subacute invasive disease. Azoles increasingly play a role in the management of Aspergillus diseases. Itraconazole is commonly used for the treatment of chronic and allergic conditions (Denning et al., 2003), while voriconazole is first choice therapy for invasive aspergillosis (Herbrecht et al., 2002, Walsh et al., 2008). The triazole posaconazole has been shown to be effective in preventing invasive aspergillosis in patients with certain hematologic malignancies (Cornely et al., 2007, Ullmann et al., 2007). Besides azoles, only amphotericin B and the echinocandins (caspofungin, micafungin, anidulafungin) have any useful clinical activity, with better evidence supporting amphotericin B for primary therapy of invasive aspergillosis (Walsh et al., 2008).

Although Aspergillus species are generally susceptible to the above mentioned compounds, intrinsic and acquired resistance has been documented. Amphotericin B has limited activity against Aspergillus terreus (Johnson et al., 1999, Walsh et al., 2003, Steinbach et al., 2004), and A. nidulans (Kontoyiannis et al., 2002), while A. calidoustus (previously known as A. ustus) appears to be intrinsically resistant to triazole compounds (Varga et al., 2008). Furthermore, several species in the Aspergillus fumigatus complex (A. lentulus, A. pseudofisheri and A fumigatiaffinis) appear to be intrinsically resistant to azoles, and in the case of A. lentulus and A. fumigatiaffinis resistant to amphotericin B as well (Balajee et al., 2005).

Several studies indicate that acquired resistance of Aspergillus species to triazoles is uncommon (Pfaller et al., 2008, Guinea et al., 2008). However, in reference mycology laboratories in both Manchester, United Kingdom, and Nijmegen, The Netherlands, azole-resistance in Aspergillus species has emerged since 1998 (Fig. 1) (Verweij et al., 2007, Snelders et al., 2008, Howard et al., 2009a, Howard et al., 2009b). In these centres acquired resistance was primarily observed in A. fumigatus, although in Manchester resistance was also observed in other species such as A. niger complex (Howard et al., 2006a, Howard et al., 2008). In addition, azole-resistance was found in clinical A. fumigatus isolates from all eight University Hospitals in the Netherlands (Snelders et al., 2008, Van der Linden et al., 2008), and resistance was reported in Spain (Mellado et al., 2007), Belgium (Lagrou et al., 2008), Denmark (Arendrup et al., 2008), Sweden (Chryssanthou, 1997), France (Dannaoui et al., 2001), and Norway (Snelders et al., 2008). The isolates originally described with itraconazole resistance were from the USA and isolated in the late 1980s (Denning et al., 1997a). Therefore, it appears that azole resistance might be more common than currently acknowledged and clinical microbiology laboratories should determine the in vitro susceptibility of clinically relevant Aspergillus isolates, at least against the triazoles, which is currently not common practice.

Numerous resistance mechanisms have been found in azole-resistant A. fumigatus isolates, most of which consist of point mutations in the cyp51A gene, which is the target for antifungal azoles, as well as alteration in expression of CYP51A. The corresponding phenotype depends on the particular base substitution and often the activity of more than one triazole is affected, and in the absence of interpretative breakpoints this has resulted in various nomenclatures in the literature. Azole-resistant isolates have been reported as “multidrug resistant” (Warris et al., 2002), “multi-azole resistant” (Howard et al., 2006b), “azole cross-resistant” (Pfaller et al., 2008), and “multiple-triazole resistant” (Verweij et al., 2007, Snelders et al., 2008). Therefore, there is an increasing need to develop breakpoints in order to obtain consistency in the nomenclature of resistance in A. fumigatus isolates, for clinical purposes. Here we propose interpretative breakpoints based on a review of the literature and on our own experience.

Section snippets

Establishing interpretative breakpoints

The normal means of establishing interpretative breakpoints for culturable microorganisms requires a number of preconditions to be met at the start. A key requirement is a reproducible means of establishing an endpoint, which is usually an inhibitory (static) endpoint rather than a cidal endpoint. A second key requirement is the availability of isolates which are resistant to the agent(s) in question. In this context, documentation by clinical failure (including breakthrough on treatment) is

Methods for MIC testing in Aspergillus species

Various formats have been developed to test the susceptibility of Aspergilli; including macro/micro broth dilution, disc diffusion and Etest (Lass-Florl and Perkhofer, 2008). In recent years there have been advances with the development of Clinical Laboratory Standards Institute (CLSI; formerly NCCLS) M38-A (NCCLS, 2002), and European Committee for Antibiotic Susceptibility Testing (EUCAST) methods for filamentous fungi (SAST, 2007). Both methods include a microtitre format, 48-h incubation at

Mechanisms of resistance in Aspergillus species

Several mechanisms of resistance have been described in Aspergilli. Azole resistance has most commonly been associated with alterations in cyp51A, the gene encoding the target enzyme of the azoles. The most frequently characterized hot spots are at codons 54, 98 and 220, although several other single nucleotide polymorphisms (SNPs) have been reported (Verweij et al., 2007, Snelders et al., 2008, Mellado et al., 2004, Mellado et al., 2007, Howard et al., 2006b, Chen et al., 2005). Codon 98

In vivo data on Aspergillus resistance

Numerous data indicate that it is possible to distinguish itraconazole susceptible and resistant isolates of A. fumigatus in animal models of infection (Arendrup et al., 2008, Denning et al., 1997a, Denning et al., 1997b, Oakley et al., 1997, Dannaoui et al., 2001). Confidence in the results require a model using isolates with widely differing clear-cut numerical endpoints for susceptible and resistant isolates, which has historically been mortality, but increasingly is colony forming units or

Proposed breakpoints/nomenclature

The emergence of azole resistance in Aspergilli has lead to confusing nomenclature in the literature which primarily relates to the fact that SNP-containing isolates might be fully resistant to one azole but show full or reduced susceptibility to other azoles. Therefore it is important to establish breakpoints that will help us to interpret the MIC. As the formal procedure for determination of interpretative breakpoints will take many years to complete, we propose to follow a pragmatic approach

Conclusion

As with virtually all antimicrobial agents, azole resistance has emerged in the Aspergilli and has launched a new phase in our handling of aspergillosis. The proposed breakpoints and nomenclature will help us interpret the different azole-resistant forms and communicate within the scientific community. Continued surveillance for resistance is required, as is continued search for new resistance mechanisms. Genotypic methods of resistance identification may provide more confidence, given the

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