Antifungal drug resistance mechanisms in pathogenic fungi: from bench to bedside

REVIEW 10.1111/1469-0691.12495 Antifungal drug resistance mechanisms in pathogenic fungi: from bench to bedside M. Cuenca-Estrella National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain Abstract The phenotypic methods for identification of antifungal resistance are reliable procedures, and MIC determination by reference techniques is the gold standard to detect resistant clinical isolates. In recent years, progress has been made towards the description of resistance mechanisms at molecular level. There are methods of detection that can be useful for clinical laboratories, but lack of standardization precludes their full and effective integration in the routine daily practice. The molecular detection of Candida resistance to azoles and to echinocandins and of Aspergillus resistance to triazoles can be clinically relevant and could help to design more efficient prevention and control strategies. This text reviews the present state of the detection of mechanisms of resistance at the molecular level in Candida spp. and Aspergillus spp. and its relevance to clinical practice. Keywords: Aspergillus, Candida, mutations, mycosis, tandem repeat Article published online: 21 December 2013 Clin Microbiol Infect 2014; 20 (Suppl. 6): 54 59 Corresponding author: M. Cuenca-Estrella, Spanish National Center for Microbiology, Ctra. Majadahonda-Pozuelo Km2, Majadahonda, Madrid 28220, Spain E-mail: mcuenca-estrella@isciii.es Introduction The standardization of antimicrobial susceptibility testing (AST) is a long process that must comply with several requirements. To this end, it is essential to develop reliable reference procedures. Antifungal susceptibility testing has been standardized in last two decades, and it is still under development for some compounds and fungal species [1,2]. There are two standards for antifungal susceptibility testing both based largely on broth microdilution methods: One created by the European Committee on Antibiotic Susceptibility Testing (EUCAST) and the other one created by the Clinical Laboratory Standard Institute (CLSI, former NCCLS). Both organisations have led standardization processes, have performed reproducibility studies and recommended methods for quality control assurance [2 5]. In addition, the EUCAST and the CLSI have developed breakpoints and epidemiological cut-off values (ECVs) that are now established for Candida spp. and Aspergillus spp. [6 9]. The procedure to develop interpretative breakpoints is a multistep process based on the analysis of the MIC distributions and the clinical relationship between MIC values and efficacy. There are currently available breakpoints to interpret AST results of amphotericin B, azoles and echinocandins for Candida and amphotericin B and azoles for Aspergillus. After standardization of AST and setting breakpoints, reliable but more practical techniques of AST should be validated for use in clinical laboratories as the dilution standard reference procedures are rather complex methods for routine susceptibility testing [1,10]. There are several commercial and disc diffusion techniques that exhibit a high correlation with results of reference procedures and that are already used in many clinical laboratories. The extended use of AST methods has defined the prevalence of strains with high MIC values and some resistance mechanisms at the molecular level [11,12]. A number of molecular studies are currently in progress to ascertain the frequency of these mutants among wild-type populations [13,14]. The results of those surveys could be very useful to design strategies of prevention and control of emergence of resistance. Clinical Microbiology and Infection ª2013 European Society of Clinical Microbiology and Infectious Diseases

CMI Cuenca-Estrella Antifungal resistance from bench to bedside 55 This text reviews the present situation of the detection of mechanisms of resistance at the molecular level in Candida spp. and Aspergillus spp. and their relevance to the clinical practice. The reliability of molecular tools for the detection of resistance is also analysed as well as their performance to monitor the incidence of mutants with resistance to antifungal agents. Detection of Molecular Resistance to Amphotericin B Table 1 shows stages of the standardization process of AST by antifungal agent and by fungal species. In the case of amphotericin B, the system for MIC determination is able to detect strains exhibiting high MIC values [15]. Several studies reported, however, that reference procedures are not completely reliable to classify correctly some amphotericin B-resistant strains [16]. The reference techniques yield a range of amphotericin B MIC values that spans only three of four twofold serial dilutions precluding reliable discrimination between susceptible and resistant populations. Nevertheless, as some rare isolates were described as intrinsically resistant to the polyenes and they exhibit very high amphotericin B MIC values, breakpoints were proposed and set for Candida and Aspergillus some years ago [5,17]. Resistance to amphotericin B is an uncommon phenomenon in Candida (1 3%) and Aspergillus, although a proportion of A. terreus and A. flavus has higher MIC values [13,15,18]. Because the shortage of clinical strains with resistance to amphotericin B, not many studies on the molecular description of resistance mechanisms have been reported. It has been published that resistance is associated with mutants with low levels of ergosterol and disturbances of levels and composition of phospholipids in the membrane. Some of these changes have been associated with mutations in genes ERG2, ERG3 and ERG11. In addition, the polyenes induce oxidative stress in fungal cells, and resistant isolates can have higher levels of antioxidative enzymes and/or alterations in the production of free radicals [19 22]. Table 2 shows a summary of the current state of the molecular detection of resistance mechanisms by antifungal compound and by Candida and Aspergillus species, from the point of view of clinical laboratories. Determining resistance mechanisms of amphotericin B at the molecular level is clinically irrelevant. The number of strains exhibiting resistance in vitro is low, and mutants harbouring DNA changes related to rises in the MIC value of amphotericin B are hardly ever found in clinical samples [11,15]. There are no reliable molecular tools to detect amphotericin B resistance. Some reports have included ergosterol quantification or determinations of catalase activity and production of free radicals, but these procedures are phenotypic techniques that have not been standardized so far [23]. It can be concluded that currently molecular determination of amphotericin B resistance is not more useful than MIC determination for susceptibility testing and for the management of patients. Molecular detection is not TABLE 2. Current situation of the molecular detection of resistance mechanisms by antifungal compound and by species from the point of view of clinical laboratories Antifungal agents Fungal species Clinical relevance of molecular testing Availability of reliable molecular tools Integration in routine daily practice Amphotericin B Candida spp. No No No Aspergillus spp. No a No No Azoles Candida spp. Yes Yes Possible Aspergillus spp. Yes Yes Possible Echinocandins Candida spp. Yes Yes Possible b Aspergillus spp. No No No a Some strains of A. terreus exhibit high MIC values of amphotericin B, and it could have clinical relevance. b A significant number of resistance mechanisms are still unknown, and number of resistant strains is low. TABLE 1. Current situation of the antifungal susceptibility testing field by antifungal agent, by species and by stages of standardization process Antifungal agents Fungal species Availability of reference procedures Breakpoint setting Integration of AST in clinical laboratories in routine daily practice Molecular description of resistance Prevention and control strategies to avoid emergence of resistance Amphotericin B Candida spp. Yes Yes Yes No e No Aspergillus spp Yes Yes b Yes No No Azoles Candida spp. Yes Yes c Yes In progress No Aspergillus spp Yes Yes b Yes In progress No Echinocandins Candida spp. Yes Yes d Yes In Progress No Aspergillus spp Yes a No In Progress No No a No totally standardized yet for testing filamentous fungi. b The Clinical Laboratory Standard Institute (CLSI) has not proposed yet breakpoints for Aspergillus. c The CLSI has not proposed posaconazole breakpoints for Candida. d The European Committee on Antibiotic Susceptibility Testing is in process of setting caspofungin breakpoints for Candida. e Irrelevant as number of resistant mutants is very low.

56 Clinical Microbiology and Infection, Volume 20 Supplement 6, June 2014 CMI a reliable approach to design strategies of prevention and control of resistance to amphotericin B. Detection of Molecular Resistance to Azole Agents It has been proven that strains and species exhibiting high MIC values to azoles are unlikely to respond to treatments with azoles. Breakpoints to interpret the results of testing are available by both CLSI (for Candida only) and EUCAST (for some species of Candida and Aspergllus). They are species specific and are periodically revised [2,6,7,24]. Resistance to azole agents has been described quite commonly in Candida spp. Candida glabrata, a species with decreased susceptibility to fluconazole and other triazoles, is the second most common cause of invasive candidiasis in many countries and geographical areas. Candida krusei is the fifth cause of candidemia and seems to be insensible to fluconazole. Resistance has been also found in C. albicans, C. tropicalis and C. parapsilosis. Several population-based surveys of invasive candidiasis have reported rates of resistance to fluconazole between 10% and 25%. Many of these fluconazole-resistant isolates have cross-resistance to other azole agents [14,15]. Resistance to triazoles is not common in Aspergillus spp., being estimated below 5% in most of countries [13,25 27]. However, triazole resistance in A. fumigatus appears to be increasing in several European countries in recent years. It has been proposed that resistance has evolved in the environment under the selective pressure of azole fungicides widely used for crop protection and material preservation in Europe [27,28]. Antifungal susceptibility testing of azole agents is highly recommended for patient management in the ESCMID Guideline for the Management and Diagnosis of Candida Diseases 2012 [6]. The target of the azoles is the 14-alpha lanosterol demethylase enzyme generated by the gene ERG11 in yeasts and the CYP51 in filamentous fungi. Several genetic disturbances have been related to azole resistance. In the case of Candida, resistance is largely caused by overexpression of genes that code efflux pumps. The overexpression increases the capability of the fungal cell of expelling harmful substances such as antifungal agents. Mutants harbouring alterations in the ABC pumps (ATP binding cassette), the MFS pumps (major facilitator superfamily) or both have been found. In addition, mutations of ERG11 have been related to disturbances in the tridimensional structure of the protein, which can cause alterations in the site of docking between the demethylase and the antimicrobial agent increasing the MIC values. Other genetic changes such as chromosomal duplication, genetic conversion and mitotic recombination have been also associated with azole resistance in Candida. It should be noted that several of mechanisms described above can coexist in the same fungal cell, a phenomenon observed in some isolates after long-term therapy (several months) with azoles [29 31]. Mechanisms of resistance to triazoles in Aspergillus spp. were described some years ago [32,33]. The identification and sequencing of two different sterol demethylase genes (CYP51A and CYP51B) in A. fumigatus led experts to describe mutations in the CYP51A responsible for resistance in vitro to azoles [34]. The point mutations cause structural changes in the active site of the demethylase, decreasing the affinity to its ligands [35]. A number of mutations have been described and related with different profiles of resistance in Aspergillus. Mutations in some codons of CYP51A have been associated with resistance to itraconazole and posaconazole, and changes in other codons (220) to cross-resistance to all triazoles [32,33,36,37]. The mutation L98H coupled with a duplication of a fragment of DNA sequence in the promoter of CYP51A (also known as tandem repeat) has been proven to upregulate the expression of the enzyme, increasing eight times its ability to function and causing resistance to the azoles [38]. Resistance due to the tandem repeat has been linked to the use of azole fungicides in agriculture and gardening [27,28,39]. The resistance due to point mutations is related to long-term treatments with azoles. It has been described in patients suffering from chronic lung diseases treated with itraconazole as prophylaxis or empirical therapy [26]. Resistance to triazoles has been also described in A. flavus and A. terreus, but it is an uncommon phenomenon [13,40,41]. It seems that the gene coding the azole target cyp51a could be a hotspot for mutations that confer phenotypic resistance and that selective pressure with triazoles and pesticides can result in increasing numbers of resistant isolates [42,43]. Recently, the link between pesticides and azole resistance has been described also in C. tropicalis, although further studies should be carried out to verify this environmental hypothesis [44]. Regarding the clinical relevance of determining molecular mechanisms of resistance to azoles, it should be noted that the underlying mechanism remains unknown in a number of resistant isolates. Some new mechanisms such as increase in expression and mutations related to efflux pumps and mutations in the CCAAT-binding transcription factor complex subunit HapE have been reported in Aspergillus [42,45 47]. However, the prevalence of resistant strains and their correlation with clinical failures make clinically relevant the molecular description of resistance. The probable link to selective pressure due to prophylactic treatments and pesticides suggests that some strategies of prevention and control

CMI Cuenca-Estrella Antifungal resistance from bench to bedside 57 could be already implemented. The main concerns about molecular determination of azole resistance are lack of standardization of techniques of detection. MIC determination is still the most reliable procedure for surveillance of clinical isolates, but molecular methods allowing detection of resistance also in culture-negative specimens must be further developed and propagated [26]. Detection of Molecular Resistance to Echinocandins Resistance to echinocandins is not common among clinical isolates of Candida and Aspergillus. [14,15]. Candida spp. are susceptible to the three compounds licensed although C. parapsilosis exhibits higher MIC values to echinocandins due to a naturally occurring amino acid change in its Fks1, a glucan synthase, the target of these antifungal agents [48]. However, the reduced echinocandin susceptibility of C. parapsilosis does not appear to have clinical relevance. Aspergillus spp. are also susceptible in vitro to echinocandins. The procedures of AST of echinocandins are able to detect high MIC values in yeasts, and these resistant isolates in vitro have been related to clinical failures and breakthrough invasive mycosis in patients receiving echinocandins [49]. Species-specific breakpoints have been proposed to interpret the results of AST of echinocandins in Candida. The CLSI has set breakpoints for Candida spp. and the three echinocandins and the EUCAST for anidulafungin and micafungin so far [3,5]. Unlike yeasts, the AST systems currently available do not yield reliable results to evaluate the activity in vitro of echinocandins against Aspergillus and other moulds [4]. The echinocandins inhibit only partially the growth in vitro of filamentous fungi. Experts recommend the use the minimal effective concentration (MEC) in an attempt to address the problem, but MEC determination is a subjective procedure. As a result, breakpoints for AST of filamentous fungi have not been set. There are some ECVs available, but it is not clear yet if they can be used to interpret the AST results [1,2,11]. The rate of resistance to echinocandins in most of species of Candida is very low. However, there are reports describing clinical cases with echinocandin-resistant isolates [50 52]. Several resistance mechanisms to echinocandins have been published. There are adaptive stress responses that result in elevated cell wall chitin content and natural amino acid changes that confer elevated MIC values. Some of isolates with high MIC values associated with clinical failures, which have been molecularly analysed, harboured a mutation in the FKS1 gene. There are two hotspots in this gene, and a number of mutations that confer reduced glucan synthase sensitivity have been published [12,53]. Table 2 shows a summary of the current situation of the molecular detection of resistance for clinical laboratories. Determining resistance mechanisms of echinocandins at the molecular level may have some relevance. The number of Candida strains exhibiting resistance in vitro is low, but there are already available lists of mutations causing resistance in vitro to these compounds. In addition, there are reliable molecular tools to detect echinocandin resistance although standardization and external party validation should be done before recommending for clinical use. The extended use of echinocandins in daily clinical practice and the presence of the hotspots lead to the conclusion that echinocandin resistance could be an emerging problem in yeasts, and therefore, epidemiological surveillance of molecular resistance could be very useful soon to design prevention programmes. However, it should be noted that currently molecular detection of echinocandin resistance is not more useful than MIC determination for knowing the incidence of resistant strains and for the management of patients. Regarding filamentous fungi, further efforts should be taken to improve the reliability of AST and then to analyse the prevalence of resistance at molecular level. Conclusions The phenotypic methods for identification of antifungal resistance are reliable, and MIC determination by reference procedures is the gold standard to detect resistant clinical isolates. In recent years, progress has been made towards the description of resistance mechanisms at molecular level. There are methods of detection that can be useful for clinical laboratories, but lack of standardization precludes their full and effective integration in the routine daily practice. Multicentre studies including third party validation and reproducibility assessment should be designed. The molecular detection of Candida resistance to azoles and to echinocandins and of Aspergillus resistance to triazoles can be clinically relevant and could help to design more efficient prevention and control strategies. New automated and massive sequencing technique could change AST procedures in the upcoming years. Transparency Declaration In the past 5 years, M. Cuenca-Estrella has received grant support from Astellas Pharma, biomerieux, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Schering Plough, Soria

58 Clinical Microbiology and Infection, Volume 20 Supplement 6, June 2014 CMI Melguizo SA, Ferrer International, the European Union, the ALBAN programme, the Spanish Agency for International Cooperation, the Spanish Ministry of Culture and Education, The Spanish Health Research Fund, The Instituto de Salud Carlos III, The Ramon Areces Foundation, The Mutua Madrile~na Foundation. He has been an advisor/consultant to the Panamerican Health Organization, Astellas Pharma, Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough. He has been paid for talks on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, Astellas Pharma and Schering Plough. References 1. Cuenca-Estrella M, Rodriguez-Tudela JL. The current role of the reference procedures by CLSI and EUCAST in the detection of resistance to antifungal agents in vitro. Expert Rev Anti Infect Ther 2010; 8: 267 276. 2. Rodriguez-Tudela JL, Arendrup MC, Cuenca-Estrella M, Donnelly JP, Lass-Flörl C. 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