Curr Fungal Infect Rep (2013) 7:224–234 DOI 10.1007/s12281-013-0143-0

ADVANCES IN DIAGNOSIS OF INVASIVE FUNGAL INFECTIONS (U BINDER, SECTION EDITOR)

Update on Antifungal Resistance and its Clinical Impact Brunella Posteraro & Patrizia Posteraro & Maurizio Sanguinetti

Published online: 4 June 2013 # Springer Science+Business Media New York 2013

Abstract Candida and Aspergillus species are important causes of opportunistic infection in an ever-growing number of vulnerable patients, and these infections are associated with high mortality. This has partly been attributed to the emerging resistance of pathogenic fungi to antifungal therapy, which potentially compromises the management of infected patients. Multi-azole resistance of Aspergillus fumigatus is a current health problem, as well as is the co-resistance of Candida glabrata to both azoles and echinocandins. In most cases, negative clinical consequences of reduced in vitro fungal susceptibility to azoles and/or echinocandins can be traced to acquisition of particular resistance mechanisms. While strategies using antifungal combinations or adjunctive agents that maximize the efficacy of existing antifungals may limit treatment failures, new therapeutic approaches, including antifungal agents with novel mechanisms of action, are urgent. In the meantime, more efforts should be devoted to close monitoring of antifungal resistance and its evolution in the clinical setting. Keywords Invasive fungal infection . Fungemia . Bloodstream infection . Candida species . Aspergillus species . Antifungal drug resistance . Multidrug resistance . B. Posteraro Institute of Hygiene, Università Cattolica del Sacro Cuore, Largo F. Vito, 1, 00168 Rome, Italy e-mail: [email protected] P. Posteraro Clinical Laboratory, Ospedale San Carlo, Via Aurelia 275, 00165 Rome, Italy e-mail: [email protected] M. Sanguinetti (*) Institute of Microbiology, Università Cattolica del Sacro Cuore, Largo F. Vito, 1, 00168 Rome, Italy e-mail: [email protected]

Azoles . Echinocandins . Efflux-pump gene overexpression . Lanosterol 14α-demethylase gene mutation . β-1,3-D-glucan synthase inhibition . Breakthrough infection . Antifungal treatment . Clinical outcome

Introduction While global health data identify escalating levels of bacterial drug resistance that threaten the advances of modern medicine [1], it is as a result of the medical progress that the resistance against antifungal drugs gained interest in the last decade [2••]. This paradox has been linked to the everincreasing diversity of opportunistic fungi causing invasive fungal infection (IFI)—along with decreased antifungal susceptibility or intrinsic resistance characters—that has paralleled the ever-growing population of immunocompromised or seriously ill hospitalized patients [3, 4••, 5••, 6]. In certain medical conditions such as solid-organ and hematopoietic stem cell transplantation (HSCT) or intensive chemotherapy of hematological malignancies, antifungal prophylaxis and concomitant use of medical devices has altered the epidemiology of fungal infections [7•, 8]. For a notable instance, widespread use of fluconazole determined the emergence of Candida glabrata since the early 1990s, with unprecedented clinical consequences owing to the difficulty to treat infections with drug-resistant non-albicans Candida species [2••]. Likewise, but more recently, prophylactic use of posaconazole—a second-generation, broadspectrum triazole agent [9]—led to a significant proportion of breakthrough IFIs (bIFIs) in high-risk patients compared with historical data, and with a shift toward drug-resistant non-Aspergillus species [10]. Despite the introduction of new antifungal agents from old (triazoles) or newest classes (echinocandins), with improved efficacy/safety profiles [11, 12], IFIs are associated with substantial morbidity and mortality [13–16]. These relate not only

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to host populations, but to the emerging resistance of fungal pathogens to standard antifungal therapy [2••], so treatment with a combination of antifungal agents is often considered to improve outcomes [17, 18]. Nonetheless, the oldest antifungal drugs, polyenes, remain useful therapeutic options, particularly for immunocompromised patients with bIFIs [19•]. However, their broad-spectrum activity and low rates of resistance are counteracted by infusion-related adverse events and nephrotoxicity, which are somewhat mitigated by lipid-associated formulations of these drugs [20]. In this paper, we review the current aspects of antifungal resistance and its consequences for the management of IFIs, as well as new strategies to be implemented for the close monitoring and overcoming of such resistance, with an emphasis on invasive candidiasis and aspergillosis.

Epidemiology of Antifungal Resistance Despite emerging opportunistic fungal infections [5••, 6], candidiasis and aspergillosis remain the most prevalent systemic infections in immunocompromised patients [3, 7•]. A recent update on a 5-year survey of IFIs from the Prospective Antifungal Therapy (PATH) Alliance revealed that among 7,526 cases—identified in 6,845 patients hospitalized at 25 medical centers in North America—5,526 were caused by Candida species (73.4 %). These fungi were followed by Aspergillus species (13.3 %), other yeasts (6.2 %) (primarily Cryptococcus), other molds (2.6 %), and Mucormycetes (1.6 %) as main causes of IFI [21••]. When focusing only on 3,648 PATH Alliance-enrolled patients with candidemia, Pfaller and colleagues [22] reported that of 4,067 Candida isolates, 57.9 % were non-albicans Candida species; with C. glabrata (26.7 %), Candida parapsilosis (15.9 %), Candida tropicalis (8.7 %), and Candida krusei (3.4 %) being the most common causative species. In a multicenter prospective survey conducted by Pemán and colleagues [23], among 1,357 bloodstream infection (BSI) episodes—identified over 13 months at 44 Spanish hospitals—the incidence of C. albicans fungemia was the highest, followed by C. parapsilosis sensu stricto, C. glabrata, and C. tropicalis. Interestingly, the incidence of fungemia by C. parapsilosis outnumbered by 11 and 74 times that by Candida orthopsilosis and Candida metapsilosis, respectively. By contrast, neither Candida nivariensis nor Candida bracarensis was isolated, despite the clinical, but moderate, interest elicited from these new C. glabrata-related species [24], owing to the propensity of some isolates to exhibit antifungal resistance [25]. The epidemiologic shift toward Candida species other than C. albicans was expected to reflect changes in the antifungal susceptibility profiles of these species [26, 27]; while in the United States, the frequency of bloodstream C. glabrata isolates increased from 18 % (years 1992–2001) to 25 % (years 2001–

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2007), an increase in fluconazole resistance, from 9 % to 14 %, was observed during the same time periods [28]. Notably, 99– 100 % of these isolates were found to be susceptible to all three echinocandins (caspofungin, anidulafungin, and micafungin) at the breakpoint of≤2 μg/ml, formerly established by the Clinical and Laboratory Standards Institute (CLSI). Data from the SENTRY antimicrobial surveillance program (2008–2009) concerning the geographic distribution of echinocandin and azole antifungal resistance—assessed by the newly revised CLSI breakpoints [29, 30]—showed that the frequency of resistance in 376 invasive C. glabrata isolates was more prominent among isolates from North America (3.2 % for anidulafungin, 2.7 % for micafungin, and 5.5 to 8.2 % for azoles) rather than among isolates from Europe (1.5 %, 0.8 %, and 0.0 % to 2.3 %), Latin America (0.0 %), and the AsiaPacific region (0.0 %) [31]. It is worth noting that five of the six echinocandin-resistant C. glabrata study isolates were from episodes of breakthrough fungemia on micafungin treatment, but none of those isolates were co-resistant to azoles [31]. However, cross-resistance between azoles and echinocandins is not unusual, although mechanisms of action and resistance are markedly different between the two classes of antifungal agents, as discussed in greater detail below. A few years ago, Chapeland-Leclerc and colleagues [32] did provide substantiated evidence of fatal BSI due to C. glabrata with a multidrugresistant (MDR) phenotype—resistant to two or more classes of antifungal agents—where consecutive isolates became progressively resistant to fluconazole, voriconazole, caspofungin (and also flucytosine) during treatment courses, each with one antifungal agent. Later, in one study with 1,669 BSI isolates of C. glabrata from two large antifungal surveys—the SENTRY global surveillance program (years 2006–2010) and the Centers for Disease Control and Prevention (CDC) population-based surveillance (years 2008–2010)—Pfaller and colleagues [33••] found that 18 of 162 (11.1 %) fluconazole-resistant isolates were resistant to one or more of the echinocandins. This documents, for the first time to the authors’ knowledge [33••], the emergence over time of co-resistance to both azoles and echinocandins in clinical isolates of C. glabrata, even though attestations of MDR C. glabrata strains have been reported elsewhere [34, 35]. Again in the Pfaller’s study [33••], all the CDC isolates for which epidemiological data could be obtained [36] were from patients who had previously been exposed to an echinocandin; by contrast, no echinocandin resistance was found in the 110 fluconazole-resistant C. glabrata isolates therein studied for comparison but, as stated by the authors [33••], these isolates were all derived from the 2001–2004 time period where only caspofungin among echinocandins was available. The drug was approved in 2001 and since that time, the overall use of echinocandins in the United States had increased significantly [37]. Thus, exposure to an echinocandin has the potential not only for the emergence of higher echinocandin MICs among the more common Candida species [36, 38], but of Candida species

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against which these drugs are relatively less active in vitro (e.g., C. parapsilosis, Candida guilliermondii) [27, 39]. As previously shown with fluconazole [26, 40], exposure to caspofungin was recently recognized as a risk factor for BSIs, due to Candida species with decreased susceptibility to caspofungin [27] and, interestingly, these infections were associated with a recent (within 30 days before a fungemia episode) prescription of caspofungin [27, 39]. Different species of Aspergillus may lead to invasive disease, but Aspergillus fumigatus remains the most common causative pathogen [41]. While non-A. fumigatus Aspergillus species, including A. lentulus, A. pseudofisheri, and A. fumigatiaffinis, may be intrinsically resistant to more antifungal drug classes [42], azole-resistant A. fumigatus sensu stricto have been isolated in azole naïve patients, in azole-exposed patients, and in the environment [43]. Although acquired resistance to triazoles, with the exception of fluconazole, in Aspergillus species is to date considered relatively uncommon [44], azole-resistant A. fumigatus have currently been reported—albeit to significantly different extents—in Canada, China, the United States, and several European countries [45], with particularly high frequencies from the Netherlands (Nijmegen) and the United Kingdom (Manchester) [46, 47]. As suggested [43], an explanation for these findings might be the extensive use of azole fungicides in agricultural practice in Nijmegen [48] or the patient population studied, primarily chronic aspergillosis cases on long-term therapy in Manchester [47]. Otherwise, the prevalence of azole resistance in A. fumigatus can be largely underestimated, as many clinical mycology laboratories do not routinely perform the antifungal susceptibility testing of their mold isolates [43]. Indeed, resistance to triazoles in A. fumigatus seems to be increasing in some European locations, where it is most extensively studied [46, 47]. Bueid and colleagues [49] updated their azole resistance experience [47] with data from A. fumigatus isolates tested for the susceptibility to itraconazole, voriconazole, and posaconazole at the Mycology Reference Centre Manchester in 2008 and 2009; they noted that of 230 isolates studied, 64 (28 %) were azoleresistant, and that 50 (78 %) of 62 itraconazole-resistant isolates were multi-azole resistant, whereas two of 64 isolates were only resistant to voriconazole. In the meantime, 497 A. fumigatus isolates collected from 2008 to 2009 as part of the ARTEMIS global surveillance study were found to have elevated minimum inhibitory concentration (MIC) values to itraconazole, voriconazole, and posaconazole [50]. However, no consistent trend toward reduced triazole susceptibility was observed for the 1,312 ARTEMIS A. fumigatus isolates over a nine-time period (years 2001–2009) [51]; this was in apparent contrast with the Manchester data attesting a progressive increase in patients infected by azole-resistant A. fumigatus isolates from 5 % in 2001 to 20 % in 2009 [49], but it was in agreement with other recent data that did

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not show any temporal increase in triazole resistance in 118 clinical A. fumigatus isolates obtained from a French cohort of 89 hematological patients [52]. Echinocandin resistance in Aspergillus species is currently much less known [43], and this has partly been attributed to the scarce—and less frequent than for azoles—disposition to do antifungal susceptibility testing of Aspergillus isolates in clinical practice [53], that in turn may be linked to technical difficulties and suboptimal reproducibility of the methods currently available [43]. However, bIFIs in patients on caspofungin therapy have been reported but sporadically [54–56], and they involved A. fumigatus isolates exhibiting elevated minimum effective concentrations (MECs) to caspofungin (≥ 1 μg/ml) [57].

Mechanisms of Antifungal Resistance The largest class of antifungal drugs in clinical use, namely the azoles, functions by inhibiting lanosterol 14α-demethylase, the ergosterol biosynthetic enzyme known as Erg11 (from Candida) or Cyp51A (from Aspergillus), that leads to depletion of ergosterol, the main sterol of fungal membranes, and accumulation of toxic sterols [58]. Both these effects result in a disrupted integrity and consequent stress of the cell membrane, as well as in the growth arrest, but not cell death, for most fungal species. The azole fungistatic activity, generally exerted against yeasts including Candida species, creates favorable conditions for the evolution of drug resistance, which is more likely to proceed by strong directional selection on the surviving fungal population [59]. Thus, in response to short periods of drug exposure such as in experimental or clinical settings, high levels of azole resistance in C. albicans isolates often accumulate by the acquisition of multiple mechanisms, which include upregulation of multidrug transporters or alterations of the target enzyme [58]. Acquired resistance was also observed in A. fumigatus isolates cultured from patients with chronic Aspergillus infections or with aspergilloma (high fungal burden) during treatment with azoles [47]. Conversely, development of phenotypic resistance during treatment of patients with acute invasive aspergillosis should be regarded as highly unlikely, unless the fungus undergoes multiple generations by the asexual modality of reproduction. As opposed to hyphal growth—typically found in acute invasive aspergillosis—it was suggested that sporulation (in the lung) may facilitate the expression of the azole-resistant phenotype [48]. As an alternative to the resistance development in azole-treated patients, there is increasing evidence to support that resistance in A. fumigatus may develop by an environmental route, with the consequence that up to two-thirds of patients with azole-resistant Aspergillus disease had no history of previous azole exposure [60]. As reviewed by Sanglard and colleagues [61••], a wellestablished azole mechanism involves alteration of the target

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enzyme by amino acid substitutions caused by nonsynonymous mutations in the Erg11 gene, namely ERG11 from C. albicans, C. tropicalis, and Cryptococcus neoformans, and related ERG11-like genes such as the A. fumigatus cyp51A gene. In C. albicans, at least 12 different point mutations in ERG11 have been associated with triazole resistance, the majority of which results in an altered target with reduced affinity for or inability to bind azoles [58]. Recently, 17 mutations, seven of which were novel, encoding distinct amino acid substitutions were found in the ERG11 gene of 23 clinical C. albicans isolates [62]. Site-directed mutagenesis of ERG11 was used to verify the effect of each of these amino acid substitutions on triazole resistance, and it was shown that substitutions A114S, Y132H, Y132F, K143R, Y257H, and a new K143Q substitution contributed to significantly increasing (≥ 4-fold) the resistance to both fluconazole and voriconazole [62]. In A. fumigatus, azole resistance consists of changes in the amino acid sequence of the cyp51A gene, leading to alterations in the target protein. However, certain mutations in cyp51A (for example at codons G54, L98, G138, M220, and G448) may result in resistance to one, two, or all three triazoles (i.e., itraconazole, voriconazole, and posaconazole) [45], which is consistent with predicted structural properties of the lanosterol 14α-demethylase enzyme and its interaction with chemically different azole drugs [47, 63]. In British A. fumigatus isolates—referring to the Regional Mycology Laboratory Manchester collection of 519 clinical isolates from 1992 to 2007—a wide variety of cyp51A mutations led to different resistance patterns; but, of note, three of the azoleresistant isolates had no mutations in their cyp51A gene [47]. This finding was corroborated by further data from the Manchester group showing that no Cyp51A mutations were found in 43 % of A. fumigatus isolates from the 2008–2009 study period [49]. Thus, the efflux pump, a common mechanism in yeasts as discussed below, could also work in these isolates to overcome the pressure of triazole exposure, or it is conceivable that an unknown mechanism could be implicated to strengthen the continuing evolution of azole resistance in A. fumigatus [49]. In this context, Camps and colleagues [64] compared the complete genomes from four isogenic sequentially isolated clinical A. fumigatus strains, two of which developed resistance during prolonged azole treatment, but did not contained any mutations in the cyp51A gene. By subsequent sexual crossing and gene replacements experiments, the authors identified a novel P88L substitution in the CCAAT-binding transcription factor complex subunit HapE as a novel cause of azole resistance [64]. By contrast, the presence of a single resistance mechanism—it involves two genomic changes, a substitution at leucine 98 for histidine of the cyp51A gene and the insertion of a 34-bp tandem repeat in the gene promoter (TR/L98H)—was found in 94 % of clinical A. fumigatus isolates in the Netherlands, and these isolates showed reduced susceptibility to itraconazole, voriconazole, and posaconazole [46]. Remarkably, isolates harboring the

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TR/L98H resistance mechanism were also cultured from environmental sources in the Netherlands, and were found to be cross-resistant to certain sterol 14α-demethylation inhibitors [65]. The dominance of this mechanism and the high proportion of resistant isolates recovered from azole-naïve Dutch patients [60] would preclude resistance development through azole therapy and strongly support the possibility that patients acquired resistant isolates from the environment [48]. As opposed to the Dutch experience with TR/L98H, the cyp51A mutations identified in two studies from the United Kingdom indicate that resistance development is due primarily to pressure under azole therapy [47, 49]. On the other hand, A. fumigatus isolates with the TR/L98H mutation were also isolated from agricultural samples of soil in Denmark [66], as well as from patients in Spain, France, Norway, United Kingdom, and Belgium [47, 66, 67]. Surprisingly, in an antifungal resistance survey conducted by Lockhart and colleagues [50], the presence of the TR/L98H mutation was noted in eight of 29 A. fumigatus isolates with elevated MIC values to one or more azoles; all were originating in China and probably stemming from the environment at the source. A second major mechanism by which Candida and, to a lesser extent, Aspergillus species can acquire antifungal resistance involves the overexpression of either of two classes of multidrug transporters, which efflux triazoles, as well as many other compounds, out of the cell, thus reducing intracellular drug concentrations to levels at which the enzyme target is not inhibited [58]. Upregulation of efflux pumps belonging to the ATP-binding cassette transporter superfamily (encoded by CDR or SNQ2 genes) or the major facilitator superfamily of transporters (encoded by MDR genes) have been associated with azole resistance in C. albicans (MDR1, CDR1, CDR2), C. glabrata (CgCDR1, CgCDR2, CgSNQ2), or Candida dubliniensis (CdCDR1, CdCDR2) [61••]. Key transcription regulators have been identified in C. albicans that control the efflux pump expression in response to drug exposure; therefore, gainof-function mutations in these transcription factors [68] or enhanced dosage of the hyperactive alleles—such as the genomic alteration that amplify the copy number of ERG11 [69]—can confer resistance [58]. However, unlike C. glabrata—in this species, only efflux pump overexpression is operating to determine cross-resistance to azoles [70–72]—in many C. albicans isolates azole resistance was shown to correlate with the occurrence of both transporter gene up-regulation and nonsynonymous ERG11 mutations [61••]. Hence, MacCallum and colleagues [73] undertook the genetic dissection of the two resistance mechanisms in an azole-resistant C. albicans isolate, DSY296, and showed a correlation between different levels of resistance, as measured by the fluconazole or voriconazole MIC in vitro, and different amounts of the genetic resistance mechanisms in the DSY296-derivative C. albicans mutant strains, which were engineered to overexpress CDR efflux pumps and/or mutations in one or both the ERG11 alleles.

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In comparison with the activities of the triazoles, the echinocandins are fungicidal for Candida species but fungistatic for molds such as A. fumigatus, where they induce abnormal morphology and growth properties [74]. They inhibit the synthesis of the major cell-wall polysaccharide β-1,3-D-glucan by presumptive binding to the β-1,3-D-glucan synthase, an enzyme complex consisting of at least a catalytic subunit, Fks (encoded, in Candida, by three related genes FKS1, FKS2, and FKS3) and a regulatory subunit, Rho1. While the echinocandins are not active at clinically relevant concentrations against C. neoformans, Fusarium, and Zygomycetes, they show good in vitro activity against Candida and Aspergillus species [75], including azole-resistant pathogens [76]; however, this drug class is less effective against some Candida species, in particular C. parapsilosis and C. guilliermondii, because of increased chitin synthesis in these species in response to the echinocandins [77]. As with the azoles, echinocandin resistance has been associated with mutations of the drug target, an enzyme absent in mammalian cells that accounts for the favorable safety profile of the echinocandin drugs [78]. In clinical breakthrough isolates, these mutations were identified within two hot-spot regions of the FKS1 gene—leading to amino acid substitutions, mostly at the Ser645 position—and were shown to be dominant, yield high MIC values, confer cross-resistance to all three echinocandin, and reduce the sensitivity of the target enzyme to the drug by several thousand-fold [79]. In contrast to C. albicans and other Candida species, these mutations in C. glabrata involve both Fks1 and Fks2, with Fks2 mutations outnumbering Fks1 mutations. This Fks1–Fks2 redundancy was shown to attenuate the rate of resistance, as well as the impact of resistanceconferring mutations, whereas their differential regulation can be exploited to reverse the Fks2-mediated resistance [80]. Although breakthrough infections by A. fumigatus isolates with elevated MECs have been reported, the mechanisms of echinocandin resistance in molds, especially Aspergillus, have not yet been understood. However, the universality of Fks1 modification as a mechanism for fungal resistance to echinocandins has been demonstrated in laboratory strains [79], so introducing a S678P mutation raised the MEC of caspofungin, anidulafungin, and micafungin to>16 μg/ml, compared to 0.25 μg/ml with the susceptible wild-type strain [81]. Alternatively, a clinical isolate of A. fumigatus from a patient who failed caspofungin therapy was resistant due to overexpression of the AfFKS1 gene [82], but this mechanism was determined by gene expression profiling upon exposure of the isolate to subinhibitory concentrations of caspofungin.

Clinical Implications of Antifungal Resistance The clinical outcome for a patient with IFI is often difficult to predict, because the antifungal susceptibility of the fungal

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isolate is only one of the factors that contribute to clinical resistance or treatment failure; these terms refer to the persistence or progression of a fungal infection, despite the administration of appropriate antifungal therapy [83]. Several factors (e.g., site of infection, high fungal burden, acquisition of a strain with increased virulence, length of treatment/and or compliance, underlying disease) have been identified as key determinants of antifungal clinical resistance [83]. However, there is no doubt that patients infected with a fungal isolate for which an antifungal MIC is higher than average, or within the range designated as the resistant breakpoint for that organism, will fail antifungal treatment [84]. The impact of antifungal resistance on the clinical outcome of patients with candidiasis has recently been reviewed by Pfaller [2••], who analyzed the consequences of MIC values for fluconazole and itraconazole in patients with candidemia and mucosal candidiasis, based on the success (clinical and/or microbiological) rate and mortality. It was therefore evident that patients infected with Candida isolates with MICs to fluconazole and voriconazole classified as resistant, respectively, had a clinical outcome significantly poorer than patients infected with isolates with MICs classified as susceptible. The negative consequences of higher MICs can often be traced to acquisition of particular mechanisms of resistance [2••]. By reviewing the microbiological records of consecutive C. glabrata bloodstream isolates collected from a HSCT patient with clinical failure—a 9-year-old girl with Fanconi anemia who died after five recurrences of candidemia—the progressively increasing isolate MICs to fluconazole, voriconazole, and caspofungin were associated with the development of multiple resistances [32]. These latter were documented in isolates from fungemia episodes four and five by increased levels of CgCDR1 and, to a lesser extent, CgCDR2 expression (compared to the basal expression of bloodstream isolates 1, 2, and 3), and by the presence of a non-synonymous nucleotide mutation (T1988C) in the FKS2 gene, leading to a an S663P amino acid substitution in Fks2. The FKS1 gene sequences were similar (and wild-type) for all isolates [32]. It is plausible that C. glabrata acquires MDR through successive independent genetic events, and that this fungal pathogen is highly prone to readily mutate in vivo in a single patient, also facilitated by the haploid nature of its genome. All this, consistent with the increase of MDR C. glabrata isolates underscored by Pfaller’s above-mentioned study [33••], poses a threat for the targeted use of echinocandins in severely immunocompromised (or critically) ill patients who have a history of recent azole exposure or colonization/infection with a Candida species known to have reduced susceptibility to azoles [85]. Likewise, azole-resistant Aspergillus infections are undoubtedly associated with a higher likelihood of therapeutic failure than infections caused by isolates with a wild-type

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susceptibility [47, 86], and a more recent study from the Netherlands reported that the case-fatality rate of patients with azole-resistant invasive aspergillosis was 88 % [60]. Through a prospective surveillance study conducted in all eight Dutch University Hospitals in 2008, van der Linden and colleagues [86] identified three cases of central nervous system aspergillosis due to azole-resistant A. fumigatus. The patients were treated with combination therapy including voriconazole and caspofungin, or with liposomal amphotericin B, but none of the patients survived. Phenotypic and genotypic characterization of A. fumigatus isolates cultured from the three patients showed MICs of both itraconazole and voriconazole above the concentrations achievable in plasma and cerebrospinal fluid—that precluded the therapeutic use of these agents [87]—as well as the presence of a TR/L98H mutation in the Cyp51A azole target. In addition, posaconazole MIC values were found to be 3–5 twofold dilutions more than those in wild-type isolates, thereby indicating that posaconazole may not be therapeutically efficacious. As none of the patients described had a history of previous exposure to mold-active antifungal azoles and then with apparent risk factors for azole resistance, the authors underlined that, while phenotypic testing of Aspergillus isolates is mandatory to enable detection of all resistance mechanisms, it may delay the initiation of adequate antifungal therapy and hence contribute to complicating the outcome of invasive aspergillosis [86]. More recently, van der Linden and colleagues investigated the effects of azole resistance on patient management strategies by means of a prospective nationwide multicenter surveillance study in the Netherlands [60]. Of 1,792 A. fumigatus isolates screened during a 20-month period (June 2007–January 2009), 82 (4.6 %) isolates were resistant to itraconazole, and the per-patient analysis showed a resistance prevalence of 5.3 %. Eight patients were diagnosed with invasive aspergillosis due to an itraconazole-resistant isolate, which harbored the TR/L98H mutation; four patients were azole naïve and one had been previously treated with fluconazole—which has no activity against Aspergillus species. All eight patients received voriconazole monotherapy, but seven (87.5 %) died within 3 months of receiving a positive culture result. Only one of three patients, for whom voriconazole therapy was switched to another class of antifungal compounds, i.e., echinocandin, polyene, or both, survived. Therefore, while there is a strong recommendation to use a non-azole class drug or echinocandin/polyene combination in initial therapy of cases of azole-resistant invasive aspergillosis [45], the low survival rate of the patients whose treatment was switched from azole therapy to non-azole therapy would point to the need for additional studies to confirm the relation between azole resistance and treatment failure [60]. The median MIC of voriconazole in the eight patients with azoleresistant invasive aspergillosis was 4 μg/ml [60], which is above the proposed interpretive breakpoint for A. fumigatus and voriconazole (resistant, > 2 μg/ml) [45].

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Resistance to echinocandins is an unusual concern in C. albicans, and this seems to be linked to reduced relative fitness of the resistant strains containing homozygous fks1 hot-spot mutations, which cause them to be less pathogenic [74]. These mutations were shown to alter the kinetic catalytic capacity of the β-1,3-D-glucan synthase, and the fks1 mutants had increased cell wall chitin content, which reduced their growth rates and impaired their filamentation capacities [88]. Thus, mice infected with high-chitin cells were less susceptible to caspofungin, as indicated by higher kidney fungal burdens and greater weight loss, in contrast to mice infected with C. albicans cells with normal chitin levels [89]. After 48 hours post-infection, caspofungin treatment induced a further increase in the chitin content of C. albicans cells recovered from mouse kidneys, and interestingly, some of these clones were shown to have acquired a S645Y mutation in Fks1 [89]. Consistently, elevated echinocandin MIC values, not associated with therapeutic failure, can result from an FKS-independent adaptive cell mechanism, which involves the molecular chaperone Hsp90 and the calcineurin pathway [90]. Nonetheless, the complex relationship between fks mutations, MICs, and in vivo response [74], especially in situations for which C. glabrata is the infecting agent [91], was partly unraveled by the revised lower values of echinocandin susceptibility breakpoints [29], which take into account the FKS resistance mechanism and new pharmacokinetic and pharmacodynamic data [74]. However, while the identification of fks mutants is epidemiologically important, the phenotype (MIC) may provide the most sensitive predictor of therapeutic efficacy [91]. Consistent with this, Alexander and colleagues [92] correlated the treatment outcome with echinocandin MIC results and the presence of fks mutations in all the C. glabrata BSIs at Duke Hospital over the past decade (years 2001–2010). Of 13 episodes involving isolates with a resistant MIC and treated with echinocandin monotherapy, five (38.5 %) responded, whereas eight (61.5 %) failed to respond or responded initially but recurred. Of interest, the eight episodes that failed all were caused by an fks mutant isolate, thus suggesting that an elevated echinocandin MIC concomitant with a characteristic FKS gene mutation in a C. glabrata isolate correlates with reduced clinical BSI outcome [92].

New Strategies to Survey and Overcome Antifungal Resistance The management of antifungal-resistant Candida and Aspergillus diseases remains extremely difficult, and new therapeutic approaches for fungal disease, including agents with novel mechanisms of action, are urgently needed [93, 94]. A number of in vitro studies, animal models, and clinical

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reports have shown that combination antifungal therapy might offer improved results over antifungal monotherapy, even though a definitively accepted strategy is far from being achieved [17, 18]. Since the targets of azoles and echinocandins are unrelated, the reduced efficacy of voriconazole in azole-resistant A. fumigatus disease, as discussed earlier, might be overcome by the concomitant administration of an echinocandin. Using a non-neutropenic murine model of invasive aspergillosis, Seyedmousavi and colleagues [95] found that interaction between voriconazole and anidulafungin was synergistic in voriconazole-susceptible invasive aspergillosis, but indifferent in voriconazole-resistant invasive aspergillosis. These findings were partly confirmed by in vitro studies conducted by the same authors, who used a large collection of isolates and with a wide range of voriconazole MICs, and showed that the combination could likely be less effective in voriconazoleresistant strains with high MICs [96]. Alternatively, treatment with liposomal amphotericin B may improve therapeutic outcome in azole-resistant invasive aspergillosis, as results from an experimental murine model of disseminated aspergillosis indicated that liposomal amphotericin B was able to prolong survival in vivo independent of the presence of an azole resistance mechanism in a dose-dependent manner [97]. As the current classes of antifungal drugs target the cell wall and cell membrane, it should be possible to maximize the efficacy of the existing antifungals, to block the emergence of drug resistance, and to exert broad-spectrum activity against diverse fungal pathogens, through the use of adjunctive agents that address distinct cellular stress response or signaling pathways [18]. It was observed that harnessing the heat shock protein 90 (Hsp90) converts the fungistatic azoles into a fungicidal combination, which enhances the therapeutic efficacy of azoles in two metazoan models of disseminated C. albicans infection [98], as well as rendering the echinocandins more effective at killing fks1-containing C. albicans laboratory strains and clinical isolates [90]. Therefore, an innovative antibody-based therapy that involves efungumab, a recombinant antibody fragment targeted against the HSP90 of C. albicans, was developed [99]. In addition to showing activity against Candida species when used alone and synergism when combined with fluconazole, caspofungin, and amphotericin B, efungumab has progressed through a randomized controlled trial; the results suggested a potential advantage of efungumab in combination with liposomal amphotericin B relative to liposomal amphotericin B alone for the treatment of invasive candidiasis [99]. However, to the best of our knowledge, efungumab remained in the Novartis development pipeline [93]. Despite increasing use of echinocandins and reports of bIFIs [79], echinocandins remain key therapeutic options for invasive candidiasis and treatment-refractory invasive aspergillosis [78], but the lack of an oral formulation limits the

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role of echinocandins for the prevention and treatment of IFIs [74]. The new glucan synthase inhibitor, MK-3118, is an orally active semisynthetic derivative of the natural product enfumafungin, which is structurally different from the echinocandins; as a consequence, the sites of mutations causing decreased susceptibility to enfumafungin are distinctly different from those in fks that are associated with echinocandin resistance [100]. In two recent company studies by Pfaller and colleagues, MK-3118 was tested against a panel of 113 clinical isolates of Candida species, including fluconazole- and caspofungin-resistant isolates; and a panel of 71 Aspergillus species, including itraconazole-resistant isolates [101, 102]. In both the studies, MK-3118 showed a potent in vitro activity against contemporary wild-type and resistant strains of either Candida or Aspergillus species. Apart from clinical paradigms dictating that C. krusei and C. glabrata are generally resistant to fluconazole, while Candida lusitaniae and Aspergillus terreus are resistant to amphotericin B (for such species antifungal susceptibility testing and further molecular investigation are seldom required or performed [58]), early and accurate recognition of infections caused by pathogens with no predictable susceptibility to antifungal agents is highly desirable to optimize treatment and patient outcome. In addition, antifungal susceptibility testing is a valuable tool to assess the activity of new and experimental compounds and to investigate the epidemiology of antifungal-resistant pathogens. By means of recently refined methods for in vitro antifungal susceptibility testing [103], coupled with detection of the molecular alterations conferring to the fungus reduced antifungal drug susceptibility [104], often directly from clinical specimens [105, 106], it is now possible to guarantee a close antifungal resistance surveillance in many clinical settings worldwide. This provides clinicians with the direction for antifungal selection, facilitates prompt institution of appropriate antifungal therapy, and ultimately contributes to improve the treatment of life-threatening fungal infections.

Conclusions Despite a continuous but slow growth of therapeutic agents with fungus-specific mechanisms of action, nowadays antifungal choices are severely restricted, and only three classes, i.e., polyenes, azoles, and echinocandins, form the armamentarium of antifungal drugs commonly administered in clinical practice. Their clinical utility is potentially compromised by the emergence of drug resistance, which limits therapeutic options, especially for C. glabrata and A. fumigatus infections. While there is a compelling need for new approaches to manage patients with azole-resistant invasive aspergillosis or fungemia caused by MDR C. glabrata, strategies aimed at slowing the development and spread of antifungal resistance

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can rely on continued surveillance and continued search for new resistance mechanisms. Urgent attention should be devoted to the clinical implication of antifungal resistance in terms of management and outcome, as well as to the in-depth research necessary to define optimal therapeutic interventions. Compliance with Ethics Guidelines Conflict of Interest Brunella Posteraro, Patrizia Posteraro and Maurizio Sanguinetti declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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