Antifungal drug resistance among Candida species: mechanisms and clinical impact

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1 mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases Supplement article Antifungal drug resistance among Candida species: mechanisms and clinical impact Maurizio Sanguinetti, 1 Brunella Posteraro 2 and Cornelia Lass-Fl orl 3 1 Institute of Microbiology, Universita Cattolica del Sacro Cuore, Rome, Italy, 2 Institute of Public Health, Section of Hygiene, Universita Cattolica del Sacro Cuore, Rome, Italy and 3 Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria Summary The epidemiology of Candida infections has changed in recent years. Although Candida albicans is still the main cause of invasive candidiasis in most clinical settings, a substantial proportion of patients is now infected with non-albicans Candida species. The various Candida species vary in their susceptibility to the most commonly used antifungal agents, and the intrinsic resistance to antifungal therapy seen in some species, along with the development of acquired resistance during treatment in others, is becoming a major problem in the management of Candida infection. A better understanding of the mechanisms and clinical impact of antifungal drug resistance is essential for the efficient treatment of patients with Candida infection and for improving treatment outcomes. Herein, we report resistance to the azoles and echinocandins among Candida species. Key words: Antifungal drug resistance, azoles, Candida, echinocandins, susceptibility testing. Epidemiology of Candida infection The frequency of Candida infections has increased in recent years, largely due to the increasing size of the atrisk population, which includes transplant recipients, cancer patients and other patients who receive immunosuppressive therapy. 1 In a recent retrospective analysis of the Extended Prevalence of Infection in the Intensive Care Unit Study (EPIC II), the global prevalence of candidaemia was reported to be 6.9 cases per 1000 patients. 2 In the United States, Candida species are currently ranked fourth among hospital-acquired bloodstream infections, 3 with reported annual incidence rates ranging from 6.0 to 13.3 cases per population. 4 6 In Europe, Candida species are among the top 10 most frequently isolated nosocomial bloodstream Correspondence: Brunella Posteraro, PhD, Institute of Public Health, Section of Hygiene, Universita Cattolica del Sacro Cuore, Largo F. Vito 1, Rome, Italy. Tel.: Fax: bposteraro@rm.unicatt.it Submitted for publication 27 February 2015 Accepted for publication 23 March 2015 pathogens, 7 with reported annual incidence rates ranging from 1.9 to 4.8 cases per population Candida albicans is the predominant cause of invasive candidiasis in the majority of clinical settings, accounting for 50% to 70% of cases 1 ; however, the epidemiology of Candida infection has changed in recent years, with longitudinal studies reporting that a considerable proportion of patients are now infected with non-albicans Candida (NAC) species. 5,14 19 NAC species of clinical importance include C. glabrata, C. tropicalis, C. parapsilosis and C. krusei which, together with C. albicans, account for more than 90% of cases of invasive candidiasis. 18,19 Other less frequently reported Candida species include C. guilliermondii, C. lusitaniae, C. kefyr, C. famata, C. inconspicua, C. rugosa, C. dubliniensis and C. norvegensis. The changing epidemiology of Candida infection has been partly attributed to the selection of less sensitive Candida strains by the widespread use of the azole fluconazole as a prophylactic and therapeutic agent. A recent study by Lortholary and colleagues, which presented data from a prospective surveillance program in France, reported that Candida species that were less susceptible or resistant to fluconazole were more frequently isolated following recent treatment ( 30 days) with the drug (odds ratio [OR] = 2.17; 95% doi: /myc Mycoses, 2015, 58 (Suppl. 2), 2 13

2 Antifungal drug resistance among Candida species Table 1 Distribution of Candida species in blood samples from Europe. Isolates (%) Study Country Study period Isolates 1 (n) C. albicans C. glabrata C. parapsilosis C. tropicalis C. krusei Others 2 [110] Austria [111] Belgium [112] Belgium [113] Denmark [114] Greece [112] Greece [115] Greece [116] Greece [117] Finland [118] France [119] France [120] France [121] France [122] Italy [123] Italy [124] Italy [125] Italy [32] Italy [126] Poland [127] Portugal [9] Spain [128] Spain [129] Turkey [130] Turkey [131] Turkey [132] UK Refers to the total number of Candida isolates from blood (or the total number of candidaemia episodes where the former was not available from the original study). 2 Includes Candida species not depicted in the table and Candida species not identified to species level. 3 Patients staying 48 h after ICU admission. Reprinted from Eur Rev Med Pharmacol Sci, 18 (5), Montagna MT, Lovero G, Borghi E, Amato G, Andreoni S, Campion L, Lo Cascio G, Lombardi G, Luzzaro F, Manso E, Mussap M, Pecile P, Perin S, Tangorra E, Tronci M, Iatta R, Morace G. Candidemia in intensive care unit: a nationwide prospective observational survey (GISIA-3 study) and review of the European literature from 2000 through 2013, Pages , Copyright (2013), with permission from Verduci Editore. confidence interval [CI] = 1.51 to 3.13; P < 0.001). 20 Previous fluconazole exposure has also been shown to increase the risk of fluconazole-resistant Candida bloodstream infections 21,22 in intensive care unit patients, 23 cancer patients 24 and in cases of candidaemia caused by C. glabrata. 21,25 Other risk factors associated with the emergence of NAC species include the duration of central venous catheter use, recent gastrointestinal surgery, increasing age, glucocorticoid therapy, candiduria, diabetes and intravenous drug use. 11,21,26 30 The distribution of C. albicans and NAC species has been shown to vary in population-based studies carried out in different geographical regions (Table 1). 31,32 In a recent systematic review that evaluated the geographical distribution of Candida species in blood samples from inpatients in various parts of the world, C. albicans was found to be the most frequently isolated pathogen in Northern and Central Europe and the USA, whereas NAC species were found to predominate in Asia, Southern Europe and South America. 33 The highest proportion of C. glabrata isolates were found in Northern and Central Europe, whereas C. parapsilosis was most commonly found in Slovakia, Southern Europe, South America and Asia and C. tropicalis predominated in Eastern Asia and Argentina. 33 In contrast, the frequency of C. krusei was found to be relatively low in all geographical regions. 33 In addition to geographical variation, the frequency of Candida species may also be affected by the underlying medical conditions of patients and the antifungal agents received, as well as local hospitalrelated factors. 31 Mycoses, 2015, 58 (Suppl. 2),

3 M. Sanguinetti et al. Antifungal drug resistance Antifungal drug resistance can be characterised as microbiological or clinical. Microbiological resistance is defined as the non-susceptibility of a fungal pathogen to an antifungal agent as determined by in vitro susceptibility testing and compared with other isolates of the same species, and can be further categorised as intrinsic or acquired. Whilst intrinsic resistance is found naturally among certain fungal strains without prior exposure to drugs, acquired resistance develops in previously susceptible fungal strains following drug exposure and can often occur as a result of altered gene expression. 34 In contrast, clinical resistance refers to the persistence of a fungal infection despite treatment with adequate therapy. Although microbiological resistance can contribute to the development of clinical resistance, other factors may also be involved, such as impaired immune function, underlying disease, reduced drug bioavailability and increased drug metabolism. 35 Here, we report resistance to the azoles and echinocandins among Candida species. The Infectious Diseases Society of America guidelines currently favour echinocandins as first-line treatment for systemic candidiasis in patients with moderate-to-severe infection and in those with prior exposure to azoles, with fluconazole reserved for the treatment of patients with less severe infection. Based on more recent data, the European Society of Clinical Microbiology and Infectious Diseases recommends echinocandins as first-line treatment for all patients with systemic candidiasis. 36 Resistance to the polyene antifungals (e.g. amphotericin B) remains relatively uncommon among Candida isolates and as such, will not be covered here. 34,37 Candida species exhibit varying degrees of susceptibility to the most commonly used antifungal agents. For example whilst C. krusei is intrinsically resistant to fluconazole, with a global resistance rate of 78.3%, C. glabrata displays reduced dose-dependent susceptibility compared with other Candida species and has a global resistance rate of 15.7%. 18 Primary resistance to fluconazole is rare for C. albicans (1.4%), C. parapsilosis (3.6%) and C. tropicalis (4.1%). 18 In contrast, the echinocandins exhibit potent antifungal activity against most Candida species, with the exception of C. parapsilosis, for which higher minimum inhibitory concentrations (MICs) have been reported In recent years, an increasing number of rarer Candida species have been reported to be intrinsically less susceptible to the azoles (C. ciferrii, C. guilliermondii, C. inconspicua, C. humicola, C. lambica, C. lipolytica, C. norvegensis, C. palmioleophila, C. rugosa and C. valida) and echinocandins (C. fermentati and C. guilliermondii). 18,44,45 Acquired resistance is generally less common than intrinsic resistance; however, data from various studies suggest that it is beginning to emerge in some countries. In a recent retrospective, case-comparator study conducted at 2 tertiary-care cancer centres in Boston, MA, USA, Oxman and colleagues reported that 19% of their Candida infections involved either fluconazoleresistant strains or strains with reduced susceptibility. C. albicans, C. tropicalis and C. parapsilosis, species that are generally considered to be susceptible to fluconazole, accounted for 36% of isolates with reduced susceptibility and 48% of resistant isolates. 22 Another study by Lortholary and colleagues conducted in Paris, France, reported that Candida species that were less susceptible or resistant to caspofungin were more frequently isolated following recent treatment ( 30 days) with the drug (OR = 4.79; 95% CI = 2.47 to 9.28; P < 0.001). 20 In addition, the frequency of C. parapsilosis, a species known for its reduced susceptibility to the echinocandins, was shown to increase after treatment with caspofungin (13% 31%). 20 A significant association between recent exposure to caspofungin ( 30 days) and an increased risk of caspofungin-resistant candidaemia (OR = 5.25; 95% CI = 1.68 to 16.35; P = 0.004) was also reported in a nested case control study conducted in patients with haematological malignancies in Paris, France. 46 Cross-resistance among Candida species to multiple antifungal agents is also an important concern. Crossresistance between the azoles has been demonstrated for many Candida species, including C. glabrata, C. albicans, C. tropicalis and C. parapsilosis. 27 A high degree of crossresistance among the echinocandins has also been demonstrated for C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei. 47 Although cross-resistance among the azoles and echinocandins is generally uncommon, 48,49 reports of multidrug-resistant C. glabrata are beginning to emerge. 50 Testing methods for antifungal drug resistance In line with the increasing incidence of invasive fungal infections and the growing number of available antifungal agents, there is a need for accurate, reproducible and clinically relevant susceptibility testing of fungal isolates to enable physicians to make informed decisions regarding treatment. The Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing 4 Mycoses, 2015, 58 (Suppl. 2), 2 13

4 Antifungal drug resistance among Candida species (EUCAST) have both developed standard susceptibility testing methods for Candida species based on broth microdilution While the base medium (RPMI 1640), incubation duration (24 h) and endpoint criteria (prominent inhibition) are the same, there are some differences between the two testing methods, including inoculum size ( to CFU ml 1 [CLSI] vs to CFU ml 1 [EUCAST]), RPMI broth glucose concentration (0.2% [CLSI] vs. 2.0% [EUCAST]), microdilution wells (rounded [CLSI] vs. flat-bottomed [EUCAST]) and MIC determination (visual [CLSI] vs. spectrophotometric [EUCAST]). The methods have been harmonised so that there is categorical agreement in the interpretation of MIC results when testing both azoles and echinocandins against Candida species. 54 The CLSI has also developed an agar-based, disc diffusion testing method for Candida species, which is an attractive alternative for use in routine clinical practice as it is simple, inexpensive and suitable for water-soluble antifungal agents, such as fluconazole and voriconazole. Disc diffusion has been standardised for fluconazole, voriconazole, caspofungin and micafungin in Candida species. 52 To interpret MIC results as susceptible, susceptible dose-dependent, or resistant, the CLSI and EUCAST have both developed species-specific clinical breakpoints for the azoles and echinocandins based on MIC distributions, pharmacokinetic and pharmacodynamic parameters, resistance mechanisms, separation of wildtype from non wild-type populations and treatment outcomes. 55,56 To facilitate the reliable detection of antifungal drug resistance, commercial MIC methods have been developed as modifications of broth microdilution or agar diffusion methods, and their performance has been found to be comparable to that of the CLSI reference method. 50,57 Three methods are currently available and largely used for testing antifungal agents against Candida species: the Sensititre YeastOne colorimetric plate (TREK Diagnostic Systems, Cleveland, OH, USA), the Vitek 2 yeast susceptibility test (biomerieux, Marcy l Etoile, France) and the Etest (biomerieux, Marcy l Etoile, France). 50,57 Novel diagnostic methods based on emerging technologies, such as matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS), flow cytometry, X-plate technology and porous aluminium oxide-based culture have also been developed for the susceptibility testing of fungal pathogens. 57 De Carolis and colleagues developed a MALDI-TOF MS-based assay that facilitated the detection of fungal isolates with reduced echinocandin susceptibility following 15 h of drug exposure. 58 Endpoint readings by MALDI-TOF MS were found to be time-saving compared with the CLSI- and EUCASTbased methods (15 vs. 24 h respectively). 58 More recently, the authors described a simplified version of this approach that enabled the discrimination of susceptible and resistant isolates of C. albicans after a 3-h incubation in the presence of breakpoint level drug concentrations of caspofungin. 59 In recent years, flow cytometry has become an important adaptive technology platform in fungal diagnostics. 60,61 Using flow cytometric methods, the effects of an antifungal agent can be evaluated by observing alterations in the fungal cell viability that can be identified via changes in the measured cell fluorescence. Chadwick and colleagues have also recently described a cost-effective and rapid chromogenic agar dilution method, known as X-plate technology, for simultaneous Candida species identification and fluconazole susceptibility testing, and reported results that were consistent with the standard microdilution broth assays. 62 The ability of microbial species to grow on a support of porous aluminium oxide coupled with microscopy has also been exploited to generate an alternative to conventional phenotyping. 63 Using this technique, Ingham and colleagues were able to detect and quantify cultured Candida microcolonies within 2 h and microcolony imaging was then used to assess susceptibility to amphotericin B, anidulafungin, and caspofungin (3.5-h culture), and voriconazole and itraconazole (7-h culture). 63 Mechanisms of antifungal drug resistance Resistance to azoles Azoles act by inhibiting the target enzyme lanosterol 14-a-sterol demethylase, which is involved in the conversion of lanosterol to ergosterol, an important component of the fungal cell membrane, and leads to the accumulation of the toxic product 14-a-methyl-3,6- diol. Consequently, ergosterol content in the fungal cell membrane is reduced, leading to altered cell membrane structure and function, and ultimately inhibited fungal growth. Three principal mechanisms of azole resistance have been reported among Candida species. 34 However, it is important to note that more than one resistance mechanism may operate in any given strain and the changes resulting in resistance may have additive effects or lead to the development of cross-resistance. 64 The first mechanism associated with azole resistance is the induction of multi-drug pumps which result in reduced drug concentrations at the enzyme target Mycoses, 2015, 58 (Suppl. 2),

5 M. Sanguinetti et al. (A) (B) (C) Figure 1 Azole resistance mechanisms in Candida albicans. (A) Induction of the ABC transporter family of multidrug efflux pumps confers resistance to multiple azoles, and induction of a major facilitator transporter confers fluconazole resistance by reducing drug concentrations at the enzyme target lanosterol 14-a-sterol demethylase (also known as cytochrome P450 14DM ) in the fungal cell. (B) Alteration or upregulation of lanosterol 14a-demethylase confers resistance by preventing azole binding to the enzymatic site. (C) Loss-of-function mutation of Erg3 confers resistance by blocking the accumulation of the toxic sterol 14-a-methyl-3,6-diol that would otherwise accumulate when lanosterol 14 a-sterol demethylase is inhibited by the azoles. Eukaryot Cell, 2008, 7 (5), Pages , DOI /EC , reproduced with permission from American Society for Microbiology. lanosterol 14-a-sterol demethylase in the fungal cell (Fig. 1A). Efflux pumps in Candida species are encoded by the CDR genes of the ATP-binding cassette superfamily and the MDR genes of the major facilitator super-family. 65,66 Whilst the induction of CDRencoded efflux pumps has been shown to confer resistance to almost all azoles, induction of the MDRencoded efflux pumps appears to be selective for fluconazole resistance. 34 Up-regulation of the CDRand MDR-encoded efflux pumps have been shown to lead to azole resistance in C. albicans (MDR1, CDR1, CDR2), C. glabrata (CgCDR1, CgCDR2) andc. dubliniensis (CdMDR1, CdCDR1). 67,68 Another common mechanism of azole resistance involves the alteration or up-regulation of the enzyme target lanosterol 14-a-sterol demethylase, which is encoded by the ERG11 gene (Fig. 1B). Mutations in ERG11 have been shown to prevent the binding of azoles to the enzymatic site. 69 Indeed, the intrinsic resistance of C. krusei to fluconazole has been attributed to the reduced affinity of ERG11p for fluconazole. 70 In some patients, Candida isolates with reduced susceptibility to azoles have been shown to have higher intracellular concentrations of ERG11p compared with azole-susceptible strains, leading to a situation where the drug is overwhelmed, and normal therapeutic concentrations no longer inhibit ergosterol synthesis. 71 However, minimal up-regulation of altered target enzymes has been observed thus far, suggesting that this mechanism plays a limited role in the development of clinical resistance to the azoles. 34 The third mechanism of azole resistance involves the development of bypass pathways (Fig. 1C). As discussed previously, exposure to azoles results in reduced ergosterol content in the fungal cell membrane and leads to the accumulation of the toxic product 14-a-methyl-3,6-diol. Mutations in the ERG3 gene have been shown to prevent the formation of 14-a-methyl-3,6-diol from 14-a-methylfecosterol. 72 The replacement of ergosterol with the latter sterol leads to functional fungal cell membranes and therefore negates the membrane disruptive effects of the azoles. Resistance to echinocandins Echinocandins act by non-competitively inhibiting b(1,3)d-glucan synthase, the enzyme which is 6 Mycoses, 2015, 58 (Suppl. 2), 2 13

6 Antifungal drug resistance among Candida species (A) (B) Figure 2 Echinocandin resistance mechanisms in Candida albicans. (A) Point mutations and intrinsic mutations in specific regions of genes encoding the FKS subunits of b(1,3)d-glucan synthase confers resistance by reducing drug impact on the cell. (B) Rho1 is a positive regulator of b(1,3)d-glucan synthase and contributes to resistance by mediating stress responses, such as upregulation of the cell wall component chitin. Eukaryot Cell, 2008, 7 (5), Pages , DOI /EC , reproduced with permission from American Society for Microbiology. responsible for the biosynthesis of b(1,3)d-glucan, a key component of the fungal cell wall. 64 Inhibition of b(1,3)d-glucan synthase by the echinocandins leads to the formation of a defective fungal cell wall and ultimately cell death. 64 Two principal mechanisms of echinocandin resistance have been reported among Candida species. 73 Reduced susceptibility or resistance to echinocandins has been linked with point mutations and intrinsic mutations in specific regions of genes encoding the FKS subunits of b(1,3)d-glucan synthase (Fig. 2A). 74 Specific point mutations have been found to cluster around 2 highly conserved regions, known as hotspot regions. 73 In C. albicans, these mutations are found at amino acid positions 641 to 649 (hot-spot 1 [HS1]) and 1345 to 1365 (hot-spot 2 [HS2]) in FKS1. 75 Hot-spot mutations in FKS1 have also been reported for C. glabrata, C. krusei, C. tropicalis and C. dubliniensis. 75,76 In addition, resistance in C. glabrata can also be associated with mutations in FKS2 (paralog of FKS1). 75,76 Mutations in these hot-spot regions have been shown to result in elevated MICs, reduced b(1,3)d-glucan synthase sensitivity, and cross-resistance among the echinocandins In contrast, C. parapsilosis is intrinsically less susceptible to echinocandins because of naturally occurring FKS1 mutations. 73 Surveillance studies have shown that C. parapsilosis consistently displays higher MIC values (range lg ml 1 ) compared with other more susceptible Candida species. 43 Another potential mechanism of echinocandin resistance involves the initiation of the adaptive stress response (Fig. 2B). The fungal cell wall is highly dynamic and is able to increase the production of one or more components when another is inhibited. In vitro studies have shown that an increase in chitin synthesis occurs in Candida species in response to inhibition of b(1,3)d-glucan synthesis by the echinocandins, and this increase is mediated by protein kinase C, high-osmolarity glycerol response and Ca 2+ calcineurin signalling pathways, which have been shown to negate the lethal effects of the echinocandins. 79,80 In addition, in vitro studies have noted that some strains of C. albicans are able to grow at supra-mic concentrations of caspofungin, a process known as the paradoxical effect In the presence of supra-mic concentrations of caspofungin, these strains have significantly higher chitin content compared with isolates exposed to lower concentrations of caspofungin Antifungal drug resistance and loss of heterozygosity The genomic plasticity of C. albicans has been shown to play an important role in the development of antifungal drug resistance through loss of heterozygosity (LOH) at resistance genes and chromosomal rearrangements that amplify the copy number of genes associated with resistance. LOH is an important event in the generation of phenotypic diversity in Mycoses, 2015, 58 (Suppl. 2),

7 M. Sanguinetti et al. C. albicans. 86 As diploid cells carry two copies of each gene, and because the majority of mutations are recessive, new mutations usually have little or no effect. 87 However, recombination between pairs of chromosomes can result in LOH, which then unmasks the phenotypes of previously acquired mutations. 87 In C. albicans, LOH events have been observed in strains growing under selection for antifungal drug resistance. 88 Forche and colleagues have demonstrated that LOH events in C. albicans increased during in vitro exposure to heat stress (39 C; 1- to 40-fold increase in LOH), oxidative stress (hydrogen peroxide; 3- to 72-fold increase in LOH), and treatment with antifungal agents (fluconazole; 285-fold increase in LOH). 89 Furthermore, an increase in the severity of stress was found to correlate with increased rates of LOH. 89 Whilst LOH and chromosomal rearrangements both contribute to the development of antifungal drug resistance, the spread and persistence of antifungal drug resistance depends on the relative fitness of resistant and susceptible genotypes. Accumulated genomic changes have the potential to affect the cellular fitness of an organism, defined as reproductive output throughout a specific time period, leading to trade-offs of fitness in exchange for the acquisition of survival-enhancing features, such as resistance. Cowen and colleagues examined the changes in gene expression that occurred in 12 populations of C. albicans during 330 generations (approximately 100 days) of evolution in the presence and absence of fluconazole, and reported that fluconazole exposure led to different levels of resistance in the populations, and that resistant populations initially paid a fitness penalty for the resistance mutation, which was found to diminish with continued exposure to the drug. 90 Epidemiology of antifungal drug resistance Global surveillance programs provide important antifungal susceptibility data from a large number of hospitals and laboratories worldwide and have been used to examine the epidemiology of antifungal drug resistance. One of the largest and longest-run surveillance programs is the ARTEMIS DISK global antifungal surveillance study, the objective of which was to analyse the susceptibility of various Candida species to fluconazole and voriconazole, as determined by CLSIstandardised disc diffusion. 18,91 93 The most recent publication by Pfaller and colleagues reported the results of the 10.5-year analysis and provided susceptibility data for fluconazole ( ) and voriconazole ( ) for more than Candida isolates from 41 countries worldwide. 18 In total, 90.2% of the Candida isolates were found to be susceptible to fluconazole; however, reduced fluconazole susceptibility (defined as <75.0% susceptible) was observed in 13 of 31 species. Resistance to fluconazole was shown to increase throughout time for C. parapsilosis, C. guilliermondii, C. lusitaniae, C. sake and C. pelliculosa. Fluconazole resistance was found to be emerging in isolates of C. guilliermondii, C. inconspicua, C. rugosa and C. norvegensis. In contrast, resistance to voriconazole was generally uncommon (<5.0% resistant) during the study. Furthermore, approximately one-third of fluconazole-resistant isolates of C. albicans, C. glabrata, C. tropicalis, C. rugosa, C. lipolytica, C. pelliculosa, C. apicola, C. haemulonii, C. humicola, C. lambica and C. ciferrii remained susceptible to voriconazole. Another large and long-running surveillance program is the SENTRY antimicrobial surveillance program, which was initiated in 1997 to monitor the resistance patterns of predominant pathogens on a Table 2 Relationship between susceptibility and treatment outcomes for fluconazole and voriconazole in patients with Candida infections. Azole Infection Species Category 1 MIC breakpoint (lg ml 1 ) No. of events % success Fluconazole Voriconazole Candidaemia and mucosal candidiasis Candidaemia and invasive candidiasis C. albicans, C. tropicalis, C. parapsilosis C. albicans, C. tropicalis, C. parapsilosis Susceptible Susceptible dose-dependent Resistant Susceptible Susceptible dose-dependent Resistant According to CLSI interpretive criteria for azoles. 55 CLSI, Clinical Laboratory Standards Institute; MIC, minimum inhibitory concentration. Reprinted from Am J Med, 125 (1), Pfaller MA, Antifungal drug resistance: mechanisms, epidemiology and consequences for treatment, Pages S3 13, Copyright (2012), with permission from Elsevier. 8 Mycoses, 2015, 58 (Suppl. 2), 2 13

8 Antifungal drug resistance among Candida species global scale. 19,94 97 A recent publication by Pfaller and colleagues provided susceptibility data for 3107 clinical isolates of Candida from 2010 to During this period, resistance to fluconazole occurred in 8.8% of C. glabrata isolates but was uncommon among isolates of C. albicans (0.4%), C. tropicalis (1.3%) and C. parapsilosis (2.1%). Voriconazole was found to be effective against all Candida species with the exception of C. glabrata (10.5% non-wild-type). Resistance to the echinocandins was generally uncommon, with incidence rates ranging from 0% 1.7%. In total, 38% of echinocandin-resistant C. glabrata isolates from 2011 were also found to be resistant to fluconazole. In a recent review of the SENTRY antimicrobial surveillance program ( ) and the Centres for Disease Control and Prevention population-based surveillance ( ) conducted in Atlanta, GA, USA, and Baltimore, MD, USA, Pfaller and colleagues reported for the first time the broad emergence of fluconazole-resistant bloodstream infection isolates of C. glabrata that were also resistant to 1 or more echinocandins. 50 The authors partly attributed the emergence of this cross-resistance to the increased use of echinocandins and azoles during the study period. Although the vast majority of C. glabrata isolates remain highly susceptible to the echinocandins, the emergence of co-resistance to both azoles and echinocandins remains an important concern. Clinical impact of antifungal drug resistance Antifungal drug resistance is associated with elevated MICs that are associated with poorer clinical outcomes, breakthrough infections during treatment, and increased healthcare costs. Pfaller and colleagues reported that clinical outcome was significantly poorer for patients infected with Candida isolates with resistant MICs for fluconazole and voriconazole compared with those for which the MICs were classified as susceptible or susceptible dose-dependent (Table 2). 64 Case reports describing resistance to caspofungin and the other echinocandins among various Candida species also provide compelling evidence of the relationship between high or increasing MICs and poor clinical outcomes Antifungal drug resistance can also lead to the development of breakthrough infections in high-risk patients receiving antifungal prophylaxis. Alexander and colleagues reported eight cases of breakthrough infection in 295 adult bone marrow transplant patients receiving prophylaxis with fluconazole during the period 2002 to 2004 at Duke University Medical Centre in Durham, NC, USA. 104 Of these cases, seven were attributed to C. glabrata and 4/7 displayed cross-resistance to fluconazole, voriconazole, itraconazole and posaconazole. 94 In another study conducted at Duke University Medical Centre, Durham, NC, USA, 12 breakthrough infections were reported among 649 bone marrow transplant and solid organ transplant recipient patients receiving prophylaxis with micafungin. 105 In addition to high morbidity and mortality rates, invasive Candida infections are also associated with increased healthcare costs and prolonged length of stay (LOS) in hospital. 106,107 However, patients with resistant infections may experience treatment delays due to difficulties in diagnosis, which can substantially increase these costs and hospital LOS. Invasive Candida infections have been reported to add a total of $300 million USD to healthcare expenditures annually. 108 Although resistant infections are thought to substantially increase these costs, limited data are available on the economic impact of resistant Candida infections. A number of strategies for preventing the emergence and spread of antifungal drug resistance have been implemented. Whilst patient-specific strategies focus on reducing the negative impact of immunosuppression, drug-specific strategies include the establishment of optimal treatment regimens to prevent sub-optimal drug exposure. 109 More systematic strategies include the development of protocols for prophylaxis and/or pre-emptive therapy, aggressive resistance surveillance to detect changes in antifungal susceptibility patterns, and guidelines on the use of antifungal agents. 109 Discussion The epidemiology of Candida infections has changed in recent years. Whilst the number of patients suffering from such infections has increased, the Candida species involved have become more numerous as NAC species begin to emerge. The various Candida species vary in their susceptibility to the available antifungal agents. The intrinsic resistance to antifungal therapy observed in some species, along with the development of acquired resistance during treatment in others, is becoming a major problem in the management of Candida infections. Antifungal susceptibility testing has therefore become essential for effective patient management and resistance surveillance. Acknowledgements This review was sponsored by Pfizer Inc. Editorial support was provided by Karen Irving of Complete Medical Communications and sponsored by Pfizer Inc. Mycoses, 2015, 58 (Suppl. 2),

9 M. Sanguinetti et al. Conflicts of Interest Maurizio Sanguinetti has received grant support from Gilead Sciences, Merck Sharp and Dohme and Pfizer Inc; and has been paid for talks on behalf of Astellas Pharma, Gilead Sciences, Merck Sharp and Dohme and Pfizer Inc. Brunella Posteraro has no conflicts of interest. Cornelia Lass-Fl orl has received grant support from Astellas Pharma, Austrian Science Fund, Gilead Sciences, Merck Sharp and Dohme, MFF Tirol, Pfizer Inc and Schering Plough; has acted as an advisor/consultant for Astellas Pharma, Gilead Sciences, Merck Sharp and Dohme, Pfizer Inc and Schering Plough; and has received travel/accommodation support from and has been paid for talks on behalf of Astellas Pharma, Gilead Sciences, Merck Sharp and Dohme, Pfizer Inc and Schering Plough. References 1 Arendrup MC. Epidemiology of invasive candidiasis. Curr Opin Crit Care 2010; 16: Kett DH, Azoulay E, Echeverria PM, Vincent JL. 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