The American Journal of Medicine
Volume 125, Issue 1, Supplement , Pages S3-S13, January 2012

Antifungal Drug Resistance: Mechanisms, Epidemiology, and Consequences for Treatment

  • Michael A. Pfaller, MD

      Affiliations

    • Corresponding Author InformationRequests for reprints should be addressed to Michael A. Pfaller, MD, Medical Microbiology Division, C606 GH, Department of Pathology, University of Iowa College of Medicine, 200 Hawkins Drive, Iowa City, Iowa 52242

Medical Microbiology Division, Department of Pathology, University of Iowa College of Medicine, Iowa City, Iowa, USA

Article Outline

Abstract 

Antifungal resistance continues to grow and evolve and complicate patient management, despite the introduction of new antifungal agents. In vitro susceptibility testing is often used to select agents with likely activity for a given infection, but perhaps its most important use is in identifying agents that will not work, i.e., to detect resistance. Standardized methods for reliable in vitro antifungal susceptibility testing are now available from the Clinical and Laboratory Standards Institute (CLSI) in the United States and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in Europe. Data gathered by these standardized tests are useful (in conjunction with other forms of data) for calculating clinical breakpoints and epidemiologic cutoff values (ECVs). Clinical breakpoints should be selected to optimize detection of non–wild-type (WT) strains of pathogens, and they should be species-specific and not divide WT distributions of important target species. ECVs are the most sensitive means of identifying strains with acquired resistance mechanisms. Various mechanisms can lead to acquired resistance of Candida species to azole drugs, the most common being induction of the efflux pumps encoded by the MDR or CDR genes, and acquisition of point mutations in the gene encoding for the target enzyme (ERG11). Acquired resistance of Candida species to echinocandins is typically mediated via acquisition of point mutations in the FKS genes encoding the major subunit of its target enzyme. Antifungal resistance is associated with elevated minimum inhibitory concentrations, poorer clinical outcomes, and breakthrough infections during antifungal treatment and prophylaxis. Candidemia due to Candida glabrata is becoming increasingly common, and C glabrata isolates are increasingly resistant to both azole and echinocandin antifungal agents. This situation requires continuing attention. Rates of azole-resistant Aspergillus fumigatus are currently low, but there are reports of emerging resistance, including multi-azole resistant isolates in parts of Europe.

Keywords:  Antifungal resistance , Azoles , Candida glabrata , Clinical breakpoint , Echinocandins , Epidemiologic cutoff

 

Both the frequency of invasive fungal infections (IFIs) and resistance to antifungal therapy continue to increase despite the introduction of new antifungal agents.1 In vitro antifungal susceptibility testing is now standardized internationally2 and is becoming essential in patient management and resistance surveillance. Although in vitro susceptibility testing is often used to select antimicrobial agents likely to be clinically active for a given infection, perhaps its most important function is the detection of resistance, i.e., to determine which agents will not work. Improvements in the ability of antifungal susceptibility testing methods to detect emerging resistance patterns, coupled with molecular characterization of resistance mechanisms, provide useful adjuncts to optimize the efficacy of antifungal therapy. Epidemiologic surveys that examine local and regional data can be used to develop empiric treatment strategies and are essential in tracking resistance trends. This review examines various definitions of antifungal resistance, testing methods for antifungal resistance, mechanisms of antifungal resistance, and the epidemiology and consequences of that resistance.

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Case Study: Multidrug Resistance (MDR) in Candida glabrata 

The following is based on a case recently reported by Chapeland-Leclerc and coworkers3:

A 9-year-old girl with Fanconi anemia who underwent a hematopoietic stem cell transplant (HSCT) becomes fungemic with Candida glabrata. She is treated with antifungals from each of the major antifungal drug classes (sometimes in combination)—including flucytosine (5FC), fluconazole, voriconazole, liposomal amphotericin B, and caspofungin (CSF)—without success. Minimum inhibitory concentrations (MICs) of consecutive C glabrata isolates suggest the pathogenic organism had become progressively resistant to each of the antifungal agents during the course of therapy. Analysis of resistance mechanisms identified point mutations in C glabrata FUR1(CgFUR1) and CgFKS2, which have been respectively linked with 5FC and CSF resistance, as well as overexpression of CgCDR1 and CgCDR2, linked with fluconazole and voriconazole resistance.

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Antifungal Resistance: Definitions 

Antifungal resistance can be defined as microbiologic or clinical resistance, or as a composite of the two.4 Microbiologic resistance is said to occur when growth of the infecting organism or pathogen is inhibited by an antimicrobial agent concentration higher than the range seen for wild-type strains. Clinical resistance is defined by the situation in which the infecting organism is inhibited by a concentration of an antimicrobial agent that is associated with a high likelihood of therapeutic failure. In other words, the pathogen is inhibited by an antimicrobial concentration that is higher than could be safely achieved with normal dosing. By the composite definition, resistance is present when isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules and/or when they demonstrate MICs that fall in the range where specific microbial resistance mechanisms are likely, and where clinical efficacy against the isolate has not been reliably shown in treatment studies.

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Testing Methods for Antifungal Resistance 

The need for reproducible, clinically relevant, antifungal susceptibility testing has been prompted by the increasing number of IFIs, the expanding use of new and established antifungal agents, and recognition of antifungal resistance as an important clinical problem.5, 6, 7 Currently, there are 2 independent standards for broth microdilution (BMD) susceptibility testing of Candida and filamentous fungi: the Clinical and Laboratory Standards Institute (CLSI) methods8, 9, 10, 11 and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods.6, 12, 13, 14, 15, 16 These methods are similar in that both use BMD, although there are some differences in inoculum size and MIC endpoint determination. The 2 methods have been harmonized so that there is close agreement between MIC results obtained when testing azoles and echinocandins against Candida17, 18 and azoles against Aspergillus species.19 Furthermore, both methods have been shown to provide clinically useful results and to reliably discriminate between susceptible “wild-type” strains (no acquired resistance mechanisms) and resistant strains that exhibit intrinsic or acquired resistance mechanisms.13, 14, 20, 21, 22

The CLSI has also developed agar-based, disk diffusion testing for yeasts8, 23 and molds.24, 25 Compared with the rather cumbersome BMD method, disk diffusion testing is convenient, simple, and economical, and is particularly well suited for water-soluble antifungals such as 5FC, fluconazole, and voriconazole.5, 6 Disk diffusion testing has been standardized for fluconazole and voriconazole versus Candida species, and breakpoints have been provided for each of these agents.20, 21, 23 Disk diffusion testing can also be used to determine the in vitro susceptibility of yeasts to echinocandins, and interpretive breakpoints have been developed for CSF and micafungin (MCF) versus Candida species.22 The CLSI has also published a reference method for disk diffusion antifungal susceptibility testing of molds (nondermatophyte filamentous fungi).24, 25

Commercial development of MIC methods for antifungal susceptibility testing of yeasts that conform to CLSI standards has been gradual. Three products have been cleared by the US Food and Drug Administration (FDA) for testing antifungal drugs against Candida as part of patient care: the Sensititre YeastOne colorimetric plate (TREK Diagnostic Systems, Inc., Cleveland, OH), the Vitek 2 yeast susceptibility test (bioMerieux, Inc., Marcy l' Etoile, France) and the Etest (bioMerieux). These products typically represent modifications of agar or BMD methods and have potential advantages in terms of ease of use, flexibility, standardization, and rapidity of results.

In each case, the commercial products have been carefully validated by comparison with CLSI BMD results and employ the interpretive breakpoints established by CLSI.26, 27, 28, 29, 30, 31 In general, these products allow for the testing of amphotericin B, 5FC, fluconazole, itraconazole, voriconazole, and the echinocandins.

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Development of Breakpoints 

Both CLSI and EUCAST have established clinical breakpoints for fluconazole and voriconazole versus Candida by taking into account the MIC distributions, pharmacokinetic (PK) and pharmacodynamic (PD) parameters, resistance mechanisms, and clinical outcomes as they relate to MIC values.10, 13, 14, 32 Initially with fluconazole, the CLSI did not allow for species-specific clinical breakpoints and assigned values of ≤8 μg/mL as susceptible (S), 16-32 μg/mL as susceptible dose-dependent (SDD), and ≥64 μg/mL as resistant (R) for all species irrespective of their wild-type MIC distribution.32 As can be seen from the MIC distribution shown in Figure 1, there were problems with these breakpoints in that a breakpoint for S of ≤8 μg/mL is too high to provide a sensitive means of predicting the emergence of resistance among the more susceptible species such as Candida albicans, Candida tropicalis, and Candida parapsilosis and, at the same time, bisects the wild-type distribution of C glabrata. The latter issue is of concern in that, by splitting the wild-type MIC distribution, one runs the risk of artificially producing S, SDD, and R categorizations of isolates, all with identical susceptibilities.33 This clearly impairs the reproducibility of the test categorization and may also lead to very major (false-susceptible) and major (false-resistant) reporting errors.

Data compiled from Pfaller MA et al, 2010.20

In an effort to address the shortcoming of these earlier clinical breakpoints for fluconazole, the CLSI utilized the global antifungal surveillance MIC database as shown in Figure 1 to ascertain the wild-type MIC distribution for fluconazole and each of the 7 most common species of Candida causing bloodstream infections using the CLSI BMD method. This information allowed the CLSI to establish epidemiologic cutoff values (ECVs) that more effectively assess the emergence of strains with decreased susceptibility to this important antifungal agent and served as a necessary step in establishing species-specific CBPs. In this construct, the ECVs represent the upper limit of the wild-type MIC distribution and establish a cutoff to detect the emergence of reduced susceptibility (acquired resistance). It should be noted that ECVs are not always the same as CBPs. Whereas CBPs are used to indicate those isolates that are likely to respond to treatment with a given antimicrobial agent administered at the approved dosing regimen for that agent, the ECV may serve as the most sensitive measure for the emergence of strains with reduced susceptibility (acquired resistance mechanisms) to that agent.4

These data were used along with molecular, PD, and clinical data to revise the CBPs for fluconazole to provide species-specific interpretive criteria for C albicans, C tropicalis, and C parapsilosis that are identical to that of EUCAST: S ≤2 μg/mL, SDD 4 μg/mL, and R ≥8 μg/mL. As can be seen in Table 1, the CBPs for S are the same as or very close to the ECVs for these species. In contrast to EUCAST, the CLSI elected to assign CBPs for fluconazole and C glabrata of ≤32 μg/mL for SDD and ≥64 μg/mL for R, with the caveat that maximum doses of fluconazole (12 mg/kg/day) be used when treating C glabrata infections with fluconazole.

Table 1. Epidemiologic Cutoff Values (ECVs) and Clinical Breakpoints (CBPs) for Fluconazole and 5 Species of Candida
Adapted from Pfaller MA et al, 2010.20
SpeciesECV (μg/mL)CBP (μg/mL)
C albicans0.52
C tropicalis22
C parapsilosis22
C lusitaniae22
C glabrata3232

The ability of the revised CBPs to predict the outcome of fluconazole treatment of infections (candidemia and mucosal candidiasis) due to C albicans, C tropicalis, and C parapsilosis is shown in Table 2: 92% success rate for 550 events for which the 24-hour fluconazole MIC is ≤2 μg/mL (S), 83% success among 52 events for which the MIC is 4 μg/mL (SDD), and 37% success among 212 events for which the MIC is ≥8 μg/mL.20, 34, 35, 36 When the MIC results are read after 48 hours of incubation, one finds similar degrees of success and failure to that seen at 24 hours at the CBPs of ≤2 μg/mL and ≥8 μg/mL, respectively. These CBPs are supported by PD and data mining analysis and should prove to be more sensitive and specific in detecting emerging resistance and predicting the outcome of therapy.20 This same process was used to establish new CBPs for voriconazole and Candida species.21 For all species, with the exception of C glabrata and Candida krusei, the CBPs for voriconazole are ≤0.125 μg/mL (S), 0.25 to 0.5(Intermediate [I]), and ≥1 μg/mL (R) (Table 3).21 These categories function reasonably well in predicting therapeutic outcome when results are read at either 24 or 48 hours of incubation (Table 3). The CBP for C krusei has been set at ≤0.5 μg/mL for S and ≥2 μg/mL for R based on the wild-type MIC distribution and limited clinical data, indicating that strains with MICs as high as 0.5 μg/mL respond to voriconazole. The poor clinical response of C glabrata to voriconazole therapy and the complete lack of any relation between voriconazole MIC and outcome precludes the establishment of a CBP for voriconazole and this species. In this case the CLSI has elected to use the ECV of ≤0.5 μg/mL to differentiate wild-type from non–wild-type strains of C glabrata for purposes of resistance surveillance.21

Table 2. Summary Analysis of Clinical Fluconazole Minimum Inhibitory Concentration (MIC) Ranges in Patients with Candidemia and Mucosal Candidiasis
Adapted from Pfaller MA et al, 2010.20
Outcome at MIC
24-Houra48-Hourb
MIC (μg/mL)No. of Events% SuccessNo. of Events% Success
≤25509236692
452833090
≥82123715567

a Data compiled from Clancy CJ et al, 200534, Rex JH et al, 1997,35 Rodriguez-Tudela JL et al, 2007.36

b Data compiled from Clancy CJ et al, 2005,34 Rex JH et al, 1997.35

Table 3. Summary Analysis of Clinical Voriconazole Minimum Inhibitory Concentration (MIC) Ranges in Patients with Candidemia and Invasive Candidiasis
Adapted from Pfaller MA et al, 2011.21
Outcome at MIC
24-hr48-hr
MIC (μg/mL)No. of Events% SuccessNo. of Events% Success
≤0.12517375.715978.6
0.25–0.59100.01560.0
≥1837.51656.3

The CLSI initially proposed CBPs for Candida species tested against the echinocandins (S, ≤2 μg/mL; non-susceptible [NS], >2 μg/mL) based on an analysis of MIC distributions and the clinical relationship between MIC and efficacy, indicating that standard dosing regimens of anidulafungin, CSF and MCF may be used to treat infections due to Candida species for which MICs were as high as 2 μg/mL.37 Although PK and PD data and resistance mutations were taken into account in establishing these CBPs, the data for each of these important considerations was not nearly as robust as it is now. Furthermore, the CLSI Antifungal Subcommittee lumped all species together, assigning a single breakpoint for all species despite clear evidence that echinocandin MICs were significantly lower for some species than others. Since that time, significant progress has been made in relating MICs with resistance mutations, with case reports and case series clearly showing that clinical resistance is related to FKS1/FKS2 mutations (encodes the target enzyme) in strains for which the MICs are greater than those of WT strains but not necessarily greater than the initially proposed CBP of 2 μg/mL.22, 38, 39, 40 These findings, coupled with an improved understanding of the PD indices associated with efficacy for the echinocandins, provide the rationale to develop species-specific CBPs for both S and R.22

In revising the CBPs for the echinocandins and Candida, the CLSI Subcommittee took into account the most recent and comprehensive molecular, biochemical, microbiologic, PD and clinical data. In contrast to the previously proposed CBPs, the Subcommittee evaluated both the microbiologic and clinical data according to species in an effort to develop criteria that not only predict clinical outcome but also improve the sensitivity of the CLSI BMD method to detect emerging resistance associated with FKS mutations.22 Given these considerations, CBPs for susceptibility (S) to anidulafungin (ANF), CSF and MCF have been proposed: ≤0.25 μg/mL for C albicans, C tropicalis, and C krusei and ≤2 μg/mL for C parapsilosis and C guilliermondii; for C glabrata, the proposed CBP for S is ≤0.06 μg/mL for MCF and ≤0.12 μg/mL for ANF and CSF. Strains of C albicans, C tropicalis, and C krusei for which the MIC of each echinocandin is ≥1 μg/mL (≥0.25 μg/mL [MCF] or ≥0.5 μg/mL [ANF, CSF] for C glabrata) are almost all clinically R and possess an acquired FKS mutation. Strains of C parapsilosis and C guilliermondii for which the MICs for each echinocandin are ≥8 μg/mL are considered to be R. Those isolates for which the echinocandin MICs fall between S and R are considered to be intermediate (I). The use of an I category provides a buffer zone for antimicrobial susceptibility testing, which is necessary in order to avoid major or very major errors that may occur given the inherent variability of the BMD method. In addition, such a category may also be used to designate strains with elevated MICs that may respond clinically to a higher-than-standard dose of the drug and in situations where drug penetration is maximized. The ability of the new CBPs to differentiate WT strains of C glabrata from those with FKS mutations is shown in Table 4. In this example a total of 169 BSI isolates of C glabrata were tested for the presence of FKS1/FKS2 mutations and then submitted to BMD MIC testing using the CLSI method for each echinocandin. Among the 30 FKS mutants in this collection, 27(90%) were classified as R to ANF and 26(87%) were classified as R to MCF and CSF.22 These species-specific CBPs should prove to be much more sensitive and specific in detecting emerging resistance to these important antifungal agents.

Table 4. Potential Clinical Breakpoints for Echinocandins Versus Candida Glabrata using the Clinical and Laboratory Standards Institute Broth Microdilution Method
Data compiled from Pfaller MA et al, 2011.22
Species (N)Antifungal agentBreakpoint (S/R)Category
SIR
C glabrata (169)Anidulafungin≤0.12/≥0.5135(2)⁎4(1)30(27)
Caspofungin≤0.12/≥0.5129(1)5(3)35(26)
Micafungin≤0.06/≥0.25134(1)6(3)29(26)

I = intermediate; R = resistant; S = susceptible.

⁎ Number of strains with fks mutations shown in parentheses.

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Mechanisms of Resistance 

Each of the antifungal classes utilizes a different means to kill or inhibit the growth of fungal pathogens.41, 42, 43 Mechanisms of antifungal resistance are either primary or secondary, and are related to intrinsic or acquired characteristics of the fungal pathogen that interfere with the antifungal mechanism of the respective drug/drug class or that lower target drug levels. Resistance can also occur when environmental factors lead to colonization or replacement of a susceptible species with a resistant one. The antifungal effects of both polyene and azole antifungals are due to actions on the fungal cell membrane, while echinocandins act by disrupting the fungal cell wall.42, 43 5FC acts as an antimetabolite to interfere with DNA and RNA synthesis. As discussed in greater detail below, a variety of mechanisms of resistance have been described for azoles and yeasts as well as filamentous fungi.42 Echinocandin resistance in yeasts is mediated via point mutations resulting in target modification, and a similar mechanism appears to be operative for emerging echinocandin resistance in molds.42

Proposed mechanisms of polyene resistance in molds include decreased access to the drug target due to altered membrane ergosterol content, accumulation of other sterols and reduced intercalation, and increased catalase activity, leading to a reduction in oxidative damage.42 Currently, yeast resistance to polyenes is very low. Secondary resistance of yeasts to 5FC is mediated by enzymatic modifications that either interfere with drug uptake into the cell or the conversion of 5FC to 5-fluorouracil or 5-fluorouracil to 5-fluorouridine monophosphate.42

Azole Resistance 

Azoles inhibit fungal growth by interfering with biosynthesis of ergosterol in the fungal cell membrane. 41, 43 More specifically, azole drugs bind and inhibit the enzyme lanosterol 14-α-sterol-demethylase in yeasts and 14-α-sterol-demethylase in molds, which are involved in the conversion of lanosterol into ergosterol. As a result, ergosterol content in the cell membrane is depleted, membrane structure and functions are altered, and fungal growth is inhibited. In terms of azole resistance, four principal mechanisms have been described in Candida species.41 More than one mechanism may be operative in a given resistant strain, and the changes leading to resistance can occur in sequence and may have additive effects or lead to cross-resistance.

The various mechanisms associated with azole resistance in Candida species have been reviewed elsewhere.41, 42 The first mechanism associated with decreased susceptibility or resistance of Candida to azole antifungals is induction of efflux pumps that lead to decreased drug concentration at the enzyme target within the fungal cell. Upregulation of efflux pumps encoded by either MDR or CDR genes have been associated with azole resistance in C albicans (MDR1, CDR1, CDR2), C glabrata (CgCDR1, CgCDR2), or Candida dubliniensis (CdMDR1, CdCDR1). Induction of CDR gene-encoded efflux pumps tends to affect all azole drugs and is often sufficient for resistance in and of itself in certain species. Conversely, efflux pumps encoded by MDR genes are usually selective for fluconazole. Another common mechanism of resistance in Candida species is the acquisition of point mutations in the gene encoding for the target enzyme (ERG11), resulting in an altered target with reduced affinity for or incapacity to bind azoles. This mechanism may be compounded if there is overexpression or upregulation of the altered target enzyme, a third possible mechanism of azole resistance in Candida. This can lead to a situation in which there are targets that do not bind well with azole drugs, but there are also more of them. However, minimal upregulation of altered target enzymes has been observed to date, and this mechanism does not appear to be a major cause of azole resistance in Candida at this time. The last potential mechanism of azole resistance in Candida species involves the development of bypass pathways, which negate the membrane-disruptive effects of azole drugs that are associated with inhibited fungal growth. This has been linked with mutation of the ERG3 gene in certain resistant strains of Candida.

The impact of the first two resistance mechanisms on in vitro susceptibility of C albicans to different azoles was elucidated in a recent study by MacCallum and coworkers.44 Using a single strain of C albicans, MacCallum et al. constructed several mutants expressing different amounts of CDR efflux pumps and/or mutations in the ERG11 gene encoding the target enzyme for azoles. Table 5 presents the key findings from this study with respect to susceptibility or resistance to fluconazole or voriconazole. The top row is the “control,” as it represents susceptibility in strains with basal expression of CDR and WT ERG11 genes in both alleles. As expected, low MICs were observed for both fluconazole and voriconazole. By comparison, MICs for both azoles were very much higher in the strain with overexpression of CDR and point mutations in both ERG11 alleles. Based on MIC cutoffs discussed earlier, these strains are clearly resistant to both fluconazole and voriconazole. Intermediate MICs are apparent in strains with either basal CDR expression in concert with a mutation in one or both ERG11 alleles, or an increase in CDR expression but WT ERG11 in both alleles. Of interest, the MICs for fluconazole and voriconazole were approximately twice as high in the strain with basal CDR expression and point mutations in both ERG 11 alleles as in the strain with basal CDR expression and a point mutation in only one of the ERG 11 alleles. These data highlight the additive nature of resistance mechanisms in Candida species for azoles.

Table 5. Impact of Resistance Mechanisms on in vitro Susceptibility of Candida albicans to Fluconazole and Voriconazole
Adapted from MacCallum DM et al, 2010.44
Resistance MechanismsMIC (μg/mL)
StrainCDRERG11VRCFLU
DSY294BasalWT/WT0.0080.25
DSY296IncreaseG464S/G464S264
DSY3083BasalG464S/G464S0.134
DSY3604BasalG464S/WT0.062
DSY3606IncreaseWT/WT0.134

FLU = fluconazole; MIC = minimum inhibitory concentration; VRC = voriconazole; WT = wild-type.

Echinocandin Resistance 

Echinocandins inhibit 1,3-β-D-glucan synthase and thereby disrupt biosynthesis of 1,3-β-D-glucan, a key component of the fungal cell wall. This causes the formation of a defective cell wall associated with cellular instability and lysis in yeasts and aberrant hyphal growth in molds. Mutations in the gene encoding for elements of the 1,3-β-D-glucan synthase complex have been associated with Candida resistance to echinocandins. In particular, reduced susceptibility or resistance of Candida to echinocandins has been linked with point mutations in two “hot-spot” regions (HS1 and HS2) of FKS1, the gene encoding for the major and presumed catalytic subunit of 1,3-β-D-glucan synthase.39 This resistance mechanism has been demonstrated in C albicans and non-albicans Candida species (C glabrata, C krusei, C tropicalis, and C dubliniensis). In C glabrata, echinocandin resistance has also been associated with mutations in the FKS2 gene.45 Mutations in FKS alter the glucan synthase enzyme kinetics resulting in significantly higher 50% inhibitory concentrations (IC50), as well as the kinetic inhibition constant (Ki), for the mutant enzymes when compared with corresponding enzymes from WT strains.38 This pattern of decreased enzyme sensitivity to inhibition (increased IC50) extends across all three of the echinocandins, conferring resistance across the entire class.

The case study outlined earlier (MDR in C glabrata) demonstrated point mutations in FKS2, associated with CSF resistance, as well as overexpression of CgCDR1 and CgCDR2, linked with fluconazole and voriconazole resistance.

Azole and Echinocandin Resistance in Molds 

A detailed account of the epidemiology and the mechanisms associated with azole and echinocandin resistance in Aspergillus species is beyond the scope of this review. These topics have been reviewed more extensively elsewhere.41, 42, 46 Briefly, with the exception of fluconazole, acquired resistance of Aspergillus species to azoles or echinocandins is relatively uncommon. Azole resistance has been primarily linked with mutations in the Cyp51A gene, leading to alterations in the 14-α-sterol-demethylase target enzyme. Other possible, but not well established, mechanisms of azole resistance in Aspergillus include upregulation of efflux pumps and mutations in the promoter region of Cyp51A leading to overexpression of the protein product. Echinocandin resistance is even more rare, and has been associated with point mutations in hot spots of the FKS1 gene encoding the major subunit of 1,3-β-D-glucan synthase—similar to the mechanism associated with azole resistance.

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Consequences of Antifungal Resistance 

Antifungal resistance has consequences in terms of elevated MICs that are associated with poorer outcomes and breakthrough infections during antifungal treatment and prophylaxis. Antifungal resistance and its negative consequences can often be traced to acquisition of particular resistance mechanisms. The most obvious consequence of antifungal resistance may be seen in the results shown in Tables 2 and 3, where the clinical outcome was significantly poorer for those patients infected with isolates of Candida for which the MICs for fluconazole and voriconazole, respectively, were R compared with those for which the MICs were classified as S. Similarly, Baddley et al reported a lower mortality rate among candidemia patients for which the fluconazole MIC of the infecting isolate was ≤2 μg/mL (S) than among those for which the MIC was ≥8 μg/mL (R).47 Taken together, these data indicate that isolates with high (R) azole MICs obtained from patients with Candida infections are associated with lower success rates and higher mortality than those with low or susceptible MICs, illustrating the negative impact of antifungal resistance on clinical outcomes.

Antifungal resistance can also lead to breakthrough invasive fungal infections in high-risk patients receiving antifungal prophylaxis. For example, Alexander et al. described eight cases of breakthrough fungemia among 295 adult bone marrow transplant (BMT) recipients receiving fluconazole prophylaxis between October 2002 and June 2004 at Duke University Medical Center. Among the eight cases of breakthrough fungemia, seven were due to C glabrata, and four of the seven exhibited cross-resistance to all azoles (fluconazole, itraconazole, voriconazole and posaconazole).48 Although the resistance mechanism responsible for the pan-azole resistance was not elucidated, it was likely due to elevated CDR gene-encoded efflux pump activity, as this is prevalent in C glabrata and has been associated with cross-resistance among azole antifungals.

Another Duke University Medical Center study examined cases of breakthrough candidiasis among BMT or solid-organ transplant recipients receiving micafungin prophylaxis.49 Between February 2006 and May 2008, 649 high-risk patients received at least three doses of micafungin prophylaxis, and 12(1.8%) of these subsequently developed invasive candidiasis, 10 with candidemia. Four of the 10 cases of candidemia were due to C glabrata, and three of the four were cross-resistant to all three echinocandins. FKS gene mutations were detected in all four of the echinocandin–resistant isolates of C glabrata, establishing the mutational event as important for both an increase in MIC and clinical failure, i.e., breakthrough infection.

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Epidemiology of Antifungal Resistance 

Our understanding of the epidemiology of invasive fungal infections and associated resistance profiles has been enhanced considerably by data collected and published by various sentinel and population-based surveillance programs.1, 40, 50, 51, 52 Ever since the introduction of fluconazole in 1990 for the treatment of candidiasis, empirical antifungal therapy has been driven by fear of C glabrata.53, 54 Decreased susceptibility of C glabrata to fluconazole, and cross-resistance to other azoles, is well known.1, 51, 52, 55 In the United States, we have used the ARTEMIS Antifungal Surveillance Program to demonstrate that C glabrata has increased as a cause of invasive candidiasis from 18% of all BSI isolates in the time period of 1992-2001 to 25% in 2001-2007 with a concomitant increase in fluconazole resistance from 9% to 14%.51, 52 As noted previously in the cited case report of Chapeland-Leclerc et al,3 infecting strains of C glabrata may acquire resistance to flucytosine, fluconazole, voriconazole, and CSF through successive independent events following prolonged exposure to each class of antifungal agent. The recovery of different isolates exhibiting clonality for microsatellite markers but genetic diversity for antifungal resistance markers (three unique resistance mechanisms) demonstrates the high propensity of C glabrata to readily mutate in vivo in a single patient.3 Additional reports from medical centers in the U.S. and Denmark provide further documentation of multidrug resistance (MDR; resistant to 2 or more classes of antifungal agents) strains of C glabrata.56, 57, 58

It is worth noting that the data from the ARTEMIS study51, 52 are from a nationwide surveillance program. As such, they do not encompass every hospital in the United States and the findings may or may not be representative of epidemiology at a given United States hospital. It is important for each individual institution to know their own epidemiology, for Candida BSIs as well as infections involving Aspergillus species. This type of epidemiologic information is critical when choosing empiric therapy or deciding whether to use prophylaxis in high-risk patients, and if so, with what agent.

Another recent international study based on data from the SENTRY Antimicrobial Surveillance Program reported a total of 1239 Candida BSI isolates from 79 medical centers in 2008-2009.59 This study examined the frequency of infection by various species and associated resistance to azole and echinocandin antifungal agents across the different patient age groups. Resistance to both azoles and echinocandins was most prominent among isolates of C glabrata with the highest resistance rates to echinocandins (16.7%), fluconazole (16.7%), posaconazole (5.0%) and voriconazole (11.0%) among isolates from the 20-39-year age group. The emergence of MDR in C glabrata is a real concern given the fact that neither azoles nor amphotericin B are an optimal approach for therapy for C glabrata infection.3, 7, 40, 48 Future surveillance efforts should focus on emergence of these potentially MDR strains of C glabrata. Each institution should take into account their use of these different antifungal agents, the frequency with which they see C glabrata causing BSIs, and their drug-resistant phenotype.

In contrast to the increase in azole resistance seen with C glabrata, the picture is much more favorable for strains of Cryptococcus neoformans isolated from patients in developed countries where both access to antifungal therapy and antiretroviral therapy (ART) have modified the course of the infection among AIDS patients with cryptococcal meningitis. Although CBPs are not available for fluconazole and C neoformans, we have recently established an ECV of 8 μg/mL to allow for the separation of WT from non-WT strains.60 Clinical support for this ECV is provided by a study by Aller et al that demonstrated that patients infected with C neoformans, for which the fluconazole MIC values were ≤8 μg/mL, responded better to fluconazole treatment than those infected with strains for which MICs were ≥16 μg/mL.61 We have applied this ECV to MIC results obtained over a 19-year period to examine trends in fluconazole susceptibility over time from four geographic regions (Asia-Pacific, Latin America, Europe and North America).60, 62, 63 Notably, the proportion of MIC results that exceeded the ECV (non-WT strains) progressively decreased from 28% in 1990-1994 to only 1% in 2005-2008(Table 6).60, 62, 63 This trend towards improved susceptibility of C neoformans has been noted previously62, 64 and may reflect, in part, a decrease in overall drug pressure concomitant with a decrease in cryptococcosis among HIV-infected patients receiving ART.62, 65, 66

Table 6. Changes in Fluconazole Susceptibility of Clinical Isolates of Cryptococcus neoformans over 19 Years, 1990–2008
Data compiled from Pfaller MA et al, 2011,60 Brandt ME et al, 2001,62 and Pfaller MA et al,63
MIC (μg/mL)% at MIC (μg/mL)
Time PeriodNo. TestedRange50%90%≤8≥16
1990–19948820.25to>648167228
1995-19994070.12to644168911
2000-20045220.25to>6448964
2005-20082170.23to1648991

MIC = minimum inhibitory concentration.

Much of the concern regarding fluconazole resistance among C neoformans isolates is due to reports from Africa,67, 68 indicating that recent isolates from that geographic area exhibit decreased susceptibility to fluconazole and other azoles. In one report,67 75% of isolates from patients with a clinical relapse following treatment of cryptococcal meningitis with fluconazole exhibited decreased susceptibility (MIC ≥32 μg/mL) to this antifungal. It has been suggested that, in these clinical settings, continued host exposure to fluconazole as a single fungistatic agent coupled with multiple recurrences of meningitis and a high organism burden provides the “perfect” environment for the emergence of high-level azole resistance.67, 68

With respect to azole susceptibility of molds, epidemiologic data are beginning to accrue concerning the development and spread of azole-resistant Aspergillus fumigatus, and the mechanisms underlying this resistance. The epidemiology of azole-resistant A fumigatus is discussed in the case study and subsequent section below.

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Case Study: Azole-Resistant Central Nervous System (CNS) Aspergillosis 

The following is based on a case recently reported in the Netherlands by van der Linden and coworkers69:

An 11-year-old girl with B cell lymphoma presented with cough and fever 7 months after beginning chemotherapy. She was admitted to the hospital and treated with antibiotics (vancomycin and ceftazidime). Her fever persisted, and a repeat chest x-ray showed a lobar infiltrate. Four days postadministration, the patient experienced a seizure that persisted for 2 hours despite antiepilepsy treatment. The patient became respiratory insufficient and was transferred to the intensive care unit for further management. High-resolution computed tomography (CT) revealed lesions in both the brain and lung: 8 hypodense brain lesions and multiple nodules, and diffuse consolidation of the lower lobes of both lungs. Cultures of biopsied lung tissue grew A fumigatusranged from 88% to 99%., and galactomannan was detected in serum (galactomannan index 5.9).

Voriconazole therapy was initiated, but the fever persisted and CSF was added to the treatment regimen. The A fumigatus isolate from the lung grew on agar containing 4 μg/mL itraconazole—suggesting an azole-resistant strain—and voriconazole was replaced by liposomal amphotericin B. Repeat CT demonstrated progression of brain and lung lesions. Liposomal amphotericin B plus CSF therapy was stopped, and the patient was discharged home and later died. In vitro susceptibility tests revealed MICs of 0.25 μg/mL for amphotericin B, 0.5 μg/mL for CSF, and >16, 16, and 2 μg/mL for itraconazole, voriconazole, and posaconazole, respectively. It is notable that the MICs for each of the azoles is in the resistant range, and a TR/L98H mutation in Cyp51A was identified as the likely mechanism of azole resistance.

Aspergillosis of the CNS is the most serious form of invasive aspergillosis. Between 1972 to 2000, when primary therapy for CNS aspergillosis consisted of amphotericin B with or without surgical intervention, the case fatality rate ranged from 88% to 99%.69, 70, 71, 72 More recently, voriconazole has been recommended as primary therapy for all forms of invasive aspergillosis, including those involving the CNS or lungs, as in the case above.72 Clinical response rates as high as 35% have been observed with voriconazole treatment of CNS aspergillosis.71 The management of invasive aspergillosis is complicated by azole resistance. This is because azole resistance typically leads to a delay in initiation of adequate therapy, together with the general lack of effective alternative antifungal agents. This was illustrated in the case described above.

In vitro “reduced susceptibility” of Aspergillus species to azoles remains fairly low (0-8%) in most large surveys, often using itraconazole as the testing agent, and usually A fumigatus species complex as the pathogen.73, 74 Case reports and case series suggest that multi-azole resistant A fumigatus could emerge,75, 76, 77, 78, 79 and that there is some association with agricultural azole use.78, 79

Azole-resistant A fumigatus have been reported in China, Canada, the United States, and several European countries, with particularly high levels in the Netherlands and the United Kingdom.80 A recent report of azole resistance in A fumigatus isolates submitted to the Mycology Reference Centre Manchester, United Kingdom, noted a progressive increase in patients with azole-resistant strains from 0% in 1997 and 1998, 5% in 2004 and 2005, 17% in 2007, 14% in 2008, and 20% in 2009.80 During the 2008-2009 period, 78% of resistant isolates were multi-azole resistant. Of particular note, 43% of azole-resistant isolates in 2009 did not carry a Cyp51A mutation, historically the most common mechanism conferring resistance to azoles in A fumigatus. These data suggest that azole-resistant strains of A fumigatus are not only becoming more common, but that resistance mechanisms are evolving. Another study investigating the prevalence of itraconazole resistance in clinical A fumigatus isolates collected at a major medical center in the Netherlands also noted an increase azole resistance over time, from 0% of patients with itraconazole-resistant strains in 1994-1999, to 6% in 2007.74 In this study, a leucine for histidine substitution in Cyp51A, together with a tandem repeat of a 34-base pair sequence in the gene promoter (TR/L98H), was identified as the prominent mechanism.

With respect to the case patient, the TR/L98H mutation in Cyp51A has previously been associated with multi-azole resistant strains of A fumigatus, possibly linked to environmental (fungicide) use, and has been observed in patients with limited or no prior azole exposure.79 Until recently, A fumigatus strains containing the TR/L98H mutation have only been observed in isolates originating in various European countries, although a 2011 report from ARTEMIS Global Surveillance study noted a number of A fumigatus isolates originating in China that carried the TR/L98H mutation.81 The case patient was from the Netherlands and treated in a Dutch hospital.

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Summary 

In vitro antifungal susceptibility testing is now standardized internationally. The establishment of ECVs and new clinical breakpoints for azoles and echinocandins promises to provide a more sensitive means of detecting emerging resistance and to improve clinical utility of in vitro testing. Recent data regarding resistance mutations encompass both Candida and Aspergillus and document spread of antifungal resistance within hospitals and possibly the larger environment. Multidrug resistance in both Candida and Aspergillus is an emerging concern. Antifungal susceptibility testing is now rapidly becoming essential in patient management and resistance surveillance.

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Author Disclosures 

The author of this article has disclosed the following industry relationships:

Michael A. Pfaller, MD, has received consulting fees and grants for contracted research from Astellas, Merck & Co., Inc., Pfizer.

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Acknowledgment 

Editorial support for this publication was provided by Global Education Exchange, Inc., Freehold, New Jersey.

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 Statement of author disclosure: Please see the Author Disclosures section at the end of this article.

 This supplement is in part based on a closed roundtable meeting that was held June 7, 2011 in New York City and was jointly sponsored by Postgraduate Institute for Medicine and Global Education Exchange. through an educational grant from Merck & Co., Inc. The webinar was peer-reviewed and accepted as a free multimedia activity of The American Journal of Medicine and is available at www.antifungaltherapy2.net.

PII: S0002-9343(11)00913-2

doi:10.1016/j.amjmed.2011.11.001

The American Journal of Medicine
Volume 125, Issue 1, Supplement , Pages S3-S13, January 2012

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