Antifungal susceptibility testing

INTRODUCTION — The need for reproducible, clinically relevant antifungal susceptibility testing has been prompted by the increasing number of invasive fungal infections, the expanding use of new and established antifungal agents, and recognition of antifungal resistance as an important clinical problem [1-4].

The collaborative efforts of numerous investigators and the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical Laboratory Standards [NCCLS]) Subcommittee on Antifungal Susceptibility Testing have generated consensus documents describing standardized methods for broth- and agar-based antifungal susceptibility testing [5-9]. As a result, in vitro antifungal susceptibility testing plays an increasingly important role in guiding therapeutic decision making, as an aid in drug development studies, and as a means of tracking the development of antifungal resistance in epidemiologic studies [3,10,11].

An overview of antifungal susceptibility testing will be presented here. The pharmacology and use of antifungal agents for the treatment of specific fungal infections are discussed separately. (See "Pharmacology of azoles" and "Pharmacology of amphotericin B" and "Pharmacology of flucytosine (5-FC)" and "Management of candidemia and invasive candidiasis in adults" and "Treatment and prevention of invasive aspergillosis" and "Mucormycosis (zygomycosis)" and "Mycology, pathogenesis, and epidemiology of Fusarium infection".)

An overview of antibacterial susceptibility testing is also presented elsewhere. (See "Overview of antibacterial susceptibility testing".)

OVERVIEW

Rationale — The primary objective of in vitro susceptibility testing is to predict the impact of administration of the tested agent on the outcome of infection caused by the tested organism or similar organisms [3,12]. In this way, antifungal testing is performed for the same reasons that antibacterial testing is performed [3]:

●To provide a reliable estimate of the relative activities of two or more antimicrobial agents against the pathogen of interest

●To correlate with in vivo activity and to predict the likely outcome of therapy

●To provide a quantitative means by which to survey the development of resistance among a normally susceptible population of organisms

●To predict the therapeutic potential and spectrum of activity of newly developed investigational agents

In the clinical microbiology laboratory, the focus of testing is directed toward a specific clinical isolate causing infection in an individual patient. Studies examining the clinical use of antifungal susceptibility testing have shown that when such testing is available onsite, clinicians find the results helpful and frequently alter therapy based on the results [13-17]. As an example, one study found that susceptibility testing of Candida glabrata isolates results in lower overall treatment costs, based on deescalation in therapy from an expensive echinocandin to fluconazole for patients with documented fluconazole-susceptible C. glabrata bloodstream infection [14]. Routine antifungal susceptibility testing can serve as an adjunct in the treatment of candidemia in the same way that antibacterial testing aids in the treatment of bacterial infections [3,18,19].

Limitations — The prediction of outcome in a clinical infection from results obtained in an artificial and well-defined matrix (in vitro susceptibility test) is an inherently error-prone process in which only modest degrees of correlation can be expected [3,12,20,21]. Decades of experience with antibacterial susceptibility testing confirm the limited extent of in vitro–in vivo correlation that can be achieved [3,22-24]. Just as in vitro resistance does not always predict clinical failure, so in vitro susceptibility does not ensure successful therapy [3,25].

Clinical relevance — In order to be useful clinically, in vitro susceptibility testing of antimicrobial agents should reliably predict the in vivo response to therapy. However, the in vitro susceptibility of an infecting organism to the antimicrobial agent is only one of several factors that may influence the likelihood that therapy will be successful [3,11,25-27]. Factors related to the host immune response and/or the underlying disease, drug pharmacokinetics and pharmacodynamics, drug interactions, and factors related to the virulence of the infecting organism and its interaction with both the host and the antimicrobial agent administered all influence the outcome of treatment of an infection [3,11,25].

Definitions

Minimum inhibitory concentration — The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent that inhibits the growth of fungi, as established by a standardized endpoint.

The MIC₅₀ is the concentration of an antimicrobial agent at which 50 percent of the organisms tested are inhibited. The MIC₉₀ is the concentration of an antimicrobial agent at which 90 percent of the organisms tested are inhibited.

Minimum effective concentration — The minimum effective concentration (MEC) is defined as the lowest concentration of an echinocandin that results in growth of filamentous fungi producing conspicuously aberrant growth. Aberrant growth of hyphae is defined as small, round, compact microcolonies compared with the matt of hyphal growth in the control well that does not contain an antifungal agent.

Clinical breakpoints — Clinical interpretive MIC breakpoints for in vitro susceptibility testing are used to indicate those isolates that are likely to respond to treatment with a given antimicrobial agent administered using the approved dosing regimen for that agent [25,28]. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antifungal Susceptibility Testing establishes species-specific clinical breakpoints for the systemically active antifungal agents [6,9,26,29,30]. The clinical breakpoints serve as criteria that not only predict clinical outcome but also improve the sensitivity of the CLSI methods to detect emerging resistance associated with acquired or mutational resistance mechanisms. The clinical breakpoints sort isolates into categories of susceptible (S), susceptible dose dependent (SDD), intermediate (I), and resistant (R).

Epidemiologic cutoff values — Because of concern that the clinical breakpoints that have been used in the past may not detect mutational resistance in different species, an extensive effort has been undertaken to establish MICs that are to be expected in wild-type strains that have not been exposed to antifungal agents and that have not acquired resistance mutations. For each species and each antifungal agent, a so-called epidemiologic cutoff value that differentiates the wild-type susceptible strain from those with resistance mutations has been established [4,25,28,31-33]. Epidemiologic cutoff values are the most sensitive measure of the emergence of strains with decreased susceptibility to a given agent [29,31-37] and can be used as a means of tracking the emergence of reduced susceptibility to antifungal agents in surveillance studies [29,34,35]. Epidemiologic cutoff values can also be used to identify isolates that are less likely to respond to therapy when clinical breakpoints cannot be established because of the rarity of infection with unusual species of fungi [25,26,29-31,34,35]. Epidemiologic cutoff values have been established by CLSI for systemically active antifungal agents and several species of Candida, Cryptococcus, and Aspergillus [38,39].

INDICATIONS FOR TESTING — There are several situations in which antifungal susceptibility testing should be considered or obtained (table 1):

●All species of Candida isolated from blood or deep sites (eg, normally sterile fluids, tissues, abscesses) should be tested for susceptibility to fluconazole, voriconazole, and an echinocandin

●Mucosal candidiasis that is unresponsive to usual antifungal therapy

●Invasive disease that is unresponsive to the initial antifungal regimen

●Clinical failure in patients with invasive disease caused by species with significant rates of acquired resistance (see 'Cross-resistance' below)

●Invasive disease caused by unusual fungal species for which antifungal susceptibility patterns have not been well established or are unpredictable

For species, such as C. krusei and C. auris that have high rates of intrinsic resistance to fluconazole, antifungal susceptibility testing should be performed for other azoles, amphotericin B, and the echinocandins, but it is not necessary to test fluconazole (table 1).

STANDARDIZED METHODS — Antifungal susceptibility testing methods for yeasts are comparable with those used for bacteria [3,19,27]. The Clinical and Laboratory Standards Institute (CLSI) has developed and published approved methods for both broth dilution testing [5,6] and disk diffusion testing of yeasts [6,8]. These methods are reproducible, accurate, and available for use in clinical laboratories [11,26,30,40].

Standardized methods have also been developed for broth dilution testing [7] and disk diffusion testing of filamentous fungi [9] but require further study to establish the in vivo correlation with in vitro data [41-44].

Broth dilution methods — In the broth macrodilution method, small tubes containing 1 to 2 mL of broth are inoculated with a concentrated antifungal solution and serial twofold dilutions up to eight dilutions. All tubes are then inoculated with a specified amount of the fungal suspension of interest, suspended in an equal volume, and incubated at a given temperature and length of time. The tubes are then examined for turbidity, which represents fungal growth.

The broth microdilution method is a modification of the broth macrodilution test and allows for multiple tests to be performed simultaneously in smaller volumes in a 96-well plate.

Yeasts — Both broth macrodilution and microdilution methods for in vitro susceptibility testing of yeasts have been established by the CLSI [5,6].

The microdilution method employs a 24-hour incubation at 35°C and a minimum inhibitory concentration (MIC) endpoint of ≥50 percent inhibition (100 percent inhibition for amphotericin B) relative to the control well that has no antifungal agent added (picture 1). This standardized method provides reliable and reproducible MIC results with good separation of the MIC distribution between wild-type Candida isolates that are susceptible and isolates possessing acquired-resistance mutations [45-49]. (See 'Minimum inhibitory concentration' above.)

The MIC clinical breakpoints for systemically active antifungal agents have been developed in accordance with a "blueprint" used for all types of antimicrobial testing [26,29,30,50,51]. This blueprint takes into account data that relate the MICs of the specific agent to known resistance mechanisms, the MIC distribution profiles, consideration of cross-resistance patterns, pharmacokinetic (PK) and pharmacodynamic (PD) parameters, and the relationship between in vitro activity (ie, MIC) and clinical outcome according to individual species, as determined by the available clinical efficacy studies [11,26,30]. MIC breakpoints have been developed for six antifungal agents for the following Candida spp: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. guilliermondii (table 2).

In 2010 and 2011, the CLSI Subcommittee on Antifungal Susceptibility Testing established new Candida species-specific clinical breakpoints for fluconazole [29], voriconazole [30], and the echinocandins [26]. This change was made due to the recognition that MICs for the various agents were significantly lower for some species than others and that the previous clinical breakpoints for susceptibility of <8 mcg/mL for fluconazole, <1 mcg/mL for voriconazole and <2 mcg/mL for the echinocandins were not appropriate for all species.

The updated clinical breakpoints are shown in the table (table 2); the general patterns are as follows:

●For fluconazole, the clinical breakpoints for susceptible (S), susceptible dose dependent (SDD), and resistant (R), respectively, are the same values for C. albicans, C. tropicalis, and C. parapsilosis and much higher for C. glabrata, reflecting the need to use higher doses (12 mg/kg per day) of fluconazole if this agent is used in the management of C. glabrata infection [29].

●For voriconazole, the clinical breakpoints are the same values for C. albicans, C. parapsilosis, and C. tropicalis and are slightly higher for C. krusei. Given the low clinical response of cases of invasive candidiasis due to C. glabrata to voriconazole and the lack of any correlation between clinical response and MIC, the CLSI Subcommittee for Antifungal Testing has not set a clinical breakpoint for C. glabrata [30].

●For anidulafungin, caspofungin, and micafungin, the clinical breakpoints are similar for C. albicans, C. tropicalis, and C. krusei and are higher for C. parapsilosis and C. guilliermondii.

●For C. glabrata, the clinical breakpoints vary for each echinocandin.

Caspofungin susceptibility testing in vitro has been associated with significant interlaboratory variability, contributing to reports of false resistance when using the reference method described in CLSI document M27 [52]. When testing caspofungin, susceptible results may be reported as "susceptible"; however, laboratories should confirm "intermediate" or "resistant" results by performing additional susceptibility testing with micafungin [53] or anidulafungin [54], DNA sequence analysis of FKS genes to identify resistance hot spot mutations in FKS1 (all Candida species) and FKS2 (C. glabrata only) [55,56], or sending the isolate to a reference laboratory for confirmation. Candida species resistant to anidulafungin or micafungin or possessing characteristic FKS hot spot mutations are considered resistant to all echinocandins, including caspofungin, and should be reported as such [53,54].

These clinical breakpoints are only applicable to the species listed above due to the lack of outcomes data for the less common species.

Filamentous fungi — Antifungal susceptibility testing may be useful in guiding the selection of antifungal agents for the treatment of invasive disease [57-59]. This is especially true for isavuconazole [60], posaconazole, voriconazole, and the echinocandins, all of which have varying degrees of activity against opportunistic molds [43,57-59,61-63].

Using the broth microdilution method for yeasts as a template [5], the CLSI Subcommittee on Antifungal Susceptibility Testing developed a standardized method for filamentous fungi [7]. This method is applicable for testing most rapidly growing molds, including Aspergillus spp, Fusarium spp, Paecilomyces spp, Scedosporium spp, Trichoderma spp, several dematiaceous (brown-black) molds, and the Mucorales [7,31,42,43].

The MIC endpoint criterion for molds is the lowest drug concentration that shows complete growth inhibition when testing amphotericin B, itraconazole, isavuconazole [60], posaconazole, and voriconazole [7,43]. Several multicenter studies have documented the reproducibility of this method [41-43,64] and have shown promise in predicting antifungal efficacy [65,66].

One area of concern has been the difficulty in standardizing this method for determining the in vitro susceptibility of Aspergillus spp to the echinocandins [45,67]. It is generally agreed that the proper antifungal endpoint for the echinocandins when testing Aspergillus spp and other molds is the minimum effective concentration (MEC) [45]. (See 'Minimum effective concentration' above.)

In a comparative study, the use of the MEC endpoint criteria resulted in excellent agreement among 14 of 17 participating laboratories when a panel of 20 isolates of Aspergillus spp was tested against caspofungin [45]. Thus, for echinocandin testing with Aspergillus spp, the MEC offers an endpoint that can give generally reproducible results. However, the finding of aberrant results in 3 of the 17 laboratories suggests a need for caution and further refinement of the echinocandin test method for Aspergillus spp and other filamentous fungi [45].

Interpretive breakpoints based on the correlation of in vitro data with clinical outcome have been established for voriconazole and Aspergillus fumigatus (Susceptible ≤0.5 mg/L; Intermediate 1 mg/L; Resistant ≥2 mg/L) but have not been established for other mold-drug combinations [27,68-71]. Clinical failure with amphotericin B has been associated with MICs of >1 mcg/mL for Aspergillus fumigatus, A. terreus, Fusarium spp, and Lomentospora (formerly Scedoporium) prolificans [64,66,68]. Clinical failures of itraconazole in the treatment of aspergillosis have been associated with MICs of >8 mcg/mL [63,68]. Studies of resistance mechanisms in A. fumigatus have shown that MIC cut-offs of ≤1 mcg/mL for isavuconazole, itraconazole, and voriconazole and ≤0.25 mcg/mL for posaconazole provided separation of the susceptible population from strains with resistance mutations of the CYP51A gene [63]. This is clinically relevant because certain mutations of the CYP51A gene result in predictable patterns of cross-resistance among the azoles. (See 'Cross-resistance' below.)

Disk diffusion methods

Yeasts — Agar disk diffusion testing has been used for many years as a simple, flexible, and cost-effective alternative to broth dilution testing of antibacterial agents [72,73]. This method has also been incorporated into the CLSI-approved guidelines for antifungal susceptibility testing of yeasts [6,8]. Zone diameter reference ranges have been defined for fluconazole, voriconazole, posaconazole, micafungin, and caspofungin when tested against Candida spp [26,29,30,74-77].

Following 24 hours of incubation at 35ºC, the zone diameters surrounding disks are measured to the nearest millimeter at the point at which there is a prominent reduction in growth (picture 2).

The 24-hour zone diameters correlate well with both 24- and 48-hour reference MICs and have allowed the establishment of interpretive breakpoints for fluconazole, voriconazole, micafungin, and caspofungin [11,26,28,30,77].

Although not standardized for testing of yeasts other than Candida, the same disk test method has also been shown to be a useful approach for determining the susceptibility of Cryptococcus neoformans and other non-candidal yeasts to fluconazole and voriconazole [78-80]. The interpretation of such results remains under investigation [3].

Filamentous fungi — The CLSI Subcommittee on Antifungal Susceptibility Testing has proposed guidelines for disk diffusion testing of filamentous fungi against voriconazole, posaconazole, itraconazole, amphotericin B, and caspofungin, based upon the results of a multicenter collaborative study [9,81]. The clinical relevance of testing the filamentous fungi remains uncertain because interpretive breakpoints with proven clinical value have yet to be identified or approved by the CLSI or any other regulatory agency [9,81].

COMMERCIAL MIC METHODS — Three commercial products have been approved by the United States Food and Drug Administration (FDA) for testing the susceptibility of clinical isolates of Candida to fluconazole and voriconazole: the Etest (bioMerieux), the Sensititre YeastOne colorimetric plate (TREK Diagnostic Systems) and the VITEK 2 yeast susceptibility test (bioMerieux). The Sensititre YeastOne system and the Etest are also FDA-approved for testing susceptibility to itraconazole and flucytosine. The Sensititre and Vitek 2 systems are also approved for testing caspofungin.

Etest — Etest is the most sensitive and reliable method for detecting decreased susceptibility to amphotericin B among isolates of Candida spp and Cryptococcus neoformans [82-84]. Etest can be used for testing filamentous fungi, such as Aspergillus spp and the Mucorales [44,64]; however, it is not FDA approved for this purpose.

The Etest method is based upon the establishment of a stable concentration gradient of an antimicrobial agent following diffusion from a plastic strip into an agar medium. When an Etest strip is placed upon an agar plate that has been inoculated with a test organism and incubated for 24 to 48 hours, an ellipse of growth inhibition occurs, and the intersection of the ellipse with the numeric scale on the strip provides an indication of the minimum inhibitory concentration (MIC) (picture 3).

The Etest method is applicable to antifungal susceptibility testing of both yeasts and molds. Numerous studies demonstrate the usefulness of Etest for determining the in vitro activity of a variety of antifungal agents including amphotericin B, flucytosine, fluconazole, ketoconazole, itraconazole, voriconazole, posaconazole, and caspofungin [44,64,82,84-89]. MICs determined by Etest generally agree well with those determined by the broth dilution reference method; however, this agreement may vary depending upon the antifungal agent tested, the choice of agar medium, and the fungal species [90].

Sensititre YeastOne panel — The Sensititre YeastOne panel is available in a dry-form, 96-well tray with a colorimetric growth indicator. It has excellent accuracy and reproducibility and has been used widely in the United States and elsewhere for testing of Candida and Cryptococcus neoformans [40,91]. Following the addition of a broth inoculum suspension and 24-hour incubation, colorimetric MIC results are read at the first well showing a color change of red (growth) to purple (growth inhibition) or blue (no growth).

In addition to fluconazole, itraconazole, and flucytosine, the YeastOne panel may include amphotericin B, voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin. In a series of multicenter studies, excellent agreement was observed between broth microdilution and YeastOne MIC results for the triazoles (95 percent agreement) and the three echinocandins (100 percent agreement) [92,93].

In addition to providing highly reproducible MIC results that reliably predict the MICs determined by the reference broth microdilution method, the YeastOne system provides results for all classes of antifungal agents (polyenes, flucytosine, triazoles, and echinocandins) within 24 hours.

VITEK 2 yeast susceptibility test — The spectrophotometric approach to antifungal susceptibility testing has been shown to be valid and feasible for use in the clinical laboratory [94,95] and is an integral component of the European Committee on Antibiotic Susceptibility Testing (EUCAST) method [94-96].

The VITEK 2 yeast susceptibility system, which determines growth spectrophotometrically, is the first commercially available automated approach to antifungal susceptibility testing and provides optimal test standardization. It performs fully automated antifungal susceptibility testing of Candida spp against five antifungal agents (fluconazole, voriconazole, caspofungin, micafungin, and flucytosine) [97-100]. The VITEK 2 system has been updated to reflect revised CLSI clinical breakpoints for fluconazole [101]. This system allows clinical laboratories to perform yeast identification and susceptibility testing simultaneously [99].

Notably, the VITEK 2 system has not been shown to be capable of reliable detection of resistance to the echinocandins [102].

Multicenter evaluations have shown agreement of greater than 95 percent in comparisons of the VITEK 2 yeast susceptibility system MICs with reference broth microdilution MICs for the seven antifungal agents used [97,98,100]. The VITEK 2 system is rapid, with a mean time to results of 12 to 15 hours [97,98,100]. The availability of rapid quantitative antifungal susceptibility results will be a major step in optimizing the treatment of invasive candidal infections.

FUNGICIDAL ACTIVITY — In certain clinical situations, the ability of the antimicrobial agent to kill the pathogen may be important [103,104]. These situations involve infection of a site not easily accessed by host defenses and/or of a structure with essential anatomic or physiologic function, such as the heart, central nervous system, or bone. In addition, infections in immunocompromised hosts, especially those who are neutropenic, are often thought to require microbicidal therapy [103,104]. Furthermore, a microbicidal regimen may prevent the development of resistance [105].

These issues have given rise to a perceived need to assess the fungicidal activity of both new and established antifungal agents against opportunistic pathogens [104,106].

By rigorously following a common procedure, various laboratories may reliably perform and interpret both time-kill and minimum fungicidal concentration determinations with Candida and Aspergillus spp [104]. In standardizing in vitro fungicidal tests, considerable effort must be undertaken to ensure that agents classified as fungicidal by in vitro methods do in fact clear involved organs of the infecting organism in animal models of infection. Even if all of these requirements are met, there is no assurance that such in vitro testing will be meaningful clinically.

SUSCEPTIBILITY PATTERNS — Many fungal species have predictable antifungal susceptibility patterns. However, rising rates of resistance to antifungal agents have occurred among some fungal species due to the increase in the immunocompromised population and the frequent use of fluconazole and the echinocandins.

Candida spp

Amphotericin B — Among the various species of Candida, C. albicans remains the most susceptible to amphotericin B. The minimum inhibitory concentration (MIC) that encompasses 90 percent of isolates tested (MIC₉₀) is 0.5 mcg/mL (table 3). Although interpretive breakpoints for amphotericin B have not been established, isolates of Candida for which MICs are >2 mcg/mL are unusual and possibly resistant or, at the very least, may require high doses of amphotericin B for optimal treatment [37]. Both C. glabrata and C. krusei exhibit decreased susceptibility to amphotericin B compared with C. albicans (table 3). C. lusitaniae generally appears susceptible to amphotericin B upon initial isolation from blood, but resistance frequently develops during treatment (table 3) [107]. The emerging pathogen, C. auris, exhibits amphotericin B resistance rates of approximately 30 percent in the United States, based on an MIC breakpoint of ≥2 mcg/mL [108].

Azoles — Fluconazole MICs are usually ≤2 mcg/mL for C. albicans, C. parapsilosis, C. tropicalis, and C. lusitaniae. Reduced susceptibility to fluconazole is common among isolates of C. glabrata and C. guilliermondii (table 3). C. auris exhibits markedly reduced susceptibility to fluconazole with US resistance rates of 90 percent, defined as an MIC ≥32 mcg/ML [108].

C. krusei is considered to be intrinsically resistant to fluconazole; itraconazole, voriconazole, and posaconazole have significantly greater potency than fluconazole against Candida spp, including some species, such as C. krusei and C. guilliermondii, that have reduced susceptibility to fluconazole [107,109,110]. MIC₉₀ values are generally <1 mcg/mL for these agents for most species of Candida, with the exception of C. glabrata and C. auris (table 3) [108,111].

Echinocandins — All three echinocandins exhibit excellent potency against Candida spp (table 3) [112]. C. albicans, C. glabrata, and C. tropicalis are highly susceptible to all three agents, whereas elevated MICs are seen for C. parapsilosis and C. guilliermondii. Acquired resistance to the echinocandins remains sporadic [4,113] but has been documented for individual cases of infection with C. albicans, C. auris, C. glabrata, C. lusitaniae, C. tropicalis, and C. parapsilosis [114-121]. There is increasing concern that some C. glabrata bloodstream isolates with resistance to fluconazole and voriconazole are also resistant to the echinocandins [121,122]. This is a serious concern and argues for continued surveillance for resistance using standardized antifungal susceptibility testing. (See 'Echinocandins' below.)

Species-specific echinocandin clinical breakpoints were developed to improve the sensitivity of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method to identify strains of Candida with acquired resistance mutations in the FKS gene [26,28]. Conversely, strains for which echinocandin MICs fall into the susceptible category are considered wild-type strains that do not possess acquired resistance mutations. Mutations in FKS in isolates of C. albicans, C. glabrata, C. tropicalis, C. krusei, C. lusitaniae, C. dubliniensis, and C. kefyr have been associated with elevated echinocandin MICs and a poor clinical response [26,49,114-120].

In an international surveillance study that evaluated the frequency of echinocandin resistance using the clinical breakpoints, resistance remained quite low (0.0 to 0.7 percent) for isolates from 2006 to 2016 of all of these species, with the exception of C. glabrata [123]. Echinocandin resistance among C. glabrata isolates varied according to geographic origin from 0 percent in Latin America to 2.8 percent in North America [124]. Strains with echinocandin MICs in the resistant category were documented to contain mutations in either FKS1 or FKS2.

A 10-year study of C. glabrata bloodstream infections at a single medical center in the United States showed an increase in echinocandin resistance from 4.9 percent in 2001 to 12.3 percent in 2010 [121]. This resistance was confirmed by the presence of FKS mutations; strains categorized as susceptible did not possess acquired mutations. On multivariate analysis, echinocandin resistance was associated with prior exposure to an echinocandin. Among 118 episodes of C. glabrata infection in which the infecting strain was categorized as susceptible using the clinical breakpoints, 109 (92.4 percent) had successful outcomes at day 10 of treatment with micafungin. Conversely, among 13 episodes of C. glabrata infection in which the strain was categorized as resistant using the clinical breakpoints and treated with micafungin monotherapy, 5 (38.5 percent) did not respond or responded initially but relapsed or recurred. The CLSI clinical breakpoints differentiate wild-type strains from strains bearing clinically significant FKS mutations.

Additional studies of echinocandin resistance among C. glabrata isolates are presented separately.

Flucytosine — Flucytosine is generally quite active against most species of Candida with 95 percent of clinical isolates tested being susceptible with an MIC of ≤4 mcg/mL [125]. Flucytosine MICs are generally very low for C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. guilliermondii, and C. lusitaniae (table 3). In contrast, resistance to flucytosine is common among isolates of C. krusei.

The choice of antifungal regimen for infections caused by each of the Candida species described above is discussed separately. (See "Management of candidemia and invasive candidiasis in adults".)

Other yeasts — In contrast with Candida spp, the non-candidal yeasts comprise a very diverse collection of opportunistic pathogens [80,110]. It is important to understand that Cryptococcus, Trichosporon, Rhodotorula, and Saprochaetae (formerly Blastoschizomyces) spp are intrinsically resistant to the echinocandin class of agents [126]. Amphotericin B is generally active against these organisms with the notable exception of Trichosporon asahii, which should be considered resistant to amphotericin B (table 4). MICs for flucytosine are generally <1 mcg/mL for isolates of Rhodotorula and Saccharomyces spp but are >4 mcg/mL for C. neoformans and S. capitata. Fluconazole MICs are moderately elevated (MIC₉₀ S. capitata [formerly Blastoschizomyces] = 8 mcg/mL) for isolates of C. neoformans, Trichosporon spp, Saccharomyces spp, and S. capitata (table 4) [127-136]. Aside from Rhodotorula spp, which are resistant to all azoles, voriconazole and posaconazole are very active against the non-candidal yeasts (table 4).

Molds — Amphotericin B is generally active against Aspergillus spp (table 5); however, certain non-fumigatus Aspergillus species, such as A. terreus, are considered to be resistant to amphotericin B [137]. (See "Treatment and prevention of invasive aspergillosis", section on 'Consideration of antifungal resistance'.)

MICs for amphotericin B are low for the Mucorales, whereas Fusarium spp, S. apiospermum, and Lomentospora (formerly Scedoporium) prolificans are considered resistant to this agent (table 5).

All of the triazoles have potent activity against Aspergillus spp. [61,138]. Voriconazole, isavuconazole, and posaconazole generally are more active than itraconazole against Fusarium and L. prolificans [60,61,138,139]. Posaconazole, isavuconazole, and voriconazole are active against S. apiospermum, whereas only posaconazole and isavuconazole show activity against the Mucorales.

The MEC for caspofungin and the other echinocandins is usually quite low for Aspergillus spp but is elevated for the non-Aspergillus molds (table 5). The echinocandins are not recommended as first-line therapy for infections caused by Aspergillus spp since their efficacy for this indication has not been established. (See "Treatment and prevention of invasive aspergillosis".)

CROSS-RESISTANCE — Because the azole class of antifungal agents shares a common mechanism of action and (in most cases) of resistance, there is concern regarding the development of cross-resistance among the azoles with both Candida and Aspergillus spp [10,63,140,141].

Candida spp

Azoles — The potential for cross-resistance among the available triazoles is evident, especially among Candida spp capable of overexpression of Candida drug resistance (CDR) efflux pumps (eg, C. glabrata) and, to a lesser extent, those with overexpression of and/or mutation in the target enzyme, lanosterol 14-alpha-demethylase [140,142,143].

Among Candida isolates, there is a strong positive correlation between fluconazole minimum inhibitory concentrations (MICs) and those of itraconazole, voriconazole, and posaconazole, indicating considerable cross-resistance [140,144]. Thus, resistance to fluconazole may serve as a surrogate marker in predicting resistance to the other extended-spectrum triazoles with Candida spp. An analysis of cross-resistance among fluconazole and the other triazoles demonstrated that isolates of Candida spp for which fluconazole MICs are ≥64 mcg/mL (resistant) also tend to be less susceptible to itraconazole [140,145], voriconazole [79,140], and posaconazole [140,144]. Fluconazole MICs of ≤32 mcg/mL predict susceptibility, and MICs of ≥64 mcg/mL predict resistance of Candida spp to voriconazole and posaconazole [140,144].

Certain Candida species have predictable patterns of cross-resistance. Notably, none of the triazoles exhibit meaningful activity against fluconazole-resistant isolates of C. glabrata, whereas isavuconazole, voriconazole, and posaconazole are active against the intrinsically fluconazole-resistant C. krusei [140,146].

Evidence supporting the clinical relevance of azole cross-resistance has been documented in case reports and case series of invasive candidiasis in which clinically significant microbial resistance to voriconazole has been reported among critically ill patients and immunocompromised patients with a history of substantial recent azole exposure, such as fluconazole, before voriconazole is given [140,141]. These concerns have led some experts to suggest fluconazole susceptibility testing of initial blood isolates of Candida as a means of identifying those patients who may not respond optimally to either voriconazole or posaconazole therapy [14,16,141].

There is increasing concern that some C. glabrata bloodstream isolates with resistance to fluconazole and voriconazole are also resistant to the echinocandins [122]. (See 'Echinocandins' below.)

Echinocandins — Cross-resistance among echinocandins has been documented and is usually related to mutations in the FKS1 (glucan synthase) gene [46,47,147]. A 16- to 128-fold change in MIC relative to the MIC of a fully susceptible wild-type strain is consistently observed for all three echinocandins when tested against a Candida strain with FKS1 mutations [147]. The MICs for caspofungin and micafungin tend to be somewhat higher than those determined for anidulafungin in such strains [147].

The clinical significance of such differences remains to be determined; however, the more conservative approach would be to consider those isolates shown to be resistant to either anidulafungin or micafungin to be resistant to the other agents in the class.

There is increasing concern that some C. glabrata bloodstream isolates with resistance to fluconazole and voriconazole are also resistant to the echinocandins. Such resistance is termed co-resistance (rather than cross-resistance) because the resistance to the echinocandins involves a different mechanism than the resistance to the azoles. In a surveillance study of the in vitro susceptibility of 1669 C. glabrata bloodstream isolates collected in the United States between 2006 and 2010, 162 isolates (9.7 percent) were resistant to fluconazole, of which 98.8 percent were also not susceptible to voriconazole (MIC >0.5 mcg/mL), and 9.3, 9.3, and 8.0 percent were resistant to anidulafungin, caspofungin, and micafungin, respectively [122]. Of the 162 isolates that were resistant to fluconazole, 18 (11.1 percent) were resistant to one or more of the echinocandins; all of these isolates contained an FKS1 or FKS2 mutation. In comparison, there were no echinocandin-resistant strains detected among 110 fluconazole-resistant C. glabrata isolates tested between 2001 and 2004, years during which only one echinocandin, caspofungin, was available and echinocandins were used sparingly. At this point, it is not clear what impact these findings will have on treatment regimens for candidemia.

In a study of C. glabrata bloodstream infections at a single medical center in the United States conducted between 2001 and 2010, among 78 fluconazole-resistant isolates, 11 (14.1 percent) were resistant to one or more echinocandins and 8 (10.3 percent) were resistant to all echinocandins [121].

Filamentous fungi — Investigation of azole cross-resistance among filamentous fungi has focused on studies of Aspergillus fumigatus [63,140,148]. Cross-resistance among the triazoles is dependent upon specific mutations in the A. fumigatus CYP51A gene [63]. Certain mutations result in cross-resistance between itraconazole and posaconazole but not voriconazole [63]. Other mutations may result in resistance to itraconazole and voriconazole but not posaconazole. Still other mutations result in resistance to all four triazoles [63].

Given the lack of complete cross-resistance among the azoles for Aspergillus spp, antifungal susceptibility testing is warranted to identify resistant phenotypes in patients with invasive disease who have clinical failure of initial therapy [63]. (See 'Indications for testing' above.)

SUMMARY AND RECOMMENDATIONS

●Importance of susceptibility testing – The need for reproducible, clinically relevant antifungal susceptibility testing has been prompted by the increasing number of invasive fungal infections, the expanding use of new and established antifungal agents, and recognition of antifungal resistance as an important clinical problem. (See 'Introduction' above.)

●Development of susceptibility standards – The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antifungal Susceptibility Testing has generated consensus documents describing standardized methods for broth- and agar-based antifungal susceptibility testing. As a result, in vitro antifungal susceptibility testing plays an increasingly important role in guiding therapeutic decision making, as an aid in drug development studies, and as a means of tracking the development of antifungal resistance in epidemiologic studies. Several commercial methods for antifungal susceptibility testing are available. (See 'Introduction' above and 'Standardized methods' above and 'Commercial MIC methods' above.)

●Clinical relevance – In order to be useful clinically, in vitro susceptibility testing of antimicrobial agents should reliably predict the in vivo response to therapy. The in vitro susceptibility of an infecting organism to the antimicrobial agent is only one of several factors that may influence the likelihood that therapy will be successful. (See 'Clinical relevance' above.)

●Indications for susceptibility testing – There are several situations in which antifungal susceptibility testing should be considered or obtained (table 1):

•All species of Candida isolated from blood or deep sites (eg, normally sterile fluids, tissues, abscesses) should be tested for susceptibility to fluconazole, voriconazole, and an echinocandin. For species, such as C. krusei and C. auris that have high rates of intrinsic resistance to fluconazole, antifungal susceptibility testing should be performed for other azoles, amphotericin B, and the echinocandins, but it is not necessary to test fluconazole (table 1).

•Mucosal candidiasis that is unresponsive to usual antifungal therapy

•Invasive disease that is unresponsive to the initial antifungal regimen

•Clinical failure in patients with invasive disease caused by species with significant rates of acquired resistance (see 'Cross-resistance' above)

•Invasive disease caused by unusual fungal species for which antifungal susceptibility patterns have not been well established or are unpredictable

●Susceptibility patterns – Many fungal species have predictable antifungal susceptibility patterns. However, rising rates of resistance to antifungal agents have occurred among some fungal species due to the increase in the immunocompromised population and the frequent use of fluconazole and echinocandins. (See 'Susceptibility patterns' above.)

●Cross-resistance – Cross-resistance is a concern, particularly among the azole antifungal agents. (See 'Cross-resistance' above.)