Clinical, Cellular, and Molecular Factors That Contribute to Antifungal Drug Resistance

PolyenesThe polyenes are a class of antifungal drugs that target membranes containing ergosterol. These drugs, which include amphotericin B and nystatin, are amphipathic, having both hydrophobic and hydrophilic sides. The drugs are thought to intercalate into membranes, forming a channel through which cellular components, especially potassium ions, leak and thereby destroying the proton gradient within the membrane (201). The specificity of the drugs for ergosterol-containing membranes is thought to be due to the interactions of the amphotericin B channel with ergosterol in the membrane, although the mechanism of this interaction is unknown. The specificity of amphotericin B for ergosterol-containing membranes may also be associated with phospholipid fatty acids and the ratio of sterol to phospholipids (201). The drugs are less likely to interact with membranes containing cholesterol. It has also been suggested that amphotericin B causes oxidative damage to the fungal plasma membrane (201, 203, 204). However, recent evidence suggests that amphotericin B has an antioxidant effect in vivo, protecting fungal cells against oxidative attack from the host (135).

Ergosterol Biosynthesis InhibitorsSeveral inhibitors of the ergosterol biosynthetic pathway have been developed for use against medically important fungi, including allylamines and thiocarbamates, azoles, and morpholines (Table1). All of these drugs interact with enzymes involved in the synthesis of ergosterol from squalene, which is produced from acetate through acetyl coenzyme A, hydroxymethylglutaryl coenzyme A, and mevalonate. Ergosterol is an important sterol for fungi, since it is the predominant or “bulk” sterol in fungal plasma membranes. In addition, it has an essential “sparking” function in which trace amounts of ergosterol are necessary for the cells to progress through the cell cycle. This sparking function is independent of the bulk sterol in the fungal membranes, since certain sterols can replace the bulk sterol of the membrane without supplying the sparking function for the cell cycle (67). The ergosterol biosynthesis inhibitors disrupt both the bulk sterol of fungal membranes and the sparking function in the cell cycle.

The allylamines (i.e., naftifine and terbinafine) and thiocarbamates (i.e., tolnaftate and tolciclate) inhibit the conversion of squalene to 2,3-oxidosqualene by the enzyme squalene epoxidase (44, 123,163). This enzyme is the product of the ERG1 gene (gene designations are given in Table 2), which has recently been cloned in C. albicans (Table 1) (161). This class of drugs may inhibit the epoxidase through a naphthalene moiety common to both types of drugs (204). The drugs are noncompetitive inhibitors, and cells accumulate squalene in their presence.

The azoles, including both imidazoles (ketoconazole and miconazole) and triazoles (fluconazole, itraconazole, and voriconazole), are directed against lanosterol demethylase in the ergosterol pathway. This enzyme is a cytochrome P-450 enzyme containing a heme moiety in its active site (66, 199). The azoles act through an unhindered nitrogen, which binds to the iron atom of the heme, preventing the activation of oxygen which is necessary for the demethylation of lanosterol (77). In addition to the unhindered nitrogen, a second nitrogen in the azoles is thought to interact directly with the apoprotein of lanosterol demethylase. It is thought that the position of this second nitrogen in relation to the apoprotein may determine the specificity of different azole drugs for the enzyme (66,199). At high concentrations, the azoles may also interact directly with lipids in the membranes (71, 77). Two other antifungal drug classes, the pyridines (buthiobate and pyrifenox) and the pyrimidines (triarimol and fenarimol), inhibit lanosterol demethylase and are used extensively as antifungal agents in agriculture but are not used in medicine (201). While many fungal species are sensitive to the azoles, Mucor spp. are intrinsically resistant to these drugs (112). Candida krusei and Aspergillus fumigatus are intrinsically resistant to fluconazole and ketoconazole, but both are usually sensitive to itraconazole (112) and voriconazole (118).

The morpholines (i.e., fenpropimorph and amorolfine) inhibit two enzymes in the ergosterol biosynthetic pathway, C-14 sterol reductase and C-8 sterol isomerase. The genes for these two enzymes,ERG24 and ERG2, have not yet been cloned from any medically important fungus, but have been cloned from S. cerevisiae. However, little is known of the interaction of the morpholines with ERG24 or ERG2.

5-Flucytosine5-Flucytosine (5-FC) has an entirely distinct mode of action from the azoles. 5-FC is taken up into the cell by a cytosine permease and deaminated into 5-fluorouracil (FU) by cytosine deaminase. 5-FC is fungus specific since mammalian cells have little or no cytosine deaminase (203, 204). FU is eventually converted by cellular pyrimidine-processing enzymes into 5-fluoro-dUMP (FdUMP), which is a specific inhibitor of thymidylate synthetase, an essential enzyme for DNA synthesis, and 5-fluoro-UTP (FUTP), which is incorporated into RNA, thus disrupting protein synthesis.

Clinical Uses of Antifungal AgentsDrugs from the polyene class of antifungal agents, specifically amphotericin B, have long been considered the most effective of the systemically administered antifungal agents. The fungicidal antifungal drug amphotericin B has in vitro and in vivo activity against yeasts (Candida spp., Cryptococcus neoformans), molds (Aspergillus spp., Zygomycetes, dematiaceous fungi), and dimorphic fungi. Unfortunately, infusion-related toxicities, the frequent association of renal dysfunction, and the intravenous formulation of amphotericin B have limited the utility of this drug. Similarly, 5-FC, although generally active againstCandida spp., C. neoformans, and some molds, has limited clinical utility owing to the frequent association of hematological toxicity and the rapid development of resistance, particularly when it is used as a single agent (discussed in more detail below) (17). The azole antifungal agents, because of their relative safety and ease of delivery, have subsequently become a critical component in the antifungal armamentarium.

Inconsistent levels in blood and high toxicities limited the utility of the first azoles, until ketoconazole was developed and used to treat chronic mucocutaneous candidiasis in the early 1980s (72). Fluconazole, found to be active against C. albicans in vitro and in vivo (162), has since become the drug of choice for the treatment of the most common AIDS-associated opportunistic infection, oropharyngeal candidiasis. Convenient administration and extended activity against non-Candida fungal infections (cryptococcosis, histoplasmosis, and coccidioidomycosis) have made azoles attractive as systemically administered drugs for the therapy and prophylaxis of AIDS-associated opportunistic infections (29,51). Fluconazole is the preferred azole owing to a high oral bioavailability and good safety profile, although the less expensive ketoconazole is still frequently used for antifungal treatment in other countries (38). A recent study suggests that high doses of fluconazole might be effective therapy for mild blastomycosis in HIV-negative patients (137). Itraconazole and some of the newer azoles such as voriconazole have in vitro activity against various nonyeast fungal infections (molds, dimorphic fungi), but the clinical utility of the newer compounds is still being defined (17, 118).

Over the last several decades, aggressive anticancer therapy has resulted in another patient population that has become increasingly exposed to azole drugs for antifungal treatment and prophylaxis. An increased incidence of invasive Candida infections and attributable mortality have been documented in patients undergoing chemotherapy and bone marrow transplantation (193). Prophylactic fluconazole has been shown to decrease the incidence of local and invasive candidiasis in patients receiving chemotherapy and bone marrow transplantation, and today it is used in some centers for the prevention of candidiasis in neutropenic patients (59, 182,214).

The role of azole antifungal agents in preventing and treating yeast infections in nonneutropenic patients has been expanding. Fluconazole has been shown to be effective presumptive therapy in surgical intensive care unit patients colonized with Candida spp. (143, 188) and as treatment for candidemia in nonneutropenic patients (156). In addition, azole antifungal agents have been found to be an effective single-dose therapy for vaginal candidiasis (183, 190).

Resistance to each of the antifungal agents has been reported in clinical isolates under specific conditions. However, the increased use of azoles in immunocompromised patients has had the largest effect on the frequency, morbidity, and response to therapy of fungal infections in the hospital and in the outpatient setting. Recently, advances in clinical microbiology have enabled us to detect and define azole resistance. The epidemiology of antifungal resistance, including the incidence, prevalence, and related host risk factors, will be discussed in more detail below.

Definition of Resistance and Role of Susceptibility TestingBefore the development of susceptibility testing of yeasts as outlined by the National Committee for Clinical Laboratory Standards (NCCLS), MIC determinations were inconsistent and varied up to 50,000-fold in different laboratories (49). NCCLS document M27, first published in 1992, has recently been revised (M27-A) and subsequent studies have demonstrated that the interlaboratory reproducibility of MIC determination approximates that of antibacterial testing (159). The macrodilution method has set the groundwork for the development of less cumbersome methods adapted for the clinical laboratory. These methods, relying on microdilution with or without colorimetric indicators or agar diffusion, have been shown to be reproducible and consistent with the standardized macrodilution method according to preliminary studies (141). One complication of the NCCLS protocol is that for certain isolates, the timing of the end point determination can have a major effect (up to 128-fold) on the MIC. A recent study suggests that determining the end point after 24 instead of 48 h ensures that the MICs for these isolates correlate with the in vivo response to azoles (157). In addition, there have been significant advances in susceptibility testing for filamentous fungi (34), as well as in vitro testing of amphotericin B susceptibility. Technical advances of antifungal susceptibility testing have been recently reviewed (141).

Historically, clinical resistance has been defined as persistence or progression of an infection despite appropriate antimicrobial therapy. A successful clinical response to antimicrobial therapy typically not only depends on the susceptibility of the pathogenic organism but also relies heavily on the host immune system, drug penetration and distribution, patient compliance, and absence of a protected or persistent focus of infection (e.g., a catheter or abscess). This is particularly true for fungal infections (Table3).

The in vitro resistance of an isolate can be described as either primary or secondary. An organism that is resistant to a drug prior to exposure is described as having primary or intrinsic resistance. Secondary resistance develops in response to exposure to an antimicrobial agent. Both primary and secondary resistance to antifungal agents have been observed.

A correlation of in vitro susceptibility with in vivo response has been observed for mucosal candidal infections in HIV-infected patients. Many groups have noted that the clinical outcome is generally dependent on the in vitro susceptibility of the organism. One group performed fluconazole susceptibility testing on fungal isolates obtained from 87 HIV-infected patients. They observed that persistent oropharyngeal candidiasis correlated with infections caused by yeasts for which the MICs of fluconazole were high (24). Similar trends have been documented by a number of other investigators (9, 94, 108,126). However, host variables become important when in vivo resistance is compared with in vitro MIC testing, especially in evaluating systemic infections (Table 3). The more immunocompromised the host, the less reliable is the correlation between in vivo resistance and MIC. The ability of C. albicans to form biofilms on surfaces (catheters, teeth, and endothelial cells) has been implicated as a cause of clinical “resistance” despite microbial susceptibility in vitro. One study demonstrated an increased resistance in the cells of the biofilm compared to that in cells growing in suspension (62). The formation of layers of cells may confer protection to organisms in the inner layers, leading to failure of antifungal therapy. Alternatively, infection caused by a resistant organism does not always predict clinical failure. A clinical response to fluconazole occurred in 11 of 13 patients with oral candidiasis even though they were infected with isolates for which the MICs were 32 or 64 μg/ml (155). The correlation between in vivo response and in vitro susceptibility has been reviewed in detail, with most of the data being derived from patients infected with HIV (53).

For aid in clinical interpretation of antifungal susceptibility testing, the NCCLS Subcommittee for Antifungal Susceptibility Testing recently established interpretive breakpoints for testing of fluconazole and itraconazole for Candida infections (158). The breakpoints for fluconazole MICs are as follows: <8 μg/ml, sensitive; 8 to 32 μg/ml, susceptible dose dependent; and ≥64 μg/ml, resistant. The breakpoints for itraconazole MICs are as follows: ≤0.125 μg/ml, sensitive; 0.25 to 0.5 μg/ml, susceptible dose dependent; and ≥1.0 μg/ml, resistant. These breakpoints do not apply for C. krusei, which is intrinsically resistant to most azole drugs. The intermediate levels of resistance are denoted susceptible dose dependent since they are usually treatable with high doses of drug (at least 400 mg of fluconazole per day or doses of itraconazole that maintain the levels in plasma at ≥0.5 μg/ml). Despite the advances in testing and interpretation, the ability of susceptibility testing to guide therapeutic decision making remains controversial. In response to this dilemma, experts in the field recently proposed recommendations for antifungal testing in the clinical laboratory (141). According to these authors, susceptibility testing is not routinely indicated for any fungus. They recommend testing of Candidaspp. causing oropharyngeal candidiasis in patients with AIDS who are not responding to azole therapy and in some patients with invasive candidiasis (141).

The establishment of a reproducible method of susceptibility testing not only has resulted in advances in therapeutic decision making, but it also will enable further study of the epidemiology of antifungal resistance in yeasts by allowing comparisons between studies. Recent analyses addressing the prevalence, impact, and microbiological and host risk factors are discussed below and addressed in more detail in two recent reviews (3, 206).

Epidemiology: Prevalence, Risk Factors, and Clinical ImplicationsClinical in vitro resistance to all antifungal agents has been reported. Infections caused by azole-resistant Candida spp. have been found in AIDS patients worldwide. Resistance to the polyene antifungal agents and flucytosine have developed in yeasts and a few molds and needs to be taken into consideration during therapeutic decision making.

Change to a More Resistant SpeciesFor each fungal species, there is a distribution of MICs of each antifungal drug. The average MIC of a particular drug can be determined for each species. By using this type of analysis, it is clear thatC. krusei is intrinsically more resistant to azole drugs than is C. albicans, with most strains of C. krusei having high azole MICs. In addition, many strains ofC. glabrata have azole MICs that are significantly higher than those for most strains of C. albicans (reviewed in references 133 and 160). Recently, Candida norvegensis and Candida inconspicua have also been described as intrinsically resistant species, although these species are uncommon human pathogens (5,166).

These resistant species may be present by chance in a healthy individual as commensal organisms which can then become a problem when the individual becomes immunocompromised and is administered azole therapy. Alternately, infection with these species can be acquired from the environment or from other individuals. It is thought that in an immunocompetent individual, the endogenous species would prevent these new species from becoming established. However, in an immunocompromised patient, the selective pressure of antifungal drugs may allow the replacement of endogenous species with a more resistant superinfecting species.

The intrinsic resistance of C. krusei and some strains ofC. glabrata to azole drugs has resulted in an increased frequency of these species in patient populations that depend on azole drugs for treatment or prophylaxis (see, e.g., references 133,144, 226, and 227). It is likely that the azole drugs used by these patients suppress the growth of sensitive isolates such as those of C. albicans, and allow the growth of more resistant strains or species.

Change to a More Resistant Strain of C. albicans In addition to the resistant species such as C. kruseiand C. glabrata, there are intrinsically resistant strains of C. albicans that can be part of a commensal growth or can be acquired from the environment or other individuals. Resistant strains of C. albicans can occur as a result of the normal distribution of MICs that all species exhibit, or they can develop resistance through the mechanisms described below.

Several studies have investigated the frequency of strain replacement (one strain for another) in a single patient. In one such study (13), strain replacement was seen in the development of resistance in one of four AIDS patients (25%). Sensitive and resistant isolates were obtained from these patients over time, and the isolates were analyzed by pulsed-field gene electrophoresis (molecular karyotyping) and restriction fragment length polymorphism (RFLP). Three of the four patients maintained the same strain, while the sensitive and resistant isolates from the fourth patient were clearly different by RFLP but not by karyotyping. In a second study (120), sensitive and resistant isolates from seven patients were studied by RFLP and karyotyping. In all seven (0% replacement), the sensitive and resistant isolates were the same strain. In a third study (104), multiple sensitive and resistant isolates from three patients were studied by RFLP, karyotyping, and randomly amplified polymorphic DNA. Two of the three patients maintained the same strain (33% replacement). The fourth study used a mixed-linker PCR method to determine that strain replacement accompanied an increase in resistance in only one of five patients (20% replacement) (117). In the final study (142), karyotyping analysis was used to type sensitive and resistant isolates from 10 AIDS patients. Two patients (patients 6 and 20) exhibited sensitive and resistant isolates from the same strain at different times, while several other patients exhibited mixed infections of different strains with different sensitivities at several time points. It is difficult to determine if these mixed infections represent strain replacement over time or mixed infections in which the proportion of sensitive and resistant strains was altered by azole therapy. In one of the two patients from this study, a substrain was selected as resistance developed (225), indicating that the concept of strain replacement is dependent on the technique used to determine strain relatedness. In all of these studies, the sample size is small and so a generalization on the overall frequency of strain replacement is not possible. It is clear that development of resistance within the endogenous strain is much more common than strain replacement. In conclusion, there is a need for a large prospective survey of HIV-infected patients in which the development of resistance and strain identity are closely monitored. Such a study is in progress (139, 155).

The transfer of a strain from one person to another has been documented in several instances. In one case, a strain of C. albicanswas transferred orally from one HIV-infected man with oral candidiasis to another (121). In a larger study of 19 sexual partners, at least five pairs had identical or highly related strains of C. albicans (18). In another study, C. albicansstrains were identical in women with vaginal candidiasis and their male sexual partner in 8 of 10 cases studied (176). Transfer was not observed in sexual partners without symptoms of disease. None of these studies tested isolates for antifungal susceptibility. Still, the documented strain similarities suggest that transfer of strains can occur, at least in cases in which one of the individuals has disease symptoms.

Genetic Alterations That Render a Strain ResistantWithin a species, intrinsically resistant strains occur as part of the normal distribution of resistance levels (primary resistance). In addition, there is the possibility that a strain can be induced to be resistant by exposure to the drug over long periods (secondary resistance) (160). In these cases, it is hypothesized that the drug itself does not cause resistance but, rather, selects for growth of the more resistant cells in the population. Genetic mutation occurs by chance in all organisms at a low frequency (usually 10⁻⁶ to 10⁻⁸/gene). However, in a large population of yeast cells under selective drug pressure, specific random mutations that render the cell slightly more resistant will eventually become the dominant strain in the population. Unless the genetic mutations that render a cell resistant also reduce the overall fitness of the cell (133, 202), the mutant strain will persist even in the absence of the selective pressure of the drug.

Given the potential for the transfer of strains between individuals and the genetic stability of resistance, resistant isolates can quickly have a profound impact on the efficacy of the antifungal drug used for treatment, especially within a relatively isolated population with frequent interpersonal contacts, such as HIV-infected populations.

Transient Gene Expression That Renders a Cell Temporarily ResistantThere are recent data suggesting that strains ofCandida can become transiently resistant to a drug, a phenomenon called epigenetic resistance. That is, a cell can alter its phenotype, probably through transient gene expression, to become resistant in the presence of the drug, but the resistant phenotype can revert quickly to a susceptible phenotype once the drug pressure is eliminated. Recently, several researchers have demonstrated epigenetic resistance both in vitro (23) and in vivo (67,203). Given the transient nature of epigenetic resistance, little is known about it at this time.

Alteration in Cell Type C. albicans has a variety of different cell types which vary in their susceptibility to azoles. C. albicans can be divided into two serotypes, A and B, based on carbohydrate surface markers. The B serotype strains of C. albicans are more sensitive to azoles (specifically ketoconazole) but are more resistant to 5-FC than are the A serotype strains (reviewed in references132 and 201).

C. albicans is a dimorphic fungus. The yeast-like or blastospore cell types are spherical, budding cells which are associated with commensal growth of C. albicans. Under a variety of conditions, C. albicans can form long, slender hyphal projections, which are often associated with C. albicans pathogenicity (32). Azole drugs can interfere with hyphal production at 0.1 μM, 1/10 the therapeutic concentration of the drug in vivo (132). To date, there have been no studies monitoring the ability of resistant and sensitive clinical isolates of C. albicans to form hyphae. However, it is possible that a resistant strain with the ability to form hyphae even in the presence of azole drugs would be more pathogenic than a sensitive strain that is unable to form hyphae. This possibility needs further study.

In addition to the yeast/hypha transition that can occur in C. albicans, several species of Candida have the ability to alternate between several different forms, referred to as “switch phenotypes” (reviewed in references 185 and187). These switch phenotypes represent different states of a yeast cell where each state expresses a unique set of genes, cellular structures and cell morphologies. These switch phenotypes are defined by their ability to manifest as different colony morphologies on agar plates. At least two different laboratories have described strains of C. albicans in which two different switch phenotypes exhibit a difference in azole susceptibility (50, 54).

Alterations in the Fungal PopulationAntifungal drug resistance is not a constant among the fungal cells in a population. The number of cells that are present in an infection or as a commensal growth will have an effect on antifungal drug resistance, since an increased number of cells will increase the probability that a mutation can occur that confers resistance. Recently, several researchers have observed that there is a great deal of variation in MICs between individual colonies of the same strain from a single patient (75, 179). These differences may reflect genomic instability or variation within a strain. Finally, it is likely that population “bottlenecks” can randomly select for resistance (or susceptibility). A population bottleneck is a set of conditions that randomly selects for a small number of cells from a population. That small subset may contain genetic alterations that will shift the characteristics of a population in a different direction. Such a population “bottleneck” might occur when azole therapy drastically reduces the number of fungal cells in oral candidiasis. In such an example, the azole drug may select for more resistant cells. However, the bottleneck may not necessarily involve drug selection. The same phenomenon might be seen with the removal and/or sterilization of dentures. It is possible that the small subset of fungal cells that survive on the denture or in the oral cavity is more (or less) genetically predisposed to drug resistance.

Matched SetsIt is important in the analysis of the molecular mechanisms of resistance to use a matched set of isolates, that is, sensitive and resistant versions of the same strain, as determined by RFLP or karyotyping. This is required because C. albicans is mostly clonal; that is, it grows vegetatively without sexual reproduction. There is good evidence from Hardy-Weinberg equilibrium experiments (150) and balanced lethal experiments (220) thatC. albicans almost never undergoes a sexual cycle. Enzyme analysis shows that C. albicans is not in Hardy-Weinberg equilibrium, which results from a randomly mating population. The disequilibrium in C. albicans indicates that mating is not a common event (150). Most strains of C. albicanscontain a large number of balanced lethals (recessive null mutations) in which the cell relies on one functional copy of the gene for activity (220). These recessive lethal mutations could not persist in a population that mated frequently. The clonal nature ofC. albicans makes it imperative that matched sets of sensitive and resistant versions of a single strain be characterized when determining the molecular mechanisms of resistance.

RFLP analysis with the repetitive marker Ca3 demonstrates a wide variety of RFLP patterns, suggesting that only rarely are clinical isolates related to each other (105, 177). Specific mutations in a target gene can be the result of allelic variation or selection for a specific resistance phenotype. The difference can be determined only with a matched set of sensitive and resistant isolates from the same strain. To date, few studies have used matched sets of isolates (114, 142, 153, 170, 171, 222, 223, 225).

Drug ImportDefects in drug import are a common mechanism of drug resistance. However, it is important to emphasize the distinction between the import of a drug into a cell and the gradual accumulation of the drug in the cell, which is the result of a balance between import into the cell and efflux of the drug from the cell. Analysis of drug import is difficult at best and requires that mechanisms of import be separated from efflux mechanisms. The standard method for studying drug import is to determine the amount of drug that associates with cells in extremely short periods (less than 10 s). This method has been used extensively in studying drug import in parasitic protozoans (65). The short incubation periods are achieved by mixing the cells and the drug above a layer of mineral oil. After the short incubations, the cells are pelleted through the mineral oil and the amount of drug in the cell pellet is determined. At this time, an analysis of drug import during short periods has not been performed for any medically important fungi.

Studies of fungi have usually used labeled drug to monitor the amount of label that accumulates within the cell over several minutes. These accumulation studies have led to the identification of several fungal efflux mechanisms (see below). One study with C. albicanssuggested that accumulation of [³H]ketoconazole required glycolytically derived energy and was controlled by cell viability, environmental pH, and temperature (19). This study also showed that ketoconazole accumulation at low extracellular concentrations was saturable, implying a specific facilitator of import, while accumulation at high concentrations appeared to be by passive diffusion. Curiously, this study found no evidence for export, since the addition of unlabeled drug did not lower the concentration of labeled drug in the cells. Finally, data in this study suggested that other azole drugs and amphotericin B increased the accumulation of labeled ketoconazole in the cell (19). These results are not easily explained by the currently understood molecular mechanisms, which include an energy-dependent efflux pump for fluconazole (see below). The energy requirement for ketoconazole accumulation described in this study is inconsistent with the energy-requiring efflux mechanisms. Elimination of energy levels would inhibit energy-dependent efflux pumps, resulting in increased drug accumulation, the opposite of what is seen with ketoconazole. These results suggest that active transport may be involved in ketoconazole import. It is possible that the ketoconazole and fluconazole are imported by two different mechanisms. Further study is clearly needed.

Drug import may also be affected by the sterol composition of the plasma membrane. Several studies have demonstrated that when the ergosterol component of the membrane is eliminated or reduced in favor of other sterol components such as 14α-methyl sterols, there are concomitant permeability changes in the plasma membrane and a lack of fluidity (204). These changes may lower the capacity of azole drugs to enter the cell.

Modification or Degradation of the DrugIt is important to note that alterations in drug processing (modification or degradation) are important drug resistance mechanisms in a variety of bacterial and eukaryotic systems (20). To date, little analysis of drug modification or degradation within a resistant cell has been performed for the medically important fungi. It has been mentioned that azoles are inert to metabolism by C. albicans (67), although no studies have analyzed the sensitivity of azole drugs to metabolism in resistant strains or non-albicans species.

Modifications of the Ergosterol Biosynthetic PathwayAnother common mechanism of drug resistance is modification of the target enzyme and/or other enzymes in the same biochemical pathway. For azole drugs, that pathway is the ergosterol biosynthetic pathway. Analysis of the sterols of a cell can provide a wealth of information concerning the alterations that have occurred in a resistant strain (Table 1). Modifications in the ergosterol pathway are likely to generate resistance not only to the drug to which the cells are exposed but also to related drugs. This cross-resistance will be discussed in a later section.

A defective lanosterol demethylase (the predominant azole target enzyme) will result in the accumulation of 14α-methyl sterols, especially 14α-methyl fecosterol and the diol 14α-methyl-ergosta-8,24(28)-dien-3β,6α-diol (84). In azole-treated cells, the presence of 14α-methyl sterols can modify the function and fluidity of the plasma membrane. In addition, it appears that cells with 14α-methyl sterols have an increased sensitivity to oxygen-dependent microbicidal systems of the host (180). The diol that accumulates is known to cause growth arrest in Saccharomyces cerevisiae (84, 199) but is thought to be tolerated in C. albicans (11). However, recent studies suggest that accumulation of the diol is correlated with growth arrest even in C. albicans(86). The toxic effects of the diol in S. cerevisiae are eliminated by a mutation in ERG3, which encodes C-5 sterol desaturase (Table 1) (11, 84, 217).

Plasmid complementation of the ERG3 mutation in S. cerevisiae suggests that the ERG3 mutation alone can cause azole resistance (4, 84). Biochemical analysis suggests that ERG3 mutations are responsible for resistance in at least two clinical isolates of C. albicans(86). The sterol composition of Cryptococcus neoformans has been studied when cells are exposed to itraconazole (200). The sterol composition suggests that the drug affects both lanosterol demethylase and the C-4 sterol demethylase enzyme, 3-ketosteroid reductase.

Fungal cell extracts and labeled mevalonate can also be used to monitor the synthesis of sterol intermediates, including squalene, oxidosqualene, lanosterol, desmethyl sterols, and ergosterol (12,115). The analysis of cell extracts from fungi has been important in monitoring overall sterol synthesis and the level of inhibition of lanosterol demethylase by azole drugs (see, e.g., references98, 100, and 207). For example, cell extracts from a polyene- and azole-resistant strain of C. albicans (D10) did not contain any detectable lanosterol demethylase activity whereas the revertant strain, D10R, has lanosterol demethylase activity (102). In the absence of drug, strain D10 accumulates 14α-methyl sterols similar to an azole-treated wild-type cell. Strain D10 is also defective in the formation of hyphae, while the revertant forms hyphae at normal rates. This finding suggests that hyphal formation, which is an important virulence factor (32), can be affected by changes in the ergosterol pathway. To date, the genetic mutation in strain D10 has not been identified.

Biochemical analysis of an unrelated azole-resistant strain of C. albicans, the Darlington strain, has shown that lanosterol demethylase activity in this strain does not correlate with resistance (68). Surprisingly, drug accumulation in the Darlington strain is increased compared to that in azole-sensitive strains, suggesting that drug efflux (see below) is altered with resistance (69), although one would expect to see increased efflux in a resistant strain. An analysis of the sterol components of this strain shows an accumulation of fecosterol, suggesting that there may be a defect in ERG2 (not ERG3 as previously reported). The ERG3 gene was recently cloned and expressed in the Darlington strain. Overexpression of the ERG3 gene restored ergosterol as the major sterol of the transformant but did not render the cell susceptible (122).

Cell extracts from fungi other than C. albicans have been used to show that the lanosterol demethylase has become less susceptible in resistant clinical isolates. These includeCryptococcus neoformans (97, 211), A. fumigatus (35), and H. capsulatum(218). The molecular mechanisms for resistance in these isolates were not described. Cell extracts have also been used to characterize a resistant form of lanosterol demethylase from the plant pathogen Ustilago maydis (79).

Molecular Alterations of the ERG11 Gene As discussed above, the predominant target enzyme of the azole drugs is lanosterol demethylase. The gene encoding this protein is currently designated ERG11 in all fungal species, although it has previously been referred to as ERG16 andCYP51A1 in C. albicans. Several genetic alterations have been identified that are associated with theERG11 gene of C. albicans, including point mutations in the coding region, overexpression of the gene, gene amplification (which leads to overexpression), and gene conversion or mitotic recombination.

In one study, a point mutation in ERG11 was identified when an azole-resistant clinical isolate was compared with a sensitive isolate from a single strain of C. albicans(223). This point mutation results in the replacement of arginine with lysine at amino acid 467 of the ERG11 gene (abbreviated R467K, where R = Arg and K = Lys). The mutation is positioned near the cysteine which coordinates the fifth position of the iron atom in the heme cofactor. The mutation is thought to cause structural or functional alterations associated with the heme. Recent genetic manipulations suggest that R467K alone is sufficient to cause azole resistance (224). However, in the series of clinical isolates, several alterations occurred simultaneously (223), so it is impossible to determine how much R467K contributes to the overall azole resistance of the isolate.

Point mutations in ERG11 have been developed in laboratory strains that result in azole resistance. The point mutation T315A (the replacement of threonine [T] with alanine [A] at position 315) was constructed in the C. albicans ERG11 gene (100) based on the current understanding of the active site of the enzyme (reviewed in reference 77). The active site represents a pocket positioned on top of the heme cofactor. Substrates or inhibitors enter the active site through a channel that is accessible only with a shift in an α-helix of the apoprotein. Mutations in the active-site pocket, in the channel, and/or in the mobile helix would be predicted to affect the function of the enzyme. The T315A mutation, which is situated in the active-site pocket, was studied in S. cerevisiae, which is more amenable to genetic manipulation. T315A causes a reduction in enzymatic activity and a reduction in azole binding to the active site, resulting in fluconazole resistance. Another point mutation has been identified in the S. cerevisiae ERG11 gene from a laboratory strain that was resistant to azole drugs (73, 228). This mutation, D310G (replacement of aspartic acid [D] with glycine [G] at position 310), is located in the active site of the enzyme in close proximity to the T315A mutation and renders the enzyme inactive. However, the azole resistance of this strain is likely due to an ERG3 suppressor rather than to an inactive ERG11 gene product (84).

A survey of resistant and sensitive clinical isolates has identified seven different point mutations that are associated with azole-resistant isolates (106). However, the matched sensitive isolate was not available from these resistant isolates, which could be used to determine if the point mutations were associated with resistance or just the result of allelic variation.ERG11 genes from a variety of fungal sources have been expressed and manipulated in S. cerevisiae (98, 100,181) and in Escherichia coli (210). A recent study used functional expression in S. cerevisiae to identify and characterize five ERG11 point mutations from matched sets of sensitive and resistant isolates of C. albicans(168a).

Overexpression of ERG11 has been described in several different clinical isolates (2, 171, 222). In each case, the level of overexpression is not substantial (less than a factor of 5). It is difficult to assess the contribution of ERG11overexpression to a resistant phenotype, since these limited cases of overexpression have always accompanied other alterations associated with resistance, including the R467K mutation, and overexpression of genes regulating efflux pumps (see below). Overexpression has not been extensively evaluated, so it is difficult to assess its importance. It should be noted that low-level overexpression has also been documented in strains of S. cerevisiae (26, 41, 80, 88). In each case, the effect of overexpression on antifungal susceptibility has been minimal. It remains to be seen if overexpression ofERG11 alone will result in azole resistance.

A common mechanism of resistance in eukaryotic cells is gene amplification. The increase in the number of gene copies usually results in an increase in expression. Overexpression of theERG11 genes in C. albicans has not been associated with amplification of the ERG11 gene (171,222). However, in a clinical isolate of C. glabrata, increased levels of lanosterol demethylase were associated with a resistant phenotype (202). These increased levels of the protein were associated with ERG11 gene amplification (113, 203) and correlated with an increase inERG11 mRNA levels (201). In the same isolate, changes in drug efflux and in ERG7 activity were also observed (203). After 159 subcultures, the gene amplification, increased mRNA levels, enzyme levels, and drug efflux all reverted to normal. However, the revertant retained partial resistance to fluconazole (201). Recently, gene amplification of ERG11 in this isolate has been linked to a chromosome duplication, which results in overexpression ofERG11, as well as altered expression of a variety of other proteins (113).

Another genetic alteration associated with ERG11 has been described in the same clinical isolates in which the R467K mutation was described (223). Most, if not all, strains of C. albicans are diploid, having two alleles of each gene (132). Clinical isolates are usually clonal (105) and contain several sequence differences between the two copies of a gene (allelic differences). While analyzing the R467K mutation, it was observed that all of the allelic differences present in the sensitive isolate were eliminated in the resistant isolates containing R467K. These allelic differences were eliminated from the region of theERG11 gene including the promoter, coding region, and terminator region, and into the THR1 gene immediately downstream of the ERG11 gene (223). The simplest explanation is that the allelic differences were eliminated by a gene conversion or mitotic recombination involving the ERG11gene, although other possibilities (gene or chromosome deletion or mating) could not be completely discounted. This gene conversion or mitotic recombination resulted in a cell in which both copies of theERG11 gene contain the R467K mutation. Genetic evidence suggests that a cell with two copies of the R467K mutation is significantly more resistant to azoles than a cell in which only one allele has the R467K mutation (224). Thus, gene conversion or mitotic recombination, both of which have been found previously inC. albicans, may be an important mechanism in the generation of a resistant phenotype in diploid cells like C. albicans. Gene conversion or mitotic recombination would not be important in haploid organisms such as C. glabrata.

Deletion of the ERG11 gene in C. glabrata has been studied for its effect on enhanced oxidative killing (81). Two ERG11 deletion strains which were fluconazole resistant were more susceptible to killing by H₂O₂ and by neutrophils than two fluconazole-sensitive strains with intact ERG11 genes. This increased killing (in the absence of drug) may explain why no clinical isolates of any Candida species have yet been identified with a totally nonfunctional ERG11 gene, despite alterations in drug susceptibility due to point mutations in C. albicans.

Alterations in Other ERG Genes In addition to alterations in the lanosterol demethylase, a common mechanism of resistance is an alteration in other enzymes in the same biosynthetic pathway. The ergosterol biosynthetic pathway beginning with squalene is shown in Table 1. All of the genes listed have been cloned in S. cerevisiae (10, 83, 103). To date,ERG1, ERG2, ERG3, ERG4,ERG7, and ERG11 have been cloned from C. albicans (82, 89, 92, 96, 122, 174), ERG3and ERG11 have been cloned from C. glabrata(22, 52), and ERG11 has been cloned from C. krusei (22) and C. tropicalis(26). In addition, PRD1, the NADPH-cytochrome P-450 reductase that is important for the function of ERG11, has been cloned from C. albicans (174). Except for the R467K point mutation in ERG11 of C. albicans, no point mutations in the ergosterol pathway have been described from a matched set of azole-resistant and azole-sensitive clinical isolates. Similarly, no gene amplification has been observed for the C. albicans genes to date (171, 222).

The ERG3 and ERG11 genes of C. glabrata have been studied in some detail (52). When the ERG3 gene was deleted from a strain of C. glabrata, the cells remained viable under both aerobic and anaerobic conditions, accumulated the sterol ergosta-7,22-dien-3β-ol, remained susceptible to fluconazole and itraconazole, and became hypersensitive to amphotericin B. When the ERG11 gene was deleted, the cells were viable only under anaerobic conditions, accumulated lanosterol predominantly, became resistant to azoles, and had a reduced susceptibility to amphotericin B. The double mutant (deletion of both ERG3 and ERG11) was aerobically viable, accumulated 14α-methylfecosterol and lanosterol, was resistant to azoles, and had a reduced susceptibility to amphotericin B. The behavior of these mutations for aerobic and anaerobic growth and for sterol accumulation is similar to that described for S. cerevisiae (198). However, the antifungal resistance patterns are not similar, suggesting that the pattern of sterol control and antifungal resistance differs between closely related yeast species (52). This suggests that analysis of the ergosterol biosynthetic pathway in one species may not be applicable to other yeasts.

This analysis has also demonstrated that the ERG3 mRNA level was elevated in ERG11 deletions and that theERG11 mRNA level was elevated in ERG3 deletions. This suggests that transcriptional control of at least two genes occurred in the ergosterol biosynthetic pathway, perhaps by negative feedback from ergosterol. The lack of ergosterol in the deletion strains may have induced the transcription of ERG3 and/orERG11. This may be similar to transcriptional control ofERG genes in S. cerevisiae, whereERG11 is regulated by the carbon source, heme, and O₂ (aerobic and anaerobic growth) (198).

Sterol analysis of C. albicans clinical isolates has suggested that alterations in ERG3 may be a major cause of azole resistance (85, 86). ERG3 defects prevent the production of the diol that would cause growth arrest (see above).ERG3 defects have been suggested to be a major source of azole resistance in a number of fungi, including the plant pathogenU. maydis (79).

In Cryptococcus neoformans, the azoles appear to directly or indirectly block the C-4 sterol demethylase enzyme complex, which prevents the production of the diol without an ERG3 defect (76). Sterol analysis of fluconazole resistance in C. neoformans suggested that resistant strains may have defects inERG2 or in ERG3 (211); these strains appear to be cross-resistant to amphotericin B.

In addition to its interactions with lanosterol demethylase, itraconazole interacts with ERG24 in C. neoformans and H. capsulatum, which explains the increased potency of itraconazole against these species (203).

Recently, the ERG5 gene was cloned from S. cerevisiae and found to be another cytochrome P-450 enzyme (83). The azole drugs bind with similar affinity to this enzyme, suggesting that both ERG5 and ERG11 may be responsible for azole tolerance (Table 1).

Decreased Accumulation of DrugAs discussed above, little is known about the mechanisms by which a drug enters a fungal cell. However, in recent years, several studies have investigated the accumulation of drugs in cells for which the drug MIC is high. Using radioactively labeled drugs such as fluconazole, these studies have demonstrated that resistant isolates frequently accumulate less drug than do matched sensitive isolates. Several of these studies went on to show that the decreased accumulation is energy dependent.

Reduced accumulation of radiolabeled fluconazole has been documented for a variety of resistant clinical isolates of C. albicans(2, 99, 171, 209). Accumulation in sensitive isolates was shown to be temperature dependent and was not affected by energy inhibitors such as sodium azide or 2-deoxyglucose. In azole-resistant isolates, including a laboratory-induced isolate, fluconazole accumulation was reduced. Intracellular levels of the drug in resistant isolates were then increased by the addition of sodium azide, suggesting that an energy-dependent efflux pump was associated with resistance (2, 171).

Accumulation of fluconazole was studied by using [³H]fluconazole in matched sets of azole-sensitive and azole-resistant isolates of C. glabrata (70, 138,202). Azole-resistant isolates accumulated significantly less fluconazole than did azole-sensitive isolates from the same strain. Only small changes in ergosterol biosynthesis were detected in the resistant isolates. Metabolic or respiratory inhibitors increased the fluconazole accumulation of azole-resistant isolates, suggesting that energy-dependent efflux pumps were involved. Efflux pump modulators were tested for their effects on fluconazole accumulation. Only benomyl competed with fluconazole under these conditions (138) (benomyl resistance is discussed below).

Reduced drug accumulation has been associated with intrinsic azole resistance to fluconazole, itraconazole, and ketoconazole in C. krusei (111). An itraconazole-resistant isolate ofC. krusei did not exhibit any alteration in ergosterol biosynthesis but did show a reduced accumulation of drug compared to a laboratory strain (208). However, a matching sensitive version of the same strain was not available.

Rhodamine 123 (Rh123) is a substrate for a variety of efflux pumps in many systems and has been used to study accumulation in fungal cells. Azole-resistant strains of C. albicans, C. glabrata, and C. krusei accumulate less Rh123 than do azole-sensitive strains (28). Rh123 accumulation is dependent on the growth phase and temperature. Proton uncouplers and the efflux pump modulator reserpine increase accumulation, probably by decreasing the activity of the efflux pumps. In C. glabrata, Rh123 and fluconazole accumulation is competitive, suggesting a common efflux pump, while in C. albicans, the two drugs do not complete (28).

Reduced accumulation has also been suggested as a mechanism of resistance for a C. neoformans laboratory mutant that is resistant to both azoles and polyenes (76). The strain had no detectable change in its sterol pattern. No direct measure of drug accumulation was reported.

For the last several years, there were no reports of itraconazole resistance that were not the result of cross-resistance to fluconazole (201). However, recently, itraconazole resistance in three strains of A. fumigatus was described. One of the strains had a reduced cellular accumulation of labeled itraconazole, suggesting that resistance in this strain is mediated by an efflux pump (35).

Drug accumulation has also been studied for terbinafine inTrichophyton rubrum. Accumulation is 10-fold higher inT. rubrum than in C. albicans, which may explain the high sensitivity of T. rubrum to terbinafine (44).

Two Major Types of Efflux PumpsEukaryotic cells contain two types of efflux pumps that are known to contribute to drug resistance: ATP binding cassette (ABC) transporters (ABCT) and major facilitators (MF) (110, 119). Both types of pumps are known to cause drug resistance in other systems. The ABCT are frequently associated with the active efflux of molecules that are toxic to cells and are relatively hydrophobic or lipophilic, as is the case with most azole drugs. The MF have not been studied as extensively as the ABCT but are also associated with relatively hydrophobic molecules such as tetracycline (110).

The ABCT are composed of four protein domains: two membrane-spanning domains (MSD), each consisting of six or seven transmembrane-spanning segments, and two nucleotide binding domains (NBD) (119). The NBD of ABCT bind ATP through an ABC that consists of several conserved peptide motifs, including two Walker domains, a Signature domain and a Center domain. The ATP that is bound to the ABC is used as a source of energy for the ABCT, although the mechanism by which the ATP energy causes transport of the substrate molecule is unknown.

The MF do not contain NBD. They are composed primarily of 12 to 14 transmembrane segments (110, 140). The MF use the proton motive force of the membrane (gradient of H⁺ across the membrane) as a source of energy. In general, the MF work by antiport; that is, protons are pumped into the cell and substrate molecules are pumped out.

Recently, the complete sequencing of the genome of S. cerevisiae has allowed a determination of the total number of ABCT and MF in the genome of a eukaryotic cell. Thirty ABCT genes that contain an ABC were identified (194). Twenty-three of these genes appear to encode transmembrane segments, which suggests that they could function as drug pumps and are closely related to known ABCT drug pumps. Twenty-eight MF genes were also identified in the S. cerevisiae genome by homology to the five known MF genes of fungi (57). This suggests that S. cerevisiae contains at least 51 different genes encoding efflux pumps that could contribute to the drug resistance levels of the cell. There is good reason to believe that Candida and other fungi would have a similar number of efflux pumps in their genomes (see below).

Summary of Resistance to AzolesAll of the molecular mechanisms described above are summarized in Fig. 1. In a susceptible cell, azoles enter the cell by an unknown mechanism and target lanosterol demethylase, the target enzyme and product of the ERG11gene, which is part of the ergosterol biosynthetic pathway. Low-level expression of the CDR genes and the MDR1 gene is frequently observed. In a resistant cell, the azoles are blocked from interacting normally with the target enzyme because the enzyme can be modified and/or overexpressed. Mutations in other enzymes in the pathway (such as ERG3) can contribute to resistance. Both ABCT such as the CDR genes and MF such as theMDR1 gene can be overexpressed. The CDR genes appear to remove many azole drugs, while MDR1 appears to be specific for fluconazole.

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Fig. 1.

Molecular mechanisms of azole resistance. In a susceptible cell, azole drugs enter the cell through an unknown mechanism, perhaps by passive diffusion. The azoles then inhibit Erg11 (pink circle), blocking the formation of ergosterol. Two types of efflux pumps are expressed at low levels. The CDR proteins are ABCT with both a membrane pore (green tubes) and two ABC domains (green circles). The MDR protein is an MF with a membrane pore (red tubes). In a “model” resistant cell, the azoles also enter the cell through an unknown mechanism. The azole drugs are less effective against Erg11 for two reasons; the enzyme has been modified by specific point mutations (dark slices in pink circles) and the enzyme is overexpressed. Modifications in other enzymes in the ergosterol biosynthetic pathway contribute to azole resistance (dark slices in blue spheres). The sterol components of the plasma membrane are modified (darker orange of membrane). Finally, the azoles are removed from the cell by overexpression of the CDR genes (ABCT) and MDR (MF). The CDR genes are effective against many azole drugs, while MDR appears to be specific for fluconazole. Reprinted from reference 221 with permission of the publisher.

Resistance Is a Gradual Accumulation of Several AlterationsIn clinical isolates, it is unlikely that a single mutation will transform a susceptible strain into a highly resistant strain. Resistance usually arises after long periods in the presence of the drug, conditions that may support the gradual, stepwise development of resistance. It is more likely that resistance will gradually evolve over time as the result of several alterations due to continuous selective pressure from the drug. This suggests that several alterations will contribute to the final resistant phenotype. The accumulation of alterations has been documented for one series of 17 clinical isolates (Fig. 2) (222,223, 225). After the initial selection of a substrain of C. albicans (225), the MDR1 efflux pump was overexpressed (222). Subsequently, three simultaneous changes occurred, all of which involved the ERG11 gene: a point mutation (223), a gene conversion or mitotic recombination of the ERG11 point mutation converting both alleles to the mutant version (223), and overexpression ofERG11 (222). These changes resulted in an enzyme activity that was resistant to fluconazole. Finally, the CDRefflux pumps were overexpressed (222) (Fig. 2). Each of these alterations is likely to contribute in some way to the overall resistance phenotype of the final cell. At this time, there is no indication that the order of these alterations is important. It is likely that each would contribute to the resistance phenotype independent of the timing of the alteration. This gradual increase in resistance associated with specific alterations is an important mechanism of resistance, not only in C. albicans but probably also in other fungi, and it has been shown in a variety of other eukaryotic and prokaryotic systems (20).

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Fig. 2.

Time course of the development of fluconazole resistance in a patient with AIDS. Isolates are shown on the x axis in the order in which they were obtained from the patient. The dose that was administered to the patient (○) at the time each isolate was obtained is graphed at the bottom. Doses are given on a linear scale on the right axis in milligrams per day. MICs of fluconazole were determined by macrobroth dilution (□) and microbroth dilution (■). Only two differences were observed between the two methods. MICs of itraconazole (▴), ketoconazole (▵), and amphotericin B (⧫) were determined by E-tests and confirmed by the NCCLS macrobroth dilution method. MICs (in micrograms per milliliter) are given on a logarithmic scale on the left axis. Genetic changes that were identified are summarized above the graph and are described in detail in the text. Reprinted from reference 222 with permission of the publisher.

Cross-ResistanceAs described above, clinical isolates that are resistant to one azole are frequently cross-resistant to other azole drugs and can be cross-resistant to polyenes as well. With the current understanding of molecular mechanisms, predictions can be made about the molecular mechanisms that are associated with cross-resistance. Overexpression of the CDR genes is a common mechanism of resistance, and it appears that the CDR genes render a cell resistant to many different azoles while overexpression of MDR1 appears to be specific for fluconazole and is not associated with cross-resistance (169-171). R467K, the one point mutation inERG11 that is associated with resistance, appears to cause cross-resistance to other azoles but not to amphotericin B (222). It is not clear if specific point mutations inERG11 will always be associated with cross-resistance. Similarly, there has been insufficient analysis to determine if alterations in other enzymes in the ergosterol pathway will result in cross-resistance.

Unidentified MechanismsThe work in defining the mechanisms of antifungal drug resistance is still evolving. The mechanisms described above will probably be complemented by descriptions of other, as yet undefined mechanisms such as alterations in drug import or processing. As quickly as new drugs are developed, drug selection will favor new mechanisms that allow the cell to overcome the effects of these drugs.

Allylamine and Thiocarbamate ResistanceAllylamine resistance has not been reported for medically important fungi, although resistant strains have been described inS. cerevisiae and the plant pathogen U. maydis(201). Resistance to morpholines or thiocarbamates has not been reported. However, as with any antimicrobial agent, prolonged use will probably select for strain replacement with a resistant isolate or the development of secondary resistance in the original sensitive isolate.

5-Flucytosine ResistancePrimary or intrinsic resistance to 5-FC is a common phenomenon. Estimates suggest that 10% of C. albicans clinical isolates are intrinsically resistant and that 30% will develop secondary resistance (203). The genetics of 5-FC resistance has been investigated in several fungi. In diploid C. albicans, strains with partial resistance appear to be heterozygous for the resistance gene (FCY/fcy). Resistance appears to be a recessive characteristic, and the resistance phenotype (fcy/fcy) can result from further mutation or mitotic recombination of the heterozygote. Mutations in the haploid Cryptococcus neoformans are the most likely cause of 5-FC resistance in these yeasts. Genetic analysis has indicated that there are two genetic loci, FCY1 andFCY2, that can be mutated to produce 5-FC resistance (3).

Primary or intrinsic resistance to 5-FC is usually the result of a defect in cytosine deaminase (203). Secondary resistance to 5-FC in C. albicans is due primarily to a decrease in the activity of the uracil phosphoribosyl transferase (UPRTase), which is involved in the synthesis of FUMP and FdUMP (204, 219). Of the two serotypes of C. albicans, serotype B is more frequently associated with 5-FC resistance (201).

Resistance to 5-FC in C. neoformans can occur and usually results from the loss of UPRTase or cytosine deaminase activities (219). These enzymes make up the pyrimidine salvage pathway. The frequency of 5-FC resistance is lowest in C. albicans, intermediate in C. neoformans, and highest inAspergillus spp. (201).

Polyene ResistancePolyene resistance has not been a major clinical problem to date, although polyene-resistant isolates (resistant to nystatin or amphotericin B) have been reported and characterized. Intrinsic resistance to amphotericin B has been reported in Trichosporon beigelii, C. lusitaniae, C. guilliermondii,Pseudallescheria boydii, and certain dematiaceous fungi (3, 111). Amphotericin B-resistant clinical isolates ofC. tropicalis have also been found (112), and resistant strains of C. albicans, C. neoformans,Aspergillus nidulans, and A. fennelliae have been constructed in vitro (21, 71, 203). These strains were either exposed to chemical mutagenesis or serially passaged in media containing increasing amounts of polyene. Most polyene-resistant clinical isolates have a greatly reduced ergosterol content in their membranes (201). Based on an analysis of sterol composition, several clinical isolates of C. albicans may be defective inERG2 or ERG3 (21, 63, 87, 131, 201). Unfortunately, the stability of these mutations or the molecular defects that causes the azole resistance have not been described for any polyene-resistant mutation. One of these strains with an apparentERG2 defect appears to be unable to form amphotericin B-generated pores in the membrane (63). There is evidence to suggest that alterations in the membrane structure or in the sterol-to-phospholipid ratio in the membrane may be associated with resistance (21, 67).

At least three different types of amphotericin B-resistant mutants ofC. neoformans have been isolated, containing alterations in sterol content, alterations that cause cross-resistance to azoles, and alterations that are specific for amphotericin B (78). For most of these isolates, the molecular mechanisms that cause resistance have not been determined. Sterol analysis of one isolate suggested an alteration in ERG2 (87). Environmental isolates of C. neoformans that were serially passaged in a murine model of cryptococcosis became less susceptible to amphotericin B (31). This suggests that the resistance levels of a fungal population can be altered by interactions with the host.

DosingAt this time, antifungal drug resistance is clearly becoming a common problem in patients who have a history of antifungal drug use. However, it is not clear what dosing strategies should be used during prophylaxis or treatment to avoid the development of resistance. Azoles, for example, should probably not be used prophylactically for oral or vaginal candidiasis since they may predispose to resistance. However, azole maintenance therapy is be required to prevent the recurrence of cryptococcal meningitis in patients with HIV infection. Several variables need to be considered when trying to minimize the risk for development of resistance, including intermittent versus continuous dosing, the amount of drug administered, the length of treatment, and the immune status of the patient. At this time, none of these factors has been evaluated for its contribution to antifungal resistance, and a large clinical trial by the Mycoses Study Group and the AIDS Clinical Trial Group is under way to evaluate some of these factors for azole use in the HIV-infected population. From experience in other biological systems where drug resistance is a problem, one would predict that continuous treatment with a high dose of drug for as brief a time as possible to effect a cure would result in the least risk for development of resistance, but these factors must be evaluated in clinical trials targeted to specific drugs, infections, and patient populations.

DetectionThe NCCLS method for the determination of antifungal susceptibility is currently a standardized technique that can be used to determine the levels of resistance of a yeast strain. The current technique includes both macro- and microdilution broth methods to determine the MIC of a drug. Efforts to make the procedure applicable to a variety of medically important fungi will doubtless improve the value of this technique. However, it will probably remain a time-consuming technique. Quicker methods for the determination of resistance would clearly be welcomed in a clinical laboratory. However, the molecular mechanisms detailed in Fig. 1 suggest that a molecular assay for resistance is unlikely to be simple, because it would have to monitor all of the mechanisms so far described to date and new mechanisms as they are discovered. One alternative that should be explored is to use molecular methods to assess the overall fitness of a cell in the absence and presence of a drug. Such a technique might prove quicker and easier than monitoring all possible mechanisms.

Drug DevelopmentThere is a clear need for the next generation of antifungal agents. However, it is important to incorporate what is currently known about antifungal drug resistance into the development of new drugs if those resistance mechanisms are to be avoided. For instance, new and improved azole drugs such as voriconazole should be carefully monitored to determine if they are substrates for the efflux pumps that are already causing resistance to fluconazole (48). This will be difficult since there are probably a large number of efflux pumps in the fungal genome and it is impossible to test each of these pumps at present (see above). New classes of antifungal drugs that are active against azole-resistant isolates, such as the cationic peptide histatin (197), will clearly be important for future treatment strategies.

The broad substrate specificity of the efflux pumps is likely to be a problem even for drugs unrelated to the azoles. Clearly, any new drug should be tested as a substrate for the currently available efflux pumps. However, it is important to keep in mind that these pumps are only a small subset of the efflux pumps encoded in the genome (see above).

One important aspect to keep in mind when examining the molecular mechanisms of antifungal drug resistance is the potential for cross-resistance to the polyenes, especially amphotericin B. Since amphotericin B is the “gold standard” for antifungal drug therapy, it is important to carefully monitor isolates for cross-resistance to this drug. Any therapy or drug that is prone to cause cross-resistance to amphotericin B will have profound negative effects on our ability to treat those fungal infections.

Perhaps the most interesting new antifungal drug target would be the efflux pumps themselves. Any drug that inhibits the activity of these pumps would probably increase the intracellular concentration of drugs such as the azoles, thereby increasing the effectiveness of those drugs. Unfortunately, the currently available inhibitors of these efflux pumps are not effective against fungi, perhaps because the drugs cannot penetrate the fungal cell wall. Clearly, modification of these drugs will be necessary to get the efflux pump inhibitors into the cell.

It is likely that the future of antifungal drug therapy lies in drug combinations. Some drug combinations, such as amphotericin B and 5-FC, are currently in use (93, 216), and others, such as amphotericin B and azoles (165, 192), azoles and 5-FC (40, 127), and azoles and terbinafine (7), have been discussed and are being tested. With the development of new classes of antifungal drug, such as the cell wall inhibitors, and with the knowledge of molecular mechanisms of azole resistance, other potential combinations of drugs are likely to be tested and found effective. One combination that was alluded to above is treatment with azole drugs and an efflux pump inhibitor that is effective against fungal cells.

Other StrategiesThe success or failure of antifungal drug therapy is dependent not only on the drug, but also on several other factors (Table 3). Perhaps the best way to improve antifungal drug therapy is to improve the immune status of the host. In many cases, this is not possible, but there are situations in which improving host function may improve outcome. Cytokines such as granulocyte colony-stimulating factor may be useful in the treatment of fungal disease (3, 125). A combination of azoles and cytokines may be an important therapeutic strategy for fungal infections in immunocompromised individuals (147).

Another important strategy in treating fungal disease is the elimination of the disease focus. The removal of fungus-contaminated foreign objects or the surgical removal of abscesses can also reduce the fungal burden and allow the host and antifungal drugs to clear the infection, even with strains that are resistant to standard antifungal therapy.

Finally, new drug delivery systems such as lipid formulations of amphotericin B may have a place in the treatment of antifungal-drug-resistant infections. It is possible that different drug delivery systems will alter the way a yeast cell interacts with a drug and that this will have a negative or positive effect on the development of azole resistance.