Antifungal drug susceptibility testing and resistance in Aspergillus

Warnock et al.

Antifungal drug susceptibility testing and resistance in Aspergillus

Table 1 Drugs available and in development for the treatment of aspergillosis Class

Agent

Comments

Polyenes

Amphotericin B Nystatin

Azoles

Itraconazole Voriconazole

Systemic, injectable Systemic, injectable liposomal formulation in phase II development Systemic, oral and injectable Systemic, oral and injectable, in phase III development Systemic, oral and injectable, in phase III development Systemic, injectable, in phase III development Systemic, injectable, in phase II development

David W.Warnock, Beth A.Arthington-Skaggs, Ren-Kai Li Mycotic Diseases Branch, Centers for Disease Control and Prevention,Atlanta, USA

SCH 56592

Abstract Aspergillus species are the most common causes of invasive mold infections in immunocompromised patients.The introduction of new antifungal agents, and recent reports of resistance emerging during treatment of aspergillus infections, have highlighted the need for standardized methods of antifungal drug susceptibility testing for filamentous fungi.This review describes the methods that are now being developed for the in vitro testing of Aspergillus species, and the results of attempts to correlate in vitro findings with in vivo outcome. The mechanisms and clinical importance of resistance to the different agents used in the treatment of human aspergillosis are discussed.

INTRODUCTION olds of the genus Aspergillus are among the most common fungi in the human environment, being found in the air, in the soil, on plants and on decomposing organic matter.1 In non-immunocompromised individuals, these fungi can cause localized infection of the lungs, sinuses or other sites. In immunocompromised persons, inhalation of spores gives rise to invasive infection of the lungs or sinuses and dissemination to other organs often follows.2 This condition is termed invasive aspergillosis (IA). Despite the development of new antifungal agents, the death rate from this infection remains high, often in the order of 80% or more.3 Too often, this is because the disease is not diagnosed in sufficient time to be of help in patient management.At least 20 species of Aspergillus have been reported to cause human disease, including A. flavus, A. terreus, A. nidulans and A.niger.2 However, the most common pathogen is A. fumigatus, which accounts for about 90% of cases. Therapeutic failure due to antifungal drug resistance still appears to be an uncommon problem among patients with aspergillosis. However, the lack of reliable methods of in vitro testing has hindered the detection of drug-resistant strains of Aspergillus species. This review will describe the methods now being developed for the in vitro testing of these and other molds, and the results of attempts to correlate in vitro findings with in vivo outcome.The mechanisms and clinical importance of resistance to the various agents used in the treatment of human aspergillosis will also be discussed.

M

ANTIFUNGAL AGENTS AND ASPERGILLOSIS The antifungal agents available and in development for the treatment of aspergillus infections in humans can be divided 326

Drug Resistance Updates (1999) 2, 326–334

 1999 Harcourt Publishers Ltd

Echinocandins Caspofungin LY 303366

into three groups on the basis of their molecular mechanisms of action (Table 1). The polyene compounds, which include amphotericin B and nystatin, bind to ergosterol, the principal sterol in the cell membrane of susceptible fungi.4 This binding results in disorganization of the membrane, causing an impairment of its barrier function and rendering it permeable to protons and monovalent cations, eventually leading to cell death. To gain access to ergosterol, however, amphotericin B must first pass through the rigid cell wall, composed primarily of chitin and glucans. How this is accomplished and the role these compounds play in amphotericin B resistance has not been elucidated. It is now thought that amphotericin B may have more than one mechanism of action on susceptible fungal cells.5 There have been several reports indicating that the lethal effects of higher concentrations of the drug are not simply a consequence of its membrane-permeabilizing effects, but involve oxidative damage to the cells. This may be due to auto-oxidation of the drug with formation of free radicals, but the precise mechanism remains to be clarified.5 The azole compounds, which include itraconazole and voriconazole, inhibit the fungal cytochrome P-450 3A-dependent enzyme, lanosterol 14-alpha-demethylase, thereby interrupting the conversion of lanosterol to ergosterol.This leads to an accumulation of methylated sterols and a depletion of ergosterol in the fungal cell membrane, resulting in disruption of membrane structure and function with subsequently increased permeability and inhibition of cell growth and replication.6 Amphotericin B remains one of the most effective agents available for the treatment of IA despite the fact that its clinical use is limited by a range of serious side-effects, particularly renal damage.7 To overcome these problems, three new lipid-based parenteral formulations of amphotericin B have been developed.These preparations differ in the proportion of amphotericin B and lipid used, as well as in physicochemical form, tissue distribution, blood clearance and acute infusion-related reactions. These preparations are less nephrotoxic, but much more expensive, than the conventional deoxycholate formulation of amphotericin B.8 The

Drug resistance in Aspergillus overall success rate for amphotericin B treatment of IA is about 34%, but varies substantially between different patient groups.2 The three lipid-based formulations (in doses of 4–5 mg/kg per day) appear to be as efficaceous as the conventional preparation (in doses of 1 mg/kg per day), but not more so.2 Nystatin is another broad-spectrum polyene antifungal agent. However, toxic side-effects following parenteral administration have limited its clinical application to the topical treatment of mucosal and cutaneous forms of candidiasis.9 Recent studies have shown that incorporation of the drug into a multilamellar liposomal preparation reduces its toxic side-effects while preserving its antifungal activity in vitro.10 Other work indicates that, although liposomal nystatin is less active than amphotericin B in vitro, it is effective against A. fumigatus and A. flavus.11 Initial evaluations have indicated that liposomal nystatin is effective and well tolerated in the treatment of neutropenic animals with disseminated A. fumigatus infection.12,13 It was also reported to be well tolerated in a phase I clinical trial involving febrile neutropenic patients,14 although little is known about the effectiveness of the drug in this patient population. In 1990, the triazole agent itraconazole was licensed for the oral treatment of IA. The overall success rate with itraconazole in patients with IA is about 30%, but substantial variations have been seen between different patient groups.15 Variable absorption has been a serious problem with this agent, particularly in bone marrow transplant (BMT) recipients, but a new cyclodextrin solution formulation is better absorbed by these individuals.16 An intravenous formulation of itraconazole is now available. Voriconazole is a new triazole agent currently in phase III clinical trials. In vitro, voriconazole has a broad spectrum of antifungal activity including Aspergillus species and other molds.17–22 The favorable in-vitro activity of voriconazole against Aspergillus species has been confirmed in several animal models of infection17,23,24 as well as in several phase II clinical trials.25,26 Another new broad-spectrum triazole agent, SCH 56592, has also been reported to have good in vitro activity against Aspergillus species and other molds.27–29 In animal models, SCH 56592 has been shown to be superior to itraconazole and amphotericin B in the treatment of aspergillosis in both normal and immunocompromised animals.27,30–33 The echinocandins are a new class of lipopeptide antifungal agents that inhibit fungal β-(1,3)-glucan synthetase located in the cell membrane, leading to depletion of cellwall glucan in growing cells and lysis of the fungal cell.34 Cilofungin, the first semisynthetic echinocandin antifungal agent reached phase II clinical trials, but was abandoned because of side-effects associated with the carrier used for the parenteral formulation. It was reported that Aspergillus species were resistant to cilofungin in vitro, although this appeared to depend on the test conditions used.35,36 In contrast, tests with animal models of disseminated infection showed that cilofungin was active against A. fumigatus in vivo,37 and a correlation was obtained between the effect of the drug on A. fumigatus glucan synthetase and the effective dose 50% obtained in a murine model.38 More recently, several second-generation echinocandin derivatives have been

synthesized and studied. One of these, LY 303366, has undergone phase I and II clinical trials. In vitro, LY 303366 has been found to have a broad spectrum of activity, including A. fumigatus.39–41 It has demonstrated promising activity in murine models of disseminated aspergillosis.42–44 Another new echinocandin compound, caspofungin (MK 0991, formerly L 743872), has been selected for clinical development, and phase III clinical trials are progressing. In vitro, this agent has demonstrated potent fungicidal activity against A. fumigatus and other molds.41,45–47 In vivo, the parenteral form of caspofungin has proved as effective as amphotericin B in murine models of disseminated and pulmonary aspergillosis.48–50 IN VITRO SUSCEPTIBILITY TESTING OF ASPERGILLUS The introduction of new antifungal agents such as voriconazole, and recent reports of resistance to itraconazole developing during treatment of IA51,52 have combined to increase the need for reliable methods of in vitro testing that can assist in the clinical use of these compounds. The development of susceptibility testing methods for filamentous fungi has lagged behind that for Candida species and Cryptococcus neoformans.Among the reasons for this slow development was the low incidence of serious mold infections before the 1980s as well as the limited number of available therapeutic agents. Furthermore, because acquired drug resistance did not appear to be a major contributing factor in amphotericin B treatment failure in mold infections such as aspergillosis, there was no great interest in developing in vitro methods that could predict the clinical outcome of treatment. As with antibacterial compounds, tests designed to ascertain the minimum amount of drug needed to inhibit the growth of fungal strains in culture (minimum inhibitory concentration or MIC) are often used to determine the relative effectiveness of different antifungal agents and to detect the development of drug-resistant organisms. One of the most popular methods is the broth macrodilution method, but broth microdilution and agar dilution methods are also used for filamentous fungi.There are no defined standards to distinguish drug-resistant strains, but it is often assumed that strains that for which the MICs of a particular agent or group of agents are much higher than with other stains of the same organism, tested under similar conditions, can be regarded as resistant.The usefulness of this artificial distinction depends in major part on the extent of the correlation between MIC test results and clinical outcome. It has long been recognized that the poor correlation noted between in vitro tests and the results of treatment with antifungal agents in vivo has often been due to technical problems in achieving reproducible MICs. Numerous studies attest to the fact that results are influenced by a number of technical factors, including the concentration of the fungal inoculum, the composition and pH of the medium, the incubation temperature, and the length of incubation.53–57 Another problem encountered with azole compounds results from the fact that these drugs often cause partial inhibition of growth over a wide range of concentrations, making visual determination of endpoints in MIC tests difficult.  1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 326–334

327

Warnock et al. In vitro tests are further complicated by problems such as the slow growth rate of some molds and the ability of certain dimorphic organisms to grow either as a unicellular form or as a hyphal form, depending on the test conditions. In 1997, the National Committee for Clinical Laboratory Standards (NCCLS) published an approved reference method (M27-A) for determining the MICs of amphotericin B, flucytosine, fluconazole, and ketoconazole for Candida species and C. neoformans.58 The essential features of the procedure are the use of a broth macrodilution format, a defined culture medium (RPMI-1640) buffered to pH 7.0 with MOPS, an inoculum standardized by spectrophotometric reading to 500–2500 colony forming units (CFU) per ml, and incubation at 35°C for 48 h for Candida species or 72 h for C. neoformans.Although identical incubation conditions are used for the four antifungal agents, different definitions of endpoint are used. For amphotericin B, the endpoint is the lowest drug concentration at which no growth is visible. For flucytosine and the azoles, the endpoint is the lowest concentration at which growth is reduced to 20% of the control.The NCCLS M27-A document also describes a broth microdilution procedure that is easier to perform than the macrodilution reference method and which gives comparable results. The NCCLS M27-A reference method has been a useful starting point for the development of a standardized method for susceptibility testing of filamentous fungi.To accomplish this, studies have been performed to determine the optimum test medium, the most reproducible method of inoculum preparation, the optimum incubation conditions, and the best method of endpoint determination. Of these, perhaps the most important issue that has had to be resolved is which form of the mold should be used for inoculum preparation: ungerminated spores, germinated spores, or hyphal fragments. It is reasonable to suppose that the mechanisms involved in inhibiting conidial germination are different from those for preventing hyphal elongation. However, the difficulties of producing uniform hyphal fragment suspensions have led most investigators to use spores as the inoculum for in vitro testing with antifungal agents despite the fact that these structures are seldom found in vivo.A recent report demonstrated that germinated and ungerminated conidia of A. fumigatus give almost identical test results with various antifungal agents.59 These findings contrast with those of an earlier report that compared the MICs of five antifungal agents, including amphotericin B and itraconazole, obtained with conidial and hyphal inocula of Scopulariopsis, Paecilomyces and Cladophialophora species.60 MICs obtained with hyphal inocula were substantially higher than those obtained with conidial inocula.This may be explained, at least in part, by differences in cell wall composition at different stages of growth.Therefore, although a conidial inoculum is easier to prepare and standardize than a hyphal fragment suspension, the results may be less predictive of clinical outcome. It is well recognized that the composition of the test medium can have a profound effect on the MICs of various antifungal agents against Candida species and C. neoformans.61 To determine whether this is also an important factor for in vitro testing of molds, Denning et al.51 tested six A. fumigatus strains, two of which were judged to be resistant 328

Drug Resistance Updates (1999) 2, 326–334

 1999 Harcourt Publishers Ltd

to itraconazole, using five different media each in agar dilution and broth microdilution formats. Other test parameters, including inoculum concentration, temperature, and length of incubation, were standardized.The results suggested that either an agar dilution method with RPMI-1640 medium or a broth microdilution method with RPMI-1640 medium with 2% glucose best differentiated the itraconazole-resistant strains of A. fumigatus from the susceptible ones.51 The initial inoculum concentration is another factor that has the potential to influence the MICs of antifungal agents for Aspergillus species and other molds. Increasing the inoculum concentration has a minimal effect on the MICs of amphotericin B for A. fumigatus and A. flavus.62 In contrast, a significant inoculum effect has been observed with itraconazole,51,62 although this differs according to the species of mold under test. Gehrt et al.62 found that the MICs of itraconazole for Scedosporium apiospermum depended more on the inoculum concentration than did those for A. fumigatus, A. flavus or Fusarium solani. These findings indicate that inoculum concentration is a critical factor in developing a standardized test for molds and that the design of the test should allow for adjustment of the inoculum concentration depending on the test organism and test agent. Use of a spectrophotometer has been shown to facilitate the preparation of reproducible conidial suspensions.63–64 The NCCLS sub-committee on antifungal susceptibility testing has conducted a large multicenter investigation which confirmed that consistent MICs could be obtained with a standardized inoculum of about 10 000 CFU per ml in a microdilution format with buffered RPMI-1640 medium and an incubation period of 48–72 h at 35°C.65 These test conditions have been incorporated into a proposed NCCLS reference method (M38-P) for susceptibility testing of conidium-forming filamentous fungi, including Aspergillus species.66 The essential features of the proposed procedure include the use of a broth microdilution format, a defined culture medium (RPMI-1640) buffered to pH 7.0 with MOPS, an inoculum standardized by spectrophotometric reading to 4000–50000 CFU per ml, and incubation at 35°C for 24–72 h. For amphotericin B, the endpoint is the lowest drug concentration at which no growth is visible. For flucytosine and the azoles, the endpoint is the lowest concentration at which growth is reduced to 50% or less of the control. As with Candida species and C. neoformans, incorporation of the oxidation-reduction indicator Alamar Blue has facilitated endpoint determination.64,65 SIMPLIFIED METHODS OF IN VITRO TESTING The development of a proposed NCCLS reference method for in vitro testing of conidium-forming molds has provided a useful standard against which possible alternative methods can be evaluated. Microdilution methods are time-consuming and labor-intensive, and there is a need for simpler and more economical methods for the susceptibility testing of these organisms. The Etest (AB Biodisk, Solna, Sweden) is a patented commercial method for the quantitative determination of MICs. It is set up in a manner similar to a disc-diffusion test, but the disc is replaced with a calibrated plastic strip impregnated

Drug resistance in Aspergillus with a continuous concentration gradient of the antimicrobial agent. Several investigators have reported good agreement between Etest and broth microdilution MICs of amphotericin B and itraconazole for A. fumigatus and A. flavus.67–70 The most recent comparison70 showed 100% agreement (results within two doubling dilutions) for 10 isolates each of A. fumigatus and A. terreus tested against amphotericin B and itraconazole. The results of the two methods for A. flavus and A. niger showed 90–100% agreement for itraconazole and 60–100% agreement for amphotericin B. The Etest and broth microdilution MICs of itraconazole for three itraconazole-resistant strains of A. fumigatus were similarly high (>32 and >16 µg/ml). 70 These reports suggest that the Etest might be suitable for routine testing of Aspergillus species, but further work is needed to confirm that it can detect potential resistant isolates.

in vitro results with outcome in a neutropenic mouse model of infection showed that itraconazole was active against four isolates with MICs of 0.12–1 µg/ml but ineffective in vivo against the two treatment-failure isolates, both of which had itraconazole MICs of ≥16 µg/ml. Lass-Florl et al.78 observed a similar trend for amphotericin B. In their study, the MICs of 29 isolates from BMT recipients who developed IA (12 A. flavus infections, nine A. terreus infections, and eight A. fumigatus infections) were compared with clinical outcome to see if there was a correlation. Irrespective of the species, all six patients with isolates for which the MIC was <2 µg/ml survived, whereas 22 of the 23 patients with isolates resistant to ≥2 µg/ml died. These reports of an association between MICs and clinical outcome are encouraging, but further studies with animals and patients will be required for a more definitive evaluation and for development of interpretive MIC breakpoints.

CORRELATION OF IN VITRO TEST RESULTS WITH THERAPEUTIC OUTCOME

ANTIFUNGAL DRUG RESISTANCE IN ASPERGILLUS

The principal objective of in vitro susceptibility testing is to predict the outcome of treating a patient’s infection with a particular antimicrobial agent. For Candida spp. and C. neoformans, good correlation has been seen between MICs determined by the NCCLS broth dilution method and clinical outcome in animal models of candidiasis and cryptococcosis.71–73 There is also evidence of a correlation between MICs of fluconazole and therapeutic response in HIV-positive patients with oral candidiasis.74,75 In contrast, fluconazole MICs did not correlate with clinical outcome in non-neutropenic patients with candidemia.76 The discrepancies included treatment failure among patients with infections caused by organisms with low MICs, and success despite infection with organism with high MICs for fluconazole. Clearly, other factors are more important than MIC in determining the outcome of this infection in this patient group. The incidence of invasive mold infections is too low to permit large-scale prospective comparisons of antifungal drug MICs with the clinical outcome of treatment. For this reason, several investigators have sought to confirm the relevance of in vitro test results in animal models of infection. Odds et al.77 tested nine mold isolates, including one of A. fumigatus and one of A. flavus, in two animal models. However, because of difficulties in establishing reproducible, fatal infections with some of the molds, the number of animals that could be evaluated was fewer than expected. For the infections that could be evaluated, some degree of response to treatment with itraconazole was apparent in animals infected with the two aspergillus isolates, both of which were judged to be susceptible to this agent in vitro (MICs of 0.25 and 0.5 µg/ml). On the other hand, amphotericin B was active against the A. fumigatus isolate in vivo, but not against the A. flavus isolate, even though both isolates had amphotericin B MICs of 1 µg/ml. Several investigators have reported a general association of high MICs with an unfavorable clinical outcome in patients with aspergillosis.51,52,78 Denning et al.51,52 conducted in vitro and in vivo tests with six A. fumigatus isolates, two of which were from patients whose infection had not responded to itraconazole treatment. Comparison of the

There are many reasons why patients with IA may fail to respond to amphotericin B treatment.3 Many persons with underlying immunologic impairment do not respond to treatment until their neutrophil count recovers. Delays in starting amphotericin B also account for a number of treatment failures. Experience to date indicates that most isolates of A. fumigatus, the commonest cause of IA, are susceptible to amphotericin B in vitro, with MICs of 1 µg/ml or less.11,17–22,29,79 Only the work of Lass-Florl et al.78 suggests that a significant proportion (28%) of isolates of A. fumigatus have amphotericin B MICs of 2 µg/ml or more, and that this might correlate with clinical failure of treatment with this agent. It should be emphasized, however, that interpretive breakpoints have not been established at present. Most isolates of A. fumigatus appear to be susceptible to itraconazole and voriconazole (MICs of 1 µg/ml or less).17–22,29,79 To date, only one instance of clinical resistance to itraconazole in A. fumigatus has been reported.52 This developed in two immunocompromised patients following prolonged oral treatment with the agent.Therapeutic failure correlated with elevated MICs in vitro and in vivo resistance to itraconazole in a neutropenic mouse model of infection.51,52 In a subsequent investigation, mice infected with one of these itraconazole-resistant isolates of A. fumigatus were treated with the new triazole agent, SCH 56592.80 The lower survival rate of these animals, compared with mice infected with an azole-susceptible A. fumigatus isolate, suggests a degree of cross-resistance between the two triazole antifungal agents. In contrast, three A. fumigatus isolates that had MICs of >16 µg/ml of itraconazole were found to have MICs of 0.5–1 µg/ml of voriconazole.21 This was within the range of MICs for all isolates tested against voriconazole, suggesting no cross-resistance between this agent and itraconazole. At least 20 species of Aspergillus other than A. fumigatus have been reported to cause invasive infection, including A. flavus, A. terreus, A. niger and A. nidulans.2 In vitro studies have shown that A. terreus is less susceptible to amphotericin B than are other Aspergillus species.21,28,70,78 MICs of amphotericin B for A. terreus isolates have ranged from 4 to  1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 326–334

329

Warnock et al. 32 µg/ml.21,28,70,81 Indeed, Lass-Florl et al.78 suggest that all isolates of A. terreus are resistant to amphotericin B in vitro and that this is predictive of a fatal outcome. In vitro results suggest that voriconazole might be useful for the treatment of A. terreus infections that fail to respond to amphotericin B, but this has still to be confirmed.81 Schaffner and Bohler,82 reported a case of aspergillosis in which the patient, who had received prior treatment with itraconazole, failed to respond to amphotericin B. The authors speculated that the azole agent caused depletion of membrane ergosterol and this resulted in reduced binding of the polyene to the fungal cell membrane.The phenomenon was reproduced in vitro and in an animal model of A. fumigatus infection. In further tests, amphotericin B was found to have lost its in vitro effect on six A. fumigatus isolates after these were exposed to sub-fungicidal concentrations of itraconazole.82 There is at least one other report which suggests that azoles given prior to amphotericin B can antagonize the antifungal effect of amphotericin B in an animal model of aspergillosis. Schaffner and Frick83 demonstrated that when rats given prior treatment with ketoconazole were exposed to amphotericin B, the effect of the polyene was reduced. Clinical reports relating to potential interactions between amphotericin B and the azoles are hard to find.Apart from the case of Schaffner and Bohler,82 there appear to have been no other reports of possible antagonistic interactions between amphotericin B and itraconazole in patients with aspergillosis who had received prior treatment with the azole agent. On the other hand, several reports have noted that patients with IA who were treated with itraconazole and who had received prior amphotericin B treatment had similar outcomes to those who had not received amphotericin B.15,84 To date, there have been no reports of resistance to other classes of antifungal agents, such as the echinocandins developing during treatment. Of interest, the new echinocandin agent, LY 303366, was found to be effective against an amphotericin-B-resistant. A. fumigatus isolate in a neutropenic mouse model of infection.44 MECHANISMS OF DRUG RESISTANCE IN ASPERGILLUS The predominant mechanism of resistance to polyene antifungal agents is depletion of membrane ergosterol, the principal target of these antifungal compounds (Table 2).4 Although there have been few reports of amphotericin B resistance developing during treatment of aspergillus infection, resistance has been reported in laboratory strains of A. nidulans85 and A. fennelliae.86 These had been ‘trained’ to tolerate increasing concentrations of amphotericin B by Table 2 Mechanisms of drug resistance in Aspergillus Drug class

Mechanism

Reference

Polyenes

Depletion of membrane ergosterol Increased cell wall glucan Increased catalase Altered lanosterol 14-alpha-demethylase Decreased azole accumulation

4,87 88,89 90 52 52,92

Azoles

330

Drug Resistance Updates (1999) 2, 326–334

 1999 Harcourt Publishers Ltd

being transferred to media that contained increasingly higher concentrations of the drug. Genetic analysis of these mutants suggested that resistance was due to a single gene mutation.85 Mechanisms of amphotericin B resistance have been elucidated in both clinical and laboratory isolates of Aspergillus species. Kim et al.87 reported that polyene-resistant mutants of A. fennelliae contained delta 7,22,24(28)-ergostatrien-3-βol and delta 7,22-ergostadien-3-β-ol as the major sterols whereas the wild-type strains contained ergosterol. Mechanisms of amphotericin B resistance distinct from membrane sterol alterations have also been described.These include reduced access of the drug to the cytoplasmic membrane due to increased fungal cell wall glucan content88,89 and increased catalase activity, which may diminish oxidative damage caused by this antifungal agent.90 Amphotericin-B-resistant strains may or may not show cross-resistance to other polyenes, such as nystatin, and the cross-resistance may be complete or partial. Mutant strains of A. fumigatus showing in vitro resistance to amphotericin B have been shown to be cross-resistant to nystatin.91 On the other hand, Kim et al.86 described an A. fennelliae strain that demonstrated nystatin resistance without cross-resistance to amphotericin B.While specific resistance to amphotericin B can be theoretically explained by a specific loss of cell membrane ergosterol accompanied by the persistence of precursors to which nystatin can still bind, specific resistance to nystatin cannot solely be due to a block in ergosterol biosynthesis.This is because strains that lack the sterol precursors should be even more resistant to amphotericin B. Specific resistance to nystatin may be due to induction of an enzyme that degrades this agent, as has been observed in certain dermatophyte fungi such as Trichophyton mentagrophytes and Microsporum gypseum.92 Itraconazole and other azole agents act through inhibition of a cytochrome P-450 3A-dependent enzyme, lanosterol 14-alpha-demethylase (14DM), required for ergosterol biosynthesis.6 Resistance mechanisms to azole antifungal agents fall into several general categories:93,94 first, mutations in the 14DM gene which affect binding to the azoles; second, over-expression of 14DM as a result of increased transcription and/or increased gene dosage due to chromosome duplication; third, mutations in ergosterol biosynthetic genes other than 14DM (such as C56 sterol desaturase); fourth, reduced permeability of the drug into the cell; and fifth, increased efflux of the drug out of the cell. To date, only one report has documented clinical resistance to itraconazole in A. fumigatus.52 Two distinct mechanisms of resistance were identified among the isolates obtained from the two patients involved: in one an alteration in the 14DM gene caused reduced binding of the target enzyme to itraconazole; and in the second, reduced intracellular concentrations of itraconazole were detected, possibly due to increased drug efflux.52 A similar efflux mechanism has also been described in fenarimol-resistant mutants of A. nidulans.95 Van den Brink et al.96 transformed the 14DM gene from a Penicillium italicum strain into an azole-susceptible A. niger strain.This resulted in an azole-resistant mutant that demonstrated increased 14DM expression. Increasing the number

Drug resistance in Aspergillus of integrations of the P. italicum 14DM into the genome of the A. niger strain increased the level of azole resistance.96 However, the level of resistance achieved by over-expression of the 14DM gene tended to remain lower than that observed for other resistance mechanisms, such as altered efflux, alterations in the 14DM itself, or with other resistanceconferring lesions located elsewhere in the sterol biosynthetic pathway. NADPH-cytochrome P450 reductase (Cpr) serves as the redox partner of 14DM, supplying electrons required for oxidative removal of the C-14 methyl group from the sterol substrate by 14DM.Van den Brink et al.96 have demonstrated a potential role for Cpr in azole resistance in A. niger. In their study, an A. niger strain that over-expressed Cpr showed a modest decrease in etaconazole susceptibility. Increasing the copy number of both the 14DM gene (from P. italicum) and the Cpr gene (from A. niger) in the A. niger strain resulted in a greater decrease in susceptibility than the effect of the sum of the individual genes, suggesting the combination had a synergistic effect.Whether this resistance mechanism occurs or has a role in other Aspergillus species remains to be determined. CONCLUSIONS Treatment failure among patients with aspergillosis, attributable to the development of resistant strains of Aspergillus species, still seems to be an uncommon clinical problem. However, given the increasing number of patients requiring treatment for this infection, it is becoming more important to develop reliable methods for in vitro testing of antifungal agents that can detect resistant strains.The advent of a proposed reference method for susceptibility testing of Aspergillus species and other conidium-forming fungi is a significant step forward, but it remains to be seen whether clear correlations can be established between in vitro results and clinical outcome. Given the multifactorial nature of the host-pathogen interaction in immunocompromised patients with aspergillus infection, the prospects appear limited. Nonetheless, now that reports of resistance developing during treatment for aspergillosis are starting to appear, more aggressive performance of cultures to isolate aspergillus for species-specific identification and susceptibility testing is Received 26 June 1999; Revised 5 August 1999;Accepted 9 August 1999 Correspondence to: David W.Warnock, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop C-09,Atlanta, Georgia 30333, USA.Tel: +1 404 639 3053; Fax: +1 404 639 2780; E-mail: [email protected]

becoming appropriate. References 1. Nolard N, Detandt M, Beguin H. Ecology of Aspergillus species in the human environment. In:Vanden Bossche H, Mackenzie DWR, Cauwenbergh G, (Eds).Aspergillus and Aspergillosis. New York: Plenum Press, 1988; 35–41. 2. Denning DW. Invasive aspergillosis. Clin Infect Dis 1998; 26: 781–805.

3. Denning DW.Therapeutic outcome in invasive aspergillosis. Clin Infect Dis 1996; 23: 608–615. 4. Kerridge D. Mode of action of clinically important antifungal drugs. Adv Microb Physiol 1986; 27: 1–72. 5. Brajtburg J, Powderly WG, Kobayashi GS, Medoff G.Amphotericin B: current understanding of mechanisms of action.Antimicrob Agents Chemother 1990; 34: 183–8. 6. Vanden Bossche H. Biochemical targets for azole antifungal derivatives: hypothesis on the mode of action. In: McGinnis M (Ed). Current Topics in Medical Mycology. New York: Springer-Verlag, 1985; 313–351. 7. Gallis, HA, Drew RH, Pickard WW.Amphotericin B: 30 years of clinical use. Rev Infect Dis 1990; 12: 308–329. 8. Wong-Beringer A, Jacobs RA, Guglielmo BJ. Lipid formulations of amphotericin B: clinical efficacy and toxicities. Clin Infect Dis 1998; 27: 603–618. 9. Hamilton-Miller JMT. Chemistry and biology of the polyene macrolide antibiotics. Bacteriol Rev 1973; 37: 166–196. 10. Mehta RT, Hopfer RL, Gunner LA, Juliano RL, Lopez-Berestein G. Formulation, toxicity, and antifungal activity in vitro of liposomeencapsulated nystatin as therapeutic agent for systemic candidiasis. Antimicrob Agents Chemother 1987; 31: 1897–1900. 11. Johnson EM, Ojwang JO, Szekely A,Wallace TL,Warnock DW. Comparison of in vitro antifungal activities of free and liposomeencapsulated nystatin with those of four amphotericin B formulations.Antimicrob Agents Chemother 1998; 42: 1412–1416. 12. Wallace TL, Paetznick V, Cossum PA, Lopez-Berestein G, Rex JH, Anaissie E.Activity of liposomal nystatin against disseminated Aspergillus fumigatus infection in neutropenic mice.Antimicrob Agents Chemother 1997; 41: 2238–2243. 13. Groll AH, Gonzalez CE, Giri N et al. Liposomal nystatin against experimental pulmonary aspergillosis in persistently neutropenic rabbits: efficacy, safety and non-compartmental pharmacokinetics. J Antimicrob Chemother 1999; 43: 95–103. 14. Boutati E, Maltezou HC, Lopez-Berestein G,Vartivarian SE,Anaissie E. Phase I study of maximum tolerated dose of intravenous liposomal nystatin for the treatment of refractory febrile neutropenia in patients with hematological malignancies. In: Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1995; abstract LM22, p. 330. 15. Denning DW, Lee JY, Hostelter JS et al. NIAID Mycoses Study Group multicenter trial of oral itraconazole therapy for invasive aspergillosis.Am J Med 1994; 97: 135–144. 16. Prentice AG,Warnock DW, Johnson SAN, Phillips MJ, Oliver DA. Multiple dose pharmacokinetics of an oral solution of itraconazole in autologous bone marrow transplant recipients. J Antimicrob Chemother 1994; 34: 247–252. 17. Murphy M, Bernard EM, Ishimaru T,Armstrong D.Activity of voriconazole (UK-109,496) against clinical isolates of Aspergillus species and its effectiveness in an experimental model of invasive pulmonary aspergillosis.Antimicrob Agents Chemother 1997; 41: 696–698. 18. Clancy CJ, Nguyen MH. In vitro efficacy and fungicidal activity of voriconazole against Aspergillus and Fusarium species. Eur J Clin Microbiol Infect Dis 1998; 17: 573–575. 19. Cuenca-Estrella M, Rodriguez-Tudela JL, Mellado E, Martinez-Suarez JV, Monzon A. Comparison of the in-vitro activity of voriconazole (UK-109,496), itraconazole and amphotericin B against clinical

 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 326–334

331

Warnock et al.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

332

isolates of Aspergillus fumigatus. J Antimicrob Chemother 1998; 42: 531–533. Johnson EM, Szekely A,Warnock DW. In-vitro activity of voriconazole, itraconazole and amphotericin B against filamentous fungi. J Antimicrob Chemother 1998; 42: 741–745. Oakley KL, Moore CB, Denning DW. In-vitro activity of voriconazole against Aspergillus spp. and comparison with itraconazole and amphotericin B. J Antimicrob Chemother 1998; 42: 91–94. Verweij PE, Mensink M, Rijs AJMM, Donnelly JP, Meis JFGM, Denning DW. In-vitro activities of amphotericin B, itraconazole and voriconazole against 150 clinical and environmental Aspergillus fumigatus isolates. J Antimicrob Chemother 1998; 42: 389–392. George D, Miniter P,Andriole VT. Efficacy of UK-109496, a new azole antifungal agent, in an experimental model of invasive aspergillosis.Antimicrob Agents Chemother 1996; 40: 86–91. Martin MV,Yates J, Hitchcock CA. Comparison of voriconazole (UK-109,496) and itraconazole in prevention and treatment of Aspergillus fumigatus endocarditis in guinea pigs.Antimicrob Agents Chemother 1997; 41: 13–16. Denning DW, del Favero A, Gluckman E et al. UK-109,496, a novel, wide-spectrum triazole derivative for the treatment of fungal infections: clinical efficacy in acute invasive aspergillosis. In: Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1995; abstract F80, p. 126. Dupont B, Denning D, Lode H, Sarantis N,Troke PF,Yonren S. UK109,496, a novel, wide-spectrum triazole derivative for the treatment of fungal infections: clinical efficacy in chronic invasive aspergillosis. In:Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC: American Society for Microbiology, 1995; abstract F81, p. 127. Dupont B, Improvisi L, Dromer F. In vitro and in vivo activity of a new antifungal agent SCH56592 (SCH). In:Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1996; abstract F93, p. 116. Fothergill AW, Sutton DA, Rinaldi MG.An in vitro head-to-head comparison of Schering 56592, amphotericin B, fluconazole, and itraconazole against a spectrum of filamentous fungi. In:Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1996; abstract F89, p. 115. Oakley KL, Moore CB, Denning DW. In vitro activity of SCH56592 and comparison with activities of amphotericin B and itraconazole against Aspergillus spp.Antimicrob Agents Chemother 1997; 41: 1124–1126. Cacciapuoti A, Parmegiani R, Loebenberg D et al. Efficacy of SCH 56592 in pulmonary aspergillosis and candidiasis in mice. In: Abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1995; abstract F66, p. 124. Graybill JR, Bocanegra R, Najvar LK, Luther MF, Loebenberg D. SCH 56592 treatment of murine invasive aspergillosis. J Antimicrob Chemother 1998; 42: 539–542. Oakley KL, Morrissey G, Denning DW. Efficacy of SCH 56592 in two immunocompromised murine models of invasive aspergillosis. In:Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1996; abstract F97, p. 116.

Drug Resistance Updates (1999) 2, 326–334

 1999 Harcourt Publishers Ltd

33. Patterson TF, Kirkpatrick WR, McAtee RK, Loebenberg D. Efficacy of SCH 56592 in a rabbit model of invasive aspergillosis. In: Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1996; abstract F98, p. 116. 34. Debono M, Gordee RS.Antibiotics that inhibit fungal cell wall development.Ann Rev Microbiol 1994; 48: 471–497. 35. Pfaller MA, Gerarden T,Yu M,Wenzel RP. Influence of in vitro susceptibility testing conditions on the anticandidal activity of LY121019. Diagn Microbiol Infect Dis 1989; 11: 1–9. 36. Huang A, Edwards F, Bernard EM,Armstrong D, Schmitt HJ. In vitro activity of the new semi-synthetic polypeptide cilofungin (LY121019) against Aspergillus and Candida species. Eur J Clin Microbiol Infect Dis 1990; 9: 697–699. 37. Denning DW, Stevens DA. Efficacy of ciolfungin alone and in combination with amphotericin B in a murine model of disseminated aspergillosis.Antimicrob Agents Chemother 1991; 35: 1329–1333. 38. Beaulieu D,Tang J, Zeckner DJ, Parr TR. Correlation of cilofungin in vivo efficacy with its activity against Aspergillus fumigatus (1,3)-betaD-glucan synthase. FEMS Microbiol Lett 1993; 108: 133–137. 39. Zhanel GG, Karlowsky JA, Harding GAJ et al. In vitro activity of a new semisynthetic echinocandin, LY 303366, against systemic isolates of Candida species, Cryptococcus neoformans, Blastomyces dermatitidis, and Aspergillus species.Antimicrob Agents Chemother 1997; 41: 863–865. 40. Oakley KL, Moore CB, Denning DW. In vitro activity of the echinocandin antifungal agent LY 303,366 in comparison with itraconazole and amphotericin B against Aspergillus spp.Antimicrob Agents Chemother 1998; 42: 2726–2730. 41. Pfaller MA, Marco F, Messer SA, Jones RN. In vitro activity of two echinocandin derivatives, LY303366 and MK-0991 (L-743872), against clinical isolates of Aspergillus, Fusarium, Rhizopus, and other filamentous fungi. Diagn Microbiol Infect Dis 1998; 30: 251–255. 42. Zeckner D, Butler T, Boylan C et al. LY303366 activity against systemic aspergillosis and histoplasmosis in murine models. In: Abstracts of the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1993; abstract 364, p. 186. 43. Raab P, Zeckner D, Boyll B, Boylan C, Current W. LY303366: comparison of activity in murine models of Aspergillus fumigatus infection. In:Abstracts of the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC: American Society for Microbiology, 1994; abstract F167, p. 252. 44. Verweij PE, Oakley KL, Morrissey J, Morrisset G, Denning DW. Efficacy of LY 303366 against amphotericin B-susceptible and resistant Aspergillus fumigatus in a murine model of invasive aspergillosis.Antimicrob Agents Chemother 1998; 42: 873–878. 45. Bartizal K, Gill CJ,Abruzzo GK et al. In vitro preclinical evaluation studies with the echinocandin antifungal MK-0991 (L-743872). Antimicrob Agents Chemother 1997; 41: 2326–32. 46. Espinel-Ingroff A. In vitro studies with L-743,872, a water soluble pneumocandin: a comparative study. In:Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1996; abstract F31, p. 105. 47. Fothergill AW, Sutton DA, Rinaldi MG.Antifungal susceptibility testing of Merck L-743,872 against a broad range of fungi. In: Abstracts of the 36th Interscience Conference on Antimicrobial

Drug resistance in Aspergillus

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1996; abstract F29, p. 105. Abruzzo GK, Flattery AM, Gill CJ et al. Evaluation of the echinocandin antifungal MK-0991 (L-743,872): efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis.Antimicrob Agents Chemother 1997; 41: 2333–8. Bernard EM, Ishimaru T,Armstrong D. Low doses of the pneumocandin, L-743,872, are effective for prevention and treatment in an animal model of pulmonary aspergillosis. In: Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC:American Society for Microbiology, 1996; abstract F39, p. 106. Smith JG,Abruzzo GK, Gill CJ et al. Evaluation of pneumocandin L743872 in neutropenic mouse models of disseminated candidiasis and aspergillosis. In:Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy.Washington, DC: American Society for Microbiology, 1996; abstract F41, p. 107. Denning DW, Radford SA, Oakley KL, Hall L, Johnson EM,Warnock DW. Correlation between in-vitro susceptibility testing to itraconazole and in-vivo outcome of Aspergillus fumigatus infection. J Antimicrob Chemother 1997; 40: 401–414. Denning DW,Venkateswarlu K, Oakley KL et al. Itraconazole resistance in Aspergillus fumigatus.Antimicrob Agents Chemother 1997; 41: 1364–1368. Odds FC. Laboratory evaluation of antifungal agents: a comparative study of five imidazole derivatives of clinical importance. J Antimicrob Chemother 1980; 6: 749–761. Johnson EM, Richardson MD,Warnock DW. In-vitro resistance to imidazole antifungals in Candida albicans. J Antimicrob Chemother 1984; 13: 547–558. Doern GV,Tubert TA, Chapin K, Rinaldi MG. Effect of medium composition on results of macrobroth dilution antifungal susceptibility testing of yeasts. J Clin Microbiol 1986; 24: 507–511. Galgiani JN, Reiser J, Brass C, Espinel-Ingroff A, Gordon MA, Kerkering TM. Comparison of relative susceptibility of Candida species to three antifungal agents as determined by unstandardized methods.Antimicrob Agents Chemother 1987; 31: 1343–1347. Calhoun DL, Roberts GD, Galgiani JN et al. Results of a survey of antifungal susceptibility tests in the United States and interlaboratory comparison of broth dilution testing of flucytosine and amphotericin B. J Clin Microbiol. 1986; 23: 298–301. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard. Document M27-A.Wayne, Pa.: National Committee for Clinical Laboratory Standards, 1997. Manavathu EK, Cutright J, Chandrasekar PH. Comparative study of susceptibilities of germinated and ungerminated conidia of Aspergillus fumigatus to various antifungal agents. J Clin Microbiol 1999; 37: 858–861. Guarro J, Llop C,Aguilar C, Pujol I. Comparison of in vitro antifungal susceptibilities of conidia and hyphae of filamentous fungi.Antimicrob Agents Chemother 1997; 41: 2760–2762. Pfaller MA, Rinaldi MG, Galgiani JN et al. Collaborative investigation of variables in susceptibility testing of yeasts. Antimicrob Agents Chemother 1990; 34: 1648–1654. Gehrt A, Peter J, Pizzo PA,Walsh TJ. Effect of increasing inoculum sizes of pathogenic filamentous fungi on MICs of antifungal agents by broth microdilution method. J Clin Microbiol 1995; 33: 1302–1307.

63. Espinel-Ingroff A, Kerkering TM. Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi. J Clin Microbiol 1991; 29: 393–394. 64. Espinel-Ingroff A, Pfaller MA, Dawson K et al. Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi.Antimicrob Agents Chemother 1995; 39: 314–319. 65. Espinel-Ingroff A, Bartlett MS, Bowden R et al. Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi. J Clin Microbiol 1997; 35: 139–143. 66. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard. Document M38-P.Wayne, Pa.: National Committee for Clinical Laboratory Standards, 1998. 67. Espinel-Ingroff A, Silvius MA.The Etest for antifungal susceptibility testing of opportunistic filamentous fungi. In:Abstracts of the 96th General Meeting of the American Society for Microbiology. Washington DC.:American Society for Microbiology, 1996; abstract C262, p. 47. 68. Mills K,Wanger A. Evaluation of Etest for antifungal susceptibility testing of filamentous fungi. In:Abstracts of the 96th General Meeting of the American Society for Microbiology.Washington DC.:American Society for Microbiology, 1996; abstract F66, p. 85. 69. Mills K,Wanger A. Susceptibility testing of moulds using Etest. In: Abstracts of the 97th General Meeting of the American Society for Microbiology.Washington DC.:American Society for Microbiology, 1997; abstract C248, p. 163. 70. Szekely A, Johnson EM,Warnock DW. Comparison of Etest and broth microdilution methods for antifungal drug susceptibility testing of molds. J Clin Microbiol 1999; 37: 1480–1483. 71. Velez JD,Allendoerfer R, Luther M, Rinaldi MG, Graybill JR. Correlation of in-vitro azole susceptibility with in-vivo response in a murine model of cryptococcal meningitis. J Infect Dis 1993; 168: 508–510. 72. Graybill JR, Najvar LK, Holmberg JD, Correa A, Luther MF. Fluconazole treatment of Candida albicans infection in mice: does in vitro susceptibility predict in vivo response.Antimicrob Agents Chemother 1995; 39: 2197–2200. 73. Rex JH, Nelson PW, Paetznick VL, Lozano-Chiu M, Espinel-Ingroff A, Anaissie EJ. Optimizing the correlation between results of testing in vitro and therapeutic outcome in vivo for fluconazole by testing critical isolates in a murine model of invasive candidiasis. Antimicrob Agents Chemother 1998; 42: 129–134. 74. Rodriguez-Tudela JL, Martinez-Suarez JV, Dronda F, Laguna F, Chaves F,Valencia E. Correlation of in-vitro susceptibility test results with clinical response: a study of azole therapy in AIDS patients. J Antimicrob Chemother 1995; 35: 793–804. 75. Ruhnke M, Schmidt-Westhausen A, Engelmann E,Trautmann M. Comparative evaluation of three antifungal susceptibility test methods for Candida albicans isolates and correlation with response to fluconazole therapy. J Clin Microbiol 1996; 34: 3208–3211. 76. Rex JH, Pfaller MA, Barry AL, Nelson PW,Webb CD.Antifungal susceptibility testing of isolates from a randomized, multicenter trial of fluconazole versus amphotericin B as treatment of nonneutropenic patients with candidemia.Antimicrob Agents Chemother 1995; 39: 40–44. 77. Odds FC,Van Gerven F, Espinel-Ingroff A et al. Evaluation of possible correlations between antifungal susceptibilities of

 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 326–334

333

Warnock et al.

78.

79.

80.

81.

82.

83.

84.

85.

86.

334

filamentous fungi in vitro and antifungal treatment outcomes in animal infection models.Antimicrob Agents Chemother 1998; 42: 282–288. Lass-Florl C, Kofler G, Kropshofer G et al. In-vitro testing of susceptibility to amphotericin B is a reliable predictor of clinical outcome in invasive aspergillosis. J Antimicrob Chemother 1998; 42: 497–502. Rath PM. Susceptibility of aspergillus strains from culture collections to amphotericin B and itraconazole. J Antimicrob Chemother 1998; 41: 567–570. Oakley KL, Morrissey G, Denning DW. Efficacy of SCH-56592 in a temporarily neutropenic murine model of invasive aspergillosis with an itraconazole-susceptible and an itraconazole-resistant isolate of Aspergillus fumigatus.Antimicrob Agents Chemother 1997; 41: 1504–1507. Sutton DA, Sanche SE, Revankar SG, Fothergill AW, Rinaldi MG. In vitro amphotericin B resistance in clinical isolates of Aspergillus terreus, with a head-to-head comparison to voriconazole. J Clin Microbiol 1999; 37: 2343–2345. Schaffner A, Bohler A.Amphotericin B refractory aspergillosis after itraconazole: evidence for significant antagonism. Mycoses 1993; 36: 421–424. Schafner A, Frick PG.The effect of ketoconazole on amphotericin B in a model of disseminated aspergillosis. J Infect Dis 1985; 151: 902–10. Nucci M, Pulcheri W, Bacha PC et al.Amphotericin B followed by itraconazole in the treatment of disseminated fungal infections in neutropenic patients. Mycoses 1994; 37: 433–7. Ziogas BN, Sisler HD, Lusby WR. Sterol content and other characteristics of pimaricin-resistant mutants of Aspergillus nidulans. Pest Biochem Physiol 1983; 20: 320–329. Kim SJ, Kwon-Chung KJ. Polyene-resistant mutants of Aspergillus fennelliae: sterol contents and genetics.Antimicrob Agents Chemother 1974; 6: 102–113.

Drug Resistance Updates (1999) 2, 326–334

 1999 Harcourt Publishers Ltd

87. Kim SJ, Kwon-Chung KJ, Milne GWA, Prescott B. Polyene-resistant mutants of Aspergillus fennelliae: identification of sterols.Antimicrob Agents Chemother 1974; 6: 405–410. 88. Gale EF, Johnson AM, Kerridge D, Koh TY. Factors affecting the changes in amphotericin sensitivity of Candida albicans during growth. J Gen Microbiol 1975; 87: 20–36. 89. Gale EF, Johnson AM, Kerridge D.The effect of aeration and metabolic inhibitors on resistance to amphotericin in starved cultures of Candida albicans. J Gen Microbiol 1977; 99: 77–84. 90. Sokol-Anderson ML, Sligh JEJ, Elberg S, Brajtburg J, Kobayashi GS, Medoff G. Role of cell defense against oxidative damage in the resistance of Candida albicans to the killing effect of amphotericin B.Antimicrob Agents Chemother 1988; 32: 702–705. 91. Manavathu EK,Alangaden GJ, Chandrasekar PH. In-vitro isolation and antifungal susceptibility of amphotericin B-resistant mutants of Aspergillus fumigatus. J Antimicrob Chemother 1998; 41: 615–619. 92. Capek A, Simek A, Leiner J,Weichet J.Antimycotic activity and problems of resistance. Folia Microbiologica 1970; 15: 314. 93. Sanglard D, Ischer F, Calabrese D, de Micheli M, Bille J. Multiple resistance mechanisms to azole antifungals in yeast clinical isolates. Drug Resistance Updates 1998; 1: 255–65. 94. White TC, Marr KA, Bowden RA. Clinical, cellular, and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 1998; 11: 382–402. 95. deWaard MA, van Nistelrooy JGM.An energy-dependent efflux mechanism for fenarimol in a wild-type strain and fenarimolresistant mutants of Aspergillus nidulans. Pestic Biochem Physiol 1980; 13: 255–266. 96. van den Brink HJM, van Nistelrooy JGM, de Waard MA, van den Hondel CAMJJ, van Gorcom RFM. Increased resistance to 14-alpha demethylase inhibitors (DMIs) in Aspergillus niger by coexpression of the Penicillium italicum eburicol 14-alpha demethylase (cyp51) and the A. niger cytochrome P450 reductase (cpr A) genes. J Biotech 1996; 49: 13–18.