Eur. J. Epidemiol.0392-2990 May 1992,p. 407-421

EUROPEAN JOURNAL

VoL 8, No. 3

OF

EPIDEMIOLOGY

DRUG RESISTANCEIN HUMAN PATHOGENICFUNGI K. IWATA Department o f Bacteriology - Faculty o f Medicine - University o f Tokyo - 7-3-1 Hongo Bunkyo-ku - Tokyo - 113 Japan.

Key words: Drug resistance - Human pathogenic fungi - Antifungal agents Since the therapy of the mycoses, particularly the systemic mycoses, is relatively long-term in nature, emergence of resistance to antifungal drugs during the treatment of period would be of considerable clinical importance. However, most reports of resistance to antifungal agents among human pathogenic fungi indicate that naturally-occurring resistance is very rare, and that the induction of resistant mutants or variants is much more difficult to achieve in vitro and in vivo than with bacteria. As a matter of fact, amphotericin B and some other classic antifungals have not as yet posed a broadly significant problem relative to drug resistance despite their widespread and frequent use. Fungal resistance has thus received little attention, in contrast to the critical importance of bacterial resistance frequently caused by a variety of antibacterial chemotherapeutic agents, until a single exception to this generalization arose with the advent of flucytosine. This new development has aroused great interest in the problem of fungal resistance among the scientists involved with medical mycology. It is generally believed that fungi, like bacteria, are intrinsically capable of developing resistance to antifungal agents. As illustrated by flucytosine, inherently resistant mutants to antifungals occur within sensitive strains of human pathogenic fungi with significant frequency. Given the relatively high degree of such primary resistance, these mutants should develop secondary resistance during therapy, thus resulting in considerable limitations in the clinical usefulness of the antifungals. Virtually, all unsuccessful cases of mycoses treated with some of the recently exploited antifungal drugs, albeit scarce to date, would obviously be attributable to the occurrence of secondary resistance. The exploitation of new antifungal drugs thus requires investigations of their resistance as one of the most important research projects to be undertaken before receiving approval for use on humans. This paper reviews from various aspects the literature on resistance to various classic and novel antifungal agents among human pathogenic fungi. The resistance of some nonpathogenic fungi to these agents will also be described from genetic and biochemical points of view.

RESISTANCE TO VARIOUS ANTIFUNGAL AGENTS

Flucytosine resistance Flucytosine (5-fluorocytosine; 5-FC) has been proven to be an effective antifungal agent with relatively few adverse effects and is a desirable drug for that reason. However, enthusiasm for treating candidiasis, including C. glabrata infections, cryptococcosis, aspergillosis, and systemic phaeohyphomycosis with 5-FC, all of whose etiologic agents are essentially susceptible to the drug, has been tempered in a considerable number of cases by the occurrence of 407

primary resistance and the emergence of secondary resistance during therapy. Virtually all failures or relapses in 5-FC treatment can be explained by the development of secondary resistance among those mycoses (5, 18, 29, 73, 85, 86, 88, 102, 103, 107, 129, 153, 163, 173). In order to reduce the risk of the development of 5-FC resistance, concomitant use of other antifungals, particularly polyene antifungal agents, has been recommended. Combination therapy with oral 5-FC and intravenous amphotericin B has been most successfully applied to human systemic candidiasis for suppressing the occurrence of 5-FC-resistant mutants

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Eur. J. Epidemiol.

Contrary to those reports, synergism between 5of Candida spp. (103, 142, 144, 151, 159, 167, 174). The superiority of this combination therapy to 5-FC FC and amphotericin B in combination has not been monotherapy or to amphotericin B monotherapy has observed in the treatment of chronic mucocutaneous also been proven or suggested for the treatment of candidiasis (46), and even antagonism has, albeit cryptococcal meningitis with regard to fungal unconvincingly, been proven in a few strains of C. suppression and symptomatic improvement (14, 60, albicans (13, 158). The efficacy ofmonotherapy with 566, 129, 153, 161, 174). Similarly, the combined effect FC, including temporary clinical improvement, has of these two drugs has been confirmed in the already been proven in many cases of mycoses (45, treatment of aspergillosis (61, 103, 106) and 143). In some cases of Candida endophthalmitis, the phaeohyphomycosis (114). Other polyenes, e.g. therapeutic results with amphotericin B alone were nystatin, as a partner, also proved effective in found to be more effective (62, 87, 118). Neither suppressing the emergence of 5-FC-resistant mutants synergism nor antagonism was demonstrated against of Candida spp. in the intestine (150). Furthermore, A. fumigatus and A. niger in in vitro combinations of two other forms of the combination therapy have the two drugs (110). Although the subject of fungal been used: (a) In instances of failures of previous 5-FC synergism thus remains controversial or even treatment, subsequent application of the combination doubtful, particularly in relation to the method used with amphotericin B is successful in cure, for instance, for susceptibility determination, it is evident that of endophthalmitis (62), and (b) failures after the combination therapy with amphotericin B and 5-FC in monotherapy with either of these two drugs alone is the deep-seated or systemic mycoses is of great also improved by subsequent use of the partner, for significance at least in lowering the usual potentially instance, in cases of cryptococcal meningitis and some toxic doses of the former drug administered to patients by adding the latter antifungal agent which is other forms of candidiasis (45, 48, 188). The clinical effectiveness of combination therapy relatively lower in toxicity. Numerous reports indicate that only a minority of with 5-FC and polyene antimycotics has been supported by experimental evidence both in vitro and mutants manifested 5-FC resistance in the population in vivo. In vitro, the synergistic inhibitory effects of 5- of normally susceptible fungus strains that had neither FC and amphotericin B have been observed in C. a known contact with the drug in vitro or in vivo. Such albicans and other fungal species (13, 47, 67, 103, 116, primary resistance to 5-FC has been seen in varying 122, 137, 138, 158). Such anti-Candida effects of 5-FC degrees with a relatively wide range of clinical isolates were also shown in combination with other of various fungus species. With Candida spp., a antifungals, e.g. clotrimazole (11, 117). In vivo, the number of reports were published from 1971 to 1982 combination of 5-FC and amphotericin B has been (8, 13, 16, 34, 36, 41, 42, 50, 59, 64, 68, 69, 93, 107, 115, shown to be more effective in the treatment of 132, 138, 151, 152, 154, 156, 164, 165, 173, 175, 176). In experimental candidiasis in mice and rabbits (1, 67, those reports, the highest levels in the incidence of 136, 139, 141, 171) and of aspergillosis in mice (1) than resistant isolates of C. albicans recorded so far were 37.2% found among 175 strains in Canada (93), and with either of the drugs alone. The combined inhibitory effects have been 25.8% found in the study of 58 isolates in the United attributed to the permeability-enhancing activity of States (175); intermediate levels, e.g. 9.7% in France amphotericin B (and other polyene antifungals as (132) and 6.3°/0 in West Germany (8); and the lowest well) on fungal cell membranes by which other levels, e.g. 4.1% in New Zealand (176), and 0% in East antifungals would be facilitated to penetrate fungal Germany (16). The major reason for such strikingly cells. Biochemical clarifications of in vitro synergism different results may be attributed to the use of has been made as follows: For instance, Medoff et aL different test systems and different interpretation of (116) showed that the fungicidal activity of 5-FC on C. the MIC values. To take an appropriate example, in a albicans and other yeasts was enhanced at low report made by Stiller et al. (166), the conclusions concentrations of amphotericin B that are ineffectively from the results of their extensive investigation were fungicidal by themselves; Montgomerie et al. (122) that the 402 strains of C. albicans isolated from indicated that the fungistatic activity of 5-FC on C. patients with no known history of 5-FC treatment, albicans was also enhanced by amphotericin B, which had been collected at five medical centers from especially with strains of C. albicans that were different areas in the United States, could be relatively resistant to 5-FC; and Polak (136) also separated into four groups based on their MICs to the reported that 5-FC resistance frequency was reduced drug. Moreover, the common practice of identifying by subinhibitory concentrations of amphotericin B. as 'resistant' those isolates with MICs > 12.5 iag/ml The question of whether such combined effects are after 48 hours of incubation would yield resistance additive or potentiating (synergistic in the strict sense) rates in the United States of 11.5 to 15.5% in four has been raised. On the basis of these reports, centers and 35% in the fifth. Data on the results of 5potentiation itself appears rather weak or negligible, FC in Candida spp. collected in the United States, and there have been few or no reports indicating that Europe, Africa, and the Middle East from 1971 to 1976 the combination was capable of ensuring antifungal by Scholer (151), showed that among 2,685 clinical effects other than those obtainable with higher doses isolates, 2,473 strains (92.1%) were sensitive and 212 strains (7.9%) were resistant. Scholer indicated that, of either of the two drugs alone. 408

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among 400 clinical isolates of C. glabrata, 369 were resistant isolates of C albicans induced resistant sensitive (92.2%) and 31 resistant (7.8%). variants which were auxotrophic for lysine. They Primary resistance of Cr. neoformans to 5-FC has suggested that heterozygotes constitute a source of also been reported (13, 19, 40, 155, 157). Scholer (151) preexisting mutant alleles which determine resistance described that among 279 clinical isolates collected in and that 5-FC treatment of infections due to the United States and Europe by 1976, only 5 strains heterozygotes may result in a significant selection of were resistant (1.8%). It is noteworthy that this low resistant variants. The heterozygocity of 5-FC incidence of primary resistance in Cr. neoformans was resistance was also demonstrated by Whelan et al. not in parallel with the relatively high incidence of (181-183), who stated that spontaneous production of failure of 5-FC treatment of cryptococcal meningitis such variants by C. albicans isolates is due to due to the emergence of resistant mutants (19, 48, 129, segregation from a preexisting heterozygous state. 169). Primary resistance to 5-FC has also been proven These findings are of particular interest in for sensitive species ofAspergillus spp. in ca. 20% (149, understanding the fact that in C. albicans the 177) and for the agents of phaeohyphomycosis in ca. incidence of primary resistant strains is much higher 60% (177). A 5-FC-resistant strain of Phialophora than one would expect from the frequency of richardsiae, which had been isolated from a patient spontaneous resistance, as states above. The question with osteomyelitis, was also resistant to clotrimazole arises of how resistant mutants could be selected in (190). the absence of 5-FC, especially when viewed in The ecology of 5-FC resistance in Candida spp. connection with the role of C. albicans serotype B. has been studied in relation to serological types (6, 39, This serotype comprises a small minority in most 42, 138). Drouhet and his co-workers (42) reported collections of isolates but it is responsible for a that the percentage of primary resistant strains of majority of the instances of primary resistance, as human isolates of C. albicans occurring in vitro to 5- seen in the above-mentioned epidemiological and FC was much higher in Africa than in Europe. They serological reports. Fluctuation analysis has revealed believed, that this high incidence (85%) was related to that in C. albicans and Cr. neoformans 5-FC-resistant serotype B, which was observed among blacks, in mutants occur spontaneously by a one-step mutation contrast to a low incidence (< 1%) for serotype A, without any induction with the drug (17, 18, 99). predominantly encountered among whites. Five genes, which determine 5-FC-resistance, Concerning 5-FC resistance frequency, Polak and were demonstrated in S. cerevisiae by Grenson (63) Scholer (138) showed that the figures at a and Jund and Lacroute (99, 100). Normark and concentration of 64 lag/ml of the drug were in the Schoenebeck (126) distinguished two genotypes of 5order of 10.7 in C. albicans, 10.6 in Cr. neoformans, 105 FC resistance in C. albicans. Drouhet et al. (40) and in A. fumigatus, and as high as 104 to 10.3 in Polak and Scholer (138) found two more genotypes of dematiaceous fungi such as Cladosporium carrionii this resistance. and Fonsecaea pedrosoi. As to C. albicans and Cr. Morphological studies on 5-FC-resistant strains, neoformans, similar results were reported by other that appeared during therapy or were obtained by in workers (18, 19, 126). vitro selection or induction, have been made in In vitro induction of 5-FC resistance has been comparison with the original isolates or the parent reported to be relatively easy in C. albicans, C. glabrata, strain. Montplaisir et al. (123) reported an electron Cr. neoformans, and some of the dematiaceous fungi microscopic study of 5-FC-sensitive and -resistant (26, 33, 85, 86). The possible development of in vivo strains of Candida spp.; neither revealed any resistance to this drug was also suggested (20). significant difference in the structure of the interior of It has been considered that 5-FC resistance may be their cytoplasmic membranes. Nevertheless, the induced by the lack or deficiency of an enzyme at any sensitive strains of C. albicans serotype A and C. step of the metabolic pathways or by a surplus of de freyschussii could be distinguished from the resistant novo synthesis of normal compounds competing with strains of C. albicans serotype B and from C. the fluorinated antimetabolites (19, 37, 99, 123, 126, parapsilosis by their annular and granular electron138, 139). Jund and Lacroute (99, 100) indicated that dense material dividing in the inner stratum. This six types of resistant mutants of Saccharomyces material disappeared from the sensitive strains by cerevisiae to 5-FC, 5-fluorouracil (5-FU), and 5- enzymatic digestion with trypsin and reappeared after fluorouridine (5-FUR), which had been induced by 24 hours of further growth. ultraviolet irradiation, could be distinguished. Such Growth of 5-FC-resistant fungi is known to be biochemical types of 5-FC resistance have been found usually normal in the absence of the drug. Fasoli et al. in clinical isolates of other pathogenic yeasts such as (44) isolated 5-FC-resistant mutants of C. albicans C. parapsilosis (80) and Cr. neoformans (17, 18, 126, blocked in pyrimidine transport and salvage 138). Defever et al. (34) reported that among clinical metabolism. These mutants did not differ significantly isolates of C. albicans, some partially resistant isolates from the parent strain in growth rates and cell yields gave rise to highly resistant variants at an appreciable but had decreased ha their virulence for mice. frequency. All of the highly resistant isolates gave rise Contrary to this, Persson et al. (134) observed no to highly resistant variants as did some sensitive relationship between the degree of 5-FC sensitivity or isolates. Furthermore, they found that a partially resistance and virulence .for mice. 409

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With few exceptions, little or no cross-resistance of 5-FC-resistant strains has been experienced with any other antifungals (69, 80, 85, 107). Contrariwise, Woods et aL (188) reported that an isolate of C. tropicalis, resistant to amphotericin B, was normally sensitive to 5-FG. Polyene resistance

A few studies regarding the emergence of polyene-resistant strains during therapy and the resultant failure of treatment have been published. Bodenhoff (20) described an amphotericin B-resistant strain of C. albicans isolated from a patient subjected to prolonged therapy. Woods et al. (188) recorded a case of a patient with pyelonephritis due to C. tropicalis who was treated with amphotericin B (25-40 mg per day given intravenously) where the organisms gradually developed resistance to the antibiotic; the MIC rose from 0.098 pg/ml for the initial isolate to 500 pg/ml for the isolate 3 months after initiating therapy. Reviewing this report, Druz and Lehrer (43) emphasized that more of such amphotericin Bresistant strains would be recognized if sensitivity tests to this drug were more frequently performed during treatment. Very little has been published on primary resistance to the polyenes. Pappagianis et al. (130) isolated a strain of C. lusitaniae, associated with infection in a patient with acute leukemia, which had developed resistance to amphotericin B during therapy. It is interesting that strains of C. albicans, isolated from patients with chronic oral candidiasis who had been treated with amphotericin B, proved highly resistant to nystatin but sensitive to amphotericin B itself (81). A nystatin-resistant strain of Trichophyton mentagrophytes (28) and some dematiaceous fungi, e.g. Ph. richardsiae resistant to amphotericin B, have been isolated from patients (30, 185, 190). The paucity of reports of failure of polyene therapy suggests that resistance to polyenes is not generally a serious clinical problem. However, this subject cannot be ignored in clinics, since a variety of conditions such as the chronic nature of most systemic mycoses, the limitation on achievable concentrations of the polyenes in body fluids and the relatively prolonged administration of the drugs would favor a possible alternation of susceptibility of the etiologic organisms to the drugs. The in vitro and/or in vivo synergistic inhibitory effects of amphotericin B plus 5-FC or fifampicin (rifampin) on C. albicans, Cr. neoformans, Aspergillus spp., Histoplasma capsulatum, and Blastomyces dermatitidis have been reported (1, 104-106, 108, 117, 141). These effects would suppress the emergence of resistance to those drugs in Candida spp. and other fungi (9-12). Polyene-resistant variants or mutants of several of the Candida spp., including C. albicans and C. glabrata, have been induced or selected by a number

of investigators as follows: to amphotericin B (22, 70, 112, 113, 162, 168), to amphotericin A (168), to candicidin (168), to candidin (74, 75), to trichomycin (4, 21, 22), to perimycin (120), to hamycin (13), to nystatin (4, 22, 23, 28, 109, 112, 133, 168), to endomycin (133), to filipin (15,70), to natamycin (pimaricin) (76), and to rimocidin sulfate (168). Resistance of Cr. neoformans to amphotericin B and nystatin ( 2 1 ) and of Coccidioides immitis to amphotericin B (162) has also been observed. Very few reports on successful attempts to develop high resistance to the polyene antibiotics, however, have been published with C. albicans to nystatin (22, 38, 112) and to amphotericin B (112). Stout and Pagano (168), failed to develop resistance to nystatin in 4 out of 5 strains of C. albicans but in the fifth strain they developed single-step mutants that were three times as resistant as the parent strain. Littman et al. (112) reported similar results with some Candida species other than C. albicans. Bodenhoff (22) stated that it was relatively easy to induce resistance to nystatin and amphotericin B in most strains of Cr. neoformans. In this regard, however, it must be pointed out that this basidiomycetous yeast is an exception among various fungi susceptible to these polyenes. The speed with which resistance is induced to polyene antifungals appears generally different in drug-strain relationships. Littman et aL (112) reported that significant increases in the resistance of Candida spp. to nystatin and amphotericin B did not occur in most cases until about 30 passages in either of the two antibiotic broths used. Despite those data on the successful development of resistance, it seems justified to assume that it is generally difficult to induce resistance to polyene antifungals in pathogenic fungi, or, at any rate, to induce any high degree of resistance. Most of the polyene-resistant strains have been considered to be derived from the sensitive parents by multi-step mutation by means of repeated subculture in the presence of increasing concentrations of an antibiotic or by the gradient agar technique. On the other hand, Patel and Johnson (131) found that onestep mutants to nystatin occurred spontaneously in a strain of C. albicans at a frequency of approximately 107. Such mutants to nystatin, amphotericin B or filipin were also isolated efficiently by Hamilton-Miller (70), using N-methyl-N'-nitro-N-nitrosoguanidine. Genetic studies on polyene resistance have been carried out mainly in S. cerevisiae. Molzahn and Woods (121) demonstrated that nystatin resistance in this yeast was controlled by three recessive genes and two dominant modifiers, whereas Patel and Johnson (131) stated that at least one resistant gene is dominant, suggesting that ploidy is directly connected with this resistance. Scandella et al. (148) also reported nystatinresistant mutants of a cellular slime mould, Dictyostelium discoideum, falling into three phenotypic groups that corresponded to three genes; the mutants in t w o genes affected sterol metabolism while the mutants with the third gene were nystatin-sensitive.

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Biochemical analysis of polyene resistance has been made from various points. The resistance has been found to be generally associated with an increase of membrane sterols, particularly of ergosterol and its precursors (52, 53, 70, 71, 91, 125, 146, 187). HamiltonMiller (70), however, stated that increased membrane sterols were not always found in polyene-resistant mutants (27). Capek et al. (28) isolated a nystatinresistant strain of T. mentagrophytes and found that not only this species but also other dermatophytes, T. rubrum and Microsporum gypseum, produced an enzyme which degraded nystatin when grown in the presence of nystatin or amphotericin B. This suggested the possible responsibility of such an enzyme for resistance to these antibiotics. Capek and Simek (27) described that an intrinsic resistance of dermatophytes to the polyenes may be associated with decreased sterol content. Gale and his group (55-58, 128) reported that the phenotypic resistance to amphotericin B methyl ester in C. albicans may be accelerated or prevented, and its extent modified, by controlling such culture conditions as pH, aeration, composition of culture media or by treating the organisms with SH-reactive agents, inhibitors of protein synthesis, such as trichoderrnin, or cell-wall lytic enzymes, such as (1 ~ 3)B-D-glucanase. They thus suggested that the endogenous glucanases could play a role in the onset of phenotypic resistance to amphotericin B. The stability of induced polyene resistance has been investigated with some fungal species. HamiltonMiller (70) reported that, among the mutagen-induced polyene-resistant variants of C. albicans, there was substantial loss of resistance to nystatin and amphotericin B in two strains after one passage through chick chorioallantoic membrane and after forty-six in vitro subcultures. Little loss of filipin resistance, however, was noted after repeated subculture. In vivo passage did not cause any significant changes in resistance. A complete to partial loss of resistance was also observed in hamycinresistant strains of Candida spp. (3). Capek and Simek (27) also reported that nystatin-resistant strains of Candida spp. were obtained through an 'adaptation' mechanism as a result of direct contact of the organisms with the antibiotic. Cultivation of these strains on an antibiotic-free medium led to relatively rapid 'readaptation' and the isolates regained their original sensitivity. Morphological, physiological and other features of polyene-resistant mutants have been investigated. Hamilton-Miller (70) reported that the mutageninduced mutants of C. albicans, resistant to nystatin, were characterized by a decreased ability to form pseudohyphae, decreased growth rates and prolonged lag phases under certain cultural conditions, greater size of cells, greater eccentricity of cells, greater total volume and mass of cells, slower fermentation, lowered assimilation of the tricarboxylic acid cycle intermediates (citrate and succinate), etc. than the sensitive parent strain. Difference in agglutination 411

between the parent and mutant strains were not observed. Strains of C. albicans made resistant to amphotericin B also manifested decreased ability to form pseudohyphae and chlamydoconidia as well as reduced growth rates (113). Such alternations with regard to morph61ogical and cultural properties were also observed in hamycin-resistant strains of C. albicans and C. tropicalis (3). Cross-resistance to polyene antibiotics has been reported (3, 22, 68, 75, 112, 162, 168). Littman et aL (112) stated that exposure of Candida spp. to nystatin increased resistance to amphotericin B and vice versa. Heb6ka and Solotorovsky (75) reported that C. albicans made resistant to amphotericin B or candidin did not gain resistance to the non-polyene antibiotics such as eulicin and griseofulvin. Similar results were obtained by Athar (3). Hamilton-Miller (68) found that a 'trained' resistant mutant and four mutageninduced mutants with different degrees of increased resistance to the polyene antimycotics, nystatin, amphotericin B, and filipin, did not show crossresistance with the non-polyene antimycotics, 5-FC, clotrimazole, and pyrrolnitrin. Of added importance from a clinical point of view is the fact that the polyene-resistant mutants seemed to be, if anything, more sensitive to clotrimazole than the parent; the resistant strains showed virtually the same sensitivity as the parent. Azole resistance

Resistance to azole antifungals among medically important fungi has been less documented. One of the problems that can be encountered in studies on azole resistance is that, as with 5-FC and polyene antifungals, azoles possess a broad spectrum of activity with wide variations of MIC and MCC values. Secondary resistance to the azoles was f ~ t reported by Holt and Azmi (83) in a strain of C. albieans from a patient with urinary candidiasis and other disorders who had been undergoing prolonged oral administration of miconazole. The isolates proved resistant not only to miconazole but also to econazole and clotrimazole. Subsequently, such ketoconazole-resistant strains of C. albicans were isolated by Horsburgh et aL (88, 89), Ryley et aL (145), Warnock et aL (179), and Smith et al. (160) from patients with chronic mucocutaneous candidiasis during prolonged treatment with the drug, resulting in treatment failure. These isolates were further investigated in vitro by Johnson et al. (98) with both in vitro and in vivo models. Tavitian et aL (170) identified six patients with AIDS complicated by oral and esophageal candidiasis who had been treated with ketoconazole for more than two months. They isolated two strains of C. albicans resistant to the drug from two patients who had persistent Candida esophagitis. These investigators stated that it remains to be determined whether the failure of ketoconazole therapy in their other patients was due to host factors associated with the immunodeficiency state or to fungal resistance to the drug.

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Cross-resistance was also observed with the ketoconazole-resistant strains of C. albicans to ticonazole and miconazole (98), as with the abovementioned miconazole-resistant strains (83). In this connection, the datum of Hughes et al. (92) appears meaningful; they indicated that fluconazole was fungistatic in vivo against ketoconazole-resistant strains of C. albicans, only when high doses were applied. The anti-Candida synergistic effects of clotrima~zole plus amphotericin B or 5-FC have been studied by Beggs et al. (11). These combinations would be significant in suppressing the emergence of secondary resistance to those antifungals in Candida spp. Naturally occurring resistance to these azole compounds, among fungi of clinical interest, appears extremely uncommon in the light of the little available information. Bodenhoff (22), however, described primary resistance only in some species of Candida which were relatively resistant to clotrimazole. Induction of azole resistance in fungi is quite difficult. Plempel and Bartman (135) and Holt and Newman (84) failed to develop resistance to clotrimazole in strains of some fungal species by successive passage through increasing gradients of the drug. Attempts of Holt (82) to induce in vitro resistance to miconazole in Candida spp. were also unsuccessful. Iwata et aL (95) were not successful in developing resistance to ketoconazole and itraconazole in six strains of Cr. neoformans by serial subculturing through increasing gradients of the drugs. They were, however, capable of isolating one mutant each that was resistant to the two drugs by means of mutagenesis using N-methyl-N'nitro-Nnitrosoguanidine. Both mutants showed partial crossresistance. The itraconazole-resistant mutant was characterized by the formation of very rough colonies which varied in size and shape, production of a large number of cell clusters, complete loss of capsule formation, and major degenerative changes in the cells. In the ketoconazole-resistant mutant, these changes were less pronounced and cell clusters were not formed. It is o f particular interest that these ultrastructural changes resembled, to some extent, the observations made by Borgers (24) and Borgers and Van de Ven (25) on cells of sensitive strains of Cr. neoformans, C. albicans and some other fungi exposed to relatively high concentrations of itraconazole. Waltz et aL (178) described clotrimazole-resistant variants of C. albicans whose resistance developed slowly following prolonged incubation periods and reverted to the initial sensitivity when subcultured in a drug-free medium. Ryley et al. (145) have shown that their ketoconazole-resistant strains of C. albicans manifested abnormal resistance to inhibition of ergosterol biosynthesis in whole cells, but not in cell-free systems, and to inhibition of amino acid uptake. These isolates did not take up a radiolabelled ICI 153,066, a triazole antifungal, in contrast to normal isolates. From these result, they concluded that the

behavior of the resistant isolates is consistent with the development of drug resistance to ketoconazole and that resistance is due to changes in the properties of the cell membrane rather than internal enzymology. Hitchcock and co-workers (77-79) also described the mechanisms of resistance to polyenes and azoles by assessing the amounts of 14c~-sterol demethylase enzyme and by investigating lipid composition in relation to the permeability of the substances. They showed that a strain resistant to both classes of antifungals had a larger lipid content and lower polar lipid to neutral lipid ratio compared to other strains sensitive or resistant to azoles. Furthermore, they suggested that the altered membrane sterol pattern, based on some of their data, provides a common basis for the double resistance by preventing polyene binding and reducing azole permeability. Very little has been reported on the genetics of the azoles in human fungal pathogens. However, Portillo and Gancedo (140) reported an interesting genetic and biochemical study on a mutant of mitochondrial origin resistant to micomazole from a strain of S. cerevisiae isolated by themselves. They found that the mutation was linked to a structural gene for a subunit of ATPase on mitochondrial DNA and that miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Watson et al. (180) also isolated ketoconazole-resistant mutants of S. cerevisiae and showed that the mutation was nuclear origin. On the other hand, resistance to some benzimidazole fungicides, such as thiabendazole and methyl-2-benzimidazole carbamate, has been found to be induced in e.g. Schizosaccharomyces pombe (189), A. fumigatus (172), and has been well analyzed biochemically and genetically. Resistance to other antifungal agents

It seems likely that dermatologists generally believe that griseofulvin at present is not as effective in the treatment of dermatophyte-induced mycoses as before. Failure of these infections to respond to therapy with this oral antifungal has been considered to be attributable to the drug's inherent properties; for instance, its insufficient level in infected loci, and also to various substantial factors influencing therapeutic efficacy where the question of development of resistance to the antibiotic has been raised. Griseofulvin has been used for about three decades in the treatment of dermatophytoses; however, griseofulvin-resistant dermatophytes (65, 72, 191) and therapeutic failure with the antibiotic associated with isolated dermatophytes having a high MIC have been reported only in a few cases (49,101,119). The MIC of griseofulvin varies considerably with the test system (2, 7, 64, 111, 190). Artis et al. (2) reported that as a result of sensitivity tests using a liquid microculture method developed in their laboratory with 102 strains of dermatophytes isolated from patients having clinical histories of griseofulvin

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therapeutic failure, the range of MIC values was between 0.1 and 18 lag/ml or greater. It is noteworthy in this paper that the strains with the largest MIC values were isolated from patients with chronic infections who had been treated with griseofulvin for years. Similar data were shown by Grin et al. (65) and Young (191). These authors emphasized that, although the mean MIC values for the isolates obtained from griseofulvin-unresponsive patients were substantially greater than those for the responsive control isolates, this difference did not indicate a correlation between the clinical responsiveness of dermatophyte infections to griseofulvin with the in vitro sensitivity of the infecting molds to the drug. Similarly, Davies et al. (32) reported that the in vitro sensitivities of T. rubrum and T. mentagrophytes obtained before, during, and after griseofulvin treatment, did not show significant differences in the means of MIC values between the isolates from a group without treatment and that of patients treated with the antibiotic for a year or more. From these reports it seems justifiable to assume that resistance is not a major contributing factor in therapy failures with griseofulvin and that the in vitro determination of MIC is of no prognostic importance for the development of resistance to griseofulvin during therapy. Lenhart (111) demonstrated that griseofulvinresistant mutants of dermatophytes occurred spontaneously and could be induced by ultraviolet irradiation. This resistance was found to be controlled by at least two genes. The metabolic pathway through which these genes mediate griseofulvin resistance is unknown at present. Designs to induce griseofulvin resistance in vitro by repeated subculture of dermatophytes in media containing concentrations of the drug were successful in strains of M. canis and T. rubrum (7), but failed in those of T. rubrum (49). Aytoun et al. (7) reported that dermatophytes degraded griseofulvin. This could be due to the activity of enzymes elaborated by these molds that may be associated with the onset of resistance. Tolnaftate resistance has been shown by Iwata et al. (96, 97) to be induced in a strain of T. mentagrophytes having the perfect stage (Arthroderma benhamiae) after a long period of serial transfers into Sabouraud's dextrose broths and onto Sabouraud's dextrose agar slants both of which contained increasing gradients of the drug. The variants obtained were characterized by the formation of deep-brown pigments that diffused from the colonies into Sabouraud's agar, morphological changes such as swollen conidia and hyphae, reduced virulence for guinea pigs, and loss of the perfect stage. On the other hand, a new thiocarbamate, piritetrate (M-732) [methyl (6-methoxy-2-pyridyl) carbamothioic acid O-5,6,7,8-tetrahydro-2-naphthalenyl ester; C1j-I2oN202S], did not induce resistance in the strains tested.

Cycloheximide (actidione), a potent inhibitor of protein synthesis in eukaryotes, has extensively been used to select resistant mutants of lower eukaryotic species, besides its use as an agricultural fungicide. Cycloheximide-resistant mutants have been found in Neurospora crassa (90, 124), S. cerevisiae (186), Schizosaccharomyces pombe (94), Podospora anserina (31), Corprinus cinereus (127), and others. Genetic analysis of cycloheximide resistance in these eukaryotes has been conducted in detail. On the other hand, with regard to resistance in human fungal pathogens, only Cr. neoformans has been studied. Genetically defined primary resistance occurring in sensitive species has been documented by Ford and Klomparents (51), and induction of resistance by exposure to the agent has been reported by Whiffen (184) and Salldn and Hurd (147). CONCLUSIONS AND DISCUSSION

It is now clear from the many published studies that, with the exception of 5-FC, the emergence of resistant mutants to antifungal agents during treatment of infections due to human fungal pathogens and consequent failure of the treatment, by and large, have been very rare. The incidence of isolation of such mutants of either primary or secondary resistance to these drugs has also been very low. It has been considerably difficult to induce in vitro resistance of a high degree to the drugs in most fungi. 5-FC thus is indeed the very first and unique antifungal agent giving rise to a very high degree and a relatively frequent occurrence of resistance both in vitro and in vivo as well as in the human host. It is probably justified to consider that marked differences in resistance recognized with regard to various points between antifungal agents, excepting 5-FC, and antibacterial agents reflect differences between fungi as eukaryotes and bacteria as prokaryotes in relation to selective toxicity. The current fortunate situation that the rarity of emergence of resistant strains during the therapy of fungal infections and of failure of the therapy thereby has been confirmed thus far with all known antifungal agents other than 5-FC, does not permit assurance that undesirable resistance problems may not occur in the future as well, because several recently developed antifungal drugs have not as yet been available for a sufficiently long period of time, and because most of them have been applied topically to superficial mycoses but not to deep mycoses systemically, particularly in the light of the sudden and unexpected occurrence of 5-FC resistance that is analogous to resistance of bacteria to some antibacterial drugs such as streptomycin. This may be a warning of the possible occurrence of such problems with antifungal drugs that may be introduced and exploited in the future. Special consideration should be given to the possible occurrence of secondary resistance in individual cases during the course of therapy,

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particularly in deteriorated patients. To prevent this risk, the most appropriate regimen should be selected and instituted immediately; substitution of a given antifungal drug for another one of a different class, which is more potent toward the etiologic organisms and preferably less toxic, is mandatory where necessary. Combination therapy appears more effective than monotherapy in the synergistic, if any, suppression of secondary resistance, particularly in combination with 5-FC and amphotericin B. The interactions between antifungals as well as between antifungals and other drugs must be considered with regard to their synergistic and antagonistic properties. Undesirable effects may occur through the following mechanisms; (a) antagonism or inactivation of the antifungal effect, e.g. competitive inhibition of antifungal activity of 5-FC by cytosine arabinoside; (b) potentiation of their toxicity to the human host either by enhancement of their adverse effects or interference with organ functions, e.g. a possible increase of toxicity of 5-FC toward the hematopoietic organs when it is given to a patient receiving cytostatic agents such as azathioprine; and (c) pharmacokinetic interference, e.g. reduced excretion of 5-FC through the kidneys due to impaired glomerular filtration by amphotericin B in combination therapy, leading to the disfunction of the kidneys. Kostiala and Kostiala (107) reported that topical treatment of stomatitis and vaginitis due to C. albicans with clotrimazole did not produce any changes in the susceptibility of oral strains of C. albicans from patients with episodes of fungal stomatitis, whereas oral or intravenous treatment of deep-seated fungal infection with 5-FC resulted in a significant increase of oral strains resistant to this drug. These result are indicative of a more difficult development of resistance to 5-FC in superficial infections with its topical administration and conversely easier development of resistance in deep-seated infections by its systemic administration. This also suggests that in the latter case the organisms were affected more profoundly by antifungal and probably also by host factors. Correlations between the frequency of primary resistance and the incidence of secondary resistance vary considerably. The most outstanding correlation is observed in 5-FC versus the etiologic agents of some of the mycoses; in such cases, determination of incidence and degree of primary resistance may be of prognostic value for the possible occurrence of secondary resistance, leading to determination of the appropriateness of therapy with the most effective drug. It may be seen from the many papers cited in this review that in understanding the resistance of human pathogenic fungi to various antifungal agents, there exist various points to be determined for studies of fungal resistance including the assessment of in vitro determination of MIC as the very first procedure for the determination of relative resistance as with any other antimicrobial agent (54, 86, 93). However, it is

no exaggeration to say that the definition and terminology as well as the methodology of fungal resistance vary considerably from author to author. This is based on varying degrees of MIC values stemming from different test systems and divergent estimations of the extent of resistance therefrom. The very high MIC values of clinical isolates, as seen in the sensitivity tests of yeast pathogens to 5-FC at a considerably higher frequency than had previously been reported, must thus be considered exceptions. The isolation incidence of strains of primary and secondary resistance and their degree of resistance in human pathogenic fungi to antifungal agents by themselves vary in strain-drug relationship regardless of the test systems employed. These intrinsic strainspecific variations in susceptibility to antifungal agents and inaccurate or indefinite experimental conditions may amplify the magnitude of sensitivity or resistance to yield abnormally high MIC values and consequently an abnormally high incidence of isolation of resistant strains. An accurate determination of susceptibility thus is needed as a major premise. In this respect, Gale's method (54), for instance, is noteworthy. He assessed the susceptibility of C. albicans to polyene antibiotics monitoring the leakage of potassium ions from the organisms under standard conditions. The appropriateness of the test system is also of paramount importance. Emphasis should be laid upon an immediate, and preferably international, standardization of both in vitro and in vivo tests of antifungal agents for the purpose of studying not only drug resistance but also all other characteristics pertaining to the agents. It should be noted that, since the 1970s, the resistance of phytopathogenic fungi to fungicides has been causing serious problems in crop production. As a consequence of this, extensive studies on pesticide resistance have been performed in detail from various aspects (35). In this connection, comparative studies on the drug resistance of the different classes of antifungal drugs against various fungi pathogenic for man, animals, and plants thus might provide significant clues to solve the various difficulties inherent in this common problem that is under question. REFERENCES

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