CITATION: Hitchcock CA. 1996. Azole antifungal resistance in Candida albicans. APUA Newsletter 14(1):1, 5-6, 8.

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Azole antifungal resistance in Candida albicans
Christopher A Hitchcock
Pfizer Central Research, Sandwich, Kent, United Kingdom

The dimorphic fungus Candida albicans is a common commensal of humans responsible for a variety of superficial and invasive infections. Patients with impaired immunity because of underlying illness, including organ transplant recipients, those receiving cancer therapies and those infected with the human immunodeficiency virus (HIV), are particularly at risk for candidiasis. The latter group are especially prone to contracting candidiasis because they are permanently immunosuppressed, whereas immunosuppression in the other groups is transient. Nosocomial invasive candidiasis is a growing problem in neutropenic patients with cancer, and oropharyngeal candidiasis (OPC) is the most common fungal infection in patients with AIDS.(1) Although rarely life-threatening, OPC is a troublesome condition whose severity increases with the progressive deterioration of the immune status of the patient, thereby necessitating indefinite suppressive therapy.(2-4)

The natural product polyene antifungal antibiotic, amphotericin B, has been the mainstay of therapy for patients with serious candidiasis for many years, despite complications of nephrotoxicity and the requirement for slow intravenous infusion. The newer synthetic azole class of antifungal agents (fluconazole, itraconazole and ketoconazole) is being used with considerable success in the management of fungal infections, including candidiasis, and represents an important alternative to amphotericin B.(5) Fluconazole is particularly effective against
C. albicans infections. It is the most widely used azole for the treatment and prophylaxis of systemic candidiasis in cancer and organ transplant patients and of OPC and cryptococcal meningitis in patients with AIDS.(6-8) To date, it has been used to treat more than 50 million patients, including more than 250,000 AIDS patients. This fact reflects not only fluconazole's antifungal efficacy but also its excellent safety profile and the flexibility of oral and intravenous dosage forms. Although itraconazole is potent against C. albicans in vitro, its use is hampered by variable oral absorption leading to suboptimal plasma levels and by the absence of a commercially available intravenous dosage form.(9) However, it does show potent activity against a wide spectrum of fungi in vitro, including those with inherently low susceptibilities to fluconazole (e.g., Aspergillus spp., C. krusei and C. glabrata).(10) Ketoconazole also has a wide spectrum of activity and is prescribed for OPC, but its efficacy is reduced by variable oral absorption in immunosuppressed patients.(11) This drawback, together with the lack of an intravenous dosage form and the potential for hepatotoxicity, limits its widespread use.

Development of resistance to azole antifungals in
C. albicans was first reported in the early 1980s in patients with congenital defects in their immune systems who were predisposed to chronic mucocutaneous candidiasis (CMC). This disease is an uncommon condition, but prolonged ketoconazole treatment leading to resistance in the infecting strains and clinical failure is well documented.(12-14) More recently, azole resistance in C. albicans has been associated almost exclusively with the treatment of OPC in patients with AIDS. The typical scenario is a patient receives topical azoles (e.g., clotrimazole and miconazole) in the early stages of disease, followed by oral ketoconazole and increasing doses of oral fluconazole as the disease progresses and the number of relapses increases. Consequently, most AIDS patients have received either intermittent or continuous fluconazole therapy over long periods of time (e.g., three to four years). Given the requirement for long-term therapy and the widespread use of fluconazole in failing late-stage AIDS patients, reports of resistance in C. albicans strains isolated from this group of patients were not unexpected. However, the true extent of microbiological resistance is not known, but estimates from a number of expert centers would indicate that it occurs in less than 10% of late-stage AIDS patients. The paucity of data on itraconazole and ketoconazole precludes a meaningful comparison, a reflection of the fact that they are prescribed much less frequently than is fluconazole for OPC. Furthermore, interpretation of the literature is complicated by interlaboratory variations in susceptibility testing methods, which have a profound effect on MICs. Published MIC values for fluconazole against C. albicans may show interlaboratory variations of up to 1000-fold. Therefore, clinical failure should be ascribed to microbiological resistance only when an organism isolated during therapy shows a significant increase in MIC compared with those determined earlier in the treatment or at the start of therapy.

Significant advances in standardizing susceptibility testing have been made through the efforts of the National Committee on Clinical Laboratory Standards (NCCLS).(15) The next important step is to investigate whether the recommended susceptibility tests have value in predicting clinical outcome and in assigning breakpoints that can be used to guide therapy. In this regard, well-documented accounts of relapses in late-stage AIDS patients correlating with a gradual increase in the fluconazole MICs of the infecting
C. albicans strains when measured by the NCCLS method or close derivatives of it are of interest.(16,17) For example, using the standard NCCLS method, Redding and co-workers(18) measured the fluconazole MICs of C. albicans isolated from 14 episodes of OPC in a single patient. Candidiasis associated with MICs of 0.25 to >64 µg/mL responded to increasing fluconazole doses of 200, 400 and 800 mg/day. However, after two years of therapy for recurrent relapses, the patient failed to respond to 800 mg/day (episode 15) and received treatment with amphotericin B. In contrast to AIDS patients with OPC, correlation between MICs obtained by the NCCLS method and clinical response to fluconazole in non-neutropenic patients with candidemia is poor. These apparently contradictory observations undoubtedly reflect complex differences between the clinical status of the patients, their treatment regimens and the susceptibilities of the infecting Candida strains.

When assessing treatment failures, it is important to distinguish between microbiological resistance and host factors that can account for the failure or relapse. These host factors include the degree of immunosuppression, site and severity of infection, altered physiology, such as gastrointestinal malfunction and decreased saliva production, pharmacokinetic parameters, such as poor oral absorption and drug interactions, and patient compliance (Table 1). Permanently immunosuppressed patients on long-term azole therapy would appear to provide the ideal environment for selecting drug-resistant organisms. Moreover, DNA typing of
C. albicans isolates indicates that in some AIDS patients the same strain converts to resistance, whereas in others a new resistant strain is acquired during treatment.(16-17) In both instances, the resistant organisms are invariably cross-resistant to all of the commercially available azoles, which suggests that there is a common mechanism(s) of resistance. By contrast, azole-resistant C. albicans is rarely reported in immune competent patients and in those with transient immunosuppression, such as cancer and organ transplant patients and patients in the early stages of AIDS.

Mode of Action
The increasing importance of the azole antifungals in the treatment of fungal infections has been matched by a growing interest in their mode of action and, more recently, in the mechanisms by which
Candida spp. can mutate to resistance. The antifungal activity of the azoles is now well established as being due to the inhibition of cytochrome P-450-dependent 14¤-sterol demethylase (P-450DM), an important enzyme in ergosterol biosynthesis in fungi and in cholesterol biosynthesis in mammalian cells.(19) The clinical utility of the azoles resides in their selectivity for fungal over mammalian P-450DM. Ergosterol is the major fungal sterol and has a key role in maintaining membrane integrity and fungal cell growth. The depletion of ergosterol in azole-treated fungi is thought to inhibit their growth and morphogenesis. However, this mechanism of action is fungistatic rather than fungicidal in C. albicans, which underlines the importance of the host's immune system for eradicating the infecting organism and achieving a clinical cure.

The relatively low incidence of azole-resistant
C. albicans and its association with permanently immunosuppressed patients receiving long-term therapy has been suggested to be a consequence of the organism being genetically stable.(20) In contrast to other Candida spp., such as C. glabrata, C. albicans is diploid with no haploid sexual stage in its life cycle. Therefore, homozygous mutant alleles are necessary for a mutant phenotype to be expressed. This requirement in turn makes C. albicans much less susceptible than haploid organisms to point mutations and thereby reduces the chances of mutation to resistance. Furthermore, like other fungi, C. albicans is unable to exchange genetic material by transduction (via bacteriophages) or conjugation by plasmids (extrachromosomal DNA), both common mechanisms used by some bacteria to transfer resistance to antibacterial antibiotics.

There are several mechanisms by which
Candida spp. and Saccharomyces cerevisiae can become resistant to azoles in the laboratory, depending on the azole under investigation, the organism and the conditions in which it is cultured.(20) By contrast, only three mechanisms of resistance are apparent in the small number of clinical isolates of C. albicans and C. glabrata that have been studied to date. These mechanisms are overexpression of the target enzyme ( P-450DM), thereby reducing azole binding, mutations in other enzymes in the ergosterol biosynthetic pathway that compensate for the inhibition of P-450DM in azole-treated cells, or a reduced level of accumulation of drug.(20-23) Each mechanism confers cross-resistance to all of the commercially available azole antifungal agents. Furthermore, studies with radiolabeled fluconazole have shown that the resistance linked to reduced accumulation is due to energy-dependent drug efflux rather than to a barrier to influx.(21-23) This finding is characteristic of multi-drug resistance transporters that confer resistance to certain antibiotics in bacteria and protozoa and to a wide range of compounds in S. cerevisiae and mammalian cells. The relationship between the expression of the various azole resistance mechanisms and their effect on resistance in vivo is being examined in a number of laboratories. It would also be interesting to explore the relationship between resistance and pathogenicity because azole-resistant clinical isolates of C. albicans are much less pathogenic in vivo than their sensitive counterparts.


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