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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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