Supported by an unrestricted educational grant from Pfizer, Inc.

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Volume I, Issue 3 - June 1997

Opportunistic fungal infections resistant to antifungal agents have been increasingly documented in recent years and their frequency will likely continue to increase. This phenomenon appears due largely to the extensive use of antifungal agents to treat fungal infections that typically occur in severely immunocompromised and/or critically ill patients. Candida spp., Cryptococcus neoformans, and Aspergillus spp. are among the leading fungi responsible for these invasive infections. While antifungal resistance has been described with each of these fungi, resistance among Candida constitutes by far the most significant problem and will be the principal focus of this review.

As a prelude to any study of resistance, a clear definition is important. Since a poor therapeutic response can be due to factors other than antifungal drug resistance (Table 1), definitions that focus solely on persistence of the organism are problematic. Likewise, the ability of in vitro testing methods to label an organism as susceptible (S) or resistant (R) is limited by the arbitrary nature of susceptibility testing (see below). Our working definition is that resistance occurs when signs and symptoms of an infection persist despite adequate delivery of a tolerable concentration of drug.

This definition does not depend on susceptibility testing results and it also addresses the twin problems that in vitro susceptibility does not predict successful therapy and in vitro resistance predicts therapeutic failure only part of the time. All in vivo/in vitro correlations are limited by the multiple factors related to disease and host defense that affect the patient's clinical course. As a good example of this, a recent study of patients with cryptococcal meningitis found that fluconazole minimum inhibitory concentration (MIC) strongly predicted outcome in more severely compromised patients (those with concomitant cryptococcal fungemia) but had much less impact in less compromised patients. Similarly, a severely immunocompromised patient with invasive aspergillosis caused by a susceptible strain of Aspergillus may not respond to appropriate antifungal therapy until immunocompetence returns.

Antifungal drug resistance is often divided into two broad forms (Table 2). First, resistance can be present without prior antifungal therapy. This is known as primary or innate resistance and is typified by the innate resistance of Pseudallescheria boydii and Fusarium spp. to amphotericin B, the resistance of some Candida spp. to flucytosine, and the resistance of C. krusei to fluconazole. Alternately, resistance can develop in previously susceptible isolates during or after antifungal drug exposure. This form of resistance is termed secondary, or acquired, resistance and is most familiar in the setting of HIV-infected patients with oropharyngeal candidiasis due to C. albicans who receive multiple courses of azole antifungal therapy.

Since fungal drug resistance has become a more significant clinical problem, the development of antifungal susceptibility testing has attracted a great deal of interest. Initial data were difficult to interpret due to enormous interlaboratory variability in reported results (up to 50,000-fold differences were seen at times!). As a consequence of substantial efforts made both by the National Committee for Clinical Laboratory Standards and many collaborating laboratories, a standardized methodology known as M27 has been developed. The proposed method has steadily evolved, having been published as M27-proposed (P) in 1992 and M27-tentative (T) in 1995. Release as M27-approved (A) is expected shortly. The description of M27-T provided a more convenient microdilution methodology while M27-A includes a proposal for interpretive breakpoints for testing susceptibility of Candida spp. to fluconazole, itraconazole, and flucytosine (Table 3).

While the S and R interpretive categories used in the table will be familiar, the novel category "Susceptibility is Dose and/or Delivery Dependent" (S-DD) and its distinction from the intermediate (I) category requires explanation. For both azoles, infections caused by isolates with MICs in the S-DD range will respond only if a significant level of drug is delivered to the site of the infection. For fluconazole, use dosages of >=400 mg/d (6 mg/kg/d) in adults with normal renal function and body habitus is required. For itraconazole sufficient drug needs to be absorbed to produce a blood level of >=0.5 µg/mL. Conversely, the I interpretive category has been used both to describe isolates that can be treated with an increased dose of drug as well to describe isolates with indeterminate or uncertain clinical response to therapy.

The azoles have been widely used to treat a variety of both superficial and invasive fungal infections. Unfortunately, this broad usage led to both development of acquired resistance (especially among C. albicans) and to shifts in flora away from the C. albicans and towards the less susceptible non-albicans species.

The relative activity of the two newer systemic triazoles, itraconazole and fluconazole, against typical isolates of the major Candida species is shown in Table 4. Resistance to the older azole, ketoconazole, also occurs in a pattern that appears to parallel resistance to itraconazole but this drug's resistance patterns are less well studied and fewer data are available. For Candida isolates with reduced susceptibility to the azole antifungal agents, several mechanisms of resistance are known. The 14-alpha-demethylase sterol synthesis enzyme targeted by azoles may be overproduced or altered, the azole drug may be pumped from the cytoplasm using multidrug transporters, or other sterol synthesis enzymes may be altered to compensate for interference with 14-a-demethylase.

Currently, the most significant form of azole resistance is that seen between Candida and fluconazole. Both acquired resistance and intrinsic resistance are clinically relevant. Acquired resistance is most often seen with HIV-infected patients, where fluconazole-resistant oropharyngeal candidiasis (OPC) develops in patients with CD4+ T cell counts <100/mm³ and with a history of multiple episodes of OPC. Typically, initial episodes of OPC respond readily to a brief course of 100 mg/d of oral fluconazole. However, continuous or intermittent exposure to this antifungal agent can result in a need to gradually increase the dose of fluconazole in order to achieve an adequate clinical response or a complete lack of response even to high dosages of the antifungal agent. In both scenarios, patients with recurrent disease usually carry only one strain of C. albicans that gradually develops progressively higher MICs. While transmission of resistance strains from one person to another can occur, this does not yet appear common. Cross resistance to other azole antifungal agents is common but not universal.

Therapeutic strategies for patients with fluconazole-refractory infections are listed in Table 5. We find use of the suspensions especially helpful. When taken in a swish-and-swallow fashion, amphotericin B suspension (100 mg/mL, taken as 1 mL qid), itraconazole suspension (10 mg/mL, taken as 10 mL bid), or fluconazole suspension (10 mg/mL, taken as 10 mL bid) have often been quite effective.

Estimates of the frequency of this type of acquired resistance vary. In a recently completed survey, fluconazole-resistant (MIC >=64 µg/mL) C. albicans was isolated from 21% of a series of patients presenting with OPC in an AIDS clinic in Houston. Carriage of a resistant isolate was also correlated with prior antifungal therapy. While non-albicans isolates, usually with high MICs, are also frequently found in patients previously treated with an antifungal agent, their pathogenic role in OPC remains unclear.

In the hospital setting, extensive use of fluconazole has been correlated with increased rates of infection with intrinsically less susceptible isolates of species such as C. glabrata and C. krusei. This change in Candida species distribution has been observed in both cancer and general hospitals. However, outside the groups of patients who received fluconazole prophylaxis, had an immunosuppressed condition, or were infected with an intrinsically resistant strain of Candida, there have been very few cases of fluconazole resistance reported. The use of fluconazole for short periods of time to treat invasive Candida infections does not seem to be associated with development of acquired drug resistance. Treatment of patients infected with one of the intrinsically less susceptible species involves use of high dosages of fluconazole (for typical isolates of C. glabrata), use of amphotericin B (any species), and combinations of fluconazole or amphotericin B with flucytosine (any species). Due to its lack of parenteral preparations, use of itraconazole is not usually suitable in this setting.

Despite the widespread use of amphotericin B, resistance to this polyene antifungal agent remains an uncommon event among Candida isolates. Amphotericin B is thought to act by binding to ergosterol, and the principal mechanism of resistance to this drug is a decrease in the amount of ergosterol in the fungal cell membrane. Among Candida, amphotericin B has reliable activity against most of the species except for strains of C. lusitaniae, which is often intrinsically resistant (Table 4). In addition, reduced susceptibility appears common among isolates of C. glabrata. However, high-level resistance to amphotericin B, seen in all the major Candida species, is most common in neutropenic patients who have received prolonged courses of amphotericin B.

While flucytosine has been available for many years, its use in clinical practice has been limited due to its lack of parenteral formulation and its potential toxicity. Of even greater importance is the fact that many fungi are inherently resistant to this agent and that acquired resistance develops quickly during its use as monotherapy. The overall prevalence of resistance to flucytosine among Candida isolates is low except for C. krusei and C. lusitaniae.

Like the bacteria, the fungi appear quite capable of becoming resistant to a broad array of antifungal agents. While this review has focused on resistance among Candida spp., a small but growing body of evidence has also documented the potential for azole resistance among isolates of Cryptococcus neoformans, Aspergillus spp., and Histoplasma capsulatum; amphotericin B resistance among isolates of C. neoformans; and flucytosine resistance among a wide variety of fungal species. While it seems unlikely this process can be entirely prevented, the clinician is aided by the fact that antifungal resistance is usually found within well-defined and predictable settings. This, in combination with suitably performed and interpreted antifungal susceptibility testing results, aids in the identification and proper treatment of patients with resistant infections. In addition, new antifungal agents and new classes of antifungal agents under active development should provide additional tools for treating refractory infections.

Marcelo D. Martins, MD and John H. Rex, MD
Division of Infectious Diseases
University of Texas Medical School
Houston, Texas

Dr. Rex has a research grant from Janssen Pharmaceutica, Inc.

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