Chapter 44: Antifungal Agents and Resistance

Many fungal infections are self-limiting and require no chemotherapy. Superficial mycoses are often treated, but topical therapy can be used, thus limiting toxicity to the host. Invasive or systemic fungal infections that are not controlled by the host’s immune system require the prolonged use of antifungals. Given that most of the patients with these infections also have underlying immunosuppression, invasive mycosis can be among the most difficult of all infectious diseases to treat successfully. The characteristics of currently used antifungal agents are discussed next and summarized in Table 44–1. They are discussed in the text that follows in relation to their target of action, as illustrated in Figure 44–1.

CYTOPLASMIC MEMBRANE

Polyenes

The polyenes nystatin and amphotericin B are lipophilic and bind to ergosterol, the dominant sterol in the cytoplasmic membrane of fungal cells. After binding, they form annular channels, which penetrate the membrane and lead to leakage of essential small molecules from the cytoplasm and cell death. Their binding affinity for the ergosterol of fungal membranes is not completely specific, cross-reacting with mammalian sterols such as cholesterol. This is the basis for the considerable toxicity that limits their use. Almost all fungi are susceptible to amphotericin B, and the development of resistance is rare.

At physiologic pH, amphotericin B is insoluble in water and must be administered intravenously as a colloidal suspension. It is not absorbed from the gastrointestinal tract. The major limitation to amphotericin B therapy is the toxicity created by its affinity for mammalian as well as fungal membranes. Infusion is commonly followed by chills, fever, headache, and dyspnea. The most serious toxic effect is renal dysfunction, observed in virtually every patient receiving a prolonged therapeutic course. Experienced clinicians learn to titrate the dosage for each patient to minimize the nephrotoxic effects. For obvious reasons, use of amphotericin B is limited to progressive, life-threatening fungal infections. In such cases, despite its toxicity it retains a prime position in treatment often by administration of an initial course of amphotericin followed by a less toxic agent. Preparations that complex amphotericin B with lipids have been used as a means to limit toxicity. The even greater toxicity of nystatin limits its use to topical preparations.

Azoles

The azoles are a large family of synthetic organic compounds, which includes members with antibacterial, antifungal, and antiparasitic properties. Their activity is based on inhibition of the enzyme (14 α-demethylase) responsible for conversion of lanosterol to ergosterol, the major component of the fungal cell membrane. This leads to ergosterol depletion and lanosterol accumulation, forming defective membranes. All antifungal azoles have the same mechanism of action. The differences among them are in avidity of enzyme binding, pharmacology, and side effects. Azoles can also affect the precursors of some hormones and may therefore cause endocrinological side effects, restricting their use in pregnancy.

The systemic azoles are generally grouped based on antifungal spectrum. Fluconazole is primarily active against yeasts, including many Cryptococcus and Candida species. In contrast, newer azole compounds have extended activity against molds such as Aspergillus species, as well as the thermally dimorphic fungi (eg, Coccidioides, Blastomyces, Histoplasma). These mold-active azoles include itraconazole, voriconazole, posaconazole, and isavuconazole. Most of these agents can be given orally or parenterally. Although generally well-tolerated, the antifungal azoles can cause varying degrees of liver toxicity. Additionally, all systemic azoles, except isavuconazole, can adversely affect cardiac myocyte repolarization and therefore prolong the QTc interval on an EKG, placing patients at increased risk for cardiac arrhythmias. Topical agents such as ketoconazole, clotrimazole, and miconazole are available in over-the-counter topical preparations for superficial mycoses.

Allylamines

The allylamines are a group of synthetic compounds that act by inhibition of an enzyme (squalene epoxidase) in the early stages of ergosterol synthesis. The allylamine group includes an oral and topical agent, terbinafine used in the treatment of dermatophyte (ringworm) infections.

NUCLEIC ACID SYNTHESIS

Flucytosine

5-Flucytosine (5FC) is an analog of cytosine. It is a potent inhibitor of RNA and DNA synthesis. 5FC requires a permease to enter the fungal cell, where its action is not direct but through its enzymatic modification to other compounds (5-fluorouracil, 5-fluorodeoxyuridyic acid, 5-fluoruridine). These metabolites then interfere with DNA synthesis and RNA transcription.

Flucytosine is well absorbed after oral administration. It is active against most clinically important yeasts, including Candida albicans and Cryptococcus neoformans, but it has little activity against molds or dimorphic fungi. The frequent development of mutational resistance during therapy limits its application to mild yeast infections or its use in combination with amphotericin B for cryptococcal meningitis. The primary toxic effect of flucytosine is a reversible bone marrow suppression that can lead to neutropenia and thrombocytopenia. This effect is dose related and can be controlled by drug monitoring.

CELL WALL SYNTHESIS

The unique chemical nature of the fungal cell wall, with its interwoven layers of mannan, glucan, and chitin (Figure 44–1), makes it an ideal target for chemotherapeutic attack. The echinocandins, which block glucan synthesis, are now in clinical use and the nikkomycins, which block chitin synthesis, are in development.

Echinocandins

Echinocandins act by inhibition of a glucan synthetase (1,3-β-D-glucan synthetase) required for synthesis of the principal cell wall glucan of fungi. Its action causes morphologic distortions and osmotic instability in yeast and molds, similar to the effect of β-lactams on bacteria. The first such agent to be licensed was caspofungin, which has good activity against Candida and Aspergillus and a wide range of other fungi. Cryptococcus neoformans whose cell wall glucans have a slightly different structure is resistant. Since there are no similar human structures, toxicity is minimal. The newest echinocandins, micafungin and anidulafungin, have the same mode of action and a similar spectrum.

Nikkomycins

Nikkomycins target fungal cell wall components similar to echinocandins. These compounds inhibit chitin synthases, which polymerizes the N-acetylglucosamine subunits that make up chitin. The result is inhibition of chitin synthesis. The agent in development, nikkomycin Z, has activity against dimorphic fungi such as Coccidioides immitis and Blastomyces dermatitidis but not against yeast or Aspergillus. However, chitin synthesis remains an interesting strategy for novel antifungal development.

Other Antifungal Agents

Griseofulvin is a product of one of the Penicillium species of molds. Griseofulvin is actively taken up by susceptible fungi and acts on the microtubules and associated proteins that make up the mitotic spindle. It interferes with cell division and possibly other cell functions associated with microtubules. Griseofulvin is absorbed from the gastrointestinal tract after oral administration and concentrates in the keratinized layers of the skin. It is active only against the agents of superficial mycoses. Clinical effectiveness has been demonstrated for many causes of dermatophyte infection, but the response is slow. Prolonged therapy may be required.

Potassium iodide is the oldest known oral chemotherapeutic agent for a fungal infection. It is effective only for cutaneous sporotrichosis. Its activity is somewhat paradoxical, because the mold form of the etiologic agent, Sporothrix schenckii, can grow on medium containing 10% potassium iodide. The pathogenic yeast form of this dimorphic fungus appears to be susceptible to molecular iodine.

DEFINITION OF RESISTANCE

The concepts, definitions, and laboratory methods described in Chapter 23 for bacterial resistance are generally applicable to fungi. Quantitative susceptibility is determined by measuring the minimal inhibitory concentration (MIC) of a drug under conditions that favor the growth of fungi. The wide range of growth rates and diversity of growth forms (yeast, hyphae, conidia) in the various fungi have added technical variables to testing, but standardized methods are now available. Comparison of MICs with drug pharmacology allows classification of fungi as susceptible or resistant, but these results do not yet predict clinical outcome with the same certainty they do with bacteria. Because of its specialized nature, the availability of antifungal susceptibility testing is restricted to major centers and reference laboratories.

MECHANISMS OF RESISTANCE

Many of the same resistance mechanisms observed in bacteria are also found in fungi. Fungi tend to primarily inactivate drug activity using efflux pumps and by altering their biosynthetic pathways. In contrast to bacteria, fungi do not make hydrolytic enzymes to inactivate antibiotics to the same extent as bacteria. In part this may be due to the reduced ability for horizontal gene transfer between species.

Polyene Resistance

Because amphotericin B binds directly to the ergosterol in the fungal cell membrane, the only means to resist this action is to change the membrane sterol composition. Therefore, only a few rare fungal species are intrinsically resistant to amphotericin B.

Flucytosine Resistance

Flucytosine (5-FC) requires a permease for entry into the cell and then multiple enzymes to modify it to the active metabolites that inhibit nucleic acid synthesis. Mutation in any one of these enzymes renders the drug ineffective. This happens readily under the selective pressure of 5-FC use. It is one of the few antimicrobials in which emergence of resistance is predictable during therapy of an acute infection. This is the reason that its use is mostly limited to combination therapy with other antifungals.

Azole Resistance

There are several mechanisms by which fungi can become resistant to the azoles. The most well-characterized mechanism is through the induction of efflux pumps that transport the drug out of the cell. Some pumps act on all azoles, and others act on only one. Fungal species can also alter the subunits of the demethylase enzyme by mutation, effectively decreasing the affinity of the azole for its enzyme target. Multiple mutations can have an additive effect.

Additionally, some fungi are able to decrease the effect of the azoles by increasing the production of the drug target. Some azole-resistant strains of Candida and Cryptococcus species actually duplicate regions of their genome to increase the number of copies of the demethylase-encoding genes. This results in the requirement of higher concentrations of the azole drug to inhibit fungal growth. Some azole-resistant strains have also been shown to accomplish ergosterol synthesis by an alternate pathway, thus bypassing the azole target enzyme.

Echinocandin Resistance

Although the echinocandins are relatively new, resistance has already been observed with their use. One resistance mechanism is an altered target. Mutations in subunits of the glucan synthetase have been correlated with increases in MIC more than a thousand-fold.

SELECTION OF ANTIFUNGALS

As with all chemotherapy, the selection of antifungal agents for treatment of superficial, subcutaneous, and systemic mycoses involves balancing expected efficacy against toxicity. The factors to be considered are the following: (1) the threat of morbidity or mortality posed by the specific infection, (2) the immune status of the patient, (3) the toxicity of the antifungal, and (4) the probable activity of the antifungal agent against the fungus. In the case of superficial mycoses, the risks of appropriate therapy are small, and various topical agents may be safely used. At the other extreme, an immunocompromised patient will most likely be treated aggressively with systemic agents for proven or even suspected systemic fungal infection. Since susceptibility tests are usually not available, the decisions regarding which agents to use are usually made and sustained on an empiric basis. Even when guided by in vitro testing, treatment failures are common particularly in the immunocompromised, emphasizing the prime importance of the immune system in preventing and clearing systemic fungal infections. It is hoped that the addition of the novel antifungal agents will have a favorable effect on these outcomes.