At least 70 genera of filamentous fungi (56), yeasts (11), and dimorphic fungi (3) have been reported to cause human keratitis^{2, 3, 4, 5, 6, 7, 8, 9, 10, 11} (Table 1). The principal causes in the United States are the septate filamentous fungi, predominantly Alternaria, Aspergillus, Curvularia, and Fusarium and yeasts, predominantly Candida. Of importance to ophthalmologists are the differences in geographic prevalence, risk factors, pathogenesis, distinctive signs of keratitis, and antifungal susceptibility of filamentous fungi and yeasts. Fungi account for less than 5% of cases of microbial keratitis in developed countries and 10% to 15% or more in developing countries.¹²

TABLE 1. Reported Causes of Fungal Keratitis

  Filamentous Fungi
  Septate

  Moniliaceae (nonpigmented)
  Acremonium
  Acrostalagmus
  Arthrobotrys
  Arthrographis
  Aspergillus
  Aureobasidium
  Beauvaria
  Botryodiplodia
  Botrytis
  Calcarisporium
  Cladosporium
  Colletotrichum
  Cylindrocarpon
  Drechslera
  Epidermophyton
  Exserohilum
  Fonsecaea
  Fusarium
  Fusidium
  Geotrichum
  Glenospora
  Graphium
  Helminthosporium
  Hormodendrum
  Metarhizium
  Microsphaeropsis
  Microsporum
  Nigrospora
  Paecilomyces
  Penicillium
  Periconia
  Phaeoisaria
  Phoma
  Pithomyces
  Rhizoctonia
  Scedosporium
  Scopulariopsis
  Stachybotrys
  Syncephalastrum
  Tetraploa
  Trichoderma
  Trichophyton
  Trichosporon
  Verticillium
  Volutella
  Dematiaceae (pigmented)

  Alternaria
  Aureobasidium
  Bipolaris
  Curvularia
  Exserohilum
  Lasiodiplodia
  Phialophora
  Torula

  Absidia
  Mucor
  Rhizopus

  Candida
  Monilia
  Oospora
  Pichia
  Rhodosporidium
  Rhodotorula
  Saccharomyces
  Sporotrichum
  Torulopsis
  Trichosporon
  Ustilago

  Blastomyces
  Cryptococcus
  Sporothrix

Contact lens wear is an uncommon risk factor in fungal keratitis.²⁴ Filamentous fungi cause most infections associated with cosmetic or aphakic refractive contact lens wear. Candida is the principal cause of keratitis secondary to therapeutic contact lens wear. Pathogenesis of these infections is probably enhanced by fungal contamination of contact lens cases and paraphernalia, by the ability of fungi to adhere to soft contact lenses, and by microtrauma to the corneal epithelium, which accompanies lens wear.

The precise events that follow adherence of fungi to the corneal epithelium or exposed deeper structures and replication of the organism to produce keratitis have not been defined. The intrinsic virulence of fungi relates to their ability to proliferate within corneal tissue, resist host defenses, and produce tissue damage.²⁵ Filamentous fungi proliferate within the corneal stroma without release of chemotactic substances, thereby delaying the beneficial, inhibitory phases of the host response. Filamentous fungi multiply by apical hyphal extension, whereas yeast multiply by budding or transformation from the blastophore to the pseudohyphal phase. The large size of the true hyphae of filamentous fungi and pseudohyphae of yeasts precludes complete ingestion by neutrophils and macrophages. Certain fungi secrete enzymes that degrade organic substances into soluble nutrients, which are passively absorbed or actively transported into the cell. Candida albicans contains a phospholipase A and lysophospholipase on the surface of blastospores, which may aid invasion of tissue. Similar mechanisms probably account for the ability of Fusarium solani and other filamentous fungi to ramify within the corneal stroma and penetrate Descemet's membrane. Fungi also release various toxic substances, including proteases, hemolysins, and exotoxins. The role of mycotoxins in the pathogenesis of fungal keratitis has not been determined. Comparable to the pathogenesis of other forms of infectious microbial keratitis, the host response contributes to stromal destruction by release of lysosomal substances from polymorphonuclear leukocytes and other substances through a cascade of complex reactions.

The principal risk factors for yeast or Candida keratitis are protracted epithelial ulceration, penetrating keratoplasty, and therapeutic soft contact lens wear. Concurrent administration of topical corticosteroids enhances the replication of Candida through suppression of the host inflammatory response. Although polymicrobial keratitis is uncommon, risk factors for Candida are comparable to bacterial keratitis, and Candida is the most common genus among mixed bacterial and fungal keratitis.^26,27 Polyfungal keratitis is rare.¹⁴

The distinct biomicroscopic signs of infectious microbial keratitis are epithelial and stromal ulceration (vs. intact epithelium), necrotizing stromal inflammation (vs. nonnecrotizing inflammation), and single location (vs multifocal sites) of the stromal inflammation. These distinctive signs are determined by the strain (virulence) of the responsible organism(s); the mechanism of inoculation into the cornea; the time interval from inoculation to examination; the antecedent status of the cornea; antimicrobial or corticosteroid therapy; and other host factors, such as an ocular surface abnormality, tear dysfunction state, lid closure dysfunction, and immunosuppression. Other biomicroscopic signs are helpful in judging the severity of the inflammatory process but not the presence or type of infection. These include epithelial edema, stromal edema, loss of endothelial reflectivity (pseudoguttata), endothelial plaque, aqueous flare and cells, and hypopyon.

In the early stages of infection, filamentous fungi produce signs that are readily distinguishable from yeast or bacterial keratitis. The most distinctive sign is the presence of delicate, fine, feathery, opalescent, gray-white or yellow-white material in the anterior stroma, surrounded by scant cellular infiltrate or edema (Fig. 1). The epithelium may be intact. The overlying epithelium may be granular and the surface elevated and irregular in contour. Linear infiltrates typically extend into the adjacent stroma. Multiple discrete opacities may develop outside the perimeter of the principal focus of inflammation, either separated by clear stroma or linked by fine linear collections of inflammatory cells and material (Figs. 2 and 3). In the absence of inflammation in the adjacent stroma, branching hyphal fragments may be visualized by biomicroscopy (Figs. 4 and 5). Confocal microscopy may also detect hyphal elements within the stroma.^28,29 Peripheral infection resembles noninfectious marginal infiltrative and ulcerative keratitis (Fig. 6). Multifocal keratitis may develop after contact lens wear or injury by multiple projectiles (Fig. 7). In the early stages, iritis is present and the intraocular pressure remains normal. Inappropriate, empirical therapy of fungal keratitis with topical fluoroquinolone or aminoglycoside antibiotics may suppress or eliminate the superficial elements but allow extension of the organisms into the stroma because these agents may possess selective antifungal activity.^4,30,31

There is no distinguishing clinical sign by which to recognize the genus or species of the infectious filamentous fungus. F. solani is the most virulent organism and typically produces rapidly progressive infection characterized by epithelial and stromal ulceration, dense stromal necrosis, abundant cellular infiltrate, and edema in the adjacent stroma and hypopyon (Figs. 8 and 9). Delicate feathery components are transient. Individual hyphal fragments are rarely visualized. Infection by certain species of Aspergillus and Scedosporium (Figs. 10 and 11) resembles F. solani keratitis and progresses rapidly. Infection by less virulent organisms, such as Curvularia and Alternaria species, produces small, focal (less than 3-mm diameter) areas of nonnecrotizing stromal inflammation with delicate feathery borders (see Fig. 1 and Fig. 12). Macroscopic pigmentation may develop in keratitis caused by Alternaria, Curvularia, and other dematiaceous fungi (Fig. 13).^4,11,14 The central component may progress to dense, opaque, gray-white suppuration in the deep stroma without enlargement in total area and may be accompanied by mild inflammation in the adjacent stroma. Iritis is minimal to moderate. Infection caused by other, relatively less virulent organisms resembles herpes simplex or noninfectious keratitis (Fig. 14).

Keratitis caused by Nocardia, nontuberculous Mycobacterium, and other low-virulence bacteria resembles filamentous fungal keratitis by the presence of intact epithelium, multiple stromal infiltrates, and minimal inflammation in the adjacent stroma.

Candida infection typically produces epithelial ulceration, focal necrotizing stromal inflammation, moderate cellular infiltrate and edema in the adjacent stroma, and mild or moderate iritis in the early stages, indistinguishable from bacterial keratitis (Figs. 15, 16, and 17). Fungal elements cannot be detected by biomicroscopy. If untreated, the keratitis evolves to produce dense suppuration and necrosis of the deep stroma. Although multifocal suppuration may develop in polymicrobial keratitis, there is no distinctive sign of mixed Candida and bacterial infection (Fig. 18).

Advanced, severe filamentous fungal or yeast keratitis is indistinguishable from keratitis caused by virulent bacteria such as Staphylococcus aureus or Pseudomonas aeruginosa. The area of epithelial and stromal ulceration is large. Dense, opaque, homogenous, yellow-white stromal necrosis develop and is surrounded by confluent cellular infiltrate and full-thickness stromal edema (Figs. 19, 20, 21). Hyphal elements may penetrate Descemet's membrane and endothelium and be visualized in the anterior chamber. Fibrinous material accumulates over the endothelium, anterior chamber angle, and iris. Pain is typically severe. Secondary ocular hypertension may ensue. Progressive stromal necrosis leads to corneal perforation and, rarely, consecutive endophthalmitis.

TABLE 2. Materials and Media Used

The Kimura platinum spatula (number E1091)* is ideal for obtaining material from the cornea. The spatula tip cools rapidly after sterilization in the alcohol flame. Use of several spatulas expedites specimen collection. If a platinum spatula is not available, material may also be obtained with a disposable blade, bent needle, surgical knife, disposable cautery, or similar instrument. Proparacaine hydrochloride (0.5%) is less antiseptic than cocaine, tetracaine, and other topical anesthetics and is preferred for anesthetizing the cornea. Topical anesthetics do not interfere with cytologic techniques. Glass slides with frosted ends and etched circles facilitate labeling and recognition of smeared material. Slides should be dipped in methanol and wiped to remove dust and other material, which may also absorb stain to produce false-positive results. After transfer of material, slides should be immersed immediately in methanol (95%) in a Coplin jar and retained for 5 to 10 minutes for fixation before staining.

Blood and chocolate agar are used in the standard procedure principally for primary isolation of aerobic bacteria; however, these media support the growth of most fungal pathogens at 35°C under increased carbon dioxide.^32,33 Sabouraud's dextrose agar with gentamicin (50 μg/ml) is the primary isolation medium for fungi. The medium is nutritionally deficient but the low pH favors fungal growth. Cycloheximide should not be incorporated in Sabouraud's medium because it inhibits the saprophytic fungi principally responsible for keratitis. Thioglycollate broth is a semisolid medium that provides adequate redox potentials for the growth of aerobic fungi, as well as aerotolerant anaerobes, microaerophilic bacteria, facultative anaerobes, and aerobic bacteria. Schaedler's agar enhances the growth of anaerobic bacteria but does not support growth of filamentous fungi and yeasts.

Instillation of one or two drops of proparacaine hydrochloride (0.5%) provides adequate corneal anesthesia in adults. Sedation, akinesia, or general anesthesia may be required for children. The cooled Kimura spatula should be scraped over the surface of the area of suppuration with a series of short, moderately firm strokes in one direction, taking care not to scrape adjacent, normal intact epithelium or touch the eyelids. Each scraping should be used to inoculate only one medium or to prepare one smear. The agar plate should be inoculated by lightly streaking both sides of the spatula over the surface to produce a row of separate inoculation marks. The surface of the agar should not be penetrated because the recognition and isolation of fungi and other microorganisms in cut streaks of the agar is difficult. Thioglycollate broth should be inoculated by transferring the material from the spatula to a moistened calcium alginate swab and pushing the shaft to the bottom of the tube. Multiple samples should be obtained for each medium and for preparation of multiple slides.

The preferred stains for identification of hyphal fragments and yeasts in smears of corneal scrapings are acridine orange and calcofluor white.^{33, 34, 35, 36, 37, 38} Each of these techniques requires use of a fluorescent microscope. The optical activity of acridine orange is due to the binding of the dye to deoxyribonucleic acid (DNA) of bacteria, fungi, and amoebae to form a green-fluorescing complex. Acridine orange is more sensitive than the Gram stain in detecting microorganisms in clinical specimens³⁶ and is, therefore, a rapid and sensitive method for screening all corneal smears in suspected infectious keratitis. If microorganisms are detected by acridine orange stain, the slide can be washed and stained with Gram or other specific stains. The preferred procedure is to fix the smears in 95% methanol for 5 to 10 minutes, apply the acridine orange dye (number 3336-75)* for 2 minutes, rinse the slide with tap water, blot dry, and examine by fluorescent microscopy. Hyphal fragments stain yellow-orange or green, and yeasts stain brilliant orange (Fig. 22).

Calcofluor white is a fabric whitener used in the textile industry. The material absorbs ultraviolet light at 340 to 400 nm and emits a blue light. Calcofluor white has an affinity for cellulose and chitin, which are common compounds of hyphal fragments and yeasts.^37,38 The preferred procedure is to fix the smear in 95% methanol for 5 to 10 minutes, apply calcofluor white stain (number 17353)* to the surface of the slide, apply a coverslip over the slide, drain excessive fluid with an absorbent towel, and examine by fluorescent microscopy. Depending on the selection of filters, fungal elements stain apple green to steel gray against a rust or red background (Fig. 23).

Other stains should be used if a fluorescent microscope is not available. The Gram stain does not stain the cell wall or septa of hyphal fragments but is absorbed by the protoplasm. The stain characteristics are inconsistent and nonspecific (Fig. 24). Yeasts typically stain dark blue and can be readily distinguished from bacteria, foreign material, precipitate, and other artifacts. The Giemsa stain is a simple, rapid method for identifying fungal elements, although not as sensitive as acridine orange or calcofluor white in detecting hyphal fragments and yeasts. The Giemsa stain has an affinity for DNA and ribonucleic acid (RNA) in microorganisms. The stain must be prepared fresh daily according to standards. The preferred method is to fix the smear in 95% methanol for 5 to 10 minutes, immerse the slide in Giemsa stain for 1 hour, rinse the slide briefly in 95% ethanol, blot dry, and examine by standard light microscopy. Hyphal fragments and yeasts appear dark blue or purple.

Fungal elements may also be identified by standard light microscopy in direct smears by trichrome, Gomori's methenamine silver, and periodic acid-Schiff stains, although these techniques require 1 to 2 hours. A saline or potassium hydroxide wet mount is a simple, rapid technique.^10,39 Polymerase chain reaction may be further developed as a practical and sensitive method of detecting fungi in corneal material.^14,40,41

Most fungi responsible for keratitis generally appear on solid media or in liquid media within 2 to 5 days after inoculation. Solid media should be examined daily by direct illumination and a dissecting microscope. All media should be kept for a minimum of 21 days because of the slow growth of certain organisms. Filamentous fungi and yeasts are identified according to standard procedures. Speciation may be helpful in predicting the severity of keratitis, such as F. solani or C. albicans, but often requires consultation with a diagnostic mycologist or reference laboratory.

Corneal material is difficult to obtain with a Kimura's spatula if the epithelium is intact or if the locus of infection is deep within the corneal stroma. The preferred method is corneal biopsy with the aid of topical anesthesia (proparacaine hydrochloride 0.5%) and slit lamp biomicroscopy. A simple method is to incise the cornea to the approximate depth of the suppuration with a disposable 2- or 3-mm punch trephine Baker's biopsy punch*, deepen the incision with an unguarded diamond or metal blade, grasp the tissue with a 0.12-mm forceps, and excise with straight or angled small (Vannas) scissors. The fragment should be placed into trypticase soy broth or saline without preservative for transportation to the microbiology laboratory. The fragment is emulsified and inoculated on the same media as used for corneal scrapings (see Table 2). Smears are prepared directly from the suspension. A second biopsy specimen should be submitted to the pathology for histologic sections. Corneal fragments can also be excised with blade or scissors and inoculated directly on media and emulsified for smears. Corneal tissue obtained by superficial keratectomy or penetrating keratoplasty should be bisected by the surgeon. One half should be submitted to the microbiology laboratory and one half for histologic section.

Keratocentesis is reserved for suspected extension of organisms into the anterior chamber from deep stromal suppuration that cannot be sampled by corneal scraping or biopsy.

If fungal keratitis is suspected on the basis of the risk factors and distinctive biomicroscopic signs, antifungal therapy should be deferred until confirmation of fungal elements in smears of corneal or growth of an organism in one or more media (Fig. 25).

INITIAL THERAPY

The preferred initial therapy of early, nonsevere filamentous fungal keratitis, as suggested by the detection of hyphal fragments in corneal smears, is natamycin 5% drops administered hourly. Natamycin is a tetraene polyene antibiotic similar in mode of action to amphotericin B. Natamycin is effective in vitro against most filamentous fungi responsible for keratitis. Polyene antifungal agents achieve fungicidal activity by complexing with ergosterol in the plasma membrane, causing membrane disruption, increased permeability, leakage of cytoplasmic contents, and cell death. The compound is available as a 5% microsuspension (Natacyn*) in a 15-ml glass bottle and should be refrigerated or stored at room temperature with minimal light exposure. Corneal and intraocular penetration of natamycin following topical application is limited, although enhanced in the presence of epithelial ulceration and stromal inflammation. The compound is relatively nonirritating to the conjunctiva and cornea. It typically adheres to the stromal surface within the area of ulceration and accumulates in the inferior fornix. Natamycin is insoluble in water. Undiluted or diluted natamycin 5% suspension should not be administered by subconjunctival injectionbecause of the potential for producing severe conjunctival inflammation and necrosis. If natamycin is not immediately available, initial therapy should be amphotericin B drops (1 to 2.5 mg/ml) every 15 minutes for the initial 24 to 48 hours (see below).

The preferred initial therapy for early, nonsevere yeast keratitis as suggested by the detection of yeasts or pseudohyphae in corneal smears is topical amphotericin B drops (1 to 2.5 mg/ml) every 15 minutes for the initial 24 to 48 hours. Amphotericin B is a heptaene polyene antibiotic, similar in mode of action to natamycin and other polyenes. Amphotericin B is more active in vitro against C. albicans and other Candida species than natamycin and may be equally effective against various filamentous fungi. The initial selection of amphotericin B in yeast infections is based on comparative trials in experimental keratitis and clinical experience with keratitis in humans. Topical administration of amphotericin B drops produces low levels of the compound in the corneal stroma, which is enhanced by the absence of epithelium and the presence of stromal inflammation.

Amphotericin B drops are prepared by adding 10 ml of sterile distilled water without a bacteriostatic agent to the intravenous preparation (Fungizone Intravenous*) to produce a concentration of 5 mg/ml. This solution is further diluted by sterile distilled water to produce a final concentration of 1 to 2.5 mg/ml. Amphotericin B eye drops are irritating to the conjunctiva and cornea, presumably related to the sodium deoxycholate used as a solubilizing agent in the intravenous preparation. Amphotericin B eye drops as constituted should be protected from light and kept on ice or refrigerated. A new formulation should be prepared weekly. Topical amphotericin B ointment (5 mg/g) has been used as single or adjunctive therapy for A. fumigatus and F. solani keratitis.⁴² The safety and efficacy of topical or intravenous amphotericin B lipid complex in fungal keratitis has not been established.⁴³

The consideration of additional topical, subconjunctival, or systemic antifungal therapy in filamentous fungi or yeast keratitis is problematic. The rationale for use of one or more additional agents is based on the availability of new compounds and their in vitro activity, safety, and efficacy in keratitis in humans and relative lack of toxicity. The most promising compounds are the azole class of antifungal agents.

Azoles are synthetic chemical antifungal agents that contain a five-member azole ring that is attached by a carbon-nitrogen bond to other aromatic rings. Older imidazole drugs, such as clotrimazole, econazole, miconazole, and ketoconazole, contain two nitrogen atoms in the azole ring. The newer triazole drugs, such as fluconazole, itraconazole, and voriconazole, contain three nitrogen atoms in the azole ring. The primary mechanism of action of the azole compounds is to interact with the cytochrome P-450 enzyme to inhibit C-14 demethylation of lanosterol, thereby causing ergosterol depletion and accumulation of aberrant sterols in the fungal cell membrane.⁴⁴ Inhibition of ergosterol biosynthesis alters fungal cell membrane fluidity and modifies the function of other fungal cell membrane-associated enzymes. In high concentrations, the azoles directly damage the cell membrane by attacking phospholipids thereby exerting rapid fungicidal activity. The compounds may also inhibit other enzyme systems and block the morphogenetic transformation of Candida yeast form to pseudohyphae. Although similar in mechanism of action, the antifungal azoles differ extensively in antifungal spectrum, water solubility, tissue penetration, pharmacokinetics, and toxicity. Because systemic azole antifungal agents inhibit cytochrome P-450 dependent enzymes involved in biosynthesis of steroid hormones in mammalian cells, they may produce endocrine-related side effects such as depletion of testosterone and glucocorticoids, resulting in gynecomastia and adrenal insufficiency, respectively.⁴⁴

For the initial treatment of severe filamentous fungal or yeast keratitis or for keratitis progressing on initial topical natamycin or amphotericin B therapy, fluconazole should be considered as adjunctive therapy by oral, topical, or subconjunctival administration.^45,46 Fluconazole has broad in vitro antifungal activity but is most active against Candida and Aspergillus. Intravenous miconazole is no longer available for reconstitution for topical administration or subconjunctival injection. Oral ketoconazole is not indicated as adjunctive therapy with the availability of the newer triazoles.

Fluconazole, is a water-soluble triazole; has a relatively low molecular weight (305); and is available in oral 100-mg capsules (Diflucan oral) and intravenous solution (Diflucan IV), sterile solution in 0.9% sodium chloride, 2 mg/ml, 100 ml unit.⁴⁷ Fluconazole is weakly protein bound (11%), has a high bioavailability (half-life of approximately 30 hours), is metabolically stable, penetrates well into cerebrospinal fluid, and is excreted unchanged in the urine. Adverse reactions following oral administration are uncommon, predominantly nausea, vomiting, diarrhea, abdominal pain, and headache. Oral fluconazole produces effective drug levels in the cornea, aqueous, vitreous, and retina/choroid in animals.⁴⁸ Intraocular injection of fluconazole (100 μg) is well tolerated and has been associated with resolution of consecutive and exogenous fungal endophthalmitis. Although the minimal inhibitory concentrations of fluconazole are higher than other azoles in most susceptibility test systems, fluconazole achieves greater effectiveness in nonocular human infections on the basis of its more favorable pharmacokinetic properties.^44,47 The recommended daily dosage of oral fluconazole is 200 to 400 mg, either by oral or intravenous administration. Oral absorption of the drug is rapid and almost complete, thereby reducing the requirement for intravenous administration. The efficacy and safety of higher doses have not been determined.

Itraconazole may also be a helpful adjunctive agent in fungal keratitis,⁴⁹ based on in vitro activity, pharmacokinetic properties, and limited clinical trials in nonocular infections. Itraconazole is a triazole antifungal agent similar in spectrum of in vitro activity to that of fluconazole but is poorly soluble in water and highly protein bound, thereby limiting bioavailability in tissue. Food increases absorption of itraconazole. Following oral administration, tissue concentrations are often higher than plasma concentrations, even in patients with meningitis. Oral itraconazole has been most effective as alternate therapy to intravenous amphotericin B in invasive Aspergillus and has been superior to other azoles in the treatment of oral, esophageal, and vaginal candidiasis. Itraconazole is available in oral 100-mg capsules (Sporanox); the recommended daily dosage is 200 to 400 mg.

The most promising new triazole antifungal agent for treatment of fungal keratitis is voriconazole (UK-109,496), a derivative of fluconazole, currently in clinical trials by Pfizer Central Research. Its mechanism of action is similar to that of the other triazole agents, however, it is more selective to various mammalian cytochrome P-450 enzyme systems than other azoles. It is more highly active in vitro than fluconazole and itraconazole and equally effective as amphotericin B against Aspergillus sp., F. solani, F. oxysporum, Scedosporium apiospermum, S. prolificans, and Candida sp.^{50, 51, 52, 53, 54, 55, 56, 57, 58} Intravenous and oral administration of voriconazole in normal adults in dosages of 50 to 200 mg/day produces maximum plasma concentration between 1 and 2 hours.⁵⁷ Adverse side effects are minimal and include fully reversible visual disturbances, gastrointestinal upset, and elevation of liver function enzymes. As an investigational agent, voriconazole is available as an intravenous preparation as a cyophile, containing 200 mg voriconazole with 3200 mg sulphobutyl ether-B-cyclodextrin sodium, and 50-mg tablets (An open label, noncomparative protocol for the emergency use of Voriconazole [UK 109.496] in patients with life-threatening, invasive mycoses who are failing on currently available antifungal agents, Pfizer Central Research, September, 1999).

F. solani endophthalmitis in a 16-year-old girl resolved following intravenous (4 mg/kg twice daily), oral (6 mg/kg twice daily), topical (10 mg/ml), and intraocular (10 μg/0.1 ml) administration of voriconazole.⁵⁹ At the Cullen Eye Institute, oral voriconazole was successfully used (200 mg daily) to treat Scedosporium apiospermum keratitis in a 53-year-old woman failing on topical natamycin; topical, subconjunctival, and oral fluconazole; oral itraconazole; and oral terbinafine.

Supplementary initial therapy in fungal keratitis includes a cycloplegic-mydriatic agent, such as atropine 1% every 8 hours or scopolamine hydrobromide every 6 hours. A topical β-blocker or oral carbonic anhydrase inhibitor should be used to control secondary ocular hypertension. Topical or oral nonsteroidal anti-inflammatory agents and topical anticollagenolytic agents provide no additional benefit. Topical corticosteroids should not be used.

OTHER AGENTS

Terbinafine is an allylamine antifungal agent that inhibits the enzyme squalene epoxidase in the fungal membrane,⁶⁰ thereby interfering with the biosynthesis of ergosterol. Terbinafine has broad in vitro antifungal activity against filamentous fungi and yeasts, including Aspergillus, Fusarium, Scedosporium, and Candida,^{61, 62, 63, 64, 65, 66} and acts synergistically in vitro with amphotericin B and selected azoles against certain fungi.⁶⁵ Terbinafine is available for oral administration (Lamisil), 250 mg once daily, primarily for the treatment of onychomycosis. Adverse reactions are minimal, predominantly skin rash and gastrointestinal upset. Reports are lacking on the efficacy of oral terbinafine as adjunctive therapy of fungal keratitis, although in vitro activity, pharmacokinetic properties, and safety are promising.

Chlorhexidine gluconate is a biguanide germicide that has broad antimicrobial effect and is well tolerated by the ocular surface in concentrations less than 1%. Chlorhexidine gluconate 0.02% drops have been used extensively in single or combined therapy of Acanthamoeba keratitis in humans. Recent randomized clinical trials in human fungal keratitis suggested that chlorhexidine gluconate 0.2% drops may be as equally effective as natamycin 2.5% drops in Aspergillus and Fusarium keratitis.⁶⁷

Polyhexamethylene biguanide is a polymeric biguanide that interacts with cytoplasmic membranes to cause leakage of cellular constituents and inhibition of respiratory enzymes. The agent is active in vitro against a variety of bacteria, fungi, and Acanthamoeba. In a rabbit model of F. solani keratitis, polyhexamethylene biguanide 0.02% drops were effective in reducing the number of colony-forming units as compared with placebo controls.⁶⁸

MODIFICATION OF THERAPY

Refinement of initial therapy of fungal keratitis is hampered by multiple factors, including the slow evolution of clinical signs, uncertainty of the virulence and destructiveness of the organism based solely on the identification of genus or species, invalidity of in vitro antifungal susceptibility tests in predicting in vivo efficacy, and limited availability of safe and effective alternate antifungal agents. The rate of improvement and duration of antifungal therapy of individual genera or species of fungi cannot be predicted. If untreated, F. solani, Scedosporium, and C. albicans infections produce rapidly progressive stromal necrosis, fibrin accumulation in the anterior chamber, secondary glaucoma, and corneal perforation.

Improvement in clinical signs may be difficult to detect during the initial days of effective antifungal therapy. The most helpful biomicroscope signs are blunting of the perimeter and reduction of the density of the suppuration, reduction in cellular infiltrate and edema in the surrounding stroma, reduction in anterior chamber inflammation, and progressive re-epithelialization. The most encouraging sign of improvement is loss of the delicate, feathery perimeter of the stromal inflammation. Reduction in the area of suppuration may be difficult to detect. Frequent sketching of the cornea and measurement of the principal site of suppuration with the continuous variable slit on the Haag-Streit slit lamp or the eyepiece reticule on the Zeiss Slit lamp are helpful. The area of stromal suppuration can be estimated by measuring the greatest length and width in millimeters and multiplying the figures. Rate of epithelial healing is not a reliable sign, particularly because natamycin 5%, amphotericin B 0.1% to 0.25%, and other agents interfere with epithelial repair. Anterior chamber flare and cells diminish slowly.

For suspected filamentous fungal keratitis, natamycin 5% drops should be administered hourly for the initial 24 to 48 hours and at 2-hour intervals for the next 3 to 5 days if the corneal signs suggest stability or improvement of corneal inflammation. For yeast keratitis, amphotericin B 0.1% to 0.25% drops should be administered at 15-minute intervals for the first 24 to 48 hours and thereafter reduced during the next 4 to 6 days (e.g., 30-minute intervals for 2 to 3 days, hourly for 2 to 3 days). Cycloplegic-mydriatic drops should be continued. Antifungal therapy should be maintained for at least 4 to 6 weeks or longer if the inflammatory process is judged to be improved or inactive. Reduction in frequency of daily administration of drops should be at once- or twice-weekly intervals (i.e., eight times daily for 1 week, six times daily for 1 week, etc.). Exophytic fibroinflammatory fungal sequestration may require superficial keratectomy.¹⁴ Protracted epithelial ulceration, punctate corneal epithelial erosion, and diffuse stromal haze imply drug toxicity. Mild or moderate conjunctival hyperemia is an acceptable adverse reaction if other signs show improvement.

Identification of the fungal genus or species does not predict the clinical course and likely response to one or more antifungal agents. Decisions about alternate therapy must be based on the biomicroscopic signs and tolerance of the topical medications. The value of antifungal susceptibility testing for therapy of fungal keratitis has not been established. These procedures are not routinely performed by clinical laboratories but are available in some reference laboratories. The broth dilution method is the preferred technique for susceptibility tests of ocular yeasts and filamentous organisms and may suggest alternate therapy within a class of antifungal agents (e.g., polyenes, azoles). Antifungal susceptibility testing is highly dependent on various test conditions, including the size of the inoculum, type and pH of the medium, incubation temperature, time of reading results, and criteria for end points.

Progressive infection is characterized by an increase in the area and density of stromal suppuration, reduction in clarity of the adjacent stroma, and increase in anterior chamber flare and cells. If initial therapy was topical natamycin or amphotericin B drops alone and the stromal suppuration is judged to be progressing, topical (2 mg/ml) and oral fluconazole (200 mg twice daily) should be added for either filamentous fungal or yeast keratitis. In vitro susceptibility tests and limited animal models of nonocular infections imply that polyene and azole antibiotics may have a synergistic effect against certain fungi. If the suppuration progresses to the deep stroma with advanced corneal thinning and impending perforation, subconjunctival fluconazole (1 mg) may provide additional antifungal activity. In fungal keratitis following LASIK, management may require elevation of the flap, irrigation of the bed with amphotericin B or fluconazole, and, in some instances, excision of the flap. Consideration should also be given to acquiring voriconazole from Pfizer Central Research for compassionate use by intravenous and oral administration.

The empirical addition of a second topical antifungal agent is problematic. In vitro susceptible tests and trials in keratitis in animals judging the efficacy of combined topical antifungal agents are inconclusive. No evidence exists that combined topical natamycin and amphotericin B therapy is more efficacious than either drug alone. Administration of two polyene antibiotics may also enhance drug toxicity. Failure of topical natamycin therapy in filamentous fungal keratitis merits a trial of topical amphotericin B drops at the highest concentration tolerated (2.5 to 5 mg/ml). The possibility of polymicrobial keratitis and superinfection with other microorganisms should be considered. Interruption of antifungal therapy and repeat corneal scrapings and laboratory investigations may be warranted.

To assess the effectiveness of medical therapy and determine the requirement for surgical intervention, a careful, detailed corneal drawing should be made and the area, depth, and density of the stromal suppuration should be frequently quantitated. The extent of progression of suppuration into the deep stroma is difficult to judge in the presence of an endothelial plaque or fibrinous exudate in the anterior chamber. Abrupt withdrawal of topical corticosteroids produces increased stromal and anterior chamber inflammation, which are difficult to distinguish from signs of progressive invasion of replicating organisms. Imminent perforation is suggested by reduction in the density of the stromal suppuration and improved visualization of the anterior segment secondary to loss of stromal substance.

Small corneal perforations (less than 2 mm in diameter) occurring early in the course of medical therapy should be managed by either a therapeutic soft contact lens or tissue adhesive. These measures prolong antifungal therapy and reduce intraocular inflammation, thereby allowing penetrating keratoplasty to be performed under more ideal conditions. Application of a tissue adhesive prevents full visualization of the area of stromal suppuration and may create a partial barrier to penetration of effective antifungal agents. A trial of a soft therapeutic contact lens may effectively seal the perforation and promote restoration of the anterior chamber. The principles are to use a tight-fitting lens, continue cycloplegic-mydriatic drops (atropine 1% or hyoscine 0.25%), and avoid external pressure on the globe. A Fox shield should be used. If the anterior chamber can be restored and maintained, medical therapy should be continued. Control of fungal replication may permit stromal healing and eliminate urgent penetrating keratoplasty. In larger perforations (greater than 3 mm in diameter) or if a soft therapeutic contact lens alone fails to maintain the anterior chamber, tissue adhesive should be applied. In the presence of large areas of stromal thinning and necrosis, the likelihood of corneal perforation should be anticipated and penetrating keratoplasty performed urgently.

The goals of therapeutic penetrating keratoplasty are to restore the structure of the cornea, maintain the anterior chamber, and eliminate replicating organisms. In phakic eyes, an additional goal is to preserve the integrity of the lens and avoid additional intraoperative procedures that may enhance the spread of organisms into the vitreous to produce consecutive fungal endophthalmitis. In eyes without impending or extant perforation, akinesia and anesthesia may be accomplished by standard lid and retrobulbar block. General anesthesia, possibly supplemented by direct infiltration of extraocular muscles, is preferred in the presence of corneal perforation. Cycloplegic-mydriatic drops should be discontinued 24 to 48 hours before surgery to facilitate protective miosis. A carbonic anhydrase inhibitor (e.g., acetazolamide, 500 mg, oral or intravenous) and intravenous mannitol (1 g/kg) should be administered.

Although the decision on the size and placement of graft must be made at surgery, careful preoperative slit lamp biomicroscopy is helpful to determine the requirement for donor tissue. The trephination should provide a margin of 1 to 1.5 mm around the area of suppuration to eliminate the replicating organisms. The exact extent of organisms within the stroma adjacent to the area of suppuration cannot be accurately determined by slit lamp biomicroscopy. If the required donor cornea is estimated to be less than 12 mm, use of corneal-scleral tissue stored in standard medium is preferred and the eye bank should be advised of the need for tissue with an adequately large scleral rim. If a larger graft is required, a whole globe is preferred to simplify excision of the donor tissue.

In an intact globe, external pressure should be applied by a balloon device or hand compression to soften the eye and reduce the orbital contents. The Schott speculum* is ideal to support the eyelids, expose the globe, and minimize extrinsic pressure. A partial or 360-degree peritomy should be done if the anticipated graft-host junction requires placement of corneal sutures outside the limbus. A scleral support ring should be sutured 3 to 4 mm posterior and concentric to the limbus. To facilitate vitreous aspiration if required to reform the anterior chamber, a partial-thickness sclerotomy incision should be made 3.5-mm posterior to the limbus in the inferotemporal quadrant and partially closed with a horizontal mattress suture.

The size and placement of the graft are determined by the location and area of the stromal suppuration. The goal is to center the trephination with reference to the cornea rather than to the site of suppuration. If the area of suppuration is large and extends asymmetrically, decentration of the trephination may be required to obtain an adequate margin adjacent to the limbus (Fig. 26). If the suppuration is located in the peripheral cornea, a small, decentered graft may be preferred to provide a free margin of replicating organisms and avoid the complications of a large graft, despite the necessity of placement of the graft-host junction within the optical zone (Fig. 27). Circular trephinations are preferred; free-hand, half-circle, or annular grafts are technically difficult and unnecessary. If suppuration and necrosis extend beyond the limbus, a large 10- or 11-mm graft is required.

The size of the trephination is best determined in an intact globe. In corneal perforation, intraoperative application of tissue adhesive permits reformation of the anterior chamber through a corneal stab incision. The donor cornea should be oversized at least 0.25 mm in phakic eyes and 0.5 mm or 1.0 mm in phakic eyes for trephinations 9.5 mm or larger. The hollow trephine handle and disposable blade are ideal for trephination of the host cornea. The initial incision should be three-fourths to seven-eighths thickness. The anterior chamber is entered with an unguarded diamond or metal blade. In corneal perforation, the host cornea should be marked with the trephine and the cornea incised full thickness with the diamond or metal blade.

The excised tissue should be bisected. One half should be submitted for histopathologic study. One half should be transported immediately to the laboratory to be emulsified for inoculation on standard media as for corneal cultures. Fibrinous exudate should be removed from the anterior chamber and iris surface and inoculated directly on media or placed in tryptic soy broth for transportation to the laboratory. One or two midstromal or peripheral iridectomies should be done. Posterior synechiae should be lysed by gentle pressure on the iris surface at the pupillary margin or direct synechialysis with a blunt instrument to avoid damaging the lens. Acetylcholine should be instilled to reduce the pupil. A viscoelastic substance should be injected into the chamber angle and over the iris surface to separate the host tissue, reform the anterior chamber, and facilitate placement of the donor cornea. In corneal perforation or if extension of replicating organisms into the anterior chamber is suspected, 5 μg/ml of amphotericin B in 0.1 ml should be injected into the anterior chamber. Every effort should be made to retain the crystalline lens to reduce the likelihood of consecutive endophthalmitis. If the suppurative process involves the lens, posterior chamber, or vitreous, lensectomy, anterior vitrectomy, and intravitreal injection of amphotericin B (5 μg/ml in 0.1 ml) are required. Multiple interrupted 10-0 nylon sutures are preferred for wound closure. Inability to maintain or reform the anterior chamber necessitates aspiration of vitreous through the prepared sclerotomy site.

Postoperative medical therapy is determined by multiple factors, including the surgeon's estimate of the likelihood of excision of replicating fungi, degree of postoperative inflammation, intraocular pressure, status of other intraocular structures, results of cultures of corneal tissue and intraocular fluid, and histopathologic examination of the excised host or cornea. Topical antifungal agents may be withheld in the early postoperative period if corneal perforation did not occur, trephination adequately surrounded the area of suppuration, and organisms were not cultured from anterior chamber material. Oral fluconazole (400 mg daily) should be administered for the initial 7 to 10 days or longer. If excision of infected corneal tissue was incomplete and organisms were present in the anterior chamber based on clinical findings or culture, a topical antifungal agent (natamycin 5% or amphotericin B 0.25% to 0.5%) should be continued during the early postoperative period. Signs of corneal toxicity should be monitored carefully. Topical and oral corticosteriods should be avoided during the initial 48 to 72 hours and longer if possible. Cyclosporine A has been demonstrated to have an inhibitory effect against Fusarium oxysporum, F. solani, and Aspergillus fumigatus in vitro⁷⁰ and may be an effective alternative to topical corticosteriods following therapeutic penetrating keratoplasty. Identification of fungal elements in the corneal sections or recovery of the organism in corneal cultures does not necessarily require antifungal therapy for prophylaxis of consecutive endophthalmitis. Fungal endophthalmitis secondary to therapeutic penetrating keratoplasty of fungal keratitis in phakic eyes is uncommon. Intraocular injection of antifungal agents and other surgical measures should be reserved for eyes with confirmed fungal endophthalmitis.

Conjunctival flap or lamellar keratoplasty is not indicated in the management of fungal keratitis unresponsive to medical therapy. These procedures do not eliminate replicating organisms and may create barriers to penetration of potentially effective antifungal agents.

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