Fungal keratitis is an important cause of ocular morbidity

Transcription

1 Cornea Polyhexamethylene Biguanide and Calcineurin Inhibitors as Novel Antifungal Treatments for Aspergillus Keratitis Rachelle A. Rebong, 1 Ricardo M. Santaella, 1 Brian E. Goldhagen, 1 Christopher P. Majka, 1 John R. Perfect, 2 William J. Steinbach, 2 and Natalie A. Afshari 1 PURPOSE. To establish polyhexamethylene biguanide (PHMB) as an effective treatment for Aspergillus keratitis in a novel murine model. To determine the ability of the calcineurin inhibitors tacrolimus (FK506) and cyclosporine A (CSA) to enhance the activity of PHMB, amphotericin B (AMB), and voriconazole (VCZ) against Aspergillus keratitis. METHODS. In vitro studies: Broth antifungal susceptibility tests were performed with PHMB, AMB, VCZ, and FK506, individually and in combination against Aspergillus fumigatus. Minimum inhibitory concentrations (MIC) and fractional inhibitory concentration index (FICI) values were used to analyze antifungal activity. In vivo studies: A novel murine model was created to establish Aspergillus keratitis. Infected mice were randomly assigned to treatment groups receiving saline, CSA, AMB, VCZ, PHMB, AMB CSA, VCZ CSA, or PHMB CSA. An ophthalmologist blinded to the treatment groups assessed disease severity daily based on a grading scale. The mean end change in disease score was compared between groups. RESULTS. In vitro studies: FK506 in combination with PHMB, VCZ, or AMB enhanced fungal growth inhibition. FICI values showed an additive effect between FK506 and PHMB, AMB, or VCZ. PHMB monotherapy eliminated Aspergillus growth starting at 4 g/ml. In vivo studies: All treatment groups showed a significant improvement in disease score compared to the control group. CSA significantly worsened VCZ activity against Aspergillus keratitis. CONCLUSIONS. PHMB is an effective inhibitor of Aspergillus growth. Further investigation of the role of calcineurin inhibitors in the treatment for Aspergillus keratitis is warranted. (Invest Ophthalmol Vis Sci. 2011;52: ) DOI: / iovs Fungal keratitis is an important cause of ocular morbidity worldwide, leading to corneal ulceration and scarring, corneal transplantation, and blindness. 1,2 Whereas yeastlike Candida species predominate in more temperate climates, in tropical and subtropical areas such as Asia, Africa, and the southern From the 1 Duke University Eye Center and the 2 Division of Infectious Diseases, Duke University Medical Center, Durham, North Carolina. Supported by an Allergan Horizon Grant and Research to Prevent Blindness. Submitted for publication April 14, 2011; revised June 24, 2011; accepted July 17, Disclosure: R.A. Rebong, None; R.M. Santaella, None; B.E. Goldhagen, None; C.P. Majka, None; J.R. Perfect, None; W.J. Steinbach, None; N.A. Afshari, None Corresponding author: Natalie A. Afshari, Cornea and Refractive Surgery Service, Duke University Eye Center, DUMC Box 3802, Durham, NC 27710; natalie.afshari@duke.edu. United States, filamentous fungi such as Aspergillus species are the most common causes of fungal keratitis. 3 7 Currently available medical treatments for Aspergillus keratitis are not only largely ineffective but are also expensive. As the fungus penetrates deeper into the cornea, it becomes more difficult for the currently available treatments to reach and eradicate it. Although 15% to 27% of all patients with fungal keratitis ultimately require surgical intervention, Aspergillus species cause a particularly severe keratitis that necessitates keratoplasty in 42% to 60% of cases. 8 Given the prevalence of Aspergillus keratitis in many areas, particularly agricultural areas in developing nations, the development of new treatment options to increase effectiveness and to decrease cost is imperative. Despite the high incidence of keratomycoses globally, firstline topical polyene antifungal antibiotics, including amphotericin B (AMB) and natamycin, are not effective in completely eliminating severe keratomycoses. 7 Polyene macrolides work by binding to ergosterol, a sterol unique to fungal cell membranes, to form pores that allow the leakage of electrolytes and lead to cell death. 9 Natamycin is the only U.S. Food and Drug Administration (FDA) approved topical ocular antifungal, but it has poor corneal penetration, is cost-prohibitive, degrades easily, and is not commercially available in many regions of the world. 8,10 AMB is easier to handle and has been used to treat keratomycoses since 1959; however, it is not available as an ophthalmic preparation and needs to be compounded as a topical solution (0.15% 0.30%). 8,11 Topical AMB appears to be well tolerated at lower concentrations but causes conjunctival irritation at higher concentrations ( 0.50%), limiting its use at these concentrations. 2 Azoles are another class of antifungals used in the treatment of Aspergillus keratitis. They inhibit the cytochrome P-450 enzyme 14 -demethylase needed to convert lanosterol to ergosterol and lead to increased permeability of fungal cell membranes. 12 Voriconazole (VCZ) is a triazole antifungal agent with a broad spectrum of activity against Aspergillus species and is FDA-approved for the treatment of invasive aspergillosis. 10 The use of oral and topical VCZ, compounded as a 1% solution, has been reported in cases of fungal keratitis It has been effective in treating even Aspergillus infections resistant to AMB and has less severe side effects than AMB. Adverse effects of systemic VCZ include skin rashes and visual disturbances, which are usually mild and transient. 10 Elevation of hepatic enzymes rarely occurs. 12 However, VCZ is very costly and is not readily available in most parts of the world; like AMB, it must be compounded for use as an ophthalmic solution. Moreover, Aspergillus resistance to VCZ is a growing problem Polyhexamethylene biguanide (PHMB) is an inexpensive, readily available general biocide used as a swimming pool and contact lens disinfectant. 19 It is thought to work by disrupting the cell Investigative Ophthalmology & Visual Science, September 2011, Vol. 52, No. 10 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 7309

2 7310 Rebong et al. IOVS, September 2011, Vol. 52, No. 10 membranes of microorganisms, causing leakage of intracellular components and inhibiting the function of respiratory enzymes. 2 In vitro studies demonstrated PHMB s activity against Candida albicans, Fusarium solani, and Aspergillus niger. 19 In a rabbit model of Fusarium keratitis, 0.02% topical PHMB significantly reduced fungal growth compared to placebo. 20 Topical PHMB at concentrations of 0.02% to 0.053% has been used in the treatment of Acanthamoeba keratitis, but only one published animal study and no human studies have evaluated the use of PHMB against Aspergillus keratitis. 21 In the study evaluating PHMB treatment for Aspergillus keratitis, topical PHMB 0.02% was shown to be moderately effective against the infection in a rabbit model, showing significantly improved ulcer healing time compared with the control. 21 However, only six animals were treated in that study. Given the limited effectiveness, growing fungal resistance, and high cost associated with current treatment options for Aspergillus keratitis, the development of new treatments becomes important. Tacrolimus (FK506) and cyclosporine A (CSA) are immunosuppressive agents that target the calcineurin pathway to prevent rejection after organ transplantation. 22 Calcineurin is a serinethreonine phosphatase in the calcium signaling pathway of the conserved cell stress response. In fungi, this signaling pathway has been found to mediate growth, morphology, stress responses, and pathogenicity; it has also been shown to have a role in regulating cell wall formation. 23 Thus, calcineurin inhibitors may play a role not only in immunosuppression but also in inhibiting fungal growth. Evidence to support this hypothesis comes from previous studies which showed that calcineurin inhibitors inhibit the growth of A. fumigatus and that deletion of a gene within the calcineurin pathway of A. fumigatus reduces hyphal growth and attenuates virulence In addition, in a murine model of C. albicans keratitis, we have shown that calcineurin promotes corneal fungal infection and that calcineurin inhibitors can work synergistically with fluconazole in the treatment of Candida keratitis. 27 In fact, calcineurin inhibitors have already been shown to enhance the activity of caspofungin, an echinocandin that inhibits fungal cell wall synthesis, against Aspergillus species. 28 Our hypothesis is that similar synergy occurs between the calcineurin inhibitor FK506 and PHMB, AMB, and VCZ, respectively, against A. fumigatus. Using calcineurin inhibitors in combination with more conventional antifungals may provide a more effective and ultimately less costly treatment option for Aspergillus keratitis. METHODS Aspergillus Strain, Media, and Inoculum Preparation Wild-type A. fumigatus strain AF293 was used in all experiments. RPMI 1640, prepared according to CLSI (Clinical and Laboratory Standards Institute) standards, was used in all in vitro assays. 29 The inoculum was prepared from AF293 grown and harvested according to CLSI guideline for antifungal susceptibility testing. 29 Drug Preparation For in vitro assays, VCZ (Vfend; Pfizer, New York, NY) and AMB (Fungizone; Bristol-Myers Squibb, New York, NY) were prepared from powders according to CLSI guidelines. 29 FK506 was obtained from Astellas Pharma US, Inc. (Deerfield, IL). Polyhexamylene biguanide was obtained as a 0.02% ophthalmic solution from the Duke Pharmacy Compounding Facility (Durham, NC). It was prepared by adding 0.1 ml of a commercially available 20% PHMB solution (Baquacil; Arch Chemicals, Inc., Norwalk, CT) to 100 ml of normal saline. CSA was obtained as a 2% ophthalmic solution from the Duke Pharmacy Compounding Facility (Durham, NC). CSA was prepared by adding 0.6 ml of cyclosporine 100 mg/ml to 2.4 ml of corn oil. In Vitro Study Design Broth susceptibility testing of A. fumigatus was performed using FK506, PHMB, AMB, and VCZ individually and in combination (FK506 with PHMB, AMB, or VCZ) against wild-type A. fumigatus, according to a modified version of the CLSI guidelines. 29 A checkerboard titration was used, testing two drugs to compare the effects of the drugs alone and in combination. FK506 was used at concentrations between 0.01 and 0.08 g/ml, PHMB between 0.3 and 30 g/ml, AMB between 2 and 64 g/ml, and VCZ between and 1 g/ml. Samples were incubated for 48 hours at 37 C. At 48 hours, fungal growth was compared against that in the controls and across the various drug concentrations and combinations. Elimination of growth (MIC) and inhibition of growth, seen as the blunting of hyphal tips compared to control wells and known as the minimum inhibitory concentration (MIC), were assessed (Fig. 1). Quantitative Analysis of In Vitro Data The MIC and fractional inhibitory concentration index (FICI) were used to analyze in vitro antifungal activity and interactions against A. FIGURE 1. Examples of morphologic changes in wild-type A. fumigatus as seen in vitro. (A) Growth of untreated wild-type A. fumigatus. Note the extensively branching filaments. (B) Inhibition of growth with VCZ (0.25 g/ ml) and FK506 (40 ng/ml). (C) Inhibition of growth with PHMB (0.03 g/ml) and FK506 (2.25 g/ml). Note the blunted hyphal tips in (B) and (C) (arrows). (D) Elimination of growth with PHMB (3 g/ml). VCZ, voriconazole; FK506, tacrolimus; PHMB, polyhexamethylene biguanide. Bar, 200 m.

3 IOVS, September 2011, Vol. 52, No. 10 Novel Treatments for Aspergillus Keratitis 7311 fumigatus. The FICI is used to analyze drug interactions in combination and is calculated with the following formula: FICI [(MIC A in combination)/mic A] [MIC B in combination)/mic B]. Interpretation of the FICI can vary. In our study, interpretation was determined according to accepted standards by the following: FICI 0.5, synergistic effect; 0.5 but 1, additive effect; 1 but 4, indifferent effect; and 4, antagonistic effect. 30 In Vivo Study Design. A prospective randomized control study was performed according to a modified version of a previous protocol. 27 This protocol was approved by the Duke Institutional Animal Care and Use Committee. All study animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. A total of 158 adult male BALB/c mice, each weighing 20 to 25 g, were immunosuppressed with methylprednisolone (100 mg/kg) on days 5, 1, and 1 of inoculation, to rapidly establish infection. An intramuscular injection of a ketamine (10 mg/ml)-xylazine (1 mg/ml) mixture and a drop ( 30 L) of proparacaine 0.5% ophthalmic solution (Alcon, Fort Worth, TX) on the cornea were given for anesthesia. A drop of moxifloxacin 0.5% ophthalmic solution (Vigamox; Alcon) was then administered to the cornea to avoid bacterial contamination. The right corneas of 158 mice were de-epithelialized with a 30-gauge needle. Two 5- L suspensions, each containing 10 6 A. fumigatus spores, were evenly distributed in succession on the scratched cornea of each mouse. A previously described disease grading scale from 0 (no disease) to 4 (severe disease) was modified, and animals were graded as follows by an ophthalmologist who was blinded to the drug treatments (Fig. 2): 0, no sign of epithelial defect or infection; 1, signs of epithelial scratches but no infiltrate; 2, corneal infiltrate covering 25% to 50% of the corneal surface; 3, corneal infiltrate covering 50% to 75% of the corneal surface; and 4, corneal infiltrate covering 75% of the corneal surface. 27 Eyes that perforated received an automatic score of 4. Mice that developed clinically significant infection, indicated by reaching a grade of 2, were included in the study and started on antifungal treatment. Eligible mice were randomly assigned to one of eight treatment groups: balanced salt solution (control; Akorn, Lake Forest, IL), CSA, AMB, VCZ, PHMB, AMB CSA, VCZ CSA, or PHMB CSA. Study mice received four doses (5 L/dose) of the treatment drug each day for 10 consecutive days. For combination therapy, the drugs were administered in succession with at least 2 minutes between doses. Grading was performed daily with an operating microscope (Carl Zeiss Meditec, Dublin, CA). The results from two independent experiments were combined and analyzed. Statistical Analysis of In Vivo Data The mean changes in disease score between groups were compared using ANOVA, in which all pairwise comparisons between the eight groups were performed by using Student s t-test. We included all mice that developed a disease score of 2 and excluded mice that progressed to perforation of the cornea before treatment could be initiated. Data from mice whose corneas perforated after treatment was initiated were included (statistical analyses by JMP 8 software; SAS Institute, Cary, NC). RESULTS FK506 Enhanced PHMB, AMB, and VCZ Activity against A. fumigatus In Vitro With FK506 alone, inhibition of A. fumigatus growth occurred starting at 0.02 g/ml; fungal growth elimination did not occur, even at the highest concentration (Fig. 3). When FK506 was added to PHMB, AMB, or VCZ, fungal growth inhibition was enhanced and/or occurred at lower concentrations than when PHMB, AMB, or VCZ was used individually. PHMB eliminated growth starting at 4 g/ml but showed no effects at lower concentrations. With the addition of at least 0.01 g/ml FK506, PHMB still eliminated growth at 4 g/ml but also inhibited fungal growth at 0.5 g/ml PHMB (Fig. 3). AMB inhibited growth at the highest concentration 64 g/ml. With the addition of 0.08 g/ml FK506, fungal growth inhibition occurred at 8 g/ml AMB (Fig. 3). VCZ alone inhibited growth starting at 0.25 g/ml and eliminated growth starting at 1 g/ml. Fungal growth inhibition was enhanced at 0.25 g/ml VCZ with the addition of at least 0.02 g/ml FK506 (Fig. 3). FICI values indicated an in vitro additive effect when FK506 was combined with any of the study medications. The FICI values calculated from the individual and combined MICs of the drugs are 0.625, 0.625, and 1 for PHMB FK506, AMB FK506, and VCZ FK506, respectively (Table 1). All Murine Model Treatment Groups Showed Significant Improvement in Disease Score Compared to the Control Group A total of 103 of 158 mice achieved grade 2 infection and were included in the study, with 12 to 14 mice per group. The control group showed an increase or worsening of disease score after 10 days of treatment ( ). All FIGURE 2. Disease grading scale for evaluating mice corneas. The right eyes of BALB/c mice were scraped and inoculated with wild-type A. fumigatus. Study eyes were viewed and digitally photographed through an operating microscope. (A) A score of 0 indicated no sign of epithelial defect or infection. (B) A score of 1 showed signs of epithelial scratches but no infiltrate. (C) A score of 2 showed a corneal infiltrate covering 25% to 50% of the corneal surface. (D) A score of three showed a corneal infiltrate covering 50% to 75% of the corneal surface. (E) A score of 4 showed a corneal infiltrate covering 75% of the corneal surface or any signs of perforation, as shown in this image.

4 7312 Rebong et al. IOVS, September 2011, Vol. 52, No. 10 FIGURE 3. In vitro growth of A. fumigatus after treatment with FK506. In vitro growth inhibition occurred at lower concentrations of PHMB with the addition of FK506 and was enhanced at 0.25 g/ml VCZ with the addition of at least 0.02 g/ml FK506. In vitro growth of A. fumigatus after treatment with increasing doses of (A) 0.01, (B) 0.04 g/ml FK506 alone. Inhibition occurred at lower concentrations of PHMB and AMB, with the addition of FK506; (C) 0.5 g/ml PHMB 0.01 g/ml FK506. Note the increased area of clearing (arrow); (D) 0.5 g/ml PHMB 0.02 g/ml FK506; (E) 16 g/ml AMB 0.08 g/ml FK506; and (F) 64 g/ml AMB 0.08 g/ml FK506. In vitro growth inhibition was enhanced at 0.25 g/ml VCZ with the addition of at least 0.02 g/ml FK506; (G) 0.25 g/ml VCZ alone; and (H) 0.25 g/ml VCZ 0.08 g/ml FK506. Bar, 200 m. other treatment groups showed an improvement in disease score after 10 days of treatment, which can be seen as a decrease in the average daily disease score per group over TABLE 1. In Vitro Additive Effects of FK506 and Antifungals against Wild-Type A. fumigatus by Microdilution Checkerboard Interaction Drug MIC ( g/ml) MICs of Antifungal Agent Combination Drug A/FK506 ( g/ml) FICI FK AMB / VCZ / PHMB / FICI is used to analyze drug interactions in combination. FICI [(MIC A in combination)/mic A] [(MIC B in combination)/mic B]. FICI values were interpreted as follows: 0.5, synergistic effect; 0.5 but 1, additive effect; 1 but 4, indifferent effect; and 4, antagonistic effect. the complete course of treatment (Fig. 4) or calculated as a negative mean end change in disease score (Table 2). Of the treatment groups, AMB alone led to the largest mean end decrease in disease score from baseline ( ), whereas VCZ CSA led to the smallest mean end decrease in disease score from baseline ( ). P values for the comparison of the mean end change in disease score between the control group and the other study groups were all significant (P 0.05; Table 3). CSA significantly worsened VCZ activity against A. fumigatus infection in study mice. Significant differences were found between VCZ CSA and VCZ alone, AMB alone, and PHMB CSA (P , , and , respectively) in study mice. Mice receiving VCZ CSA showed significantly less improvement in disease score than did the mice receiving VCZ alone, AMB alone, or PHMB CSA. The rest of the comparisons between noncontrol treatment groups were not significant (Table 3).

5 IOVS, September 2011, Vol. 52, No. 10 Novel Treatments for Aspergillus Keratitis 7313 FIGURE 4. Mean daily disease scores over 10 days of treatment of the study mice. The control group showed an increase or worsening of mean daily disease score, whereas all other groups showed a decrease or improvement of mean daily disease score over the treatment course. BSS, balanced salt solution. Alopecia Was a Notable Side Effect in Study Mice All animals treated with CSA, either alone or in combination, developed alopecia around the treated eye (Fig. 5). DISCUSSION In this study, we sought to determine whether PHMB monotherapy is a viable treatment for Aspergillus keratitis and to ascertain the ability of calcineurin inhibitors to enhance the antifungal activity of PHMB, AMB, and VCZ against A. fumigatus, with the ultimate goal of providing better and less expensive treatment options for Aspergillus keratitis. To test our hypotheses, we developed a novel murine model for Aspergillus keratitis. In addition to establishing the efficacy of using calcineurin inhibitors for Aspergillus keratitis, our studies provided additional evidence to demonstrate the effectiveness of PHMB against Aspergillus. Each of the drugs tested showed antifungal activity individually against A. fumigatus. This result was expected for AMB and VCZ; even the calcineurin inhibitor FK506 had been shown in previous studies to have an effect against A. fumigatus. 24 However, data on PHMB s effectiveness against A. fumigatus were limited to a single study in a rabbit model, showing that PHMB was moderately effective but not better than natamycin in clearing Aspergillus keratitis. 21 In our study, PHMB was effective, not only in inhibiting but also in eliminating Aspergillus growth. Interestingly, unlike the other drugs tested, PHMB did not show a progression of fungal growth inhibition with increasing doses of the drug; rather, the transition between dilutions abruptly changed from no effect to complete elimination of fungal growth. Further in vitro and in vivo studies are warranted to determine PHMB s activity against Aspergillus. If shown to be effective, PHMB essentially, diluted pool cleaner could become a revolutionary and very affordable option for the treatment of Aspergillus keratitis for patients in agricultural areas of developing countries, where it is a significant problem. TABLE 2. Sample Size and Mean End Change in Disease Score after 10 Days of Treatment of Study Mice Treatment Group Sample Size End Change (Mean SD) Balanced salt solution (control) CSA AMB AMB CSA VCZ VCZ CSA PHMB PHMB CSA Based on our initial in vitro studies, FICI values indicate that the calcineurin inhibitor FK506 displays additive effects when combined with PHMB, AMB, or VCZ against A. fumigatus. An additive, as opposed to a synergistic, effect observed between these drugs may be explained in several ways. First, compared with Candida, against which CSA and fluconazole appear to show in vivo synergy, Aspergillus causes a more virulent corneal infection. 27 In Candida keratitis, even deep corneal lesions treated with medical therapy alone generally carry a favorable prognosis; in Aspergillus keratitis, deep corneal lesions generally do not respond to medical therapy and require surgical intervention. 2 The role of the calcineurin pathway may differ between Candida and Aspergillus species. More information on calcineurin-dependent genes in Candida species versus in Aspergillus species are available and differences between the two TABLE 3. Comparison of the Mean Changes in Disease Score between Two Groups of Study Mice Group A Group B P Balanced salt solution CSA < Balanced salt solution AMB < Balanced salt solution AMB CSA < Balanced salt solution PHMB < Balanced salt solution PHMB CSA < Balanced salt solution VCZ < Balanced salt solution VCZ CSA VCZ CSA AMB VCZ CSA VCZ VCZ CSA PHMB CSA PHMB AMB CSA AMB AMB CSA AMB VCZ CSA AMB CSA VCZ CSA CSA PHMB VCZ VCZ CSA PHMB CSA VCZ PHMB CSA AMB PHMB PHMB CSA AMB CSA VCZ CSA PHMB CSA VCZ AMB AMB CSA PHMB CSA PHMB AMB CSA PHMB CSA VCZ PHMB CSA CSA AMB CSA The mean end change in disease score between groups was compared using an ANOVA in which all pairwise comparisons between the eight groups were performed with Student s t-test. Significant differences are highlighted in bold.

6 7314 Rebong et al. IOVS, September 2011, Vol. 52, No. 10 FIGURE 5. All mice treated with CSA developed localized alopecia. genera with regard to gene targets as well as phenotypes attributed to calcineurin signaling have been identified. 31 Whether these differences can account for the two genera s different responses to calcineurin inhibitors is a topic that should be studied further. The additive as opposed to synergistic effects between FK506 and PHMB, AMB, or VCZ may reflect the different target sites for the drugs. Caspofungin inhibits the activity of 1,3- -Dglucan synthase, an enzyme critical for fungal cell wall synthesis. 28 AMB and VCZ, meanwhile, inhibit fungal cell membrane synthesis by interfering with ergosterol. The synergistic activity of calcineurin inhibitors with caspofungin and their additive activity with AMB and VCZ may be explained by the possibility that the calcineurin pathway plays a role in cell wall synthesis of Aspergillus species. Incidentally, although the antifungal activity of PHMB is not as well understood, its additive effect with calcineurin inhibitors supports the idea that PHMB acts at the level of the fungal cell membrane as opposed to the cell wall. Although the calculated FICI values did not meet the criteria for synergy, the observed morphologic changes clearly showed that FK506 enhanced the ability of PHMB, AMB, and VCZ to inhibit Aspergillus growth in vitro. Our in vitro studies indicate that calcineurin inhibitors like FK506 can have a role in improving the antifungal treatment of Aspergillus keratitis. Our in vivo studies showed little statistical significance when comparing noncontrol treatment groups, probably because of the low sample size. A smaller number of animals was desirable for this study, as it allowed us to establish and evaluate a novel mouse model of Aspergillus keratitis in addition to testing the effects of different drug combinations. Previous models of Aspergillus keratitis used rabbits, whose larger corneas make experimentation easier. 32,33 A mouse model of Aspergillus keratitis was created by Zhong et al., 34 who used a more complicated procedure similar to epikeratophakia compared with our method involving corneal de-epithelialization. Our studies showed that a procedure involving scratching of the cornea, as opposed to a more technically complicated procedure similar to epikeratophakia, can lead to successful infection of over 65% of inoculated mice. 34 This mouse model provides a more practical and economical option which can be used in future research on Aspergillus keratitis. We elected to use the calcineurin inhibitor CSA in our in vivo experiments rather than using FK506, as we did in our in vitro experiments. We initially chose FK506 for the in vitro experiments because it has been shown to be superior to CSA in its traditional role as an immunosuppressant after organ transplantation. 35 We then chose CSA for the in vivo experiments, because CSA is already commercially available and used to treat other ocular diseases such as dry eye syndrome. Although FK506 and CSA act on the same pathway by inhibiting calcineurin phosphatase, they bind to different members of the immunophilin family FK506 to FKBP12 and CSA to cyclophilin which, in cases of organ transplantation, leads to different risk and benefit profiles. 35 Whether these differences translate to the calcineurin pathway in fungi and whether these differences are significant are questions that have yet to be answered. If FK506 and CSA in fact behave differently in Aspergillus, it would help explain the discrepancy between our results. Key differences between in vitro studies and in vivo studies using calcineurin inhibitors may stem from the fact that calcineurin inhibitors in vivo not only exhibit an antifungal effect against Aspergillus but may also exert immunosuppressive effects on the host. During in vivo experiments, since calcineurin inhibitors are traditionally used as immunosuppressant agents, they may limit the host s ability to combat infection. Alternatively, calcineurin inhibitors may help suppress the host s inflammatory response, which at times can cause as much or more damage to the cornea than does the infection. To better define this issue, the balance between the antifungal and immunosuppressive effects of calcineurin inhibitors in host species requires further investigation. Another issue to consider with regard to our in vivo studies is that the preparation of the CSA ophthalmic solutions may have influenced their ability to be delivered effectively to the site of infection. Compared to the balanced salt solution, VCZ, AMB, and PHMB ophthalmic solutions, the CSA ophthalmic solution was more viscous due to being prepared in corn oil. CSA was more difficult to administer, not only because the viscous solution was difficult to apply but also because the animals more vigorously wiped it off after application. The alopecia resulting from CSA administration may be due to this vigorous wiping rather than or in addition to the properties of the drug itself. A viscous substance like CSA may be better applied and therefore absorbed across a larger surface area, like the human cornea, as opposed to the smaller corneas of our study animals. Considering alternative preparations for CSA may improve drug administration to and penetration of the cornea in future experiments. Future in vivo studies are needed to assess the effects of using calcineurin inhibitors to enhance the antifungal treatment of Aspergillus keratitis. If proven effective, the use of calcineurin inhibitors in conjunction with current antifungal therapies may lower the concentrations needed for the individual drugs to exert their desired effects. Using calcineurin inhibitors to enhance the treatment of Aspergillus keratitis would help avoid potential side effects at higher doses of the individual drugs and would increase the effectiveness of current treatment regimens against Aspergillus keratitis. Perhaps more importantly, the use of calcineurin inhibitors may help in avoiding the problem of growing fungal resistance when individual drugs are used alone. References 1. Klotz SA, Penn CC, Negvesky GJ, Butrus SI. Fungal and parasitic infections of the eye. Clin Microbiol Rev. 2000;13: Thomas PA. Current perspectives on ophthalmic mycoses. Clin Microbiol Rev. 2003;16:

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