Efficacy of Antifungal Agents in the Cornea /. A Comparative Study Denis M. O'Day, Richard Robinson, and W. Steven Head A standardized model of Candida albicans keratitis was developed in pigmented rabbits using a quantitative mycologic technique to evaluate the disease at intervals throughout the course. In this model, using two different infecting strains, the efficacy of five antifungal agents was compared. Amphotericin B, in concentrations of 0.5% to 0.075%, was superior to all other agents tested. Na- tamycin 5% ranked next, followed by 1% flucytosine, and 1% miconazole. Ketoconazole 1% was ineffective. Invest Ophthalmol Vis Sci 24:1098-1102, 1983 The selection of appropriate therapy for fungal cor- (minimal inhibitory concentration [MIC] > 50 /xg/ neal infection remains an unsettled question. Al- ml), whereas strain F357 is resistant to flucytosine though there is considerable clinical evidence to sup- (MIC > 50 iig/m\) but susceptible to ketoconazole port the use of certain agents,1"6 objective experi- (MIC = 0.10 Mg/ml). The discrepancy between the mental data is still lacking because of the difficulties inhibitory and fungicidal values with these agents is associated with the development of a suitable animal indicative of a fungistatic rather than a fungicidal model of fungal disease. In this paper we describe a mechanism of action. In contrast, the levels with am- model of Candida albicans infection in the rabbit eye, photericin B suggest a fungicidal mechanism in vitro. using quantitative mycologic techniques, and its ap- Animals: Pigmented outbred male rabbits, 1.5-3 plication to a comparison of efficacy of a variety of kg were used in all experiments. antifungal agents. Infection protocol: Rabbits were anesthetised with either intravenous pentobarbital (Nembutal) or in- Materials and Methods tramuscular Ketamine and xylazine hydrochloride. A retrobulbar injection of 1% xylocaine was given Development of the Animal Model and topical anesthesia was achieved with Alcaine Inoculum: Two-day-old cultures of C. albicans 0.5%. Using an operating microscope, eight wells, grown on trypticase soy agar with 5% sheep blood one-half stromal thickness in depth, were trephined (BBL) were used to inoculate the rabbit cornea. A in the cornea of each eye in a regular pattern. A glass suspension of the inoculum in normal saline in a trephine prepared from a microhematocrit tube 1 concentration of 5 X 109 per ml was prepared and mm in diameter was used. A second trephine loaded stored at 4 C over night. In preliminary studies only with C. albicans suspension was used to inoculate two of the five strains tested produced significant dis- each well. The trephine was placed in each well, ro- ease and these were used in subsequent studies (Strain tated three to four times and then removed. Each well LV and Strain F357). The susceptibility of these two was inoculated a second time with a new trephine strains to five antifungal agents, using a modification loaded with fresh inoculum. If inadvertent corneal (personal communication, Dan B. Jones, 1980) of the perforation occurred the animal was removed from method of Shadomy,7 is shown in Table 1. In vitro, the study. the LV strain is susceptible to flucytosine at a con- Isolate recovery: At predetermined intervals the centration of 0.30 ng/m\ but resistant to ketoconazole animals were sacrificed with a commercially prepared euthanasia solution T-61 (Taylor Pharmacal, Deca- tur, IL). The whole cornea was removed by excision From the Department of Ophthalmology, Vanderbilt University at the limbus, placed in a sterile petri dish, and cut School of Medicine, Nashville, Tennessee. into small pieces. These were then ground in a tissue Supported by NIH grant EY 01621 and an unrestricted grant grinder (Ultraturex Model SDT) for three 10-sec in- from Research to Prevent Blindness, Inc. Submitted for publication: November 18, 1982. tervals in 3 ml of sterile saline. Ten and 100 n\ ali- Reprint requests: Denis M. O'Day, MD, Room D-5217, Van- quots of this corneal suspension were plated in trip- derbilt Medical Center North, Nashville, TN 37232. licate on trypticase soy agar with 5% sheep blood. 0146-0404/8 3/0800/1098/$ 1.05 © Association for Research in Vision and Ophthalmology 1098 Downloaded from iovs.arvojournals.org on 09/24/2021 No. 8 EFFICACY OF ANTIFUNGAL AGENTS IN THE CORNEA / O'Doy er ol. 1099 Table 1. In vitro susceptibility (/tig/ml) Amphotericin B Natamycin Ketoconazole Flucytosine Miconazole Strain MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC LV 0.2 0.39 6.25 >50 >50 >50 0.39 >50 12.5 >50 F357 0.1 1.56 3.12 25 0.10 >50 >50 >50 12.5 >50 MIC = minimal inhibitory concentration. MFC = minimal fungicidal concentration. After 48 hrs incubation at 25 C the colony forming The following experiments were performed: units (CFU) were counted, and the number of CFU a. Candida albicans strain LV: a comparison of per whole cornea was calculated based on a 3-ml total all five agents (natamycin 5%, amphotericin B volume. 0.5%, miconazole 1%, ketoconazole 1% and flucyto- sine 1%). Evaluation of Topical Antifungal Agents in the Model b. Candida albicans strain LV: a comparison of different dilutions of amphotericin B in distilled wa- Agents evaluated: Polyenes: Amphotericin B 0.5%, ter, 0.5%, 0.15%, 0.075%. 0.15%, and 0.075% in distilled water (Fungizone®, c. Candida albicans strain F357: a comparison of E. R. Squibb & Son, Princeton, NJ) and Natamycin 1% flucytosine, 1% ketoconazole and 0.15% ampho- 5% suspension (Alcon Laboratories, Fort Worth, tericin B. TX). Imidazoles: Miconazole 1% in Cremophor (Janssen Pharmaceuticals, New Brunswick, NJ) and Results Ketoconazole 1% in polyethylene glycol 400 (Jans- sen). Pyrimidine: Flucytosine 1% solution in normal Animal Model saline (Roche Laboratories, Nutley, NJ). Within 24 hours of inoculation a ring of infiltrate Treatment protocol: Treatment was begun 30 min developed around the base of each trephination. This after inoculation. Animals were assigned randomly area of infiltrate grew larger over the next 36 hrs and to a treated or untreated control group. Six rabbit then slowly diminished as the infection resolved clin- eyes were exposed to each treatment regimen with ically over a 5-day period. With both strains the both eyes of each animal receiving either the test drug course of the disease was similar and paralleled the or no treatment. Drops were administered 10 times quantitative isolate recovery rate at the end of each per day at hourly intervals. Eighteen hours after the 24-hr period. During the first 48 hrs the isolate re- conclusion of the treatment period both treated and covery rate was high (Fig. 1), but by 72 hrs it had control animals were killed and the corneas pro- fallen to 10% of the 24-hr value and by the fifth day cessed. it was less than 1%. CANDIDA ALBICANS - NORMAL DISEASE Strain LV m Strain F357 Fig. 1. The normal course (r of disease following inocu- O lation with two different o strains of Candida albicans as measured by the number of CFU/cornea. Each data point represents six eyes + (mean and range). Inoculation 24 48 72 96 120 TIME POST INOCULATION (hours) Downloaded from iovs.arvojournals.org on 09/24/2021 1100 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / August 1983 Vol. 24 CANDIDA ALBICANS-STRAIN LV Control i*S] Treated AM "B"O,5% AM "B"O,I5% AM "B" 0.075% Fig. 2. The response of an infection to differing con- centrations of amphotericin P<O,OOOI = 0.0084 B as measured by the num- - P'O.OOOI P<O.OOOI P=O ber of CFU/cornea isolated after 24 and 48 hrs of treat- ment. Each data point rep- resents six eyes (mean and II range). 24 HOURS 48 HOURS 24 HOURS 48 HOURS 24 HOURS 48 HOURS DURATION OF TREATMENT Although the range at each sampling point was Drug Studies quite broad, the course of the disease was consistent with each isolate. Because the most active infection Due to a multiplicative error structure, the re- in the untreated model occurred in the first 48 hrs, sponse variable analyzed was loge (1 + CFU). Prelim- evaluation of the response to drug therapy was re- inary analysis showed a highly significant correlation stricted to this period. between right and left eye results (P < 0.0001) in both treated and untreated groups. Hence, a nested anal- ysis of variance was used to test differences between CANDIDA ALBICANS -STRAIN LV treatments. The error term was between rabbit mean 9 ^Control HJTreoted squares. In the experiment comparing the efficacy of five NATAMYCIN 5% FLUCYTOSINE 1% antifungal agents against the LV strain, there was a marked variation in the response to treatment (Figs. P<O.OOOI = 0.0OO2 P<O.OOOI P=0.0072 2, 3). The most effective drugs as a class were the polyenes, 0.5% amphotericin B and 5% natamycin, but only amphotericin B was able to eradicate infec- tion in 48 hours. Both 1% miconazole and 1% flu- III cytosine significantly reduced the number of CFU/ cornea at 24 and 48 hrs but were far inferior to the polyenes. Treatment with 1% ketoconazole was in- 24 HOURS 24 HOURS 48 HOURS effective with this strain. Based on the excellent response to 0.5% ampho- MICONAZOLE 1% KETOCONAZOLE 1% tericin B, a further study was performed with con- P= 0.2471 centrations of 0.15% and 0.075%. As can be seen in P =0.0370 P=0.7369 P =0.0056 Figure 2, these were also highly effective. After 48 Ifl hours of treatment infection was eliminated in most ifl eyes. The final experiment was designed to study the relationship between in vitro derived susceptibility data and the in vivo therapeutic response.