Safety and Efficacy of Intrastromal Injection of 5% Natamycin in Experimental Fusarium Keratitis

Abstract

Purpose:

To compare the efficacy of combined intrastromal injection and topical natamycin 5% to standard topical therapy alone in an experimental rabbit model of Fusarium keratitis.

Methods:

Fungal keratitis was induced in the right eyes of 12 New Zealand rabbits by stromal injection of Fusarium solani spore suspension into the cornea. Four days after inoculation, animals were randomly assigned to 2 different treatment groups (n=6 in each group). The study group received intrastromal injections of natamycin 5% on treatment day 1 and 4, combined with topical natamycin 5% eye drops given hourly between 8:00 and 20:00 for the first 2 days, followed by 4 times daily on days 3–11. The control group received only topical natamycin 5% at identical intervals. Eyes were examined clinically on days 1, 4, 7, and 11 for status of corneal healing, corneal vascularization, and hypopyon. Animals were sacrificed on day 11, and corneas were subjected to histopathological examination.

Results:

Both groups showed significant improvement in terms of conjunctival hyperemia, size and density of corneal infiltrate, corneal edema, and total clinical score. In the study group, there was a significant improvement in the height of hypopyon in the anterior chamber, while there was also an increased amount of vascularization.

Conclusions:

This study showed that intrastromal injection of natamycin 5% combined with topical treatment has little beneficial effect over topical therapy in a Fusarium keratitis rabbit model. The addition of intrastromal injection should be reserved to the most severe or recalcitrant cases.

Introduction

Until recently, fungal keratitis was considered to be prevalent mostly in developing countries.¹ In the last 4 decades, there has been an increase in the occurrence of fungal keratitis in developed countries^2–4 as a result of a broad use of antibiotics and steroids, long-term use of contact lenses, and secondary to an increasing number of surgical corneal procedures.^5–7 In addition, a relatively recent outbreak of Fusarium species was linked to the use of a specific contact lens solution.⁸ Filamentous fungi, specifically Fusarium species, are the predominant cause of fungal ulcers in tropical regions.⁹

Fungal keratitis can be a major therapeutic challenge. Indeed, modern antifungal medications appear to be less effective in fungal keratitis than modern antibacterial drugs for the treatment of bacterial keratitis.¹⁰ Adverse outcomes such as perforation of corneal ulcers,¹¹ the need for a corneal transplant, or even eye removal¹⁰ are more common when compared with bacterial infections. It also appears that tissue penetration of some antifungal drugs into the corneal stromal layers is less optimal than others.¹² Thus, cases of deep fungal keratitis can be very difficult to treat. Unlike voriconazole,¹³ topical natamycin has been shown to penetrate the cornea poorly, with effective drug levels not being achieved in both the cornea and aqueous humor.^12,14 Thus, prior debridement of the epithelium has been recommended when treating fungal keratitis with natamycin.¹⁴ However, a recent publication by Prajna et al. reported a trend toward scraping being associated with worse outcomes.¹⁵ It has been proposed that deep fungal keratitis should be treated with intrastromal or intracameral injections of the antifungal medication, and successful treatment of complicated cases of fungal keratitis has been reported.^16–20

We therefore conducted this study to compare the efficacy of intrastromal injection of natamycin 5% (Natacyn; Alcon Laboratories, Inc.) combined with topical natamycin 5% to a standard topical natamycin 5% in a rabbit model of Fusarium fungal keratitis. Specifically, we tested the hypothesis that intrastromal injection of natamycin 5% combined with topical natamycin 5% is more efficient by means of a shorter infection duration and decreased damage to the cornea than the standard therapy.

Methods

This study is a nonblinded, prospective randomized experiment conducted in a rabbit model of fungal keratitis.

Experimental model

Twelve New Zealand white male rabbits weighing 2.5–3.0 kg were selected for the experiment. Animals were treated according to the Association for Research in Vision and Ophthalmology statement, which refers to the usage of animals in medical research in this field,²¹ and the research was approved by the Institutional Review Board of Meir Medical Center. The rabbits were kept under observation for 7 days to exclude any local or systemic diseases. All examinations and interventions, during this study, were performed under general anesthesia by means of intramuscular injection of ketamine 50 mg/kg and xylazine 20 mg/kg combined with topical anesthesia with localin 0.4% (Fischer Pharmaceuticals Ltd).

Fungus isolation and production

The strain of Fusarium used for this experiment was obtained from the cornea of a human patient suffering from fungal keratitis and treated in our Department of Ophthalmology. The fungus was incubated on a Sabouraud Dextrose Agar (SDA) plate at 32°C for 48 h. A sample for injection was prepared by rinsing the SDA plate with a 10 mL solution of Tween 80 0.1% into a physiological saline solution. After scraping the plate with a dispensable plastic scraper, the sample was transferred to a sterile test tube and vortexed for 30 s. The sample was filtered twice through 2 layers of sterile gauze to remove hyphal fragments. It was then transferred to a new sterile test tube and was centrifuged at 10,000 g for 5 min to allow the conidia to sink to the bottom of the tube. After removing the supernatant fluid using a sterile pipette, the sample was rediluted with sterile normal saline to produce a sample with a concentration of 1.6×10⁵ cfu/100 μL. The sample was tested with a hemocytometer counter and its viability was tested by seeding of repetitive dilutions, which was found to be >99%. This strain was presumably susceptible to natamycin because the patient from whom it was obtained was treated with topical natamycin and healed. Moreover, in general, natamycin has good activity against Fusarium species.²²

In all animals, the right eye was selected for fungal injection. Seeding was induced through midstromal injection adjacent to the center of the cornea using a 0.1 mL sample with a concentration of 1.6×10⁵ cfu/100 μL using a tuberculin syringe and a 30-gauge-diameter needle. After the injection of the fungus, the rabbits were followed for 4 days without any treatment until a corneal infiltrate (abscess) developed.

Treatment randomization

Four days postinoculation and after a corneal infiltrate was evident, the rabbits were randomly assigned to 2 treatment groups. The study group was treated with a combined protocol that included intrastromal injections of natamycin 5% on day 1 and 4 postrandomization, combined with topical treatment with natamycin 5% drops every hour for the first 2 days (08:00–20:00) and then 4 times a day until the end of the study. The control group was treated only with local topical treatment of natamycin 5% drops using an identical protocol.

For intrastromal injection, 1 mL of natamycin 5% was drawn into a tuberculin syringe with a 30-gauge-diameter needle. Injections were performed obliquely at the junction of a clear cornea at the boundaries of the infiltrate with an overall volume of 0.5–0.7 mL. Any complications that occurred during the procedure were well documented.

Follow-up examinations

Eyes of each group were examined clinically by an operative microscope on treatment day 1, 4, 7, and 11, and corneal photographs were taken. On treatment day 11, the rabbits were sacrificed by intravenous overdose of sodium pentobarbital (Sleepaway; Fort Dodge Animal Health), and the corneas were harvested for histopathological examination. Periodic acid-Schiff staining was performed to demonstrate the fungi. Hematoxylin and eosin staining was added to characterize the extent of the inflammation and the presence of vascularization.

The following parameters were evaluated and recorded: conjunctival hyperemia, size and density of corneal infiltrate, size of the epithelial defect, corneal edema, extent of vascularization, and the presence of hypopyon in the anterior chamber. The evaluation of the parameters was based on the Schreiber score.²³

Conjunctival hyperemia was graded as 0, no hyperemia; 1, light hyperemia; 2, moderate hyperemia; and 3, severe hyperemia. Corneal infiltrate density was graded as 0, no opacity; 1, nebular opacity; 2, macular opacity; and 3, leucoma opacity. Corneal edema was graded as 0, clear cornea; 1, minor edema (less than half of the cornea); 2, moderate edema (more than half of the cornea); and 3, severe edema (total corneal clouding). The extent of vascularization was graded as 0, no vascularization; 1, peripheral vascularization (up to 2 mm from the limbus); 2, midperipheral vascularization (more than 2 mm from the limbus); and 3, central vascularization (until the center of the cornea). The size of the corneal infiltrate, epithelial defect, and hypopyon level were measured using the largest diameter in millimeter.

The main measurements to evaluate the efficacy of treatment included the size of the infiltrate, infiltrate density, area of corneal epithelial defect, evaluation of anterior chamber reaction (hypopyon height), response time to treatment, and histopathological comparison of corneal tissues between the 2 groups. We calculated the total clinical score as a sum of the clinical grading or measurements of the clinical parameters, which were documented on the different treatment days in both groups (size of infiltrate, density of infiltrate, hyperemia of conjunctiva, corneal edema, and size of hypopyon). We used this parameter to better describe the process of clinical healing throughout the treatment period.

The main measurements to evaluate safety included the extent of toxic effects of the medication on the ocular surface and the cornea such as conjunctival hyperemia and epithelial defects.

Statistical analysis

Changes of clinical parameters over time were analyzed using the nonparametric repeated measures test (Friedman test). Data are expressed both as mean±SD and median (range). A P-value of <0.05 was considered to be statistically significant.

Results

Fusarium solani colony injection into the corneal stroma was successful in establishing an animal model of fungal keratitis. Four days after fungal inoculation, there were corneal edema and infiltration, hypopyon, and swelling of the 12 rabbit eyes.

Table 1 depicts the results of the Friedman analysis showing that over time both groups showed significant improvement in terms of conjunctival hyperemia, size and density of corneal infiltrate, corneal edema, and total clinical score. Neither group showed a significant difference over time in the size of the corneal epithelial defect. In the study group, there was a significant improvement in the height of hypopyon in the anterior chamber while there was also an increased amount of corneal vascularization.

Table 1.

Friedman Test Demonstrating Changes Over Time for All Parameters of the Study in Both Groups

	Study group		Control group
	Chi square	P-value	Chi square	P-value
Conjunctival hyperemia	9.058	0.029	9.133	0.028
Size of infiltrate	13.632	0.003	11.727	0.008
Infiltrate density	12.529	0.006	9.750	0.021
Corneal edema	12.763	0.005	11.327	0.010
Corneal epithelial defect	3.000	0.392	3.000	0.392
Corneal vascularization	13.776	0.003	2.027	0.567
Anterior chamber hypopyon	10.800	0.013	4.875	0.181
Total clinical score	12.763	0.005	11.327	0.010

Table 2 depicts the mean±SD and median (range) of the outcome variables studied in both groups on each day of the study. The difference between the total clinical score on day 1 versus day 11 was minor and not significantly different from 0 (P=0.715). Assuming that this difference would be real, we calculated at a power of 0.8 that 160 rabbits would be required in each group for this difference to reach the 0.05 significance level.

Table 2.

Clinical Parameters in Both Groups on Days 1, 4, 7, and 11

	Study group		Control group
	Mean±SD	Median (range)	Mean±SD	Median (range)
1st day
Conjunctival hyperemia	2.8±0.4	3.0 (2–3)	2.4±0.5	2.0 (2–3)
Size of infiltrate	3.5±1.3	3.6 (2–5.69)	3.1±1.4	3.0 (1.25–4.75)
Infiltrate density	2.3±0.5	2.0 (2–3)	2.6±0.5	3.0 (2–3)
Corneal edema	2.0±0.9	2.0 (1–3)	1.8±0.4	2.0 (1–2)
Corneal epithelial defect	0.4±1.0	0.0 (0–2.5)	0.4±0.9	0.0 (0–2)
Corneal vascularization	1.0±0	1.0 (1–1)	1.0±0	1.0 (1–1)
Anterior chamber hypopyon	2.2±1.8	2.5 (0–4)	1.5±1.7	1.0 (0–3.5)
Total clinical score	13.3±3.6	13.5 (7–17.19)	11.8±3.7	12.0 (7.25–15.75)
4th day
Conjunctival hyperemia	2.2±1.0	2.5 (1–3)	2.0±1.0	2.0 (1–3)
Size of infiltrate	3.7±2.8	3.4 (1–8.67)	3.5±1.1	4.0 (1.87–4.48)
Infiltrate density	1.7±1.0	1.0 (1–3)	2.2±0.8	2.0 (1–3)
Corneal edema	1.5±1.0	1.5 (0–3)	1.4±1.1	1.0 (0–3)
Corneal epithelial defect	0.0±0.0	0.0 (0–0)	0.2±0.4	0.0 (0–1)
Corneal vascularization	1.2±0.4	1.0 (1–2)	1±0.7	1.0 (0–2)
Anterior chamber hypopyon	0.8±1.3	0.0 (0–3)	0.1±0.2	0.0 (0–0.5)
Total clinical score	9.8±6.3	7.1 (3.50–20.67)	9.4±3.4	8.3 (6–13.48)
7th day
Conjunctival hyperemia	1.8±0.4	2.0 (1–2)	1.4±0.5	1.0 (1–2)
Size of infiltrate	1.4±1.8	0.5 (0–3.81)	2.4±1.3	2.3 (1–4.38)
Infiltrate density	0.8±1.0	0.5 (0–2)	2.0±1.0	2.0 (1–3)
Corneal edema	1.3±1.2	1.5 (0–3)	1.0±1.2	1.0 (0–3)
Corneal epithelial defect	0.0±0.0	0.0 (0–0)	0.2±0.4	0.0 (0–1)
Corneal vascularization	2.0±0.0	2.0 (2–2)	1.4±0.5	1.0 (1–2)
Anterior chamber hypopyon	0.0±0.0	0.0 (0–0)	0.6±1.3	0.0 (0–3)
Total clinical score	5.4±4.0	4.0 (2–10.81)	7.6±3.9	6.5 (4–12.38)
11th day
Conjunctival hyperemia	1.3±0.8	1.5 (0–2)	0.8±1.1	0.0 (0–2)
Size of infiltrate	0.8±1.0	0.4 (0–2)	2.0±1.1	1.7 (1–3.33)
Infiltrate density	0.5±0.5	0.5 (0–1)	1.6±0.5	2.0 (1–2)
Corneal edema	1.5±0.8	1.0 (1–3)	0.8±1.1	0.0 (0–2)
Corneal epithelial defect	0.0±0.0	0.0 (0–0)	0.1±0.1	0.0 (0–0.25)
Corneal vascularization	2.3±0.8	2.5 (1–3)	1.0±1.4	0.0 (0–3)
Anterior chamber hypopyon	0.0±0.0	0.0 (0–0)	0.0±0.0	0.0 (0–0)
Total clinical score	4.1±2.2	4.0 (1–7)	5.3±3.7	4.0 (2–9.33)

No complications occurred related to the procedures or treatments. No toxic reaction in the conjunctiva, sclera, or the corneal epithelium was witnessed after the injections. No defects or damage to the epithelium were documented throughout the entire study.

On histopathological examination, no presence of fungi was detected in the specimens. A mixed inflammatory reaction, including the presence of polymorphonuclear cells, lymphocytes, and plasma cells both inside and around the infiltrate, was documented both in study and control specimens. A superficial and deep vascular reaction was also documented in both groups reaching up to two-thirds of the thickness of the cornea.

Discussion

In this study, both treatment regimens were found to be effective for the treatment of fungal keratitis. Both treatment groups showed a statistically significant improvement over time in the following parameters: conjunctival hyperemia, size and density of corneal infiltrate, corneal edema, and total clinical score. However, only the study group improved in terms of hypopyon at the cost of an increased amount of vascularization.

Infections involving the deep layers of the cornea are a therapeutic challenge especially in cases of fungal corneal infections. The limited efficacy and the low capability of the medications to penetrate the deep layers of the cornea place patients with fungal abscesses at risk of developing a spontaneous perforation and spread of the infection into the eye cavity causing severe endophthalmitis.^24,25

In this study, both treatment modalities were effective in eradicating the fungus. However, the intrastromal injection only led to a significant improvement in the anterior chamber hypopyon size over time. In contrast, there was a significant increase in the corneal vascularization only in the intrastromal injection group. This apparent disadvantage of the intrastromal injection might not be relevant in humans as the propensity to develop corneal vascularization appears to be highly specific to rabbits.²⁶

Existing literature contains a relatively small number of cases in which intrastromal injection of antifungal medication was used to treat corneal fungal abscesses with conflicting results. Garcia-Valenzuela and Song described a complicated case of recurrent fungal keratitis with endophthalmitis following a contaminated penetrating keratoplasty.¹⁹ Ultimately, the patient was treated with intrastromal corneal injections combined with intravitreal injection of amphotericin B that led to the eradication of the corneal fungal plaques and the intraocular infection. A recent case series of 12 patients with recalcitrant fungal keratitis to topical and systemic treatment, who underwent intrastromal injection of voriconazole, showed improvement in the size of corneal infiltrate and final visual acuity in 83% of patients.²⁷ One small case series of 3 patients reported that the intrastromal injection of voriconazole together with topical voriconazole effectively reduced the infiltration size and controlled the infection in patients with Fusarium keratitis.²⁸ Recently, a randomized clinical trial found no differences between topical natamycin 5% combined with topical 1% voriconazole versus topical natamycin 5% combined with intrastromal injections of voriconazole in recalcitrant cases of fungal keratitis.²⁹

The findings of this study are supported by previous studies that found no advantage when combining intrastromal antifungal injections with topical treatment.^29,30

In this study, the first to compare combined topical and intrastromal natamycin versus topical natamycin alone, no complications related to the injection itself were documented in the study group. No severe allergic reactions or inflammatory responses associated with the drug itself were documented. An advantage of the intrastromal injection was the significant decrease over time in the height of hypopyon. This isolated finding may imply a certain advantage for combined intrastromal injection and topical treatment over topical treatment alone in cases of fungal keratitis with severe hypopyon.

Our study had several limitations. First of all, we used a total clinical score, which was essentially the sum of all the clinical parameters considered, to evaluate the clinical response to treatment. Obviously, if this score was to be used clinically in humans, it would have to undergo careful validation. Second, our sample size was relatively small (n=12). The total clinical score in the study group was greater (although not statistically significant) than that in the control group on the 1st day, but by the 11th day, the total clinical score in the study group was less than that in the control group. The difference between the initial total clinical score and the last total clinical score was not statistically significant and was relatively small. For this difference to become significant, it would have required a sample size of 320 rabbits (160 in each group), an enterprise that would be unrealistic in view of the very little clinical difference between the 2 groups.

Third, fungal keratitis requires a longer treatment period when compared with bacterial keratitis. During the study period, both groups showed significant improvement over time in most parameters; however, a study with a longer follow-up period may have led to other findings (superiority of 1 method over the other).

Finally, a limitation of our study is that we have used for intrastromal injection the same natamycin medication that is used for topical application. This topical natamycin formulation is more of an emulsion than a solution, and frequently leaves a precipitate on the cornea after topical application. It is conceivable that the nature of the natamycin preparation used, being basically meant for topical application, was not optimal or ideal for intrastromal injection. However, it must be noted that pharmaceutical-grade powder is very difficult to obtain, especially for drugs that are not frequently used, such as natamycin.³¹

In summary, this experimental study shows that intrastromal injection of natamycin 5% combined with topical treatment has little beneficial effect over topical therapy alone in the treatment of Fusarium keratitis. We speculate that the addition of intrastromal injection should be reserved to the most severe or recalcitrant cases.

Footnotes

Author Disclosure Statement

All authors declare no conflicts of interest.

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