Evaluation of Corneal Cross-Linking for Treatment of Fungal Keratitis: Using Confocal Laser Scanning Microscopy on an Ex Vivo Human Corneal Model

Abstract

Purpose: Some previous reports have established the use of photoactivated chromophore-induced corneal cross-linking (PACK-CXL) in treating fungal keratitis. The results of these case reports have often been conflicting. To systematically study the effect of PACK-CXL in the management of Fusarium keratitis, we have developed an ex vivo model of human corneal infection using eye-banked human corneas.

Methods: Sixteen healthy ex vivo human corneas were divided into four study groups: (1) untreated control, (2) cross-linked, (3) infected with fungal spores, and (4) infected with fungal spores and then cross-linked. All infected corneas were inoculated with Fusarium oxysporum spores. The PACK-CXL procedure was performed 24 hours post inoculation for group 4. For PACK-CXL treatment, the corneas were debrided of epithelium; then 1% (wt/vol) isotonic riboflavin was applied dropwise at 5-minute intervals for 30 minutes and during the course of UV-A cross-linking for another 30 minutes. The corneas were imaged using a confocal microscope at 48 hours post inoculation, and the Fusarium hyphal volume and spore concentration were calculated.

Results: The infected and then cross-linked group had a significantly lower volume of Fusarium hyphae, compared to the infected (P = 0.001) group. In the infected and then cross-linked group there was significant inhibition of Fusarium sporulation compared with the infected (P = 0.007) group.

Conclusions: A model of human corneal infection was successfully developed for investigation of the effects of PACK-CXL on fungal keratitis. A treatment regimen of combined UV-A/riboflavin–induced corneal cross-linking appears to be a valuable approach to inhibit the growth and sporulation of Fusarium and suppress the progression of fungal keratitis.

Fungal keratitis is a destructive corneal infection with a high level of ocular morbidity,^1,2 which is particularly common within the developing world and subtropical areas, in patients with compromised corneal integrity. In the developing world, the annual number of recorded corneal ulcers is swiftly nearing between 1.5 and 2 million, and the actual figure is likely to be more. For most of these infections the final outcome is usually corneal opacity, or possibly even more devastating results such as corneal perforation, endophthalmitis, or phtisis.³ Fungal keratitis is viewed as a key blinding eye disease, in particular in agriculture-based geographic areas that have hot, humid, subtropical, and tropical climates. Fungi have been reported to cause 44% of all central corneal ulcers in South India, 36% in Bangladesh, and 17% in Nepal, in addition to 35% in South Florida and 37.6% in Ghana.^4–9 

Fungal keratitis has been documented to be caused by over 70 species covering 40 fungal genera.¹⁰ The key etiologic agents of fungal keratitis are the filamentous fungi, with Fusarium sp. comprising the most frequently occurring corneal keratitis–associated filamentous fungal genus.^11–15 Fusarium sp. are the key pathogens in 37% to 62% of instances of fungal keratitis.^16–19 

Fungal keratitis appears as ulcerative lesions²⁰ and is usually managed using topical antifungal medications, occasionally integrated with subconjunctival injections, although therapeutic keratoplasty may be necessary for patients whose corneal infection persists.¹³ This type of corneal infection presents a problem for the ophthalmologist as its control is limited by the availablility of effective antimycotics and the level to which they may infiltrate the corneal tissue. In addition, fungal infection is inclined to mimic other kinds of infectious keratitis,²⁰ where it is often misdiagnosed or diagnosed only at a very late stage, following the failure of extensive treatments for viral or bacterial keratitis. Unfortunately, this delay in diagnosis and treatment can cause an irreparable loss of sight.² Additionally, some microorganisms have displayed resistance to antimicrobial medications¹³; therefore, the infections could progress through corneal ulceration leading to corneal melting or perforation, even after provision of the necessary treatment. Consequently, current research is aimed at finding an innovative treatment that actively controls and manages infectious keratitis, particularly in infections in which the microorganism is difficult to identify or those caused by drug-resistant microorganisms. 

Ultraviolet (UV) light has been identified as an efficient method for disinfection, particularly of water, and as an alternative to chemical treatment; this has resulted a large demand for UV treatment globally. Regardless of variations in modes of inactivation, disinfection by UV is effective against a range of pathogens encompassing bacteria, protozoa, and viruses.^21,22 Riboflavin comprises a natural substance and is part of the vitamin B group, which is readily available in various foods. Photoactivated riboflavin has been utilized to minimize the microbial burden of liquids over many years. A good example is within the sphere of transfusion medicine, where concentrates of platelets are regularly treated with combined riboflavin and UV-A light.²³ The mixture of riboflavin and UV-A light results in irreparable damage to the DNA and RNA of pathogenic organisms, preventing genome replication and thus preventing infection.²⁴ 

Ultraviolet-induced corneal cross-linking (CXL) is a noninvasive procedure established for the management of keratoconus and corneal ectasia.^25,26 This technique relies upon using the photosensitizer riboflavin and UV-A to create photochemical cross-links within the anterior stromal collagen fibers and thus raise the biochemical and mechanical strength of the cornea,^27–29 which in keratoconus halts the progression of the corneal thinning. 

Photoactivated riboflavin and UV-A light have been trialed as potential adjuvant treatment for the management of corneal infections, particularly for the antibiotic-resistant forms of infectious keratitis. This treatment may also be beneficial where corneal melting occurs. Corneal stiffening resulting from this treatment could reduce melting and reduce or prevent corneal perforation.^30–32 The term photoactivated chromophore (PACK-CXL) was utilized instead of simply CXL in order to distinguish between using the CXL for treatment of infectious keratitis and for keratoconus.³² 

The aim of this study was to establish an ex vivo human corneal infection model by means of utilizing donor postmortem human corneas, infecting them with a fluorescently labeled Fusarium oxysporum, and then investigating the effect of PACK-CXL treatment in the management of fungal keratitis. 

Materials and Methods

Ex Vivo Human Cornea

Sixteen healthy postmortem human corneal buttons from nine male (M) and seven female (F) donors were used in this study. The corneas provided by the Manchester Eye Bank (NHS Blood and Transplant, UK) were preconsented for research if they were not suitable for transplantation. They were from donors in the age range 58 to 83 years (mean ± SE, 67.5 ± 7.6 years). At the Manchester Eye Bank, the corneas, along with a 3-mm scleral rim, were extracted from eyes less than 24 hours after death and placed into Eagle's minimal essential medium containing 2% (vol/vol) fetal bovine serum (FBS), 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 μg/mL amphotericin B (all Sigma-Aldrich Ltd, Poole, UK) and maintained at 34°C. The corneas used in this research had been released for research because the endothelial cell count was below that required for transplantation or for other reasons that did not directly affect the eye, such as inadequate medical history. The project had full ethical approval from the National Health Research Committee (No. 040811) and adhered to the terms of the Declaration of Helsinki. 

The corneas were randomly assigned to one of the four groups with four corneas in each group as follows: 

•  
  C: Control (not infected, not treated with CXL procedure) (one M and three F, 59–83 years)
•  
  X: Cross-linked (not infected, treated with CXL) (three M and one F, 59–71 years)
•  
  I: Infected (infected, not treated with PACK-CXL) (two M and two F, 59–76 years)
•  
  IX: Infected and then cross-linked (infected and then treated with PACK-CXL) (three M and one F, 58–71 years)

Corneal Tissue Culture

The human corneal buttons were transferred into six-well cell culture plates (Costar, Corning Ltd, Ewloe, UK) with the outer surface of the cornea uppermost and submerged in fresh culture medium (Dulbecco's modified Eagle's medium [DMEM], 1000 mg/L glucose [Sigma-Aldrich Ltd], supplemented with 10% [vol/vol] FBS [Labtech International Ltd, Uckfield, UK], 2 mM glutamine, 1% [vol/vol] penicillin with streptomycin [all GE Healthcare Life Sciences, Little Chalfont, UK], and 0.1% [vol/vol] hydrocortisone [H4126, Sigma-Aldrich Ltd]). The corneas were incubated at 34°C in 5% CO₂ for a minimum of 72 hours prior to the infection and CXL processes (Fig. 1). The manipulation of corneal tissue, subsequent infection, and CXL treatment processes were performed in a recirculating Class II Microbiological Safety Cabinet (Labcaire SC-R; Labcaire Systems Ltd, Clevedon, North Somerset, UK). 

Figure 1

 

The main steps of the methodology. The flow chart shows the main procedures, culturing, scratching, infection, cross-linking, and imaging processes, which were carried out for the different experimental groups, along with the time scale (peach color: procedure; green color: time scale).

Fungal Strains

The infecting fungus used was Fusarium oxysporum f. sp. lycopersici strain 4287 (race 2) expressing a cytosolic green fluorescent protein (GFP). The strain was kindly provided by Antonio Di Pietro (Department of Genetics, University of Cordoba, Cordoba, Spain).³³ 

The F. oxysporum GFP strain was cultured in Potato Dextrose Broth (PDB) (BD Difco, Oxford, UK) for 5 days at 28°C. On the day of corneal inoculation, the spores (microconidia) were harvested. The fungal spore suspension was filtered using a filter cloth (Miracloth; Millipore Limited, Watford, Hertfordshire, UK), and a spore pellet was collected by centrifugation at 3260g for 5 minutes followed by washing with sterile distilled water and further centrifugation. The spore pellet was then resuspended in 1 mL sterile distilled water, diluted 1/1000 with sterile distilled water, and the concentration of this spore suspension was determined using a Fuchs-Rosenthal hemocytometer (Scientific Laboratory Supplies, Nottingham, UK). The spore concentration was adjusted to 5 × 10⁵ spores/mL using fresh culture medium, and 10 μL of this suspension was used to inoculate each of the scratched corneas as detailed below. 

Corneal Infection Process

Seventy-two hours post corneal culturing, two of the human corneas, the I and IX, were inoculated with the spores of F. oxysporum expressing cytosolic GFP: The GFP fluorescence facilitated the tracking of the fungal hyphal invasion through the corneal layers. 

As an ex vivo model of corneal insult and fungal infection, corneal buttons were mounted upon artificial anterior chambers (Moria SA, Antony, France; SD Healthcare, Manchester, UK) and held in place by the retainer and locking ring. Then, the anterior surface was scratched in an asterisk shape using a 0.6 × 25-mm-gauge needle (BD Microlance 3; BD Biosciences, Oxford, UK). They were transferred into a fresh, dry six-well tissue culture dish; and two of them, previously assigned for infection, were inoculated with 5 × 10⁵ Fusarium oxysporum spores in 10 μL culture medium. The viability of the spores was checked by inoculating a further 10-μL spore suspension into growth medium. Inoculated corneas were left for few minutes to aid spore attachment to the corneal tissue. Next, a small volume of culture medium was added around the periphery of each cornea to prevent washing off the fungal spores. The corneas were incubated for 24 hours at 34°C prior to the initial assessment using confocal laser scanning microscopy and the treatment with the CXL procedure. 

CXL Procedure

The corneas in the X and IX groups were treated with the corneal cross-linking technique according to a standard CXL protocol.²⁵ The X and IX corneas were subjected to the cross-linking procedure 24 hours post inoculation with spores. 

The corneal button was mounted on an artificial anterior chamber as described above. Phosphate-buffered saline (PBS) was infused through a tube, valve, and syringe to fill the interior of the cornea to mimic the in vivo intraocular pressure and maintain corneal stability and shape throughout the CXL procedure (Fig. 2). 

Figure 2

 

A human corneal button fixed onto an artificial anterior chamber. The figure shows a human corneal button fixed onto an artificial anterior chamber and filled with PBS via a tube, valve, and syringe (A) to mimic intraocular pressure, which maintains the shape and stiffness of the corneal surface (B).

Debridement of the corneal epithelium layer was performed to allow adequate riboflavin penetration to the corneal stroma. The central 7- to 9-mm-diameter area of the corneal epithelium was debrided with a single-use surgical blade (size 10; Swann-Morton, Sheffield, England), then wiped using a cotton eye spear (bviMerocel; Beaver Visitec, International Ltd., Abingdon, Oxfordshire, UK). Subsequently, isotonic riboflavin eye drops (0.1% [wt/wt] riboflavin, 20% [wt/wt] dextran T500) were dropped onto the anterior surface of the cornea at 5-minute intervals for 30 minutes. The prepared cornea was irradiated with UV-A light utilizing a medical electrical UV-A light emitter (370 nm, irradiance of 3 mW/cm²; VEGA, C.S.O. srl, Florence, Italy), which delivered a total UV-A irradiation dose of 5.4 J/cm² to the corneal surface over a period of 30 minutes, along with application of further isotonic riboflavin, again at 5-minute intervals. Following this procedure, the cornea was rinsed several times with sterile PBS and placed in a fresh six-well tissue culture plate along with fresh culture medium and incubated at 34°C in 5% CO₂. 

Imaging

Twenty-four hours post inoculation, the corneas were inverted and placed on a two-well chamber slide (Thermo Fisher Scientific, Rochester, NY, USA) and imaged using a Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems Ltd., Breckland, Linford Wood, Milton Keynes, UK) with a ×20 objective. At 48 hours post inoculation, the corneas were imaged using a Canon camera (Canon Powershot S5IS, zoom lens ×12, 8.0 megapixel; Canon Ltd., Reigate, Surrey, UK) and the confocal microscope. The confocal microscopy images were analyzed with the Surpass module of the Imaris v8.0 software (Bitplane Scientific software module; Bitplane AG, Zurich, Switzerland). The percentage of the total volume occupied by the fungal hyphae was calculated in five different z-stack images, each of 100-μm thickness, for each study group from each experimental set: The hyphae were visualized from the GFP signal within corneal tissues. After imaging, the number of fungal spores per milliliter of corneal culture medium at 48 hours post inoculation was counted for all infected corneas. 

Results

Fusarium keratitis-like infection developed successfully in all inoculated corneas. The effect of PACK-CXL treatment on Fusarium was determined by measuring fungal hyphal volume within the corneal tissues. 

Twenty-four hours after infection, all inoculated corneas developed Fusarium infection, whereas the C and X corneas remained intact and uninfected. Forty-eight hours after the initial inoculation, the fungal infection had progressed in the I group, while IX corneas showed less severe infection. Figure 3 shows the reduced progress of Fusarium infection at 48 hours post inoculation in the IX group (Fig. 3B) compared to the I (Fig. 3A) group. The Fusarium infection can be seen as branching, green fluorescent fungal hyphae. 

Figure 3

 

Confocal microscopy images of infected corneal buttons 48 hours post inoculation following PACK-CXL treatment. Each image is a projection of a z-stack of confocal images (×20 magnification) that is 100 μm thick. The images of the different infected corneas show the extent of the branching hypha growth of the fungus, which is expressing GFP in its cytoplasm at 48 hours post inoculation. Extensive fungal growth is visible in the I (A) group, while there are fewer hyphae evident in the IX group (B).

The percentage of the total volume occupied by the fungal hyphae and the quantity of fungal spores in the culture medium from each infected group are shown in Figures 4 and 5, respectively. There is variation in the volume occupied by Fusarium hyphae between the infected groups at 48 hours after inoculation. The I group showed greater amounts of Fusarium hyphae and, consequently, more severe fungal infections compared to the IX group. Analysis of variance (ANOVA) of the Fusarium colony volume data showed a significant difference between the I and IX groups (P = 0.001). These results show that the PACK-CXL treatment controlled the fungal infection in the IX group, where the growth of fungus was inhibited by the PACK-CXL procedure. 

Figure 4

 

The percentage of the total volume occupied by the fungal hyphae. The graph shows percentage of the total volume occupied by the fungal hyphae, as visualized by the GFP signal from the fungal hyphae within the corneal tissues for the different infected groups at 48 hours post inoculation. The I group exhibited greater Fusarium hyphal volume compared to that of the IX group.

Figure 5

 

The quantity of Fusarium spores in the infected groups. The graph shows the number of Fusarium spores per milliliter of corneal culture medium at 48 hours post inoculation for the infected groups. The quantity of Fusarium spores of the IX group was significantly less than that of the I group (P = 0.007).

There was also variation in the quantity of Fusarium spores that were present in the culture medium at 48 hours post inoculation between the infected groups. The IX group shows a considerable decrease in the number of Fusarium spores present compared to the I group. Analysis of variance of the Fusarium spore quantity data shows that there is a significant difference in fungal spore density between the I and IX groups (P = 0.007). These observations show that the combined riboflavin and UV-A cross-linking treatment had an inhibitory effect on the Fusarium sporulation. 

Discussion

This study aimed to establish a model of human corneal fungal infection using human donor corneas infected with F. oxysporum expressing cytosolic GFP, which permits the tracking of hyphal growth within the corneal tissue. With the model established, the combined UV-A/riboflavin–induced corneal cross-linking as a primary therapeutic technique for the management of fungal keratitis could be evaluated. To our knowledge, this study is the first ex vivo attempt to model the treatment of human fungal keratitis using this PACK-CXL technique. 

Fusarium oxysporum was selected for this study due to its aggressive destruction of the cornea and its regularity and frequency of appearance as a clinical pathogen. Fungal keratitis caused by Fusarium spp. is recognized as one of the most sight-threatening corneal infections.^{10,18,19,34–36} The control of fungal keratitis presents a challenging problem mostly as a result of belated diagnosis and limited options for therapy.³⁷ As current antifungal medications display little corneal infiltration and restricted effectiveness, the treatment of ocular diseases caused by fungi is presently unsatisfactory.^1,38 In addition, most antimycotics utilized in the management of Fusarium keratitis require a long course of treatment and frequently fail to preserve vision.³⁹ 

Introduction of a therapy based upon the photosensitizer riboflavin and UV-A light was first described by Wollensak et al.^25,40 for the induction of corneal cross-linking for corneal ectasia. Recently, indications for the use of the CXL procedure have expanded to include Fuch's corneal dystrophy⁴¹ and pseudophakic bullous keratopathy⁴² as well as infectious keratitis.^{30,31,43–45} The use of the CXL technique for treatment of corneal ectasia and advanced keratoconus is well established, while the efficacy of the PACK-CXL procedure in the management of infectious keratitis is still subject to appraisal. 

In this study, a human ex vivo corneal Fusarium keratitis model was established. The results show that there was a significant difference in the volume of invading fungus within the infected corneal tissue between the groups, with the infected corneas treated with PACK-CXL showing a significant decrease in the fungal volume compared to the other experimental group. This is a good indication that the PACK-CXL procedure is effective in suppressing the progression of fungal infection. The favorable outcomes obtained in this study are contradicted by some published studies, which report that the PACK-CXL procedure is not effective in managing fungal keratitis.^37,46,47 Nevertheless, these observations are supported by numerous in vivo studies that attempted to examine the influence of combined riboflavin/UV-A in the management of infectious fungal keratitis. It is suggested that this combination has positive antimicrobial effects, which assist in inhibiting the growth of pathogens and managing the infection.^30,48–50 The beneficial antimicrobial effect of the PACK-CXL procedure in suppressing infection by Fusarium is likely to be attributable to several mechanisms. Inactivation of ribonucleic acids of organisms may occur by the combined UV-A/riboflavin–induced cross-linking that may have a cytotoxic effect on the pathogens.^23,51 In addition, it has been shown that the PACK-CXL technique brings about cross-linking in the corneal collagen fibers, thus increasing the strength and simultaneously reducing its penetrability by fungal hyphae. Furthermore, the cross-linked collagen is more resistant to enzymatic digestion by microbial pathogens, which in turn reduces corneal melting.^52,53 In this study, the direct antifungal activity of the UV-A/riboflavin combination is perhaps the greater contributory factor. It is also possible that cross-linking might entrap the fungal hyphae within the collagen matrix, thereby reducing the growth rate still further. The proposal that the direct antifungal effect of the UV-A/riboflavin treatment is the major component in reducing corneal infection is supported by the fact that the IX group showed a significant reduction in the number of Fusarium spores present after the PACK-CXL treatment compared with the I group. This is indicative of the riboflavin/UV-A treatment having an inhibitory effect on Fusarium sporulation. On the other hand, debriding the epithelial layer post inoculation and prior to the PACK-CXL treatment may have played a role in reducing the infection by removing some of the Fusarium hyphae growing within the corneal epithelial tissue. Nevertheless, the reduction in spore numbers in the culture medium post PACK-CXL treatment lends some support to the hypothesis that the riboflavin/UV-A combination has an antifungal action. 

Iseli et al.³⁰ set out the first published clinical trial to investigate the effect of PACK-CXL application on a series of five cases, with infectious melting keratitis caused by pathogens including fungi that showed no response to intensive antibiotic therapies. They found that following PACK-CXL, corneal melting was arrested in all cases, and that there was no need for emergency corneal transplantation as would normally have been the case. Another clinical trial presented results that support the efficacy of the PACK-CXL procedure.⁴⁸ The study looked at seven eyes with infectious keratitis associated with corneal ulcers and melting, some of which were caused by fungi. After PACK-CXL treatment, it was noted that the corneal infections had been well controlled; in all cases, progression of the corneal melting had stopped and ulcers had healed with no severe consequences, and no further surgery was necessary. In recent studies, the therapeutic efficacy of the PACK-CXL technique was assessed by Hafez⁵⁰ on a series of five eyes with resistant fungal corneal ulcers. Even though notable vision improvement was not achieved post PACK-CXL treatment, the findings showed that the PACK-CXL procedure can be effective in the management of resistant corneal ulcers, as all cases showed improvement on infection signs and complete ulcer healing. In addition, Li et al.⁴⁹ presented the clinical findings in eight patients with fungal keratitis who received PACK-CXL treatment. After the PACK-CXL procedure, resolution of infection and ulcer healing were achieved in all cases, and none required further surgical intervention. Li et al.⁴⁹ suggested that the PACK-CXL procedure is a viable option for treating fungal keratitis. 

However, confounding outcomes were found in a clinical trial conducted by Escarião et al.⁴⁶ on 11 eyes with bacterial or fungal keratitis that were nonresponsive to the medications administered for at least a period of 1 week. The bacterial keratitis cases showed relief in the symptoms post PACK-CXL treatment, whereas in the fungal keratitis cases no improvement was observed, which indicated that the effectiveness of PACK-CXL procedure is related to the causative agent. This finding is consistent with a study by Vajpayee et al.³⁷ in which 41 cases of fungal keratitis were divided into two groups. The first group was treated with the PACK-CXL technique along with antifungal therapy, while the second group received antifungal medications only. It was observed that infection resolution was achieved in 90% and 85.7% of cases, the recovery time rate was 31 ± 27 and 31 ± 20 days, and the best-corrected vision rate was 1.13 ± 0.55 and 1.25 ± 0.46 (logMAR) in the first and second group, respectively. Moreover, keratoplasty surgery was carried out in two cases from the cross-linked group and in three cases from the control group. Vajpayee et al.³⁷ concluded that there was no beneficial effect of the combined antifungal/PACK-CXL treatment over the antifungal agent alone. Additionally, Uddaraju et al.⁴⁷ evaluated the efficacy of PACK-CXL treatment in resistant deep stromal keratitis caused by fungi, with 13 cases were included, which showed no response to antifungal medications administered for 2 weeks. The cases were randomly divided into cross-linked and control groups; the cross-linked group was treated with UV-A/riboflavin combination along with the medications, while the control group received antifungal therapy only. Uddaraju et al.⁴⁷ concluded that the PACK-CXL procedure is not an effective treatment in the management of advanced fungal keratitis, as the cross-linked group experienced a higher perforation rate than the control group. Notably, the majority of the clinical studies were carried out on resistant forms of corneal infections; the antimicrobial medications had been administered for various durations and mostly in cases in which the infections had failed to resolve. In such cases, the PACK-CXL technique was implemented only after the infections had progressed to late stages and had caused further serious damage to the cornea. Thus, late intervention could have resulted in a great reduction in the effectiveness of PACK-CXL procedure, minimization of the healing rate, or even a treatment failure. 

In conclusion, the treatment of Fusarium keratitis with combined UV-A/riboflavin–induced corneal cross-linking is effective in inhibiting Fusarium sporulation and hyphal growth, and thus reduces the intensity of infection. 

Acknowledgments

The authors thank the Manchester Eye Bank for providing the human corneal buttons for this study. 

Supported by Ministry of Health, Riyadh, Saudi Arabia (JMA). DC-L was supported by a grant to David W. Denning (The University of Manchester, The National Aspergillosis Centre, University Hospital of South Manchester, Manchester, UK) and from the Global Action Fund for Fungal Infections (GAFFI) to NDR. 

Disclosure: J.M. Alshehri, None; D. Caballero-Lima, None; M.C. Hillarby, None; S.G. Shawcross, None; A. Brahma, None; F. Carley, None; N.D. Read, None; H. Radhakrishnan, None