|Year : 2022 | Volume
| Issue : 2 | Page : 43-48
Evaluation of the efficacy of high-fluence corneal collagen cross-linking in fusarium corneal ulcer in rabbits
Ramy A.M Elbassiouny1, Alaa A Ghaith2, Hafezi Farhad3, Manal M Baddour4, Sheta Eman5, Ahmed A Elmassry2
1 Department of Ophthalmology, Alexandria General Ophthalmology Hospital, Alexandria, Egypt
2 Department of Ophthalmology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
3 Department of Ophthalmology, Faculty of Medicine, Geneva University, Geneva, Switzerland
4 Department of Microbiology and Medical Immunology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
5 Department of Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
|Date of Submission||14-Jan-2021|
|Date of Acceptance||27-Jun-2021|
|Date of Web Publication||08-Jul-2022|
MD, PHD, MRCS Ed Ramy A.M Elbassiouny
Department of Ophthalmology, Alexandria General Ophthalmology Hospital, 31 Elmoaameron B Tower, Hilton Street, Somouha, Alexandria, 21684
Source of Support: None, Conflict of Interest: None
Background Accelerated photoactivated chromophore for keratitis–corneal collagen cross-linking (PACK-CXL) was investigated to be effective in the treatment of fungal keratitis in many studies. High-fluence CXL was used effectively in the treatment of progressive keratoconus but not tested in PACK-CXL for fungal infection.
Aim To evaluate the antifungal efficacy of high-fluence CXL in the treatment of induced fusarium corneal ulcer in rabbits.
Design This is an experimental interventional study.
Patients and methods This is an experimental interventional study conducted on 16 eyes of eight rabbits for which fusarium corneal infection was induced by intrastromal corneal injection. The rabbits were divided into two groups: group A received no treatment and was left as a control group. Group B received a single high-fluence PACK-CXL session. All rabbits were followed up for 6 days and then the eyes were enucleated. Culture was performed for seven corneas and histopathology for the remaining cornea.
Results There was statistically significant deterioration in group A (P=0.003), while a statistically significant clinical improvement was seen in group B (P<0.001). The median colony-forming units was more in group A (4×104) than in group B (2×102). Histopathological examination also revealed less inflammatory signs in high-fluence PACK-CXL-treated group.
Conclusions High-fluence PACK-CXL total ultraviolet A fluence of 7.2 J/cm2 is effective in the treatment of mycotic keratitis.
Keywords: high fluence, mycotic keratitis, photoactivated chromophore for keratitis–corneal collagen cross-linking
|How to cite this article:|
Elbassiouny RA, Ghaith AA, Farhad H, Baddour MM, Eman S, Elmassry AA. Evaluation of the efficacy of high-fluence corneal collagen cross-linking in fusarium corneal ulcer in rabbits. J Egypt Ophthalmol Soc 2022;115:43-8
|How to cite this URL:|
Elbassiouny RA, Ghaith AA, Farhad H, Baddour MM, Eman S, Elmassry AA. Evaluation of the efficacy of high-fluence corneal collagen cross-linking in fusarium corneal ulcer in rabbits. J Egypt Ophthalmol Soc [serial online] 2022 [cited 2022 Sep 28];115:43-8. Available from: http://www.jeos.eg.net/text.asp?2022/115/2/43/350258
| Introduction|| |
Corneal collagen cross-linking (CXL) leads to an increase in corneal stiffness and promotes corneal stromal mechanical and biological stability. Those mechanisms were proposed to decrease invasion of microorganisms and minimize corneal melting. Therefore and since 2008, CXL was intensely studied in the management of all types of microbial keratitis .
Clinical studies reported varied outcomes regarding the efficacy of photoactivated chromophore for keratitis–CXL (PACK-CXL). Several studies reported PACK-CXL to have a good outcome in the management of bacterial and fungal keratitis. In vitro, in vivo, and clinical studies have confirmed that PACK-CXL has beneficial antimicrobial efficacy, especially for moderate ulcers. It accelerates healing, speeds up epithelization, and decreases the risk of perforation ,,.
Studies claim that the accelerated PACK-CXL procedure is as safe and efficient as the conventional infectious keratitis-treatment procedure. On the other hand, no study currently evaluated the efficacy of high-fluence PACK-CXL on fungal keratitis .
The aim of the current study is to evaluate the antifungal efficacy of high-fluence CXL in the treatment of induced fusarium corneal ulcer in rabbits.
| Materials and methods|| |
This is an interventional experimental study that was conducted in Alexandria University, animal lab in collaboration with Ophthalmology, Microbiology, and Pathology departments, Alexandria Main University Hospitals (AMUH). The study was conducted in accordance with the ethical committee, Alexandria University guidelines on March 20, 2020.
The study included 16 eyes of eight adult-male New Zealand albino rabbits weighing 2.5–3 kg. All rabbits were of the same age. Both eyes of all rabbits were included in the study.
The rabbits were divided into two groups: group A (control group) includes eight eyes of four rabbits with fusarium keratitis and received no treatment. Groups B (PACK-CXL-treated group) with fusarium keratitis received riboflavin 0.1% drops every 5 min for 30 min, then high-fluence ultraviolet A (UVA) (30 mW/cm2) for 4 min with total UVA fluence of 7.2 J/cm2.
Induction of mycotic keratitis
On day 0, mycotic keratitis was induced in all eyes using 0.1 ml of prepared fusarium suspension in a concentration of 105 colony-forming units (CFU/ml) injected intrastromal into the cornea of both eyes of all rabbits. The fusarium suspension was prepared in the microbiology lab of Alexandria University Hospital and acquired from mycotic keratitis patient’s culture.
The procedures of intrastromal injection of fusarium were performed in the animal lab, Alexandria University, under general anesthesia. Topical anesthesia, Oxybuprocaine HCL 0.4% (Benox; EIPICO Pharmaceuticals, Nasr city, Cairo, Egypt) was also used to decrease corneal sensations. We then injected the corneal stroma of all eyes with 0.1 ml of prepared fusarium suspension after removal of the central 5 mm of the corneal epithelium. We then performed three oblique deep stromal scratches using bent-tipped needle.
On day 4, signs of fungal keratitis were evident clinically with ciliary injection, corneal ulceration, and discharge, hypopyon was also present in most eyes. We confirmed the diagnosis of the ulcer with microscopic examination of the corneal specimen seen on a glass slide with KOH 20% and examined under the microscope that revealed fungal hyphae and spores.
Group A received no treatment and was kept as a control group to detect the natural history of the disease. Group B received a single high-fluence PACK-CXL session that was performed under general and topical anesthesia. The procedures were performed in the animal lab, Alexandria University, under completely aseptic conditions.
After removal of the corneal epithelium and scraping of the necrotic tissues with a hockey knife, riboflavin 0.1% drops were instilled and spread over the affected area for 30 min (one drop every 4–5 min). High-fluence UVA irradiation 30 mW/cm2 was then focused over the corneal surface using Peschke CXL system (CCL-Vario Crosslinking; Peschke Meditrade GmbH, Zurich, Switzerland). We extend the exposure time to 4 min to get a total UVA fluence of 7.2 J/cm2.
All eyes were followed up twice daily for 6 days. The eyes were examined and grades of clinical signs recorded (ciliary injection, corneal infiltration, corneal vascularization, and hypopyon). Any complications observed were also assessed and recorded.
On day 10, all rabbits were sacrificed, considering euthanasia. The cornea of seven rabbits in each group was removed from the limbus and divided into two halves. One half was cultured on blood agar and the other half was cultured on sabouraud dextrose agar plates. Plates were followed on microbiology laboratory and fungal growth was assessed and colony counts were recorded.
On day 10, one eye from each group (A2, B2) was enucleated and assessed in the Pathology Department, Alexandria University. The eyes were processed into paraffin blocks after sagittal dissection. Four-μm-thick sections were fixed on glass slides. Hematoxylin and eosin and periodic acid–Schiff stains were used for staining of the specimens. The degree of corneal infiltration, edema, and vascularization was then assessed and recorded. The presence of fungal hyphae was also reported.
| Results|| |
There is evident and statistically significant improvement of the mean clinical sign score in the group treated with high-fluence PACK-CXL, while in comparison with the control group, the score was increasing that indicates clinical deterioration ([Figure 1]). A clinically significant difference between both groups was evident starting from day 7 forward ([Table 1]).
|Figure 1 Comparison between the different studied times according to average grade for recorded clinical signs for the two evaluated groups.|
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|Table 1 Representing comparison between the two studied groups according to the score of clinical signs on day 4 and day 10|
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There was also a decrease in the average CFU in the high-fluence PACK-CXL-treated group in relation to the control group ([Table 2]).
|Table 2 Representing comparison between the two studied groups according to colony-forming units /ml|
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Regarding histopathological examination, group A ([Figure 2]a and b) showed ulceration of surface epithelium. Dense stromal inflammatory infiltrate was seen affecting near-full thickness of the cornea. The infiltrate composed mainly of neutrophils admixed with lymphocytes, plasma cells, and eosinophils. Stromal edema and collagen destruction were seen along all corneal thicknesses. Fungal hyphae were detected below the surface epithelium and extend downward to the stroma. Corneal neovascularization was also seen. On the other hand, group B ([Figure 3]a and b) showed surface epithelial hyperplasia. Inflammatory cells were detected only in the anterior part of corneal stroma. Mild edema was detected in corneal stroma.
|Figure 2 Histopathologic examination of the control group: (a) H&E stain, ×200. (b) PAS stain, ×200. Ep, epithelium; Stroma, corneal stroma. Red arrow=neovascularization. Black arrow=inflammatory cells. *=edema. Arrowhead=fungal hyphae. Magnification ×200, scale bar 100 mm. H&E, hematoxylin and eosin; PAS, periodic acid–Schiff.|
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|Figure 3 Histopathologic examination of the high-fluence PACK-CXL-treated group: (a) H&E stain, ×200. (b) PAS stain, ×200. Ep, epithelium; Stroma, corneal stroma. Red arrow=neovascularization. Black arrow=inflammatory cells. *=edema. Arrowhead=fungal hyphae. Magnification ×200, scale bar 100 mm. H&E, hematoxylin and eosin; PACK-CXL, photoactivated chromophore for keratitis–corneal collagen cross-linking; PAS, periodic acid–Schiff.|
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| Discussion|| |
Mycotic keratitis is a common corneal infection that carries a high risk of corneal necrosis, perforation, and spread of the infection into intraocular structures, leading to endophthalmitis. The healing of the ulcer if not treated early is associated with dense corneal scarring and vascularization .
In this study, the antimicrobial efficacy of high-fluence-accelerated CXL was evaluated in the treatment of induced mycotic keratitis in rabbits’ eye. We investigated the clinical, microbiological, and pathological signs of infection in comparison with a control group that received no treatment.
In the present study, we found that PACK-CXL alone can effectively suppress fungal infection and leads to improvement in the clinical manifestations of fungal keratitis. Significant clinical improvement can be seen while comparing the PACK-CXL treatment group (group B) with the control group (group A). Also, microbiological evaluation detected a significant decrease in fungal CFU/ml for PACK-CXL-treated group in comparison with the control group. In histopathological examination, PACK-CXL-treatment group shows less inflammatory signs and lower hyphae density than the control group.
Spoerl et al.  reported decreased risk for corneal melting and perforation due to change in the structure of collagen fibers as a result of CXL increasing corneal resistance and reducing the effect of the activated enzymes on the corneal collagen. Decreased inflammatory cell infiltration was explained by the direct cytotoxic effect of CXL on inflammatory cells besides damage of the fungal RNA, leading to elimination of the organisms that elaborates inflammatory mediators responsible for inflammatory cell infiltration .
Wei et el.  in their study for the evaluation of PACK-CXL as adjuvant therapy for mycotic keratitis found that CXL reduced the need for medications and decreased the need for surgical intervention in fungal keratitis cases. They also noticed accelerated corneal healing and decrease in the duration of treatment of their cases.
Uddaraju et al.  in their study found that fungal keratitis with deep corneal ulcer did not respond to PACK-CXL. Corneal ulcer may continue to progress even after treatment and may result in corneal perforation. They also noted that better results with scarring and healing were associated with fusarium species, while cases caused by Aspergillus showed poor healing.Most of the studies  in the literature have used the conventional CXL Dresden protocol (3 mW/cm2 for 30 min, total fluence 5.4 mJ/cm2). Few reports described successful use of accelerated CXL protocol (9 mW/cm2 for 10 min, total fluence 5.4 mJ/cm2) . No studies in the literature evaluated higher total fluence of UVA 7.2 J/cm2 in fungal keratitis.
Kling et al.  in their study tested the efficacy of PACK-CXL with high fluence (J/cm2) of UVA on different bacterial strains. They used two fluences of UVA 5.4, 7.2 mW/cm2 (18 mW/cm2 for 5 min and 6 min 40 s, respectively). They concluded that increasing UVA fluence leads to two-fold increase in the killing effect of CXL (from 50 to 100% bacterial killing) in low-bacterial concentration. However, they did not study the side effects of increasing UVA fluence, especially on corneal endothelium and crystalline lens in their in vitro study.
Further studies were performed to evaluate higher UVA fluence’s safety and efficacy in fungal keratitis and to monitor the possible long-term complications.
| Conclusion|| |
PACK-CXL in high-fluence level 30 mW/cm2 for 4 min (total UVA fluence of 7.2 J/cm2) is effective in the treatment of mycotic keratitis. It decreases fungal load and hastens the signs of inflammation, accelerates healing, and decreases the risk of complications.
Financial support and sponsorship
Conflicts of interest
There are no conflits of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]