Abstract
PURPOSE:
To evaluate the effectiveness of ultraviolet (UV)-A/Riboflavin corneal cross-linking (CXL) for the treatment of the refractory cases of fungal keratitis.
METHODS:
In this prospective interventional study, 9 patients with the diagnosis of fungal keratitis that were referred to our emergency eye center were included. These patients were resistant to conventional treatment and underwent therapeutic UV-A/Riboflavin CXL. Response to the treatment was considered as good if rapid epithelialization and rapid decrease in stromal infiltration was occurred after PACK-CXL, and poor when the emergency transplantation was necessary to eradicate the infection.
RESULTS:
Nine patients treated with CXL due to recalcitrant fungal keratitis. Culture of the corneal scrapings showed Aspergillus species in 4 patients, Candida albicans in 1 patient and Fusarium species in the remainder of them. CXL was performed from 1 to 20 days after the presentation of corneal ulcers (Mean: 9.12 ± 4.02; range: 5–20 days). Postoperatively, the mean time to epithelialization was 14.25 ± 2.38 days, and mean time to resolution of stromal infiltration was 22.5 ± 7.29 days, in responsive cases. Four out of 9 eyes showed good response, and five patients showed no response, and corneal transplantation was performed to eradicate the infection. There was no statistically significant difference in mean depth of infiltration and mean size of ulcer between responsive and unresponsive patients (P = 0.86 and 0.08, respectively).
CONCLUSION:
Although UV-A/Riboflavin CXL is not a definite treatment for all of the fungal keratitis, it seems promising in the management of some refractory cases.
Keywords: Corneal cross-linking, fungal, keratitis, ultraviolet-A/riboflavin
Introduction
Fungal keratitis is a significant cause of the corneal ulcer, especially in tropical climates and one of the leading cause of monocular blindness in these regions.[1,2] Contact lens usage, previous ocular trauma, or surgeries are counted out as predisposing factors.[3] The species of fungal infection have been found to be different in relation to the predisposing factors. While candida is considered as a predominant fungus in tropical climates, Fusarium keratitis has been highlighted in relation to contact lenses.[4] The variety of organisms makes growing the idea of specifying treatment. Although it is easier recently to detect the susceptibility of organisms, the choice of antifungal therapy remains as a challenging subject.
Fungal ulcers tend to have a poor prognosis and are believed to be more difficult to treat than bacterial corneal ulcers, and most of the fungal keratitis downhill toward thinning and corneal melting even in a situation of proper diagnosis and specifying susceptibility of the organisms.[5,6] Treatment of fungal keratitis with systemic and topical antifungal agents has been only partially successful, and there has been an alarming resistance to antifungal agents. In addition, the choice of antifungal therapy remains a challenging subject, and the lack of definitive data on the efficacy of antifungals may lead clinicians to use multiple therapies in the hope of maximizing potential benefit, and this may result in medication toxicity. Furthermore, there is a significant variation in the management of fungal corneal ulcers.[5,6] Hence, there is a continuing quest for an effective treatment for fungal corneal ulcers, especially in recalcitrant cases that provide rapid and complete resolution of infection with minimal toxic effects and less susceptibility to mechanisms of resistance.
In addition to keratoconus and other ecstatic disorders of cornea, riboflavin/ultraviolet A (UVA) corneal cross-linking (CXL) has been used empirically for the treatment of other different disorders of the cornea such as corneal edema, bullous keratopathies, Fuchs dystrophy, nonhealing corneal ulcers, corneal erosive disorders, moderate bacterial corneal ulcers, and infectious melting keratitis.[7,8,9,10,11,12,13,14,15,16,17]
The combination of UV light/riboflavin and the radicals induced during cross-linking may synergistically play against microorganisms to protect the cornea. In addition, previous studies have found that CXL with Riboflavin/UVA increases resistance of cornea against enzymatic digestion with enzymes.[18,19,20] In this survey, we are going to report the results of CXL with Riboflavin/UVA in patients with isolated fungal keratitis with no concurrent bacterial infection, who were resistant to conventional treatment. In the present study, we also determined the type of pathogenic fungus and evaluated the effectiveness of CXL by Riboflavin UVA in the treatment of this entity.
Methods
In this case series study, nine patients with the diagnosis of refractory fungal keratitis who were referred to Khalili hospital eye emergency room, affiliated to “Shiraz” University of Medical Sciences were included. These patients were resistant to conventional treatment and underwent therapeutic CXL. Patients with perforated corneal ulcer, those with a corneal ulcer that had produced a descematocele, patients with collagen vascular disease, and immunocompromised patients were excluded from the study. This study was performed in accordance with the Declaration of Helsinki and approved by our institution's ethics committee. Written informed consent was obtained from all subjects before inclusion. All patients underwent complete ophthalmologic examination, including corrected visual acuities and slit-lamp biomicroscopy. Corneal ulcers were graded according to their location, size, depth, and severity of anterior segment infiltration. Grade 1 ulcers were non-axial, <2 mm in size, involved superficial one-third of the cornea with mild anterior segment reaction. Ulcers were graded as 2 if their area was between 2 and 6 mm, involved superficial two-third of the cornea and had moderate to severe fibrinous exudates in the anterior chamber. Grade 3 ulcers were more than 6 mm in size, extending to the inner one-third of the cornea with severe hypopyon. As a routine approach, smear and culture were prepared from the corneal lesions. Then, patients were received standard medical treatment of empirical therapy. Further changes in medications were done according to laboratory results. Subsequently, daily evaluation of corneal ulcer was done. If the ulcer was resistant to conventional treatment and there was a progression to corneal melting and thinning, CXL was performed. After treatment with CXL, the patients received preoperative medical therapies and were examined daily to evaluate their response to treatment. The time to re-epithelialization and time to resolution of stromal infiltrate were recorded. Response to the treatment was considered as good if rapid epithelialization and rapid decrease in stromal infiltration (significant change in the course of the disease) occurred after CXL. If there was no change in the course of healing, if the ulcer deteriorated after treatment and if surgical treatment was necessary for eradication of infection despite enough time after CXL, response of the patient was defined as poor (no response).
The data were presented as mean ± standard deviation. The statistical analyses of differences between responsive and unresponsive groups were performed using the t-test. P < 0.05 was considered as significant. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) software, version 19 (IBM, USA).
Treatment
With the patient in the supine position, the cornea was anesthetized with topical Tetracaine 0.1% drops every 3 min for at least 15 min. After inserting a lid speculum, corneal epithelium within a 8 mm-diameter zone that included all microbial infiltrates were removed by means of thin blunt metal (Hockey knife) and then 0.1% Riboflavin in Dextran500 20% drops (MEDIOCROSSTM Medio Haus edizinprodukte GmbH, Behrensbrook 6, D-24214 neudorf) were administered every 3 min for 30 min. Thereafter, the cornea was exposed to UV-A rays (365 nm) in an optical zone of 8 mm for 30 min with an irradiance of 3 mW/cm2 (UV-XTM; Peschke Meditrade, Cham, Switzerland).
Light distance from the corneal apex was about 50 mm. During the procedure, the cornea was moistened every 5 min with 0.1% Riboflavin and Tetracaine drops. After the CXL, the eye was addressed with a therapeutic soft contact lens (T-lens) and artificial tear and previous topical medications.
Results
A total of nine patients with fungal keratitis were treated with CXL. All of them were male and their age was ranged from 29 to 69 years. Seven out of 9 patients did not have any predisposing factor. Accordingly, one patient had a history of trauma with plants and one of them had been received a previous corneal graft due to keratoconus. One patient was a farmer, but he denied any previous trauma with vegetative matter. Visual acuities ranged from 20/200 to light perception. Corneal ulcer was graded as 2 in 2 and as 3 in 7 out of 9 patients.
Culture of corneal scrapings demonstrated heavy growth of Aspergillus species in 4 out of 9 patients, candida species in one patient, and Fusarium in the remainder of them. CXL was performed from 1 to 20 days after admission in our center. The patient who underwent CXL 1 day after his admission, had received conventional treatment at another center and had been referred to our center due to progression to corneal melting and thinning. Postoperatively, mean time to epithelialization was 14.25 ± 2.38 (range 738 days) and mean time to resolution of stromal infiltration was 22.5 ± 7.29 (range 7–38 days), in responsive cases. Four out of 9 eyes showed good response after treatment and infection was completely halted and scared down. Five out of 9 patients showed no response, and therapeutic penetrating keratoplasty was performed to preserve the globe. The mean depth of infiltration was 57.50 ± 26.30% of corneal thickness in cases with response to treatment compared to 61+/32.09% in unresponsive patients. This difference was not statistically significant (P = 0.86). The mean size of ulcer as approximated by multiplying the horizontal and vertical diameters was 8 ± 1.4 and 22.55 ± 14.09 mm2 in responsive and nonresponsive patients, respectively. Statistical analysis showed no difference (P = 0.08).
In the responsive group, 2 cases were caused by Aspergillus species and another 2 were caused by Fusarium species. All cases in the nonresponsive group were graded as grade 3, with only half of the cases of the responsive group placed in the same category. Other 2 cases were graded as grade 2.
Time from admission to CXL + Riboflavin treatment was 7–20 days in the responsive group (mean 15.5 days) and 1–6 days (mean 3.5 days) in the unresponsive group. No significant statistical difference was seen.
We describe two patients in detail; a responsive and a non-responsive one.
Case 1
A 29-year-old man referred to us with an impression of recalcitrant bacterial keratitis in her right eye. The patient complained of ocular pain and red-eye, and had received fortified cefazolin and gentamycin for one week prior to referral to our hospital. Visual acuities were 2 meters counting fingers (CF) in the right eye and 20/25 in the left eye. Slit-lamp examination showed a 3 mm × 2 mm, feathery border central corneal ulcer with 70% depth stromal infiltration, 1 mm hypopyon and a 3 mm × 4 mm corneal epithelial defect. KOH smear was in favor of the presence of fungal elements and 5 days later, culture showed heavy growth of Aspergillus spp. Therefore, natamycin 5% eye drop and oral fluconazol were started; however, no acceptable response was achieved with the continuation of medication. CXL was performed 11 days later due to the deterioration of the ulcer. Postoperatively, stromal infiltration decreased gradually, and 17 days after CXL, keratitis totally became scared down. Moreover, no recurrence was occurred in about 6 months of follow-up and visual acuity was improved to 4 m CF.
Case 2
A 31-year-old man was referred because of a refractory fungal corneal ulcer of the left eye despite 20 days usage of natamycin eye drops. On arrival, visual acuities were 20/80 in the right eye and light perception in the left eye. A 6 mm × 7 mm central corneal ulcer and 5 mm hypopyon was detected. Lab exams confirmed the presence of Fusarium infection. Despite systemic fluconazole administration and topical natamycin5% (Q1 h), the corneal ulcer progressed over the next 3 days. As an alternative to immediate corneal transplantation, the patient was offered corneal CXL. Corneal ulcer was progressed. Penetrating keratoplasty was performed to eradicate the infection 10 days later due to aggressive nature. Histopathologic study of the cornea showed fungal elements associated with inflammatory cells. At 3 months of follow-up, no recurrence was occurred, and visual acuity was 3 meters CF in the left eye.
Discussion
Since 14 years ago, there is a growing clinical experience with Riboflavin/UVA CXL in the management of ectatic disorders of the cornea, especially keratoconus. Because CXL has been an effective and a relatively safe modality in the management of keratoconus, it has been used for the treatment of other different disorders of the cornea such as corneal edema, bullous keratopathies, Fuchs dystrophy, nonhealing corneal ulcers, corneal erosive disorders, and infectious melting keratitis.[7,8,9,10,11,12,13,14,15,16] The present study was conducted to evaluate the effectiveness of Riboflavin/UVA CXL in the treatment of refractory cases of fungal keratitis. According to our results, four out of nine patients that were treated for refractory fungal keratitis showed a good response. Five patients showed no response and penetrating keratoplasty was necessary to eradicate the infection in these patients.
Although the exact mechanisms underlying therapeutic effects of CXL in the treatment of fungal keratitis are unclear, several mechanisms may underlie these therapeutic effects. The direct antimicrobial effect of CXL on infectious agents has been demonstrated. This effect has been proved against a wide range of pathogens, including bacteria, viruses, fungi and parasites in platelet concentrates, fresh frozen plasma, and in red blood cells[21,22,23,24,25,26] These effects are the consequence of damage of DNA, RNA through several mechanisms such as the production of singlet oxygen and generation of hydrogen peroxide with the formation of hydroxyl radicals induced by Riboflavin/UV light.[21,22,23,24,25,26,27] Antimicrobial efficacy of riboflavin/UVA combination against the most common offending pathogens of microbial keratitis has also been shown in culture plates.[28,29,30]
Although Martins et al. demonstrated that at the riboflavin concentrations tested, combined riboflavin/UVA treatment has no effect on Candida albicans, the synergistic effects of riboflavin/UV-A with amphotericin B against fungal pathogens has been shown by Sauer et al.[28,30] They demonstrated that compared with amphotericin alone, riboflavin/UV-A combined with previous treatment with amphotericin B is more effective on three groups of fungi (Candida albicans, Fusarium sp, and Aspergillus fumigatus) selected from severe cases of keratomycosis. They also proposed that cross-linking of the cornea may facilitate diffusion of amphotericin B into the cornea.[30] More in vitro work is necessary to improve our knowledge about the direct antifungal effects of riboflavin/UV-A.
Besides the mentioned antifungal effects of CXL, an increase in biomechanical and thermal stability of the cornea induced by cross-linking process is another possible mechanism that may underlie beneficial effects of CXL in the treatment of infectious corneal ulcers, including fungal keratitis.[18,19,20] Fungi can produce proteolytic enzymes such as adherence and matrix metalloproteinase that digest human connective tissue in the cornea, inducing tissue melting.[31,32] Photochemical cross-linking of the cornea using Riboflavin and UVA results in a markedly increased resistance against enzymatic digestion.[20] Therapeutic effects of CXL in noninfectious ulcers of the cornea such as erosive corneal processes and melting ulcers of the cornea of various origins, including sterile keratitis that has been demonstrated in several studies also provide evidence that CXL may reduce a load of proteolytic enzymes in the diseased cornea.[12,13,16]
To our knowledge, there is very limited clinical experience with CXL in the management of fungal keratitis; one responsive patient reported in the study of Iseli et al. and two unresponsive patients in the study performed by Ehlers et al.[12,13] In the study conducted by Iseli et al., five patients with infectious keratitis associated with corneal melting were treated with CXL. In this study, one patient had suffered from a contact lens-induced fungal keratitis and had progressed despite intensive topical and systemic treatment. After CXL, there has been an initial improvement, but the corneal infiltrate has progressed 3 weeks after the CXL, and the patient has undergone penetrating keratoplasty 1 month after cross-linking. Because culture results have been negative, and the histologic workup has shown an infiltrate with granulocytes and macrophages without intact fungal cells, authors have stated that progressive keratitis was caused by an immune reaction without persistent active fungal disease, and this patient could have been treated with topical corticosteroids alone. Because no cases of recurrent infection occurred after cessation of specific therapy, they concluded that the CXL procedure can destroy microbes.[13] In a clinical study performed by Ehlers et al., from fourteen eyes that were treated for chronic, nonhealing corneal ulceration, two patients had fungal keratitis, and CXL was ineffective in the treatment of these two patients. According to their results, six of the 14 eyes healed after the treatment and among these were 2 eyes with marginal keratitis, 2 eyes with acanthamoeba Keratitis, one eye with diabetic corneal ulcer, and one eye with corneal ulcer after drug abuse. Three patients had some healing. These eyes were two patients with bacterial keratitis and a patient with chronic epithelial defect after lamellar grafting. In fives eyes, there was no clear effect from CXL.
These eyes were non-healing Varicella zoster virus keratitis, acanthamoeba recurrence after keratoplasty, Fusarium recurrence after keratoplasty, fungal keratitis after keratoplasty and an eye with paralytic cornea.[12]
Some studies state that CXL is an effective procedure and adjuvant therapy for managing fungal ulcers.[33] Our study exclusively includes patients with refractory fungal keratitis to evaluate the effectiveness of Collagen cross-linking by Riboflavin/UVA in the treatment of this entity. Our results clearly showed that CXL is effective in treatment of some patients with refractory fungal keratitis and this finding has clinical significance because in severe and unresponsive cases of fungal keratitis, an emergency keratoplasty is usually needed to eradicate the infection and to preserve the eye. Emergency keratoplasty has several disadvantages, such as high rates of re-infection and rejection.
CXL by Riboflavin/UVA may allow us to avoid an emergency keratoplasty and provide the opportunity to perform elective keratoplasty with a lower risk of complications.[34,35] In addition, irradiation of fungal infection by Riboflavin/UVA may be useful in the initial stages of keratitis, when the offending pathogen has not yet been identified, especially in slow-growing fungi species.
According to our results, Riboflavin/UVA was ineffective in the treatment of some patients with refractory fungal keratitis, and in two patients, the disease aggravated after treatment. Aggravation of keratitis after CXL may be a coincidental occurrence and related to the natural course of the disease, but it may also be attributed to the procedure complications. Corneal thickness was not measured in our study because of difficulties in the measurement of corneal thickness in the setting of corneal ulcer and its possible complications such as corneal perforation in a thinned cornea. In addition, the effect of UV-A light and or riboflavin on fortified antibiotics is actually unknown, and they may have toxic effects on an already diseased cornea. Super-infection of the ulcer is another possible mechanism that may underlie aggravation of the disease after treatment. Several cases of bacterial keratitis, such as Pseudomonas keratitis, Escherichia coli keratitis, and polymicrobial keratitis, have also been reported after UA/Riboflavin CXL.[36,37,38,39] Although Keratitis following CXL have been attributed to several factors such as the postoperative use of a soft bandage contact lens and topical corticosteroids, and the presence of an epithelial defect, as trials of corneal collagen cross-linking in the treatment of corneal disorders are increasing, it seems necessary to conduct studies to evaluate the possible adverse effects of the UV light on the immune mechanisms of the cornea and the corneal wound healing processes.
Although the size of the specimen is small and no definite conclusion can be drawn, comparison of responsive and nonresponsive patients showed that depth of infiltration does not influence the response to treatment.
Although the mean size of ulcer was smaller in responsive patients (8 ± 1.4 mm2) compared to non-responsive patients (22.55 ± 14.09 mm2), statistical analysis showed no difference. (P = 0.08).
Conclusion
CXL with UV-A and Riboflavin is effective in some cases of fungal keratitis and it can be added to the armamentarium of antimicrobial agents for the management of severe and refractory cases of fungal keratitis. Because both the experimental and clinical results are still preliminary and UV-A/Riboflavin may potentially aggravate the course of keratitis, we recommend its use only in refractory cases of fungal keratitis to avoid emergency keratoplasty. There is clearly a demand for further studies to evaluate the efficacy and safety of this modality as the initial therapy for patients with fungal keratitis.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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