Abstract: In this review, recent studies regarding riboflavin-ultraviolet A (UVA) collagen cross-linking for the treatment of acanthamoeba keratitis (AK) were reviewed. English written studies about acanthamoeba, keratitis, riboflavin and collagen cross-linking were retrieved from PubMed search engine (
First reported in the U.S. and the U.K. in 1973 acanthamoeba keratitis (AK) is among the most intractable infectious diseases of the human cornea (1,2). The incidence of AK is widely variable and ranges from 0.15 per million in United States to 1.4 per million in United Kingdom (2). Contact lens use, trauma and exposure to contaminated water are the most common risk factors for AK (2). More than 85% of AK occurs in contact lens users, especially in countries where the prevalence of contact lens wear is high (2).
In general, early diagnosis and prompt delivery of effective antimicrobial agents is a key for successful treatment of infectious keratitis. Typically, AK is unilateral and progresses slowly. Therefore, the diagnosis of AK is usually delayed, unless there is keen awareness among clinicians (3,4). During the early phase of AK, the clinical feature is similar to other mild corneal diseases such as punctate epitheliopathy or pseudodendrites mimicking herpes keratitis (1). Since the toxicity of anti-Acanthamoeba medication is very strong, many are hesitant to begin treatment without clear diagnosis. In addition, the resistance of Acanthamoeba cysts to antimicrobial chemotherapy is another significant impediment to successful treatment (1,2,4).
Currently, the mainstay of AK treatment is combined use of two biguanides such as polyhexamethylene biguanide (PHMB) 0.02% to 0.06% and Chlorhexidine 0.02% to 0.2% (2,4). The action of biguanides is to damage the cytoplasmic membrane of Acanthamoeba and the inhibition of its respiratory enzymes (5). In European countries, commercial diamidine eyedrop [propamidine isethionate 0.1% (Brolene?)] is available for the treatment of AK. The mechanism of action of diamidines (hexamidine and propamidine) is to jeopardize DNA synthesis inside Acanthamoeba species by inhibiting S-adenosylmethionine decarboxylase (6).
Despite this regimen, there are many cases of AK treatment of failure, and novel treatment options are urgently needed (4).
Riboflavin-ultraviolet A (UVA) assisted corneal collagen cross-linking (CXL) has been widely used to enhance corneal mechanical strength in various ectatic corneal diseases such as keratoconus, pellucid marginal degeneration and progressive ectasia following photorefractive surgeries (7,8). The treatment is well established as a minimally invasive procedure to slow or halt progression of these diseases (8).
In addition to enhancing corneal mechanical strength, recent findings have revealed a novel treatment role of riboflavin-UVA CXL (9-12). Previous studies have repeatedly verified that riboflavin-UVA CXL is an effective treatment for infectious keratitis which is refractory to conventional topical antibiotic treatment. Reports of successful recovery from refractory fungal and amoebic keratitis after riboflavin-UVA CXL have been reported (11,13). Riboflavin-UVA CXL for the management of corneal infection is now known as PACK-CXL (photoactivated chromophore for keratitis-CXL) (14,15).
In this review, in vivo and in vitro evidence regarding the role of CXL in the treatment of AK is examined and summarized. In addition, a future direction of investigation is proposed.
Riboflavin-UVA CXL was first introduced by researchers at the Technical University of Dresden, Germany in the early 1990s. Through a series of photochemical reactions such as photosensitization, photo-oxidation, and photopolymerization, UVA is thought to activates riboflavin and generate singlet oxygen, which then facilitates cross-linking between adjacent collagen fibrils in corneal stroma via various pathways such as imidazolone production, triggering of endogenous populations of carbonyl groups, and releasing free radicals from riboflavin (7). In detail, the underlying photochemical processes consist of both aerobic (Type II photochemical kinetic mechanism) and anaerobic (Type I photochemical kinetic mechanism) phases. The role of riboflavin is to act as a photosensitizer. It is activated by UVA to an excited singlet or reactive triplet state and interacts with triplet oxygen species in the atmosphere to generate active singlet oxygen (aerobic phase). Active singlet oxygen induces the crosslink between carbonyl groups of collagens. In the anaerobic phase, the oxygen depletion induces triplet riboflavin to form riboflavin free radicals such as 2,3-butanedione which interacts with corneal stroma and induces crosslinks between collagen molecules. Because the depth of penetration of UVA is limited, the typical increase in the diameter of type I collagen fibers is mainly confined to the anterior stromal layer.
CXL improves corneal resistance to proteolytic enzymes secreted by the microorganisms. The stiffening effect of CXL on the corneal stroma increasing the resistance of corneal tissue to enzymatic digestion combined with the toxic action against micro-organisms offers great promise for a possible role in the treatment of microbial keratitis (7). In the majority of intractable microbial keratitis cases reported, progression of the melting process was halted within a few days of treatment and emergency keratoplasty was avoided. There seems to be a consensus forming among ophthalmologists that riboflavin-UVA CXL is a novel and effective treatment modality for multi-drug resistant bacterial keratitis (15). However, the effect of riboflavin-UVA CXL on AK is still debatable. Interestingly, in a recent survey, more than half (52%) of clinical experts responded that riboflavin-UVA CXL is not effective in AK and may even be detrimental (15).
The safety of CXL has been extensively studied. UVA can be toxic to corneal endothelium, lens and macula. However, previous study has shown that riboflavin absorption of UVA prevents damaging UVA penetration to the corneal endothelium (16). Using an irradiance of 3 mW/cm2 of UVA and 0.1% riboflavin, as much as 95% of UVA light is absorbed within the cornea before reaching the anterior chamber. This corneal UVA absorbance results in a 20-fold reduction of the original irradiance of 3 mW/cm2 of UVA (at the corneal surface) down to 0.15 mW/cm2 (at the endothelial level), which is below 0.36 mW/cm2, the threshold considered cytotoxic for the endothelium (17,18).
There have been several treatment protocols developed for riboflavin-UVA CXL. The conventional Dresden protocol for CXL uses irradiance of either 3 mW/cm2 or 5.4 J/cm2 of 370 nm of UVA for 30 minutes after 30 minutes of instillation of 0.1% riboflavin in 20% dextran solution (19)]. The riboflavin solution is applied to the corneal surface every 3–5 minutes during UVA irradiation. As an alternative treatment, several accelerated CXL protocols were suggested based on the Bunsen-Roscoe law of photochemical reciprocity where the same photochemical effect has to be maintained while applying a higher irradiation dose for a shorter period of time (20). Most accelerated protocols treat the cornea using 9 to 30 mW/cm2 of UVA irradiation for 3 to 10 minutes. For example, current accelerated protocols use irradiance of 9 mW/cm2 for 10 minutes, 18mW/cm2 for 5 minutes and 30mW/cm2 for 3 minutes in order to achieve a similar effect as Dresden protocol (20).
In vitro studies have shown that riboflavin and UVA (3 mW/cm2 for 30 or 60 minutes) does not eradicate Acanthamoeba strains (21-23). Kashiwabuchi et al. observed no antitrophozoite (clinically isolated Acanthamoeba species) effect of riboflavin and UVA combination treatment in vitro and no improvement of clinical signs in AK animal model (Chinese hamster models) (22). del Buey et al. cultured two strains of Acanthamoeba (Acanthamoeba species 65 and 7376) on non-nutrient agar plates covered with Escherichia coli as a food source (21). They applied combined riboflavin and UVA treatment (5.4 and 10.8 J/cm2, 370 nm, 30 or 60 minutes) to the central Acanthamoeba inoculation zone and measured the viability zone. They found live trophozoites and cysts were observed more than 5 mm away from the treatment center even at 24 hours after treatment, indicating no amoebicidal effect of the treatment. Berra et al. created AK in rabbit eyes and applied riboflavin-UVA CXL (3 mW/cm2 for 30 minutes) for the treatment (23). They found that rabbits treated with CXL showed no significant clinical difference compared to the no-treatment control rabbits. Furthermore, the protozoa count was even higher in the CXL group than in the control group.
Although riboflavin and UVA treatment failed to show amoebacidal effect in previous studies, there was another report that another cross-linking photosensitizer, rose bengal, showed promising effect when green light irradiation was used. Atalay et al. applied rose bengal solution (0.1% or 0.2%) to culture wells containing Acanthamoeba with subsequent irradiation with green light (523 nm, 5.4 J/cm2). Their interesting finding was that rose Bengal-green light CXL eradicated 66% of Acanthamoeba trophozoites. The control wells with riboflavin-UVA CXL showed no significant amoebicidal effect (24).
Regarding bacterial and fungal keratitis, many promising reports regarding Riboflavin-UVA CXL effect have been published. In a case series reported by Shetty et al., CXL controlled non-resolving microbial keratitis treated previously with conventional antimicrobial agents (10). Six of nine bacterial and three of six fungal keratitis patients were fully resolved 4 to 6 weeks after CXL treatment. The time required for epithelial healing in these cases were 14 to 28 days. They also reported the resolution of pain on the first postoperative day of CXL due to the depletion of the corneal subepithelial nerve plexus. Of interest, sometimes, hypopyon increased after CXL and took longer periods of time for resolution than epithelial or stromal healing. Said et al. studied the effect of riboflavin-UVA CXL in advanced infectious keratitis (25). They divided 40 eyes of advanced infectious keratitis into two groups: medical treatment only vs. riboflavin-UVA CXL plus medical treatment. Although the time to corneal healing was not significantly shortened with CXL, the incidence of complications such as corneal perforation and recurrent infection was lowered with riboflavin-UVA CXL.
Although there are many reports on the efficacy of riboflavin-UVA CXL on bacterial keratitis, there have been only anecdotal reports about the effect of riboflavin-UVA CXL in the treatment of AK. In many reports, riboflavin-UVA CXL was used as adjuvant treatment in intractable AK treated with conventional topical biguanides. However, topical treatment is almost always continued after riboflavin-UVA CXL. Therefore it is difficult to isolate the effect of CXL. For clear demonstration of riboflavin-UVA CXL, comparison between groups with or without concomitant use of anti-acanthamoeba medication is necessary. Furthermore, bacterial superinfection is not uncommon in AK and should be suspected when the clinical response to anti-acanthamoeba treatment is limited or symptoms worsen (26).
The clinical effect of riboflavin-UVA CXL in AK was first reported by Ehlers et al. in 2009 (27). They applied riboflavin-UVA CXL in 3 eyes and 2 eyes completed the healing of corneal ulcers. Subsequently, Mor?n et al. reported l case of successful treatment of AK by riboflavin-UVA CXL in 2010 (28). Although they did not confirm acanthamoeba infection either by culture or by histology, the clinical features of their case suggested AK. The clinical features worsened even with topical PHMB, and so they applied riboflavin-UVA CXL (5.4 J/cm2). After the treatment, keratitis resolved slowly over 2 months. Shortly after Mor?n et al.’s first case report, Khan et al. reported 3 cases of successful treatment of AK using CXL (13). After that, Chan et al. reported 1 case of successful treatment (29).
Price et al. applied 365 nm UVA light (3 mW/cm2) for 15 to 45 minutes in 40 infectious keratitis patients (11). The causative organisms were bacteria (24 eyes), fungal (7 eyes), acanthamoeba (2 eyes), virus (1 eye) and no identified organism (6 eyes). Six cases (2 bacterial, 3 fungal, and 1 without growth) failed to resolve even after riboflavin-UVA CXL and received penetrating keratoplasty. However, 2 cases of AK resolved after riboflavin-UVA CXL.
Hager et al. (30) raised a serious question about the amoebicidal effect of riboflavin-UVA CXL. They performed histological examination of corneal buttons obtained after penetrating keratoplasty due to intractable AK (30). These corneas had undergone riboflavin-UVA CXL (2 eyes with 5.4 J/cm2 and 5 eyes with 32.4 J/cm2) during the anti-acanthamoeba treatment before penetrating keratoplasty. The surprising finding was that acanthamoeba cysts and trophozoites persisted in the corneal tissue following riboflavin-UVA CXL in six out of seven corneal buttons.
Table 1 summarizes the case reports regarding riboflavin-UVA CXL in AK patients. It is noteworthy that most cases of AK eventually resolved with the treatment except Hager et al.’s cases.
| Year | Authors | Case number | Healed | Treatment protocol | Clinical outcomes | Complications | Note |
|---|---|---|---|---|---|---|---|
| 2009 | Ehlers |
3 | 2 | 5.4 J/cm2 | 2 eyes completed healing; 1 eye underwent enucleation | – | – |
| 2010 | Mor?n |
1 | 1 | 5.4 J/cm2 | Complete epithelialization in 32 days | – | Clinically suspected AK case |
| 2011 | Khan |
3 | 3 | 5.4 J/cm2 | Complete epithelialization in 2 months | Two needed PK due to dense central corneal scar | repeated CXL at 1 or 2 weeks interval |
| 2011 | Gardu?o-Vieyra |
1 | 1 | 5.4 J/cm2 | Clinical symptom improved 24h after CXL; disease was controlled at 3weeks after CXL | No | Culture positive AK |
| 2012 | Price |
2 | 2 | 5.4 J/cm2 | Complete epithelialization in 54 in one eye and 145 days in the other eye | No | repeated CXL |
| 2012 | Panda |
1 | 1 | 5.4 J/cm2 | Complete epithelialization in 8 days; complete resolution in 4 weeks | No | Culture positive AK |
| 2012 | Müller |
1 | 1 | 5.4 J/cm2 | – | – | – |
| 2013 | Rosseta at al. ( |
1 | 1 | 5.4 J/cm2 | Complete epithelialization in 10 days | – | Culture positive AK |
| 2013 | Demirci |
1 | 1 | 5.4 J/cm2 | Complete epithelialization in 10 days | No | Confocal microscopy |
| 2014 | Arance-Gil |
1 | 1 | 5.4 J/cm2 | Anti-acanthamoeba medication stopped at 3 months after CXL; amniotic membrane transplantation for persistent epithelial defect at 6 months after CXL; penetrating keratoplasty at 8 months after CXL | Glaucoma and cataract | 1 year of topical anti-acanthamoeba treatment with no improvement before CXL |
| 2014 | Said |
1 | 1 | 5.4 J/cm2 | Complete epithelialization in 26 days | – | Faster epithelial healing compared to medication only control eyes |
| 2014 | Chan |
1 | 1 | 5.4 J/cm2 | Significant symptomatic improvement and epithelial healing at 1 week | – | Confocal microscopy |
| 2016 | Hager |
7 | 0 | 5 eyes: 32.4 J/cm2; 2 eyes: 5.4 J/cm2 | Proceed to PK due to uncontrolled infection | – | Persistence of acanthamoeba cysts in 6 corneal buttons |
It is noteworthy that there have been continuous anecdotal reports of effective treatment of AK with riboflavin-UVA CXL. However, it is hard to find any in vitro and in vivo animal studies that clearly demonstrate amoebicidal or cysticidal effects of riboflavin-UVA CXL. Human eyes mount an active immune reaction to AK that is hard to reproduce in a laboratory environment or in animal models. Differences in the acanthamoeba burden in human disease and experimental setting can also contribute to this discrepancy.
Given the severity and intractability of AK, it is very important to establish an effective and systematic treatment protocol through the discovery of any possible treatments candidates and the demonstration of their effects. Therefore, it is necessary to conduct a prospective case-control study, ideally using three groups: topical anti-acanthamoeba medication only, riboflavin-UVA CXL only, and combined anti-acanthamoeba medication and riboflavin-UVA CXL.
