Collagen Crosslinking for Infectious Keratitis

Key Concepts

Corneal crosslinking (CXL) could be considered an adjunct treatment in infectious keratitis.
CXL should be considered in cases of severe unresponsive infectious keratitis.
Indirect benefits of CXL, such as corneal melting improvement, may help clinical and visual outcomes of treatments for corneal infections.
CXL potentially shortens corneal reepithelization time and reduces pain symptoms.
Although published studies cover mostly ultraviolet-A (UVA)-light/riboflavin-mediated CXL, green light/rose Bengal-mediated CXL has been lately employed with promising results.

Corneal infections due to bacteria, fungi, and Acanthamoeba are an important cause of vision loss. Difficulties in treatment can be related to microbial resistance and drug penetration among other factors. Over the last 35 years, no major classes of antibiotics and antifungals have been developed. A recent study isolated resistant bacteria in up to 50% of cases in samples from patients chronically using fourth-generation fluoroquinolones. Also, during the global report of the World Health Organization, antimicrobial resistance has already been placed as an urgent issue. In this scenario, treatment alternatives to antimicrobial use are most welcome.

Corneal crosslinking (CXL) has been safely and effectively used for progressive forms of keratoconus and other forms of ectasia for approximately 15 years. CXL with photoactivated chromophore had been tested for treatment of infectious keratitis and named as photoactivated chromophore for keratitis (PACK)-CXL. It is considered a new modality in the therapeutic arsenal for the treatment of corneal infections. ^, The combination of ultraviolet-A light (UVA) and riboflavin have already been used for disinfection purposes, such as in blood banks and for drinking water purification.

There are three hypotheses for potential mechanisms in which the PACK-CXL procedure would reduce pathogen infectivity and improve corneal infiltrates or ulcers:

Oxidative stress would cause disruption in cell membranes;
Direct damage to microbial nucleic acid and proteins, which would stop the replication;
Steric hindrance would bring increased resistance to digestion.

Bacterial Susceptibility

In Vitro Experimental Assays

Martins et al. reported the in vitro effects of riboflavin and UVA on susceptible and resistant bacteria: oxacillin-resistant Staphylococcus epidermidis , penicillin-resistant Streptococcus pneumoniae , pan-resistant Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus ; oxacillin-susceptible S. epidermidis, S. aureus, and P. aeruginosa . Although the use of UVA alone promoted significant inhibition of in vitro bacterium growth, the combination of UVA and riboflavin were more effective against all bacteria. The cytotoxic effect of this combination has also been shown to be superior, with a 10-fold increase in the effect in relation to the use of UVA only.

Makdoumi et al. found that 60 minutes (10.8 J/cm ² ) of UVA exposure achieved higher eradication of bacteria in vitro compared with 30 minutes (5.4 J/cm ² ). On the other hand, Kashiwabuchi et al. did not observe antimicrobial activity against oxacillin susceptible S. aureus using total energy of 5.4 J/cm ² . Whereas more studies are required to determine clinical safety, unpublished data from the Laboratory of Ocular Cell Biology of the University of Zurich, Switzerland, suggest that the currently applied riboflavin-mediated PACK-CXL treatment protocols do not achieve the maximal possible bacterial killing rate efficacy and could be further improved by using higher UVA fluence.

While further studies on rose Bengal’s safety are required for clinical use, in vitro analysis has demonstrated its efficiency in multidrug-resistant strains. Green light with 0.1% rose Bengal was more effective than 0.03% rose Bengal and 0.1% riboflavin with UVA for growth inhibition of two different methicillin-resistant S. aureus strains.

Fungal Susceptibility

Candida albicans

Studies show that riboflavin PACK-CXL decreases C. albicans diameter. This effect could be explained by a reduction of catalase activity and an impairment of antioxidant activity and growth regulation. Other reasons may include a partial or complete inactivation of genes due to oxidative stress and a reduction of β-1,3-glucan synthesis.

Fusarium solani

Morphologic and phenotypic changes observed with Fusarium solani suggest that it is more susceptible to riboflavin than C. albicans. The main hypothesis is related to CXL induction of senescence in filamentous cells, which may reduce diameter size and compromise fungal growth. Both CXL and isolated exposure to riboflavin may have an inhibitory effect on Fusarium growth.

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