martins et al . , first reported on the antimicrobial effect of riboflavin and uva in vitro .
an inhibition of growth was seen in both drug sensitive as well as drug resistant bacteria , but no effect was observed on the growth of candida albicans .
subsequently , schrier et al . , reported the effectiveness of a combination of riboflavin and uva exposure for 30 minutes against staphylococcus aureus as well as pseudomonas aeruginosa .
makdoumi et al . , tested the effects of riboflavin and uva on bacterial suspensions in a fluid solution .
they demonstrated that exposure for 60 minutes achieved a high degree of eradication of bacteria in vitro , whereas an exposure for 30 minutes achieved only a limited eradication . in vitro and animal - model studies from multiple centers failed to show a beneficial effect of this treatment against acanthamoeba . using a rabbit model , galperin et al .
in contrast , no effect of a similar treatment was observed on fusarium solani and c. albicans isolates in an in vitro experiment performed by kashiwabuchi et al .
the modality has been studied in bacterial , fungal , viral , and acanthamoeba keratitis .
case selection appears arbitrary . a lack of homogeneity in terms of predisposing factors , clinical presentation and responsible organisms makes interpretation of results difficult .
treatment protocols range from a single 5-minute exposure of uva to multiple sessions lasting up to 45 minutes .
surgical procedures performed during active infection include keratoplasty , amniotic membrane transplantation , phototherapeutic keratectomy , flap amputation , intracorneal voriconazole injection , and enucleation .
outcome assessment is largely subjective , as a clear definition of what constitutes resolution or healing is missing in a majority of reports .
almost all results are confounded by the concurrent use of standard of care medical therapy . though these reports seem to indicate that cxl may be an option in the treatment of infection , one can not conclusively say it is effective as no control groups are available to compare against .
we have carried out in vitro experiments to test the effects of a combination of riboflavin and uva exposure on drug sensitive as well as multi - drug resistant bacteria , fungi , and acanthamoeba .
we used the standard dresden protocol including 30 minutes of soaking time with 0.1% riboflavin in dextran and a 30-minute exposure of 3 mw / cm to 370 nm uv light .
we conducted our experiments at lv prasad eye institute in four arms , control , riboflavin only , uv only , and combined riboflavin + uv . we found the group with combined riboflavin and uv exposure had the greatest efficacy in reducing growth of the exposed microbes . in our experience ,
the treatment was most effective against bacterial isolates , with drug resistant strains requiring multiple exposures .
we have not been able to demonstrate arrest the growth of fungi or acanthamoeba 
 in vitro with this treatment .
looking at the mixed published results as well as our own experimental data , we have been hesitant thus far to use cxl as a therapeutic option in cases of microbial keratitis .
we have , however , managed cases treated elsewhere , which seemed to show equivocal results .
we havent yet seen a patient with either fungal or acanthamoeba keratitis where cxl helped in resolution of the infectious process following failure of specific therapy .
we believe the next steps should be aimed at evaluating the response of the cornea with active keratitis to cxl and changes that occur over time rather than a cross - sectional observation . the ideal model for this kind of data would be an animal model of keratitis treated with cxl to observe changes in histology at various stages following the exposure .
the promise of a simple , effective , and safe alternative to anti - microbial medication or keratoplasty is somewhat of a holy grail in the management of microbial keratitis .
putative mechanisms include the genome damage resulting from intercalation of activated flavins with nucleic acids and direct free radical insult to microbial deoxyribose nucleic acid ( dna ) , in addition to the presumably increased resistance of the cornea to melting due to cxl - induced stiffening .
proof of principle exists , as demonstrated by the successful use of this technology in transfusion medicine . in vitro studies show mixed results .
the modality seems to be able to inhibit growth of bacteria , with drug - resistant strains requiring greater exposure time .
studies on fungi have conflicting results , and activity against acanthamoeba seems even less convincing .
most cases reported include ulcers that would probably heal well with adequate duration of standard of care therapy .
ethical constraints preclude the use of cxl in isolation , without first using anti - microbial drugs .
potential concerns include endothelial damage in corneas that are already thin , as well as corneal melts and reactivation of viral keratitis .
gray areas include optimum concentration of riboflavin and uva exposure duration needed to combat microbes .
based on available evidence , it is difficult to say whether cxl is effective in cases of microbial keratitis where we need it the most - drug resistant organisms , advanced keratitis and keratitis caused by organisms such as fungi and acanthamoeba , which are refractory to conventional medical therapy . an ideal study would be prospective , with an adequate sample size , well - defined inclusion and exclusion criteria , appropriate outcome measures , and unbiased assessment as well as a robust interpretation of data .
the preliminary results reported by price et al . , are part of an ongoing prospective , multi - center study .
parallels in transfusion medicine and data from laboratory experiments indicate the photochemical reaction used in cxl holds promise as a future therapeutic option for microbial keratitis .
well - designed studies investigating the safety and efficacy of this modality in appropriately chosen cases of microbial keratitis are sorely needed .
 the success of collagen cross - linking as a clinical modality to modify the clinical course in keratoconus seems to have fueled the search for alternative applications for this treatment . 
 current clinical data on its efficacy is limited and laboratory data seems to indicate that it performs poorly against resistant strains of bacteria and against slow growing organisms . 
 however , the biological plausibility of crosslinking and the lack of effective strategies in managing infections with these organisms continue to focus attention on this potential treatment . 
 well - conducted experimental and clinical studies with controls are required to answer the questions of its efficacy in future . 
Evidence from Laboratory Studies
Clinical Reports
Our Experience
Discussion
Conclusion