High Fluence Increases the Antibacterial Efficacy of PACK Cross-Linking

PURPOSE: Photoactivated chromophore for keratitis cross-linking (PACK-CXL) is used as an adjunct therapy to antibiotic medication in infectious keratitis. This experimental study aimed at quantifying the PACK-CXL efficacy as a function of UV fluence using several bacterial strains and irradiated volumes. METHODS: Six distinct bacterial strains, including standardized strains and clinically isolated strains from patients with keratitis, were analyzed. Bacterial concentrations between 10 and 10 cells/mL were used (simulating small corneal ulcers). Volumes of either 11 μL (≈285 μm stromal thickness) or 40 μL (≈1000 μm stromal thickness) were irradiated within a microtiter plate at different fluences (5.4-27 J/cm) and irradiances (3, 9 and 18 mW/cm). The ratio of bacterial killing (B†) was determined to evaluate the antimicrobial efficacy of PACK-CXL. RESULTS: B† was similar (51 ± 11%) in bacterial concentrations between 10 and 10 per ml. In 11 μL volume, Staphylococcus aureus (SA) 8325-4 ATCC 29213, Bacillus subtilis (BS) 212901, and Pseudomonas aeruginosa (PA) 2016-866624 were most sensitive to PACK-CXL at 5.4 J/cm (on average B† = 49 ± 8%), whereas Klebsiella oxytoca (KO) 2016-86624 (B† = 25%) was least sensitive. When irradiating a larger volume, B† was on average lower in 40 μL (19 ± 18%), compared with 11 μL (45 ± 17%, P < 0.001). By contrast, applying a higher UV fluence increased B† of SA ATCC 29213, from 50% at 5.4 J/cm to 92% at 10.8 J/cm, to 100% at 16.2 J/cm and above. CONCLUSIONS: Applying higher UV fluences substantially increases the bacterial killing rates. Safety limits for clinical application require further investigation.