High fluence PACK-CXL as adjuvant treatment for advanced Acanthamoeba keratitis.

PURPOSE: To describe the outcome of adjuvant high fluence photoactivated chromophore for infectious keratitis cross-linking (PACK-CXL) used to treat an advanced form of refractory Acanthamoeba keratitis (AK) diagnosed several months after initial presentation. OBSERVATIONS: An otherwise healthy 24-year old female presented with a severe unilateral keratitis. The diagnosis eluded clinicians for several months and when finally confirmed as AK, anti-amoebic therapy was instated and only appeared to be effective after addition of high fluence PACK-CXL. CONCLUSION AND IMPORTANCE: In this case of advanced AK, high fluence PACK-CXL treatment given adjuvant to pharmacologic anti-amoebic therapy resulted in lasting pain relief, re-epithelization and eradication of the Acanthamoeba parasite. Given adjuvant to anti-amoebic pharmacotherapy, high fluence PACK-CXL might be a useful method for treating typically refractory advanced AK.

The efficiency of cross-linking methods in eradication of bacteria is influenced by the riboflavin concentration and the irradiation time of ultraviolet light.

PURPOSE: To determine bacterial eradication using numerous riboflavin concentrations and different ultraviolet light A (UVA) radiant and exposure time in an experimental model. METHODS: Dilutions of Staphylococcus epidermidis were mixed with riboflavin at varying concentrations (0.007­0.09%). Effects on bacterial growth were evaluated after 0, 3, 6, 30 and 60 min of UVA exposure (irradiance 30 and 3 mW/cm2). Standard settings of UVA were compared with high-power UVA approach. Different fluid thicknesses of the exposed dilutions were also examined to improve the model. RESULTS: Bacterial eradication (%) was increased after 60 compared with 30 min of UVA exposure for concentrations of 0.03­0.07% but not for 0.09% riboflavin.There was a significant difference between the efficacy between 0.03 and 0.09% and eradication dropped from 80%to 50%(p = 0.01).A correlation could be calculated for the amount of riboflavin at 60 min of UVA and the ability to kill bacteria(p = 0.01). The antibacterial effect was more pronounced when the tested bacterial suspension thickness was reduced. High-power UVA method was less potent in microbial elimination, eradicating only 60%of bacteria after 6 min versus 97­99%after 60 min in the low-power setting, compared with respective controls (p = 0.02). CONCLUSIONS: In these in vitro experiments, a longer UVA exposure time in combination with lower riboflavin levels were found to be favourable in killing bacteria as compared to the standard cross-linking settings. Further studies are needed to evaluate the clinical relevance of these findings.

Corneal cross-linking in 9 horses with ulcerative keratitis.

BACKGROUND: Corneal ulcers are one of the most common eye problems in the horse and can cause varying degrees of visual impairment. Secondary infection and protease activity causing melting of the corneal stroma are always concerns in patients with corneal ulcers. Corneal collagen cross-linking (CXL), induced by illumination of the corneal stroma with ultraviolet light (UVA) after instillation of riboflavin (vitamin B2) eye drops, introduces crosslinks which stabilize melting corneas, and has been used to successfully treat infectious ulcerative keratitis in human patients. Therefore we decided to study if CXL can be performed in sedated, standing horses with ulcerative keratitis with or without stromal melting. RESULTS: Nine horses, aged 1 month to 16 years (median 5 years) were treated with a combination of CXL and medical therapy. Two horses were diagnosed with mycotic, 5 with bacterial and 2 with aseptic ulcerative keratitis. A modified Dresden-protocol for CXL could readily be performed in all 9 horses after sedation. Stromal melting, diagnosed in 4 horses, stopped within 24 h. Eight of nine eyes became fluorescein negative in 13.5 days (median time; range 4-26 days) days after CXL. One horse developed a bacterial conjunctivitis the day after CXL, which was successfully treated with topical antibiotics. One horse with fungal ulcerative keratitis and severe uveitis was enucleated 4 days after treatment due to panophthalmitis. CONCLUSIONS: CXL can be performed in standing, sedated horses. We did not observe any deleterious effects attributed to riboflavin or UVA irradiation per se during the follow-up, neither in horses with infectious nor aseptic ulcerative keratitis. These data support that CXL can be performed in the standing horse, but further studies are required to compare CXL to conventional medical treatment in equine keratitis and to optimize the CXL protocol in this species.

Comparison of UVA- and UVA/riboflavin-induced growth inhibition of Acanthamoeba castellanii.

PURPOSE: To investigate whether ultraviolet light (UVA) at 365 nm can inhibit/eliminate Acanthamoeba growth and if riboflavin would potentiate such an association. METHODS: Acanthamoeba castellanii in a fluid medium with a concentration of approximately 1.7 × 10(4) protozoa/ml were prepared with (0.01 %) and without riboflavin. Exposure of UVA (dose 5.475 J/cm(2)) took place twice, with each illumination period followed by culturing of 10 µl in peptone yeast-extract glucose (PYG) medium for 7 days. Every suspension prepared had a non-exposed control solution. Determination of Acanthamoeba was conducted daily, by count in Burker chamber days 4 through 7 after exposure. Statistical analysis was done by repeated-measurement ANOVA and post-hoc analysis for unpaired samples. RESULTS: The exposure of ultraviolet light resulted in an inhibited growth of Acanthamoeba compared to the non-exposed solutions, with a statistically significant reduction over time (p = 0.0003). The addition of riboflavin did not amplify the effect, and there were no tendencies for an interaction effect between UVA and riboflavin. CONCLUSIONS: The antiprotozoal effect of the UVA wavelength, utilized in CXL, is solely mediated by ultraviolet light, and riboflavin does not seem to amplify the antimicrobial efficacy.

UVA-riboflavin photochemical therapy of bacterial keratitis: a pilot study.

BACKGROUND: The aim of this work as to investigate the photochemical interaction used in corneal crosslinking (CXL) as the primary therapy for bacterial keratitis. METHODS: A prospective non-randomized study was conducted including 16 patients with a clinical diagnosis of bacterial keratitis. No patient had any prior antibiotic treatment for the current infection. Photography and microbial culturing of the infected cornea were performed. Riboflavin was topically administered for 20 min and ultraviolet light (UVA) exposure settings for treatment of keratoconus were used. After the procedure, clinical examinations were done at least once daily until signs of improvement had been established. The frequency of examinations was thereafter reduced. Antibiotic therapy was initiated if infectious progression was suspected. The trial was registered at ISCRTN.org (no: 21432643). RESULTS: All eyes responded to the photochemical treatment with improvement in symptoms and signs of reduced inflammation. Epithelial healing was achieved in all cases. Antibiotic administration was necessary in two cases. One patient required a human amniotic membrane transplant. CONCLUSIONS: This trial illustrates that photosensitization of riboflavin using UVA at 365 nm has the potential to induce healing in patients with microbial keratitis. The results from the treatment of these 16 patients with corneal ulcers indicate that UVA-riboflavin photochemical therapy merits a controlled study in order to assess its efficacy and safety compared to antibiotics.

Infectious keratitis treated with corneal crosslinking.

PURPOSE: To describe 7 eyes with severe infectious keratitis treated using collagen crosslinking (CXL) with riboflavin. MATERIALS AND METHODS: Seven eyes of 6 patients with severe infectious keratitis were treated with corneal crosslinking. Three patients were contact lens users. Symptom duration before CXL ranged between 0 and 7 days. Corneal melting was present in all cases. Photodocumentation of the keratitis was carried out and repeated at follow-up. All but 1 patient received topical antibiotic treatment in addition to the CXL treatment. CXL was conducted according to the standardized protocol for keratoconus. RESULTS: In all but 1 eye, patients experienced improvement in symptoms within 24 hours. Two patients reported no symptoms whatsoever at this time. Corneal melting was arrested and complete epithelialization was achieved in all cases. In the 2 eyes with hypopyon, this regressed completely within 2 days after the CXL. Follow-up ranged between 1 and 6 months. DISCUSSION: Our experience based on the above and other cases suggest that CXL could be an effective tool in battling difficult cases of infectious keratitis. This treatment could present many advantages but will need further investigation.

Evaluation of antibacterial efficacy of photo-activated riboflavin using ultraviolet light (UVA).

BACKGROUND: To evaluate the antibacterial efficacy of photo-activated riboflavin using Ultraviolet A (UVA) on three bacterial strains commonly detected in keratitis. METHODS: Three bacterial strains (Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa) were cultured on blood/hematin-agar plates and dispersed in PBS. Dispersion was done of 10 microl of bacterial stock-solutions in 90 microl of RPMI, where different riboflavin molarities had been added, to achieve a bacterial concentration of 1-4 x 10 (4)/ml. Riboflavin end molarities before illumination were 0, 100, 200, 300 and 400 microM. Each solution had a negative control. The solutions were illuminated with UVA (365 nm) for 30 minutes (5.4 J/cm(2)) and then continued for a total time of 60 minutes (10.8 J/cm(2)). A count of CFU was conducted after incubation and results compared. RESULTS: In all tested strains, a slight decrease of bacteria was seen when exposed to UV for 30 minutes. A doubling of the UV dose showed a marked decrease of bacterial count in all bacteria tested. The combination of UV and riboflavin showed a more extensive reduction of CFU, confirming an interaction effect between UV and riboflavin. CONCLUSION: Riboflavin photo-activation using UVA (365 nm) can achieve an extensive eradication of bacteria, and the combination is more potent in reducing bacterial number than UV alone.

Riboflavin and ultraviolet a collagen crosslinking of the cornea for the treatment of keratitis.

PURPOSE: To describe riboflavin and ultraviolet light (UV) collagen crosslinking as an effective treatment for infectious keratitis. METHODS: A 25-year-old previously healthy female contact lens user was diagnosed with unilateral severe keratitis with unclear pathogenesis, although the clinical presentation suggested acanthamoeba as the infectious agent. A 4-mm diameter, annular, semi-opaque infiltrate was found on the paracentral parts of the cornea in the left eye (OS). Laboratory examinations for bacteria, herpes simplex, and acanthamoeba were performed, but no specific pathogen could be detected. Best corrected visual acuity (BCVA) at presentation was 20/1000. Treatment was initialized with broad-spectrum antibiotics also covering acanthamoeba. During the first month of treatment the keratitis progressed and the corneal thickness diminished. Therefore, treatment with riboflavin and UV collagen crosslinking was initiated. RESULTS: After riboflavin and UV collagen crosslinking therapy, there was a rapid decrease of pain and necrotic material. Reepithelialization of the cornea started within a few days and was complete within a month. After 2 months, the wound had healed completely. Nine months after the UV treatment, BCVA was 20/30. CONCLUSIONS: This case illustrates the positive effects of riboflavin and UV collagen crosslinking on presumed infectious keratitis with a satisfactory final visual outcome. This may be a promising new treatment for keratitis, although this remains to be elucidated in detail in future studies. Until more data are available this treatment should only be considered in therapy-refractive keratitis or ulceration and not in the first line of defence since it may have cytotoxic side effects.