CLINICAL SCIENCE

Ultraviolet A/Riboflavin Collagen Cross-Linking for Treatment of Moderate Bacterial Corneal Ulcers Shahram Bamdad, MD, Hossein Malekhosseini, MD, and Amir Khosravi, MD

Purpose: To evaluate the therapeutic effect of UV-A/riboflavin collagen cross-linking (CXL) on moderate bacterial corneal ulcers.

Methods: Thirty-two patients with moderate bacterial keratitis were selected. All patients were treated according to the standard medical treatment protocol. The patients were randomly allocated to 2 groups: case and control groups of 16 patients each using a numerical randomization table. The case group received CXL treatment. In the CLX group, corneal epithelium was removed and 0.1% riboflavin drops were applied. Then the corneas were irradiated with UV-A (365 nm) with an irradiance of 3 mW/cm2 for 30 minutes. The grade of ulcers, size of epithelial defects, and area of infiltrates were recorded on days 1, 7, and 14 of treatment. Results: There was no statistically significant difference between the groups 1 day after the treatment. The mean treatment duration was 17.2 6 4.1 days in the CXL group and 24.7 6 5.5 days in the control group. The epithelial defects were smaller in the CXL group at 7 days (P = 0.001) and 14 days (P = 0.001) after the beginning of treatment. The area of infiltrates in CXL group was smaller than the control group at both 7 days (P = 0.001) and 14 days (P , 0.001) after the start of treatment. Conclusions: Our results support the beneficial effect of CXL in patients with moderate bacterial keratitis. In addition to accelerating epithelialization, this method shortens the course of treatment and may minimize or remove the need for surgery or other serious sequelae, such as corneal perforation. Key Words: UV-A/riboflavin, collagen cross-linking, corneal ulcer, moderate bacterial keratitis (Cornea 2015;34:402–406)

B

acterial ulcerative keratitis may have a devastating impact on ocular tissue and is a sight-threatening condition. It requires skilled management and effective chemotherapy to preserve vision. Corneal ulceration leads to activation of proteolytic enzymes, which digest collagen thereby facilitating corneal melting and perforation. Some virulent bacteria Received for publication August 5, 2014; revision received December 8, 2014; accepted December 18, 2014. Published online ahead of print February 13, 2015. From the Poostchi Eye Research Center, Department of Ophthalmology, Shiraz University of Medical Sciences, Shiraz, Iran. The authors have no funding or conflicts of interest to disclose. Reprints: Amir Khosravi, MD, Poostchi Eye Research Center, Zand St, Shiraz 7134997446, Iran (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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can accelerate the corneal destruction to be completed within 24 to 48 hours.1 If appropriate antimicrobial treatment is not administered on time, it is estimated that only 50% of the eyes would heal with a good visual outcome.1 Once melting has developed, treatment options are limited and often surgery is the only therapeutic option. Treatment of corneal ulcers with topical antimicrobial agents has been confounded by the ability of microbes to develop resistance to the drugs used. Hence, there is a need for an agent which provides complete and rapid antimicrobial activity with minimum toxicity. The UV-A/riboflavin collagen cross-linking (CXL) procedure was introduced primarily by Wollensak et al2 in 2003 to stabilize progressive keratoconus by improving the biomechanical characteristics of the stroma. Riboflavin and UV light CXL of the cornea induces a change in properties of the collagen and has a stiffening effect on the corneal stroma, leading to its stabilization and increased resistance to enzymatic degradation.3 Although corneal CXL was originally introduced as a treatment for corneal ectasia, it has been used in the treatment of a variety of other disorders such as symptomatic Fuchs corneal dystrophy,4 pseudophakic bullous keratopathy,5 and more recently, infectious keratitis.6,7 Iseli et al8 used CXL successfully to treat 5 patients with infectious melting resistant to conservative treatment. They hypothesized that the combination of the anticollagenase effect of CXL and the antimicrobial effect of UV light might act synergistically to protect corneas from the damage caused by infection. It is known that UV irradiation has a double antimicrobial action. It causes irreversible damage to the RNA and DNA of microorganisms, thereby preventing them from replicating. UV irradiation and free oxygen radicals interfere with cell membrane integrity leading to direct destruction of bacteria.8 In the present study, we aimed to evaluate the effectiveness of CXL by riboflavin/UV-A in the treatment of patients with bacterial keratitis.

MATERIALS AND METHODS In this prospective interventional study, we included 32 patients with bacterial corneal ulcers, who were referred to Khalili Hospital, affiliated to Shiraz University of Medical Sciences. We excluded patients with any kind of corneal perforation, corneal descemetocele, collagen vascular disease, or immunocompromising diseases, and those who needed any kind of emergency keratoplasty. All patients who met the inclusion criteria were included in the study from December Cornea  Volume 34, Number 4, April 2015

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2013 to May 2014. Written informed consents were obtained from all patients before inclusion. Research was approved by the Ethics Committee of Shiraz University of Medical Sciences. Complete ophthalmologic examination was performed for each patient including evaluation of visual acuities and slitlamp biomicroscopy. A single examiner initially graded all the patients and all follow-up visits as well. Corneal ulcers were graded according to their size, depth, and severity of anterior segment infiltrates. Ulcers were grouped as follows: grade 1 (mild), if they were nonaxial, less than 2 mm in size, involved the superficial one third of the cornea with mild anterior chamber reaction; grade 2 (moderate ulcers), if they were 2 to 6 mm in size, involved the superficial two thirds of the cornea, and had 4+ anterior chamber reactions. Ulcers more than 6 mm in size, extending to the inner one third of the cornea or with severe hypopyon were considered as grade 3, or severe. Only patients with moderate corneal ulcers at the time of admission to our hospital were selected. We used the slit lamp for estimating the area. We averaged vertical (90°), horizontal (180°), and 4 oblique (30°, 60°, 120°, and 150°) diameters of the ulcers and assumed that the shape of the ulcers was circular. Smear and culture were obtained from corneal ulcers. Patients were randomly allocated to 2 groups: case and control groups of 16 patients each using simple randomization with a numerical randomization table. The case or CXL group was treated with CXL and then with standard medical therapy, and the control group was treated with standard medical therapy only. In our center, initial standard medical therapy for moderate bacterial ulcers are lubrication, fortified cefazolin (50 mg/mL) every 1 hour, fortified gentamicin (15 mg/mL) every 1 hour, and systemic doxycycline every 12 hours after loading doses of fortified cefazolin and gentamicin (every 5 minutes for 30 minutes). Initial medical therapy was continued if a positive clinical response was seen and doses of the medications were tapered based on clinical responses. However, if the clinical response was not suitable, further changes in medications were carried out according to laboratory results. For patients in the CXL group, on the day of admission and before starting any kind of medication, CXL with UV-A and riboflavin were performed under sterile conditions in the operating room. The patients were supine. Their corneas were anesthetized with topical tetracaine 0.1% drops. After inserting a lid speculum, an 8-mm diameter zone of corneal epithelium over the microbial infiltrates was removed by a blunt knife (hockey knife) and was sent for smear and culture. Then 0.1% riboflavin in Dextran 500 20% drops (MedioCROSS; Medio-Haus Medizinprodukte GmbH) were applied every 3 minutes for 30 minutes. Then, the corneas were irradiated with UV-A rays (365 nm) in an optical zone of 8 mm for 30 minutes with an irradiance of 3 mW/cm2 (UVX; Peschke Meditrade, Cham, Switzerland). The distance between the light and the corneal apex was approximately 50 mm. During irradiation, the cornea received 0.1% riboflavin every 5 minutes. After treatment, the eyes were given a therapeutic soft contact lens (T-lens). The patients were then admitted to the ward and empirical therapies were started soon after. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

Treatment of Moderate Bacterial Corneal Ulcers

Subsequently, corneal ulcers were evaluated daily. The patients were checked with respect to the dose of drugs and were advised to use medications correctly. The examiner was masked to the groups the patients were allocated to and we designed a data collecting form, but unfortunately some of the patients informed the examiner of their operation. Therefore, the examiner was not masked completely. The grade of ulcers, the size of epithelial defects, and the area of infiltrates were recorded on the 1st, 7th, and 14th days after the beginning of treatment. Our criteria for discontinuing medical therapy were uniform among the patients and included disappearance of severe conjunctival injection, epithelial defects, active corneal infiltrates, hypopyon, and severe anterior chamber reaction. Amniotic membrane transplantation (AMT) was performed for patients with good corneal optical potential showing improvement in their corneal ulcer healing process but who had a persistent epithelial defect, which was not responsive to medical therapy, punctal occlusion, or tarsorrhaphy. If they did not respond to AMT, a conjunctival flap was performed.

RESULTS Thirty-two patients were enrolled in our study, including 22 men (65.6%) and 11 women (34.4%). Cultures of corneal scrapings showed growth of bacterial species and no growth of fungal species in all patients. Age (mean 6 SD) of the patients in the CXL and control groups were 39.6 6 16.8 and 40.3 6 14.9 years, respectively (P = 0.91). Duration of treatment (mean 6 SD) was 17.2 6 4.1 days in the CXL group and only 1 patient required AMT after 25 days. In the control group, duration (mean 6 SD) of treatment was 24.7 6 5.5 days, 1 patient required AMT after 28 days and 1 patient required a conjunctival flap after 30 days. There was no other complication requiring medical or surgical intervention in either of the groups. The duration of treatment for patients in the CXL group was shorter than those of patients in the control group (P , 0.001). The grade of ulcers, size of epithelial defects, and area of infiltrates in both CXL group and control group are given in Table 1. One day after the beginning of treatment, no significant difference was noted between the grade of ulcers (P . 0.99), size of epithelial defects (P = 0.16), or area of infiltrates (P = 0.89) neither in the CXL nor control groups. The differences between the grades of ulcers were not significant 7 days after the beginning of treatment (P = 0.56), whereas it became significant 14 days after starting treatment (P = 0.001; Fig. 1). Epithelial defects were smaller in the CXL group on day 7 (P = 0.001) and day 14 (P = 0.001) after beginning of treatment (Fig. 2). The area of infiltrates in the CXL group was smaller than control group on day 7 (P = 0.001) and day 14 (P , 0.001) after treatment (Fig. 3).

DISCUSSION Infectious keratitis is one of the leading causes of monocular blindness worldwide. Various microorganisms, including bacteria, may cause infectious keratitis. Bacterial infections and inflammatory reactions may lead to corneal www.corneajrnl.com |

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Bamdad et al

TABLE 1. CXL Group (Treated Initially With CXL and Then With Standard Medical Therapy) and Control Group (Treated Only With Standard Medical Therapy) Day After Beginning of Treatment 1 7 14

Area of Infiltrates, Mean 6 SD, mm2

Area of Epithelial Defects, Mean 6 SD, mm2

Grade of Ulcers, Mean CXL Group

Control Group

P

CXL Group

Control Group

P

CXL Group

Control Group

P

2 1.81 0.69

2 1.93 1.70

.0.99 0.56 0.001

17.00 6 5.39 7.94 6 4.47 1.25 6 1.77

19.63 6 4.99 14.94 6 6.05 8.31 6 6.75

0.16 0.001 0.001

19 6 5.15 9.31 6 3.84 3.63 6 2.83

19.25 6 5.02 14.88 6 1.11 9.63 6 1.21

0.89 0.001 ,0.001

ulceration, melting, and perforation if not treated sufficiently. The increasing resistance to antibiotics can lead to destructive outcomes.9 Therefore, it seems necessary to focus on alternative and complementary treatment options for the management of bacterial keratitis. Several studies have revealed that CXL is an effective and a relatively safe modality in the management of different disorders of the cornea such as keratoconus, corneal edema, bullous keratopathies, Fuchs dystrophy, nonhealing corneal ulcers, corneal erosive disorders, and infectious keratitis associated with corneal melting.4,5,7,8,10–12 In this study, 16 patients with moderate bacterial keratitis were treated with CXL followed by standard medical therapy and 16 patients were treated only with standard medical therapy. The mean duration of treatment in the CXL group was shorter than in the control group. Skaat et al13 reported that after CXL therapy 5 of the 6 patients showed rapid reduction in symptoms and decreased infiltrate size. In our study, the number of patients in the CXL group who needed surgery was lower than the control group. Panda and co-workers14 treated patients with keratitis-associated corneal melting with CXL. They claimed that melting was stopped, and emergency keratoplasty was prevented in all 7 eyes. Müller et al15 used CXL for 6 patients with corneal melting of variable origins (including bacterial, fungal, and Acanthamoeba keratitis). They reported healing without any need for further interventions in 4 patients and stabilization of the melting cornea and facilitated additional surgical procedures in 2 patients. Sa glk et al16 reported on a 68-year-old man with diabetes mellitus and unilateral severe corneal ulcer

and Al-Sabai et al17 reported on a 70-year-old woman with severe infectious ulcerative keratitis caused by Pseudomonas aeruginosa, who were treated with CXL. Corneal melting was stopped and CXL treatment was successful in both patients. They concluded that CXL might be considered in the management of corneal ulcers unresponsive to medical management. Spiess et al18 presented 3 cats and 3 dogs with corneal melting, which were treated with CXL. Forty days after CXL, all eyes presented a quiescent corneal state without signs of active inflammation and with initial scar formation. They concluded that CXL might be a cost-efficient and safe treatment for corneal melting. Makdoumi et al6 used CXL as primary treatment in 7 eyes of 6 patients with bacterial keratitis and reported symptomatic relief and arrest of progression of melting in all patients. Iseli et al8 performed CXL in 5 patients with resistant bacterial or fungal ulcerative keratitis and reported immediate regression of the corneal melting process and a significant decrease in the area of infiltrates. CXL also has been performed successfully in post– laser in situ keratomileusis keratitis, fungal keratitis, and in a patient with Escherichia coli keratitis.19–21 UV-A alone can prevent the growth of fungi and bacteria.22–24 Martins et al25 reported that a combination of UV-A–riboflavin had antibacterial properties in vitro against microorganisms such as Staphylococcus epidermidis, S. aureus, methicillin-resistant S. aureus, drug-resistant Streptococcus pneumoniae, P. aeruginosa, as well as multidrugresistant P. aeruginosa. During cross-linking, free radicals are produced and interfere with the microbial cell wall.26,27 The by-products of

FIGURE 1. Grade of ulcers in CXL and control groups.

FIGURE 2. Mean area of epithelial defects (mm2) in CXL and control groups.

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Treatment of Moderate Bacterial Corneal Ulcers

ACKNOWLEDGEMENTS This article has been derived from the thesis by Hossein Malekhosseini - Grant No : 5685 REFERENCES

FIGURE 3. Mean area of infiltrates (mm2) in CXL and control groups.

riboflavin after UV-A exposure can inactivate pathogens. Direct electron transfer, production of singlet oxygen, and generation of hydrogen peroxide with the formation of hydroxyl radicals damage nucleic acids. Pathogen DNA/ RNA may even be affected in the absence of oxygen.28,29 Bacteria can produce enzymes that digest human connective tissue in the cornea and induce tissue melting.30 Spoerl et al31 found that CXL resulted in a marked increase in collagen resistance to digesting enzymes. Although the exact mechanism of stabilization of the infectious process in the cornea has not been fully understood, it seems that both antimicrobial power and increased resistance against enzymatic digestion play important roles in reducing the severity of corneal ulcers. To the best of our knowledge, almost all previous studies have investigated the efficacy of CXL in the treatment of severe keratitis. In this study, we investigated CXL as an adjuvant therapy in the treatment of patients with moderate bacterial keratitis. Moreover, the number of patients in our study was higher than most of the previous studies in this regard. We observed a rapid improvement in symptoms and found that the sizes of epithelial defects and areas of infiltrates in the CXL group were smaller 7 and 14 days after starting treatment. One patient in the CXL group required AMT but other patients’ eyes healed rapidly without any complications. By shortening the course of treatment, CXL can reduce the toxic effects of topical antibiotics on the cornea and prevent healthcare-associated infections. The fact that our examiner was not masked completely is one of the limitations of our study. We did not check the final visual acuity and did not compare the changes in acuity between the 2 groups and this can be considered as another imperfection in our study. In conclusion, our results support the beneficial effect of CXL in cases of moderate bacterial keratitis. This treatment accelerates epithelialization, shortens the course of treatment, and may minimize or remove the need for surgery and reduce the risk for severe complications, such as corneal perforation. However, the effects of CXL on moderate bacterial corneal ulcers need further investigation both using in vitro and in vivo studies to obtain more information especially about its potential side effects. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

1. Jones DB. Decision-making in the management of microbial keratitis. Ophthalmology. 1981;88:814–820. 2. Wollensak G, Spoerl E, Seiler T. Riboflavin/UV-A-induced collagen cross-linking for the treatment of keratoconus. Am J Ophthalmol. 2003; 135:620–627. 3. Spoerl E, Wollensak G, Seiler T. Increased resistance of crosslinked cornea against enzymatic digestion. Curr Eye Res. 2004;29:35–40. 4. Hafezi F, Dejica P, Majo F. Modified corneal collagen cross-linking reduces corneal oedema and diurnal visual fluctuations in Fuchs dystrophy. Br J Ophthalmol. 2010;94:660–661. 5. Ghanem RC, Santhiago MR, Berti TB, et al. Collagen crosslinking with riboflavin and ultraviolet-A in eyes with pseudophakic bullous keratopathy. J Cataract Refract Surg. 2010;36:273–276. 6. Makdoumi K, Mortensen J, Crafoord S. Infectious keratitis treated with corneal crosslinking. Cornea. 2010;29:1353–1358. 7. Schnitzler E, Sporl E, Seiler T. Irradiation of cornea with ultraviolet light and riboflavin administration as a new treatment for erosive corneal processes, preliminary results in four patients [in German]. Klin Monbl Augenheilkd. 2000;217:190–193. 8. Iseli HP, Thiel MA, Hafezi F, et al. Ultraviolet A/riboflavin corneal cross-linking for infectious keratits associated with corneal melts. Cornea. 2008;27:590–594. 9. Neu HC. The crisis in antibiotic resistance. Science. 1992;257: 1064–1073. 10. Snibson GR. Collagen cross-linking: a new treatment paradigm in corneal disease—a review. Clin Experiment Ophthalmol. 2010;38: 141–153. 11. Ashwin PT, McDonnell PJ. Collagen cross-linkage: a comprehensive review and directions for future research. Br J Ophthalmol. 2010;94: 965–970. 12. Goldich Y, Marcovich AL, Barkana Y, et al. Safety of corneal collagen cross-linking with UV-A and riboflavin in progressive keratoconus. Cornea. 2010;29:409–411. 13. Skaat A, Zadok D, Goldich Y, et al. Riboflavin/UVA photochemical therapy for severe infectious keratitis. Eur J Ophthalmol. 2014;24:21–28. 14. Panda A, Krishna SN, Kumar S. Photo-activated riboflavin therapy of refractory corneal ulcers. Cornea. 2012;31:1210–1213. 15. Müller L, Thiel MA, Kipfer-Kauer AI, et al. Corneal cross-linking as supplementary treatment option in melting keratitis: a case series. Klin Monbl Augenheilkd. 2012;229:411–415. 16. Saglk A, Uçakhan OO, Kanpolat A. Ultraviolet A and riboflavin therapy as an adjunct in corneal ulcer refractory to medical treatment. Eye Contact Lens. 2013;39:413–415. 17. Al-Sabai N, Koppen C, Tassignon MJ. UVA/riboflavin crosslinking as treatment for corneal melting. Bull Soc Belge Ophtalmol. 2010;315: 13–17. 18. Spiess BM, Pot SA, Florin M, et al. Corneal collagen cross-linking (CXL) for the treatment of melting keratitis in cats and dogs: a pilot study. Vet Ophthalmol. 2014;17:1–11. 19. Li Z, Jhanji V, Tao X, et al. Riboflavin/ultraviolet light mediated crosslinking for fungal keratitis. Br J Ophthalmol. 2013;97:669–671. 20. Kymionis GD, Kankariya VP, Kontadakis GA. Combined treatment with flap amputation, phototherapeutic keratectomy, and collagen crosslinking in severe intractable post-LASIK atypical mycobacterial infection with corneal melt. J Cataract Refract Surg. 2012;38:713–715. 21. Micelli Ferrari T, Leozappa M, Lorusso M, et al. Escherichia coli keratitis treated with ultraviolet A/riboflavin corneal crosslinking: a case report. Eur J Ophthalmol. 2009;19:295–297. 22. Yoshimura M, Namura S, Akamatsu H, et al. Antimicrobial effects of phototherapy and photochemotherapy in vivo and in vitro. Br J Dermatol. 1996;135:528–532. 23. Wikler JR, Janssen N, Bruynzeel DP, et al. The effect of UV-light on pityrosporum yeasts: ultrastructural changes and inhibition of growth. Acta Derm Venereol. 1990;70:69–71. 24. Alam ZB, Otaki M, Furumai H, et al. Direct and indirect inactivation of Microcystis aeruginosa by UV-radiation. Water Res. 2001;35:1008–1014.

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25. Martins SA, Combs JC, Noguera G, et al. Antimicrobial efficacy of riboflavin/UVA combination (365 nm) in vitro for bacterial and fungal isolates: a potential new treatment for infectious keratitis. Invest Ophthalmol Vis Sci. 2008;49:3402–3408. 26. Maisch T, Baier J, Franz B, et al. The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria. Proc Natl Acad Sci U S A. 2007;104:7223–7228. 27. Jeong J, Kim JY, Yoon J. The role of reactive oxygen species in the electrochemical inactivation of microorganisms. Environ Sci Technol. 2006;40:6117–6122.

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riboflavin collagen cross-linking for treatment of moderate bacterial corneal ulcers.

To evaluate the therapeutic effect of UV-A/riboflavin collagen cross-linking (CXL) on moderate bacterial corneal ulcers...
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