Corneal Collagen Cross-Linking Window Absorption

TECHNIQUES

Corneal Collagen Cross-Linking Window Absorption Pietro Rosetta, MD, Riccardo Vinciguerra, MD, Mario R. Romano, MD, PhD, and Paolo Vinciguerra, MD

Purpose: To evaluate the safety and efficacy of treatment of severe infectious corneal ulcers with hypoosmolar riboflavin solution and UV-A collagen cross-linking (CXL) without deepithelizing the cornea.

Methods: Four eyes of 3 patients with severe keratitis and associated corneal melting, were treated with a new protocol of corneal CXL for infectious keratitis, named cross-linking window absorption (CXLWA). CXL-WA was performed when the infection did not respond to systemic and topical antibiotic therapy. The protocol entails the use of hypoosmolar riboflavin before the irradiation with UV-A, and the penetration is obtained through the epithelial defect overlying the ulcer.

Results: In all cases, CXL halted the progression of the ulcer. After a minimum follow-up of 3 months, none of the patients had recurrence of the infection. Conclusion: CXL-WA is a promising technique of treatment of infectious corneal ulcers, delaying or preventing more invasive approaches. Key Words: ultraviolet corneal cross-linking, corneal infectious keratitis, riboflavin (Cornea 2012;0:1–5)

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orneal collagen cross-linking (CXL) with UV-A and riboflavin is an accepted technique for the treatment of progressive keratoconus,1–7 aimed at increasing corneal rigidity and arresting the progression of ectatic disease. Through the production of reactive oxygen species, the procedure induces intrafibrillar and interfibrillar covalent bonding,8 with oxidation of nucleic acids leading to apoptosis of keratocytes and an increased resistance to collagen-degrading enzymes.9 Moreover, these actions have prompted the recent use of CXL for the treatment of infectious keratitis and corneal melting10 because photoactivation of riboflavin damages the RNA and DNA of bacteria, fungus, and virus. This effect has been well known for many years and has been already used to inactivate virus.11 Furthermore, UV light alone is already in use as an antibacterial and antiviral procedure for disinfection of drinking water.12 Received for publication November 14, 2011; revision received March 28, 2012; accepted April 8, 2012. From the Department of Ophthalmology, Istituto Clinico Humanitas, Milan, Italy. The authors have no funding or conflicts of interest to disclose. Reprints: Paolo Vinciguerra, Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy (e-mail: [email protected]). Copyright © 2012 by Lippincott Williams & Wilkins

Cornea Volume 0, Number 0, Month 2012

A recent publication showed the efficacy of CXL UV-A against many microbes and viruses, including those that are multidrug resistant,13 and many case reports and publications have reported the efficacy of CXL in treating keratitis,14,15 even associated with severe corneal melting.10 The procedure used in these reports is the gold standard for the treatment of keratoconus with epithelial debridement. We would like to introduce a novel technique, named cross-linking window absorption (CXL-WA), for the treatment of keratitis with CXL carried out without epithelial removal.

MATERIALS AND METHODS Four eyes of 3 patients with severe keratitis and associated corneal melting were treated with corneal CXL. Patients were, respectively, 15, 55, and 72 years of age. Case 1, a 15-year-old girl, presented a bilateral pseudomonas keratitis associated with corneal melting in 1 eye because of cosmetic contact lens use. Case 2, a 55-year-old woman, soft contact lens user, presented severe ulcerative Acanthamoeba keratitis in 1 eye. Case 3 was a 72-year-old woman with an ulcerative keratitis caused by Streptococcus pneumoniae infection of a transplanted cornea. All patients were treated with topical and systemic antibiotics, oral or intravenous, according to the antibiogram. Microbial culturing was performed. The size of the ulcer was estimated at the slit-lamp evaluation and with optical coherence tomography (OCT; Cirrus Carl Zeiss Meditec, Dublin, CA) measurement. In all patients, signed informed consent was obtained.

Technique: CXL-WA Thirty minutes before the surgery, pain medication was administered. All patients underwent CXL treatment under topical anesthesia with oxybuprocaine hydrochloride 0.2% and lidocaine 4% administered 5 minutes before surgery. The procedure was conducted in sterile surgical conditions. After topical anesthesia, the patient was draped, the ocular surface was rinsed with sterile physiologic balanced salt solution, and a lid speculum was applied. The center of the ulcer was gently swiped to eliminate all cellular debris. The cornea was not deepithelized. Before beginning irradiation, a hypoosmolar 0.1% riboflavin solution, produced by diluting 0.5% vitamin B2-riboflavin-5-phosphate (G. Streuli & Co. AG, Uznach, Switzerland) with physiologic salt solution (sodium chloride 0.9% solution, 310 mOsmol/l; B. Braun Medical AG, Sempach, Switzerland), was instilled www.corneajrnl.com |

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for 30 minutes to obtain stromal swelling. A calibrated laser meter at 1.0 cm (LASER TEST UVA Peschke Meditrade GmbH) was used before the treatment to check for the accuracy of irradiance. The cornea was exposed to UV-A light with the UV-X System (Peschke Meditrade GmbH, Huenenberg, Switzerland), which emits light at a wavelength of l 370 ± 5 nm and an irradiance of 3 mW/cm2 or 5.4 J/cm2. Exposure lasted for 30 minutes, during which time the riboflavin solution was again applied 6 times, every 5 minutes. After surgery, patients received cyclopentolate (Ciclolux; Allergan, Rome, Italy) and levofloxacin drops (Oftaquix; Tubilux Pharma, Pomezia, Rome, Italy). A soft therapeutic contact lens was not applied to avoid leaving a device that could promote bacterial growth, particularly anaerobes. In addition, all patients received oral amino acid supplements (Trium; Sooft, Montegiorgio, Italy) for 7 days. Follow-up visits entailed photodocumentation with and without fluorescein to evaluate epithelial growth, anterior segment OCT, and analysis of the anterior chamber with Pentacam (Oculus Optikgerate GmbH, Wetzlar, Germany).

RESULTS The pachymetry in the thinnest point, acquired by ultrasound, in the first case was 347 mm, in the second case was 277 mm, and in the third case was 304 mm.

days. One month after treatment, uncorrected visual acuity in the right eye was 20/20, and in the left eye, the best-corrected visual acuity was 20/40. The visible OCT demarcation line was 207 mm (Fig. 1).

Case 2 A 55-year-old woman, soft contact lens user with a bestcorrected visual acuity of 20/20 (before keratitis), presented a ring-shaped corneal ulcer of 4 mm in diameter in the right eye as well as Descemet folds, diffuse peripheral corneal epitheliopathy, and perikeratic conjunctival injection (Fig. 2). Based on the clinical aspect of the ulcer, an immediate empiric topical therapy of propamidine isethionate 0.1%, moxifloxacin 0.5%, and chloramphenicol tetracycline eye drops was prescribed, together with oral fluconazole 200 mg twice daily. The microbial culture result was Acanthamoeba. After 5 days of therapy, there was no improvement in symptoms or clinical findings and corneal thickness had decreased to 304 mm. CXL treatment was conducted the same day. After the procedure, the ulcer and infiltrates gradually disappeared, and on day 10, a complete epithelialization had occurred. After 6 months, no signs of recurrence were reported (Fig. 2) and the bestcorrected visual acuity was 20/50, with an endothelial cell count of 1789 cells per square millimeter (preoperative 1978 cells/mm2). With OCT examination, a clear demarcation line was detected at 240 mm (Fig. 2).

Case 1 Case 1 was a 15-year-old girl, a cosmetic contact lens user. Before the development of keratitis, she was bilaterally emmetropic (uncorrected visual acuity 20/20). On examination, the patient had a 5-day history of redness, pain, and epiphora, with more intense symptoms in the left eye. The patient admitted having worn contact lenses, ignoring the pain for the first 3 days. Severe bilateral stromal infiltrates were found, extending deeply into the temporal quadrant and covering 2 mm of the central cornea in the left eye. The patient was prescribed wide‐spectrum therapy with moxifloxacin hydrochloride 5 mg/mL eye drops every hour, chloramphenicol 10 mg; colistimethate sodium 180,000 IU; tetracycline 5 mg/g eye drops every hour, atropine sulfate 1% eye drops twice daily, and oral fluconazole 150 mg twice daily. The following day (day 6 after the onset), the ulcer in the right eye was significantly worse, and in the left eye, the infiltrate was 4 mm wide, with severe corneal melting and ground glass stroma (Fig. 1) and 1-mm hypopyon. The culture was positive for Pseudomonas aeruginosa. We decided to perform a bilateral CXL, off-centering temporally the irradiation in the right eye, to better target the ulcer. One day after treatment, the patient experienced an improvement in subjective symptoms; in the right eye, the cornea was noticeably clearer. On day 3, the epithelium was already healed in both eyes, and in the left eye, the hypopyon was no longer present. On day 15, the infiltrate and perilesional ulcer were resolved (Fig. 1). However, a wide central leukoma persisted after treatment (Fig. 1). The postoperative therapy, modified according to the antibiogram, lasted for 15

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Case 3 The third case is a 72-year-old woman, with an ocular history of a perforating keratoplasty in the right eye 3 years before and phacoemulsification 2 years before the current infection. She currently had a partially decompensated graft with a best-corrected visual acuity of 20/200 and an endothelial cell count of 767 cells/mm2. On examination, the patient had a 10-day history of pain and redness in the right eye. Clinically, a severe, infectious, ulcerative keratitis of the transplanted cornea, which still had sutures, was diagnosed. Slit-lamp examination revealed infiltrates 3 mm in size at the periphery of the flap; no Seidel or hypopyon was observed (Fig. 3). After the ulcer was cultured, an empiric antibiotic treatment was initiated with vancomycin conjunctival injection and topical eye drops of moxifloxacin 0.5%, chloramphenicol, tetracycline, and propamidine isethionate 0.1%, together with oral cefotaxime 500 mg every 12 hours. The bacterial culture was indicative of S. pneumoniae. After 8 days of therapy without improvement, associated with a reduction of pachymetry to 277 mm, a CXL was performed. After 1 week, the infiltrate was reduced in size, and after 15 days of treatment, it had completely disappeared. However, the perilesional ulcer was slow to heal and antibiotic treatment was continued for another month. After 6 months, the best-corrected visual acuity was 20/200; bullous keratopathy and a persisting corneal scar were still present (Fig. 3). The maximum visible OCT demarcation line was 308 mm (Fig. 3). Ó 2012 Lippincott Williams & Wilkins


Cross-Linking Window Absorption

FIGURE 1. Case 1: Pseudomonas aeruginosa corneal ulcer after cosmetic contact lens use in left eye. Slit-lamp examination and OCT evaluation (pre-CXL) show residual corneal scar and demarcation line at 280 mm through 15 days, 1-month follow-up after CXL.

DISCUSSION CXL for the treatment of infectious keratitis is a promising technique that can delay or prevent having to perform keratoplasty. UV-A CXL can slow or, frequently, stop bacterial growth. As reported by different authors,11,16 UV-induced CXL damages the RNA and DNA of microorganisms, causing lesions in chromosomal strands; other researchers subsequently reported the efficacy of UV light, associated with riboflavin, to deactivate pathogens in plasma, platelets, and red blood cell products.17 The antibacterial activity of riboflavin and UV-A was tested in vitro on different types of bacterial and fungal isolates and has proven to be effective against Staphylococcus aureus, P. aeruginosa, multidrug-resistant P. aeruginosa, and drug-resistant S. pneumoniae and Staphylococcus epidermidis.13 Spoerl et al9 showed that corneas that underwent CXL were more resistant to enzymatic digestion. Furthermore, in some cases of keratitis, corneal melting is a self-perpetuating process by the activated antigen-presenting cells; UV-A CXL can reduce this process through the induced apoptosis of the antigen-presenting cells.18 This led to the hypothesis that in parallel with the ability of CXL to induce lysis of bacterial cells through direct

damage to DNA and RNA, because of the formation of reactive oxygen species, there was also a beneficial effect because of the increased resistance to enzymatic digestion of stromal collagen. After these reports, the use of CXL for infectious keratitis, which is unresponsive to medical therapy, has increased rapidly and many case reports have reported the efficacy of this treatment in slowing the progression of keratitis and preventing the need to do an urgent keratoplasy before perforation.10,14,15 The standard protocol of CXL, defined by the University of Dresden, is performed with an isoosmolar 0.1% solution of riboflavin with dextran T500 20%. After a 9-mm abrasion of the corneal epithelium, the riboflavin solution is applied every 3 to 5 minutes for 30 minutes. To ensure a minimal stromal thickness of 400 mm, ultrasound pachymetry is performed during the treatment.19 After ensuring by slit-lamp examination the successful penetration of riboflavin into the anterior chamber, the eye is irradiated for 30 minutes with UV-A (Table 1). In the modified protocol, a hypoosmolar solution of riboflavin is used to treat corneas thinner than 400 mm.20,21 This solution induces stromal swelling and a consequent increase of thickness that permits treatment. This hypoosmolar solution

FIGURE 2. Case 2: corneal melting from Acanthamoeba in the right eye of a 55-year-old woman, a soft contact lens user. Preoperative slit-lamp examination showed a ring-shaped corneal ulcer, edema, and corneal melting in the OCT image. After 3 and 6 months, no signs of recurrence were reported. At month 6, the visible demarcation line was at 240 mm. Ó 2012 Lippincott Williams & Wilkins

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FIGURE 3. Case 3: preoperative slitlamp image of a severe Streptoccoccus pneumoniae infectious ulcerative keratitis of a transplanted corneas. The OCT image showed severe corneal melting and edema. After 1 month, the healing of the perilesional ulcer is visible. At month 6, no recurrences were reported, and the maximum visible demarcation line was at 308 mm.

does not contain dextran, and it is generated by diluting vitamin B2-riboflavin-5-phosphate with physiologic salt solution (sodium chloride 0.9% solution, 310 mOsm/L; Braun Medical AG). In this protocol, also, the epithelium is removed before instillation of riboflavin (Table 1). The protocol used for CXL in cases of infectious keratitis is usually the standard one10,14,15 with isoosmolar solution and removal of epithelium, although in 2 studies, the epithelium was not removed.14,22 CXL-WA is a new protocol for the treatment of infectious keratitis with corneal collagen CXL. As reported in the literature, in the presence of compromised ocular surface defense, such as corneal infection, chronic inflammation may lead to persistent epithelial defects.23 To reduce the risk of delayed epithelial healing in CXL-WA, the epithelium is not removed. The penetration of riboflavin is obtained

TABLE 1. Difference in Preoperative and Postoperative Medications and Surgical Parameters Between Standard CXL and CXL-WA Modified CXL With Hypoosmolar Riboflavin

Surgical Parameters

Standard CXL

Prior medication Epithelial removal Impregnation

Anesthetic and miotic Yes

Anesthetic and miotic Yes

30 min with epithelium off

30 min with epithelium off

Standard with dextran 3 mW/cm2

Hypoosmolar, no dextran 3 mW/cm2

Only anesthetic No (penetration through ulcer) 30 min with absorption through ulcer window Hypoosmolar, no dextran 3 mW/cm2

30 min

30 min

30 min

Antibiotic

Antibiotic

Antibiotic + mydriatic

Solution UV-A source Time of irradiation Postoperative medication

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CXL-WA

through the epithelial defect overlying the ulcer and the absence of the tight junctions of the epithelium, which are solved. Before the beginning of the surgery, only topical anesthetic is administered to avoid postoperative pain because of ciliary muscle spasm. For the same reason, at the end of CXL, mydriatic eye drops are applied together with the antibiotic (Table 1). Another difference from the standard protocol is the riboflavin solution used. In the presence of an acute infection and corneal ulcer, it is not possible to determine what part of the residual pachymetry is attributable to the stroma and what is attributable to melting. For this reason, a hypoosmolar riboflavin solution is recommended to induce corneal swelling and to avoid the risk of treating a residual stromal bed with a thickness of less than 400 mm. By plumping up the cornea with a hypoosmolar solution, it is possible to treat keratitis with a residual pachimetry inferior to 400 mm. The compiled results of these 3 cases were promising: They showed that the demarcation line was between 200 and 300 mm, no adverse effects of the swelling were reported, and all patients had a complete resolution of the infective process. After a minimum follow-up of 3 months, none of the patients had recurrence of the infection. Unfortunately, as reported, stromal scars can persist after the treatment, probably because CXL is normally used only for advanced infectious keratitis unresponsive to medical therapy. More studies are needed to understand if it is possible to obtain better results combining medical therapy with CXL as a first choice treatment, when the infective process is first diagnosed. However, it is not advisable to use CXL for the treatment of herpes simplex virus and herpes zoster virus keratitis.23 Based on our experience in treating infectious keratitis, we suggest using this new protocol, CXL-WA, which entails no removal of the epithelium and a hypoosmolar riboflavin infusion. The parameters used in CXL-WA are similar to those used for keratoconus; however, more studies are needed to understand at which riboflavin level peak eradication is achieved and if a CXL time of 30 minutes is Ó 2012 Lippincott Williams & Wilkins


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