Purpose
To report the long-term results of 44 keratoconic eyes treated by combined riboflavin ultraviolet A collagen cross-linking in the first Italian open, nonrandomized phase II clinical trial, the Siena Eye Cross Study.
Design
Perspective, nonrandomized, open trial.
Methods
After Siena University Institutional Review Board approval, from September 2004 through September 2008, 363 eyes with progressive keratoconus were treated with riboflavin ultraviolet A collagen cross-linking. Forty-four eyes with a minimum follow-up of 48 months (mean, 52.4 months; range, 48 to 60 months) were evaluated before and after surgery. Examinations comprised uncorrected visual acuity, best spectacle-corrected visual acuity, spherical spectacle-corrected visual acuity, endothelial cells count (I Konan, Non Con Robo; Konan Medical, Inc., Hyogo, Japan), optical (Visante OCT; Zeiss, Jena, Germany) and ultrasound (DGH; Pachette, Exton, Pennsylvania, USA) pachymetry, corneal topography and surface aberrometry (CSO EyeTop, Florence, Italy), tomography (Orbscan IIz; Bausch & Lomb Inc., Rochester, New York, USA), posterior segment optical coherence tomography (Stratus OCT; Zeiss, Jena, Germany), and in vivo confocal microscopy (HRT II; Heidelberg Engineering, Rostock, Germany).
Results
Keratoconus stability was detected in 44 eyes after 48 months of minimum follow-up; fellow eyes showed a mean progression of 1.5 diopters in more than 65% after 24 months, then were treated. The mean K value was reduced by a mean of 2 diopters, and coma aberration reduction with corneal symmetry improvement was observed in more than 85%. The mean best spectacle-corrected visual acuity improved by 1.9 Snellen lines, and the uncorrected visual acuity improved by 2.7 Snellen lines.
Conclusions
The results of the Siena Eye Cross Study showed a long-term stability of keratoconus after cross-linking without relevant side effects. The uncorrected visual acuity and best spectacle-corrected visual acuity improvements were supported by clinical, topographic, and wavefront modifications induced by the treatment.
Keratoconus is a degenerative, noninflammatory disease of the cornea. Changes in corneal collagen structure and organization, alterations of the extracellular matrix, as well as keratocyte apoptosis involving the anterior stroma and Bowman lamina partially explain the biomechanical corneal weakening typical of the disease. Biochemical alterations with increased expression of proteolytic enzymes and decreased concentrations of protease inhibitors, decreased stromal thickness, and modified configuration of collagen lamellae also have been reported among the pathophysiologic mechanisms of keratoconus. The technique of corneal collagen cross-linking, conceived in Dresden, consists of a photopolymerization of stromal collagen fibers induced by the combined action of a photosensitizing substance (riboflavin or vitamin B 2 ) and ultraviolet (UV) A light that induces corneal stiffening by increasing the number of intrafibrillar and interfibrillar covalent bonds and corneal collagen resistance against enzymatic degradation. This therapy was introduced for the first time in Italy by us in 2004 at the Department of Ophthalmology of Siena University to slow the progression of keratoconus and to reduce the demand for penetrating keratoplasty.
Methods
To date, 363 eyes with progressive keratoconus have been treated in Siena with the riboflavin UV A corneal collagen cross-linking procedure. The Siena Eye Cross Study (phase II nonrandomized open trial) included 44 patients with keratoconus between 10 and 40 years of age with disease progression documented clinically and instrumentally in the last 6 months, minimum corneal thickness of 400 μm in the thinnest point evaluated by Orbscan IIz (Bausch & Lomb, Inc., Rochester, New York, USA), topographic mean K value of less than 55 diopters (D), clear cornea by slit-lamp examination (absence of Vogt striae, subepithelial, and stromal scars), and absence of eye infections, herpetic clinical history, autoimmune disease, and pregnancy. All patients reported in this series were enrolled in the first 6 months of the study and reached a minimum follow-up of 48 months (mean, 52.4 months; range, 48 to 60 months). Statistical analysis was conducted by the Mann–Whitney U test for nonparametric data (uncorrected visual acuity [UCVA] and best spectacle-corrected visual acuity [BSCVA]) and by the paired t test for parametric data (refraction, mean curvature power, central corneal thickness, and intraocular pressure [IOP]). Patients gave their specific informed consent before inclusion in the study. Riboflavin UV A corneal collagen cross-linking was performed according to the Siena protocol using the CSO Vega CBM X linker (CSO, Florence, Italy), developed in Italy at the Department of Ophthalmology of Siena University by Mazzotta and associates. The treatment was conducted under topical anesthesia (4% lidocaine drops). After applying the eyelid speculum, a 9-mm diameter marker was used to mark the corneal epithelium in a central circle, then epithelium was removed with a blunt metal spatula. After epithelial scraping, a disposable solution of riboflavin 0.1% and dextrane 20% (Ricrolin Sooft, Montegiorgio, Italy) was instilled for 15 minutes of corneal soaking before starting UV A irradiation. The riboflavin and dextrane solution was administered every 2 minutes for a total of 30 minutes of UV A exposure (3 mW/cm 2 ). Treated eyes were dressed with a soft contact lens bandage for 4 days and were medicated with antibiotics (ofloxacin drops 4 times/day), nonsteroidal anti-inflammatory drugs (diclofenac drops 4 times/day), and lacrimal substitutes (phospholipidic microemulsion drops tapered 4 times/day). In the first postoperative month, after therapeutic corneal lens removal, fluorometholone 0.2% drops (4 times/day) and lacrimal substitutes (phospholipidic microemulsion drops, 4 times/day) were administered for 4 to 6 weeks. Preoperative and follow-up examinations included: UCVA, BSCVA, spherical spectacle-corrected visual acuity measured by Snellen decimal equivalent, biomicroscopy (CSO, 990 SL, Florence, Italy), endothelial cells count (I Konan, Non Con Robo V; Konan Medical, Inc., Hyogo, Japan), IOP (Medtronic Tono-Pen; Medtronic Ophthalmics, Jacksonville, Florida, USA), optical pachymetry (Visante OCT; Zeiss, Jena, Germany), ultrasound (US) pachymetry (DGH Pachette; DGH Technology, Inc., Exton, Pennsylvania, USA), corneal topography (CSO EyeTop, Florence, Italy) and tomography (Orbscan IIz; Bausch & Lomb, Inc.), surface aberrometry (CSO EyeTop), macular optical coherence tomography (Stratus OCT; Zeiss, Jena, Germany), and in vivo confocal microscopy (HRT II, Rostock Cornea Module; Heidelberg Engineering, Rostock, Germany).
Results
The mean preoperative pachymetric value measured by central US pachymetry was 450 ± 14.54 μm (range, 422 to 512 μm) and that by optical Orbscan IIz system in the thinnest point was of 438 ± 13.87 μm (range, 408 to 503 μm). During follow-up, the Orbscan II z measurement showed significant underestimation of corneal thickness compared with US and confocal microscopic pachymetric examinations, with a mean of −120 μm in the first 6 months and −70 μm between 6 and 12 months. US and confocal postoperative pachymetric data did not show significant differences with respect to preoperative values. Because the Visante OCT system became available in our department 24 months ago, we checked the same data measured by US and confocal pachymetric examinations, confirming that preoperative and postoperative thickness data were superimposable ( Figure 1 ).
Preoperative mean endothelial cell density was 2451 ± 130.444 cells/mm 2 (range, 2092 to 3016 cells/mm 2 ). A statistically insignificant reduction in endothelial cells count with respect to physiologic reduction was observed after treatment, namely a mean of 2% per year ( Figure 2 , Top). Preoperative mean US central corneal pachymetry was 450 ± 14.54 μm (range, 422 to 512 μm). Statistically nonsignificant corneal thinning was recorded in the first 2 postoperative months (438.177 ± 15.118 μm in the first month and 443.955 ± 15.349 μm in the second month). No statistically significant difference in central corneal thickness measured by US pachymetry was observed over the third 3-month of follow-up ( Figure 2 , Middle). Mean preoperative IOP measured by Tono-Pen II XL (Medtronic, Jacksonville, Florida, USA) was 14.773 ± 1.696 mm Hg (range, 11 to 18 mm Hg). No statistically significant modifications were observed in IOP values during the entire follow-up ( Figure 2 , Bottom).
No persistent early or late side effects were observed after the cross-linking procedure. Stromal edema, clinically detectable by slit-lamp examination in 70% of patients, occurred in the first 30 postoperative days. Temporary haze occurred in 9.8% of cases, 14 cases in the first 3 months and 2 cases after 6 months, disappearing progressively after topical preservative-free steroid therapy (fluorometholone preservative-free drops for 1 to 3 months). No delayed re-epithelialization or endothelial damage was detected during follow-up. No adverse events were recorded during the mean follow-up of 52.4 months (range, 48 to 60 months). Topographic analysis conducted 1 year after treatment by the CSO EyeTop system showed a mean reduction of −1.96 ± 0.63 D (range, −0.92 to −3.24 D) in mean K readings. The mean K reduction increased to −2.12 ± 0.65 D after 2 years, −2.24 ± 0.61 D after 3 years, and −2.26 ± 0.68 D after 4 years of follow-up ( Figure 3 ). On the contrary, comparative topographic study of fellow eyes (control group) showed a mean K increase of +1.2 ± 0.96 D and 2.2 ± 1.24 D, respectively, after 1 and 2 years, against a similar decrease in mean K power in treated eyes. Fellow control eyes (initially untreated) were treated after 24 months of observation and showed a decrease in corneal power similar to that observed after treatment of the first (worst) eyes in the first and second year of follow-up ( Figure 4 ). According to topographic data, the decrease in corneal curvature and spherical refraction, calculated in the spectacle plane, showed a mean hyperopic shift of +1.62 ± 1.03 D at 1 year of follow-up (range, 0 to 3.75 D), increasing at +1.87 ± 1.06 D (range, 0.25 to 3.75 D) after 2 years and maintaining this value in the longer period (+1.86 ± 0.97 D after 3 years and 1.87 ± 0.98 D after 4 years). Cylinder refraction showed a similar course, but with a reduction smaller than the spherical one: at 1 year, we observed a mean reduction of −0.52 ± 0.38 D (range, 0.75 to −2 D) that maintained similar values after 2, 3, and 4 years: −0.53 ± 0.37 D, −0.53 ± 0.38 D, and −0.55 ± 0.38 D, respectively.
The spherical equivalent resulted in hyperopic shift with the following values at 1, 2, 3, and 4 years of follow-up, respectively: +1.87 ± 1.24 D (range, 0.5 to 4.37 D), +2.12 ± 1.27 D (range, 0.5 to 4.00 D), +2.13 ± 1.12 D (range, 0.5 to 4.12 D), and 2.15 ± 1.19 D (0.87 to 4.12 D; Figure 5 ). Statistical analysis with the paired t test showed a significant difference in sphere values between preoperative evaluation and 6, 12, 24, and 48 months of follow-up ( P value, 3.7 × 10 −6 at 6 months, 1.8 × 10 −7 at 12 months, 1.4 × 10 −9 at 24 months, 1.1 × 10 −9 at 36 months, and 5.1 × 10 −10 at 48 months; Figure 5 ).
UCVA improved by a mean of +2.41 ± 0.88 Snellen lines after 12 months, +2.75 ± 0.79 Snellen lines after 24 months, +2.80 ± 0.76 Snellen lines after 36 months, and +2.85 ± 0.81 Snellen lines after 48 months ( Figure 6 ). BSCVA improved by a mean of +1.34 ± 1.13 Snellen lines after 12 months, +1.93 ± 1.04 Snellen lines after 24 months, +1.91 ± 1.03 Snellen lines after 36 months, and +2.03 ± 1.04 Snellen lines after 48 months ( Figure 6 ).
Clinical and topographic improvements were recorded between the second and third month after the operation and continued thereafter, reaching reliable stability after 24 months. Refractive stability was confirmed in our series after 4 years without progression of keratoconus. A major finding recorded in all treated eyes during follow-up was reduction in the difference between superior and inferior hemimeridians (flattest vs steeper) expressed by preoperative and postoperative comparative values of the topographic symmetry index ( Figure 7 ).
Statistical analysis comparing the preoperative and postoperative superior–inferior index provided by the CSO EyeTop system showed a nonsignificant reduction in this parameter in the first 3 months ( P = .136 after 1 month; P = .053 after 3 months), whereas the reduction in superior–inferior topographic index became statistically significant after 6 to 48 months with a constant increase (from P = 1.8 × 10 −4 comparing the preoperative and sixth postoperative month, to P = 2.4 × 10 −8 comparing preoperative and forty-eighth postoperative month; Figure 7 ). Other important data provided by topographic analysis were the surface wavefront results ( Figure 8 ). A statistically significant reduction in coma aberration between preoperative and postoperative values ( P = .032, paired t test on 44 eyes) was recorded 1 month after treatment with a constant statistically significant increase up to 24 months of observation ( P = 1.4 × 10 −8 , paired t test on 44 eyes). The statistical significance of these values was maintained at 36 and 48 months of follow-up. After the third postoperative month, a reduction in total wavefront higher order aberrations was recorded, starting with a low statistical significance ( P = .044, paired t test on 44 eyes), which became high at 1 year ( P = 2.5 × 10 −5 , paired t test on 44 eyes) and further increase at 2 years ( P = 1.3 × 10 −5 ). Nonsignificant changes in these data were observed at 3 years ( P = 3.6 × 10 −7 ) without appreciable modification at 4 years of follow-up ( P = 4.3 × 10 −7 ). We did not observe any significant changes in spherical aberration in our analysis ( Figure 8 ).
