Keratoconus is a progressive, frequently bilateral disease that causes asymmetrical steepening and thinning of the cornea.1 Thanks to new technologies, its diagnosis has become possible at an earlier stage when it is still possible to treat this ectatic disease conservatively with corneal cross-linking (CXL). This photochemical reaction employs riboflavin with ultraviolet light, aiming to increase corneal stiffness and reduce or halt further worsening of the corneal shape.2 Long-term studies have already proved that CXL is not only able to avoid progression, but also can induce improvement in visual acuity and morphological and functional parameters.3–5 The original Dresden protocol involves epithelial removal to allow riboflavin penetration inside the stroma.6 Epithelial debridement has the drawback of causing postoperative pain,7 delaying visual recovery,4,5,8 and increasing the risk of infection.
Transepithelial CXL was introduced to try to overcome these disadvantages, but the original protocol was ineffective.9–11 Conversely, CXL with the aid of iontophoresis with12 and without13 epithelial removal has showed promising results even when compared to standard Dresden protocol (S-CXL).14–17 However, to the authors' knowledge, there is no study comparing the outcomes of transepithelial iontophoresis (I-CXL), iontophoresis with epithelial removal (I-SCXL), and the S-CXL.
The aim of our study was to report the 2-year follow-up results of three groups of patients with keratoconus who were treated with I-CXL, I-SCXL, and S-CXL.
Patients and Methods
Sixty eyes of 60 patients were enrolled at the Eye Center, Humanitas Clinical and Research Center (Rozzano, Milan, Italy) for a comparative, prospective, non-randomized, single center clinical study. The inclusion criteria for enrollment were progressive keratoconus (demonstrated with differential maps as a change of 1.00 diopter [D] maximum curvature and/or thinning of 20 µm in minimum thickness) over a maximum period of 12 months and patients older than 9 years.5 Exclusion criteria were previous herpetic keratitis, dry eye, corneal infection, concomitant ocular or systemic autoimmune pathologies, pregnancy or breastfeeding, and the presence of central or paracentral scars.
The institutional review board of the ethical committee of Humanitas Clinical and Research Center approved this study and it was conducted according to the ethical standards set in the 1964 Declaration of Helsinki, as revised in 2000. All patients provided informed consent.
At the baseline visit and all follow-up visits (1, 3, 6, 12, and 24 months), the following parameters were measured and evaluated: corrected distance visual acuity (CDVA), corneal topography and corneal aberrometry for the evaluation of low and higher order aberrations (Costruzione Strumenti Oftalmici [C.S.O.], Florence, Italy), optical coherence tomography (Cirrus HD-OCT; Carl Zeiss Meditec, Dublin, CA), pachymetry with Pentacam (Oculus Optikgeräte, Wetzlar, Germany), and endothelial biomicroscopy (Konan Specular Microscope; Konan Medical Inc., Hyogo, Japan) preoperatively and 1 month postoperatively.
Table 1 summarizes the different CXL protocols used. S-CXL was previously described5 and includes 9-mm epithelial debridement with an Amoils brush (Amoils Brush Epithelial Scrubber; Vision Technology Co., Seoul, Korea). The 0.1% riboflavin with 20% dextran (Ricrolin; Sooft, Montegiorgio, Italy) solution is then instilled every minute for 30 minutes to soak the corneal stroma. If corneal thickness was reduced under 400 µm by the dextran solution, hypo-osmolar 0.1% riboflavin solution was instilled to induce corneal swelling. Ultraviolet-A (UV-A) irradiation parameters were: irradiance power of 3 mW/cm2 (UV-X System; Peschke Meditrade GmbH, Huenenberg, Switzerland) for 30 minutes for a total energy dose of 5.4 J/cm2.
I-CXL was also previously described.13 The corneal iontophoresis electrode (I-ON XL; Sooft) produces a constant current set at 1 mA (the total dose of 5 mA/5 minutes is monitored by the generator) for 5 minutes. The riboflavin solution used has no dextran but includes the presence of enhancers (Ricrolin+; Sooft). UV-A irradiation parameters were: 10 mW (UV-X 2000; IROC Innocross AG, Switzerland) for 9 minutes for a total energy dose of 5.4 J/cm2.13
I-SCXL includes a 9-mm epithelial debridement with the Amoils brush followed by 5 minutes of impregnation with Ricrolin+ and iontophoresis current generator set, as I-CXL, at 1 mA for 5 minutes. UV-A irradiation is, similarly, 10 mW (UV-X 2000; IROC Innocross AG, Switzerland) for 9 minutes.12
A soft therapeutic contact lens was applied in all groups. Postoperatively, an ophthalmic gel of 0.15% sodium hyaluronate, 1% xanthan gum, and 0.3% netilmicin (Xanternet; SIFI SpA, Catania, Italy) was prescribed four times a day until complete epithelial healing (epithelial integrity was assessed with fluorescein staining). After contact lens removal, dexamethasone 21-phosphate 0.15% drops (Etacortilen; Sifi, Lavinaio, Italy) were used four times a day for 10 days, and 0.15% sodium hyaluronate drops (BluYal; Sooft) six times daily for 45 days. Additionally, to promote epithelial healing all patients received oral amino acid supplements (Aminoftal; Sooft) for the first 7 postoperative days.18
Statistical analysis was performed using SPSS software (version 24; IBM Corporation, Armonk, NY).
Given that in our previous study we showed the 1-year follow-up of S-CXL,4 I-CXL,14 and I-SCXL12 to assess the long-term safety and efficacy of these treatments, the preoperative values were compared with the 2-year values. Refractive outcomes were analyzed using the methods of Kaye and Harris19,20 after transforming the data into Long's matrix formalisms.21
A generalized linear model was used to compare the groups. Outcome measures were refractive error, maximum keratometry, minimum pachymetry, corneal symmetry index, and surface asymmetry index. Fixed and random factors were age, gender, laterality and outcome measures, and group. Covariation of the change in refractive error with preoperative refraction was tested. A P value of less than .05 was considered significant. Changes in aberrometry were evaluated with the Wilcoxon signed-rank test.
Box plots were made to illustrate the distribution of parameters. The box represents the 25th to 75th percentile range, and the internal line represents the median. The whiskers are limited by upper and lower adjacent values. Outside the whiskers, outliers can be seen.
Gender and age distribution of the three groups are summarized in Table 2. All patients in the S-CXL group completed the 1, 3, 6, 12, and 24 months of follow-up. In the I-CXL group, 1 patient was lost to follow-up at month 3, 4 patients at month 12, and 3 patients at month 24. Conversely, all patients in the I-SCXL group completed all follow-up visits except 3 patients who missed the 3-month follow-up.
Comparison at baseline demonstrated no significant difference in terms of visual acuity, maximum keratometry, and pachymetry (P > .008, Bonferroni correction), but I-CXL had significantly higher comatic and higher order aberrations (P < .0001). There was also no significant difference in the preoperative refraction between the groups.
The statistical analysis showed a significant increase in CDVA compared to baseline in all groups (S-CXL P = .0004, I-SCXL P = .0045, and I-CXL P = .004) at 2 years postoperatively (Figure 1A). Conversely, there was no statistical difference in the improvement in CDVA between the groups (P = .635) (Table 3).
Box and whiskers plot for (A) corrected distance visual acuity (CDVA), (B) comatic aberrations (COMA), (C) maximum keratometry (Kmax), and (D) minimum pachymetry at baseline (T0) and 24 months postoperative (T24) for transepithelial iontophoresis (I-CXL), iontophoresis with epithelial debridement (I-SCXL), and standard Dresden protocol (S-CXL).
There were no significant differences in the change in refractive error following CXL in all groups or between groups (Table 4).
Refractive Error Before (T0) and After (T24 +24 Months) CXL
Aberrometric results are summarized in Table 3. There was a significant improvement at the 2-year follow-up for higher order aberrations (P = .0007) and comatic aberrations (P = .0007, Figure 1B) in I-CXL. I-SCXL induced a significant improvement of higher order aberrations (P = .0049). S-CXL did not change significantly from preoperative values when considering aberrometric results (P > .05). The difference in the induced change in comatic aberrations and higher order aberrations between the groups was significant (P < .0001) (Table 3).
Topographic results are summarized in Table 3. The comparative analysis of the morphological indices suggests a stabilization of keratoconus in all groups. There was no significant difference in the change of all evaluated indexes (including maximum keratometry, Figure 1C) at 24 months of follow-up between the groups.
Pachymetry outcomes for S-CXL, I-CXL, and I-SCXL are summarized in Table 3 and Figure 1D. The main result of this analysis is the confirmation of previous findings that S-CXL and I-SCXL induce a significant decrease in minimum pachymetry compared to baseline (P = .0003 and .0064, respectively), whereas I-CXL, even if with significantly lower preoperative values, does not cause thinning (P = .7485). This difference between the groups was statistically significant (P = .038).
None of the patients developed infection or haze, progressed, or had other complications.
S-CXL is a well-known treatment for progressive keratoconus and there is an extensive literature showing its efficacy in both pediatric and adult patients with long-term follow-up.3,5,22,23 However, S-CXL has two main drawbacks: it is time-consuming (total time of 60 minutes) and it includes epithelial removal. The first disadvantage was overcome by the new accelerated protocols that have shown similar results (but with less flattening effect) with reduced surgical time.24,25 Conversely, previous attempts of transepithelial CXL showed contradictory results9–11 until the introduction of I-CXL, which showed promising outcomes.14–17 On the other side, ICXL with epithelial debridement was intended to be a further improvement in terms of time (5 minutes of impregnation and 10 minutes of irradiation for a total of 15 minutes) of the validated S-CXL.12
The main outcome of the study was the non-statistically significant difference between the three protocols in induced change in most of the parameters, including visual acuity, topographic indexes, and maximum keratometry after 2 years of follow-up. Conversely, I-CXL induced less corneal thinning and induced a significantly higher reduction of higher order aberrations and coma. All protocols were equally safe in terms of complications and progression rate (none in all protocols).
It is known that more than 1 year of follow-up is needed to be able to judge the effect of a CXL treatment. The original transepithelial protocol was showing promising results after 1 year,26 but when longer follow-up was assessed it showed disappointing outcomes.9
Our study provides further evidence that I-CXL is able to effectively and safely treat progressive keratoconus. Moreover, the lack of inferiority of I-CXL compared to either S-CXL and I-SCXL in most of the parameters evaluated together with the greater and quicker improvement of visual acuity might be an important advantage of this protocol. This last finding is surprising when considering the previous preclinical evidence showing an inferior riboflavin concentration compared to S-CXL27 and the known shield effect of the epithelium for UV-A (approximately 15% to 20%).28
However, it must be noted that in this study patients treated with S-CXL did not show the well-known flattening effect previously described in many studies3,5; for this reason, the comparison between S-CXL and I-CXL might be flawed. As a matter of fact, if the S-CXL group had shown the usual reduction of curvature, the difference between the groups could be significant. This should be considered as a possible limitation of the study.
However, it is also known that not all patients treated with S-CXL show flattening, so we hypothesize that this was a “unfortunate” cohort of patients that did respond to the treatment (did not progress) but also did not regress. Another possible explanation could be the fact that all patients included in this prospective study happened to be not advanced (more than 400 µm of minimal thickness). Indeed, we previously showed that these patients are less likely to have an improvement after CXL compared to patients with a corneal thickness of more than 400 µm.29
Our findings are partially in agreement with Bikbova and Bikbov,16 who reported a significant difference in CDVA between S-CXL and I-CXL at month 6 but no significant difference in any of the parameters evaluated between the two protocols after 24 months, even if S-CXL was showing a tendency to increased flattening. Additionally, 1 patient in the I-CXL group progressed despite the treatment. It must be noted that the protocol described used another iontophoresis device and riboflavin.
Conversely, using the same commercial device as our study, Cantemir et al.17 demonstrated with a 3-year follow-up study that I-CXL is not inferior to S-CXL for stopping the progression of keratoconus but is able to induce faster recovery of visual acuity. In their 2-year follow-up study, Jouve et al.30 reported that I-CXL halted the progression of keratoconus but was more efficient compared to S-CXL with regard to flattening effect.
This study adds the comparison with S-CXL and I-SCXL. To our knowledge, this is the first comparative prospective clinical study in which refractive, topographic, tomographic, and aberrometric outcomes have been analyzed in eyes with progressive keratoconus treated with I-CXL, I-SCXL, and S-CXL with 2 years of follow-up.
The previously described limitation of this study is the relatively low number of patients in each group. The total energy dose of 5.4 J/cm2 of I-CXL was used to be comparable to previous publications of S-CXL1,2 and TE-CXL6 studies that chose this dose. However, considering the epithelial shield effect, it could be advisable to increase the energy dose in I-CXL by 20% to 6.5 J/cm2.
This comparative clinical study will continue the follow-up of the patients to assess the long-term results of these two new protocols (I-CXL and I-SCXL). The 2-year results of this comparative, prospective clinical study are providing further evidence of the efficacy and safety of transepithelial CXL with iontophoresis to treat progressive keratoconus and overcome the limitations of CXL with epithelial debridement.
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- Wollensak G, Spoerl E, Seiler T. Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking. J Cataract Refract Surg. 2003;29:1780–1785. doi:10.1016/S0886-3350(03)00407-3 [CrossRef]
- Caporossi A, Mazzotta C, Baiocchi S, Caporossi T. Long-term results of riboflavin ultraviolet a corneal collagen cross-linking for keratoconus in Italy: the Siena eye cross study. Am J Ophthalmol. 2010;149:585–593. doi:10.1016/j.ajo.2009.10.021 [CrossRef]
- Vinciguerra P, Albe E, Trazza S, et al. Refractive, topographic, tomographic, and aberrometric analysis of keratoconic eyes undergoing corneal cross-linking. Ophthalmology. 2009;116:369–378. doi:10.1016/j.ophtha.2008.09.048 [CrossRef]
- Vinciguerra R, Romano MR, Camesasca FI, et al. Corneal cross-linking as a treatment for keratoconus: four-year morphologic and clinical outcomes with respect to patient age. Ophthalmology. 2013;120:908–916. doi:10.1016/j.ophtha.2012.10.023 [CrossRef]
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- Ghanem VC, Ghanem RC, de Oliveira R. Postoperative pain after corneal collagen cross-linking. Cornea. 2013;32:20–24. doi:10.1097/ICO.0b013e31824d6fe3 [CrossRef]
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- Caporossi A, Mazzotta C, Paradiso AL, Baiocchi S, Marigliani D, Caporossi T. Transepithelial corneal collagen crosslinking for progressive keratoconus: 24-month clinical results. J Cataract Refract Surg. 2013;39:1157–1163. doi:10.1016/j.jcrs.2013.03.026 [CrossRef]
- Leccisotti A, Islam T. Transepithelial corneal collagen cross-linking in keratoconus. J Refract Surg. 2010;26:942–948. doi:10.3928/1081597X-20100212-09 [CrossRef]
- Soeters N, Wisse RP, Godefrooij DA, Imhof SM, Tahzib NG. Transepithelial versus epithelium-off corneal cross-linking for the treatment of progressive keratoconus: a randomized controlled trial. Am J Ophthalmol. 2015;159:821–828. doi:10.1016/j.ajo.2015.02.005 [CrossRef]
- Vinciguerra P, Romano V, Rosetta P, et al. Iontophoresis-assisted corneal collagen cross-linking with epithelial debridement: preliminary results. Biomed Res Int. 2016;2016:3720517. doi:10.1155/2016/3720517 [CrossRef]
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|Fluence (total) (mJ/cm2)||5.4||5.4||5.4|
|Soak time and interval (minutes)||5||5||30 (q2)|
|Iontophoresis current||1 mA||1mA||NA|
|Treatment time (minutes)||9||9||30|
|Epithelium status||Iontophoresis on||Iontophoresis off||Off|
|Chromophore carrier||Trometamol and sodium-EDTA||Trometamol and sodium-EDTA||Dextran|
|Chromophore osmolarity||Slightly hypo-osmolar||Slightly hypo-osmolar||Iso-osmolar and hypo-osmolar|
|Light source||UV-X System, Peschke Meditrade GmbH, Huenenberg, Switzerland||UV-X System, Peschke Meditrade GmbH, Huenenberg, Switzerland||UV-X System, Peschke Meditrade GmbH, Huenenberg, Switzerland|
|Irradiation mode (interval)||Continuous||Continuous||Continuous|
|Protocol modification||Iontophoresis impregnation||Iontophoresis impregnation||–|
|Abbreviation in the manuscript||I-CXL||I-SCXL||S-CXL|
|Gender (M)||12 (60.0%)||16 (80.0%)||14 (70.0%)||.442|
|Mean age (y)||27.4 ± 6.6||30.3 ± 6.7||31.9 ± 9.3||.182|
| I-CXL||0.25 ± 0.15||0.15 ± 0.18||.0122|
| I-SCXL||0.17 ± 0.12||0.11 ± 0.12||.0064|
| S-CXL||0.18 ± 0.09||0.10 ± 0.10||.0017|
| I-CXL||59.1 ± 3.9||58.7 ± 4.4||.8945|
| I-SCXL||56.7 ± 5.0||56.3 ± 5.4||.6103|
| S-CXL||56.6 ± 4.2||56.6 ± 4.6||.8946|
| I-CXL||434 ± 38||439 ± 38||.7485|
| I-SCXL||474 ± 37||456 ± 51||.0064|
| S-CXL||468 ± 34||411 ± 103||.0003|
| I-CXL||1.05 ± 0.40||0.33 ± 0.19||.0007|
| I-SCXL||0.32 ± 0.16||0.28 ± 0.12||.0049|
| S-CXL||0.69 ± 0.27||0.61 ± 0.32||.2627|
| I-CXL||2.41 ± 1.03||0.67 ± 0.32||.0007|
| I-SCXL||0.52 ± 0.34||0.51 ± 0.37||.0705|
| S-CXL||1.62 ± 0.86||1.31 ± 0.99||.1084|
| I-CXL||7.80 ± 3.86||6.67 ± 3.33||.4694|
| I-SCXL||6.17 ± 3.66||5.70 ± 3.36||.2802|
| S-CXL||6.20 ± 3.40||5.65 ± 3.31||.1862|
| I-CXL||8.98 ± 5.06||8.95 ± 5.02||.1815|
| I-SCXL||7.36 ± 2.83||7.35 ± 3.20||.9612|
| S-CXL||7.72 ± 3.77||8.08 ± 4.59||.5068|
Refractive Error Before (T0) and After (T24 +24 Months) CXL
|Mean||95% CI||Mean||95% CI||Mean||95% CI|
|I-CXL||−3.37 1.21 × 19||−0.64 5.20 × 154 to −4.62 −5.75 × 142||−3.45 1.85 × 20||0.47 3.21 × 149 to −9.84 5.41 × 38||−0.09 0.64 × 22
(P = .58)||2.34 6.35 × 152 to −1.70 −6.72 × 146|
|I-SCXL||−4.20 1.17 × 169||−0.09 5.86 × 139 to −12.61 5.08 × 37||−3.85 2.38 × 176||0.82 5.79 × 138 to −12.28 6.50 × 26||0.58 1.33 × 14
(P = .88)||1.61 5.88 × 137 to −5.72 7.35 × 38|
|S-CXL||−2.8 1.89 × 9||0.38 4.00 × 148 to −7.87 5.16 × 34||−2.52 1.36 × 175||0.55 4.14 × 146 to −8.11 3.60 × 36||−0.48 0.94 × 120
(P = .77)||7.20 −6.19 × 45 to −1.81 −4.64 × 141|