paediatric age at the time of diagnosis represents a negative prognostic factor for keratoconus progression , with increased probability of corneal transplant .
particularly younger patients represent a population at high risk for more rapid progression of the disease [ 1 , 2 ] .
the long - term results reported in literature [ 35 ] have demonstrated the ability of cross - linking to slow the progression of keratoconus by a photo - polymerization reaction of stromal collagen fibres .
cross - linking photodynamic reaction is induced by the combined action of a photosensitizing substance ( riboflavina o vitamin b2 ) and ultraviolet ( uv ) a light , allowing a corneal stiffening by increasing the number of intrafibrillar , interfibrillar covalent bonds , and corneal collagen resistance against enzymatic degradation [ 68 ] .
the long - term effects of the technique are related also to a process of collagen neosynthesis with a different structure and higher molecular weight [ 912 ] which confers to the corneal stroma an increased resistance and lamellar compaction responsible of the variable functional modifications recorded after the treatment [ 1114 ] .   according to international results [ 35 ] ,
cross - linking should be the primary choice in young patient with   progressive keratoconus .
to report a comparative prospective long - term functional analysis after cross - linking in three different age groups ( 18 years , between 1926 years , and   27 years ) of patients affected by progressive keratoconus .
since 2004 to date more than 610 patients were treated by combined riboflavin uv a corneal collagen cross - linking .
present prospective nonrandomized open study comprised 516 eyes of 413 patients aged between 10 and 40 years , affected by progressive keratoconus .  
the comparative functional analysis comprised the following :   paediatric group ( 18 years and under ) included 152 eyes of 105 patients ( 29.5% ) , intermediate group ( 1926 years ) included 286 eyes of 243 patients ( 55.4% ) , adult group ( 27 years ) included 78 eyes of 65 patients ( 15.1% ) .
all patients included in the treatment protocol were affected by progressive keratoconus with a documented clinical and instrumental worsening at least in the last three months of observation .
the parameters we considered to establish keratoconus progression were worsening of ucva / bscva > 0.50 snellen lines , increase of sph / cyl > 0.50  d , increase of topographic symmetry index
sai / si > 0.50  d , increase of maximum k reading > 0.50  d , reduction of the thinnest point at optical pachometry 10  m , clear cornea at biomicroscopic examination , absence of reticular dark striae at confocal laser microscopy in vivo .
we considered  significant  for the inclusion in the study the variation of at least 3 of the parameters listed above ( one clinical plus two instrumental ) .
statistical analysis was conducted by the mann - whitney u test for nonparametric data ( ucva and bscva ) and by the paired t test for parametric data ( maximum curvature power , symmetry indices and coma values ) .
the surgical procedure of corneal cross - linking with riboflavina uva was performed in all patients according to the siena protocol   using the vega cbm ( caporossi - baiocchi - mazzotta ) x linker ( cso , florence , italy ) developed in italy at the department of ophthalmology of siena university by the same authors , under intellectual property of siena university , italy .
the treatment was conducted under topical anaesthesia ( 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 10 minutes   of corneal soaking before starting uv a irradiation .
the riboflavin and dextrane solution was administered every 2.5 minutes for a total of 30 minutes of uva exposure ( 3  mw / cm ) .
treated eyes were dressed with a therapeutic soft contact lens bandage for 4 days and medicated with antibiotics ( ofloxacin drops 4  times / day ) , nonsteroidal anti - inflammatory drugs ( diclofenac drops 4  times / day ) and lachrymal substitutes , until contact lens removal .
after therapeutic corneal lens removal , fluorometholone 0.2% drops ( 3  times / day ) and lacrimal substitutes were administered for 6 to 8 weeks .
( 1 ) 152 eyes of 105 patients 18 years ( 91 eyes with followup of 12 months , 74 eyes at 24 months , 25 eyes at 36 months , 7 at 48 months ) . ( 2 )
286 eyes of 243 patients between 19 and 26 years ( 108 eyes with followup 12 months , 83 eyes at 24 months , 56 eyes at 36 months , 11 at 48 months ) .
( 3 ) 78 eyes of 65 patients   27 ( 35 eyes with followup 12 months , 25 eyes at 24 months , 12 eyes at 36 months , 8 at 48 months ) .
pre- and postoperative examination included uncorrected visual acuity ( ucva ) , best spectacle corrected visual acuity ( bscva ) , corneal topography and surface aberrometry ( cso eye top , florence , italy ) , optical pachometry ( visante oct , zeiss meditech , jena , germany ) , and in vivo confocal microscopy ( hrt ii , heidelberg , rostock cornea module , germany ) .
according to epidemiology findings , we found a male / female ratio in the whole sample of 4  :  1 ; a male / female ratio in paediatric group of 6  :  1 , and a male / female ratio of 3  :  1 in patients between 19 and 40 years old .
these results , even if in a small group , differ from epidemiological data reported in the literature   were a male / female rate of 2  :  1 is reported .
there was no statistical difference in the incidence of keratoconus between right and left eye in the whole population of 516 eyes and age - related groups .
comparative ucva in patients 18 years showed a mean gain of + 0.14 ( p = 0.0037 ) , + 0.17 ( p = 0.0043 ) , + 0.16 ( p = 0.0051 ) and + 0.2 ( p = 0.006 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively .   
patients between 19 and 26 years showed a mean gain of + 0.13 ( p = 0.0034 ) , + 0.16 ( p = 0.0041 ) , + 0.12 ( p = 0.0032 ) , and + 0.14 ( p = 0.0073 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively .   patients 27 years showed a mean gain of + 0.08 ( p = 0.0036 ) , + 0.09 ( p = 0.005 ) , + 0.12 ( p = 0.0047 ) and + 0.12 ( p = 0.0071 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively , ( figure 1 ) .  
comparative bscva in patients 18 years showed a mean gain of + 0.15 ( p = 0.0056 ) , + 0.19 ( p = 0.0031 ) , + 0.18 ( p = 0.0059 ) and + 0.21 ( p = 0.0079 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively .    patients between 19 and 26 years showed a mean gain of + 0.10 ( p = 0.0052 ) , + 0.12 ( p = 0.0045 ) , + 0.13 ( p = 0.0056 ) , and + 0.2 ( p = 0.0075 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively .  
patients 27 years showed a mean gain of + 0.07 ( p = 0.0054 ) , + 0.06 ( p = 0.0067 ) , + 0.08 ( p = 0.0069 ) , and + 0.10 ( p = 0.0075 ) snellen lines at 12 , 24 , 36 , and 48 months of followup , respectively , ( figure 2 ) . 

k 
 max in paediatric group varied by a mean of 0.7  d ( p = 0.006 ) , 0.8  d ( p = 0.0045 ) , 1.1  d ( p = 0.051 ) , and 0.9  d ( p = 0.071 ) ; in the intermediate group ( patients between 19 and 26 years ) varied by a mean of 0.6  d ( p = 0.0053 ) , 0.5  d ( p = 0.0051 ) , 0.3  d ( p = 0.0045 ) , and 0.6  d ( p = 0.0091 ) ; in adult group ( patients 27 years ) varied by a mean of 0.4  d ( p = 0.0065 ) , 0.6  d ( p = 0.0074 ) , 0.5  d ( p = 0.0095 ) , and 0.5  d ( p = 0.0091 ) , at 12 , 24 , 36 , and 48 months of follow - up , respectively for each group ( figure 3 ) .   surface asymmetry index ( sai  m ) in paediatric patients improved by a mean value of , 0.42  d ( p = 0.0054 ) , 0.18  d ( p = 0.0066 ) , 0.24  d ( p = 0.091 ) , and  0.10  d ( p = 0.096 ) ;   in the intermediate group improved by a mean of , 1.05  d ( p = 0.0032 ) , 1.14  d ( p = 0.0021 ) , 0.84  d ( p = 0.0036 ) , and 0.65  d ( p = 0.076 ) ; in adult group improved by a mean of , 0.52  d ( p = 0.0067 ) , 1.0  d ( p = 0.0077 ) , 0.17  d ( p = 0.0081 ) , and  1.11  d ( p = 0.0094 ) , ( figure 4 ) .  
topographic superior - inferior symmetry index ( si  m ) in paediatric patients varied by a mean of , + 0.3  d ( p = 0.0098),+ 0.6  d ( p = 0.011 ) , + 0.2  d ( p = 0.017 ) , and + 1.5  d ( p = 0.021 ) ; in the intermediate group changed by a mean of , 0.42  d ( p = 0.0086 ) , 0.55  d ( p = 0.0079 ) , + 0.90  d ( p = 0.091 ) , and + 0.40  d ( p = 0.099 ) ; in adult patients varied by a mean of , 0.26  d ( p = 0.0059 ) , 0.21  d ( p = 0.0048 ) , + 1.17  d ( p = 0.012 ) , and 0.21  d ( p = 0.0011 ) at 12 , 24 , 36 , and 48 months of followup , respectively , ( figure 5 ) . coma values in paediatric patients improved by a mean of 0.47  m ( p = 0.0034 ) , 0.52  m ( p = 0.0025 ) , 0.47  m ( p = 0.0022 ) , and 0.45  m ( p = 0.0054 ) ; in the intermediate group coma values decreased by a mean of 0.89  m ( p = 0.0034 ) , 0.96  m ( p = 0.0065 ) , 0.93  m ( p = 0.0074 ) , and 0.91  m ( p = 0.0081 ) ; in old patients we recorded a mean postoperative value of 0.2  m ( p = 0.0056 ) , 0.18  m ( p = 0.0045 ) , 0.21  m ( p = 0.0034 ) , and 0.19  m ( p = 0.0067 ) at 12 , 24 , 36 and 48 months of followup respectively , ( figure 6 ) .
according to the literature in   and our previous reports , keratoconus progression is more frequent and faster in younger patients under 18 years old at the time of diagnoses , with higher probability to undergo a corneal transplantation [ 1 , 3 ] .
therefore paediatric patients represent the goal of photo - induced riboflavin uv a corneal collagen cross - linking .   the italian pilot study  siena cxl paediatrics
 , conducted on a large cohort of patients with a long - term follow up , demonstrated that there was a significant and fast functional improvement in younger patients after riboflavin uv a corneal cross - linking . as we recently published , it is however impossible to exactly predict the distribution of cross - links and the geometric redistribution of newly formed collagen [ 12 , 14 ] .  
long - term comparative analysis showed that functional results after riboflavin uv a corneal collagen cross - linking among paediatric patients were slightly better , but without statistically significant differences with the results recorded in the intermediate group patients . on the other hand , patients over 27 years showed a positive but poorer functional response compared with other age groups .   the mean kmax   variation and topographic surface asymmetry index results were statistically significant in the paediatric sample , particularly in the postoperative 24 months .
after the 24th month , until the 48th month , the mean data results were statistically not significant , reasonably due to reduced number of patients in the longitudinal analysis .  
the comparative aberrometric data of coma values showed a significant improvement in all analyzed groups , justifying the rapid improvement of visual acuity in all treated patients . in the paediatric group of our cohort
, there was a minority of patients   ( about 5% ) that , despite the treatment , showed a worsening trend or at least an instability of keratoconus .   in our opinion , this concept
should be remarked because the disease in this age group is more aggressive and the possibility of progression higher than in the others age groups .  
the instability of certain cases should be explained by the different genetic patterns of keratoconus [ 1719 ]   with relative biochemical modifications   potentially occurring in corneal stroma associated with negative influences of some environmental factors ( allergy , atopy ) [ 2124 ] .  
every time we decide to treat a paediatric patient under 18 years affected by progressive keratoconus , parents and patient himself should be well informed about the possibility that the treatment in a minority of cases could not warrant a total and long - lasting stabilization of the disease , with the possibility to repeat the cross - linking or to undergo alternative treatments .
pilot study demonstrated the effective ability of corneal cross - linking to retard keratoconus progression in all age groups with better functional response in patients under 26 years .
treatment ensured   a long - term keratoconus stabilization in over 90% of treated cases . the lower functional response observed in patients over 27 years
plasticity  in the adult age , as well demonstrated in the literature [ 25 , 26 ] .  
cross - linking treatment may result in less effectiveness with increased failure and complication rate particularly in adult patients over 35 years , as reported in the literature . according to our long - term comparative age - related analysis and results
, the standard riboflavin uv a cross - linking with epithelium removal should be the first choice therapy of progressive keratoconus in paediatric and under-26-year old patients with corneal thickness at least of 400  m in the thinnest point .
 
 purpose . to report a comparative prospective long - term functional analysis after riboflavin uv a corneal cross - linking ( cxl ) in three different age groups of patients affected by progressive keratoconus ( kc ) . methods . functional analysis 
 comprised paediatric patients ( 18 years ) included 152 eyes ( 29.5% ) ; intermediate group ( 1926 years ) 286 eyes ( 55.4% ) , and adults ( 27 years ) 78 eyes ( 15.1% ) . 
 cxl was performed according to the siena protocol by using the vega cbm ( caporossi - baiocchi - mazzotta ) x linker ( cso , florence , italy ) at siena university by the same authors . 
 pre- and post - op examinations included ucva , bscva , corneal topography , and surface aberrometry ( cso eye top , florence , italy ) , at 48 months followup . results . 
 at 48 months followup paediatrics , intermediate , and adult patients showed a mean gain in ucva of + 0.2 , + 0.14 and + 0.12 snellen lines . 
 bscva gained by a mean of + 0.21 , + 0.2 , and + 0.1 snellen lines .   
 kmax was reduced by a mean value of   0.9  d , 0.6  d , and 0.5  d , respectively . 
 coma values improved by a mean of 0.45  m , 0.91  m , and 0.19  m , respectively . 
 treatment ensured a long - term keratoconus stabilization in over 90% of treated patients . 
 conclusion . according to our long - term comparative results 
 , epithelium - off riboflavin uv a cross - linking should be the first choice therapy of progressive kc , particularly in paediatric age and patients under 26 years . 
1. Introduction
2. Purpose
3. Methods
4. Results
5. Discussion