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 facilitat-
ing 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: amir11khosravi@gmail.com).
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
402 | www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
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 anticollage-
nase 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.
M ATERIALS AND M ETHODS
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
 Cornea Volume 34, Number 4, April 2015
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 slit-
lamp 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 randomiza-
tion 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 insert-
ing 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 (UV-
X; 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.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
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 transplanta-
tion (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, includ-
ing 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 | 403
 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)
Gr ade of Ulcer s, Mean
Day After Beginning
of Tr eatment CXL Gr oup Contr ol Gr oup P CXL Gr oup
1 2 2 . 0.99
7 1.81 1.93 0.56
14 0.69 1.70 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 proce-
dures in 2 patients. Saglk et al16 reported on a 68-year-old
man with diabetes mellitus and unilateral severe corneal ulcer
FIGURE 1. Grade of ulcers in CXL and control groups.
404 | www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 4, April 2015
Ar ea of Epithelial Defects, Ar ea of Infiltr ates,
Mean 6 SD, mm 2 Mean 6 SD, mm 2
Contr ol Gr oup P CXL Gr oup Contr ol Gr oup P
17.00 6 5.39 19.63 6 4.99 0.16 19 6 5.15 19.25 6 5.02 0.89
7.94 6 4.47 14.94 6 6.05 0.001 9.31 6 3.84 14.88 6 1.11 0.001
1.25 6 1.77 8.31 6 6.75 0.001 3.63 6 2.83 9.63 6 1.21 , 0.001
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 Strep-
tococcus pneumoniae, P. aeruginosa, as well as multidrug-
resistant P. aeruginosa.
During cross-linking, free radicals are produced and
interfere with the microbial cell wall.26,27 The by-products of
FIGURE 2. Mean area of epithelial defects (mm 2) in CXL and
control groups.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
 Cornea Volume 34, Number 4, April 2015
FIGURE 3. Mean area of infiltrates (mm 2) 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 enzy-
matic 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.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Treatment of Moderate Bacterial Corneal Ulcers
ACKNOWLEDGEM ENTS
This article has been derived from the thesis by Hossein
Malekhosseini - Grant No : 5685
REFERENCES
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 keratop-
athy. 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.
www.corneajrnl.com | 405
 Bamdad et al
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.
406 | www.corneajrnl.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Cornea Volume 34, Number 4, April 2015
28. Baier J, Maisch T, Maier M, et al. Singlet oxygen generation by UVA light
exposure of endogenous photosensitizers. Biophys J. 2006;91:1452–1459.
29. Hiraku Y, Ito K, Hirakawa K, et al. Photosensitized DNA damage and its
protection via a novel mechanism. Photochem Photobiol. 2007;83:
205–212.
30. Iakovleva M, Kozel’tsev V. Proteolysis of collagen by several species of
micromycetes and spore-forming bacteria [in Russian]. Prikl Biokhim
Mikrobiol. 1994;30:121–126.
31. Spoerl E, Mrochen M, Sliney D, et al. Safety of UVA-riboflavin
crosslinking of the cornea. Cornea. 2007;26:385–389.
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.