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Photoactivated Chromophore for Moderate to

Severe Infectious Keratitis as an Adjunct

Therapy: A Randomized Controlled Trial


 PURPOSE: To evaluate the efficacy of photoactivated leading to corneal perforation or endophthalmitis is not
chromophore for infectious keratitis (PACK-CXL) in uncommon. A new paradigm-changing treatment able to
the treatment of patients with moderate to severe infec- enhance microbial eradication and improve treatment out-
tious keratitis as adjunct therapy to the topical medication comes with fewer side effects needs to be established.
treatment. Corneal collagen cross-linking is a procedure in which
 DESIGN: Randomized clinical trial. the photosensitizer riboflavin and ultraviolet A (UVA)
 METHODS: Thirty eyes from 30 patients with moderate irradiation are used. This procedure preliminarily aims to
to severe infectious keratitis were randomized to receive strengthen the corneal stroma, thereby improving the
either standard treatment plus PACK-CXL (n [ 15) or corneal biomechanics in ectatic corneal disorders.1 Soon
standard treatment alone (control group, n [ 15). The after the acceptance of this concept, corneal cross-linking
primary outcome was the sizes of stromal infiltrates was proposed to be effective for treating infectious keratitis
measured on slit-lamp photographs 30 days after treat- based on the disinfectant properties of photoactivated
ment. The secondary outcomes were the sizes of epithelial chromophore. The possible mechanisms include inhibition
defects, the complication rates, and best pinhole- of microbial replication, intercalation of the chromophore
corrected visual acuity (BPVA). with microbial nucleic acid,2 direct damage to the path-
 RESULTS: The median (interquartile range [IQR]) sizes ogen cell walls by reactive oxygen free radicals,3,4
of stromal infiltrates at day 30 were 5.0 mm2 increased resistance of the cross-linked cornea to enzymatic
(0–23.0 mm2) in the PACK-CXL group and 10.6 mm2 damage, and changing of the ocular surface environment.5
(1.1–16.3 mm2) in the control group (median difference However, the clinical evidence in terms of collagen cross-
0, 95% CI L7.0 to 0, P [ .66). The median (IQR) sizes linking efficacy for keratitis is still inconclusive.6–9
of epithelial defects were 0.7 mm2 (0–6.3 mm2) and In this study, we evaluated the efficacy of photoactivated
4.6 mm2 (0–10.2 mm2) in the PACK-CXL group and chromophore for infectious keratitis (PACK-CXL) as an
control group, respectively (median difference L3.0, adjunct to medical treatment for patients with moderate
95% CI L0.8 to 0, P [ .41). The complication rates to severe infectious keratitis.
and BPVA after treatment were comparable between
 CONCLUSIONS: Standard treatment combined with
PACK-CXL did not provide any advantageous effect METHODS
over standard treatment alone in moderate to severe in-
fectious keratitis over a 30-day period. (Am J THE INSTITUTIONAL REVIEW BOARD, FACULTY OF MEDI-
Ophthalmol 2016;165:94–99. Ó 2016 Elsevier Inc. All cine, Chulalongkorn University approved and monitored
rights reserved.) this randomized controlled trial, which adhered to the te-
nets of the Declaration of Helsinki. The trial was registered
with (NCT01831206). The sample size

NFECTIOUS KERATITIS IS A LEADING CAUSE OF RAPID for the study was calculated using a superiority design for-
and devastating visual loss worldwide, especially in mula with power of 0.8 and a 2-tailed significance level
developing countries. Despite topical broad-spectrum of .05 to detect 7 mm2 difference in areas of stromal infiltra-
medical therapies being used initially, infectious keratitis tion with standard deviation of 6. This provided a sample
size of 15 patients per group. Written informed consent
was obtained from all participants.
Supplemental Material available at The participants were recruited from the Department of
Accepted for publication Feb 24, 2016.
From the Department of Ophthalmology, Faculty of Medicine, Ophthalmology, King Chulalongkorn Memorial Hospital,
Chulalongkorn University, and King Chulalongkorn Memorial Hospital, Bangkok, Thailand from March 2013 to December 2014.
Bangkok, Thailand. All patients presenting with infectious keratitis underwent
Inquiries to Vannarut Satitpitakul, Department of Ophthalmology,
Faculty of Medicine, Chulalongkorn University, 1873 Rama 4 Road ophthalmic examination including best pinhole-corrected
Pathuwan, Bangkok, Thailand 10330; e-mail: visual acuity (BPVA), slit-lamp biomicroscopy, anterior

94 Ó 2016 ELSEVIER INC. ALL RIGHTS RESERVED. 0002-9394/$36.00
TABLE 1. Baseline Characteristics of Participants With Moderate to Severe Infectious Keratitis in the Medical Therapy Plus
Photoactivated Chromophore Group and Medical Therapy Group

Parameter PACK-CXL Group (n ¼ 15) Control Group (n ¼ 15)

Mean age (range), y 44.60 (17–73) 53.93 (15–84)

Male-to-female ratio 11:4 10:5
Moderate to severe infectious 2:13 4:11
keratitis ratio
Median size of epithelial defect 31.29 (13.48–41.61) 31.11 (19.13–45.94)
(IQR), mm2
Median size of stromal infiltration 31.89 (28.52–62.78) 31.07 (17.93–54.88)
(IQR), mm2
Hypopyon 9/15 (60%) 7/15 (46.67%)
Mean initial BPVA (logMAR) 1.75 6 0.22 1.68 6 0.32
Etiologic organisms
- Bacteria 7/15 (46.67%) 5/15 (33.33%)
- 1 Pseudomonas spp - 4 Pseudomonas spp
- 1 Enterobacter spp - 1 negative laboratory result
- 5 negative laboratory results
- Fungus 8/15 (53.33%) 10/15 (66.77%)
- 2 Fusarium spp - 5 unidentified septate hyphae
- 1 Aspergillus spp - 1 unidentified budding yeast
- 1 Purpureocillium spp - 1 Pythium spp
- 1 Pythium spp - 3 negative laboratory results
- 2 negative laboratory results

BPVA ¼ best pinhole-corrected visual acuity; IQR ¼ interquartile range.

PACK-CXL group ¼ medical therapy plus photoactivated chromophore; Control group ¼ medical therapy alone.

segment photography, and posterior segment ultrasonogra- medical therapy for bacterial keratitis included hourly instil-
phy. The severity of keratitis was graded by slit-lamp bio- lation of fortified cefazolin (50 mg/mL; BIOLAB, Samutpra-
microscopy using a modification of Jones’s grading.10 karn, Thailand) and fortified amikacin (20 mg/mL; Atlantic
Ulcers that were 2-6 mm in size and infiltration that Lab, Bangkok, Thailand); the primary medical therapy for
involved the mid stroma but not beyond the posterior fungal keratitis included hourly topical application of
one third of the corneal stroma were graded as moderate in- amphotericin B (1.0 mg/mL; Bharat Serums and Vaccines,
fectious keratitis. Ulcers either involving the posterior one Maharashtra, India) and topical natamycin (50 mg/mL;
third of the cornea or that were more than 6 mm in size Alcon, Bangkok, Thailand). In case of positive clinical
were graded as severe infectious keratitis. response, the medications were tapered based on the judg-
Consecutive cases of patients aged older than 6 years ment of 1 clinician (N.K.). However, if the ulcers
with moderate to severe infectious keratitis were enrolled progressed, the regimens were changed according to the re-
in the study. Pregnant patients or patients with a history sults of the microbiological evaluation. All participants were
or evidence of herpetic keratitis, parasitic keratitis, corneal treated as inpatients until the medications were tapered to
perforation, autoimmune diseases, endophthalmitis, or applications of fewer than 4 times a day. No topical or sys-
corneal thickness less than 400 mm by ultrasound pachy- temic corticosteroids were used during the study period.
metry were excluded. For the participants randomized to PACK-CXL, corneal
After enrollment, participants were randomized to collagen cross-linking with UVA and riboflavin was
receive standard treatment with or without PACK-CXL us- performed under topical anesthesia on the first day of pre-
ing simple randomization. Sealed envelopes, used to sentation. The corneal limbus was shielded by a Merocel
conceal the randomization, were opened after enrollment ring (Medtronic, Inc, Dublin, Ireland). Riboflavin (Medio-
of each participant. A microbiological evaluation included CROSS [Peschke Meditrade GmbH, Germany] 0.1% ribo-
Gram stain, KOH preparation, and cultures; the samples flavin/20% dextran solution) was administered to the
were obtained by corneal scraping in all participants. cornea every 2 minutes for an initial period of 30 minutes
Participants randomized to standard treatment received and then every 5 minutes for a further 30 minutes during
standard medical therapy according to the patient’s history, UVA illumination. The epithelium was not removed as
clinical findings, and initial laboratory results. The primary there were epithelial defects overlying the ulcers. The


UV-X lamp (Peschke Meditrade GmbH, Hünenberg,
Switzerland) was used to deliver UVA (365 nm with 3.0

P Valuea


TABLE 2. Areas of Stromal Infiltration in Participants With Moderate to Severe Infectious Keratitis at Day 7 and Day 30 After Treating With Medical Therapy Plus Photoactivated
mW/cm2) for 30 minutes. After the PACK-CXL treat-
ment, the participants received standard medical treatment

1.1 (16.9 to 0)
Median Difference
as described previously.
Fungal Keratitis Cases (Median, IQR; mm2)

6.0 (12.4
The results of measurement of the BPVA, slit-lamp ex-

(95% CI)

to 10.4)
amination, and anterior segment photography with and
without fluorescein staining were recorded at the initial
presentation and on day 7 and day 30. The logarithm of
the minimal angle of resolution visual acuity values equal
Control Group

40.5 (12.5
to 59.7)

to 23.9)
(n ¼ 10)

12.6 (4.4
to or below the counting fingers level were recorded as fol-
lows: counting fingers, 1.7; hand movements, 1.8; light
perception, 1.9; and no light perception, 2.0. All anterior
9.1 (0 to 27.4)

segment photography was masked and 1 investigator

Group (n ¼ 8)

(V.S.) randomly assessed the images for the sizes of stromal

30.2 (19.9
to 64.8)

infiltrates and epithelial defects using Image-Pro version

7.0 (Media Cybernetics, Rockville, Maryland, USA).
The primary outcome was the size of the stromal infiltrates;
P Valuea

the secondary outcomes were the sizes of the epithelial de-



fects, the complication rates after treatment, and BPVA at

day 30. The results were compared between the 2 groups us-
0.73 (7.8 to 4.6)
Chromophore Compared to Medical Therapy Alone

Median Difference
Bacterial Keratitis Cases (Median, IQR; mm2)

ing Mann-Whitney U test for both the sizes of the stromal

(95% CI)

infiltrates and epithelial defects; the unpaired t test was

10.3 (3.6
to 17.1)

PACK-CXL group ¼ medical therapy plus photoactivated chromophore; Control group ¼ medical therapy alone.

used for the complication rates and BPVA (IBM SPSS sta-
tistics, Version 22.0; IBM Corp, Armonk, New York,
USA). An alpha value of 0.05 was considered significant.
21.7 (8.1 to 25.4)

8.6 (0 to 14.9)
Control Group
(n ¼ 5)

Group (n ¼ 7)

18.1 (13.6

to 44.6)

to 19.1)
0.8 (0.3


enrolled in this study. The ulcers in all participants
involved the visual axis. The baseline characteristics
P Valuea

were comparable in both groups (Table 1). The clinical



pictures suggested that 7 patients in the PACK-CXL group

and 5 patients in the control group had bacterial keratitis.
0 (7.0 to 0)
Median Difference

Eight and 10 cases, respectively, in the PACK-CXL group

4.76 (0.74
(95% CI)

to 6.36)

and control group were treated for fungal keratitis. Five of

All Cases (Median, IQR; mm2)

the 15 participants (33.33%) in each group had a history of

contact lens wear. Seven (46.67%) and 8 (53.33%) partic-
ipants in the PACK-CXL and control groups, respectively,
25.4 (10.3 to 48.6)

10.6 (1.1 to 16.3)

had a history of infection with a vegetative origin. Both

Control Group
(n ¼ 15)

participants with a Pythium infection had been exposed

to contaminated water. The microbiological laboratory re-
sults are shown in Table 1.
The median sizes of stromal infiltrates in the PACK-
Mann-Whitney U test.

CXL group at days 7 and 30 were 22.1 mm2 (interquartile

5.0 (0 to 23.0)
Group (n ¼ 15)

range [IQR], 16.0–53.6 mm2) and 5.0 mm2 (IQR,

22.1 (16.0
to 53.6)

0–23.0 mm2) and those in the control group were

25.4 mm2 (IQR, 10.3–48.6 mm2) and 10.6 mm2 (IQR,
1.1–16.3 mm2), respectively. There was no significant
Day 30

(P ¼ .50 and P ¼ .66) differences between the groups. Sub-

Day 7

group analysis in the bacterial and fungal keratitis samples

also showed no significant differences (Table 2).


The median sizes of the epithelial defects at days 7 and
30 in the PACK-CXL group were 23.0 mm2 (IQR, 13.5–

P Valuea


41.6 mm2) and 0.7 mm2 (IQR, 0–6.3 mm2), respectively;
TABLE 3. Areas of Epithelial Defect in Participants With Moderate to Severe Infectious Keratitis at Day 7 and Day 30 After Treating With Medical Therapy Plus Photoactivated the median sizes of the epithelial defects in the control

1.48 (33.7 to 0)
group were 16.9 mm2 (IQR, 6.3–39.5 mm2) and 4.6 mm2

Median Difference
Fungal Keratitis Cases (Median, IQR; mm2)
(IQR, 0–10.2 mm2), respectively. However, there were

5.3 (12.2
(95% CI)

to 0.62)
no significant (P ¼ .68 and P ¼ .41) differences between
the 2 groups. Subgroup analysis regarding etiology also
showed no significant differences (Table 3).
Therapeutic keratoplasty was performed in 2 cases in the
Control Group

34.4 (6.6

3.8 (0.7
to 47.1)

to 22.8)
(n ¼ 10)

PACK-CXL group and 3 cases in the control group owing

to uncontrolled infection with corneal perforation. One
case in the control group underwent evisceration owing
1.42 (0 to 13.6)

to endophthalmitis. Recurrent stromal infiltrates into the

Group (n ¼ 8)

corneal graft with development of endophthalmitis

to 31.8)
23.7 (9.8

occurred in 1 case in the PACK-CXL group and the eye

was eviscerated.
Among cases without further intervention, the BPVA at
P Valuea



day 30 improved from baseline in 8 of 12 cases (66.7%) in

the PACK-CXL group and 7 of 11 cases (63.6%) in the
4.2 (13.2 to 7.3)

control group. The mean BPVAs was 1.48 6 0.64 in the

Chromophore Compared to Medical Therapy Alone

Median Difference

0 (0 to 2.9) 7.7 (0 to 9.9) 8.0 (6.5 to 0)

Bacterial Keratitis Cases (Median, IQR; mm2)

standard treatment plus PACK-CXL group and 1.20 6

(95% CI)

0.67 in the standard treatment group. There was no signif-

PACK-CXL group ¼ medical therapy plus photoactivated chromophore; Control group ¼ medical therapy alone.

icant difference in the mean BPVAs on day 30 after treat-

ment (P ¼ .32, 95% CI 0.29 to 0.85).
Control Group

to 16.87)
12.4 (7.23
(n ¼ 5)

Group (n ¼ 7)

to 44.23)
31.3 (14.1


ity to treat infectious keratitis through various proposed
mechanisms.2–5 Moreover, the microbial eradicating
property of PACK-CXL against Pseudomonas aeruginosa,
P Valuea



Staphylococcus aureus, Staphylococcus epidermidis, Strepto-

coccus pneumoniae, Candida albicans, Fusarium spp, and
Aspergillus fumigatus including multidrug-resistant strains
1.3 (10.0 to 0.8)
Median Difference

was also verified by various in vitro studies.11–16

3.0 (8.0 to 0)
(95% CI)

In the current randomized controlled trial, we compared

All Cases (Median, IQR; mm2)

the exact sizes of stromal infiltrates in patients treated with

medical treatment with or without PACK-CXL in the early
period after cross-linking. Primary medical therapies were
chosen according to the common isolated microorganisms,
16.9 (6.3 to 39.5)

Day 30 0.7 (0 to 6.3) 4.6 (0 to 10.2)

Control Group

which were P aeruginosa, S pneumoniae, Fusarium spp,

(n ¼ 15)

Aspergillus spp and Curvularia spp.17,18

Initially, we believed that PACK-CXL treatment that
induced massive and simultaneous death of microorgan-
Mann-Whitney U test.

isms might debulk the infectious load. This could be used

Group (n ¼ 15)

as a 1-time adjunctive treatment to hasten the resolution


23.0 (13.5
to 41.6)

of moderate to severe infectious keratitis. The antimicro-

bial efficacy of PACK-CXL is shown in many case series.
Systematic reviews and meta-analysis of reported cases sug-
gested that from the pooled results of 12 articles, CXL had a
Day 7

favorable effect on the blocking of corneal melting in 85%

of eyes (77%–91%), with very few complications.6


As far as we know, the current study is the first to report a oomycete, is a fungal-like organism that displays charac-
randomized, single-masked, controlled trial. We compared teristics of sparse septate or aseptate hyphae. The clinical
the exact areas of corneal infiltrate changes 30 days after response to antifungal therapy of P insidiosum keratitis is
PACK-CXL and we found that the adjunctive PACK- poor because of the lack of chitin or ergosterol in the
CXL with standard medical treatment did not speed up cellular wall. Unfortunately, the response of PACK-CXL
healing of the infiltrate sizes. The complication rates and in P insidiosum keratitis was nonsignificant. However,
BPVA after treatment were comparable between groups. there was no re-infection in this patient after therapeutic
Re-infection after therapeutic keratoplasty was found in 1 keratoplasty.
of 3 cases in the PACK-CXL group, which was approxi- On the first day after PACK-CXL treatment, 11 partici-
mately the same as reported previously in patients not pants had increased conjunctival injection, cells, and flare
treated with PACK-CXL.19 in the anterior chamber. Eight participants also had
The current results agreed with those from a randomized increased hypopyon. No significant increase in pain
study of advance infectious keratitis with coexisting corneal severity or corneal melting was observed and all signs
melting by Said and associates.7,20 They concluded that evenly improved within 2 days. These could be explained
PACK-CXL did not shorten the healing time in advance in- by 2 mechanisms, including extensive death of microorgan-
fectious keratitis with corneal melting. The study also isms that induced release of endotoxins or a Jarisch-
showed a clear but nonsignificant trend in decreasing the Harxheimer reaction21; and a phototoxic and thermal ef-
rate of corneal perforation and recurrent infection. In fect of UVA on the ocular surface.22
contrast, the randomized study of Uddaraju and associates9 The lack of effect of PACK-CXL in the current study
on recalcitrant deep stromal fungal keratitis and after the may be due to the fact that most infiltrates were deep to
initial 2 weeks of topical antifungal therapy, the study was posterior corneal stroma, as shown in the report of
stopped before complete enrollment because of a signifi- Abbouda and associates.23
cantly higher rate of corneal perforation in the PACK- This trial had some important limitations. The trial
CXL group vs the no treatment group. However, the authors enrolled a relatively small number of participants. Despite
suggested that further studies be undertaken in which the the trend of smaller sizes of infiltrates and epithelial defects
fungal keratitis is limited only to the anterior stroma. in the PACK-CXL group, we cannot report any significant
Until now, only 1 randomized study has reported the ad- advantages of the treatment over the 30-day trial period.
vantages of PACK-CXL in patients with moderate bacterial Anterior segment optical coherence tomography can be
keratitis. Bamdad and associates8 reported that PACK-CXL used to identify the extent and depth of the inflammation.
helped to shorten the healing time regarding epithelializa- However, the stromal demarcation line after PACK-CXL
tion and areas of infiltration on days 7 and 14 after treatment. may be difficult to evaluate owing to hyperreflective stroma
Compare to our study, the studies of Said and associates,7 corresponding to the infiltrates. PACK-CXL combined
Uddaraju and associates,9 and Bamdad and associates8 were with medical therapy is still limited. To improve the
unmasked and the corneal epithelial tissue was removed outcome of sight-threatening infectious keratitis, especially
before PACK-CXL treatment. Epithelium removal itself in advanced cases, future studies of other PACK-CXL reg-
might enhance topical drug penetration and enlarged the imens with increased UVA exposure time and/or energy
initial sizes of epithelial defects in the PACK-CXL group should be evaluated.
only. Therapeutic contact lens wear after PACK-CXL in In conclusion, the standard cross-linking technique as
the study of Bamdad and associates8 also confounded the adjunct to medical therapy is not superior to standard med-
epithelium healing rate. ical therapy alone in patients with moderate to severe in-
To the best of our knowledge, the current study is the fectious keratitis over a 30-day period. The appropriate
first to report a case of Pythium insidiosum keratitis cross-linking technique should be evaluated, especially in
managed with PACK-CXL. P insidiosum, a pathologic advanced cases.

financial disclosures: Ngamjit Kasetsuwan, Usanee Reinprayoon, and Vannarut Satitpitakul. All authors attest that they meet the current ICMJE criteria
for authorship.

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