Rose Bengal Electromagnetic Activation With Green Light for Infection Reduction
Phase: PHASE3
Conditions: Acanthamoeba Keratitis, Fungal Keratitis
Sponsor: University of California, San Francisco

Steroids and Cross-linking for Ulcer Treatment (SCUT II), 
Rose Bengal Electromagnetic Activation with Green 
light for Infection Reduction (REAGIR), 
and 
Rose Bengal Electromagnetic Activation with Green 
light for Infection Reduction II (REAGIR II) 
Trials 
Protocol Number: 2.0 
National Clinical Trial (NCT) Identified Numbers:  NCT04097730, NCT05110001 
Principal Investigators: Tom Lietman and Jennifer Rose-Nussbaumer 
Sponsor: University of California, San Francisco 
Funded by: National Eye Institute 
Version Number:  6.0 
3 September 2024 
 
 
Summary of Changes from Previous Version: 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
Throughout 
Addition of UNIFESP as an enrollment center 
for REAGIR II: increasing REAGIR II target 
enrollment to 60, and including prednisolone 
acetate 1% as study drug 
 
Section 9 
Added Bayesian Analysis 
 

  
NIH-FDA Clinical Trial Protocol for SCUT II – v6.0  3 September 2024 
ii 
Table of Contents 
STATEMENT OF COMPLIANCE ............................................................................................................................ 1 
1 
PROTOCOL SUMMARY .............................................................................................................................. 1 
1.1 
Synopsis ............................................................................................................................................... 1 
1.2 
SchemA ................................................................................................................................................ 8 
1.3 
Schedule of Activities (SoA) .............................................................................................................. 9 
2 
INTRODUCTION ........................................................................................................................................... 9 
2.1 
Study Rationale ................................................................................................................................... 9 
2.2 
Background ........................................................................................................................................ 11 
2.3 
Risk/Benefit Assessment ................................................................................................................. 14 
2.3.1 
Known Potential Risks .................................................................................................. 14 
2.3.2 
Known Potential Benefits ............................................................................................. 14 
2.3.3 
Assessment of Potential Risks and Benefits ............................................................. 14 
3 
OBJECTIVES AND ENDPOINTS ............................................................................................................. 15 
4 
STUDY DESIGN .......................................................................................................................................... 17 
4.1 
Overall Design ................................................................................................................................... 17 
4.2 
Scientific Rationale for Study Design ............................................................................................. 18 
4.3 
Justification for Dose ........................................................................................................................ 18 
4.4 
End of Study Definition ..................................................................................................................... 18 
5 
STUDY POPULATION ................................................................................................................................ 19 
5.1 
Inclusion Criteria ............................................................................................................................... 19 
5.2 
Exclusion Criteria .............................................................................................................................. 19 
5.3 
Lifestyle Considerations ................................................................................................................... 19 
5.4 
Screen Failures ................................................................................................................................. 20 
5.5 
Strategies for Recruitment and Retention ..................................................................................... 20 
6 
STUDY INTERVENTION ............................................................................................................................ 20 
6.1 
Study Intervention(s) Administration .............................................................................................. 20 
6.1.1 
Study Intervention Description .................................................................................... 20 
6.1.2 
Dosing and Administration ........................................................................................... 23 
6.2 
Preparation/Handling/Storage/Accountability ............................................................................... 25 
6.2.1 
Acquisition and accountability ..................................................................................... 25 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ............................................... 25 
6.2.3 
Product Storage and Stability ...................................................................................... 25 
6.2.4 
Preparation ..................................................................................................................... 25 
6.3 
Measures to Minimize Bias: Randomization and Blinding .......................................................... 26 
6.4 
Study Intervention Compliance ....................................................................................................... 26 
6.5 
Concomitant Therapy ....................................................................................................................... 26 
6.5.1 
Rescue Medicine ........................................................................................................... 26 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL ................................................................................................................... 27 
7.1 
Discontinuation of Study Intervention ............................................................................................ 27 
7.2 
Participant Discontinuation/Withdrawal from the Study .............................................................. 27 
7.3 
Lost to Follow-Up .............................................................................................................................. 27 
8 
STUDY ASSESSMENTS AND PROCEDURES ..................................................................................... 27 
8.1 
Efficacy Assessments ...................................................................................................................... 27 
8.2 
Safety and Other Assessments ...................................................................................................... 28 
8.3 
Adverse Events and Serious Adverse Events .............................................................................. 28 

  
NIH-FDA Clinical Trial Protocol for SCUT II – v6.0  3 September 2024 
iii 
8.3.1 
Definition of Adverse Events (AE) ............................................................................... 28 
8.3.2 
Definition of Serious Adverse Events (SAE) ............................................................. 28 
8.3.3 
Classification of an Adverse Event ............................................................................. 29 
8.3.4 
Time Period and Frequency for Event Assessment and Follow-Up ...................... 29 
8.3.5 
Adverse Event Reporting ............................................................................................. 30 
8.3.6 
Serious Adverse Event Reporting ............................................................................... 30 
8.3.7 
Reporting Events to Participants ................................................................................. 30 
8.3.8 
Events of Special Interest ............................................................................................ 30 
8.3.9 
Reporting of Pregnancy ................................................................................................ 30 
8.4 
Unanticipated Problems ................................................................................................................... 31 
8.4.1 
Definition of Unanticipated Problems (UP) ................................................................ 31 
8.4.2 
Unanticipated Problem Reporting ............................................................................... 31 
8.4.3 
Reporting Unanticipated Problems to Participants ................................................... 31 
9 
STATISTICAL CONSIDERATIONS .......................................................................................................... 32 
9.1 
Statistical Hypotheses ...................................................................................................................... 32 
9.2 
Sample Size Determination ............................................................................................................. 33 
9.3 
Populations for Analyses ................................................................................................................. 34 
9.4 
Statistical Analyses ........................................................................................................................... 35 
9.4.1 
Analysis of the Primary Efficacy Endpoint ................................................................. 35 
9.4.2 
Analysis of the Secondary Endpoints ......................................................................... 36 
9.4.3 
Safety Analyses ............................................................................................................. 36 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .............................. 37 
10.1 
Regulatory, Ethical, and Study Oversight Considerations .......................................................... 37 
10.1.1 
Informed Consent Process .......................................................................................... 37 
10.1.2 
Study Discontinuation and Closure ............................................................................. 37 
10.1.3 
Confidentiality and Privacy ........................................................................................... 38 
10.1.4 
Future Use of Stored Specimens and Data ............................................................... 39 
10.1.5 
Key Roles and Study Governance .............................................................................. 39 
10.1.6 
Safety Oversight ............................................................................................................ 39 
10.1.7 
Clinical Monitoring ......................................................................................................... 39 
10.1.8 
Quality Assurance and Quality Control ...................................................................... 40 
10.1.9 
Data Handling and Record Keeping ........................................................................... 40 
10.1.10 
Protocol Deviations ....................................................................................................... 41 
10.1.11 
Publication and Data Sharing Policy .......................................................................... 41 
10.1.12 
Conflict of Interest Policy .............................................................................................. 42 
10.2 
Additional Considerations ................................................................................................................ 42 
10.3 
Abbreviations ..................................................................................................................................... 43 
10.4 
Protocol Amendment History ........................................................................................................... 44 
11 
REFERENCES ............................................................................................................................................. 45 
 

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1 
STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonisation Good 
Clinical Practice (ICH GCP) and the following:  
 
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR 
Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of NIH-funded clinical trials have 
completed Human Subjects Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
1 
 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS  
Title: 
Steroids and Cross-linking for Ulcer Treatment (SCUT II), Rose Bengal 
Electromagnetic Activation with Green light for Infection Reduction 
(REAGIR), and Rose Bengal Electromagnetic Activation with Green light for 
Infection Reduction II (REAGIR II) 
 
 
Study Description: 
 
The Steroids and Cross-linking for Ulcer Treatment (SCUT II) and Rose 
Bengal Electromagnetic Activation with Green light for Infection Reduction 
(REAGIR) trials are international, randomized, double-masked, sham and 
placebo-controlled, clinical trials; and Rose Bengal Electromagnetic 
Activation with Green light for Infection Reduction II (REAGIR II) is a 
randomized, double-masked feasibility study.  
The purpose of these studies is to determine differences in 6-month visual 
acuity between medical antimicrobial treatments alone versus 
antimicrobial treatment plus corneal cross-linking (CXL), as well as to 
further evaluate findings from subgroup analyses of SCUT.  
 
SCUT II 
Patients presenting to one of the Aravind Eye Hospitals in India, to the 
University of California San Francisco (UCSF), or to Bascom Palmer Eye 
Institute at the University of Miami with smear-positive typical (i.e. non-
Nocardia or Mycobacteria) bacterial corneal ulcers and moderate to 
severe vision loss, defined as Snellen visual acuity of 20/40 or worse, will 
be eligible for inclusion. Those who agree to participate will be 
randomized to one of three treatment groups:  

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2 
• 
Group 1, Standard therapy: topical 0.5% moxifloxacin plus topical 
placebo plus sham UVX 
• 
Group 2, Early steroids: topical 0.5% moxifloxacin plus topical 
difluprednate 0.05% plus sham UVX  
• 
Group 3, UVX plus early steroids: topical 0.5% moxifloxacin plus 
topical difluprednate 0.05% plus UVX 
 
REAGIR 
Patients presenting to one of the Aravind Eye Hospitals in India or to the 
Federal University of São Paulo in Brazil (UNIFESP) with either smear or 
culture positive fungal or acanthamoeba keratitis or smear and culture 
negative corneal ulcers and moderate to severe vision loss, defined as 
Snellen visual acuity of 20/40 or worse, will be eligible for inclusion. Those 
who agree to participate will be randomized to one of two treatment 
groups:  
• 
Group 4, Sham RB-PDT: topical chlorhexidine gluconate 
0.02%/PHMB 0.02% (acanthamoeba), moxifloxacin 0.5% 
(smear/culture negative) or natamycin 5%/Amphotericin B 0.15% 
(fungal keratitis) plus sham RB-PDT 
• 
Group 5, RB-PDT: topical chlorhexidine gluconate 0.02%/PHMB 
0.02% (acanthamoeba), moxifloxacin 0.5% (smear/culture 
negative) or natamycin 5%/Amphotericin B 0.15% (fungal keratitis) 
plus RG-PDT 
 
REAGIR II 
Patients presenting to one of the Aravind Eye Hospitals in India or to the 
Federal University of São Paulo in Brazil (UNIFESP) with smear-positive 
and/or culture positive typical (I.e. non-Nocardia or Mycobacteria) 
bacterial corneal ulcers and moderate to severe vision loss, defined as 
Snellen visual acuity of 20/40 of worse, will be eligible for inclusion. Those 
who agree to participate will be randomized to one of two treatment 
groups: 
• 
Group 6, RB-PDT Plus Early Steroids: topical 0.5% moxifloxacin plus 
topical difluprednate 0.05%/prednisolone acetate 1% plus RB-PDT 
• 
Group 7, Sham RB-PDT Plus Early Steroids: topical 0.5% 
moxifloxacin plus topical difluprednate 0.05%/prednisolone 
acetate 1% plus sham RB-PDT 
 
Objectives: 
 
Specific Aim 1: To determine if corneal cross-linking with riboflavin (UVX) 
is a beneficial adjuvant in the treatment of smear- and/or culture-
positive bacterial ulcers. 
a. We anticipate that UVX will result in better best spectacle 
corrected visual acuity (BSCVA) at 6 months compared with 
antibiotic alone.  
b. We anticipate that UVX will result in faster microbiological 
cure rates, smaller scar size and lower rate of corneal 
perforation compared with antibiotic alone.   

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3 
Specific Aim 2: To determine if early topical steroids are a beneficial 
adjuvant in the treatment of smear- and/or culture-positive bacterial 
ulcers. 
a. We hypothesize that those randomized to early topical 
steroids will have improved BSCVA at 6 months compared 
with antibiotic alone.  
Specific Aim 3: To determine which ulcer characteristics predict the most 
benefit from the addition of adjuvant corneal cross-linking and/or early 
steroids. 
a. We hypothesize that culture positive bacterial ulcers with drug 
resistant organisms, as measured by MIC50, will benefit more 
from adjuvant UVX than those with antibiotic susceptible 
organisms.  
 
 
Specific Aim 4: To determine if cross linking with rose bengal (RB-PDT) is 
a beneficial adjuvant in the treatment of fungal, acanthamoeba, and 
smear or culture negative corneal ulcers. 
a. We hypothesize that there will be improved visual acuity at 6 
months among those randomized to adjuvant RB-PDT after 
controlling for baseline visual acuity. 
b. We anticipate that there will be smaller infiltrate/scar size and 
decreased rate of perforation and/or need for TPK at 6 months 
among RB-PDT treated patients. 
 
Specific Aim 5: To determine if corneal cross-linking with rose bengal 
(RB-PDT) is a beneficial adjuvant in the treatment of smear- and/or 
culture-positive bacterial ulcers. 
a. We hypothesize that study participants treated with topical 
antibiotics plus adjunctive RB-PDT will have better best spectacle 
corrected visual acuity at 6 months compared to those treated 
with antibiotics alone.  
 
Endpoints: 
Primary Endpoint: Best spectacle-corrected visual acuity at 6 months 
Secondary Endpoints:  
• 
Microbiological cure on repeat smear and culture at Day 2  
• 
Interaction of minimum inhibitory concentration (MIC) and CXL on 
best spectacle-corrected visual acuity 
• 
Infiltrate/scar size/depth, as measured by clinical exam, clinical 
photographs, Pentacam and OCT at week 3 and months 3, 6, and 
12 
• 
Adverse events including rate of perforation/need for TPK 
• 
BSCVA at Day 1, 3 weeks, 3 months, and 12 months 
• 
Astigmatism, higher order aberrations, topography, and 
densitometry as measured on Pentacam at week 3 and months 3, 
6, and 12  

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4 
• 
Corneal thickness and scar size/depth as measured by AS-OCT at 
week 3 and months 3, 6, and 12  
• 
Basal nerve plexus, white blood cell count, and keratocyte density 
on confocal microscopy at week 3 and months 3, 6, and 12 
• 
Visual function questionnaire (VFQ) will be compared between 
groups at 6 months, controlling for Day 1 VFQ 
• 
Pain scale at baseline, Day 1, and Day 3  
• 
Subgroup analysis of study participants receiving prior topical 
antimicrobial therapy 
 
Study Population: 
Participants with bacterial corneal ulcers (SCUT II n= 279; REAGIR II n = 60) 
and 330 participants with fungal, acanthamoeba, or smear/culture 
negative corneal ulcers (REAGIR) will be enrolled at Aravind Eye Hospitals 
in Coimbatore, Madurai, Pondicherry, and Tirunelveli in Tamil Nadu, South 
India, at the Federal University of São Paulo, Brazil, at the University of 
California San Francisco, USA, and at the University of Miami.  We 
anticipate that participants from India will be native to India, an ethnic 
group often reported as “South Asian”. Previous studies at Aravind suggest 
that approximately 39-50% of presenting ulcers will be in women. At UCSF, 
patients will be a mixture of White (non-Hispanic), Hispanic, African-
American, and Asian, and will be an estimated 50% female. The original 
SCUT trial had a median age of 53 years, with interquartile range of 40-61 
years, and we expect a similar age range in this study. Participants from 
Bascom Palmer Eye Institute will be patients that live in South Florida and 
the Caribbean islands around South Florida. South Florida has a mixture of 
Hispanic patients, White (non-Hispanic), and African American. Previous 
studies from Bascom Palmer have had a patient population younger than 
the SCUT trial. Participants from Brazil will be a mixture of White (non-
Hispanic), Hispanic, African American, and Asian, and will be an estimated 
50% female. Our most recent epidemiological study of infectious keratitis 
cases at Hospital Sao Paulo at the Federal University of Sao Paulo showed 
that the population had a mean age of 48 ± 21 years, and we expect a 
similar age range in this study. 
  
 
Phase: 
N/A 
Description of 
Sites/Facilities Enrolling 
Participants: 
Participants will be enrolled at four sites in India, one site in Brazil, and 
two sites in the United States. In India, participants will be enrolled at 
Aravind Eye Hospitals in Tamil Nadu: Coimbatore, Madurai, Pondicherry, 
and Tirunelveli. Patients will be enrolled at the Cornea Clinic in each 
hospital. In the United States, participants will be enrolled at the Proctor 

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5 
Foundation clinic at the University of California, San Francisco in San 
Francisco, CA, and at Bascom Palmer Eye Institute at the University of 
Miami in Miami, FL. In Brazil, participants will be enrolled at Hospital Sao 
Paulo, the University hospital of Escola Paulista de Medicina at the Federal 
University of Sao Paulo (Universidade Federal de Sao Paulo – UNIFESP).   
 
  
Description of Study 
Intervention: 
SCUT II 
Participants with bacterial corneal ulcers will be randomized in a 1:1:1 
fashion to one of three arms: 
 
• 
Group 1, Standard therapy: topical 0.5% moxifloxacin plus topical 
placebo plus sham UVX 
• 
Group 2, Early steroids: topical 0.5% moxifloxacin plus topical 
difluprednate 0.05% plus sham UVX  
• 
Group 3, UVX plus early steroids: topical 0.5% moxifloxacin plus 
topical difluprednate 0.05% plus UVX 
 
Participants in Groups 1, 2, & 3 will begin study drug (topical difluprednate 
0.05% or placebo) within 24 hours after enrollment. Participants in Group 
3 will receive UVX (modified Dresden protocol), and Groups 1 & 2 will 
receive sham UVX. All participants will receive a 30-minute loading dose of 
topical 0.1% riboflavin and 20% dextran T500 drops every 2 minutes. For 
Group 3, this will be followed by exposure to UV-A light at a wavelength of 
365 nm with an irradiance of 3 mW/cm2 for 30 minutes for a total dose of 
5.4 J/cm2 (UV lamp:  PESCHKE Meditrade GmbH, Hueneberg, Switzerland 
for India; Avedro KXL System, Waltham, MA, USA for USA). During 
irradiation patients will continue to receive topical riboflavin at 5-minute 
intervals. For Groups 1 & 2, sham UVX simulates this experience however 
the light will be shined adjacent to the patient, careful to avoid exposure 
to the cornea. In place of riboflavin we will use either saline drops or saline 
drops dyed with fluorescein. All study participants will have repeat corneal 
cultures 30 minutes after the UVX or sham UVX procedure. 
After UVX/sham UVX and a repeat culture, antibiotics will be initiated. All 
participants will receive 0.5% topical moxifloxacin drops every 1 hour for 2 
days, and then every 2 hours while awake until resolution of the epithelial 
defect. 
 
Participants in Groups 2 & 3 will receive one drop of 0.05% difluprednate 
four times daily beginning 24 hours after the initiation of antibiotics for 1 
week, decreased by 1 drop weekly for a total of 4 weeks of steroid 
therapy. Participants in Group 1 will receive topical placebo in place of 
steroids, with the same medication schedule described above.  
 
REAGIR 

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6 
Participants with fungal, acanthamoeba, or smear/culture negative 
corneal ulcers will be randomized in a 1:1 fashion to one of two arms: 
• 
Group 4, Sham RB-PDT: topical biguanide (acanthamoeba), 
moxifloxacin 0.5% (smear/culture negative) or polyene (fungal 
keratitis) plus sham RB-PDT 
• 
Group 5, RB-PDT: topical biguanide (acanthamoeba), moxifloxacin 
0.5% (smear/culture negative) or polyene (fungal keratitis) plus 
RG-PDT 
 
Participants in Groups 4 and 5 will begin topical antimicrobial at 
enrollment. We will use every hour topical biguanide for acanthamoeba, 
moxifloxacin 0.5% for smear/culture negative, and polyene for fungal 
keratitis. Topical agents will be applied to the affected eye every hour 
while awake for the first week, then every 2 hours while awake until 3 
weeks after enrollment. Thereafter, application will be tapered at the 
discretion of the treating physician based on response to therapy. 
 
RB-PDT/sham RB-PDT will occur within 48 hours of randomization. For 
patients with acanthamoeba keratitis, de-epithelialize central 9mm of the 
cornea. For Group 5, Rose Bengal (0.1% RB in 0.9% sodium chloride) will 
be applied in 3-minute intervals to the de-epithelialized cornea for 30 
minutes followed by irradiation with a 6mW/cm2 custom-made green LED 
source for 15 minutes (5.4J/cm2). They will undergo repeat cornea cultures 
within 24 hours after the procedure. Participants in Group 4 will undergo a 
sham procedure using topical balanced salt solution and pen light covered 
with a green filter. 
 
REAGIR II 
With SCUT II closed to enrollment, participants with typical bacterial 
corneal ulcers will be randomized to one of two possible treatment arms: 
• 
Group 6, RB-PDT Plus Early Steroids: topical 0.5% moxifloxacin plus 
topical difluprednate 0.05%/prednisolone acetate 1% plus RB-PDT 
• 
Group 7, Sham RB-PDT Plus Early Steroids: topical 0.5% 
moxifloxacin plus topical difluprednate 0.05%/prednisolone 
acetate 1% plus sham RB-PDT 
 
Participants in Groups 6 & 7 will begin topical difluprednate 0.05% (India) 
or prednisolone acetate 1% (Brazil) within 24 hours after enrollment. The 
affected eye will receive one drop of 0.05% difluprednate/prednisolone 
acetate 1% four times daily beginning 24 hours after the initiation of 
antibiotics for 1 week, decreased by 1 drop weekly for a total of 4 weeks of 
steroid therapy. 
 
RB-PDT/sham RB-PDT will occur within 48 hours of randomization. For 
Group 6, Rose Bengal (0.1% RB in 0.9% sodium chloride) will be applied in 
3-minute intervals to the de-epithelialized cornea for 30 minutes followed 
by irradiation with a 6mW/cm2 custom-made green LED source for 15 
minutes (5.4J/cm2). They will undergo repeat cornea cultures within 24 

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7 
hours after the procedure. Participants in Group 7 will undergo a sham 
procedure using topical balanced salt solution and pen light covered with a 
green filter. 
  
Study Duration: 
5 years 
Participant Duration: 
 
12 months 
  
  
 

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8 
1.2 
SCHEMA 
Prior to  
Enrollment 
 
 
 
 
 
 
 
 
 
 
 
 
 
Day 0 
 
 
Day 1 
 
 
Day 2 
 
 
Day 3 
 
 
 
Week 3 
 
 
 
3 Month 
12 Month 
 
 
 
6 Month 
 
 
 
13 Month 
12 Month 
Screen potential participants by inclusion and exclusion criteria; obtain history, document. 
Screening assessments include: 
pinhole visual acuity, culture/smear, AS-OCT, and confocal microscopy 
Perform additional baseline assessments: 
Baseline form, slit lamp examination, Pentacam, clinical photography, IOP, pain scale 
Group 1: 
Standard 
Therapy 
N=93 
 Slit lamp examination, begin study drug, BSCVA, IOP, VFQ, pain scale 
Randomize 
Group 3: 
UVX 
N=93 
Group 2: 
Early  
Steroids 
N=93 
Pain scale 
 CXL or sham CXL, repeat scraping 
Follow-up assessments of study endpoints and safety: 
Follow-up form, IOP, AS-OCT, confocal microscopy, Pentacam, clinical photography, slit 
lamp examination, BSCVA 
Follow-up assessments of study endpoints and safety: 
Follow-up form, IOP, AS-OCT, confocal microscopy, Pentacam, clinical photography, slit 
lamp examination, BSCVA 
Follow-up assessments of study endpoints and safety: 
Follow-up form, Final form, IOP, AS-OCT, confocal microscopy, Pentacam, clinical 
photography, slit lamp examination, BSCVA 
Follow-up assessments of study endpoints and safety: 
VFQ, Follow-up form, IOP, AS-OCT, confocal microscopy, Pentacam, clinical photography, 
slit lamp examination, BSCVA 
Obtain informed consent 
Group 4: 
Sham RB-PDT 
N= 165 
 
Group 5: 
RB-PDT 
N= 165 
Group 6:  
RB-PDT + 
Early 
Steroids 
N= 30 
Group 7: 
Sham RB-
PDT + Early 
Steroids 
N=30 

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9 
1.3 
SCHEDULE OF ACTIVITIES (SOA) 
 
 
Visit 1 
Day 0 
Visit 2 
Day 1 
Visit 3 
Day 2 
Visit 4 
Day 3 
Visit 5 
3-Week 
Follow-
Up 
Visit 6 
3-Month 
Follow-
Up 
Visit 7 
6-Month 
Follow-
Up 
Visit 8 
12-
Month 
Follow-
Up 
Forms 
 
 
 
 
 
 
 
 
Consent & Authorization 
X 
 
 
 
 
 
 
 
Baseline Form  
X 
 
 
 
 
 
 
 
Clinical Drawing 
X 
X 
 
 
X 
X 
X 
X 
VFQ 
 
X 
 
 
 
 
X 
 
Follow-up Form  
 
X 
 
 
X 
X 
X 
X 
Final Form 
 
 
 
 
 
 
 
X 
Procedures 
 
 
 
 
 
 
 
 
 
CXL/Sham CXL 
 
 
X 
 
 
 
 
 
Tests 
 
 
 
 
 
 
 
 
IOP 
X 
X 
 
 
X 
X 
X 
X 
Pain Scale 
X 
X 
 
X 
 
 
 
 
AS-OCT 
X 
 
 
 
X 
X 
X 
X 
Confocal Microscopy 
X 
 
 
 
X 
X 
X 
X 
Pentacam Topography 
X 
 
 
 
X 
X 
X 
X 
Clinical Photography* 
X 
 
 
 
X 
X 
X 
X 
Slit Lamp Examination   
X 
X 
 
 
X 
X 
X 
X 
BSCVA/ETDRS/MRx 
 
X 
 
 
X 
X 
X 
X 
Pinhole Visual Acuity  
X 
 
 
 
 
 
 
 
Culture/Smear 
X 
 
X+ 
 
 
 
 
 
Total Visit Time 
2 hours 
2 hours 
3 hours 
0.5 
h
1 hour 
1 hour 
1 hour 
1 hour 
*Clinical photographs also taken upon adverse events 
+Repeat scraping and culture of corneal ulcer done at time of CXL for Participants in Groups 1, 2,  3, 6, and 7; and within 24 hours of CXL 
for Groups 4 and 5 
 
 
2 
 
INTRODUCTION 
2.1 
STUDY RATIONALE  
Although antibiotics are successful at achieving microbiological cure in infectious keratitis, outcomes 
are often poor due to corneal scarring. Randomized trials comparing different antibiotic treatments 
have not been able to demonstrate superiority of one antibiotic over another.1 During acute infection 
pathogens, keratocytes and other inflammatory cells secrete enzymes that promote protein degradation 
and keratolysis with resultant opacity and irregular astigmatism. Corneal perforation can also result, 
requiring urgent surgical intervention with therapeutic penetrating keratoplasty (TPK), which has a poor 
prognosis compared with penetrating keratoplasty (PKP) performed for visual rehabilitation.2,3,4 Ideal 
treatment of corneal ulcers would address both the infection and inflammation.  
 

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10 
The Steroids for Corneal Ulcer Trial (SCUT U10EY015114) Investigated adjuvant topical steroids in 
addition to antibiotics to reduce the inflammatory response in bacterial ulcers. The trial failed to find 
benefit or harm overall; however, pre-specified subgroup analyses suggested that earlier steroid 
treatment of large, central, non-Nocardia ulcers led to better clinical outcomes.5,6 These subgroup 
analyses have led some to conclude that topical corticosteroids may be beneficial for specific subgroups 
of culture positive bacterial ulcers, and that they were most effective when administered early with 
appropriate antibiotics.5,6  
 
Corneal cross-linking (CXL) is a novel prospective therapy that may simultaneously reduce both 
inflammatory cells and bacterial pathogens.7-9 UV-A + riboflavin is effective in vitro against common 
bacterial ocular pathogens, such as Pseudomonas aeruginosa and Streptococcus pneumoniae.10 Multiple 
case reports have suggested potential benefits of UVX for treatment of bacterial keratitis, including 
resolution of resistant infection, halting of progressive melting and symptomatic improvement.11-14 In 
one small case series bacterial infections resolved even though patients were treated exclusively with 
photo-chemically activated riboflavin.15 Therapies to reduce inflammation may improve outcomes, 
however there are concerns about potentiating infection and poor healing. The possibility that UVX may 
immediately reduce the burden of infectious organisms makes subsequent anti-inflammatory treatment 
safer.  
 
Recently another CXL method has been proposed that uses rose bengal (RB) as the photosensitizer 
and green light (532 nm) and is termed RB-PDT.16 RB-PDT appears to have similar effects on corneal 
biomechanical properties, is safe for limbal stem cells and endothelium, and demonstrates less toxicity 
to keratocytes in vitro than UVX.17-22 In vitro studies have demonstrated limited benefit of UVX for fungal 
or acanthamoeba keratitis, and one randomized clinical trial also did not show a benefit of adjuvant UVX 
in filamentous fungal keratitis patients.23 In vitro RB-PDT appears to be much more effective against 
fungal and acanthamoeba isolates.24,25 CXL for infectious keratitis is identified in the literature as Rose 
Bengal Photodynamic Therapy (RB-PDT) and Photoactivated Chromophore for Infectious Keratitis (PACK-
CXL).26-28 
 
Cross-linking may reduce the risk of progressive corneal melting and perforation that can complicate 
the management of corneal ulcers. Corneal melting occurs in response to proteolytic enzymes released 
both from pathogens and leukocytes sent to combat infection.3,4 Corneal perforation is a devastating 
complication, often treated with surgical interventions such as therapeutic penetrating keratoplasty 
(TPK) which have a poor prognosis compared with penetrating keratoplasty (PKP) performed for visual 
rehabilitation.2 UVX corneas show increased in vitro resistance to keratolysis by collagenase A.29 One 
non-randomized prospective series of 40 patients found a decreased rate of perforation among those 
treated with UVX compared with controls despite the fact they had on average larger baseline ulcer 
size.28 A recent meta-analysis concluded that the probability that UVX was beneficial in inhibiting 
melting in patients with infectious keratitis was 85% (95% CI 0.77 to 0.91).7 One ex vivo study 
demonstrated that RB-PDT treated corneas have similarly increased resistance to enzymatic degradation 
by collagenase A compared to UVX despite less penetration of RB into the corneal stroma.30 
 
Clinical hypotheses are sometimes best addressed through international research collaborations. 
While corneal ulceration is an important cause of disability in technologically advanced countries, it 
occurs 10-fold more frequently in developing countries.8,31,32 Approximately 2 million people form a 
corneal ulcer every year in India alone.33,34 The high burden of disease in South Asia permits 
investigation of the optimal treatment of infectious keratitis to avoid corneal opacification, a leading 
cause of blindness worldwide.35,36 Here, we propose an international, 3-arm randomized controlled 

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clinical trial to investigate corneal crosslinking and early topical steroids as adjuvant therapies in the 
treatment of bacterial corneal ulcers.  
 
2.2 
BACKGROUND  
Infectious keratitis is a leading cause of monocular blindness worldwide.37 In the United States bacteria 
are the most common etiology for corneal ulcers, and they are often associated with contact lens use.38 
Despite appropriate antibiotic treatment, severe cases can progress rapidly, and cause permanent vision 
loss requiring corneal transplantation.38 It has been estimated that the incidence of all forms of 
infectious keratitis is 28 per 100,000 person-years,39 with bacterial keratitis affecting approximately 
30,000 individuals in the United States annually.40 The monocular vision loss associated with corneal 
ulceration has been shown to reduce vision-related quality of life.41  
The first step to the treatment of bacterial infection is to achieve microbiological cure. Clinicians weigh 
many factors when choosing an antibiotic regimen: broad-spectrum coverage, toxicity, availability and 
cost, and region -specific epidemiology of pathogens and resistance patterns. We surveyed the Cornea 
Society listserv regarding empiric antibiotic choice for presumed bacterial ulcers. Despite its toxicity, 
55% (N=57) of US physicians used fortified topical vancomycin as their first choice due to concerns over 
the emergence of resistant organisms such as Methicillin-Resistant Staphylococcus Aureus (MRSA).42 
However, a recent Cochrane review of high quality, randomized, controlled, clinical trials on the 
management of bacterial keratitis with topical antibiotics identified no antibiotic strategy that produced 
a significant difference in the relative risk of treatment success defined as complete re-epithelialization 
of the cornea or on time to cure.1 Therefore, we may not be able to dramatically improve clinical 
outcomes by antibiotic choice alone. 
The best treatment strategies for fungal keratitis have not been well characterized. Topical natamycin, 
a polyene, is the only antifungal agent approved by the Food and Drug Administration (FDA) for 
treatment of fungal keratitis. The Mycotic Ulcer Treatment Trials (MUTT) I and II, were two NEI-funded 
randomized double-masked clinical trials that found topical natamycin to be superior to topical 
voriconazole and no additional benefit of adjuvant oral voriconazole. Two recent randomized clinical 
trials also failed to demonstrate a benefit of adjuvant intrastromal voriconazole or adjuvant UVX in the 
treatment of fungal keratitis.43,44 However, natamycin is fungistatic and has limited penetration into the 
corneal layers.45 Furthermore, outcomes of fungal keratitis with topical natamycin are extremely poor as 
demonstrated in MUTT II where approximately 50% of patients had full thickness corneal perforation or 
required TPK despite topical natamycin, topical voriconazole, and adjuvant oral voriconazole.  
Although much less common, acanthamoeba keratitis (AK) may have the most prolonged and severe 
course of any corneal infection. AK is typically related to contact lens use and the incidence of these 
infections varies from as low as 1% to 4-8% of culture positive microbial keratitis cases in countries 
where contact lens use is common.46 Topical biguanides such as chlorhexidine 0.02% and 
Polyhexamethylene biguanide (PHMB) 0.02% are thought to be the most effective available medical 
therapy. However, large series suggest that only 60% of patients achieve complete cure with medical 
therapy alone by one year and that almost 50% end up with a poor outcome, defined as requiring TPK or 
having visual acuity less than 20/80.47 Furthermore, these medications are highly toxic and cause 
permanent damage to delicate ocular structures such as limbal stem cells and trabecular meshwork.48 
 
Even if infectious organisms are eliminated, poor vision can result from corneal opacity and irregular 
astigmatism. The use of adjuvant corticosteroids has long been debated in the treatment of bacterial 

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keratitis.49-51 Proponents argue that they decrease inflammation and reduce scarring, 
neovascularization, and stromal melt.51-54 However, others argue that corticosteroids delay epithelial 
healing and prolong infection.55-58 Three small randomized controlled trials examining the benefit of 
adjuvant topical steroids for the treatment of corneal ulcers found no difference in visual acuity 
outcomes or healing times between those randomized to topical antibiotic alone versus topical 
antibiotic plus topical steroid.59-61 
 
The Steroids for Corneal Ulcers Trial (SCUT U10EY015114) is by far the largest randomized controlled 
trial to have evaluated the role of adjuvant steroids for bacterial ulcers.62 Five hundred study 
participants with culture-positive bacterial ulcers were enrolled at UCSF, Aravind Eye Hospitals in 
Madurai, Coimbatore, and Tirunelveli India, and at the Dartmouth-Hitchcock Medical Center in New 
Hampshire. Patients were randomized to receive either topical prednisolone sodium phosphate 1.0% or 
topical placebo (sodium chloride 0.9%), started after 48-hours of topical moxifloxacin 0.5%. All patients 
received 1 drop of their assigned treatment 4 times daily for the first week after enrollment, then 2 
times daily for the second week, and then 1 time daily for the third week. After controlling for baseline 
BSCVA a multiple linear regression showed that corticosteroids provided no significant improvement in 
3-month BSCVA over placebo (P=0.82). Similarly, there was no difference between arms in secondary 
outcomes such as rate of re-epithelialization (P=0.25), infiltrate/scar size (P=0.40) or the number of 
perforations observed (P>0.99). It is also important to note that corticosteroids did not cause an 
increase in adverse events.62  
 
Pre-specified subgroup analyses have suggested that earlier treatment of large, central, non-Nocardia 
ulcers did have improved visual acuity outcomes compared with antibiotic alone (Figure 1).6 A 12-
month SCUT analysis excluding Nocardia ulcers found a 1-line visual acuity benefit among those 
randomized to topical steroid.63 We also found that those treated with steroid earlier, within 2 to 3 days 
of antibiotics, had 1-line better visual acuity at 3 months.5 These subgroup analyses have led some to 
conclude that topical corticosteroids may be beneficial for specific subgroups of culture positive bacterial 
ulcers, and that they were most effective when administered early with appropriate antibiotics.5,6  
 
Corneal cross-linking may benefit patients with infectious corneal ulcers through direct anti-microbial 
and anti-inflammatory effects, as well as increased resistance of corneal tissue to enzymatic 
degradation.7-9 Photoactivation of riboflavin with UV light results in release of reactive oxygen species 
that promote chemical covalent bond formation between adjacent collagen molecules. Reactive oxygen 
species are also thought to have an antiseptic effect against a broad range of pathogens.64 UVX is 
currently used as a treatment for corneal ectatic disorders such as keratoconus and post-LASIK ectasia 
and has been shown to strengthen the cornea and allow it to retain its normal shape.65-68 Immediately 
after UVX there is a decrease in the sub-epithelial nerve plexus and loss of keratocytes in the anterior 
one-third of the corneal stroma, although this recovers after a few months.69,70 UVX would presumably 
destroy inflammatory cells in the anterior stroma by similar mechanisms, although this does not appear 
to have been studied previously. 
 
 
To date, three small prospective clinical trials have been conducted to assess the effect of CXL in the 
treatment of infectious keratitis. Bamdad et al randomized 32 patients with moderate bacterial keratitis 
to receive either UVX plus standard therapy versus standard therapy alone.71 Two weeks after the 
treatment, those receiving UVX had a lower mean grade of ulcer (0.69 vs 1.70; P=0.001), smaller area of 
epithelial defect (P=0.001), and smaller area of infiltrate (P<0.001) than those receiving standard 
therapy alone. Mean treatment duration was also shorter in the UVX group (P<0.001). Another trial 
randomized patients with bacterial, fungal, Acanthamoeba, or mixed origin keratitis to UVX versus 

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antimicrobial treatment alone.28 While this trial found no difference between groups, it had multiple 
issues, including inappropriate randomization, vastly different etiologies of infection, and insufficient 
power.72 A third, small randomized clinical trial investigating cross-linking as adjuvant therapy for deep 
fungal ulcers at Aravind Eye Hospital in Madurai, India stopped after only enrolling 13 patients due to 
concern that UVX could increase the rate of perforation in severe fungal ulcers.73 Given the limitations of 
these clinical trials and mixed results, it is not known whether UVX is a beneficial adjuvant therapy for 
infectious keratitis and a well-designed, larger scale randomized clinical trial is warranted.   
In vitro Rose Bengal Photodynamic Therapy (RB-PDT) appears to be much more effective against 
fungal and acanthamoeba isolates.24,25 Rose Bengal (RB) is one of the most commonly used dyes in the 
diagnosis of ocular surface disease.74 Rose Bengal is an effective photosensitizer, readily converting 
triplet oxygen (3O2) to produce high singlet oxygen (1O2) yields with exposure to green light.75 Although 
RB dye penetration is to approximately 100µm into the stroma, subsequent free radical formation 
occurs up to 1/3 of the corneal stromal depth.76,77 The ability of RB to continue free radical formation is 
self-limited after photo-irradiation has ceased.78 Multiple In vitro and ex vivo studies have suggested 
that RB-PDT may be safer than UVX. Wound healing studies found more corneal haze and slower wound 
healing after UVX compared with RB-PDT.17 Rabbit studies have demonstrated the safety of RB-PDT on 
limbal stem cells and endothelium and found anterior stromal keratocyte damage in RB-PDT comparable 
to epithelial debridement alone.21,22 By contrast, UVX causes an immediate decrease in the sub-
epithelial nerve plexus and loss of keratocytes in the anterior one-third of the corneal stroma, although 
this recovers after a few months.69,70 
 
Smear and culture negative ulcers represent another therapeutic challenge for clinicians. Up to 60% of 
corneal cultures are smear and culture negative.79 When these patients do not improve with topical 
antibiotics alone, clinicians must decide what alternative medical therapy to introduce. There is little 
guidance in the literature on how to manage these patients. These cases are challenging to study since 
they represent different underlying aetiologies and one medical therapy is unlikely to address all of 
them. RB-PDT is unique in its potential to address bacterial, fungal, and parasitic infections making it a 
particularly attractive novel therapy.   
  
CXL may also reduce the risk of corneal perforation in corneal ulceration. Corneal melting occurs in 
response to proteolytic enzymes released both from pathogens and leukocytes sent to combat 
infection.3,4 Corneal perforation is a devastating complication, often treated with therapeutic 
penetrating keratoplasty (TPK) which has a poor prognosis compared to non-emergent penetrating 
keratoplasty (PKP) performed for visual rehabilitation.2 Cross-linked corneas show increased in vitro 
resistance to keratolysis by collagenase A.29 One non-randomized prospective series of 40 patients 
found a decreased rate of perforation among those treated with UVX compared with controls despite 
the fact they had on average larger baseline ulcer size.28 A recent meta-analysis concluded that the 
probability that UVX was beneficial in inhibiting melting in patients with infectious keratitis was 85% 
(95% CI 0.77 to 0.91).7 UVX may even provide a new option for treatment of corneal melt in select auto-
inflammatory conditions.  
It will still be important to measure the effect of UVX on clinical outcomes such as visual acuity and 
scar size. A number of studies have demonstrated the safety and efficacy of UVX for the treatment of 
keratoconus with follow up in the 5-10 year range.80 However, the observed corneal flattening 
associated with improved visual acuity outcomes in keratoconus, could result in unexpected topographic 
changes in infectious keratitis and it is not known what effect UVX has on corneal scarring in these 
cases. 

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2.3 
RISK/BENEFIT ASSESSMENT   
2.3.1 KNOWN POTENTIAL RISKS  
The main risk of steroid (difluprednate, prednisolone) use is IOP elevation, which may be associated with 
optic nerve damage or even permanent vision loss if not monitored. We believe that the possible risk of 
increased IOP is outweighed by the potential benefits for this treatment and will be monitoring IOP 
routinely while the patients are inpatient during the first 3 days of treatment. There was no evidence in 
SCUT that steroids potentiated infection.  
The process of corneal cross-linking is painless. Adverse events associated with corneal cross-linking 
appear to be exceedingly rare. A recent publication of 3-year follow-up of 100 keratoconus eyes, found 
two adverse events: one instance of corneal infection and one case of corneal edema, which resolved 
within one week.81 In a series of 16 patients with bacterial ulcers and presenting average visual acuity of 
approximately 20/100 (including one patient with 20/20 vision) no complications or side effects of 
treatment were observed.15 Additionally, a meta-analysis of the literature concerning cross-linking for 
infectious keratitis concluded that the available evidence supports the use of cross-linking for the 
treatment of infectious keratitis.7 All procedures will be performed by ophthalmologists board certified in 
the study country who are well versed in all planned treatment procedures. 
There may be some discomfort during follow-up testing (BSCVA, IOP, slit lamp, Pentacam, confocal 
microscopy, and slit lamp imaging of the eye), but this will be kept to a minimum. The participant will be 
asked to tell the doctor if any of this testing feels painful. There may be a medication reaction such as eye 
irritation, swelling, pain, redness, or discharge. If this occurs the risks of stopping the medication must be 
weighed against the severity of infection and other treatment options.  
 
2.3.2 KNOWN POTENTIAL BENEFITS  
Blindness from infectious corneal ulcers is a worldwide public health problem. Although antibiotics are 
successful at achieving microbiological cure, outcomes remain poor. Acute infection can result in 
scarring, irregular astigmatism, and corneal perforation requiring surgery. There is reasonable evidence 
that our interventions (UVX, RB-PDT and early steroid treatment) will be beneficial in the treatment of 
bacterial keratitis. If our hypotheses are correct, there could potentially be a profound societal benefit 
from adjuvant steroid, UVX, and RB-PDT.  
 
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
Knowledge gained from this study may help reduce the burden of corneal blindness in countries around 
the world. Given the minimal risks to participants in this study, we feel the benefits of the important 
knowledge we expect to gain from this study outweigh the risks.   
 
 

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3 
OBJECTIVES AND ENDPOINTS 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR ENDPOINTS 
Primary 
 
 
Specific Aim 1: To determine 
the effect of UVX on best 
spectacle-corrected visual 
acuity 
Best spectacle-corrected 
visual acuity at 6 months 
 
The most clinically important outcome 
for patients 
Specific Aim 2: To determine 
the effect of early topical 
steroids on best spectacle-
corrected visual acuity 
Best spectacle-corrected 
visual acuity at 6 months 
 
The most clinically important outcome 
for patients 
Specific Aim 4: To determine 
the effect of RB-PDT on best 
spectacle-corrected visual 
acuity for acanthamoeba and 
fungal keratitis 
Best spectacle-corrected 
visual acuity at 6 months 
 
The most clinically important outcome 
for patients 
Specific Aim 5: To determine 
the effect of RB-PDT on best 
spectacle-corrected visual 
acuity for bacterial keratitis 
Best spectacle-corrected 
visual acuity at 6 months 
 
The most clinically important outcome 
for patients 
Secondary 
 
 
Specific Aim 1: To assess the 
effect of UVX on 
microbiological cure 
Repeat culture at Day 2 
Culture status is highly related to 
clinical outcomes. This will allow us to 
assess for development of drug 
resistance during treatment. 
To assess the effect of early 
topical steroids on 
microbiological cure 
Repeat culture at Day 2 
Culture status is highly related to 
clinical outcomes and one concern 
with topical steroids is that it might 
prevent resolution of infection.  
Specific Aim 4: To assess the 
effect of RB-PDT on 
microbiological cure 
Repeat culture at Day 2 
Culture status is highly related to 
clinical outcomes. This will allow us to 
assess for development of drug 
resistance during treatment. 
Specific Aim 3: To assess the 
relationship between UVX and 
drug resistant organisms 
Interaction of minimum 
inhibitory concentration 
(MIC) and UVX on best 
spectacle-corrected visual 
acuity  
Drug resistance is a concern, and the 
Infectious Disease Society of America 
recommends looking for novel 
treatments for bacterial infections. 
UVX does not use antibiotics. 
 
 
Specific Aim 4: To assess the 
relationship between RB-PDT 
and drug resistant organisms  
Interaction of minimum 
inhibitory concentration 
(MIC) and RB-PDT on best 
spectacle-corrected visual 
acuity 
Culture status is highly related to 
clinical outcomes. This will allow us to 
assess whether RB-PDT is more 
effective when organisms are resistant 
to topical antimicrobials  

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR ENDPOINTS 
To determine the effect of CXL 
and/or early topical steroids 
on corneal healing  
Scar size and infiltrate, % 
epithelial healing, over the 
12-month follow-up 
period 
Corneal healing is the primary goal of 
treatment 
To determine the effect of CXL 
and/or early topical steroids 
on adverse events 
Adverse events including 
corneal perforation and 
need for therapeutic 
penetrating keratoplasty, 
glaucoma over the 12-
month follow-up period 
To ensure safety of the intervention 
To determine the effect of CXL 
and/or early topical steroids 
on quality of life 
Visual function 
questionnaire at 6 months 
as compared to Day 1 
questionnaire  
Quality of life is an important patient 
outcome and will help determine if any 
differences in BSCVA are clinically 
relevant 
Subgroup analysis of study 
participants receiving prior 
topical antimicrobial therapy 
MIC, Visual acuity, scar 
size, % epithelial healing, 
corneal perforation/TPK 
We will evaluate whether antimicrobial 
resistance as measured by MIC leads to 
worse visual acuity, scar size, epithelial 
healing, and higher risk of 
complications such as TPK. 
Tertiary/Exploratory  
 
 
To evaluate the effect of 
corneal infections and study 
interventions at the cellular 
level on the cornea using 
confocal microscopy 
Automated corneal nerve 
fiber analysis and manual 
quantitative analysis of 
epithelial cells, 
inflammatory cells, and 
keratocytes, fungal 
hyphae or acanthameoba 
cells from confocal 
microscopy images at 
baseline, 3 weeks, 3 
months, 6 months, and 12 
months 
This objective will help us to learn 
more about the pathophysiology of 
corneal infections as well as the effects 
of study interventions on keratocytes 
and white blood cell trafficking; to 
identify findings on confocal which 
would guide clinicians regarding the 
best management of bacterial keratitis; 
and to validate these findings as 
potential outcomes for future clinical 
trials. 
To assess the effect of study 
interventions on corneal 
thinning, scarring, and 
irregularity using Pentacam 
Scheimpflug topography 
 
Steep and flat 
keratometry readings, 
total astigmatism in 
diopters, root mean 
square Zernike 
polynomials in the central 
4mm of the pupil, and 
densitometry in gray scale 
units, at baseline, 3 
weeks, 3 months, 6 
months, and 12 months 
While CXL might kill bacteria, there are 
unknown effects on astigmatism. 
Steroids may decrease the amount of 
astigmatism.  
To assess the effect of study 
interventions on corneal 
thinning and scarring using 
Central corneal thickness, 
point of maximal thinning, 
infiltrate/scar size, and 
We can look for baseline ulcer 
characteristics which predict response 
to a particular intervention, for 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR ENDPOINTS 
high resolution anterior 
segment OCT 
infiltrate/scar depth at 
baseline, 3 weeks, 3 
months, 6 months, and 12 
months 
example deep infiltrates may not 
benefit from CXL 
 
4 
STUDY DESIGN  
4.1 
OVERALL DESIGN 
The Steroids and Cross-linking for Ulcer Treatment (SCUT II) and Rose Bengal Electromagnetic Activation 
with Green light for Infection Reduction (REAGIR) trials are international, randomized, double-masked, 
sham and placebo controlled, clinical trials; and Rose Bengal Electromagnetic Activation with Green light 
for Infection Reduction II (REAGIR II) is a randomized, double-masked feasibility study.  The purpose of 
these studies is to determine differences in 6-month visual acuity between medical antimicrobial 
treatments alone versus antimicrobial treatment plus corneal cross-linking (CXL), as well as to further 
evaluate findings from subgroup analyses of SCUT.  
 
SCUT II 
Patients presenting to one of the Aravind Eye Hospitals in India to the University of California San 
Francisco (UCSF), or to Bascom Palmer Eye Institute at the University of Miami with smear-positive 
typical (i.e. non-Nocardia or Mycobacteria) bacterial corneal ulcers and moderate to severe vision loss, 
defined as Snellen visual acuity of 20/40 or worse, will be eligible for inclusion. Those who agree to 
participate will be randomized to one of three treatment groups:  
 
Group 1, Standard therapy: topical 0.5% moxifloxacin plus topical placebo plus sham UVX 
Group 2, Early steroids: topical 0.5% moxifloxacin plus topical difluprednate 0.05% plus sham UVX  
Group 3, UVX plus early steroids: topical 0.5% moxifloxacin plus topical difluprednate 0.05% plus UVX 
 
We hypothesize that both UVX and early topical steroids will result in better best spectacle-correct 
visual acuity at 6 months compared with antibiotic alone. The patient, physicians, microbiologist and 
refractionist performing the best spectacle-corrected visual acuity (BSCVA) assessment will be masked 
to the treatment group. Due to the nature of the intervention, the surgeon and technician performing 
cross-linking will not be masked. All study medications and placebo will be labelled identically to ensure 
adequate masking of study physicians and patients. An interim analysis will be performed once primary 
outcome data is available for one third of the patients (see SCUT II Statistical Analysis Plan (SAP)–- 
Section 1.5.6 for details).  
 
REAGIR 
Patients presenting to one of the Aravind Eye Hospitals in India, or to the Federal University of São Paulo 
in Brazil (UNIFESP) with either smear or culture positive fungal or acanthamoeba keratitis or smear and 
culture negative corneal ulcers and moderate to severe vision loss, defined as Snellen visual acuity of 
20/40 or worse, will be eligible for inclusion. Those who agree to participate will be randomized to one 
of two treatment groups:  
 
Group 4, (Sham RB-PDT): chlorhexidine gluconate 0.02% (acanthamoeba), moxifloxacin 0.5% 
(smear/culture negative) or natamycin 5% (fungal keratitis) plus sham RB-PDT 
Group 5, RB-PDT: chlorhexidine gluconate 0.02% (acanthamoeba), moxifloxacin 0.5% (smear/culture 
negative) or natamycin 5% (fungal keratitis) plus RB-PDT 

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We hypothesize that study participants randomized to RB-PDT will have improved BSCVA, achieve 
microbiological cure more quickly, have smaller scar sizes and lower perforation rates compared with 
those who receive topical antibiotic alone. The patient, physicians, microbiologist and refractionist 
performing the best spectacle-corrected visual acuity (BSCVA) assessment will be masked to the 
treatment group. Due to the nature of the intervention, the surgeon and technician performing cross-
linking will not be masked. As in the SCUT II trial, an interim analysis will be performed once primary 
outcome data is available for one third of the patients (see REAGIR Statistical Analysis Plan (SAP)–- 
Section 3.5 for details). 
 
REAGIR II 
Patients presenting to Aravind Eye Hospitals – Madurai (in India) or to the Federal University of São 
Paulo in Brazil (UNIFESP) with smear-positive and/or culture positive typical (I.e. non-Nocardia or 
Mycobacteria) bacterial corneal ulcers and moderate to severe vision loss, defined as Snellen visual 
acuity of 20/40 of worse, will be eligible for inclusion. Those who agree to participate will be randomised 
to one of two treatment groups: 
• 
Group 6, RB-PDT Plus Early Steroids: topical 0.5% moxifloxacin plus topical difluprednate 
0.05%/prednisolone acetate 1% plus RB-PDT 
• 
Group 7, Sham RB-PDT Plus Early Steroids: topical 0.5% moxifloxacin plus topical difluprednate 
0.05%/prednisolone acetate 1% plus sham RB-PDT 
 
We hypothesize that study participants treated with topical antibiotics plus adjunctive RB-PDT will have 
better best spectacle corrected visual acuity at 6 months compared to those treated with antibiotics 
alone. The patient, physicians, microbiologist and refractionist performing the best spectacle-corrected 
visual acuity (BSCVA) assessment will be masked to the treatment group. Due to the nature of the 
intervention, the surgeon and technician performing cross-linking will not be masked. 
 
 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
Randomized control trials are known to be the least biased form of evidence; we have chosen to do a 
randomized control trial with placebo and sham controls to introduce as little bias as possible. Sham 
cross-linking and placebo with study drug vehicle are included to maintain masking of the study 
participant and treating physician. No group is receiving less than the standard of care, which is topical 
antibiotic or antimicrobial treatment.  
 
4.3 
JUSTIFICATION FOR DOSE 
Route of administration and dosage of study medications are consistent with standard practice. For 
SCUT II, topical placebo will be administered in the same way as topical difluprednate to mask the 
patient, physician, and study staff. The topical placebo solution will have the same clear appearance as 
difluprednate to maintain masking.  
 
4.4 
END OF STUDY DEFINITION 
A participant is considered to have completed the study if he or she has completed all phases of the 
study including the final 12-month visit, as shown in the Schedule of Activities (SoA), Section 1.3. 
 
 

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5 
STUDY POPULATION 
5.1 
INCLUSION CRITERIA 
In order to be eligible to participate, an individual must meet all of the following criteria: 
 
SCUT II and REAGIR II: 
• 
Corneal ulcer that is smear positive and/or culture positive (within 24 hours) for typical bacteria 
(i.e. non-Nocardia or Myobacteria) 
• 
Moderate to severe vision loss, defined as Snellen visual acuity of 20/40 (6/12) or worse 
• 
Corneal thickness ≥350 µm, as measured on AS-OCT 
• 
Age over 18 years 
• 
Basic understanding of the study as determined by the physician 
• 
Commitment to return for follow up visits 
 
REAGIR:  
• 
Presence of smear or culture positive fungal or acanthamoeba ulcer; smear or culture negative 
ulcer; or any atypical bacteria (such as Nocardia)   
• 
Moderate to severe vision loss, defined as Snellen visual acuity of 20/40 (6/12) or worse 
• 
Corneal thickness ≥350 µm, as measured on AS-OCT 
• 
Age over 18 years 
• 
Basic understanding of the study as determined by the physician 
• 
Commitment to return for follow up visits  
 
5.2 
EXCLUSION CRITERIA 
An individual who meets any of the following criteria will be excluded from participation in this study: 
• 
Evidence of concomitant infection on exam, gram stain, or confocal microscopy (i.e. herpes, 
both bacteria and acanthamoeba on gram stain) 
• 
Impending or frank perforation at recruitment 
• 
Involvement of sclera at presentation 
• 
Presence of desmetocele at recruitment 
• 
Non-infectious or autoimmune keratitis 
• 
History of corneal transplantation 
• 
History of intraocular surgery within the last three months*  
• 
Pinhole visual acuity worse than 20/200 in the unaffected eye  
• 
Participants who are decisionally and/or cognitively impaired 
 
*Recent intraocular surgery is an exclusion to avoid ulcers related to the surgery itself (e.g.: wound 
infection). Three months post-surgery is generally acceptable, although the ophthalmologist can judge 
whether, in their opinion, the ulcer is related to the surgery. For example, one month after an 
uncomplicated cataract surgery might be considered acceptable.  
 
5.3 
LIFESTYLE CONSIDERATIONS 
Patients at some Aravind Eye Hospital enrollment centers will be admitted to the inpatient setting for 
the first 3 days of the study.  
 

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5.4 
SCREEN FAILURES 
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently randomly assigned to the study intervention or entered in the study. A minimal set of 
screen failure information is required to ensure transparent reporting of screen failure participants, to 
meet the Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes demography, screen 
failure details, eligibility criteria, and any serious adverse event (SAE). In this study, all patients who are 
enrolled will be randomized and included in the primary analysis, regardless of whether or not they 
actually receive the assigned intervention.  
 
5.5 
STRATEGIES FOR RECRUITMENT AND RETENTION 
Patients presenting to recruitment sites with smear-positive typical bacterial corneal ulcers, smear or 
culture positive fungal or acanthamoeba ulcers, or smear or culture negative ulcers with moderate to 
severe vision loss will be approached for possible inclusion in the study. For eligible patients, the study 
will be explained in the local language (Tamil at the Aravind Eye Clinics, English or Spanish at UCSF and 
University of Miami, and Portuguese at UNIFESP) in addition to the risks and benefits of participating in 
the study. Patients at some Aravind sites will be admitted to the hospital for the first 3 days of the study, 
ensuring minimal loss to follow-up through day 3. Patients will schedule their follow up visits with the 
study coordinator while they are in inpatient care. The study coordinator will give the patient written 
documentation of their upcoming visits, and will follow-up with a phone call as their appointments 
approach. Previous studies with Aravind and UCSF, including the original Steroids for Corneal Ulcers 
Trial, the Mycotic Ulcer Treatment Trials I & II, and the small cross-linking for bacterial keratitis feasibility 
assessment study we performed, have had high retention and leave us confident that this study will 
have high retention as well.   
6 
STUDY INTERVENTION 
6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
6.1.1 STUDY INTERVENTION DESCRIPTION 
 
SCUT II: Corneal Cross-Linking with Riboflavin 
Participants in Groups 1, 2, & 3 will begin study drug (topical difluprednate 0.05% or placebo) within 24 
hours after enrollment.  
Participants in Group 3 will receive UVX (modified Dresden protocol), and Groups 1 & 2 will receive 
sham UVX within 48 hours of enrollment. All participants will receive a 30-minute loading dose of topical 
0.1% riboflavin and 20% dextran T500 drops every 2 minutes. For Group 3, this will be followed by 
exposure to UV-A light at a wavelength of 365 nm with an irradiance of 3 mW/cm2 for 30 minutes for a 
total dose of 5.4 J/cm2 (UV lamp:  PESCHKE Meditrade GmbH, Hueneberg, Switzerland for India; Avedro 
KXL System, Waltham, MA, USA for USA). During irradiation patients will continue to receive topical 
riboflavin at 5-minute intervals. For Groups 1 & 2, sham UVX simulates this experience however the light 
will be shined adjacent to the patient, careful to avoid exposure to the cornea. In place of riboflavin we 
will use either saline drops or saline drops dyed with fluorescein.  

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Moxifloxacin 0.5% 
Topical moxifloxacin 0.5% is a fluoroquinolone antibiotic that is used to treat bacterial infections. This is 
standard therapy for bacterial keratitis. Immediately after UVX/sham UVX and repeat culture, all 
participants will receive topical moxifloxacin drops every 1 hour for 2 days, and then every 2 hours while 
awake until resolution of the epithelial defect. 
 
Difluprednate 0.05% 
Difluprednate 0.05% is a corticosteroid used to reduce inflammation in the eye. Participants in Groups 2 
& 3 will receive one drop of 0.05% difluprednate four times daily beginning 24 hours after the initiation 
of antibiotics for 1 week, decreased by 1 drop weekly for a total of 4 weeks of steroid therapy.  
 
Placebo 
Participants randomized to Group 1 will receive topical placebo in place of topical difluprednate. The 
placebo will be the vehicle used in difluprednate. Group 1 will receive the topical placebo on the same 
medication schedule described above for difluprednate. 
 
REAGIR: Corneal Cross-Linking with Rose Bengal  
Participants in Groups 4 and 5 will begin topical antimicrobial at enrollment.  
 
Participants in Group 5 will receive RB-PDT and Group 4 will receive sham RB-PDT within 48 hours of 
randomization. Group 5 participants will receive a 30-minute loading dose of topical Rose Bengal (0.1% 
RB in 0.9% sodium chloride) which will be applied in 3-minute intervals to the de-epithelialized cornea. 
This will be followed by irradiation with a 6mW/cm2 custom-made green LED source for 15 minutes 
(5.4J/cm2). Participants in Group 4 will undergo a sham procedure using topical balanced salt solution 
and pen light covered with a green filter. Participants in both groups will undergo repeat cornea cultures 
within 24 hours after the procedure. 
 
Rose Bengal 0.1% 
Rose Bengal (RB) is one of the most commonly used dyes in the diagnosis of ocular surface disease.74 
Rose Bengal is an effective photosensitizer, readily converting triplet oxygen (3O2) to produce high 
singlet oxygen (1O2) yields with exposure to green light.75 Although RB dye penetration is to 
approximately 100µm into the stroma, subsequent free radical formation occurs up to 1/3 of the 
corneal stromal depth.76,77 The ability of RB to continue free radical formation is self-limited after photo-
irradiation has ceased.78 
 
Chlorhexidine gluconate 0.02% or polyhexamethylene biguanide 0.02%  
Topical biguanides such as chlorhexidine gluconate 0.02% or PHMB 0.02% are thought to be the most 
effective available medical therapy for acanthamoeba. Participants in Groups 4 and 5 with 
acanthamoeba ulcers will receive topical chlorhexidine drops every 1 hour for 2 days, and then every 2 
hours while awake, tapering at the clinician’s discretion.  
 
Moxifloxacin 0.5% 
At enrollment, participants with smear/culture negative ulcers will receive topical moxifloxacin drops 
every 1 hour for 2 days, and then every 2 hours while awake, tapering at the clinician’s discretion.  
 
Natamycin 5%  
Topical natamycin, a polyene, is the only antifungal agent approved by the Food and Drug 
Administration (FDA) for treatment of fungal keratitis. Participants in Groups 4 and 5 with fungal ulcers 

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will receive topical natamycin every 1 hour for 2 days, and then every 2 hours while awake, tapering at 
the clinician’s discretion.  
 
Amphotericin B 0.15% 
In Brazil, participants in Groups 4 and 5 with fungal ulcers will receive topical amphotericin B 0.15% 
every 1 hour for 2 days, then every 2 hours while awake, tapering at the clinician’s discretion.   
 
REAGIR II: Corneal Cross-Linking with Rose Bengal  
Participants in Groups 6 and 7 will begin topical difluprednate 0.05% within 24 hours after enrollment.  
 
RB-PDT/sham RB-PDT will occur within 48 hours of randomization. For Group 6, Rose Bengal (0.1% RB in 
0.9% sodium chloride) will be applied in 3-minute intervals to the de-epithelialized cornea for 30 
minutes followed by irradiation with a 6mW/cm2 custom-made green LED source for 15 minutes 
(5.4J/cm2).  Participants in Group 7 will undergo a sham procedure using topical balanced salt solution 
and pen light covered with a green filter. Participants in both groups will undergo repeat cornea cultures 
within 24 hours after the procedure. 
 
Rose Bengal 0.1% 
Rose Bengal (RB) is one of the most commonly used dyes in the diagnosis of ocular surface disease.74 
Rose Bengal is an effective photosensitizer, readily converting triplet oxygen (3O2) to produce high 
singlet oxygen (1O2) yields with exposure to green light.75 Although RB dye penetration is to 
approximately 100µm into the stroma, subsequent free radical formation occurs up to 1/3 of the 
corneal stromal depth.76,77 The ability of RB to continue free radical formation is self-limited after photo-
irradiation has ceased.78 
 
Moxifloxacin 0.5% 
Topical moxifloxacin 0.5% is a fluoroquinolone antibiotic that is used to treat bacterial infections. This is 
standard therapy for bacterial keratitis. Immediately after UVX/sham UVX and repeat culture, all 
participants will receive topical moxifloxacin drops every 1 hour for 2 days, and then every 2 hours while 
awake until resolution of the epithelial defect. 
 
Difluprednate 0.05%/Prednisolone acetate 1% 
Difluprednate 0.05% and Prednisolone acetate 1% are both corticosteroids used to reduce inflammation 
in the eye. Participants in Groups 2 & 3 will receive one drop of 0.05% difluprednate (India) or 
prednisolone acetate 1% (Brazil) four times daily beginning 24 hours after the initiation of antibiotics for 
1 week, decreased by 1 drop weekly for a total of 4 weeks of steroid therapy.  
 
 
 

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6.1.2 DOSING AND ADMINISTRATION 
The schedule of medications for the studies are below: 
 
SCUT II: Dosing schedule for medications 
 
Group 1:  
Standard Therapy 
Group 2: 
Early Steroids 
Group 3: 
UVX 
 
Dose 
Duration 
Dose 
Duration 
Dose 
Duration 
Moxifloxacin 
0.5%  
Q 1 hours x 
2 days, then 
Q 2 hours 
while 
awake  
Until 
resolution of 
epithelial 
defect 
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake  
Until resolution of 
epithelial defect 
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake 
Until resolution of 
epithelial defect 
Difluprednate 
0.05%* 
 
 
QID  
Starting at 24 
hours after 
initiation of 
antibiotics for 1 
week, then 
decreasing by 1 
drop weekly for a 
total of 4 weeks of 
steroid therapy 
QID  
Starting at 24 
hours after 
initiation of 
antibiotics for 1 
week, then 
decreasing by 1 
drop weekly for a 
total of 4 weeks of 
steroid therapy 
Riboflavin 0.1% 
 
 
 
 
Q2 min 
60 minutes 
Homatropine 
TID  
3 days 
TID  
3 days 
TID  
3 days 
*for patients randomized to Groups 2 & 3 only 
 
Participants randomized to receive topical placebo instead of topical steroids will receive the placebo on 
the same medication schedule as difluprednate outlined above. If at any time the masked treating 
physician deems it appropriate to change the patient’s treatment, he/she may do so.  
 
 
 

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REAGIR: Dosing schedule for medications 
 
Group 4: 
Sham RB-PDT 
Group 5: 
RB-PDT 
 
Dose 
Duration 
Dose 
Duration 
Biguanide 
(Chlorhexidine 
gluconate 
0.02%  
or  
PHMB 0.02%) 
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake  
Tapered at 
clinician’s 
discretion 
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake  
Tapered at 
clinician’s 
discretion 
Moxifloxacin 
0.5% 
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake  
Tapered at 
clinician’s 
discretion 
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake  
Tapered at 
clinician’s 
discretion 
Polyene 
(Natamycin 5% 
or 
Amphotericin B 
0.15%) 
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake  
Tapered at 
clinician’s 
discretion 
Q 1 hours x 
2 days, then 
Q 2 hours 
while 
awake  
Tapered at 
clinician’s 
discretion 
Rose Bengal 
0.1% RB in 
0.9% sodium 
chloride 
 
 
Q 3 min 
45 minutes 
Homatropine 
TID  
3 days 
TID  
3 days 
 
 
 

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REAGIR II: Dosing schedule for medications 
 
Group 6: 
RB-PDT +  
Early Steroids 
Group 7: 
Sham RB-PDT +  
Early Steriods 
 
Dose 
Duration 
Dose 
Duration 
Moxifloxacin 
0.5%  
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake  
Until resolution of 
epithelial defect 
Q 1 
hours x 
2 days, 
then Q 2 
hours 
while 
awake 
Until resolution of 
epithelial defect 
Steroid 
(Difluprednate 
0.05% 
or 
Prednisolone 
Acetate 1%) 
QID  
Starting at 24 
hours after 
initiation of 
antibiotics for 1 
week, then 
decreasing by 1 
drop weekly for a 
total of 4 weeks of 
steroid therapy 
QID  
Starting at 24 
hours after 
initiation of 
antibiotics for 1 
week, then 
decreasing by 1 
drop weekly for a 
total of 4 weeks of 
steroid therapy 
Rose Bengal 
0.1% RB in 
0.9% sodium 
chloride 
Q2 min 
60 minutes 
 
 
Homatropine 
TID  
3 days 
TID  
3 days 
 
 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
Medications and masked placebos will be purchased and distributed in the country of enrollment. At 
Aravind Eye Hospitals, study medications will be compounded and distributed by Aurolab.  
 
For UCSF and University of Miami, Rancho Park Compounding Pharmacy in Los Angeles, CA will assist 
with compounding and distributing study medications.  Information on current medications and drug 
allergies will be collected in REDCap and reviewed by Rancho Park before study medication is dispensed.  
 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
Transfer of topical study medication or vehicle (placebo) to an identical bottle by the compounding 
pharmacy will ensure patient and physician remain masked to treatment arm.  
 
6.2.3 PRODUCT STORAGE AND STABILITY 
All study medications should be stored at 15-25°C and should be protected from sunlight. Expiration 
dates will be clearly labeled.  
 
6.2.4 PREPARATION 
All preparation will be performed by the compounding pharmacy and delivered directly to the patient. 
To that end, patient mailing addresses will be provided to Rancho Park.  

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6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
The DCC at UCSF will create the REDCap database to house the randomization list and store data. Once a 
patient has been identified for inclusion in the study, the study coordinator will review the 
inclusion/exclusion criteria checklist, and then input the study eye into the REDCap database.  
 
Patients with bacterial ulcers are eligible for randomization to Groups 1, 2, and 3, while patients with 
fungal, acanthamoeba, and smear/culture negative ulcers are eligible for randomization to Group 4 or 5. 
Each study eye will be randomly assigned to a treatment group by REDCap. Randomization will be 
stratified by site. Stratified, block randomization will ensure that an approximately equal number of 
patients are randomized to each treatment group by site. In SCUT II (groups 1, 2, 3) exact block size will 
be randomly permuted (block size of 3 with probability 0.7 and 6 with probability 0.3) and will only be 
known to the biostatistician. In REAGIR (Groups 4, 5) exact block size will be randomly permuted (block 
size of 4 with probability 0.7 and 6 with probability 0.3) and will only be known to the biostatistician. Block 
randomization will be performed using a computer program (Statistical package R; R Foundation for 
Statistical Computing, Vienna, Austria) by the coordinating site.  
 
Once an eye is enrolled (and only one eye can be enrolled) in the study, the study coordinator will assign 
the study participant’s eye an ID (alpha-numeric code) and organize the procedure in the operating room 
within 48 hours of enrollment. Once the study participant has been assigned a study participant ID and 
randomized to treatment group, they will be included in the intent to treat analysis. 
 
The patient, physician performing repeat scraping and clinical follow up, microbiologist and refractionist 
performing the BSCVA will be masked to treatment arm. Due to the nature of the surgical intervention, 
the surgeon and technician performing cross-linking will not be masked. All study medications and 
placebo will be labeled identically to ensure adequate masking of study physicians and patients.  
 
6.4 
STUDY INTERVENTION COMPLIANCE 
We will have several measures in place to assess compliance with medications. All Aravind patients will 
be hospitalized for the first three days of the study, and during this time there will be direct observation 
of medication administration by study staff. At follow-up visits, patients will be asked to bring their 
medication bottles to assess compliance. We will also have inpatient and outpatient medication logs to 
track compliance and missed doses, to be filled out by study staff and the patient, respectively.  
 
6.5 
CONCOMITANT THERAPY 
The masked treating physician will be allowed to change antibiotics or antimicrobials based on 
sensitivity data from culture results and clinical response to treatment. These changes will be recorded 
and reported on the follow up form.  
 
6.5.1 RESCUE MEDICINE 
The masked treating physician will be allowed to add or change any therapy deemed necessary, 
including surgery.  
 
 
 

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7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
Discontinuation from assigned study intervention does not mean discontinuation from the study, and 
remaining study procedures should be completed as indicated by the study protocol. If a clinically 
significant finding is identified (including, but not limited to changes from baseline) after enrollment, the 
investigator or qualified designee will determine if any change in participant management is needed. 
Any new clinically relevant finding will be reported as an adverse event (AE). 
 
The data to be collected at the time of study intervention discontinuation will include the following: 
• 
Reason for study intervention discontinuation 
• 
New prescribed treatment 
 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Participants are free to withdraw from participation in the study at any time upon request. The reason 
for participant discontinuation or withdrawal from the study will be recorded on a dropout form, and 
the patient will be presented with an Informed Consent Form Addendum. With the patient’s consent, 
we will monitor their surgical outcomes through collection of follow-up data from their medical provider 
and medical record.  
 
Patients who sign the informed consent form and are randomized but do not receive the study 
intervention may not be replaced and will be included in the intent to treat analysis. Patients who sign 
the informed consent form, and are randomized and receive the study intervention, and subsequently 
withdraw, or are withdrawn or discontinued from the study, will not be replaced and will be included in 
the primary analysis.  
 
7.3 
LOST TO FOLLOW-UP 
A participant will be considered lost to follow-up if he or she fails to return for scheduled visits and is 
unable to be contacted by the study site staff.  
 
The following actions must be taken if a participant fails to return to the clinic for a required study visit: 
• 
The site will attempt to contact the participant and reschedule the missed visit and counsel the 
participant on the importance of maintaining the assigned visit schedule and ascertain if the 
participant wishes to and/or should continue in the study. 
• 
Before a participant is deemed lost to follow-up, the investigator or designee will make every 
effort to regain contact with the participant (where possible, 3 telephone calls and, if necessary, 
a certified letter to the participant’s last known mailing address or local equivalent methods). 
These contact attempts should be documented in the participant’s medical record or study file.  
• 
Should the participant continue to be unreachable, he or she will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
  
8 
STUDY ASSESSMENTS AND PROCEDURES 
8.1 
EFFICACY ASSESSMENTS  
The following masked efficacy assessments will be performed: 
• 
Slit lamp examination. Performed by the treating physician. 

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• 
Visual acuity assessment. Both pinhole visual acuity and BSCVA/ETDRS/Mrx will be used 
depending on the time point. Visual acuity will be assessed only by a trained, masked 
optometrist to ensure proper procedures are followed across sites. See MOP Section 4.4 for the 
visual acuity protocol.  
• 
Imaging assessments. Non-contact imaging will be performed with HR AS-OCT, Pentacam 
topography, and slit lamp photography, and contact imaging with confocal microscopy. See 
MOP Section 4.6 for detailed descriptions of imaging procedures. Performed by trained study 
staff. 
• 
Corneal Gram Stain and Culture. Performed by the treating physician. See MOP Section 4.2 for 
detailed description of corneal scraping procedures.   
• 
Visual function questionnaire (VFQ). The National Eye Institute Visual Function Questionnaire 
(NEI-VFQ) and the Indian Visual Function Questionnaire (IND-VFQ) will be administered at 
UCSF/UM and Aravind, respectively. Both are validated questionnaires to evaluate the effect of 
vision on the patient’s quality of life. Performed by trained study staff.  
 
Please see the SoA in Section 1.3 of this protocol for a detailed schedule of all study activities.  
All of the above assessments, with the exception of the VFQ, will be performed during the screening 
process. These are standard of care procedures that the patient would receive even if they were not 
being screened for the study.  
The masked treating physician may use the results of these assessments to change the participant’s 
treatment in any way they deem necessary.  
 
8.2 
SAFETY AND OTHER ASSESSMENTS 
The following assessments will be administered to monitor patient safety: 
• 
Interval history from the patient to ask about side effects, etc. 
• 
Visual acuity assessment. 
• 
Intraocular pressure. 
• 
Slit lamp exam to identify complications. Ongoing AEs and SAEs will be followed until resolved. 
• 
Assessment of study intervention adherence. See Study Intervention Compliance, Section 6.4. 
   
8.3 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
 Adverse event means any untoward medical occurrence associated with the use of an intervention in 
humans, whether or not considered intervention-related (21 CFR 312.32 (a)). 
 
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of either the 
investigator or sponsor, it results in any of the following outcomes: death, a life-threatening adverse 
event, inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant 
incapacity or substantial disruption of the ability to conduct normal life functions, or a congenital 
anomaly/birth defect. Important medical events that may not result in death, be life-threatening, or 
require hospitalization may be considered serious when, based upon appropriate medical judgment, 
they may jeopardize the participant and may require medical or surgical intervention to prevent one of 
the outcomes listed in this definition. 
 

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8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
8.3.3.1 
SEVERITY OF EVENT 
The following guidelines will be used to describe AE severity.  
 
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• 
Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”. 
 
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION 
All adverse events (AEs) – ocular and non-ocular -must have their relationship to study intervention 
assessed by the clinician who examines and evaluates the participant based on temporal relationship 
and his/her clinical judgment. The degree of certainty about causality will be graded using the categories 
below. In a clinical trial, the study product must always be suspect.  
 
• 
Related – The AE is known to occur with the study intervention, there is a reasonable possibility 
that the study intervention caused the AE, or there is a temporal relationship between the study 
intervention and event. Reasonable possibility means that there is evidence to suggest a causal 
relationship between the study intervention and the AE. 
• 
Not Related – There is not a reasonable possibility that the administration of the study 
intervention caused the event, there is no temporal relationship between the study intervention 
and event onset, or an alternate etiology has been established. 
 
8.3.3.3 EXPECTEDNESS  
The treating physician will be responsible for determining whether an adverse event (AE) is expected or 
unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of the event is 
not consistent with the risk information previously described for the study intervention. 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the attention of 
study personnel during study visits and interviews of a study participant presenting for medical care, or 
upon review by a study monitor. 
 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured on the 
adverse event form. Information to be collected includes event description, time of onset, clinician’s 
assessment of severity, relationship to study product (assessed only by those with the training and 
authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs occurring while 
on study must be documented appropriately regardless of relationship. All AEs will be followed to 
adequate resolution. 
 
Any medical condition that is present at the time that the participant is screened will be considered as 
baseline and not reported as an AE. However, if the study participant’s condition deteriorates at any 
time during the study, it will be recorded as an AE.  

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Changes in the severity of an AE will be documented to allow an assessment of the duration of the event 
at each level of severity to be performed. AEs characterized as intermittent require documentation of 
onset and duration of each episode. 
 
The masked treating physician will record all reportable events with start dates occurring any time after 
informed consent is obtained until 7 (for non-serious AEs) or 30 days (for SAEs) after the last day of 
study participation.  At each study visit, the investigator will inquire about the occurrence of AE/SAEs 
since the last visit.  Events will be followed for outcome information until resolution or stabilization. 
 
8.3.5 ADVERSE EVENT REPORTING  
All AEs will be recorded in both the follow-up form and in a specific adverse event form. The SCUT 
II/REAGIR Data and Safety Monitoring Committee will review Adverse Events at twice yearly committee 
meetings. 
 
8.3.6 SERIOUS ADVERSE EVENT REPORTING  
All AEs should be followed until they are resolved or until a stable clinical endpoint is reached. Each AE is 
to be classified by the Investigator as SERIOUS (SAE) or NONSERIOUS (NSAE). Examples of ocular SAE: 
TPK, corneal perforation, endophthalmitis, IOP>30. Examples of non-ocular SAE: death, hospitalization, 
major medical event (eg. MI, stroke).  
 
If an Adverse Event occurs, the investigator must submit the adverse event form in REDCap within 24 
hours of the occurrence of the Adverse Event. The investigator must provide written follow-up reports 
until the SAE or clinically significant AE has resolved or until a stable clinical endpoint is reached. The 
medical monitor, Stephen McLeod (UCSF), will automatically be notified by the REDCap system when an 
Adverse Event or Severe Adverse Event is submitted. Because TPKs and corneal perforations are 
expected outcomes, they will be reported to Dr. McLeod monthly.   
 
Notification of an SAE or clinically significant AE must also be submitted to the Institutional Review 
Board (IRB) in accordance with its requirements. All Serious Ocular Adverse Events will be reported to 
the SCUT II/REAGIR DSMC on a monthly basis. All AEs must be reported from the time that the patient 
provides informed consent through the last study visit. 
 
The study sponsor will be responsible for notifying the Food and Drug Administration (FDA) of any 
unexpected fatal or life-threatening suspected adverse reaction as soon as possible, but in no case later 
than 7 calendar days after the sponsor's initial receipt of the information.   
 
 
8.3.7 REPORTING EVENTS TO PARTICIPANTS  
AEs and SAEs will be discussed on an individual level with the masked treating physician. At the end of 
the study, all study participants will be given a report of the results in their native language. 
 
8.3.8 EVENTS OF SPECIAL INTEREST  
 Not applicable. 
 
8.3.9 REPORTING OF PREGNANCY  
Not applicable. 

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8.4 
UNANTICIPATED PROBLEMS 
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
participant population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
8.4.2  UNANTICIPATED PROBLEM REPORTING  
The investigator will report unanticipated problems (UPs) to the reviewing Institutional Review Board 
(IRB) and to the Data Coordinating Center (DCC)/lead principal investigator (PI). The UP report will 
include the following information: 
 
• 
Protocol identifying information: protocol title and number, PI’s name, and the IRB project 
number; 
• 
A detailed description of the event, incident, experience, or outcome;  
• 
An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP;  
• 
A description of any changes to the protocol or other corrective actions that have been taken or 
are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline:   
 
• 
UPs that are serious adverse events (SAEs) will be reported to the IRB and to the DCC/study 
sponsor within 7 days of the investigator becoming aware of the event.  
• 
Any other UP will be reported to the IRB and to the DCC/study sponsor within 30 days of the 
investigator becoming aware of the problem.  
• 
All UPs should be reported to appropriate institutional officials (as required by an institution’s 
written reporting procedures), the supporting agency head (or designee), and the Office for 
Human Research Protections (OHRP) within 30 days of the IRB’s receipt of the report of the 
problem from the investigator. 
 
8.4.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
 
Participants will be notified of UPs in aggregate. When UPs affect an individual, they will be notified 
individually by the masked treating physician.  
 
 

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9 
STATISTICAL CONSIDERATIONS  
9.1 
STATISTICAL HYPOTHESES 
Specific Aim 1: To determine if corneal cross-linking with riboflavin (UVX) is a beneficial adjuvant in 
the treatment of smear- and/or culture-positive bacterial ulcers. 
a. We anticipate that UVX will result in better best spectacle corrected visual acuity (BSCVA) at 6 
months compared with antibiotic alone.  
b. We anticipate that UVX will result in faster microbiological cure rates, smaller scar size and 
lower rate of corneal perforation compared with antibiotic alone.   
Specific Aim 2: To determine if early topical steroids are a beneficial adjuvant in the treatment of 
smear- and/or culture-positive bacterial ulcers. 
a. We hypothesize that those randomized to early topical steroids will have improved BSCVA at 6 
months compared with antibiotic alone.  
Specific Aim 3: To determine which ulcer characteristics predict the most benefit from the addition of 
adjuvant corneal cross-linking and/or early steroids. 
a. We hypothesize that culture positive bacterial ulcers with drug resistant organisms, as measured 
by MIC50, will benefit more from adjuvant UVX than those with antibiotic susceptible organisms.   
 
Specific Aim 4: To determine if cross linking with rose bengal (RB-PDT) is a beneficial adjuvant in the 
treatment of fungal, acanthamoeba, and smear or culture negative corneal ulcers. 
a) We hypothesize that there will be improved visual acuity at 6 months among those randomized 
to adjuvant RB-PDT after controlling for baseline visual acuity. 
b) We anticipate that there will be smaller infiltrate/scar size and decreased rate of perforation 
and/or need for TPK at 6 months among RB-PDT treated patients. 
 
Specific Aim 5: To determine if corneal cross-linking with rose bengal (RB-PDT) is a beneficial adjuvant 
in the treatment of smear- and/or culture-positive bacterial ulcers. 
a. We hypothesize that study participants treated with topical antibiotics plus adjunctive RB-PDT 
will have better best spectacle corrected visual acuity at 6 months compared to those treated 
with antibiotics alone.  
 
Primary Endpoint: Best spectacle-corrected visual acuity at 6 months 
• 
Prior to publishing the SCUT II primary outcomes, optometrist experts will be surveyed 
regarding the outcomes of the study for a Bayesian analysis. The results of the questionnaire will 
serve as the prior when performing Bayesian analysis of the clinical trial results. 
 
Secondary Endpoints:  
• 
Microbiological cure on repeat smear and culture at Day 2  
• 
Interaction of minimum inhibitory concentration (MIC) and CXL on best spectacle-corrected 
visual acuity 
• 
Infiltrate/scar size/depth, as measured by clinical exam, clinical photographs, Pentacam and OCT 
at week 3 and months 3, 6, and 12 

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• 
Adverse events including rate of perforation/need for TPK 
• 
BSCVA at Day 1, 3 weeks, 3 months, and 12 months 
• 
Astigmatism, higher order aberrations, topography, and densitometry as measured on 
Pentacam at week 3 and months 3, 6, and 12 
• 
Corneal thickness and scar size/depth as measured by AS-OCT at week 3 and months 3, 6, and 
12 
• 
Basal nerve plexus, white blood cell count, and keratocyte density on confocal microscopy at 
week 3 and months 3, 6, and 12 
• 
Visual function questionnaire (VFQ) will be compared between groups at 6 months, controlling 
for Day 1 VFQ 
• 
Pain scale at baseline, Day 1, and Day 3  
• 
Subgroup analysis of study participants receiving prior topical antimicrobial therapy 
 
See Sections 9.4.1 and 9.4.2 for prespecified statistical analyses for Primary and Secondary Endpoints. 
 
9.2 
SAMPLE SIZE DETERMINATION 
 
Sample Size Calculation for SCUT II Primary Analysis: BSCVA. We power the study for each pairwise 
comparison. Specifically, we will be comparing (Specific Aim 1) CXL plus early steroids (arm 3) versus 
steroids and sham cross linking (arm 2), and (Specific Aim 2) early steroids with sham CXL (arm 2) versus 
standard therapy (arm 1).  The power calculation was based on the primary outcome, 6-month BSCVA. 
We informed the calculation with measurements from the first Steroids for Corneal Ulcers Trial (SCUT), 
among patients enrolled with between 20/60 and 20/400 vision. The SCUT trial measured BSCVA at 
baseline, 3-months, and 12-months. We conservatively used the 12-month outcome measure for the 
calculations since there was no 6-month measurement. The standard deviation of BSCVA at 12 months 
was 0.293. Since the primary analysis will adjust for baseline BSCVA, we used an estimate of the residual 
standard deviation, which is 𝑆𝐷! = 𝑆𝐷√1 −𝑟", where r is the correlation between the baseline 
measure and primary endpoint. In SCUT, the correlation between baseline and 12-month BSCVA among 
patients with between 20/60 and 20/400 vision at enrollment was 0.216. We thus assumed a residual 
standard deviation of  0.293√1 −0.216" = 0.286. Assuming a significance level of 0.05/2=0.025, and 
allowing for approximately 15% loss to follow up, we estimate that we will have 80% power to detect a 
1.4-line difference (logMAR 0.14) between groups with 93 study participants per arm (279 total). This 
calculation applies to each of the two prespecified primary outcomes for this trial (corresponding to the 
two separate research questions), and is based on the standard power formula for the T-test (using an 
estimated residual standard deviation).  Note that if the trial were to enroll 60 patients per arm, the 
minimum detectable effect under all the same assumptions would be 1.8 lines (logMAR 0.18). See SCUT 
II SAP Section 1.5.6 for details on interim power analysis.  
 
SCUT II Sample Size Calculation for Secondary Outcome: Microbiological Cure. Studies have suggested 
that in addition to providing an initial diagnosis, repeated culture can be used to assess response to 
treatment and is highly correlated with clinical outcomes such as visual acuity. We will re-culture all 
study participants 30 minutes after CXL or sham CXL (Day 2) to assess the effect of CXL on rate of 
microbiological cure. We hypothesize that those in the CXL group (Arm 3) will have a higher rate of 
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Steroid Group (Arm 2). Participants in both arms 1 and 2 will serve as the comparison group increasing 
the power in this analysis.  
 
For microbiological outcomes, we will be comparing sham cross-linking to actual cross-linking, with 
specimens collected just prior to any administration of antibiotics (as discussed in the Manual of 
Operations).  This comparison will be conducted contrasting Arm 3 to Arms 1 and 2 combined (which 
undergo sham cross-linking).  We expect that approximately 70% of patients will show positive baseline 
cultures; only culture positive patients are likely to contribute information to the microbiological 
outcome. Taking into account loss to follow-up, we anticipate 80% power to detect at least a 26% 
difference in absolute terms between those experiencing cross linking (Arm 3) and those who did not 
receive cross linking (Arm 1 and Arm 2). The calculation assumes a comparison of two proportions. This 
is a prespecified secondary outcome. 
 
Note that for both of these prespecified secondary analyses, the final hypothesis test will be conducted 
at an alpha of 0.025 (thereby applying a Bonferroni correction for two hypothesis tests). 
 
SCUT II Effects Stratified by Presence of Drug resistant Organisms. This analysis is similar to the 
microbiological outcome in the secondary analyses, in that we contrast culture-positive patients from 
Arm 3 to culture-positive patients from Groups 1 and 2 (Specific Aim 3).  Prior studies suggest a drug 
resistance to moxifloxacin probability of between 0.34 and 0.43, depending on prior exposure to 
moxifloxacin.  We hypothesize that when drug resistant organisms are present, outcomes will be more 
favorable when cross-linking is conducted. The outcome variable is visual acuity. We anticipate 
approximately 80% power to detect a difference of approximately 3 lines in outcomes, assuming 35% of 
individuals are drug resistant at baseline (based on simulation; details available upon request). The 
calculation assumed 15% loss to follow-up. This is a prespecified secondary analysis. 
 
See Section 9.4.1 for details regarding pre-specified analyses.  
 
REAGIR Primary outcome, BSCVA at 6 months. The sample size calculation for REAGIR used the same 
assumptions as the SCUT II sample size calculation for BSCVA.  Assuming a significance level of 0.05, 
allowing for approximately 15% loss to follow up, we estimate that we will have 90% power to detect a 
1.1-line difference (logMAR 0.11) between groups with 165 study participants per arm (330 total). For 
the same sample size and under the same assumptions, the detectable difference at 80% power is 1.0-
lines (logMAR 0.10). These calculations were based on the standard power formula for the T-test (using 
an estimated residual standard deviation).   
REAGIR II Primary outcome, BSCVA at 6 months. The sample size calculation for REAGIR II uses the 
same assumptions as the SCUT II sample size calculation for BSCVA. Assuming a significance level of 
0.05, allowing for approximately 15% loss to follow up, we estimate that we will have 80% power to 
detect a 5.3-lines difference (logMAR 0.53) between groups with 30 study participants per arm (60 
total). For the same sample size and under the same assumptions, the detectable difference at 90% 
power is 6.1-lines (logMAR 0.61). These calculations were based on the standard power formula for the 
T-test (using an estimated residual standard deviation).   
 
 
9.3 
POPULATIONS FOR ANALYSES 

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Our analysis will adhere strictly to the intent-to-treat principle. All participants randomized will be 
analyzed according to their allocation.   
Missing Outcomes: We distinguish two kinds of missing data: missing data in covariates, and missing 
data for outcomes. For the latter, we distinguish simple loss to follow-up (as when a patient has needed 
to move out of the area) from data missing because of the occurrence of a medical event (such as the 
loss of an eye). We will always report the results of complete case analysis, although such results are 
known to be biased.82 In the event of missing baseline covariates among >10% of patients, we will use 
regression based multiple imputation (with at least 100 replicates) to impute these missing covariates 
(based only on other variables available at baseline). To compare with the complete case analysis, we 
will also report multiple-imputation based analysis for missing outcomes (or equivalently, likelihood 
methods which integrate over the missing outcome data based on the same assumption of missingness 
at random); such analyses are important to report because of the intent to treat principle. Finally, we 
conduct sensitivity analyses in which the data are not assumed missing at random, to assess how 
extreme the missing values would need to be to change the conclusions of the study. We propose use of 
the R package mice (Multiple Imputation by Chained Equations) for multiple imputation; other choices 
are available and would serve equally well.  
 
9.4 
STATISTICAL ANALYSES 
 
9.4.1 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINT 
 
The Statistical Analysis Plans for SCUT II, REAGIR, and REAGIR II include more detail about planned 
analyses.  The primary outcome will be 6-month BSCVA, and the primary analysis of each trial will use 
the same approach. We will use multiple linear regression models to evaluate BSCVA measured at 6 
months with covariates for treatment arm (expressed as a binary indicator variable for steroids and a 
binary indicator variable for cross-linking), indicators for study site (randomization strata), and BSCVA 
measured on Day 1 (before treatment). We propose a two-sided alpha of 0.025 for each pairwise 
primary comparison. Specifically, we propose to contrast Groups 1 and 2 using a linear model in which 
we use baseline visual acuity and a binary indicator for assignment to Group 2 as predictors, and BSCVA 
as the outcome variable. We then propose to contrast Groups 2 and 3 separately using a model with 
baseline visual acuity and a binary indicator for assignment to Group 3 as predictors, with BSCVA as the 
outcome variable. 
 
For study participants who experience perforation or the need for TPK this will be noted and BSCVA will 
be recorded prior to performing surgery (either corneal glue or TPK). The last observation (at 3 weeks or 
3 months) will be carried forward (LOCF) as the 6-month BSCVA, but a separate secondary analysis will 
include these study participants actual 6-month visual acuity.  
 
A supplementary analysis using linear mixed effects regression will be conducted to analyze all BSCVA 
data (including 3-week and 3-month observations). The model will include baseline BSCVA and 
treatment assignment as covariates (fixed effects) plus a random effect for patient to allow for repeated 
measures over time. Such analyses will be sharply distinguished from the primary pre-specified analysis. 
Permutation testing will be the basis of inference. Specifically, we will conduct 10,000 random 
permutations of treatment assignment, and conduct the primary regression analysis on these re-

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randomized data sets. We will then compare the observed regression coefficients to the randomization 
distribution, reporting the quantiles. 
 
9.4.2 ANALYSIS OF THE SECONDARY ENDPOINTS 
Microbiological cure. Studies have suggested that in addition to providing an initial diagnosis, repeated 
culture can be used to assess response to treatment and is highly correlated with clinical outcomes such 
as visual acuity.83-86 We will re-culture all study participants at Day 2, prior to starting antibiotics to 
assess the effect of CXL on rate of microbiological cure. We hypothesize that those in the CXL group 
(Group 3) will have a higher rate of microbiological cure on Day 2 cultures than those randomized to the 
Standard Therapy (Group 1) or Early Steroid Group (Group 2). Participants in both group 1 & 2 will serve 
as the comparison group increasing the power in this analysis.  
We propose the primary analysis to be a Fisher’s exact test comparing the proportion of positivity at 
follow-up between initially culture positive individuals who were assigned to cross linking (Group 3) 
versus initially culture positive individuals assigned to sham cross linking (Groups 1 and 2). Additionally, 
we will report the results for initially culture negative individuals as a supplementary analysis in a logistic 
regression with assignment, indicators for site (randomization strata), and initial culture results as 
covariates. 
Scar/infiltrate. The analysis for scar/infiltrate size will follow the templates for visual acuity given above. 
Multiple linear regression models will be used to evaluate 12-month scar size by treatment arm (a 3-
level categorical variable) while correcting for baseline measurements. As in the primary analysis, In 
SCUT II (groups 1, 2, 3) we will perform pairwise comparisons between arms with a significance level of 
0.05/2=0.025. In REAGIR (groups 4, 5) we will compare the two arms with significance level of 0.05. 
Corneal thinning and scarring will be evaluated similarly using Anterior Segment Optical Coherence 
Tomography (AS-OCT) correcting for baseline values.  
Corneal Perforation. A Cox proportional hazards model will estimate the hazard of perforation, defined 
as perforation (flat anterior chamber with presence of iris plugging a defect in the cornea or seidel 
positivity) or the need for TPK while correcting for baseline infiltrate depth. 
Visual Function Questionnaire (VFQ). VFQ will be compared between groups controlling for Day 1 VFQ. 
NEI-VFQ in the US and the Indian-VFQ (IND-VFQ) in India. This will be conducted using linear regression 
with baseline and assignment variables. 
 
9.4.3 SAFETY ANALYSES 
Interim reports for the DSMC will be prepared by the Data Coordinating Center at Proctor. These reports 
will include (a) recruitment overall, and by study site, (b) compliance, and (c) retention. The reports will 
also list study outcomes, including 6-month BSCVA and microbiological outcomes, and all adverse 
outcomes, including mortality and perforations.  See Sections 9.4.1 and 9.4.2 of this protocol for a 
discussion of primary and secondary analyses to be performed.  
All adverse events will be tabulated and reported. Statistical comparisons will be conducted using 
Fisher’s exact test, but with the caution that failure to find a statistically significant difference cannot be 
used to infer a lack of risk difference, since the study is not powered to examine rare outcomes. 
Procedures for reporting both adverse events and serious adverse events, including notification of the 
Medical Monitor, will be reviewed by the DSMC prior to opening enrollment. We will categorize adverse 
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trial monitoring and reporting. The Statistical Analysis Plan – Section 1.5.6 (SCUT II) and Section 3.5 
(REAGIR) include details about interim analyses and stopping guidelines.87 
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO 
PARTICIPANTS 
Consent forms describing in detail the study intervention, study procedures, and risks are given to the 
participant and written documentation of informed consent is required prior to starting 
intervention/administering study intervention.  Consent forms will be given to the participant in English 
or Spanish (US sites), Tamil (India sites), or Portuguese (Brazil site).   
 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the 
study and continues throughout the individual’s study participation. Consent forms will be Institutional 
Review Board (IRB)-approved and the participant will be asked to read and review the document. The 
investigator will explain the research study to the participant and answer any questions that may arise. 
A verbal explanation will be provided in terms suited to the participant’s comprehension of the 
purposes, procedures, and potential risks of the study and of their rights as research participants.  
Participants will have the opportunity to carefully review the written consent form and ask questions 
prior to signing. The participants should have the opportunity to discuss the study with their family or 
surrogates or think about it prior to agreeing to participate. The participant will sign the informed 
consent document prior to any procedures being done specifically for the study. Participants must be 
informed that participation is voluntary and that they may withdraw from the study at any time, without 
prejudice. A copy of the informed consent document will be given to the participants for their records. 
The informed consent process will be conducted and documented in the source document (including the 
date), and the form signed, before the participant undergoes any study-specific procedures. The rights 
and welfare of the participants will be protected by emphasizing to them that the quality of their 
medical care will not be adversely affected if they decline to participate in this study. 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
 This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause.  Written notification, documenting the reason for study suspension or termination, will be 
provided by the suspending or terminating party to study participants, investigator, funding agency, 
sponsor, and regulatory authorities. The Statistical Analysis Plan – Section 1.5.6 includes details of 
interim stopping rules based on efficacy or futility. If the study is prematurely terminated or suspended, 
the Principal Investigator (PI) will promptly inform study participants, the Institutional Review Board 
(IRB), and sponsor and will provide the reason(s) for the termination or suspension.  Study participants 
will be contacted, as applicable, and be informed of changes to study visit schedule. 
  
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
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• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, 
and satisfy the sponsor, IRB and/or Food and Drug Administration (FDA). 
 
10.1.3 CONFIDENTIALITY AND PRIVACY  
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their 
staff, and the sponsor(s) and their interventions. This confidentiality is extended to cover testing of 
biological samples and genetic tests in addition to the clinical information relating to participants. 
Therefore, the study protocol, documentation, data, and all other information generated will be held in 
strict confidence. No information concerning the study or the data will be released to any unauthorized 
third party without prior written approval of the sponsor.  
 
All research activities will be conducted in as private a setting as possible. 
 
The study monitor, other authorized representatives of the sponsor, representatives of the Institutional 
Review Board (IRB), regulatory agencies or pharmaceutical company supplying study product may 
inspect all documents and records required to be maintained by the investigator, including but not 
limited to, medical records (office, clinic, or hospital) and pharmacy records for the participants in this 
study. The clinical study site will permit access to such records. 
 
The study participant’s contact information will be securely stored at each clinical site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location for as 
long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. 
 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will 
be transmitted to and stored at the Data Coordinating Center. This will not include the participant’s 
contact or identifying information. Rather, individual participants and their research data will be 
identified by a unique study identification number. The study data entry and study management 
systems used by clinical sites and by the Data Coordinating Center research staff will be secured and 
password protected. At the end of the study, all study databases will be de-identified and archived at 
the Data Coordinating Center at UCSF. 
 
Certificate of Confidentiality  
To further protect the privacy of study participants, a Certificate of Confidentiality will be issued by the 
National Institutes of Health (NIH). This certificate protects identifiable research information from 
forced disclosure. It allows the investigator and others who have access to research records to refuse to 
disclose identifying information on research participation in any civil, criminal, administrative, legislative, 
or other proceeding, whether at the federal, state, or local level. By protecting researchers and 
institutions from being compelled to disclose information that would identify research participants, 
Certificates of Confidentiality help achieve the research objectives and promote participation in studies 
by helping assure confidentiality and privacy to participants. 
 
 
 

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10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA  
Data collected for this study will be analyzed and stored at the Data Coordinating Center at UCSF. After 
the study is completed, the de-identified, archived data will be transmitted to and stored on the 
Research Electronic Data Capture system (REDCap).  
 
With the participant’s approval and as approved by local Institutional Review Boards (IRBs), de-
identified biological samples will be stored at the enrollment site. These samples could be used to 
research the relationship between minimum inhibitory concentration and clinical outcomes, as well as 
drug resistance.  
 
During the conduct of the study, an individual participant can choose to withdraw consent to have 
biological specimens stored for future research. However, withdrawal of consent with regard to 
biosample storage may not be possible after the study is completed.  
 
When the study is completed, access to study data will be provided through REDCap.  
 
10.1.5 KEY ROLES AND STUDY GOVERNANCE 
Co-Principal Investigator 
Co-Principal Investigator 
Medical Monitor 
Tom Lietman, MD 
Director and Professor 
Jennifer Rose-Nussbaumer, MD 
Stephen McLeod, MD 
Chair and Professor 
Francis I. Proctor Foundation, 
University of California, San 
Francisco   
Stanford University   
Department of 
Ophthalmology, University of 
California, San Francisco   
490 Illinois Street 
San Francisco, CA 94158 
 
2452 Watson Court 
Palo Alto CA 94303 
490 Illinois Street 
San Francisco, CA 94158 
 
415-502-2662 
650-722-7422 
415-476-1922 
Tom.Lietman@ucsf.edu 
rosej@stanford.edu 
Stephen.McLeod@ucsf.edu 
 
Please see MOP Section 2 for detailed descriptions of study investigators and staff.  
 
10.1.6 SAFETY OVERSIGHT 
A Data and Safety Monitoring Committee (DSMC) has been empaneled by the NEI. The committee 
consists of six individuals, including cornea specialists; an independent biostatistician; and a bioethicist. 
The committee will meet in person at least once per year and will convene biannual teleconferences for 
progress reports. Ad hoc meetings as needed may also be convened. All study protocols will be subject 
to review and approval by Institutional Review Boards. Please refer to the Statistical Analysis Plan – 
Section 1.5.6 for a detailed description of our planned interim analyses. 
10.1.7 CLINICAL MONITORING 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of 
the trial is in compliance with the currently approved protocol/amendment(s), with International 
Conference on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory 
requirement(s).  
 

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Clinical monitoring will be conducted by the Clinical Coordinating Center (CCC) at Stanford University. A 
two-person team will make site visits to all enrollment sites three times per year to monitor study 
activities. At each visit, the CCC will check the quality of photographs, sit in refractions to ensure the 
proper refraction protocol is followed, and visit the microbiology lab. During each visit the CCC will 
conduct a complete chart review of all patient charts to ensure data is being recorded in a complete 
fashion. The Data Coordinating Center will conduct regular weekly off-site reviews of data entered in 
REDCap to ensure 100% data verification.  
 
10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL 
Each clinical site will perform internal quality management of study conduct, data and biological 
specimen collection, documentation and completion.  An individualized quality management plan will be 
developed to describe a site’s quality management. 
 
Quality control (QC) procedures will be implemented beginning with the data entry system and data QC 
checks that will be run on the database will be generated. Any missing data or data anomalies will be 
communicated to the site(s) for clarification/resolution. 
 
Following written Standard Operating Procedures (SOPs), the monitors will verify that the clinical trial is 
conducted and data are generated and biological specimens are collected, documented (recorded), and 
reported in compliance with the protocol, International Conference on Harmonisation Good Clinical 
Practice (ICH GCP), and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good 
Manufacturing Practices (GMP)).  
 
The investigational site will provide direct access to all trial related sites, source data/documents, and 
reports for the purpose of monitoring and auditing by the sponsor, and inspection by local and 
regulatory authorities. 
 
10.1.9 DATA HANDLING AND RECORD KEEPING  
10.1.9.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
investigator. The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. 
 
Clinical data (including adverse events (AEs), concomitant medications, and expected adverse reactions 
data) and clinical laboratory data will be entered into Research Electronic Data Capture (REDCap), a 21 
CFR Part 11-compliant data capture system provided by the Data Coordinating Center at UCSF. The data 
system includes password protection and internal quality checks, such as automatic range checks, to 
identify data that appear inconsistent, incomplete, or inaccurate.  
 
Any hardcopies of the study visit worksheets will be provided for use as source document worksheets  
for recording data for each participant enrolled in the study. All source documents should be completed 
in a neat, legible manner to ensure accurate interpretation of data. Clinical data will be double data-
entered directly from the source documents.  
  
 
 
 
 

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10.1.9.2 STUDY RECORDS RETENTION  
Study documents should be retained for a minimum of 2 years after the last approval of a marketing 
application in an International Conference on Harminosation (ICH) region and until there are no pending 
or contemplated marketing applications in an ICH region or until at least 2 years have elapsed since the 
formal discontinuation of clinical development of the study intervention. These documents should be 
retained for a longer period, however, if required by local regulations. No records will be destroyed 
without the written consent of the sponsor, if applicable. It is the responsibility of the sponsor to inform 
the investigator when these documents no longer need to be retained. 
 
10.1.10 
PROTOCOL DEVIATIONS  
A protocol deviation is any noncompliance with the clinical trial protocol, ICH GCP, or MOP 
requirements. The noncompliance may be either on the part of the participant, the investigator, or the 
study site staff. As a result of deviations, corrective actions are to be developed by the site and 
implemented promptly.  
 
These practices are consistent with ICH GCP:  
• 
4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
• 
5.1 Quality Assurance and Quality Control, section 5.1.1  
• 
5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
 
It is the responsibility of the site investigator to use continuous vigilance to identify and report 
deviations within 7 working days of identification of the protocol deviation, or within 7 working days of 
the scheduled protocol-required activity.  All deviations must be addressed in study source documents, 
reported to National Eye Institute Program Official and the Coordinating Center at Stanford.  Protocol 
deviations must be sent to the reviewing Institutional Review Board (IRB) per their policies. The site 
investigator is responsible for knowing and adhering to the reviewing IRB requirements.  
 
10.1.11 
PUBLICATION AND DATA SHARING POLICY 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. As 
such, this trial will be registered at ClinicalTrials.gov, and results from this trial will be submitted and 
published on ClinicalTrials.gov. In addition, every attempt will be made to publish results in peer-reviewed 
journals. Consistent with the collaborative nature of the proposed research, the PI anticipates sharing all 
data generated by the study with collaborators. Analytic datasets that will be developed through the 
project will comply with the NIH Data Sharing Policy. The analytical datasets from this project will include 
patient-level data generated from the study visits. 
 
This study will adhere to the National Institutes of Health (NIH) Public Access Policy, which ensures that 
the public has access to the published results of NIH funded research. It requires scientists to submit 
final peer-reviewed journal manuscripts that arise from NIH funds to the digital archive PubMed Central 
upon acceptance for publication. External investigators can contact the study PIs to initiate a request for 
study data to support new study proposals or manuscripts. Approval of such requests and initiation of 
collaborations will consider the following criteria: 
1. The proposed project must be of high scientific merit.  
2. The proposed project must be consistent with the overall goals and objectives of the parent 
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3. The proposed ancillary project must meet certain participant burden criteria (for any new 
primary data collection involving subjects), including:  
a. Acceptable to the subjects (e.g., risks, time, discomfort, privacy); and, 
b. Not hinder or disrupt clinical care provided by study sites. 
4. The proposed project’s investigators must plan for adequate resources to effectively complete 
the project, including:  
a. Sufficient budget to cover costs of personnel and supplies; and  
b. Staff possessing the requisite expertise to meet the objectives of the project.  
5. The proposed project should document any involvement of parent study investigators as part of 
the research team.  
Approved requests for data will follow data sharing agreements that UCSF has with NIH. Data will be de-
identified prior to release for sharing.  However, there remains the possibility of deductive disclosure of 
subjects with unusual characteristics and disclosure of UCSF proprietary information. Thus, researchers 
who seek access to individual level data will be required to sign a data sharing agreement prior to 
release for sharing. The agreement provides for: (1) a commitment to using the data for research 
purposes only and not to identify any individual participants or to disclose proprietary information; (2) a 
commitment to securing the data using appropriate computer technology; (3) a commitment to 
destroying or returning the data after analyses are completed; and (4) a commitment to meet any 
requirements that might be stipulated by the IRB at UCSF.  
 
10.1.12 
CONFLICT OF INTEREST POLICY 
 The independence of this study from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical.  Therefore, any actual conflict of interest of persons who have a role in the design, 
conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, 
persons who have a perceived conflict of interest will be required to have such conflicts managed in a 
way that is appropriate to their participation in the design and conduct of this trial.  The study 
leadership in conjunction with the National Eye Institute has established policies and procedures for all 
study group members to disclose all conflicts of interest and will establish a mechanism for the 
management of all reported dualities of interest. 
 
10.2 ADDITIONAL CONSIDERATIONS 
None 
 
 

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10.3 ABBREVIATIONS 
 
AE 
Adverse Event 
AS-OCT 
Anterior Segment Optical Coherence Tomography 
BSCVA 
Best Spectacle-Corrected Visual Acuity 
CFR 
Code of Federal Regulations 
CCC 
Clinical Coordinating Center 
CONSORT 
Consolidated Standards of Reporting Trials 
CXL 
Corneal Cross-Linking 
DCC 
Data Coordinating Center 
DSMC 
Data Safety Monitoring Committee 
ETDRS 
Early Treatment Diabetic Retinopathy Study 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practices 
GMP 
Good Manufacturing Practices 
ICH 
International Conference on Harmonisation  
IDE 
Investigational Device Exemption 
IOP 
Intraocular Pressure 
IND 
Investigational New Drug Application 
IND-VFQ 
Indian Visual Function Questionnaire 
IOP 
Intraocular Pressure 
IRB 
Institutional Review Board 
LASIK 
Laser-Assisted in situ Keratomileusis 
LOCF 
Last Observation Carried Forward 
logMAR 
Logarithm of the Minimum Angle 
MIC 
Minimum Inhibitory Concentration 
MOP 
Manual of Operations and Procedures 
MRSA 
Methicillin-Resistant Staphylococcus Aureus 
MRx 
Manifest Refraction 
NCT 
National Clinical Trial 
NEI 
National Eye Institute 
NEI-VFQ 
National Eye Institute Visual Function Questionnaire 
NIH  
National Institutes of Health 
NSAE 
Non-Serious Adverse Event 
OCT 
Optical Coherence Tomography 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
PKP 
Penetrating Keratoplasty 
PRC 
Proctor Reading Center 
QA 
Quality Assurance 
QC 
Quality Control 
QID 
Four times daily 
RB 
Rose Bengal 
RB-PDT 
Rose Bengal Photodynamic Therapy  
REAGIR 
Rose Bengal Electromagnetic Activation with Green light for Infection Reduction 
REDCap 
Research Electronic Data Capture 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SCUT 
Steroids for Corneal Ulcer Trial 

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SCUT II 
Steroids and Cross-linking for Ulcer Treatment 
SoA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
TID 
Three times daily 
TPK 
Therapeutic Penetrating Keratoplasty 
UCSF 
University of California, San Francisco 
UNIFESP 
Federal University of São Paulo 
UP 
Unanticipated Problem 
US 
United States 
UV 
Ultra-violet 
UV-A 
Ultra-violet A 
UVX 
Corneal Cross-Linking with riboflavin 
VFQ 
Visual Function Questionnaire 
 
10.4 PROTOCOL AMENDMENT HISTORY 
The table below is intended to capture changes of IRB-approved versions of the protocol, including a 
description of the change and rationale. A Summary of Changes table for the current amendment is 
located in the Protocol Title Page.  
 
Version 
Date 
Description of Change  
Brief Rationale 
2.0 
17 July 2020 
Addition of Specific Aim 4 
 
2.0 
21 July 2021 
Added detail about baseline 
information collected/entered in 
REDCap for Rancho Park 
Compounding Pharmacy 
A pharmacist at Rancho Park 
will review current medications 
and any drug allergies prior to 
dispensing study medication. 
Patient addresses are needed 
for shipment of study 
medication directly to 
participants. 
2.0 
23 February 2022 
Clarification that patients 
randomized to Groups 1 & 2 receive 
saline drops during UVX 
 
3.0 
15 March 2023 
Addition of Federal University of Sao 
Paulo as REAGIR enrollment center 
 
 
 
Correction to concentration of Rose 
Bengal: 0.1% RB in 0.9% sodium 
chloride 
 
 
 
Clarification: Tumbling E chart only 
used for patients who don’t read 
letters 
 
4.0 
31 January 2024 
Addition of Specific Aim 5 
REAGIR II 
 
 
Addition of Informed Consent Form 
Addendum 
For patients who elect to drop 
out of the study, signing the 
addendum allows the study 
team to collect follow-up data 
through 12 months post-op 

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5.0 
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Added Bayesian Analysis to Section 
9 
 
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