Conditions: Corneal Ulcer Infectious Keratitis Bacterial Ulcer Fungal Ulcer
Keywords: Cornea Ulcer Infectious keratitis Bacterial Fungal
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 147
Healthy volunteers: no

Brief summary:
The primary purpose of this study is to determine if patients randomized to corneal collagen cross-linking plus medical therapy will have a lower prevalence of positive bacterial or fungal cultures immediately after the procedure than patients who received medical therapy alone.

The secondary purpose of this study is to determine if patients randomized to corneal collagen cross-linking will have a better visual acuity at 3 and 12 months than patients who receive medical therapy alone.

Detailed description:
The proposed study is a randomized controlled trial to determine whether collagen cross-linking improves outcomes in microbial keratitis. Patients presenting to the Aravind Eye Hospitals in Madurai, India for treatment of microbial keratitis will be recruited for the proposed study. Approximately 266 patients will be enrolled in the study. Subjects presenting with bacterial keratitis will be randomized to receive medical therapy plus corneal collagen cross-linking at presentation or to receive medical therapy alone. All subjects with bacterial ulcers will receive standard topical antibiotic medications as required after the procedure.

Subjects with fungal keratitis will be randomized into one of four groups:

1. collagen cross-linking + amphotericin;
2. collagen cross-linking + natamycin;
3. amphotericin alone;
4. natamycin alone.

All study subjects will be followed for 12 months to evaluate response to treatment.

Investigators from the University of California, San Francisco (UCSF) will assist Aravind Eye Hospital with the study design, implementation, and analysis of the research, and will help fund the study. The investigators plan to visit Aravind to help with the study implementation. UCSF will play an important role in this study by assisting with the study design, implementation, analysis, and funding.

Arms:
arm 1: Standard of care topical treatment for bacterial ulcer plus cross-linking arm 2: Standard of care topical treatment for bacterial ulcer arm 3: Standard of care topical treatment for fungal ulcer with natamycin plus cross-linking arm 4: Standard of care topical treatment for fungal ulcer with natamycin arm 5: Standard of care topical treatment for fungal ulcer with amphotericin plus cross-linking arm 6: Standard of care topical treatment for fungal ulcer with amphotericin

Interventions: intervention 1: Corneal Cross-linking intervention 2: Anti Fungal Drug
Intervention descriptions: intervention 1: For those subjects randomized to receive collagen cross-linking, the procedure will be performed as per the routine at the hospital (UV-X machine; strict aseptic precautions; corneal epithelium debrided and 0.1% riboflavin applied for 30 minutes, then UV-A radiation applied for 30 minutes at 370nm with 3mW/cm2). Corneal cross-linking is a routine procedure performed by Aravind Eye Hospital for infectious keratitis. intervention 2: Topical Amphotericin B vs Topical Natamycin

Protocol document:
ICF_002.pdf: 
Cross-linking for Corneal Ulcers Treatment Trial (CLAIR)
NCT02570321
May 12, 2016


 
ARAVIND CROSSLINKING-V6; 12 MAY 2016 
 
PAGE 1 OF 5 
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO 
ARAVIND EYE HOSPITAL, MADURAI 
CONSENT TO PARTICIPATE IN A RESEARCH STUDY 
 
Study Title: Cross-Linking for Corneal Ulcers Treatment Trial 
 
This is a medical research study.  Your study doctor(s), Dr. Jennifer Rose-Nussbaumer from the 
University of California and Dr. NV Prajna from the Aravind Eye Hospital will explain this 
study to you. 
 
Medical research studies include only people who choose to take part.  Take your time to make 
your decision about participating.  You may discuss your decision with your family and friends 
and with your health care team.  If you have any questions, you may ask your study doctor. 
 
You are being asked to take part in this study because you have a corneal ulcer. 
 
Why is this study being done? 
 
The purpose of this study is to determine the effect that corneal cross-linking has on corneal 
ulcers.  Cross-linking is a procedure where a special type of light is applied to the front of the eye 
to try to clear the eye infection and make your eye stronger. The cross-linking device is called 
the UV-X, and is not yet approved in India. We want to find out what effects, good and/or bad, it 
has on you and your corneal infection. 
 
For corneal infections caused by fungus, there are two standard of care topical medications used 
for treatment: natamycin and amphotericin B. Amphotericin B has been shown to be effective 
when used with cross-linking in non-human studies. We would also like to find out what effects, 
good and/or bad, the combination of these topical medications with collagen cross-linking has on 
you and your corneal infection, and learn whether there are differences between these two 
medications when used with cross-linking.  
 
How many people will take part in this study? 
 
About 266 people will take part in this study.   
 
What will happen if I take part in this research study? 
 
If you take part in this study, we will first record your vision and take pictures of the front of 
your eye. You will be "randomized" into one of the study groups described below.  
Randomization means that you are put into a group by chance.  A computer program will place 
you in one of the groups.  Neither you nor your doctor can choose the group you will be in. 
Regardless of which group you are placed in, you will receive standard of care topical 
medications that are routinely used in the treatment of your infection. Collagen cross-linking is 
not currently standard of care. 
 
If you have a corneal ulcer caused by bacteria, you will be randomized into one of two groups: 
 Group 1: You will receive standard of care eye drops called moxifloxacin and the 
corneal cross-linking procedure. During this procedure a blue-colored light will be 


 
ARAVIND CROSSLINKING-V6; 12 MAY 2016 
 
PAGE 2 OF 5 
directed toward your eye for 30 minutes. As part of the procedure, your cornea will be 
gently scraped to test for bacteria, fungus, and acanthamoeba after the procedure has been 
completed.  
 Group 2: You will receive standard of care eye drops called moxifloxacin and the mock 
corneal cross-linking procedure. During this procedure a blue-colored light will be 
directed near your eye for 30 minutes. As part of the procedure, your cornea will be 
gently scraped to test for bacteria, fungus, and acanthamoeba after the procedure has been 
completed.  
 
If you have a corneal ulcer caused by fungus, you will be randomized into one of four groups: 
 Group 1: You will first receive standard topical antifungal eye drops call amphotericin 
B. You will then receive another type of eye drops called riboflavin and the corneal 
cross-linking procedure. During this procedure a blue-colored light will be directed 
toward your eye for 30 minutes. As part of the procedure, your cornea will be gently 
scraped to test for bacteria, fungus, and acanthamoeba after the procedure has been 
completed. 
 Group 2: You will first receive standard topical antifungal eye drops call natamycin. 
You will then receive another type of eye drops called riboflavin and the corneal cross-
linking procedure. During this procedure a blue-colored light will be directed toward 
your eye for 30 minutes. As part of the procedure, your cornea will be gently scraped to 
test for bacteria, fungus, and acanthamoeba after the procedure has been completed. 
 Group 3: You will first receive standard topical antifungal eye drops call amphotericin 
B. You will then receive another type of eye drops and the mock corneal cross-linking 
procedure. During this procedure a blue-colored light will be directed near your eye for 
30 minutes. As part of the procedure, your cornea will be gently scraped to test for 
bacteria, fungus, and acanthamoeba after the procedure has been completed. 
 Group 4: You will first receive standard topical antifungal eye drops call natamycin. 
You will then receive another type of eye drops and the mock corneal cross-linking 
procedure. During this procedure a blue-colored light will be directed near your eye for 
30 minutes. As part of the procedure, your cornea will be gently scraped to test for 
bacteria, fungus, and acanthamoeba after the procedure has been completed. 
 
If you have a corneal ulcer caused by acanthamoeba, you will be randomized into one of two 
groups: 
 Group 1: You will receive standard of care eye drops called polyhexamethylene 
biguanide (PHMB) and the corneal cross-linking procedure. During this procedure a 
blue-colored light will be directed toward your eye for 30 minutes. As part of the 
procedure, your cornea will be gently scraped to test for bacteria, fungus, and 
acanthamoeba after the procedure has been completed.  
 Group 2: You will receive standard of care eye drops called polyhexamethylene 
biguanide (PHMB) and the mock corneal cross-linking procedure. During this procedure 
a blue-colored light will be directed near your eye for 30 minutes. As part of the 
procedure, your cornea will be gently scraped to test for bacteria, fungus, and 
acanthamoeba after the procedure has been completed.  
 
These procedures will all occur in the operating theater.   


 
ARAVIND CROSSLINKING-V6; 12 MAY 2016 
 
PAGE 3 OF 5 
After the cross-linking procedure, you will receive the normal, standard of care treatment for 
your eye infection.  
Three days after the procedure, your cornea will be gently scraped once more to test for bacteria, 
fungus, and acanthamoeba. 
You will have two follow-up study visits, one at 3 weeks and one at 3 months after the 
procedure. At these visits, you will have photographs taken of your eye and your vision checked. 
How long will I be in the study? 
Participation in the study will take a total of about six hours over a period of three months. The 
first study visit will last approximately four hours, and the follow-up study visits will last 
approximately one hour each. 
 
Can I stop being in the study? 
Yes.  You can decide to stop at any time.  Tell the study doctor if you are thinking about 
stopping or decide to stop.  He or she will tell you how to stop your participation safely.  
The study doctor may stop you from taking part in this study at any time if he/she believes it is in 
your best interest, if you do not follow the study rules, or if the study is stopped. 
What side effects or risks can I expect from being in the study? 
You may have side effects while on the study.  Everyone taking part in the study will be watched 
carefully for any side effects.  However, doctors don't know all the side effects that may happen.  
Side effects may be mild or very serious.  Your health care team may give you medicines to help 
lessen side effects.  Many side effects go away soon after you complete the cross-linking 
procedure. In some cases, side effects can be serious, long lasting, or may never go away.   
You should talk to your study doctor about any side effects you experience while taking part in 
the study. 
Cross-linking is a commonly used procedure worldwide and is thought to be very safe. Possible 
risks include secondary infections including corneal infection and corneal edema. If you 
experience one of these side effects, you will be treated with standard of care treatments. The 
experimental treatments may have side effects that no one knows about yet.  The researchers will 
let you know if they learn anything that might make you change your mind about participating in 
the study. For more information about risks and side effects, ask your study doctor. 
Are there benefits to taking part in the study? 
Taking part in this study may or may not make your health better.  While doctors hope corneal 
crosslinking in addition to use of topical antibiotic, antifungal, or antiamoebic eye drops will be 
more effective than the standard (usual) treatment, there is no proof of this yet. 


 
ARAVIND CROSSLINKING-V6; 12 MAY 2016 
 
PAGE 4 OF 5 
There will be no direct benefit to you from participating in this study.  However, this study will 
help doctors learn more about corneal crosslinking and it is hoped that this information will help 
in the treatment of future patients with corneal ulcers. 
What other choices do I have if I do not take part in this study? 
Your other choices may include: 
 
Getting no treatment 
 
Getting standard treatment for your condition with medications, without being in a study.  
Please talk to your doctor about your choices before deciding if you will take part in this study. 
How will information about me be kept confidential? 
 
Participation in research involves some loss of privacy.  We will do our best to make sure that 
information about you is kept confidential, but we cannot guarantee total privacy.  Some 
information from your medical records will be collected and used for this study. People involved 
with your future care may become aware of your participation and of any information added to 
your medical record as a result of your participation.  Study tests that are performed by research 
labs, and information gathered directly from you by the researchers will be part of your research 
records and will also be added to your medical record.  Your personal information may be given 
out if required by law.  If information from this study is published or presented at scientific 
meetings, your name and other personal information will not be used. 
Organizations that may look at and/or copy your medical records for research, quality assurance, 
and data analysis include: 
 Aravind Eye Care System 
 University of California 
 Government agencies involved in keeping research safe for people.  
What are the costs of taking part in this study? 
You will not be charged for any of the study activities. 
Will I be paid for taking part in this study? 
You will not be paid for taking part in this study. 
What happens if I am injured because I took part in this study? 
It is important that you tell your study doctor, Dr. Prajna, if you feel that you have been injured 
because of taking part in this study.  You can tell the doctor in person or call him at +91 452 435 
6100. If you are injured as a result of being in this study, the costs of the treatment may be billed 
to you or your insurer just like any other medical costs, or covered by Aravind Eye Hospital, 
depending on a number of factors. The University and the study sponsor do not normally provide 
any other form of compensation for injury.  


 
ARAVIND CROSSLINKING-V6; 12 MAY 2016 
 
PAGE 5 OF 5 
What are my rights if I take part in this study? 
Taking part in this study is your choice.  You may choose either to take part or not to take part in 
the study.  If you decide to take part in this study, you may leave the study at any time.  No 
matter what decision you make, there will be no penalty to you and you will not lose any of your 
regular benefits.  Leaving the study will not affect your medical care.  You can still get your 
medical care from our institution.  
We will tell you about new information or changes in the study that may affect your health or 
your willingness to continue in the study. 
In the case of injury resulting from this study, you do not lose any of your legal rights to seek 
payment by signing this form. 
Who can answer my questions about the study? 
You can talk to your study doctor about any questions, concerns, or complaints you have about 
this study.  Contact your study doctor Dr. Prajna at +91 452 435 6100. 
If you wish to ask questions about the study or your rights as a research participant to someone 
other than the researchers or if you wish to voice any problems or concerns you may have about 
the study, please call the Aravind Medical Research Foundation at +91-452-435 6550. 
 
CONSENT 
PARTICIPATION IN RESEARCH IS VOLUNTARY.  You have the right to decline to 
participate or to withdraw at any point in this study without penalty or loss of benefits to which 
you are otherwise entitled. 
 
If you wish to participate in this study, you should sign below. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Date 
 
 
Participant's Signature for Consent 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Date 
 
 
Person Obtaining Consent 
 
 
 
 
 

Prot_000.pdf: 
Cross-linking for Corneal Ulcers Treatment Trial (CLAIR)
NCT02570321
October 4, 2016


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
 
 
 
 
 
 
Cross-Linking Assisted Infection Reduction 
CLAIR 
Manual of Operations and Procedures (MOP) 
 
 
Aravind Eye Hospital, Madurai, Coimbatore, and Pondicherry, India 
Francis I. Proctor Foundation, University of California San Francisco, USA 
 
 
 
Investigators: 
N. Venkatesh Prajna, DO DNB FICO1 
Dionna Fry, MPH2 
Tom Lietman, MD2 
Jeremy Keenan, MD MPH2 
Jennifer Rose-Nussbaumer, MD2 
Ariana Austin, MS2 
 
 
 
 
 
1Aravind Eye Hospital, Madurai, Pondicherry, and Coimbatore, India   
2 Francis I. Proctor Foundation, University of California, San Francisco, USA 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
 
 
 
 
CONTENTS 
1. 
INTRODUCTION AND BACKGROUND ......................................................................................................................... 1 
1.1 
Specific Aims:................................................................................................................................................................. 1 
1.2 
Study Outcomes: ........................................................................................................................................................... 2 
1.3 
Study Design: ................................................................................................................................................................. 2 
2. 
ORGANIZATION ......................................................................................................................................................... 2 
2.1 
Collaborating Institutions: ............................................................................................................................................. 3 
3. 
PATIENT FLOW ........................................................................................................................................................... 3 
3.1 
Study Timeline ............................................................................................................................................................... 3 
3.2 
Eligibility Requirements ................................................................................................................................................. 4 
3.3 
Randomization .............................................................................................................................................................. 4 
3.4 
Study visits..................................................................................................................................................................... 4 
3.5 
Study Schedule .............................................................................................................................................................. 6 
3.6 
Adverse Events (AEs) ..................................................................................................................................................... 6 
4. 
PROCEDURES ............................................................................................................................................................. 6 
4.1 
Masking ......................................................................................................................................................................... 6 
4.2 
Ulcer Classification .......................................................................................................... Error! Bookmark not defined. 
4.3 
Procedures ..................................................................................................................................................................... 7 
4.4 
Pentacam Protocol ........................................................................................................................................................ 7 
5. 
STUDY MEDICATIONS ................................................................................................................................................ 8 
5.1 
Medical management protocol & Treatment Schedule ................................................................................................ 8 
5.2 
Possible Side Effects of Study Medications .................................................................................................................... 9 
6. 
PROTECTION OF HUMAN SUBJECTS ........................................................................................................................... 8 
6.1 
Institutional Review Board Approval ............................................................................................................................. 8 
6.2 
Informed Consent .......................................................................................................................................................... 9 
6.3 
Risks to Study Participants .......................................................................................................................................... 10 
6.4 
Privacy, Confidentiality and Data Security .................................................................................................................. 10 
6.5 
Exclusion of Vulerable Populations ............................................................................................................................. 11 
6.6 
Compensation to Participants ..................................................................................................................................... 11 
7. 
DATA COLLECTION AND MANAGEMENT .................................................................................................................. 11 
7.1 
Data Collection and Entry............................................................................................................................................ 11 
7.2 
Data Consistency, Validity, and Monitoring ................................................................................................................ 11 
7.3 
Data Storage and Security........................................................................................................................................... 11 
8. 
STATISTICAL ANALYSIS PLAN ................................................................................................................................... 11 
9. 
References ............................................................................................................................................................... 12 
 


CLAIR MOP 
Version 1.6; Revised 3 May 2016 
 
1 | P a g e  
 
1. INTRODUCTION AND BACKGROUND  
Cross-linking (CXL), a process by which collagen fibrils form covalent bonds with adjacent molecules, was 
first discovered in the corneas of diabetic patients who cross-link through glycosylation and have a 
decreased incidence of keratoconus.1 It is currently used as a treatment for corneal ectatic disorders such as 
keratoconus and post-LASIK ectasia. CXL involves applying riboflavin to the cornea and photo-chemically 
activating it with UV light. This has been shown to strengthen the cornea and allow it to retain its normal shape.1-4 
There has been recent interest in corneal CXL as an adjuvant therapy for infectious keratitis.5 There are two 
mechanisms by which CXL may benefit patients with infectious corneal ulcers; antimicrobial effects, and increased 
resistance of corneal tissue to enzymatic degradation.  
In vitro efficacy of UV-A + riboflavin against the most common bacterial ocular pathogens, such as 
Pseudomonas aeruginosa and Streptococcus pneumonia has been demonstrated.6 In addition to these in vitro 
data, multiple case reports have suggested potential benefits of collagen CXL for treatment of bacterial and fungal 
keratitis, such as symptomatic improvement, resolution of resistant infection, and halting of progressive melting.7-
10 One small study treated a series of patients with bacterial keratitis exclusively with photo-chemically activated 
riboflavin, which resolved infection in all patients.11 Interestingly, only 2 of the 16 study participants required 
topical antibiotic treatment. Thus, riboflavin + UV-A could potentially decrease antibiotic resistance and ocular 
surface toxicity that can complicate the current management of bacterial keratitis. 
Filamentous fungal corneal ulcers continue to have a poor prognosis and further investigation into their 
treatment is warranted. Unfortunately, in vitro collagen CXL alone has failed to demonstrate fungal pathogen 
inactivation.6,12 However, one study showed improved inhibition of fungus in vitro with amphotericin plus 
riboflavin+UV-A compared with amphotericin alone.6 The authors suggest that CXL may improve diffusion of 
antifungal medications or inhibit fungal proliferation through more complex mechanisms. Although the evidence 
for cross-linking as a treatment for fungal keratitis is less robust, clinicians have already begun using UV-A + 
riboflavin in conjunction with antifungals in hopes that there will be some benefit in this challenging disease. Thus, 
scientific investigation into this treatment approach is necessary.   
Research Question: Is collagen cross-linking a useful adjuvant in the treatment of infectious corneal ulcers? 
1.1 Specific Aims: 
1. To determine if patients with culture positive bacterial and fungal keratitis clear their repeat cultures 
more quickly with the addition of collagen cross-linking. 
 
We hypothesize that there will be an increased rate of negative cultures immediately post procedure in the 
CXL group compared with sham CXL. 
2. To determine if CXL can decrease enzymatic degradation and prevent corneal thinning and perforation.  
 
Hypothesis 2A) There will be a decreased rate of perforation among study participants treated with CXL.  
 
Hypothesis 2b) There will be less corneal thinning as measured by optical coherence tomography in the 
CXL group at 3 months. 
 
3. To determine what effect CXL has on visual acuity, corneal astigmatism, and 3-month infiltrate/scar size.  


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 2 
 
 
Hypothesis 3A) There will be improved visual acuity with reduced 3-month infiltrate/scar size among 
study participants treated with CXL. 
 
Hypothesis 3B) There will be no difference in irregular astigmatism (as measured by topography) 
between participants in the CXL group versus antibiotics alone. 
 
1.2 Study Outcomes: 
Primary Outcome: 
 
Microbiological cure on repeat culture 
o CLAIR I. Bacterial ulcers – Culture at 4 hours  
o CLAIR II Fungal ulcers – Culture at 24 hours  
Secondary Outcomes: 
 
BSCVA at 3 months 
 
Scar size/depth, as measured by clinical exam, slit lamp photographs and OCT  
 
Adverse events including rate of perforation/need for TPK 
 
Thinning as measured by pachymetry and OCT  
 
Topography and corneal higher-order aberrations  
 
Change in IND-VFQ from baseline to 3 months.  
1.3 Study Design: 
Cross-Linking Assisted Infection Reduction (CLAIR) I for bacterial ulcers and CLAIR II for fungal ulcers is a series 
of randomized, masked, clinical trials. The purpose of these studies is to determine differences in microbiological 
cure for 1-day repeat cultures between different medical antimicrobial treatments alone versus antimicrobial 
treatment plus collagen cross-linking. There will be 1:1 randomization to each of these treatment groups: 
CLAIR I. Bacterial Ulcers: 
1) Topical 0.5% Moxifloxacin alone  
2) Topical 0.5% Moxifloxacin plus cross-linking  
CLAIR II. Fungal Ulcers: 
1) Topical 5% natamycin alone  
2) Topical 5% natamycin plus cross-linking  
3) Topical 0.15% amphotericin alone 
4) Topical 0.15% amphotericin plus cross-linking 
2. ORGANIZATION 
Aravind Eye Hospital, Madurai along with the University of California San Francisco (UCSF) will jointly execute 
these clinical trials. Aravind Eye Hospital will mainly be responsible for recruitment and enrollment, intervention 
implementation, and follow-up visits. UCSF will take the lead on all data analysis, writing of study-related 
materials, and writing journal publications. 
 
 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 3 
 
2.1 Collaborating Institutions: 
Aravind Eye Hospital, India  
Dr. N. Venkatesh Prajna will be the lead investigator at Aravind Eye Hospital. All recruitment, 
treatment/intervention, and follow-up visits will be done at the Aravind Eye Hospitals. All study personnel 
assisting with the research will be adequately informed about the protocol, the research procedures, and their 
duties and functions through ongoing communication between the 2 sites.   
 
Aurolab at Aravind Eye Hospital in Madurai will be responsible for preparing and distributing study medications. 
The microbiology lab in Madurai will perform the cultures and serve as a bank for all specimens collected. Madurai 
microbiology lab will serve as a reference lab that will assist with any difficult microbiological identification. 
Francis I. Proctor Foundation, University of California, San Francisco, USA 
 
The Proctor Foundation is an organized research unit at the University of California, San Francisco. The 
Foundation has a 56-year history of research in ocular infectious and inflammatory disease and runs one of the 
leading corneal fellowship training programs in the United States.  Proctor Foundation faculty have been involved 
in prevention of blindness research in developing countries since the Foundation’s inception. The impetus for 
establishing the Foundation in 1947 was to eradicate trachoma in the American Southwest and other parts of the 
world. 
 
UCSF will perform the data analysis. UCSF will not consent any participants, perform surgeries, or collect study 
data. UCSF will have the most current protocol, consent documents and HIPAA authorization for reference. UCSF 
has received IRB approval for this study (IRB number: 14-14918). All modifications to either IRB of record will be 
communicated between sites. UCSF will ensure protection of all study-related data by using de-identified 
information over a secure server. All non-compliance with the study protocol or applicable requirements will be 
reported in accordance with local policy. There will be ongoing communication of problems, interim results, and 
study closure between both sites. 
 
 
Jennifer Rose-Nussbaumer, MD is the co-PI at UCSF for this study. Dr. Rose-Nussbaumer is a Cornea fellowship 
trained Ophthalmologist at F. I. Proctor Foundation. In addition to her clinical work in cataract and corneal 
transplant surgery, she is an NIH funded clinical researcher. She is studying corneal ulcer treatment in India and 
Nepal and corneal transplant outcomes in Ethiopia. She is the principle investigator on the Corneal Preservation 
Time Study (CPTS) and Descemets Endothelial Thickness Comparison Trial (DETECT) at UCSF. Her previous vision 
research in Ophthalmology includes work with the World Health Organization on Trachoma, as well as 
investigating the ocular manifestations of HIV disease. Dr. Rose-Nussbaumer’s role in this study will be to perform 
data analysis, to collaborate with surgery site and eye bank regarding study design and protocols. 
 
3. PATIENT FLOW 
3.1 Study Timeline 
When the patient presents to clinic with infectious keratitis and undergoes corneal scraping by the treating 
ophthalmologist, a specimen will be submitted for microbiological testing (KOH and Gram stain bacterial or 
fungal). This is a routine practice already in place at the hospital. If the patient has a positive KOH or Gram stain, 
the patient will be approached by study personnel to be enrolled in study.   


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 4 
 
If enrolled, study subjects will follow the procedures below - if bacteria positive on Gram stain, the subject will 
undergo procedures outlined for CLAIR I. Bacterial ulcers; if positive for fungus on KOH, subject will undergo 
procedures outlined for CLAIR II. Fungal ulcers. 
Study participants will be required to have 4 follow-up visits, one for the cross-linking procedure and re-scraping, 
one at 3 days for re-scraping, one at 3 weeks  (2.5 – 5 weeks) and one at 3 months (visit window: 2.5 -3.5 months). 
Additional visits may be needed and will be determined by the physician/investigator.  
 
3.2 Eligibility Requirements 
Only those who meet all of the inclusion criteria and none of the exclusion criteria will be enrolled in this study.  
Inclusion criteria: 
 
Corneal ulcer that is smear positive for either bacteria, filamentous fungus  
 
Pinhole visual acuity worse than 6/21 (20/70) in the affected eye  
 
Age over 18 years 
 
Basic understanding of the study as determined by the physician 
 
Commitment to be hospitalized for 3 days 
 
Commitment to return for follow up visits 
 
Exclusion criteria: 
 
No evidence of concomitant infection on exam or gram stain (i.e. herpes, both bacteria and acanthameoba 
on gram stain) 
 
Impending or frank perforation at recruitment 
 
Involvement of sclera at presentation 
 
Non-infectious or autoimmune keratitis 
 
History of corneal transplantation or recent intraocular surgery 
 
No light perception in the affected eye 
 
Pinhole visual acuity worse than 20/200 in the unaffected eye  
 
Participants who are decisionally and/or cognitively impaired 
 
Involvement of posterior 1/3 of stroma at presentation for fungal patients 
3.3 Randomization 
Each study eye will be randomly assigned to the treatment group. Block randomization will be performed using a 
computer program (Statistical package R; Version 2.12; R Foundation for Statistical Computing, Vienna, Austria) by 
the coordinating site.  
 
Once an eye is 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 3 hours for bacterial ulcers, and within 24 
hours for fungal ulcers. Once the study participant has been assigned a study participant ID and randomized to 
treatment group, they will be included in the intention to treat analysis.  
 
3.4 Study visits 
3.3.1 Baseline Visit. During this visit, eligible patients will be enrolled in the study and will give consent. The 
baseline patient form will be completed and baseline topography will be obtained. 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 5 
 
CLAIR I. Bacterial Ulcers: 
Study participants will be randomized to one of two possible treatment arms: 1) topical 0.5% moxifloxacin alone 
2) topical 0.5% moxifloxacin plus CXL.  Those randomized to CXL will undergo the procedure in the operating 
theatre within 4 hours. Both arms will undergo repeat scraping at 4 hours post enrollment. Thereafter, all study 
participants will start 0.5% moxifloxacin every hour. All patients will be admitted to the inpatient setting until the 
3rd day. 
CLAIR II. Fungal Ulcers: 
They will be randomized to one of four possible treatment arms 1) topical 5% natamycin alone, 2) topical 5% 
natamycin plus CXL, 3) topical 0.15% amphotericin alone or 4) topical 0.15% amphotericin plus CXL. All study 
participants will begin medical therapy immediately (either natamycin or amphotericin) every hour and those 
randomized to CXL will undergo the procedure in the operating theatre within 24 hours. All study participants will 
undergo repeat scrapping at 24 hours from enrollment. All patients will be admitted to the inpatient setting until 
the 3rd day.  
3.3.2 Procedure visit. All study participants randomized to CXL will have the procedure in the operating theater. 
This will occur within 4 hours for bacterial ulcers and within 24 hours of the baseline visit for fungal ulcers. Once a 
study participant has been randomized to treatment, he will have the treatment even if there is a delay in the 
operating theatre that results in delayed treatment, however re-scraping will occur at 4 hours for bacterial ulcers 
and 24 hours for fungal ulcers even if they have not yet had cross-linking.  If the cross-linking occurs after repeat 
scrapping, a third smear and culture should be performed.  
3.3.3 Day 3 visit. During this visit, study participants will have BSCVA recorded, IND-VFQ, slit lamp examination and 
undergo repeat scraping and culture of the corneal ulcer. Thereafter, all additional treatments including but not 
limited to topical antimicrobials, or oral antimicrobials, will be at the clinician’s discretion.  At this point patients 
may be discharged from the inpatient setting.  
3.3.4 3 week visit. During this visit study participants will have BSCVA recorded, slit lamp examination, and they 
will have digital photography. 
3.3.5 Final Visit. At the 3- Month visit study participants will have their BSCVA and slit lamp examination recorded, 
in addition to repeat topography and be administered the IND-VFQ.  
 
 
 
 
 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 6 
 
3.5 Study Schedule 
Visit 1 
Baseline Exam 
Visit 2 
Procedure & Repeat 
Culture 
Visit 3 
3-Day Repeat 
Culture 
Visit 4 
3 week Follow-up 
Visit 5 
Month 3 Final 
Forms 
 
 
 
 
 
Consent and Authorization form  
X 
 
 
 
 
Baseline form  
X 
 
 
NEI-VFQ 
 
 
X 
 
X 
Follow-up form  
 
 
X 
X 
X 
Final form 
 
 
 
 
X 
Procedures 
 
 
 
 
 
 
Cross-linking 
X 
 
Tests 
 
 
 
 
 
IOP 
X 
 
X 
X 
X 
Pachymetry U/S 
X 
 
X 
X 
X 
Pentacam Topography 
X 
 
 
 
X 
Slit Lamp Photography* 
X 
 
 
X 
X 
BSCVA/ETDRS/MRx 
 
X 
 
X 
X 
Total visit time 
2 hours 
2 hours 
2 hours 
1 hour 
1 hour 
 
 
* Slit lamp photography also taken upon an adverse event 
 
 
3.6 Adverse Events (AEs) 
During each study visit, the subject will be questioned about AEs in a non-leading manner. All AEs, whether 
observed by the Investigator, elicited by the Investigator, or spontaneously reported by the subject, will be 
documented in the subject's chart and the adverse event form. Slit lamp photography will also be completed after 
any adverse event takes place.  
 
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). 
 
If a SAE occurs, the investigator at Aravind Eye Hospital, Madurai must fill out the serious adverse event form and 
email it to Stephen Mcleod at UCSF within 24 hours of the occurrence of the SAE. 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. Notification of an SAE or clinically significant AE must also be submitted to the Institutional Review Board 
(IRB)/Ethics Committee (EC) in accordance with its requirements. All AEs must be reported from the time that the 
subject provides informed consent through the last study visit.  
4. PROCEDURES 
4.1 Masking 
Due to the nature of the intervention, the study participant, surgeon and technician performing cross-linking will 
not be masked, however, the physician performing repeat scraping as well as the microbiologist will be masked to 
treatment arm. The refractionist performing the BSCVA will also be masked. UCSF study personnel will not be 
given any identifying information.  


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 7 
 
4.2 Procedures  
4.2.1 
Corneal Gram Stain and Culture  
After patient history and slit lamp examination at the enrollment visit (Day 0), corneal scraping is performed to determine 
the eligibility of the patient (positive fungal stain and negative bacterial stain). Corneal scraping is recommended as 
standard of care for all corneal ulcer patients at the Aravind Eye Hospitals. There is a small risk to patients of inducing a 
perforation, worsening an epithelial defect, or contaminating an ulcer, and patients are duly informed of these risks prior 
to the taking of the corneal scrape.  
 
Corneal scraping will be obtained as follows:  
 
A drop of topical anesthetic (0.5% tetracaine or 4% lidocaine) is administered to the eye to be examined.  
 
Aseptic technique is used to obtain each corneal scrape. A flame-sterilized Kimura spatula is used, with the aid of 
slit lamp magnification, to obtain a scrape from the leading edge and base of the corneal ulcer. The Kimura 
spatula is again flame sterilized between the takings of each sample.  
 
Two scrapings are smeared directly on to two separate glass microbiology slides for Gram stain and for KOH wet 
mount (if necessary, Giemsa or Gram Stain can be used to identify fungal elements as well). 
 
Three further scrapings are taken and directly inoculated on to sheep’s blood agar, chocolate agar, potato dextrose 
agar or Sabouraud’s agar for bacterial and fungal culture. 
 
 
4.2.2 
Collagen Cross-Linking 
All study participants randomized to collagen cross-linking will have cross-linking within 4 hours for bacterial 
ulcers and within 24 hours for fungal ulcers. They will be taken to the operating theatre and the procedure will 
be performed under sterile conditions.  
Cross-linking will be performed as follows: 
 
A drop of topical anesthetic (0.5% tetracaine or 4% lidocaine) is administered to the eye.  
 
A 30-minute loading dose of 0.1% topical riboflavin and 20% dextran T500 drops will be administered 
every 2 minutes. 
 
The cornea will be exposed to UV-A light at a wavelength of 365nm with irradiance of 3mW/cm2 for a 
total of 30 minutes. 
 
Throughout the UV-A treatment the study participant will continue to receive topical riboflavin Q5 min 
intervals during treatment. 
 
The study participant will be transferred back to the microbiology laboratory for repeat scraping and 
culture (see section 4.4.1). 
4.3 Pentacam Protocol 
Cornea densitometry and topography will be documented using the Pentacam HR (high resolution) 
Scheimpflug camera at baseline and 3 months.  The images will be captured using standardized protocols by 
study certified ophthalmic technicians. The data will then be shared with UCSF via Dropbox. 
 
In order to obtain the highest quality Pentacam HR Scheimpflug camera examination, the technician will scan 
the appropriate eye according to the Pentacam HR instruction manual.  
 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 8 
 
4.3.1 
Imaging Procedures 
Corneal densitometry and topography measurement 
 
The examination will be performed in a designated diagnostic room with a uniform ambient light level.
 
To begin the corneal densitometry measurement, the technician will select the examination scan for 
cornea densitometry measurement, per the Pentacam HR manual.  
 
The cornea densitometry measurement will consist of a measurement protocol of 25 images under the 
examination mode of 3D scan.  
4.3.2 
Check Scan Quality 
 
If Quality Specifications (“QS”) field reads “OK,” then the measurement is correct and reproducible.  
 
If the “QS” field is highlighted YELLOW, the examination will be repeated until an “OK,” reading is
obtained.  
 
If the “QS” field is RED, then the examination must be repeated.  
 
If an “OK” reading is not obtainable, then the best of 3 examinations will be saved.  
4.3.3 
Saving Image 
 
Download data to excel or save to USB drive  
 
De-identify data 
o Patient names or any other identifying information such as birthday should not appear within 
any of the image files.  
 
Rename data 
o The image files will be designated with: 1) the eye ID number, 2) the timing of visit  ‘3m’, ‘6m’, or
‘12m’ respectively for the 3, 6, and 12-month follow-up visits, and 3) image number of the series. 
An image renamed to C101_3m_1.jpg will be the first image for eye number C101’s 3 month 
follow-up visit.  An image renamed to C222_12m_3.jpg will be the third image for eye 222’s 12 
month follow-up visit.  
 
Batch upload images to Dropbox. Procedures for uploading images to Dropbox:  
o Nagivate to Dropbox  eye’s folder  relevant study visit 
o Upload images to the eye’s relevant study visit folder 
 
5. STUDY MEDICATIONS 
5.1 Medical management protocol & Treatment Schedule  
All study participants will receive the same medical management in the initial 3 day study period. Table 2 outlines 
the dosing schedule for medications for CLAIR I and CLAIR II. Study participants will be randomized to medical 
management plus CXL versus medical management alone. This study requires 5 visits over 3 months including 
baseline visit, procedure visit, 3-day follow-up, 1 and 3-month follow-up. After the 3-day repeat culture visit 
additional treatments may be added per clinician preference including but not limited to topical and oral 
antimicrobials. 
 
 
 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 9 
 
 
 
Table 2: Dosing schedule for Medications in the first 3 days. 
 
CLAIR I 
CLAIR II 
Moxifloxacin 0.5%  
Q 1 hours x 3 days 
 
Natamycin 5%  
 
Q 1 hour x 3 days*  
Amphotericin 0.15% 
 
Q 1 hour x 3 days* 
Homatropine 
TID x 3 days  
TID x 3 days 
*50% randomized to Natamycin 5% and 50% randomized to Amphotericin 0.15% 
 
5.2 Possible Side Effects of Study Medications 
 
Amphotericin: eye irritation, swelling, pain and/or redness, discharge 
 
Homatropine: eye irritation, swelling, pain, or redness; Eye dilation may result in temporary blurred vision, light 
sensitivity, and increased heart rate.  
 
Moxifloxacin: eye irritation, swelling, pain, or redness 
 
Natamycin: eye irritation, swelling, pain and/or redness, discharge 
 
6. PROTECTION OF HUMAN SUBJECTS 
6.1 Institutional Review Board Approval 
6.1.1 Aravind Eye Hospital, Ethics Review Committee The Ethics Committee with review the study protocol annually 
for ethical approval. 
 
6.1.2 University of California, San Francisco Committee on Human Research (CHR)  The UCSF CHR will review the 
study protocol annually for ethical approval. 
 
6.2 Informed Consent   
Aravind personnel will obtain full written consent from each patient. The primary surgeon will screen participants 
and determine their eligibility. He/she will clearly explain the process and risks involved and will also ask the 
patient to sign any necessary consent documents. The study participant will have 4 visits total per eye included in 
the study over 3 months; visits include hospitalization for the first 3 days and a procedure visit. The patient has the 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 10 
 
ability to withdraw at any time and will not be forced into anything with which he/she is not comfortable.  Consent 
documents have been uploaded to the IRB application. 
 
6.3 Risks to Study Participants 
Blindness from infectious corneal ulcers is a worldwide public health problem. If our hypothesis is correct, and 
corneal crosslinking provides an additive benefit to topical antimicrobials in the treatment of corneal infections, 
there could potentially be a profound societal benefit. 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.13 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.11 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.5 All procedures will be performed by ophthalmologists board certified in the study country who are well 
versed in all planned treatment procedures. 
We anticipate minimal risk for study patients. This fact, coupled with the potential benefit the data provided by the 
study could provide in terms of guiding future therapies for corneal infections, makes the minimal risks posed to 
the patient entirely reasonable. 
 
There may be some discomfort during follow-up testing (BSCVA, IOP, slit lamp, Pachymeter, Topography, and photo 
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.  
6.4 Privacy, Confidentiality and Data Security  
All data will be stored and transferred using a secure server with de-identified information. 
 
We will take steps to keep the participant’s personal information confidential. Health information will be kept 
secure and separate from information which identifies participants.  Upon enrollment, participants will be 
assigned a code that will be used instead of their name, medical record number or other personally identifying 
information. Electronic files for data analysis will contain only the participant code. The code will be stored at 
Aravind on a secure server and will only be accessed by study personnel – the PI, study coordinator, or other 
member of the study team). Access to study-related patient information will be limited only to members of the 
study team. 
 
Only a small number of researchers related to this study will have direct access to the participant’s medical 
information. Paper files will be stored in locked filing cabinets in restricted access offices at Aravind. Access to 
data/specimens is restricted to study personnel. 
 
Information may be released to others outside of Aravind who are involved in coordinating or overseeing research, 
but information which identifies the participant will be kept secure. To ensure the success of this study, Aravind 
has partnered with University of California, San Francisco (UCSF)’s   F.I. Proctor Foundation, which is a center that 
specializes in ophthalmology research. The coordinating center at UCSF will have access to de-identified 
information using a code number. UCSF’s role is to support Aravind on the data analysis portion of this study and 
assisting with the development of consent and study-related materials. UCSF has obtained IRB approval to take 
part in this study (IRB number: #14-14918). 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 11 
 
6.5 Exclusion of Vulnerable Populations 
The following populations will not be enrolled in this study: children (18 years and under), pregnant women, 
decisionally impaired adults, and prisoners. 
6.6 Compensation to Participants 
There is no additional cost or compensation for the study participant.  
7. DATA COLLECTION AND MANAGEMENT 
7.1 Data Collection and Entry 
All data will be collected using hardcopy patient forms at Aravind during baseline and follow-up visits. De-
identified data will be uploaded to a Box account in order to share the data with the UCSF study coordinator. De-
identified data will be input into a database using double data entry at UCSF.  
7.2 Data Consistency, Validity, and Monitoring 
Data monitoring reports will be prepared using STATA and Excel. If the forms are not filled out completely, 
the UCSF study coordinator will contact the person responsible for completing the form to provide the missing 
data, or clarify any inconsistent data. The Aravind study coordinator is the only person who is authorized to add 
missing data or make any changes to the study forms.  All changes should be made with a red ink pen, and then 
signed and dated. 
7.3 Data Storage and Security 
De-identified data will then be input into a database using double data entry at UCSF. Data will be stored at UCSF 
for about 10 years. 
8. STATISTICAL ANALYSIS PLAN 
Please see the Statistical Analysis Plan (SAP) for details on the statistical analysis. 
 
 


CLAIR MOP 
Version 1.8; Revised 4 October 2016 
 
Page | 12 
 
9. REFERENCES 
1. 
Keating A, Pineda R, 2nd, Colby K. Corneal cross linking for keratoconus. Seminars in ophthalmology. Sep-Nov 
2010;25(5-6):249-255. 
2. 
Lamy R, Netto CF, Reis RG, et al. Effects of corneal cross-linking on contrast sensitivity, visual acuity, and corneal 
topography in patients with keratoconus. Cornea. May 2013;32(5):591-596. 
3. 
Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen crosslinking with riboflavin and ultraviolet-A light in 
keratoconus: long-term results. Journal of cataract and refractive surgery. May 2008;34(5):796-801. 
4. 
Vinciguerra P, Albe E, Trazza S, Seiler T, Epstein D. Intraoperative and postoperative effects of corneal collagen 
cross-linking on progressive keratoconus. Archives of ophthalmology. Oct 2009;127(10):1258-1265. 
5. 
Alio JL, Abbouda A, Valle DD, Del Castillo JM, Fernandez JA. Corneal cross linking and infectious keratitis: a 
systematic review with a meta-analysis of reported cases. Journal of ophthalmic inflammation and infection. 
2013;3(1):47. 
6. 
Martins SA, Combs JC, Noguera G, et al. Antimicrobial efficacy of riboflavin/UVA combination (365 nm) in vitro 
for bacterial and fungal isolates: a potential new treatment for infectious keratitis. Investigative ophthalmology 
& visual science. Aug 2008;49(8):3402-3408. 
7. 
Panda A, Krishna SN, Kumar S. Photo-activated riboflavin therapy of refractory corneal ulcers. Cornea. Oct 
2012;31(10):1210-1213. 
8. 
Iseli HP, Thiel MA, Hafezi F, Kampmeier J, Seiler T. Ultraviolet A/riboflavin corneal cross-linking for infectious 
keratitis associated with corneal melts. Cornea. Jun 2008;27(5):590-594. 
9. 
Makdoumi K, Mortensen J, Crafoord S. Infectious keratitis treated with corneal crosslinking. Cornea. Dec 
2010;29(12):1353-1358. 
10. 
Shetty R, Nagaraja H, Jayadev C, Shivanna Y, Kugar T. Collagen crosslinking in the management of advanced non-
resolving microbial keratitis. The British journal of ophthalmology. Aug 2014;98(8):1033-1035. 
11. 
Makdoumi K, Mortensen J, Sorkhabi O, Malmvall BE, Crafoord S. UVA-riboflavin photochemical therapy of 
bacterial keratitis: a pilot study. Graefe's archive for clinical and experimental ophthalmology = Albrecht von 
Graefes Archiv fur klinische und experimentelle Ophthalmologie. Jan 2012;250(1):95-102. 
12. 
Sauer A, Letscher-Bru V, Speeg-Schatz C, et al. In vitro efficacy of antifungal treatment using riboflavin/UV-A 
(365 nm) combination and amphotericin B. Investigative ophthalmology & visual science. Aug 2010;51(8):3950-
3953. 
13. 
Wittig-Silva C, Chan E, Islam FM, Wu T, Whiting M, Snibson GR. A randomized, controlled trial of corneal 
collagen cross-linking in progressive keratoconus: three-year results. Ophthalmology. Apr 2014;121(4):812-821. 
 
 

SAP_001.pdf: 
Cross-linking for Corneal Ulcers Treatment Trial (CLAIR)
NCT02570321
May 28,2019


CLAIR SAP version 1.4
Last edited May 28, 2019
1 
 
 
Cross-Linking Assisted Infection Reduction 
Statistical Analysis Plan 
 
 
Aravind Eye Hospital, Madurai and Pondicherry, India 
Francis I. Proctor Foundation, University of California San Francisco, USA 
 
 
 
Investigators: 
N. Venkatesh Prajna, DO DNB FICO 
Naveen*** 
Tom Lietman, MD 
Jeremy Keenan, MD 
Jennifer Rose-Nussbaumer, MD 
 
 
 
 
 
 
 
 
 
*Principal Investigator 
1Aravind Eye Hospital, Pondicherry, India   
2 Francis I. Proctor Foundation, University of California San Francisco, San Francisco, California, USA 
 


 
 
Table of Contents 
1. 
Background ........................................................................................................................................... 1 
2. 
Statistical Considerations, Sample Size ................................................................................................. 1 
3. 
Interim Monitoring ............................................................................................................................... 2 
 
 


CLAIR Statistical Analysis Plan 
 
 
1 | P a g e  
 
Version 1.4 
 
 
Revised: 28 May 2019 
 
1. Background 
Cross-Linking Assisted Infection Reduction (CLAIR) I for bacterial ulcers,  CLAIR II for fungal ulcers 
are two randomized, masked, clinical trials. The purpose of these studies is to determine 
differences in microbiological cure for repeat cultures between different medical antimicrobial 
treatments alone versus antimicrobial treatment plus collagen cross-linking. There will be 1:1 
randomization to each of these treatment groups: 
CLAIR I. Bacterial Ulcers: 
1) Topical 0.5% Moxifloxacin alone  
2) Topical 0.5% Moxifloxacin plus cross-linking  
CLAIR II. Fungal Ulcers: 
1) Topical 5% natamycin alone  
2) Topical 5% natamycin plus cross-linking  
3) Topical 0.15% amphotericin alone 
4) Topical 0.15% amphotericin plus cross-linking 
2. Statistical Considerations, Sample Size  
We will perform separate analyses for each specific aim for (I) the bacterial ulcers, (II) the fungal 
ulcers.  Enrollment criteria require all study participants to have bacteria on smear from corneal 
scrapping. Previous studies have shown that 88% of bacterial cultures are positive when initial 
gram stain is positive.5 This is consistent with the experience of the microbiology lab at our own 
institution. We feel that this is a conservative estimate based on prior studies in a similar south 
Indian setting that have found approximately 90-95% of corneal scrapings with a positive 
Gram/KOH stain are also culture positive.  
 
There is little current data to inform us about the effect of repeat scrapping on the culture status. In 
the Mycotic Ulcer Treatment Trial II, approximately 55% of fungal ulcers were culture positive 6 
days after enrolling in the trial and receiving treatment. This is an approximately 7.5% reduction is 
culture positivity on average per day. If we consider initial scrapping to have a similar effect as one 
day of treatment, we can estimate that there would be an approximately 8% reduction in culture 
positivity due to repeat scraping in our trial.  
 
SPECIFIC AIM 1 analysis:  
CLAIR I Bacterial Trial: McNemar’s two-sample paired-proportions test will be used to compare 
pre- and post- cross-linking cultures on smear-positive bacterial corneal ulcers. In order to detect a 
30% difference in repeat culture status (80% in the control group vs. 50% in the crosslinking 
group), we would need to enroll 78 study participants, assuming a significance level of 0.05 and no 
loss to follow-up (since the primary outcome occurs the same visit as enrollment).  
CLAIR II Fungal Trial: Given the 4 arms of the trial, we have estimated a sample size based on a 
significance level of 0.0125. In order to detect a 30% difference (80% in control versus 50% in the 
crosslinking group), we would need to enroll 110 study participants. This assumes no loss to 
follow-up, since the primary outcome occurs at the same visit as enrollment. In reality, we should 
have more statistical power since we will also take into account the results of the baseline culture, 
which we expect to be highly correlated with the follow-up culture.  
 


 
 
SPECIFIC AIM 2 analysis: 3-month best spectacle-corrected visual acuity (BSCVA) will be examined 
in a multiple linear regression model with terms for treatment (dichotomous variable for bacterial 
and 4-level categorical variable for fungal ulcers) and baseline BSCVA. Similar to above, for the 
fungal ulcers we will first test for an overall difference between the 4 groups, then pairwise 
comparisons as indicated, using a Bonferroni correction. 3 and 12-month best spectacle-corrected 
visual acuity (BSCVA) will be examined in a repeated measures multiple linear regression model 
with terms for treatment (dichotomous variable for bacterial ulcers and 4-level categorical variable 
for fungal ulcers) and baseline BSCVA. Similar to above, for the fungal ulcers we will first test for an 
overall 12-month BSCVA difference between the 4 groups, then pairwise comparisons as indicated, 
using a Bonferroni correction. 
 
For study participants who experience perforation or undergo therapeutic penetrating keratoplasty 
(TPK) this will be noted and a BSCVA will be performed prior to performing further surgery. This 
last observation will be carried forward (LOCF) as the 3-month BSCVA. An enhanced analysis using 
standard longitudinal modeling methods will be used to handle data from study participants who 
are lost to follow up.  Additional secondary analyses will include: 
 
Scar size as measured on clinical exam and photographs 
 
Corneal topography  
 
Corneal Thickness and scar size as measured by OCT  
 
IND-VFQ will be compared between the two groups controlling for baseline VFQ 
 
Analyses of perforations or other adverse events 
 
A cost effectiveness analysis will be performed.  
3. Interim Monitoring 
Due to the uncertainty regarding the effect of repeat culture on culture positivity, a masked interim 
analysis will be conducted to re-evaluate sample size based on day-1 repeat culture positivity when 
one-third of the data have been collected (note that this interim analysis will not look at outcome by 
treatment arm). A medical monitor will be appointed at UCSF to monitor any serious adverse 
events. A small DSMC at UCSF will be appointed to conduct an un-masked interim analysis to 
evaluate for futility or harm. Either trial may be stopped early without unmasking for temporal 
reasons or due to low enrollment.