Clinical trial NCT02731638
Conditions: Corneal Ulcer Fungal Keratitis
Keywords: cornea ulcer fungal keratitis
Sponsor: University of California, San Francisco
Start date: 2016-09-01; Completion date: 2018-07-01
Enrollment: 70 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Mycotic Antimicrobial Localized Injection (MALIN) is a randomized, masked, two-arm clinical trial investigating intrastromal voriconazole in the treatment of fungal corneal ulcers. There is currently little evidence to guide the treatment of fungal keratitis beyond topical anti-fungal drops, though intrastromal voriconazole and oral antifungal treatments are used as well. This study will provide evidence to guide the treatment of fungal keratitis in the future. The purpose of this study is to determine differences in microbiological cure for 3-day repeat cultures between different antifungal treatments. For this study, there will be 1:1 randomization to one of these two treatment groups: 1) topical natamycin plus intrastromal voriconazole injection or 2) topical natamycin alone.

Detailed description:
The proposed study is a randomized controlled trial to determine whether intrastromal voriconazole improves outcomes in fungal keratitis. Patients presenting to the Aravind Eye Hospitals in Pondicherry, India for treatment of fungal keratitis will be recruited for the proposed study. Approximately 70 patients will be enrolled in the study. Subjects presenting with fungal keratitis will be randomized to receive topical natamycin plus intrastromal voriconazole or to receive topical natamycin alone. Subjects may also receive oral antifungals after day 3 at the discretion of the treating clinician. All study subjects will be followed for 3 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 will 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: Intrastromal voriconazole plus standard of care topical treatment for fungal keratitis arm 2: Standard of care topical treatment for fungal keratitis
Interventions: intervention 1: Intrastromal voriconazole intervention 2: Natamycin
Intervention descriptions: intervention 1: Subjects will receive three intrastromal voriconazole injections over 10 days. Intrastromal voriconazole injection is a routine procedure performed by Aravind Eye Hospital for fungal keratitis. intervention 2: Subjects will receive topical 5% natamycin drops hourly for 3 days.

Protocol documents:
Prot_000.pdf: 
 
 
Mycotic Antimicrobial Localized Injection 
Manual of Operations and Procedures 
 
 
Aravind Eye Hospital, Pondicherry, India 
Francis I. Proctor Foundation, University of California San Francisco, USA 
 
 
 
Investigators: 
Tiruvengada Krishnan, DO DNB FICO1 
Shivananda Narayana, DO DNB FICO1 
Seema Prabu, DO DNB FICO1 
Tom Lietman, MD2 
Jennifer Rose-Nussbaumer, MD2 
Ariana Austin, MS2 
 
 
 
 
 
 
 
 
 
 
 
 
 
1Aravind Eye Hospital, Pondicherry, India   
2 Francis I. Proctor Foundation, University of California San Francisco, San Francisco, California, USA 


MALIN Manual of Operations and Procedures 
 
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Version 2.0 
 
 
Revised: 17 February 2017 
CONTENTS 
1. INTRODUCTION AND BACKGROUND ................................................................................................................................ 3 
1.1 Specific Aims: ..................................................................................................................................................................... 3 
1.2 Study Outcomes: ................................................................................................................................................................ 4 
1.3 Study Design: ..................................................................................................................................................................... 4 
2. ORGANIZATION ............................................................................................................................................................... 4 
2.1 Collaborating Institutions: ................................................................................................................................................. 4 
3. PATIENT FLOW ................................................................................................................................................................. 5 
3.1 Study Timeline ................................................................................................................................................................... 5 
3.2 Eligibility Requirements ..................................................................................................................................................... 5 
3.3 Randomization................................................................................................................................................................... 5 
3.4 Study visits ......................................................................................................................................................................... 6 
3.5 Study Schedule ................................................................................................................................................................... 7 
3.6 Adverse Events (AEs) ......................................................................................................................................................... 7 
4. PROCEDURES ................................................................................................................................................................... 7 
4.1 Masking ............................................................................................................................................................................. 7 
4.2 Ulcer Classification ............................................................................................................................................................ 8 
4.3 Medical management protocol ......................................................................................................................................... 8 
4.4 Procedures ......................................................................................................................................................................... 8 
5. STUDY MEDICATIONS ...................................................................................................................................................... 9 
5.1 Treatment schedule for study medications ....................................................................................................................... 9 
5.2 Voriconazole Dosage and Administration ....................................................................................................................... 10 
5.3 Possible Side Effects of Study Medications ...................................................................................................................... 10 
6. PROTECTION OF HUMAN SUBJECTS ............................................................................................................................... 10 
6.1 Institutional Review Board Approval ............................................................................................................................... 10 
6.2 Informed Consent ................................................................................................................ Error! Bookmark not defined. 
6.3 Risks to Study Participants ............................................................................................................................................... 11 
6.4 Privacy, Confidentiality and Data Security ...................................................................................................................... 11 
6.5 Exclusion of Vulnerable Populations ................................................................................................................................ 12 
6.6 Compensation to Participants ......................................................................................................................................... 12 
7. DATA COLLECTION AND MANAGEMENT ........................................................................................................................ 12 
7.1 Data Collection and Entry ................................................................................................................................................ 12 
7.2 Data Consistency, Validity, and Monitoring .................................................................................................................... 12 
7.3 Data Storage and Security ............................................................................................................................................... 12 
8. STATISTICAL ANALYSIS PLAN ......................................................................................................................................... 12 
9. REFERENCES ................................................................................................................................................................... 12 
 
 
 
 
 


MALIN Manual of Operations and Procedures 
 
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Revised: 17 February 2017 
1. INTRODUCTION AND BACKGROUND  
Fungal corneal ulcers, because of their poor outcomes and the lack of evidence to guide treatment, 
present a therapeutic challenge to clinicians.1 The incidence of fungal keratitis varies by region, which is 
thought to be due to differences in climate as well as risk of corneal trauma.1,2 In the tropics, fungal infection 
can account for upwards of 50% of corneal ulcers.1,3,4 In the United States fungal keratitis ranges from 35% of 
corneal ulcers in South Florida5 to 4% in temperate climates such as Los Angeles.6 The Mycotic Ulcer Treatment 
Trial I (MUTT I), a NEI-funded randomized double masked clinical trial found that natamycin is superior to 
voriconazole for the treatment of filamentous fungal ulcers and in particular for fusarium species.7 However, 
natamycin is fungistatic and has limited penetration into the corneal layers.8  
Voriconazole may still be an important adjunct in the treatment of fungal ulcers. In vitro studies suggest 
that voriconazole should have good efficacy against Aspergillus and Fusarium.9 Intrastromal injection of 
voriconazole may provide steady state drug concentrations at the site of infection and avoid intervals of sub-
theraputic drug dosing. Secondly, the identity of the organism may play an important role in guiding treatment. 
Ulcers caused by Aspergillus species did equally well with topical voriconazole and natamycin, whereas those 
caused by Fusarium species had 4 lines worse visual acuity at 3 months. Possibly, Aspergillus ulcers will fare 
better when oral voriconazole is added. Thirdly, there may be presenting characteristics, such as deep stromal 
involvement, which could predict a benefit from intrastromal voriconazole injection.  
Studies of intrastromal injection of voriconazole have yielded mixed results. Several case series have 
found intrastromal voriconazole to be beneficial.10-12 However, one randomized controlled trial, comparing 
intrastromal injection to topical voriconazole found significantly improved 3-month visual acuity in the topical 
voriconazole group.13 However, in the intrastromal voriconazole arm there were more central ulcers than in 
the topical voriconazole arm and scar size between the two groups was comparable implying that intrastromal 
injection does not lead to worse scarring. In this study we will evaluate the effectiveness of intrastromal 
voriconazole injection in addition to topical natamycin treatment for the treatment of moderate to severe 
fungal keratitis.  
Research Question: Is intrastromal injection of voriconazole beneficial in addition to topical natamycin in the 
treatment of filamentous fungal corneal ulcers? 
1.1 Specific Aims: 
1) Does the addition of intrastromal injection of voriconazole decrease the rate of repeat 3-day 
culture positivity compared with medical management? We hypothesize that there will be decreased 
3-day repeat culture positivity in the intrastromal voriconazole injection group. 
  
2) Does intrastromal injection of voriconazole improve best spectacle corrected visual acuity at 3 
months compared with medical management? We hypothesize that intrastromal injection with 
voriconazole will improve 3-month visual acuity compared with topical natamycin alone. 
 
3) Does the addition of intrastromal injection of voriconazole improve 3-month scar size, days to re-
epithelialization and rate of perforation/need for Therapeutic penetrating keratoplasty (TPK) 
compared with medical management? We hypothesize that there will be improved 3-month scar size 
and decreased perforation rate in the intrastromal voriconazole group, but no difference in days to re-
epithelialization. 
 
 


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Revised: 17 February 2017 
1.2 Study Outcomes: 
Primary Outcome: 
• 
Microbiological cure on 3-day repeat cultures 
Secondary Outcomes: 
• 
BSCVA at 3 months 
• 
Microbiological cure on 7-day repeat cultures 
• 
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 
• 
Difference in IND-VFQ at 3 months between groups 
• 
Corneal neovascularization, as measured by clinical exam & slit lamp photographs 
1.3 Study Design: 
Mycotic Antimicrobial Localized Injection (MALIN) is a randomized, masked, two-arm clinical trial. The 
purpose of the study is to determine differences in microbiological cure for 3-day repeat cultures between 
different antifungal treatments. There will be 1:1 randomization to one of these two treatment groups: 1) 
Topical natamycin plus intrastromal voriconazole injection or 2) Topical natamycin alone. The enrollment 
period will be 3 months.  
2. ORGANIZATION 
Aravind Eye Hospital, Pondicherry along with the University of California, San Francisco (UCSF) will jointly 
execute this clinical trial. Aravind Eye Hospital, Pondicherry 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. 
2.1 Collaborating Institutions: 
Aravind Eye Hospital, Pondicherry, India  
Dr. Shivananda Narayana will be the lead investigator and Drs. Tiruvengada Krishnan and Seema Prabu will be 
the co-investigator for this study in Pondicherry. Administration for the Pondicherry site will be done through 
Aravind Eye Hospital, Madurai, under the direction of Dr. N. V. Prajna. All recruitment, treatment/intervention, 
and follow-up visits will be done at the Aravind Eye Hospital at Pondicherry. 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 Pondicherry 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. 


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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. 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 
The enrollment period will be 3 months. Study participants will be required to have 4 follow-up visits at 3 days, 
7 days, 1 month, and 3 months (visit window: 2.5 -3.5 months). All study participants will be admitted to the 
hospital for 7 days. 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: 
• 
Moderate to severe corneal ulcer that is smear positive for filamentous fungus  
• 
Pinhole visual acuity worse than 20/70 in the affected eye  
• 
Age 18 years or greater, and less than or equal to 70 years 
• 
Basic understanding of the study as determined by the physician 
• 
Commitment to return for follow up visits 
 
Exclusion criteria: 
• 
Gram stain positive for bacteria or evidence of other concomitant infection (i.e. herpes, acanthameoba) 
• 
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  
• 
Pregnant women 
• 
Participants who are decisionally and/or cognitively impaired 
3.3 Randomization 
Each study eye will be randomly assigned to the 1) Intrastromal voriconazole plus topical natamycin or 2) 
Natamycin alone. 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.  


MALIN Manual of Operations and Procedures 
 
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Revised: 17 February 2017 
 
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 24 hours. 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 
3.4.1 Baseline Visit. During this visit, eligible patients at Pondicherry will be enrolled in the study and will give 
consent. The baseline patient form will be completed and BSCVA obtained. All study participants will begin 
topical therapy with hourly 5% natamycin immediately. They will have baseline photography and OCT. They 
will then be randomized to either intrastromal voriconazole plus natamycin or topical natamycin alone. Those 
randomized to intrastromal injection will undergo this procedure in the operating theatre within 24 hours. 
They will have an additional 2 stromal injections: one on the 3rd day and one on the 5th day. All patients will be 
admitted to the inpatient setting until the 7th day.  
3.4.2 Procedure visit. Within 24 hours of the baseline visit study participants randomized to intrastromal 
voriconazole will undergo injection in the operating theatre. They will have 2 additional rounds of intrastromal 
voriconazole injections: one on the 3rd day and one on the 5th day of the study.  
3.4.3 Day 3 visit. During this visit, study participants will have slit lamp examination and undergo repeat scraping 
and culture of the corneal ulcer. Adverse events will also be assessed and recorded. Thereafter, all additional 
treatments including but not limited to topical voriconazole, or oral ketoconazole, will be at the clinician’s 
discretion. The treating physician will remain masked to treatment arm. After the scraping and culture have 
been performed, participants randomized to receive intrastromal voriconazole injections will receive an 
additional injection of voriconazole in the operating theatre.  
3.4.5 Day 5 visit. On day 5 participants randomized to receive intrastromal voriconazole injections will receive 
their final injection of voriconazole in the operating theatre.  
3.4.6 Week 1 visit. Approximately one week from enrollment patients will undergo repeat scraping and culture. 
No other study visit information will be gathered during this visit, therefore any further examination, testing, or 
treatment will be at the discretion of the treating physician. After the Day 7th day, patients may be discharged 
from the inpatient setting. 
3.4.7 One Month visit. During this visit, study participants will have their BSCVA, slit lamp examination, and OCT 
performed during this visit. Photography will also be performed. Adverse events will be reviewed and 
recorded. 
3.4.8 Final Visit. At the 3- Month visit study participants will have their BSCVA and slit lamp examination 
recorded, in addition to repeat photography, topography and OCT and be administered the IND-VFQ. 
Endothelial cell count will also be measured. Adverse events will be reviewed and recorded.  
 
 
 
 


MALIN Manual of Operations and Procedures 
 
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Revised: 17 February 2017 
3.5 Study Schedule 
 
Visit 1 
Baseline Exam 
Visit 2 
Procedure  
Visit 3 
Day 3 
Visit 4 
Day 5 
Visit 5 
Week 1 
Visit 6 
Month 1 
Visit 7 
Month 3 
Forms 
 
 
 
 
 
 
 
Consent and 
Authorization form  
X 
 
 
 
 
 
 
Baseline form  
X 
 
 
 
 
 
 
Procedure form 
 
X 
X 
X 
 
 
 
IND-VFQ 
 
 
 
 
 
 
X 
Follow-up form  
 
 
X 
 
X 
X 
X 
Final form 
 
 
 
 
 
 
X 
Procedures 
 
 
 
 
 
 
 
Intrastromal  
 
X 
X 
X 
 
 
 
Smear/culture 
X 
 
X 
 
X 
 
 
Tests 
 
 
 
 
 
 
 
IOP 
X 
 
X 
 
 
X 
X 
Topography 
 
 
 
 
 
 
X 
OCT 
X 
 
 
 
 
X 
X 
Slit lamp photography* 
X 
 
 
 
 
X 
X 
BSCVA/ETDRS/MRx 
X 
 
 
 
 
X 
X 
Endothelial cell count 
 
 
 
 
 
 
X 
Total visit time 
2 hours 
2 hours 
2 hours 
2 hours 
1 hour 
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, Pondicherry 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 
All study participants will be masked to their intervention. The refractionist performing the BSCVA will also be 
masked. Due to the nature of the intervention, the surgeon and technician performing intrastromal injection 
will not be masked, however, the physician performing repeat scraping as well as the microbiologist will be 
masked to treatment arm. UCSF study personnel will not be given any identifying information. 
 
 
 


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Revised: 17 February 2017 
4.2 Ulcer Classification 
Factor 
Mild 
Moderate 
Severe 
Location Nonaxial 
Central or peripheral Central or peripheral 
Area 
2 mm 
2–6 mm 
6 mm 
Depth 
Superficial one third 
of stroma 
Superficial two thirds 
of stroma 
Extending to inner one third 
of stroma and/ or endothelial plaque 
Any two of three criteria should be fulfilled. 
4.3 Medical management protocol 
In the first 3 days all patients will receive hourly 5% Natamycin, 0.5% Moxifloxacin for prophylaxis every two 
hours while awake, and 2% homatropine TID. They will be randomized to intrastromal injection with 
voriconazole versus sham injection. This study requires 7 visits over 3 months including baseline visit, 
procedure visit, 3-day follow-up and procedure visit, 5-day procedure visit, 1-week follow-up, 1-month 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 voriconazole and oral Ketoconazole. 
4.4 Procedures   
4.4.1 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. 
4.4.2 Intrastromal Voriconazole Injection 
The intrastromal injection will be performed in the operating theatre under the operating microscope by the 
cornea surgeon under peribulbar anesthesia and with full aseptic precautions. This will be performed within 24 
hours of the baseline visit. Voriconazole 0.5 mg/ml solution will be loaded in a 1 ml tuberculin syringe with a 
30-gauge needle. With the bevel down, the needle will be inserted obliquely starting in the adjacent uninvolved 
stroma to reach the inﬁltrate at the mid-stromal level (as the intended level for drug deposition) in each case. 
The drug is then injected and the amount of hydration of the cornea is used as a guide to assess the area 
saturated with medication. Once the desired amount of hydration is achieved, the plunger is withdrawn slightly 
to ensure discontinuation of the capillary column and thus prevent back-leakage of the drug. Four to six divided 
doses are given around the inﬁltrate/s to surround the entire circumference of the lesion.  
4.4.3 Additional Injections 
An additional two rounds of intrastromal injections will be performed at 3 days and 5 days post enrollment in 
the voriconazole injection arm only. This MUST be performed after the 3-day repeat culture visit. The study 
coordinator and treating physician will be aware of the treatment assignment; however, the follow-up 
physician and microbiologist will remain masked to treatment arm.  
 


MALIN Manual of Operations and Procedures 
 
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Revised: 17 February 2017 
4.4.4 TPK 
For patients requiring TPK, the corneal button will be sent to microbiology and pathology. Patients should be 
photographed prior to surgery.  
4.5 Anterior Segment Optical Coherence Tomography Protocol 
OCT will be documented at baseline, 1 and 3 months.  The images will be captured with the Visante using 
standardized protocols by study certified ophthalmic technicians. The data will then be shared with UCSF via 
Dropbox. 
4.5.1 OCT Scan Procedures 
In order to obtain the highest quality anterior segment optical coherence tomography (OCT), the technician will 
scan the appropriate study eye according to the Visante instruction manual. 
All OCT scans will be performed in a designated diagnostic room with a uniform ambient light level. 
An initial scan of the global pachymetry will be obtained. Three scans should then be obtained at the pupillary 
geometric center on the Quad setting (4 line scans through the cornea). Subsequently, a dense scan centered 
over the area of greatest thinnest (as determined by the global pachymetry) will be performed using the Quad 
setting. Quantitative data will be obtained using the caliper function by an examiner masked to the results of 
clinical examination. 
4.5.2 Saving Scan 
• 
Save the raw images on the Visante OCT machine 
• 
Download PDF images and save to a USB drive  
o Images over the area of thinning should include the caliper measurement 
• 
De-identify images 
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 study ID number, 2) the timing of visit  ‘1d’, “1m’, 
or ‘3m’ respectively for the 1 day, 1, and 3-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_1m_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 
The following medications will be administered for treatment purposes: Natamycin 5%, Moxifloxacin 0.5%, 
Homatropine 2%, and Voriconazole 0.5mg/ml injection.  
5.1 Treatment schedule for study medications 
In the first 3 days all patients will receive hourly 5% Natamycin drops, 0.5% Moxifloxacin drops for prophylaxis 
every two hours while awake, and 2% homatropine drops TID. The aforementioned drugs will be used for FDA 
approved indications. Study participants will then be randomized to intrastromal injection with voriconazole 
plus natamycin versus natamycin. All medications will be protected and only appropriate personnel will 
administer the drugs.  
 


MALIN Manual of Operations and Procedures 
 
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Revised: 17 February 2017 
 
 
 
Table 2: Dosing schedule for Medications 
Medication 
Day 1-3 
Day 1 Procedure 
Day 3 Procedure  
After Day 3 
Scraping 
Day 5 Procedure 
Natamycin 5% 
Every hour 
 
 
Physician 
determined  
 
Moxifloxacin 
0.5% 
6 times/day 
 
 
Physician 
determined  
 
Homatropine 
2% 
3 times/day 
 
 
Physician 
determined  
 
Voriconazole 
0.5 mg/ml 
 
50mg/0.1ml 
administered via 
intrastromal 
injection 
50mg/0.1ml 
administered via 
intrastromal 
injection  
Physician 
determined  
50mg/0.1ml 
administered via 
intrastromal 
injection  
 
5.2 Voriconazole Dosage and Administration  
Voriconazole is an FDA approved triazole antifungal agent. The primary mode of action of voriconazole is the 
inhibition of fungal ergosterol biosynthesis by inhibiting the cytochrome P450 mediated 14 alpha-lanosterol 
demethylation. Voriconazole has demonstrated in vitro activity against Apsergillus and Fusarium species and is 
an approved treatment for serious fungal infections. Intrastromal Voriconazole injection is practice for the 
treatment of moderate to severe filamentous fungal corneal ulcers at Aravind Eye Hospital, Pondicherry.  
Voriconazole is available as 1 mg of white lyophilised powder in a glass vial (VOZOLE PF; Aurolab, India). The 
powder is reconstituted with 2 mL of lactated Ringer’s solution to a concentration of 0.5 mg/mL (50 mg/0.1 
mL). A dose of 50 mg/0.1 mL reconstituted intrastromal voriconazole is prepared just prior to administration. 
5.3 Possible Side Effects of Study Medications 
Natamycin: eye irritation, swelling, pain and/or redness, discharge 
 
Voriconazole: eye irritation, swelling, pain and/or redness, discharge 
 
Moxifloxacin: eye irritation, swelling, pain, or redness 
 
Homatropine: eye irritation, swelling, pain, or redness; Eye dilation may result in temporary blurred vision, 
light sensitivity, and increased heart rate.  
6. PROTECTION OF HUMAN SUBJECTS 
6.1 Institutional Review Board Approval 
6.1.1 Aravind Eye Hospital, Ethics Review Committee  
The Ethics Committee will 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. 
 


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Revised: 17 February 2017 
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 7 visits total per 
eye included in the study over 3 months; visits include hospitalization for the first 7. The patient has the 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 
Fungal keratitis is a very serious sight threatening infection. The best therapy is still not known; therefore many 
treatments including topical and oral antifungals are often administered. Intrastromal Voriconazole injection, in 
addition to oral and topical antifungals, is current practice for the treatment of moderate to severe filamentous 
fungal corneal ulcers at Aravind Eye Hospital, Pondicherry.  Participants in the study have the same risks as 
those undergoing treatment of their fungal keratitis who choose not to participate in the study.  
It is not known whether or not the addition of oral antifungal is of benefit to these patients. The Mycotic Ulcer 
Treatment Trial II is an ongoing large prospective randomized controlled two arm clinical trial looking at 
whether or not oral voriconazole is of benefit in addition to topical antifungals. In this study we are asking 
patients to delay oral therapy for 3 days.   
As with any surgery, there are risks associated with intrastromal injection which include risk of infection, or 
damage to the cornea, which could result in vision loss. There is a very small risk of full thickness perforation 
with the needle. If this were to occur, it would most likely be self-sealing, but may increase the risk of 
endophthalmitis, a severe sight threatening intraocular infection. Sterile precautions will be taken to ensure 
decreased risk of infection.  
 
There may be some discomfort during follow-up testing (BSCVA, IOP, slit lamp, Pachymeter, Topography, and 
OCT testing 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 


MALIN Manual of Operations and Procedures 
 
_______________________________________________________________________________________
 
 
Version 2.0 
 
 
Revised: 17 February 2017 
portion of this study and assisting with the development of consent and study-related materials. UCSF will 
obtain IRB approval to take part in this study (IRB number: 15-17347). 
 
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. There will be no compensation for 
research-related injury.  
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 Dropbox 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. 
9. REFERENCES 
1. 
Gopinathan U, Garg P, Fernandes M, Sharma S, Athmanathan S, Rao GN. The epidemiological 
features and laboratory results of fungal keratitis: a 10-year review at a referral eye care center in 
South India. Cornea. Aug 2002;21(6):555-559. 
2. 
Shah A, Sachdev A, Coggon D, Hossain P. Geographic variations in microbial keratitis: an analysis 
of the peer-reviewed literature. The British journal of ophthalmology. Jun 2011;95(6):762-767. 
3. 
Deorukhkar S, Katiyar R, Saini S. Epidemiological features and laboratory results of bacterial and 
fungal keratitis: a five-year study at a rural tertiary-care hospital in western Maharashtra, India. 
Singapore medical journal. Apr 2012;53(4):264-267. 
4. 
Srinivasan M, Gonzales CA, George C, et al. Epidemiology and aetiological diagnosis of corneal 
ulceration in Madurai, south India. The British journal of ophthalmology. Nov 1997;81(11):965-971. 
5. 
Liesegang TJ, Forster RK. Spectrum of microbial keratitis in South Florida. American journal of 
ophthalmology. Jul 1980;90(1):38-47. 


MALIN Manual of Operations and Procedures 
 
_______________________________________________________________________________________
 
 
Version 2.0 
 
 
Revised: 17 February 2017 
6. 
McLeod SD, Kolahdouz-Isfahani A, Rostamian K, Flowers CW, Lee PP, McDonnell PJ. The role of 
smears, cultures, and antibiotic sensitivity testing in the management of suspected infectious 
keratitis. Ophthalmology. Jan 1996;103(1):23-28. 
7. 
Prajna NV, Krishnan T, Mascarenhas J, et al. The mycotic ulcer treatment trial: a randomized trial 
comparing natamycin vs voriconazole. JAMA ophthalmology. Apr 2013;131(4):422-429. 
8. 
O'Day DM, Head WS, Robinson RD, Clanton JA. Corneal penetration of topical amphotericin B and 
natamycin. Current eye research. Nov 1986;5(11):877-882. 
9. 
Hariprasad SM, Mieler WF, Lin TK, Sponsel WE, Graybill JR. Voriconazole in the treatment of 
fungal eye infections: a review of current literature. The British journal of ophthalmology. Jul 
2008;92(7):871-878. 
10. 
Kalaiselvi G, Narayana S, Krishnan T, Sengupta S. Intrastromal voriconazole for deep recalcitrant 
fungal keratitis: a case series. The British journal of ophthalmology. Feb 2015;99(2):195-198. 
11. 
Prakash G, Sharma N, Goel M, Titiyal JS, Vajpayee RB. Evaluation of intrastromal injection of 
voriconazole as a therapeutic adjunctive for the management of deep recalcitrant fungal keratitis. 
American journal of ophthalmology. Jul 2008;146(1):56-59. 
12. 
Sharma N, Agarwal P, Sinha R, Titiyal JS, Velpandian T, Vajpayee RB. Evaluation of intrastromal 
voriconazole injection in recalcitrant deep fungal keratitis: case series. The British journal of 
ophthalmology. Dec 2011;95(12):1735-1737. 
13. 
Sharma N, Chacko J, Velpandian T, et al. Comparative evaluation of topical versus intrastromal 
voriconazole as an adjunct to natamycin in recalcitrant fungal keratitis. Ophthalmology. Apr 
2013;120(4):677-681. 
 

SAP_001.pdf: 
Mycotic Antimicrobial Localized Injection 
Statistical Analysis Plan 
 
 
Aravind Eye Hospital, Pondicherry, India 
Francis I. Proctor Foundation, University of California San Francisco, USA 
 
 
 
Investigators: 
Tiruvengada Krishnan, DO DNB FICO*1 
Shivananda Narayana, DO DNB FICO*1 
Seema Prabu, DO DNB FICO*1 
Tom Lietman, MD2 
Jennifer Rose-Nussbaumer, MD*2 
 
 
 
 
 
 
 
 
 
 
 
*Principal Investigator 
1Aravind Eye Hospital, Pondicherry, India   
2 Francis I. Proctor Foundation, University of California San Francisco, San Francisco, California, USA 


MALIN Statistical Analysis Plan 
 
 
 
Version 2.7 
 
 
Revised: 26 May 2016 
 
Table of Contents 
1. 
Background ........................................................................................................................................... 1 
2. 
Sample Size and Randomization ........................................................................................................... 1 
3. 
Baseline Characteristics ........................................................................................................................ 1 
4. 
Primary Analysis ................................................................................................................................... 1 
5. 
Secondary Analyses .............................................................................................................................. 1 
6.     Interim Analysis…………………………………………………………………………………….... 2 
 
 


MALIN Statistical Analysis Plan 
 
 
1 | P a g e  
 
Version 2.7 
 
 
Revised: 26 May 2016 
 
1. Background 
Mycotic Antimicrobial Localized Injection (MALIN) is a randomized, double-masked, two-arm 
clinical trial. The purpose of this study is to determine if intrastromal injection with voriconazole 
in combination with topical natamycin is superior to topical natamycin alone. Study participant’s 
eyes will be randomized to one of two treatment groups – 1.) Topical natamycin alone or          
2.) Topical natamycin plus intrastromal voriconazole injections. 
2. Sample Size and Randomization 
If we enroll 70 study participants we estimate that we will have over 80% power to detect a 25% 
difference in repeat culture status (50% in the control group vs. 25% in the voriconazole group), 
assuming a two-tailed alpha 0.05% and no loss to follow up (since participants will be 
hospitalized after enrollment until the 3-day repeat cultures are taken). Participants will be 
randomized using random block sizes in Microsoft Excel (KJR).  
3. Baseline Characteristics  
Baseline characteristics between the 2 arms will be compared using Fisher exact test for 
categorical variables and Wilcoxon rank sum test for continuous variables.  
4. Primary Analysis  
The primary study analysis will be microbiological cure at 3 days. We will compare 3-day 
culture positivity between the two groups using a logistic regression model (dichotomous culture 
positive or culture negative outcome) controlling for baseline culture status. Protocol for 
obtaining cultures is outlined in the MOP.   
5. Secondary Analyses  
• We will compare BSCVA between the two groups, at the 3-month time period (between 
2.5-3.5 months) using the Wilcoxon rank sum with a 2-sided alpha of 0.05. For study 
participants who experience perforation or undergo 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.  
• We will compare 7-day culture positivity between the two groups using a logistic 
regression model (dichotomous culture positive or culture negative outcome) controlling 
for baseline culture status 
• 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 


MALIN Statistical Analysis Plan 
 
 
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Version 2.7 
 
 
Revised: 26 May 2016 
 
6. Interim Monitoring 
A masked interim analysis will be conducted to re-evaluate sample size based on 3-day culture 
positivity when one-third of the data have been collected. 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.