Effects of Carboxymethylcellulose Artificial Tears on the Eye Microbiome: a Randomized, Controlled, Double-Blind Study
Phase: PHASE4
Conditions: Dry Eye Syndromes
Sponsor: University of Florida

Clinical Trial: Effects of Carboxymethylcellulose Artificial Tears on the Eye Microbiome 
NCT: 05292755 
 
Study Protocol and Statistical Plan 3/31/2022 
 
• Principal Investigator: Yujia Zhou, M.D. 
• Co-Investigator: Elizabeth Dawson 
• Co-Investigator: Gary Wang, M.D., Ph.D. 
• Supervisor: Walter Allan Steigleman, M.D. 
 

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University of Florida IRB-01 
Protocol 
 
 
1. Title: 
Effects of Carboxymethylcellulose Artificial Tears on the Eye Microbiome: A 
Randomized, Controlled, Double-Blind Study 
2. Investigators: 
• Principal Investigator: Yujia Zhou 
• Co-Investigator: Elizabeth Dawson 
• Co-Investigator: Gary Wang, M.D., Ph.D. 
• Supervisor: Walter Allan Steigleman, M.D. 
3. Abstract: 
Carboxymethylcellulose (CMC), a common component in artificial tears, modifies the gut 
microbiome when ingested. We will identify its effects on the eye microbiome of adult UF 
eye clinic patients in this randomized, controlled, double-blind study. Effects of CMC on 
the eye microbiome are unknown and have implications for ocular disease and artificial 
tear choice. Participants will be given either CMC artificial tears or artificial tears without 
CMC, complete symptom surveys, and undergo conjunctival swabs. Investigators will 
conduct chart reviews, bacterial genome sequencing, and analyze microbiomes using 
Shannon’s diversity index and principal coordinate analysis on R statistical packages. 

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4. Background:  
Carboxymethylcellulose (CMC) is a thickener frequently included in FDA-approved 
artificial tears. A study by Chassaing et al. in 2021 entitled “Randomized Controlled-
Feeding Study of Dietary Emulsifier Carboxymethylcellulose Reveals Detrimental 
Impacts on the Gut Microbiota and Metabolome” demonstrated that CMC ingestion 
alters the gut microbiome. The eye also has a microbiome distinct from other organ 
systems. The ocular microbiome influences eye diseases including dry eye disease 
and keratitis, as summarized in an article entitled “Microbes of the human eye: 
Microbiome, antimicrobial resistance and biofilm formation” by Ranjith et al. in 2021. 
Interventions such as contact lens wear can change the diversity and composition of 
eye bacteria, as demonstrated in an article entitled “Changes in the Eye Microbiota 
Associated with Contact Lens Wearing” by Hakdong et al. in 2016. 
Therefore, we hypothesize that the use of CMC in artificial tears may affect the eye 
microbiome and influence ocular diseases. Similar studies on microbiome composition 
used bacterial ribosomal RNA sequencing and statistical analysis with existing R 
packages such as phyloseq and DADA2. A UF investigator, Rachel Newsome, recently 
conducted a microbiome study entitled “The gut microbiome of COVID-19 recovered 
patients returns to uninfected status in a minority dominated United States cohort” at 
the University of Florida in 2021. For these reasons, we are conducting a similar study 
on the use of artificial tears containing CMC compared to control artificial tears without 
CMC. Results of this study will lead to a better understanding of the risks and benefits 
associated with CMC artificial tears for eye lubrication and dry eye disease. 
5. Specific Aims: 
We aim to examine whether carboxymethylcellulose in artificial tears changes the eye 
microbiome and identify which bacterial genera are enriched. 
6. Research Plan:  

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• Recruitment: During the spring of 2022, 80 patients of the UF Oaks Eye Clinic who 
meet inclusion criteria will be identified by study staff prior to their visit by chart 
review using the Epic electronic medical record and clinic scheduler on a secure 
encrypted UF computer. Up to 20 participants are expected to drop out over the 
course of this study. When patients have completed their clinical workup, Dr. 
Steigleman will approach patients without exclusion criteria to ascertain 
willingness/interest in the study, then recruit them. Study staff will give willing 
participants the appropriate informed consent form and counsel participants for 
consent to enroll in a private examination room. Completed consent forms will 
labelled with the participant’s randomly selected numerical identifier as described 
below and stored securely in the UF Oaks Eye Clinic Office. 
• Participant Criteria: This study includes adults above age 18 of either sex who 
can self-administer artificial tears and are scheduled to return for clinical follow-up 
in 1 week. This study excludes individuals with active eye infections, have 
prosthetic eyes, are immunocompromised, or are diagnosed with autoimmune 
diseases or malignant neoplasms about the eye. Individuals who take 
immunomodulatory therapy, steroids, antibiotics, medicated eyedrops, or are 
already using CMC artificial tears within 1 week of the study will also be excluded. 
These criteria maximize the number of self-consenting participants while minimizing 
participants whose compliance, conditions, or medications may change the eye 
microbiome. 
• Research Intervention: Participants will be given either artificial tears without CMC 
(control) or commercial artificial tears containing CMC (treatment) and told that they 
may have received either CMC or a placebo. Both are FDA-approved for over-the-
counter use and for treatment of dry eye. Both interventions are standard therapies 
for dry eye disease. Prospective participants have the option to not enroll in the 
study, and the alternative is to complete a normal clinic visit. Individuals for whom 
artificial tears do not constitute standard therapy are excluded from this study. 
• Assignment: This study design will include an experimental control, random 
assignment, and double blinding of staff and participants. The principal investigator 
will create a securely saved Excel spreadsheet with unique randomly generated 
numerical identifiers keyed to either the control group receiving artificial tears 
without CMC or the treatment group receiving CMC artificial tears. The principal 
investigator will then create plain cardboard boxes with the identifier on removable 
adhesive labels. Boxes will contain a copy of the informed consent form, participant 
instructions, and 21 plastic vials of either artificial tears without CMC or CMC 
artificial tears as assigned on the identifier spreadsheet. Patients will be blinded to 
their assigned intervention group. Study staff other than the principal investigator, 
who are blinded to box contents, will give participants a randomly selected box and 
an electronic survey (PDF) on an encrypted clinic computer in their private 
examination room. Labels will be placed on consent forms and identifiers will 
replace personal identifying information on electronic surveys. 
• Data Collection: Electronic surveys will contain the Ocular Surface Disease Index 
and a box for entry of the patient’s unique identifier. After completion of the survey, 
licensed physicians serving as study staff will swab the participant’s right and left 
eye conjunctiva with a single cotton tip applicator, seal the applicator in a sample 

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tube, attach a label, and freeze it for storage in dry ice. The participant will complete 
a normal clinical schedule afterwards and will be scheduled to return in 1 week. 
Upon 1 week follow-up, participants will be swabbed again and complete a second 
survey. The second survey will be identical to the first survey but contain an 
additional question quantifying eye drop use to measure compliance. Patients will 
complete a normal clinical schedule afterwards. Study staff will correlate identifiers 
to patients using the informed consent forms and record ophthalmic history, 
ophthalmic medications, age, gender, tobacco usage, diabetes status, and race by 
chart review during this study. 
• Sample Processing and Data Analysis: Samples will be frozen at -80 degrees 
Celsius and transported to a BSL-2 lab at UF for processing. During processing, 
samples will be lysed, amplified, and sequenced for bacterial 16S RNA using 
protocol supplied by Illumina MiSeq. Swabs and container tubes will be destroyed 
after sequencing. The sequenced bacterial microbiome of each sample will then be 
trimmed, error-corrected, and classified using the DADA2 R package as described 
in the article described above (Newsome 2021). After all samples are sequenced, 
microbiomes will be grouped by principal coordinate analysis using the phyloseq R 
package. Shannon’s diversity indices will be calculated using the phyloseq R 
package. Differences in alpha diversity before and after treatment will be tested by 
PERMANOVA using the vegan R package, and differences in beta diversity will be 
tested by ANOVA using the nlme R package. 
• Data Security: The original consent forms with unique identifier stickers will be 
kept securely in a folder and stored in a locked office within the UF Oaks Eye Clinic. 
Surveys will be accomplished on a pdf and do not contain any participant 
identification aside from the study-assigned numerical identifier. All other 
documents and data files will be deidentified and stored on an encrypted computer 
within the UF IT environment. Only medical students, resident physicians, and 
attending physicians serving as study staff will access participant medical records 
on the Epic EMR using a secure encrypted computer or smartphone. This study will 
not have a Data Safety Monitoring Board. Dr. Steigleman will serve as oversight for 
this research project and will meet weekly with study staff to monitor study conduct 
and patient safety. 

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7. Possible Discomforts and Risks: 
Enrolled study participants may more commonly encounter mild eye discomfort during 
conjunctival swabs, anxiety regarding assignment to a treatment group, short delays in 
clinic flow (1-2 minutes), and inconvenience of completing surveys during the study. 
Rare discomforts or risks may include breach of protected patient data, eye pain, dry 
eye, eye infection, and eye trauma including corneal abrasions and conjunctival 
hemorrhage because of methods used in this study. Participants will not be charged for 
any time, medication, or procedure used in this study. 
To minimize anxiety and medical risks, patients will receive counseling and treatment 
interventions in the setting of the UF Oaks Eye Ophthalmology Clinic by a licensed 
attending ophthalmologist or an ophthalmology resident capable of mitigating and 
treating such harms. The inconvenience of enrolling in the study will be mitigated by 
utilizing the wait time (10 to 40 minutes) during a routine clinic appointment. The 
conjunctival swab is a routine technique practiced by attending and resident 
ophthalmologists with minimal risk. Eye discomfort and pain during this procedure will 
be reduced with a soft cotton-tip applicator, and patients may be given routine topical 
anesthetics or eye lubricants after the procedure if needed. Topical anesthesia prior to 
swabbing and other methods of collecting microbiome samples such as eye irrigation 
will not be used due to their anticipated negative effect on sample quality. 
8. Possible Benefits: 
The participants will receive free FDA-approved treatment (artificial tears) for dry eye 
during the study and improved guidance on artificial tear choice after the study’s 
conclusion based on the study results. This may not benefit all participants directly but 
may directly help some participants alleviate dry eye. There are no other direct benefits 
to participants. 
To date, there is no data on whether this common artificial tear ingredient may have 
deleterious or favorable effects on the eye microbiome. The composition of eye 
microbiomes has been shown to have associations with eye diseases, including dry 
eye disease. This information may guide artificial tear choice in the future, as opposed 
to the status quo, where all brands of preservative-free artificial tears are treated as 
equivalent. 
These benefits outweigh the minimal risk to enrolled study participants, who will be in 
the care of a trained ophthalmologist during the length of this study in the unlikely 
event that harm occurs to them. Our study methods use minimally invasive techniques 
to gather data and are routine in the setting in an ophthalmology clinic. We designed 
the study such that the risk of unreasonable harms such as billing for research time, 
eye infections, and patient discomfort are minimized or eliminated. 
9. Conflict of Interest: 
We do not have any conflicts of interest to disclose. Neither the principal investigator, 
co-investigators, or UF hold patents, licenses, or stock in the artificial tears used. We 
have no external sponsors.