Potential Role of Ocular Surface Microbiome in Dry Eye: Microbial Interactions and Symptom Alleviation

Study design and sample collection This study is randomized, double-blind, and prospective trial. The inclusion criteria were as follows: patients aged 19 years or older with symptoms of dry eye syndrome. A total of 50 participants were randomly assigned to either the CsA group (n = 25, cyclosporine A twice a day and hyaluronic acid twice daily) or the control group (n = 25, hyaluronic acid four times daily). 0.05% cyclosporine A (Cyporin N Eye Drops, Taejoon Pharm, Republic of Korea) and 0.15% hyaluronic acid (New Hyaluni Eye Drops, Taejoon Pharm) were used in this study. Blinding was maintained by separating the examiner and the sample collector.

Exclusion criteria included the use of topical ocular drops or systemic medication (such as systemic steroids, immunomodulators, and tetracyclines) that could influence study outcomes, as well as the presence of conjunctivitis, anterior blepharitis, Demodex infestation, parasitic eye infections, unresolved ocular trauma, healing disorders of the ocular surface (OS), a history of penetrating keratoplasty, contact lens use, or any ophthalmic surgical procedure within three months before baseline. Additionally, subjects with a single functional eye, pregnant or breastfeeding women, and those at risk of pregnancy without contraception were excluded.

Clinical data and OS samples were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation (Fig. 1a). Of the 50 participants at V1, 21 participants from each group completed follow-up at V2, while 14 participants in the CsA group and 18 participants in the Newhyaluni group remained at V3. OS samples for microbiome analysis were obtained from the inferior conjunctival sac using sterile mixed cellulose ester (MCF) membrane (Millipore, Merck). OS samples were collected from 13 participants in the CsA group and 13 participants in the Newhyaluni group. All OS samples were stored at -80 °C until DNA extraction for microbiome analysis.

Clinical data collection Symptoms were assessed using the Ocular Surface Disease Index (OSDI), Visual Analogue Scale (VAS) for pain, and the modified Standard Patient Evaluation of Eye Dryness (SPEED) questionnaire. Tear film stability was evaluated by measuring the tear break-up time (TBUT). Fluorescein staining of the cornea was performed by applying a drop of sterile saline to a sterile fluorescein strip. After the subject blinked naturally three times, the time between a normal blink and the first appearance of a dry spot in the tear film was recorded. The average of three repeated measurements was used for analysis.

OS inflammation was assessed using the fluorescein staining score (FSS) graded according to the Oxford scoring system. The severity of meibomian glands and eyelid inflammation, as well as lid margin hyperemia, was graded as none, mild, moderate, or severe. Tear secretion was measured using the Schirmer strip without anesthesia. A Schirmer strip was placed at the temporal third of the lower eyelid, between the lower palpebral conjunctiva and the lower bulbar conjunctiva. After 5 min, the length of tear fluid absorbed by the strip was measured in millimeters.

DNA extraction and whole metagenome sequencing Total DNA was extracted from the collected OS samples using the RNeasy PowerMicrobiome Kit (Qiagen, Inc., Valencia, CA, USA). DNA concentration was measured with a BioPhotometer D30 using a μCuvette G1.0 (Eppendorf, Hamburg, Germany). To eliminate potential contaminants during the experimental process, 12 negative controls were included, comprising sampling membranes, DNA-free water added to the DNA extraction kit, and DNA-free water added to the sequencing library preparation kit. These negative controls were sequenced alongside the 61 OS samples.

For whole metagenome sequencing, extracted DNA was fragmented using NEBNext dsDNA Fragmentase (New England Biolabs, Inc., Ipswich, MA, USA). Library preparation was performed with a Swift 2S Turbo DNA Library Kit (Swift Biosciences, Inc., USA) following the manufacturer's protocol. Purification and size selection were conducted using HiAccuBead (AccuGene, San Diego, CA, USA). Index PCR was carried out with the Swift 2S Turbo Combinatorial Dual Indexing Primer Kit (Swift Biosciences, Inc.), followed by additional purification and size selection using HiAccuBead. The library concentration for each sample was quantified using the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). Equimolar concentrations of each library (4nM) were pooled and sequenced on an Illumina NovaSeq 6000 system (250-bp paired end).

Raw sequence data obtained from the NovaSeq system were processed as previously described. Adapter sequences were trimmed, and quality filtering was performed using Trimmomatic. Paired-end sequences were merged using PEAR v.0.9.11. Human-derived sequences in the metagenome data were identified and removed using BBMap with a reference human genome. Taxon

Study Overview

• Status: Completed
• Conditions: Dry Eye Syndrome (DES)
• Intervention / Treatment: Drug: topical cyclosporine eyedrops and hyaluronic eyedrops

• Study Type: Interventional
• Enrollment (Actual): 50
• Phase: Not Applicable

Contacts and Locations

Study Locations

• South Korea
  • Seoul
    • Seoul, Seoul, South Korea, 07441
      • Hallym University, Kangnam Sacred Heart Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

• patients aged 19 years or older
• with symptoms of dry eye syndrome.

Exclusion Criteria:

• the use of topical ocular drops or systemic medication (such as systemic steroids, immunomodulators, and tetracyclines) that could influence study outcomes
• the presence of conjunctivitis, anterior blepharitis, Demodex infestation, parasitic eye infections, unresolved ocular trauma, healing disorders of the ocular surface (OS),
• a history of penetrating keratoplasty, contact lens use, or any ophthalmic surgical procedure within three months before baseline.
• a single functional eye, pregnant or breastfeeding women, and those at risk of pregnancy without contraception

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Treatment
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Triple

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: CsA group; topical cyclosporine A twice a day and hyaluronic acid twice daily	Drug: topical cyclosporine eyedrops and hyaluronic eyedrops; topical cyclosporine eyedrops and hyaluronic eyedrops
Placebo Comparator: Newhyaluni group; topical 0.15% hyaluronic acid (New Hyaluni Eye Drops, Taejoon Pharm) four times a day.	Drug: topical cyclosporine eyedrops and hyaluronic eyedrops; topical cyclosporine eyedrops and hyaluronic eyedrops

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
microbiome	NGS sequencing of microbiome obtained from ocular surface	Clinical data and ocular surface (OS) samples were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Ocular Surface Disease Index (OSDI)		Clinical data were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation.
Visual Analogue Scale (VAS) for pain		Clinical data were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation.
modified Standard Patient Evaluation of Eye Dryness (SPEED) questionnaire		Clinical data were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation.
tear break-up time (TBUT)	After the subject blinked naturally three times, the time between a normal blink and the first appearance of a dry spot in the tear film was recorded. The average of three repeated measurements was used for analysis. ability	Clinical data were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation.
The severity of meibomian glands and eyelid inflammation, as well as lid margin hyperemia,		Clinical data were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation.
Tear secretion	A Schirmer strip was placed at the temporal third of the lower eyelid, between the lower palpebral conjunctiva and the lower bulbar conjunctiva. After 5 min, the length of tear fluid absorbed by the strip was measured in millimeters.	Clinical data were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation.
Fluorescein staining of the cornea	Oxford score system. minimum score 0 to maximum score 4.Higher scores mean a worse outcome.	Clinical data were collected at baseline before eye drop administration (V1) and at 4 weeks (V2) and 12 weeks (V3) after treatment initiation.

Collaborators and Investigators

• Sponsor: Hallym University Kangnam Sacred Heart Hospital

Study record dates

Study Major Dates

• Study Start (Actual): January 20, 2020
• Primary Completion (Actual): January 28, 2024
• Study Completion (Actual): January 28, 2024

Study Registration Dates

• First Submitted: April 13, 2025
• First Submitted That Met QC Criteria: April 13, 2025
• First Posted (Actual): April 20, 2025

Study Record Updates

• Last Update Posted (Estimated): September 3, 2025
• Last Update Submitted That Met QC Criteria: August 26, 2025
• Last Verified: April 1, 2025

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Eye Diseases; Lacrimal Apparatus Diseases; Dry Eye Syndromes; Anti-Infective Agents; Antifungal Agents; Immunosuppressive Agents; Immunologic Factors; Physiological Effects of Drugs; Molecular Mechanisms of Pharmacological Action; Enzyme Inhibitors; Antirheumatic Agents; Dermatologic Agents; Pharmaceutical Solutions; Calcineurin Inhibitors; Cyclosporine; Cyclosporins; Ophthalmic Solutions
• Other Study ID Numbers: 2019-01-01

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No