Effectiveness of Low Level Light Therapy and Intense Pulse Light on Mite Count as Adjuntive Therapies in Demodex Blepharitis Using Artificial Intelligent Program (Ai-Demodex): A Factorial Randomized Sham-Controlled Trial
Phase: NA
Conditions: Demodex Blepharitis, Meibomian Gland Dysfunction (MGD)
Sponsor: Chulalongkorn University

Version2 Date 20241201 
 
 1 
Effectiveness of Low Level Light Therapy and Intense 
Pulse Light on Mite Count as Adjuntive Therapies in 
Demodex Blepharitis Using Artificial Intelligent Program 
(Ai-Demodex): A Factorial Randomized Sham-
Controlled Trial 
 
 
 
 
 
 
 
 
 
Department of Ophthalmology 
King Chulalongkorn Memorial Hospital 
Tel (02) 256-4000 #61635 
 
 
 

Version2 Date 20241201 
 
 2 
Full Proposal 
1. Proposal Title 
English : Effectiveness of Low Level Light Therapy and Intense Pulse Light on Mite Count as 
Adjuntive Therapies in Demodex Blepharitis Using Artificial Intelligent Program (Ai-Demodex): A 
Factorial Randomized Sham-Controlled Trial 
 
2. Investigators 
Authors   
Master of Science in Clinical Sciences (International Program), Faculty of Medicine, Chulalongkorn 
University  
Department of Ophthalmology, Faculty of Medicine, Chulalongkorn University  
Advisor  
Department of Preventive and Social Medicine, Faculty of Medicine, Chulalongkorn University  
Co-advisor  
Cornea and Refractive Surgery Unit, Department of Ophthalmology, Faculty of Medicine, 
Chulalongkorn University  
Excellence Center of Cornea and Limbal Stem Cell Transplantation, Department of 
Ophthalmology, King Chulalongkorn Memorial Hospital  
  
 
 
 
 
 
 
 
 
 
 
 

Version2 Date 20241201 
 
 3 
3. Rationale  
Demodex blepharitis is a prevalent ocular condition arising from an overgrowth of 
Demodex mites, part of the normal ocular flora, on the eyelids. Although these mites typically 
reside on the skin, an increased population can lead to inflammation and eyelid infection. A 
robust correlation exists between demodex blepharitis and meibomian gland dysfunction (MGD) 
which is the primary cause of evaporative dry eye. 
Kasetsuwan et al. in 2017  from KCMH,Thailand. The prevalence of ocular Demodex 
blepharitis in Thailand, detected by microscopic examination, was 42% (Demodex folliculorum). 
Among patients with ocular Demodex infestation, 69% had cylindrical dandruff. A higher detection 
rate of 79% was achieved using semi-nested PCR.(1) 
Flores de Venecia et al. documented that demodex blepharitis has strong correlation with 
MGD. An 85% incidence of Demodex in MGD patients compared to 34% in healthy controls 
(without lid margin disease) (2). The severity of Demodex infestation aligns with the extent of MG 
damage, indicating a direct association(3). 
Historically, demodex blepharitis and MGD were treated together using mechanical 
treatment like warm compression and lid scrubbing and pharmacological treatment as Tea Tree 
Oil (TTO)(4). An FDA-approved solution for the disease is lotilaner ophthalmic solution, 0.25% (5), 
but its high cost at 1,957 USD per 10 ml is a notable drawback. 
Hyun et al. (2012) found that only 7% of patients were Demodex-free after eyelid scrubs 
without the use of TTO. Therefore, in practice, we now recommend that patients with Demodex 
blepharitis treat their condition with eyelid scrubs containing TTO for higher treatment efficacy.(6) 
Patients often find eyelid hygiene tedious and time-consuming, leading to poor treatment 
compliance (7). Recent innovations in in-office MGD management include Intense Pulsed Light 
(IPL), proposed as an adjunctive therapy for demodicosis. IPL's heat transfer may kill Demodex 
mites and soften meibum, reducing inflammatory mediators. Limitations include cost and 
inapplicability to patients with darker skin tones (Fitzpatrick V or VI), light-intolerant individuals, or 
pediatric patients (4). 
An alternative cellular-level therapy overcoming IPL limitations is Low-Level Light Therapy 
(LLLT), using near-infrared light to induce mitochondrial light absorption and cell photoactivation, 
causing changes in inflammatory protein expression (8). This technology, recognized by NASA for 

Version2 Date 20241201 
 
 4 
plant growth and astronaut wound healing (9), has diverse applications, including dermatology for 
Androgenetic Alopecia (AGA), skin rejuvenation, body contouring (10), and orthopedics for reducing 
edema, inflammation, and relieving pain and stiffness. 
For direct effect to demodex mite Avci et al. (2013) and Sulek et al  found that blue light 
activates porphyrins, leading to an antibacterial effect(11, 12). Rhee et al. (2023) conducted a 
comprehensive review revealing that Demodex mites can carry Streptococcus and 
Staphylococcus species on their surface, while Bacillus oleronius is present in their abdomen.(4) 
 
Recently, LLLT has been used for MGD, with devices allowing combined (13) or separate 
(14) treatment with IPL or LLLT emerging as alternatives for MGD management. 
Evaporative dry eye in dry eye disease (DED) is a long-term, multifactorial condition 
affecting the tears and the ocular surface, with a huge worldwide impact. It encompasses two 
main types: aqueous deficient and evaporative dry eye(15). In the USA, DED incurs an indirect 
annual cost of US $73.12 billion, equating to US $15,596 per person, due to diminished 
productivity(16). Treatment costs vary by DED severity, ranging from US $300 to US $1100 per 
person across different countries (17). The global DED market, valued at USD 5.22 billion in 2019, 
is anticipated to reach USD 6.54 billion by 2027, with an annual growth rate of 4.7%. 
Beyond financial concerns, various studies highlight DED as a burdensome condition 
associated with depression, which is a hidden problem often overlooked. 
Artificial intelligent technology has revolutionized the field of ophthalmology, particularly 
in the analysis of meibomian gland images. By incorporating AI-based meibography, we can 
improve the accuracy and consistency of assessments related to glandular health, a crucial factor 
in managing MGD associated with Demodex blepharitis. Yuan et al. (2021) have demonstrated a 
reliable AI-based methodology for normalizing and segmenting meibomian gland images, offering 
substantial advancements over conventional techniques(18) 
Our study pioneers a Factorial Randomized Control Trial (RCT) investigating Low-Level 
Laser Therapy (LLLT) for the treatment of demodex blepharitis, comparing it with Intense Pulsed 
Light (IPL), IPL combined with LLLT, and mechanical with pharmacological approaches. The aim 
is to address the limitations of mechanical with pharmacological treatment for demodex 
blepharitis, particularly in terms of the efficacy of innovative laser treatments, improved 
compliance, and reduced disease burden, ultimately leading to better treatment outcomes and 

Version2 Date 20241201 
 
 5 
used AI-based meibography to evaluate the meibography photos which taken from demodex 
blepharitis patient before and after treatment. 
 
Novel questions  
1.Will Low-Level Light Therapy (LLLT) and/or Intense Pulsed Light (IPL) therapy synergistically or 
antagonistically improve the mean reduction of Demodex mite count in patients with Demodex 
blepharitis? 
2.Will Low-Level Light Therapy (LLLT) and/or Intense Pulsed Light (IPL) therapy synergistically or 
antagonistically improve the Meibomian Gland Dysfunction (MGD) stage in patients with Demodex 
blepharitis? 
 
 
 
4. Literature Review 
4.1) Treatment Parameters for Demodex count  : Primary outcome 
No previous study has reported the count of demodex mites through Low-Level Light Therapy 
(LLLT). It has been mentioned that Intense Pulsed Light (IPL) improves ocular surface parameters, 
while studies on the treatment parameters of demodex blepharitis have predominantly focused 
on the following outcomes: 
Advantages of Using Demodex Count as a Primary Outcome 
Objective Outcome: The Demodex count provides a quantifiable and objective 
measurement, reducing the potential for subjective bias in assessing treatment efficacy. 
Correlation with Recurrence: A high Demodex count has been shown to correlate with 
the recurrence of a series of refractory ocular surface diseases, indicating its potential as 
a predictive marker for disease management and long-term outcomes.(19) 
Disadvantages of Using Demodex Count as a Primary Outcome 
Poor Correlation with Symptoms: The Demodex count may not correlate well with the severity 
of symptoms experienced by patients. As a result, a reduction in Demodex count may not 
necessarily translate to symptomatic relief, potentially limiting its effectiveness as a standalone 
primary outcome measure. (AAO , (20)) 

Version2 Date 20241201 
 
 6 
 
4.2) Meibomian Gland Dysfunction Treatment Parameter: Secondary outcome 
 
Regional Demodex blepharitis is strongly associated with the severity of meibomian gland 
dysfunction. For instance, Hyun Koo et al. in 2012 demonstrated that TTO treatment could 
improve both Demodex count and OSDI score(6), while Zhang et al. in 2019 showed a significant 
statistical improvement in TBUT.(21) 
Only one preliminary study has suggested that LLLT, with four weekly sessions lasting 15 
minutes each and combined with non-preservative tear application, might improve tear meniscus 
height. 
There is currently no research on the effects of blue light LLLT on ocular surface 
parameters in Meibomian Gland Dysfunction. Therefore, if our study is successful, it will provide 
valuable insights into this knowledge gap. 
 
 
4.3) Epilation strategies 
The lashed epilation technique lacks a standardized method. Numerous studies 
conducted from 1967 to 2024 have demonstrated that approximately 40% of these studies utilize 
the Modified Coston method as outlined by Gao et al. in 2005. In this method, two lashes 
containing cylindrical dandruff (CD) were extracted from each eyelid of every participant using 
fine forceps. These lashes were then individually placed at opposite ends of a glass slide for 
observation under a slit-lamp biomicroscope (SL220; Carl Zeiss, Oberkochen, Germany) at a 
magnification of ×25. This process resulted in eight lashes (4 per each eye) being prepared across 
four slides for each participant. A coverslip was applied to each lash, followed by the slow 
pipetting of 20 μL of saline at the coverslip's edge until it surrounded the lash. Subsequently, 
20 μL of 100% alcohol (Sigma-Aldrich, St. Louis, MO) was pipetted at the same edge, extending 
the counting period for up to 20 minutes to facilitate the migration of embedded Demodex from 
the CD. The Demodex count was performed thrice under the microscope, and all samples were 
photographed using standard procedures by the same specialist. The presence of Demodex in at 
least one of the eight eyelashes was considered Demodex-positive.(22) 
Regarding epilation techniques, there are three primary methods: 

Version2 Date 20241201 
 
 7 
A) Direct pulling, 
B) Rotating the lash before epilation, and 
C) Removing the cylindrical dandruff from the base of the lash before epilation. 
Bitton et al. in 2024 concluded that Technique B, involving rotating the lash before 
epilation, yielded the highest Demodex count. (23) 
In our study, we intend to use Technique B along with the Modified Coston method for 
counting Demodex mites. These distinct parameters and methodologies contribute to the 
understanding of demodex blepharitis treatment, but a comprehensive investigation of LLLT in 
this context is notably absent in the existing literature. 
4.4) Eyelid scrubb compliance 
Hyun Koo et al. (2012) demonstrated that good compliance with eyelid scrubbing using 
tea tree oil (TTO) (defined as more than 10 times per week with over 70% compliance) resulted 
in significant improvements in both Demodex count and the Ocular Surface Disease Index (OSDI) 
scores. In contrast, the moderate and poor compliance groups did not show statistically 
significant improvements compared to the control group.(6) 
 For our study,we will exclude patients with moderate and poor compliance. By focusing 
on patients who strictly adhere to the treatment protocol, we aim to more accurately assess 
the effectiveness of the intervention. 
 
4.5) FDA approved medication for Demodex blepharitis : Lotilaner ophthalmic solution 0.25% 
is the only medication for Demodex blepharitis that was FDA-approved in July 2023(5). A recent 
meta-analysis showed that lotilaner significantly reduces Demodex mite density and mean 
collarette scores compared to a placebo(24). However, the cost per bottle is $2,031 for 10 
milliliters, and it is not available in Thailand. Consequently, we did not select this medication as 
an intervention in our study. 
 
4.6) Several imaging modalities are used to diagnose MGD, with meibography emerging as a 
key tool for evaluating the structure and function of the meibomian glands. Traditional 
meibography employs infrared light to visualize gland architecture and assess gland dropout, but 
its reliance on manual interpretation can introduce subjectivity. The advent of artificial intelligence 

Version2 Date 20241201 
 
 8 
(AI) in ophthalmology has opened new opportunities for more precise, automated, and objective 
assessments of meibomian gland health. AI-assisted meibography systems, particularly those 
based on deep learning algorithms, offer the potential to enhance the accuracy of MGD diagnosis 
and monitoring by providing automated segmentation, analysis, and grading of meibomian 
glands(25) 
   
Recent studies have demonstrated the effectiveness of AI in analyzing meibography 
images to detect structural changes in the glands, including gland dropout and atrophy, with high 
sensitivity and specificity. Dr. Yuan et al. described the use of deep learning models in 
meibography for the automatic segmentation of meibomian glands, offering a significant 
improvement over manual grading in terms of both speed and consistency. Their software 
integrates multiple steps, including image normalization, automatic segmentation, and manual 
refinement, to ensure high-quality analysis (18) By providing quantitative data on gland 
morphology, AI-based tools can assist clinicians in more accurately diagnosing MGD and tailoring 
treatment plans (26) 
Low-level light therapy (LLLT) and intense pulsed light (IPL) have gained traction as 
adjunctive treatments for MGD. Both therapies aim to improve meibomian gland function by 
reducing inflammation, improving glandular secretion, and decreasing Demodex mite counts 
Previous Studies suggest that combining LLLT and IPL with conventional therapies for MGD and 
blepharitis can lead to better clinical outcomes than monotherapy. However, limited research 
has evaluated the specific impact of these therapies on meibomian gland structure, especially 
when assessed using AI-assisted meibography  
Our study aims to fill this gap by comparing pre- and post-treatment meibomian gland 
parameters in patients with Demodex blepharitis, using AI-assisted meibography to evaluate the 
effectiveness of LLLT, IPL, and combined treatments. By leveraging AI technology, this research 
intends to offer more objective and quantifiable insights into the impact of these treatments on 
meibomian gland health, which could ultimately improve patient outcomes in MGD 
management. 

Version2 Date 20241201 
 
 9 
4.7) AI-Assisted Meibography Analysis: Our study plans to utilize an AI-assisted meibography 
developed system (18) to evaluate and compare the severity of meibomian gland dysfunction 
before and after low-level light therapy (LLLT) treatment for Demodex blepharitis. What sets this 
study apart is the integration of advanced AI-driven image analysis, specifically designed to assess 
the effectiveness of light-based therapies, which are playing an increasingly significant role in the 
treatment of meibomian gland dysfunction. This novel approach allows for precise, quantitative 
evaluation of treatment outcomes, offering a cutting-edge method to measure improvements in 
gland function.  
 
5. Objectives 
Study objectives to fulfill the knowledge gap  
Primary objective: 
To evaluate and compare the efficacy of mechanical with pharmacological treatment, LLLT, IPL 
and IPL + LLLT with Demodex Mite Count in MGD-related Demodex Blepharitis  
Secondary objectives: 
To evaluate and compare efficacy of mechanical with pharmacological treatment LLLT, IPL, IPL 
+ LLLT for ocular surface parameters in MGD-related Demodex Blepharitis 
 
6. Hypothesis 
Null Hypothesis (H0): 
There is no significant difference in the reduction of Demodex mite count among patients with 
Demodex Blepharitis treated with Low Level Light Therapy (LLLT), Intense Pulse Light Therapy 
(IPL), Combined LLLT + IPL, and Mechanical with pharmacological Treatment. 
Alternative Hypothesis (H1): 
There is a significant difference in the reduction of Demodex mite count among patients with 
Demodex Blepharitis treated with at least one of the following therapies: Low Level Light Therapy 
(LLLT), Intense Pulse Light Therapy (IPL), Combined LLLT + IPL, greater than Mechanical with 
pharmacological Treatment. 
 
 

Version2 Date 20241201 
 
 10 
Hypotheses  
- LLLT or IPL, will be equally effective in reducing Demodex mite count and improving 
ocular surface parameters. 
- IPL + LLLT will be more effective than LLLT or IPL alone in reducing Demodex mite count 
and improving ocular surface parameters. 
 
7. Keywords 
Demodex blepharitis, MGD, LLLT, IPL, Demodex mite count, Demodex mite eradication, Ocular 
surface parameters 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Version2 Date 20241201 
 
 11 
Conceptual framework  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  
Assessed for eligibility 
 Excluded 
Not meeting inclusion criteria 
Refused to participate 
Other reasons  
Randomized (1:1:1:1) 
Allocation 
Enrollment 
Follow up 
Analysis 
Allocated to mechanical with 
pharmacological Treatment (n 
= 22)  
Warm compression  
+Lid scrubb  
+1%TTO twice daily   
+ShamIPL + ShamLLLT 
follow up  
1st (0 wk)= 0-14 days before 
treatment session 
2nd (8th wk)= 1 months after   
treatment session 
3rd (16th wk)= 3 months after 
treatment session 
Allocated to LLLT 
(n = 22)  
Warm compression  
+ Lid scrubb  
+1%TTO twice daily   
+ ShamIPL 
 
 
follow up  
1st (0 wk)= 0-14 days before 
treatment session 
2nd (8th wk)= 1 months after   
treatment session 
3rd (16th wk)= 3 months after 
treatment session 
 
Allocated to IPL 
(n = 22) 
Warm compression  
+ Lid scrubb  
+ 1%TTO twice daily   
+ ShamLLLT 
 
Allocated to IPL + LLLT  
(n = 22) 
Warm compression  
+ Lid scrubb  
+ 1%TTO twice daily   
 
follow up  
1st (0 wk)= 0-14 days before 
treatment session 
2nd (8th wk)= 1 months after   
treatment session 
3rd (16th wk)= 3 months after 
treatment session 
 
follow up  
1st (0 wk)= 0-14 days before 
treatment session 
2nd (8th wk)= 1 months after   
treatment session 
3rd (16th wk)= 3 months after 
treatment session 
 
Analyzed 
Analyzed 
Analyzed 
Analyzed 

Version2 Date 20241201 
 
 12 
Variables  
Independent variables: 
the different treatment modalities: 
1.Low Level Light Therapy (LLLT) 
2.Intense Pulse Light Therapy (IPL) 
3.Combined LLLT + IPL 
4.Mechanical with pharmacological treatment 
Dependent variables: Mean Demodex Count, MGD Staging, OSDI Questionnaire, Meibum 
Quality, Meibum Expressibility, Telangiectasia Grading (0-3), Surface Staining (NEI Grading System), 
% Meibomian Gland Loss (MGL), Lipid Layer Thickness, Noninvasive Break-Up Time (NIBUT), Tear 
Meniscus Height (TMH),  
 
8. Research Design 
Single Center, 2x2 Factorial Randomized Control Trials 
 
 
9. Research Methodology  
Target Population: Demodex blepharitis patients at the Department of Ophthalmology, King 
Chulalongkorn Memorial Hospital. 
 
This is a single-center, prospective, 2x2 factorial Randomized Control Trials involving outpatients 
with Demodex blepharitis presenting at the Department of Ophthalmology, King Chulalongkorn 
Memorial Hospital (KCMH) from Nov 2024 to Nov 2025. 
 
The approach to participants: involves patients under the care of the research physician. They 
are recruited through advertisements and contacted through the patient’s physician to introduce 
the researcher and seek volunteer participation. 
 
 
 

Version2 Date 20241201 
 
 13 
The inclusion criteria consist of: 
1. All patients who received a definite diagnosis of demodex blepharitis based on cylindrical 
dandruff at the base of the lashes and the presence of at least one alive demodex mites 
on eyelid margin by microscopy 
2. Age >18 years. 
3. Patients who can understand and follow the study instructions and can adhere to the 
scheduled follow-up plan. 
 
The exclusion criteria consist of: 
1. Patients who received previous treatment IPL or LLLT 
2. Poor compliance with eyelid scrubbing using tea tree oil (TTO)  
3. Dead demodex mite on lash. 
4. Contraindication of IPL: Skin Fitzpatrick scale V/VI, patients with pigmented lesions on the 
treatment area (Eyelid, Cheek). 
5. Pregnancy or breastfeeding. 
6. Active Skin disease:  facial skin cancer, graft-versus-host disease, systemic lupus 
erythaematosus 
7. Active eye infection: recurrent herpes simplex 
8. Other lid disease ocular trauma, ocular deformity scar, exophthalmos, eyelid insufficiency 
9. Patients with pigmented lesion on area of treatment.   
 
We will use computer-generated random number allocation into 4 groups in a 1:1:1:1 ratio 
including seed number, with the outcome accessor blind and participant blind with allocation 
concealment: 
 
1.) ShamLLLT + ShamIPL 
2.) LLLT + ShamIPL 
3.) IPL + ShamLLLT 
4.) LLLT + IPL 

Version2 Date 20241201 
 
 14 
***All groups receive mechanical with pharmacological treatment (Warm compression + 
Lid hygiene + 1% TTO) for 90 days with google form logbook for record compliance and 
prevent co-interventional treatments. 
***All group with participant/investigator/interpreter blind. 
Blinding and Allocation Concealment in Research Team:  
1st Research Assistant : Allocates patients according to computer-generated random number 
allocation, ensuring allocation concealment, Evaluated VA , IOP , OSDI score by reads the 
questionnaire to the participant 
2nd Research Assistant (The treatment administrator) : Administers IPL, LLLT, or sham treatments. 
3rd Research Assistant : Conducts assessments using Keratograph and Lipiview2. 
Main Researcher : Evaluates primary and secondary outcomes using case record forms, remaining 
blind to the treatment groups. 
 
Intervention Description 
Mechanical with pharmacological treatment  : Warm compression 15 minutes + Lid hygiene + 
1% TTO  for 90 days 
1%TTO : OCuSOFT Oust Demodex Eyelid Cleanser Foam Bottle 50ml (OCuSOFT, 
Richmond, TX) is the only commercially available medication in Thailand that contains 
1% tea tree oil. 
IPL : The Eye-light device (Espansione Marketing S.p.A., Bologna, Italy,waveleggths 1,200-1,500 nm, 
Delivery system 12cm2) (Thai FDA No 66-2-2-2-0012685) :  
• Protective eye shields 
• 5 flashes of light were applied for each eye (3 along the inferior orbital rim, 1 at 
the lateral canthus, and 1 applied horizontally along the inferior orbital rim) 
• Therapeutic energy level (10–16 joules/ cm2) based on the degree of skin 
pigmentation. 
LLLT : The blue MY MASK-E (Espansione Marketing S.p.A., Bologna, Italy) (Thai FDA No 66-2-2-2-
0012685) 
• No eye shields 
• Applying a special mask for 15 minutes, once a week for 4 weeks 

Version2 Date 20241201 
 
 15 
ShamLLLT : the MY MASK-E (Espansione Marketing S.p.A., Bologna, Italy) – No eye shields, applying 
a power off special mask for 15 minutes , once a week for 4 weeks 
ShamIPL : the Eye-light device (Espansione Marketing S.p.A., Bologna, Italy) - Protective eye 
shields, 5 time for each eye with flashes of light off. 
 
 
Epilation Strategies 
 
the Modified Coston method as outlined by Gao et al. in 2005. In this method, two lashes 
containing cylindrical dandruff (CD) then additional rotated around itself  
were extracted from each eyelid of every participant using fine forceps. These lashes were then 
individually placed at opposite ends of a glass slide for observation under a slit-lamp 
biomicroscope (SL220; Carl Zeiss, Oberkochen, Germany) at a magnification of ×25. This process 
resulted in four lashes of right eye being prepared across two slides for each participant. A 
coverslip was applied to each lash, followed by the slow pipetting of 20 μL of saline at the 
coverslip's edge until it surrounded the lash. Subsequently, 20 μL of 100% alcohol  was pipetted 
at the same edge, extending the counting period for up to 20 minutes to facilitate the migration 
of embedded Demodex from the CD. The Demodex count was performed thrice under the 
microscope, and all samples were photographed using standard procedures by the same 
specialist. The presence of Demodex in at least one of the eight eyelashes was considered 
Demodex-positive 
 
10. Data Collection: 
Primary Outcome:  
Mean of Demodex Count - the number of Demodex mites per eyelash on a total of 4 collarettes 
lashes epilation (2 from upper eyelid ,2 from lower eyelid and identification by anatomical 
imaginary line of nasal and temporal limbus) per right eye in 30 days after end of treatment. 
(8th week) 
 
Secondary Outcomes: 
1.Mean of Demodex Count - per right eye in 90 days after end of treatment.(16th week) 

Version2 Date 20241201 
 
 16 
2.MGD staging (Severity) 
OSDI questionnaire used to evaluate ocular dry eye symptoms. 
 
Meibum Quality. 
Meibum Expressibility. 
Telangiectasia grading: 0-3 
 
 
 
 

Version2 Date 20241201 
 
 17 
 
3.Surface Staining: NEI Grading System. 
4.Lipid Layer Thickness by LIPIVIEW 
5.Noninvasive break-up time (NIBUT), tear meniscus height (TMH) , % Meibomian Gland Loss (MGL)  
by Keratograph 5M (OCULUS, Germany) 
 
 
% Meibomian Gland Loss (MGL) 
 
6.Corrected one times baseline , including BCVA, IOP. 
7. After complete datacorrection : Meibography images will be masked and analyzed using an 
AI-assisted program to assess the following: 
   
     -    Meibomian gland dysfunction staging (severity): MGD severity level and 
scored with 1-5 according to the 2011 MGD workshop (27) (Figure1) 
- Meibomian gland area ratio: The proportion of the total visible meibomian gland 
area to the overall tarsal area in the eyelid 
- Meibomian gland length: The linear measurement from the proximal to distal end 
of a meibomian gland, reflecting its elongation or shortening due to disease. 
- Meibomian gland width: The horizontal width of individual meibomian glands, 
which helps assess gland atrophy or hypertrophy. 

Version2 Date 20241201 
 
 18 
- Meibomian gland diameter deformation index: A metric for measuring irregularities 
in the shape and thickness of the glands, indicating glandular structural 
deformation. 
- Meibomian gland tortuosity index: A measure of the curvature or twisting of the 
meibomian glands, used to assess changes in gland architecture. 
Meibomian gland signal index: The intensity or brightness of the meibomian gland signal in 
meibography images, indicating gland density and activity 
Visiting schedule:  
1st visit       - Before treatment within 0-14 days (1st Data correction+Epilation)  
2nd-5th visit  - 1st – 4th week  
: Treatment session 1 per week x 4 weeks 
 
 
- 8th week   
 
: 1 month after treatment (2nd Data correction+Epilation) 
6th visit  
- 16th week   
: 3 months after treatment(3rd  Data correction+Epilation) 
 
All data will be collected using Microsoft Excel version 16.65 (Microsoft Inc, USA) and analyzed 
using SPSS Statistics. 
Informed consent process: The study includes an informed consent (Board consent) process 
that allows the use of research data for future studies without the need for additional consent. . 
Data will be collected after this process and recorded by the investigator in a manner that subjects 
cannot be identified 
 
Log book:  
Provide a paper logbook to all participants to record compliance and prevent any potential co-
intervention by patients or caregivers. 
 
11. Data Analysis and Statistics 
Sampling technique and Sample size: N=22 in each arm by  
1-Way ANOVA Pairwise, 2-Sided Equality 
 
𝑛= 2(σ
 "!" #
$%
#"!"&
$'%$(
)2 

Version2 Date 20241201 
 
 19 
1−β=Φ(z−z1−α/(2τ))+Φ(−z−z1−α/(2τ) ) , 𝑧=
$'%$(
&'$
)
 
𝑛= 2(3.56
 "!" +.+-
$%
#"!"&
(.*+%*.,- )2 
1−β=Φ(z−z1−α/(2τ))+Φ(−z−z1−α/(2τ) ) , 𝑧=
(.*+%*.,-
(.,.'$
)
 
 
n = Sample size (per pair) = 36, Each arm = 18 , 20% drop out = 21.6 = 22 per arm  
Φ  = the standard Normal distribution function 
Φ −1 = the standard Normal quantile function 
a = Type I error,  Significance level = 5% 
β = Type II error, Power (1 – β): 80%  
τ  = the number of comparisons to be made = 6 
Relative difference (δ) = 50% (or 0.50) by our expert opinion of minimal clinical significant 
difference  
From previous study(28) 
Standard deviation (σ)=  Assume an estimated value based on available data or literature (28) 
= 3.56  
(SD Demodex count at baseline) 
uA = 3.18 
uB = 1.59 
 
Statistical analysis, plan, and data management  
Demographic and baseline clinical characteristics will be presented as percentages for 
categorical data and as mean with standard deviation or median with interquartile range (IQR) for 
continuous data.  

Version2 Date 20241201 
 
 20 
For outcome ,Continuous data : Meibomian gland area ratio, Meibomian gland length and 
width,Meibomian gland diameter deformation index ,Meibomian gland tortuosity index, 
Meibomian gland signal index should be analyzed using ANOVA, while binary outcomes and 
ordinal data Meibomian gland dysfunction staging (severity) should be assessed using the chi-
square test. 
Any unavailable data will be treated as missing and will not be included in any 
calculations. The report will specify the percentage of missing data in each category. Statistical 
significance will be determined by a p-value < 0.05. 
 
 
 
12.Ethical Consideration 
The submission of the research proposal to the Institutional Review Board on Human Research at 
the Faculty of Medicine, Chulalongkorn University marks a pivotal step in ensuring the ethical 
oversight and approval of the prospective Randomized Control Trial (RCT)  
Respect for Person: 
Informed Consent: The principle of respect for persons underscores the importance of obtaining 
informed consent. Ensuring that participants have a clear understanding of the study's purpose, 
procedures, potential risks, and benefits is crucial. 
Researchers employ transparent and comprehensive communication in the informed consent 
process, elucidating the randomization process, details of each intervention, and the freedom to 
withdraw at any point without repercussions. 
 
Researchers will provide comprehensive information to ensure that volunteers understand the 
research well and make independent decisions regarding participation, without coercion or undue 
influence. If volunteers have legal representatives or proxies, they will be the ones reviewing the 
informed consent document and providing witness signatures. Researchers will seek permission 
from the hospital director at Chulalongkorn Hospital and will anonymize data using codes without 
disclosing patients' names, surnames, or hospital identification numbers. 
 
 

Version2 Date 20241201 
 
 21 
Beneficence/Non-maleficence: 
Risk-Benefit Assessment: Ethical research necessitates a careful evaluation of potential benefits 
against risks. Researchers must strive to maximize the positive outcomes of the study while 
minimizing any harm to participants. 
Continuous monitoring of the interventions for unexpected risks and a commitment to modify or 
halt the trial if harms outweigh benefits are imperative to uphold the principles of beneficence 
and non-maleficence. 
 
Patients will benefit from receiving treatment for Demodex Blepharitis through eyelid scrubbing 
+ 1% TTO (tea tree oil) and eyelid scrubbing + Low-Level Blue Light Therapy (LLLT) + Intense 
Pulsed Light (IPL) performed by specialized ophthalmologists. Volunteers will not receive any 
other benefits from participating in the research. All trial procedures will be tested by researchers 
and the team beforehand to identify potential issues. Safety will be paramount during the 
experiment, with basic first aid procedures in place and guidelines for referring participants in case 
of injuries or adverse effects 
Justice: 
Equitable Participant Selection: Justice demands fairness in participant selection, avoiding bias 
or discrimination. All eligible individuals should have an equal opportunity to participate, 
promoting inclusivity. 
Researchers design the recruitment process to ensure diversity and representation across various 
demographics. The selection criteria based on scientific relevance, fostering inclusivity in the study 
population. 
 
This research has clear inclusion and exclusion criteria, equally distributes risks and benefits, and 
employs a stratified study group allocation method. 
 
 
Equitable Access to Benefits: The principle of justice extends to ensuring that the benefits 
resulting from the study are accessible to a broad population, preventing disparities in access to 
potential interventions. 

Version2 Date 20241201 
 
 22 
Researchers proactively consider the implications of the study outcomes and work towards 
facilitating equal access to any effective interventions emerging from the trial, minimizing 
healthcare disparities. 
 
Outcomes   
Primary Outcome: Mean of Demodex Count - the number of Demodex mites per eyelash on a 
total of 4 collarettes lashes epilation per one eye in 30 days after end of treatment. 
Secondary Outcomes: 
- Mean of Demodex Count - the number of Demodex mites per eyelash on a total of 4 
collarettes lashes epilation per one eye in 90 days after end of treatment. 
- MGD Stage 1-5. 
OSDI questionnaire used to evaluate ocular dry eye symptoms. 
Meibum Quality. 
Meibum Expressibility. 
Telangiectasia grading: 0-3  
- Surface Staining: NEI Grading System. 
- LIPIVIEW used to measure Lipid Layer Thickness. 
- The Keratograph 5M (OCULUS, Germany) used to measure noninvasive break-up time 
(NIBUT), tear meniscus height (TMH),  % Meibomian Gland Loss (MGL)  
13. Expected or Anticipated Benefit Gain 
This research marks a pioneering and robust investigation into demodex blepharitis 
globally, utilizing a meticulously designed study approach tailored to the Thai population.  
Aims to unveil unprecedented insights into the new less invasive , less session , better compliance 
optimal treatment modalities for demodex blepharitis. The expected benefits, applications, and 
utilization of the study findings are multifaceted, offering valuable contributions to both the 
scientific community and clinical practice. 
 
Contributes to the optimization of light base treatment strategies for demodex 
blepharitis, helping clinicians tailor interventions based on their relative efficacy. 
Provides a foundation for further research on the sustained effects of these interventions 
and informs the development of extended treatment protocols. 

Version2 Date 20241201 
 
 23 
Generates new knowledge that extends beyond demodex mite eradication, 
encompassing the broader landscape of ocular surface health, ethical considerations in clinical 
trials, and the application of innovative therapeutic modalities. This knowledge contributes to 
the ongoing evolution of demodex blepharitis and meibomian gland dysfunction management 
and sets the stage for further advancements in ocular surface research. 
 
14.Challenges and Limitations 
1. Limited Generalizability 
• Population Specificity: The study's focus on the Thai population may limit the 
generalizability of findings to broader demographic groups, making it challenging to apply 
results to populations with different genetic, environmental, and cultural backgrounds. 
2. Long-Term Effects and Follow-Up 
• Sustainability and Longevity: The relatively short follow-up period (90 days) may restrict 
the ability to capture long-term effects of the interventions. Extended longitudinal 
studies are necessary to assess treatment sustainability over time. 
3. Blinding Constraints 
• Blinding in Light-Based Interventions: Achieving complete blinding in light-based 
interventions (IPL and LLLT) is challenging, potentially introducing bias. The visible 
nature of these treatments makes it difficult to conceal allocation from both participants 
and investigators. 
4. Outcome Measure Subjectivity 
• Subjective Assessments: Reliance on patient-reported symptoms may introduce 
variability in study outcomes. While objective measures, such as Demodex mite count, 
offer a more standardized evaluation, they remain subject to interpretation. 
5. Adherence and Compliance 
• Patient Adherence: The effectiveness of interventions depends on patient adherence to 
treatment protocols. Challenges may arise if patients perceive the procedures as 
burdensome or experience difficulties maintaining compliance. 
6. Limitations of AI Technology 

Version2 Date 20241201 
 
 24 
• Algorithm Performance: AI-driven meibomian gland analysis is dependent on the quality 
of algorithms and training data. Variability in clinical conditions and diverse populations 
may impact the accuracy and effectiveness of AI models. 
7. Integrating AI Insights into Clinical Practice 
• Clinical Implementation: Translating AI-supported research findings into practical clinical 
guidelines and integrating them into routine practice requires extensive validation. 
Additional verification and refinement of study results may be necessary before AI-driven 
approaches can be widely adopted in clinical settings. 
 
15. Risk and Investigator’s Responsibility  
1. Patient Privacy Protection: 
Risk: The collection of sensitive health information necessitates the protection of patient privacy. 
Investigator’s Responsibility: The investigator must adhere to data protection regulations, 
implement robust anonymization practices, and establish secure data storage and transmission 
procedures to safeguard patient privacy. 
2. Participant Discomfort during Interventions: 
Risk: Participants undergoing Photobiomodulation (LLLT) and Intense Pulsed Light (IPL) therapies 
may experience mild discomfort or temporary side effects. 
Investigator’s Responsibility: The investigator must thoroughly explain potential discomfort during 
the informed consent process and promptly address any participant concerns or adverse 
reactions. 
3. Selective Reporting Bias: 
Risk: There is a risk of selective reporting bias. 
Investigator’s Responsibility: The investigator must commit to transparently reporting all 
outcomes, fostering an unbiased and comprehensive understanding of the interventions' efficacy. 
4. Bias and Accuracy in AI Analysis 
Risk: AI models may have inherent biases or limitations that affect the accuracy of meibomian 
gland analysis, potentially leading to incorrect diagnoses or assessments. 

Version2 Date 20241201 
 
 25 
Investigator’s Responsibility: Researchers must rigorously monitor and evaluate the performance 
of AI systems, actively identifying and addressing biases or errors to ensure the reliability and 
credibility of study outcomes. 
 
16. Timeline  
The research will take a total of 12 months to complete.The recruitment of volunteers to 
participate in the research will begin after the research protocol has been approved by the 
Research Ethics Committee and the Medical Faculty. 
 
17. Venue of the study  
Department of Ophthalmology, King Chulalongkorn Memorial Hospital 
 
18. Tabulation of Research Activities and Timeline 
Month  
1 
2 
3-4 
5-6 
7-8 
9-10 
11-12 
Research design  
  
  
  
  
  
 
  
Proposal 
writing 
& 
defense  
  
 
  
  
  
 
  
IRB submission  
  
  
  
  
  
 
  
Data collection  
  
  
  
  
  
 
  
Data analysis  
  
  
  
  
  
 
  
Manuscript writing  
  
  
  
  
  
 
 
Publication  
  
  
  
  
  
 
  
 
 
New Knowledges 
- Contributes to the optimization of light base treatment strategies for demodex blepharitis, 
helping clinicians tailor interventions based on their relative efficacy. 

Version2 Date 20241201 
 
 26 
- Provides a foundation for further research on the sustained effects of these interventions 
and informs the development of extended treatment protocols. 
- Generates new knowledge that extends beyond demodex mite eradication, encompassing 
the broader landscape of ocular surface health, ethical considerations in clinical trials, and 
the application of innovative therapeutic modalities. This knowledge contributes to the 
ongoing evolution of demodex blepharitis and meibomian gland dysfunction management 
and sets the stage for further advancements in ocular surface research. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Version2 Date 20241201 
 
 27 
 
 
20.References  
1. 
Kasetsuwan N, Kositphipat K, Busayarat M, Threekhan P, Preativatanyou K, Phumee A, 
et al. Prevalence of ocular demodicosis among patients at Tertiary Care Center, Bangkok, 
Thailand. Int J Ophthalmol. 2017;10(1):122-7. 
2. 
de Venecia Iii A, Siong RL. Demodex sp. infestation in anterior blepharitis, meibomian-
gland dysfunction, and mixed blepharitis. Philippine Journal of Ophthalmology. 2011;36(1). 
3. 
Cheng S, Zhang M, Chen H, Fan W, Huang Y. The correlation between the microstructure 
of meibomian glands and ocular Demodex infestation: A retrospective case-control study in a 
Chinese population. Medicine (Baltimore). 2019;98(19):e15595. 
4. 
Rhee MK, Yeu E, Barnett M, Rapuano CJ, Dhaliwal DK, Nichols KK, et al. Demodex 
Blepharitis: A Comprehensive Review of the Disease, Current Management, and Emerging 
Therapies. Eye Contact Lens. 2023;49(8):311-8. 
5. 
Syed YY. Lotilaner Ophthalmic Solution 0.25%: First Approval. Drugs. 2023;83(16):1537-
41. 
6. 
Koo H, Kim TH, Kim KW, Wee SW, Chun YS, Kim JC. Ocular surface discomfort and 
Demodex: effect of tea tree oil eyelid scrub in Demodex blepharitis. J Korean Med Sci. 
2012;27(12):1574-9. 
7. 
Alghamdi YA, Camp A, Feuer W, Karp CL, Wellik S, Galor A. Compliance and Subjective 
Patient Responses to Eyelid Hygiene. Eye Contact Lens. 2017;43(4):213-7. 
8. 
Kim WS, Calderhead RG. Is light-emitting diode phototherapy (LED-LLLT) really effective? 
Laser Ther. 2011;20(3):205-15. 
9. 
Whelan HT, Smits RL, Jr., Buchman EV, Whelan NT, Turner SG, Margolis DA, et al. Effect 
of NASA light-emitting diode irradiation on wound healing. J Clin Laser Med Surg. 
2001;19(6):305-14. 
10. 
Glass GE. Photobiomodulation: The Clinical Applications of Low-Level Light Therapy. 
Aesthet Surg J. 2021;41(6):723-38. 
11. 
Sułek A, Pucelik B, Kobielusz M, Barzowska A, Dąbrowski JM. Photodynamic Inactivation 
of Bacteria with Porphyrin Derivatives: Effect of Charge, Lipophilicity, ROS Generation, and 
Cellular Uptake on Their Biological Activity In Vitro. Int J Mol Sci. 2020;21(22). 
12. 
Avci P, Gupta A, Sadasivam M, Vecchio D, Pam Z, Pam N, et al. Low-level laser (light) 
therapy (LLLT) in skin: stimulating, healing, restoring. Semin Cutan Med Surg. 2013;32(1):41-52. 
13. 
Castro C, Marques JH, Marta A, Baptista PM, José D, Sousa P, et al. Comparison of Light-
Based Devices in the Treatment of Meibomian Gland Dysfunction. Cureus. 2023;15(7):e41386. 
14. 
Giannaccare G, Pellegrini M, Carnovale Scalzo G, Borselli M, Ceravolo D, Scorcia V. Low-
Level Light Therapy Versus Intense Pulsed Light for the Treatment of Meibomian Gland 
Dysfunction: Preliminary Results From a Prospective Randomized Comparative Study. Cornea. 
2023;42(2):141-4. 
15. 
Craig JP, Nichols KK, Akpek EK, Caffery B, Dua HS, Joo CK, et al. TFOS DEWS II Definition 
and Classification Report. Ocul Surf. 2017;15(3):276-83. 

Version2 Date 20241201 
 
 28 
16. 
Yu J, Asche CV, Fairchild CJ. The economic burden of dry eye disease in the United 
States: a decision tree analysis. Cornea. 2011;30(4):379-87. 
17. 
McDonald M, Patel DA, Keith MS, Snedecor SJ. Economic and Humanistic Burden of Dry 
Eye Disease in Europe, North America, and Asia: A Systematic Literature Review. Ocul Surf. 
2016;14(2):144-67. 
18. 
Xiao P, Luo Z, Deng Y, Wang G, Yuan J. An automated and multiparametric algorithm for 
objective analysis of meibography images. Quant Imaging Med Surg. 2021;11(4):1586-99. 
19. 
Cheng AMS, Sheha H, Tseng SCG. Recent advances on ocular Demodex infestation. 
Current Opinion in Ophthalmology. 2015;26(4):295-300. 
20. 
Nicholls SG, Oakley CL, Tan A, Vote BJ. Demodex species in human ocular disease: new 
clinicopathological aspects. International Ophthalmology. 2017;37(1):303-12. 
21. 
Zhang X, Song N, Gong L. Therapeutic Effect of Intense Pulsed Light on Ocular 
Demodicosis. Curr Eye Res. 2019;44(3):250-6. 
22. 
Gao Y-Y, Di Pascuale MA, Li W, Liu DT-S, Baradaran-Rafii A, Elizondo A, et al. High 
Prevalence of Demodex in Eyelashes with Cylindrical Dandruff. Investigative Ophthalmology & 
Visual Science. 2005;46(9):3089-94. 
23. 
Bitton E, Wittich W. Double-Blind Comparison of Eyelash Epilation Techniques for the 
Purpose of Demodex folliculorum Mite Retrieval. Eye Contact Lens. 2024;50(3):152-7. 
24. 
Talha M, Haris Ali M, Fatima E, Nadeem A, Ahmed A, Nashwan AJ. Efficacy and Safety of 
Lotilaner Ophthalmic Solution (0.25%) for the Treatment of Demodex Blepharitis: A GRADE 
Assessed Systematic Review and Meta-Analysis of Observational & Experimental Studies. Am J 
Ophthalmol. 2024;264:8-16. 
25. 
Zhang Z, Wang Y, Zhang H, Samusak A, Rao H, Xiao C, et al. Artificial intelligence-assisted 
diagnosis of ocular surface diseases. Front Cell Dev Biol. 2023;11:1133680. 
26. 
Wang J, Li S, Yeh TN, Chakraborty R, Graham AD, Yu SX, et al. Quantifying Meibomian 
Gland Morphology Using Artificial Intelligence. Optom Vis Sci. 2021;98(9):1094-103. 
27. 
Tomlinson A, Bron AJ, Korb DR, Amano S, Paugh JR, Pearce EI, et al. The international 
workshop on meibomian gland dysfunction: report of the diagnosis subcommittee. Invest 
Ophthalmol Vis Sci. 2011;52(4):2006-49. 
28. 
Huo Y, Mo Y, Wu Y, Fang F, Jin X. Therapeutic effect of intense pulsed light with optimal 
pulse technology on meibomian gland dysfunction with and without ocular Demodex 
infestation. Ann Transl Med. 2021;9(3):238. 
 
 
 
 
 
 
 
 

Version2 Date 20241201 
 
 29 
 
Staging the Severity of MGD and Individual Clinical Parameters
Severity Level
Level 0
Normal
Level 1 Subclinical
Level 2 Symptomatic Minimal
Level 3 Symptomatic Mild
Level 4 Symptomatic Moderate
Level 5 Symptomatic Severe
Symptom frequency and
severity
No
symptoms
Asymptomatic or occasional
symptoms
Some of the time; precipitated by
environmental factors
Half of the time; some limitation
of activity
Most of the time; frequent limitation of
activity
All of the time Severe/disabling/constant
OSDI grade range (0–100)
0
0–12
0–12
13–22
23–32
33–100
MGD Grade
Clear
Subclinical, nonobvious MGD; altered
quality, only on expression; no gland
loss
Minimally altered quality of expressed
meibum from scattered glands; None to
minor gland loss
Mildly altered meibum quality;
occasional lid margin signs; mild
gland loss
Moderately increased opacity and
viscosity of meibum; plugging; increased
marginal vascularity; loss of oriﬁce
deﬁnition; moderate gland loss
Marked, diﬀuse MGD; cicatricial or
noncicatricial; multiple lid margin signs; lid
deformity and marked lid margin hyperaemia;
Severe gland loss
Quality of expressed meibum
grade range 0–3, LL, 8 glands,
Range (0–24)
0
1–5
6–10
11–15
16–20
21–24
Treatment of MGD based on
symptoms and gland status
+ General advice about MGD, the
potential inﬂuence of diet, home and
work environment
+ Hygienic measures, heat and massage
±Topical ATs
+Oral tetracycline derivatives
± Anti-inﬂammatories
±Hygienic measures
± Emollient lubricant or
liposomal spray
± Topical azithromycin
± Consider oral tetracycline
derivatives
This table should be read in conjunction with Table 11, which provides a staging scheme for MGD-related ocular surface disease. Severity levels for each parameter are graded 1–5. A subclinical severity level has been introduced to accommodate asymptomatic MGD with normal lid mar‐
gin features (nonobvious MGD) diagnosed only after gland expression. Note that this MGD scoring system does not provide a score for totally obstructed glands. Alternative systems for grading MGD exist and should be considered (see Appendices 5–7). Arita et al.  graded meibomian
dropout in the combined upper and lower lids, using noninvasive meibography, with a scale range of 0–6 (Table 3). de Paiva et al.
 used a composite system combining dropout, lid signs and meibum expressibility, with a scale range of 0–11 (Table 3). Korb and Blackie  record the num‐
ber of glands in a zone of 8, which yield a liquid secretion after standardized expression (MGYLS score 0–8). General treatment concepts, summarized here, are adapted from the Report on Management and Therapy. Recommended treatments are additive. At each clinical assessment, lack
of response to treatment at the previous level moves treatment to the next level. ±, the decision to use this treatment is based on clinical judgment; +, treatment is recommended at this level. LL, lower lid; OSDI, Ocular Surface Disease Index.
The increase in severity of MGD with increase in grade is denoted by a reduced quality of expressed meibum. Meibum quality (clarity and consistency) is assessed in eight glands of the central third of the lower lid on a 0–3 scale for each gland: 0 = clear; 1 = cloudy; 2 = cloudy with
debris; 3 = thick, like toothpaste (total score range 0–24).
*
3
106
52
*
75
Figure1