Efficacy of Terpinen-4-ol Combined With Eyelid Deep Cleaning for the Treatment of Demodex Blepharitis: A Randomized, Open-Label Trial

Abstract

Purpose: We aimed to evaluate the clinical efficacy of a combined treatment regimen involving terpinen-4-ol (T4O) and eyelid deep cleaning for managing Demodex blepharitis.

Methods: In this randomized, open-label trial, 40 patients diagnosed with Demodex blepharitis at the Cornea Specialty Clinic of Hankou Aier Eye Hospital were enrolled. Participants were randomly assigned to either a T4O or a combination treatment group. The T4O group used T4O cleaning wipes exclusively for two months. The combination group used T4O cleaning wipes and underwent deep eyelid cleaning at three scheduled visits (initial, first-, and second-month time points). A final follow-up was conducted one month after completion of treatment (three months after the beginning of treatment). At each visit, we assessed the number of Demodex mites, the ocular surface disease index (OSDI), eyelid margin sign scores, and corneal fluorescein staining scores.

Results: After two months of treatment, both the T4O and combination groups exhibited significant reductions in Demodex mite counts of 5.65 ± 6.03 and 7.75 ± 6.91, respectively. The T4O group, however, showed an increase in mite counts one month after ceasing treatment. Conversely, the combination group maintained and enhanced the reduction in mite counts post-treatment, with a significant difference at the third-month follow-up (8.70 ± 6.81 vs. 4.15 ± 6.91, P = 0.043). Although both groups demonstrated overall declines in OSDI scores post-treatment, only the combination group showed significant improvements at the second- and third-month follow-ups. No adverse events were reported in either group.

Conclusions: Treatment with T4O wipes for two months significantly reduces Demodex mite counts, although there is a notable risk of mite rebound when treatment is ceased. Combining T4O wipes and eyelid deep cleaning offers a more effective reduction in Demodex mite counts and improvement in ocular symptoms compared to T4O treatment alone.

Translational Relevance: The study finds that T4O combined with eyelid deep cleaning is more effective in treating Demodex blepharitis, highlighting the need for appropriate treatment duration and adjunctive therapies.

Introduction

Demodex blepharitis is a chronic and recurrent inflammation of the skin and mucous membranes of the eyelid margin caused by Demodex infestation, involving the follicles of the eyelashes and the meibomian glands.¹ Demodex blepharitis can lead to meibomian gland dysfunction, resulting in abnormal quantity or quality of meibum secretion. This, in turn, can disrupt the normal composition of the tear film lipid layer, leading to the development of dry eye syndrome. In severe cases, the condition may extend to the conjunctiva and even the cornea, causing significant impairment of visual function.² Therefore timely and effective treatment of Demodex blepharitis is crucial to protect eye health. 

Current treatment methods for Demodex blepharitis include topical acaricidal medication and physical therapies. Topical acaricidal medications are commonly used, such as terpinen-4-ol (T4O), tea tree oil (TTO), and aureomycin. These medications have been shown to effectively eradicate Demodex mites on the eyelids.³ Topical TTO is associated with known side effects, including contact dermatitis, ocular irritation, and allergic reactions.^4,5 Extensive research has demonstrated that T4O, the active component of TTO, effectively eliminates Demodex mites.^6,7 A concentration of 1% of T4O has been proven sufficient for efficacy.^3,8,9 However, in vitro studies indicate that T4O may be toxic to human meibomian gland epithelial cells.¹⁰ Thus determining the appropriate duration and frequency of T4O treatment is essential for ensuring both effectiveness and safety in treating Demodex blepharitis. In addition, physical modalities such as eyelid deep cleaning, eyelid warm compress, massage, and intense pulsed light therapy are beneficial for removing foreign bodies or debris from the base of the eyelashes, improving blood circulation in the meibomian glands, softening and eliminating meibum secretions. Among these, eyelid deep cleaning, primarily performed with an electric brush head, is a novel method for thoroughly cleaning the eyelids and the base of the eyelashes. The electric brush head can effectively remove eyelid scales and keratinized epithelial tissue, eliminate the biofilm on the eyelids, and restore the balance of the ocular surface microenvironment. The biofilm provides a barrier defense mechanism for microbial colonies.¹¹ Epstein et al. found that the use of eyelid deep cleaning in combination with T4O significantly reduces the level of microbial colonization in eyelash follicles.¹² Ultimately, these treatments aim to restore the balance of ocular ecology.¹³ 

The optimal treatment approach for Demodex blepharitis continues to be a significant topic of debate among experts in the field.¹ Recent research indicates that eyelid deep cleaning, when used with T4O wipes or control wipes, can diminish Demodex mite counts on the eyelids after one month. However, using T4O alone does not significantly reduce eyelid mite counts compared to controls.¹² The individual or combined effects of deep cleaning and T4O products, as well as the optimal treatment duration for Demodex blepharitis, are still not fully understood. This study assesses the clinical efficacy of T4O wipes used alone or in combination with eyelid deep cleaning over two months through a randomized, open-label trial. The findings of this study may provide critical real world evidence in support of the development of effective treatment protocols for Demodex blepharitis. 

Methods

Participants

In this randomized controlled trial, we recruited 40 patients with Demodex blepharitis who met the inclusion criteria at the Cornea Specialty Clinic of Hankou Aier Eye Hospital from March 5, 2023, to April 30, 2023. The inclusion criteria were as follows: (1) age ≥18 years; (2) eye discomfort with symptoms such as eye redness, itching, foreign body sensation, and recurrent, treatment-resistant chalazion; (3) abnormal eyelashes, with greasy cuff-like secretions at the base of the eyelashes, accompanied by eyelid redness and thickening; and (4) positive Demodex examination, defined as a count of at least three mites/three eyelashes in any one of the four eyelid margins confirmed by confocal laser scanning microscopy examination. 

The exclusion criteria were as follows: (1) history of eye surgery or infection within six months; (2) allergy to the cleansing solution or T4O; (3) inability to understand or comply with the treatment; (4) patients with severe systemic diseases who were intolerant to treatment; and (5) patients who were pregnant or breastfeeding. 

This study was approved by the Ethics Committee of Hankou Aier Eye Hospital (Approval No.: HKAIER2023IRB-001-01) and registered on chictr.org.cn with the clinical trial registration number ChiCTR2300076443. All patients were provided with informed consent, and the study adheres to the principles of the Helsinki Declaration. 

Study Design

The overall workflow of this study was showed in Figure 1. Eligibility for inclusion was based on medical history, slit-lamp biomicroscopy, and confocal microscopy examination results. Enrolled patients were then randomly divided into two groups equally using a random number table: a T4O group and a combination group, each consisting of 20 patients. Although both eyes were treated, only the right eye of each patients was selected as the study eye. 

Figure 1.

 

Treatment and follow-up protocol for each group. Each patient was treated with T4O twice daily for two months. The T4O + DC group underwent three DC treatment sessions at one-month intervals. Both groups were subjected to clinical assessment before treatment at each visit and one month after the final treatment. Examinations included (1) total Demodex load; (2) eyelid margin condition score; (3) OSDI; (4) corneal fluorescein staining score; and (5) ocular surface signs. DC, eyelid deep cleaning.

The T4O group received treatment with T4O cleaning wipes (OCUFACE; Guangzhou Ocuface Biotechnology Co., Ltd., Guangzhou, China). Patients were instructed to clean their eyelid margins twice daily for two months. The combination group followed the same regimen using T4O cleaning wipes, augmented by monthly deep eyelid cleaning at the initial visit, one and two months post-enrollment, totaling three sessions. Both groups underwent a treatment period of two months. Follow-up examinations were conducted at baseline, one month, and two months after enrollment, and one month after discontinuation of treatment. 

The procedure for deep eyelid cleaning was completed as follows: An electric handle and disposable sponge brush (OCU-001, OCUFACE; Guangzhou Ocuface Biotechnology Co., Ltd.) were assembled. Patients were instructed to lie flat on the treatment bed. Eyelid cleansing solution (0.01% hypochlorous acid solution, OCUFACE; Guangzhou Ocuface Biotechnology Co., Ltd.) was sprayed onto the sponge brush, allowing time for even and thorough absorption of the solution. The eyelid was then adequately exposed, and the brush was positioned perpendicular to the eyelid so that the eyelid margin and roots of the eyelashes could be brushed three times, moving from the inner to the outer canthus. The speed of the brush could be adjusted based on the degree of accumulated eyelid debris. This was then repeated for the contralateral eyelid, using a new cleaning brush each time. After the procedure, the conjunctival sac was rinsed with saline solution, and a slit-lamp examination was performed each time to check for corneal damage or other complications. 

Assessments

Examinations were conducted to evaluate the efficacy and safety of the treatments. After enrollment, the doctors and subjects were blinded during each examination, and the blinding was not revealed until the final analysis of the results. At each process, the examination was done before the treatment. 

Mite Quantification

Eight eyelash follicles on the central upper eyelid were examined using confocal microscopy. Images were taken to document mite infestation in each follicle, and mite counts were performed. The severity of mite infestation per eye was calculated by summing the counts from the eight eyelash follicles.¹⁴ Such assessment was repeated at each follow-up visit. 

Scoring for Eyelid Margin Condition

The condition of the eyelid margin was assessed based on a previous publication.¹⁵ The status of eyelid margin was evaluated at each follow-up visit. This assessment included grading the degree of eyelid margin hyperemia (0–3 points), abnormalities at the meibomian gland orifice (0–3 points), irregularity (0–2 points), thickening (0–2 points), and the characteristics of meibum secretion (0–3 points). 

Ocular Surface Disease Index (OSDI)

The OSDI questionnaire was utilized,¹⁶ consisting of 12 questions, with a total score ranging from 0 to 100. According to the scoring criteria, scores of ≤20 indicate mild symptoms, 21 to 45 indicate moderate symptoms, and scores >46 indicate severe symptoms. This assessment was performed at each follow-up visit. 

Corneal Fluorescein Staining Scoring

Corneas were stained using moistened fluorescein sodium test strips at each follow-up visit. The cornea was divided into four quadrants under slit-lamp microscopy. Each quadrant was scored on a scale of 0 to 3. Corneal staining was evaluated based on the number, morphology, and distribution of staining spots. The scoring criteria included the following: 0 points indicated no staining spots, 1 point indicated one to five staining spots, 2 points indicated six to 30 non-coalescent staining spots, and 3 points indicated more than 30 staining spots with coalescence or the presence of filamentous material adhering to the cornea. The maximum score for corneal staining in a single eye was 12.¹⁷ 

Ocular Surface Signs

Before treatment and at the second month of follow-up, we measured noninvasive tear break-up time, noninvasive tear meniscus height, meibography, and ocular redness analysis using the ocular surface analyzer (Oculus Keratograph M5; Oculus, Wetzlar, Germany). 

Statistical Analysis

The Shapiro-Wilk test was used to assess the normality of quantitative data, such as age. For continuous data after a normal distribution, results were presented as mean ± standard deviation. Non-normally distributed data were represented by the median and quartiles. Categorical data, such as gender, were shown as frequency and percentage. Independent sample t-tests were utilized for intergroup comparisons of normally distributed continuous data, whereas paired sample t-tests were used for within-group comparisons before and after treatment. Wilcoxon rank-sum tests or paired rank-sum tests were applied for other comparisons. The χ² tests or Fisher's exact tests were used for categorical data, depending on the expected count. The significance threshold was set at P < 0.05 for two-tailed tests. Given the inherent consistency between both eyes, this study only focused on right-eye data. Statistical analyses were conducted using R software (version 4.2.2). 

Sample Size Calculation

To satisfy the requirement of achieving a confidence level of over 80% and setting the significance level at 0.05 for a two-tailed test, sample size estimation was performed using PASS11 software. It was determined that each group would require at least 19 subjects.¹⁸ 

Results

Characteristics of Participants

This study enrolled 40 patients, with 6 males and 14 females in the T4O group and five males and 15 females in the combination group. The median age of the two groups was 32 and 34.5 years, respectively. There were no significant differences in age (P = 0.579) or gender distribution (P = 1.000) between the two groups. Before treatment, the number of Demodex mites in the T4O group was 12.0 (7.00, 19.2) and 14.0 (8.75, 24.0) in the combination group. However, this difference was not statistically significant (P = 0.323; Table 1). 

Table 1.

 

Baseline Characteristics

 

Comparison of Treatment Efficacy Between Groups

We subtracted post-treatment values from baseline values for each time point in both groups to compare various prognostic indicators before and after treatment. Our results showed that at the third-month follow-up visit, average reduction in Demodex mite counts in the T4O group was 4.15 ± 6.91, and 8.70 ± 6.81 (P = 0.043) in the combination treatment group. At the first month follow-up, the combination group showed a greater decrease in eyelid margin signs than the T4O group (P = 0.008). Other parameters such as corneal fluorescein staining grade, noninvasive tear break-up time, noninvasive tear meniscus height, ocular redness index, and OSDI showed no significant intergroup differences (P > 0.05; Table 2). 

Table 2.

 

Comparison of the Change of Prognostic Indicators Between the T4O Group and Combination Group

 

Intragroup Comparisons of Demodex Mites

The overall trend in the number of mites decreased in both groups (Fig. 2). In the T4O group, the number of mites significantly decreased after two months of treatment but returned to baseline one month after treatment was ended. In the combination group, the number of mites decreased significantly compared to baseline at all follow-up visits (Fig. 3). 

Figure 2.

 

Mean changes in the mite counts in the T4O and combination group. The yellow line and points indicate the T4O group. The black line and points indicate the combination group. DC, deep cleaning.

Figure 3.

 

Comparison of mite counts at each follow-up point in the T4O group (A) and combination group (B). ns, non-significant; *P < 0.05; **P < 0.01.

Intragroup Comparisons of OSDI

There was an overall trend of reduction in OSDI scores in both groups (Fig. 4). In the T4O group, although a slight decrease in OSDI was noted after treatment, the difference at each follow-up point was not significant compared to baseline. In the combination group, OSDI scores were significantly lower than baseline at the second- and third-month follow-up visits (Fig. 5). 

Figure 4.

 

Mean changes in the OSDI in the T4O group and combination group. The yellow line and points indicate the T4O group. The black line and points indicate the combination group. DC, deep cleaning.

Figure 5.

 

Comparison of OSDI at each follow-up point in T4O group (A) and combination group (B). ns, non-significant; *P < 0.05; **P < 0.01.

Figure 6.

 

The appearance and counting of mites under confocal microscope examination.

Additional Indicator Comparisons

During the follow-up period, both groups showed a trend of reduction in eyelid margin condition scores after treatment, but the differences at each time point were not significant compared to baseline. There were no significant differences in tear meniscus height, ocular redness index, NIBUT, or meibomian gland scores before and after treatment. Neither group of patients experienced complications such as eye redness, eye pain, itching, foreign body sensation, or corneal damage during the follow-up period. 

Discussion

Previous studies have found that 84% of patients with ocular discomfort have Demodex infestation, and the number of Demodex mites significantly correlates with OSDI scores.⁴ Although T4O treatment is effective in Demodex blepharitis, there is no consensus on the optimal protocol.¹ The recurrence of Demodex blepharitis is still common in clinical practice. Our study shows that the duration of T4O treatment requires a minimum of two months. Furthermore, T4O, in combination with eyelid deep cleaning, is necessary to enhance the clearance of Demodex mites and prevent rebound in one month. 

Considering that the lifecycle of Demodex mites from egg to adult is approximately 14–18 days, treatment aiming to clear mites should cover at least two of these lifecycles. In previous studies, the duration of T4O or TTO treatment was generally one month.^13,19 Jacobi et al.⁷ used 2.5% concentration of T4O wipes for eyelid cleansing over four weeks, followed by 0.2% hyaluronic acid wipes for the next four weeks to sustain eyelid hygiene and prevent mite proliferation and symptom recurrence. Demodex mite counts were noted to decrease (baseline vs. fourth week vs. eighth week = 3.2 vs. 1.6 vs. 0.9), and both products were well tolerated. In addition, Arici et al.²⁰ performed a clinical trial using T4O wipes for daily lid scrubbing twice a day for eight weeks. Their results showed that patients with 100% initial Demodex infestation had a 29.2% and 4.2% decrease after four and eight weeks of T4O treatment, respectively. The results of the above studies suggest that a four-week treatment with T4O may reduce Demodex mite counts, but a longer treatment duration may achieve better results. Therefore T4O wipes were used for two months in our current study. Consistent with previous studies, the decrease in mite counts was more significant in the second month than in the first. Additionally, at one month after treatment cessation, patients showed a rebound in mite counts in the T4O group. 

The frequent rebound in mite counts indicates that the occurrence of Demodex blepharitis is a complex, multifactorial pathological process, often accompanied by bacterial infections, immunological dysfunction, meibomian gland dysfunction, unstable tear film, and an imbalance in the ocular surface microenvironment.²¹ Topical medications alone have not been able to fully eliminate Demodex mites. The changes in mite counts observed in our study align with findings from Epstein's study, which suggested that Demodex infestation in patients with Demodex blepharitis is often challenging to eradicate, and a two-month treatment duration with T4O may be more effective in reducing mite counts.¹² However, the result of our study indicates that T4O treatment alone is insufficient, and alternative treatments are needed. 

The results of our study support the superiority of combination therapy in terms of Demodex eradication and symptom improvement. Our results indicate that eyelid deep cleaning in combination with T4O can effectively enhance the ability to eliminate Demodex mites, improve the condition of the eyelids, and provide sustained relief of symptoms. There are several possible reasons for this finding. First, eyelid deep cleaning can remove harmful substances such as scales, secretions, and keratinized epithelium from the eyelid margin, reducing irritation and blockage of meibomian gland orifaces.²² Second, deep cleaning can promote blood circulation in the eyelid margin, thereby improving meibomian gland function and additionally facilitating the penetration and absorption of T4O.¹³ Most importantly, deep cleaning effectively inhibits the growth of pathogenic micro-organisms such as bacteria by removing the biofilm, thereby improving the ocular surface microenvironment. However, there is no specific guideline on the duration of the eyelid cleaning procedure, as well as the treatment span. To our knowledge, no articles have been published comparing the efficacy of eyelid deep cleaning alone with other therapies for mite elimination. A previous study¹³ showed that a single eyelid deep cleaning combined with nightly use of lid scrubs was better than nightly use of lid scrubs in mite reduction in two weeks follow-up, but with the continuous use of lid scrubs for four weeks the effects were comparable. None of the methods completely eradicated mites in all subjects. This showed that a single deep cleaning could reduce the number of mites in two to four weeks, but the effect of mite removal was limited. The reason could be that although a single cleaning could remove bacteria and mites on the eyelid margin, the action time was short and has minimal impact on mites in the hair follicles, and so these mites could gradually move up to the eyelid margin. Therefore the combined treatment in our study was designed to treat once a month with three deep cleanings combined with T4O wipes for two months to achieve better treatment outcomes. 

In this study, no complications such as eye redness, eye pain, itching, foreign body sensation, or corneal damage were observed in any patients. This demonstrates that eyelid deep cleaning is a safe and effective treatment method. When combined with T4O wipes, it can have a synergistic effect in treating Demodex blepharitis. In this study, we did not use foam cleansers or baby wash for deep cleaning as was done in previous studies¹²; we used hypochlorous acid instead. Hypochlorous acid (HOCL) can effectively kill pathogenic micro-organisms, including bacteria, and reduce the number of mites.⁴ Stroman et al.²³ used a 0.01% HOCL to clean the eyelid margin and observed a decrease of over 90% in the load of various bacterial populations after 20 minutes. It significantly reduced bacterial burden without altering the diversity of bacterial species and did not result in significant adverse reactions. As for mite reduction, Li et al.²⁴ used 0.01% HOCL for 20 minutes every five days through ultrasonic atomization around the eyes, and the average survival time of mites in the HOCL group was 75.25 minutes after 55 days, shorter than that in the placebo group. This indicates that 0.01% HOCL can reduce the survival time of adult Demodex mites, but its killing effect is limited with 20-minute contact time. In our study, with an even shorter contact time of five minutes, such effect should be weaker, and it is even less likely to affect mites in other life cycle stages inside the hair follicles. It is known that T4O has its own acaricidal effect. The combination of HOCL and T4O may have a synergistic effect to some extent. For example, HOCL can help clean the lid margin and reduce the number of mites and bacteria on the surface, creating a better environment for T4O to act on the remaining mites. Additionally, the frequency of our deep cleaning treatment is once a month. This relatively long interval means that new mites may still appear and reproduce during this period, so it is impossible to achieve complete mite removal through HOCL alone or a synergistic effect. 

In contrast to previous studies, this study utilized confocal microscopy to confirm the quantity of Demodex mites (Fig. 6). Randon et al. reported infection rates of Demodex mites in patients with anterior blepharitis, dry eye without blepharitis, and healthy individuals as 100%, 60%, and 12%, respectively, using confocal microscopy, which are significantly higher than those obtained using the epilation method (100%, 50%, and 0%).¹⁴ Compared to examining epilated eyelashes under a light microscope, confocal microscopy enables not only accurate detection and counting of multiple hair follicles in a live state, but also the detection of Demodex mites in follicles without eyelashes.²⁵ This technique improves patient compliance, especially in cases requiring multiple examinations to assess the change in mite population. Higher detection rate and the inclusion of eight hair follicles on the upper eyelid in each eye, contributed to higher quantity of mites in our study than most previous studies,¹⁴ thus providing a more representative assessment of Demodex mite infection comparing to sampling three or four epilated eyelashes in one eyelid. 

There are several limitations in this study. First, the sample size was relatively small, and the follow-up period was short, which limited our ability to assess the long-term efficacy and recurrence rates of Demodex mite infection. Additionally, this study did not investigate the effectiveness of different types and concentrations of T4O products in mite elimination, nor did it compare the impact of various frequencies of eyelid deep cleaning. Therefore future research is necessary with an extended follow-up period, to optimize treatment protocols and to explore additional ocular surface indicators related to Demodex mite infection. A comprehensive approach will enable a more thorough and effective evaluation of the treatment efficacy and clinical significance of Demodex blepharitis. 

In conclusion, including eyelid deep cleaning in the treatment protocol significantly enhances the therapeutic efficacy of T4O for Demodex blepharitis. Future research should further explore the optimal adjunctive medications and frequency of eyelid deep cleaning. 

Acknowledgments

Supported by the scientific research project of the Clinical Research Institute of Aier Eye Hospital Group (No. AF2204D04). 

Author Contributions: Hao-Yu Wang conceived the study and was the major contributor in design and coordination, in collecting data, and in writing the manuscript. Dan shen and Meng-Ying Qi helped to collect the data. Chen Qiao and Lan Ke made the statistical analysis. Qing-Yan Zeng contributed in its design and coordination, and made the analysis and interpretation of data. All authors have read and approved the final manuscript. 

Disclosure: H.-Y. Wang, None; D. Shen, None; M.-Y. Qi, None; C. Qiao, None; L. Ke, None; M. Wang, None; Q.-Y. Zeng, None