The association of demodex infestation with pediatric chalazia

Purpose This study aimed to investigate the association of Demodex infestation with pediatric chalazia. Methods In a prospective study, 446 children with chalazia and 50 children with non-inflammatory eye disease (controls) who underwent surgical treatment were enrolled from December 2018 to December 2019. Patient ages ranged from 7 months to 13 years old. All patients underwent eyelash sampling for light microscope examination, and statistical correlation analysis between Demodex infestation and chalazia, including the occurrence, recurrence, and course of disease, morphological characteristics, and meibomian gland dysfunction (MGD) in chalazia patients was performed. Results Demodex was found in 236 (52.91%) patients with chalazia and zero control patients. Demodicosis was significantly more prevalent in chalazia patients than the control group (P < 1 × 10− 14). Recurrent chalazia (P = 0.006) and skin surface involvement (P = 0.029) were highly correlated with Demodex infestation. Demodicosis was also associated with multiple chalazia (P = .023) and MGD(P = .024). However, Demodex infestation was comparable in the course of disease (P = 0.15), seasonal change (P = 0.68) and blepharitis subgroups (P = 0.15). Within the group of chalazia patients who underwent surgical removal of cysts, 4 (0.9%) patients with concurrent demodicosis experienced recurrence. Conclusions Demodex infestation was more prevalent in pediatric chalazia patients than healthy children, and was associated with recurrent and multiple chalazia. Demodicosis should be considered as a risk factor of chalazia. In children with chalazia, Demodex examination and comprehensive treatment of Demodex mites should be applied to potentially prevent recurrence.


Introduction
Chalazia is a common lid disease that is characterized by chronic granulomatous inflammation of meibomian glands, and is prone to recurrence. Occasionally, cysts on the skin surface may spontaneously rupture, leaving obvious scarring. Preventing recurrence and reducing treatment costs remains challenging. Previous studies have found that chronic conjunctivitis, blepharitis, excessive secretion from sebaceous or sweat glands, and vitamin A deficiency are common causes of chalazia, but controlling these conditions did not reduce the incidence or recurrence of chalazia [1,2]. In recent years, the pathogenicity of ocular demodicosis was emphasized, with studies indicating that Demodex significantly impacted the onset of anterior blepharitis, including refractory and scaly blepharitis [3,4]. Posterior blepharitis is associated with the meibomian glands, but has not been the focus of research. We aimed to analyze the association of Demodex infestation with chalazia in pediatric patients.

Patients
This study was approved by the ethics committee of Guangzhou Women and Children's Medical Center and all methods in this research were performed in accordance with the relevant guidelines and regulations. In this Prospective, observational, comparative designed study, all patients were randomly selected, and informed consent was obtained from their parents/guardians prior to enrollment. The study consisted of 496 pediatric patients with ocular diseases who underwent surgery between December 2018 and December 2019. The average age was 3.36 ± 1.61 years (range, 7 months to 13 years). The patients were divided into a study group of 446 chalazia patients, and an age-and gender-matched control group of 50 patients who underwent surgery for strabismus or orbital dermoid cysts (38 and 12 cases, respectively). Inclusion criteria were age under 14 years, and no acute inflammation, to avoid potential microbial infections. Patients taking immunosuppressants, and those with rosacea based on medical history and clinical signs, were excluded because of the potential association with Demodex infection [5][6][7].

Eyelash sampling and microscopic Demodex examination
Eyelash sampling and microscopic Demodex examination were performed as previously described [8][9][10][11]. In brief, 3 lashes were epilated from each eyelid under general anesthesia before surgery. Eyelashes located near meibomian gland cysts or where cylindrical dandruff was visible, and where meibomian glands were dilated were prioritized to increase the chance of detection. The eyelashes were placed separately on a glass slide and mounted with a coverslip; 1 drop of saline solution was applied to the edge of the coverslip before microscopic examination. All samples were taken by the same operator and Demodex detection was performed by an independent masked technician who had no knowledge about each patient's clinical information. At least one Demodex mite (including adult, larva, nymph or egg) in a 100X magnified field was considered a positive result (Fig. 1).

Treatment of Demodex infestation and follow-up
Considering the biological pathogenicity and the contagiousness of Demodex, the children with demodicosis were recommended for treatment in both eyes. The comprehensive treatment for demodicosis was initiated 10 days after chalazia removal surgery. A warm compress (warm towel or hot compress eyeshade, 40-45 °C, 10-15 min) was applied first, followed by using a terpinen-4-ol wipe to scrub the lash roots from one end to the other in one stroke, which was repeated for at least 30 s. Metronidazole gel was manually applied on the lash roots with the eyes closed. This procedure was repeated

Statistical analyses
All statistical analyses were performed using SPSS software, version 19.0 (SPSS, Inc. Chicago, Illinois, USA). Continuous data are reported as the mean ± standard deviation and discrete data are expressed as rates (%). Multivariate logistic regression modeling was used to analyze the correlation strength between relevant factors and Demodex infestation. The Pearson chi-square test was used for comparative and correlative analyses between subgroups. The Fisher's exact test was used for data with low prediction frequency to avoid statistical bias. P < 0.05 was considered statistically significant.
The MGD subgroup specifically referred to meibomian glands that appeared healthy, with the exception of cyst formation, such as meibomian gland expansion, viscosity of the meibomian gland secretion increased, secretion changes from transparent to yellowish-white and ooze toothpaste-like excretion when squeezing the meibomian gland. However, Demodex infestation was comparable in unilateral or bilateral (P = 0.59), course of disease (P = 0.15), seasonal changes (P = 0.68), and anterior blepharitis (P = 0.15).

Discussion
Demodex mites are common in nature, and people are generally susceptible to them. There are two species of Demodex parasites that can infest human skin. Demodex follicularis lives in eyelash follicles, whereas Demodex brevis burrows into meibomian and sebaceous glands of the eyelid [12]. Both species Demodex were clearly observed in patients with demodicosis and chalazia. Most patients with demodicosis are asymptomatic mite carriers. Recently, it has been reported that demodicosis was prevalent in cases of blepharitis, and that Demodex mites played an important role in chronic inflammation of the skin, hair follicles, and glands of the eyelid [3]. Although the mechanism by which Demodex induce pathogenic damage is unclear, it likely involves direct damage from Demodex mites and their metabolites, delayed hypersensitivity induced by these metabolites, local infiltration of inflammatory cells, and secondary infection of pathogenic microorganisms, especially bacteria [8,13]. Chalazia is common in pediatric patients, and presents with multiple and recurrent chalazia. Chalazia exhibits poor treatment coordination and is difficult to prevent. This study showed that Demodex infestation was more prevalent in patients with chalazia than the control group. Furthermore, demodicosis was not found in the control group. Demodicosis is commonly age dependent [11,[14][15][16], with more frequent detection in individuals over 71 years old than in children between 3 and 15 years old [11]. This was consistent with the observations that demodicosis was rare in healthy children [11,17,18], and suggesting that Demodex infestation of pediatric patients is a risk factor for chalazia.
Previous studies [2,19] commonly used the presence of mites to determine Demodex infection. Even though Demodex mites were counted in some articles, the mite count was not used as the diagnostic criterion. There have not, to our knowledge, been specific studies in children with demodicosis, and the diagnosis and treatment for demodicosis in domestic populations are as follow: 2 mites/3 lashes in each eyelid is suspiciously positive and ≥ 3 mites/3 lashes is definitively positive, requiring clinical treatment [11]. In addition, Demodex lives in eyelash follicles, meibomian glands, and sebaceous glands [12]. Any mites attached to the eyelash root are removed simultaneously when eyelashes are removed for sampling, but it is important to consider that Demodex mites also lay eggs in the eyelash follicle, indicating that there may be more Demodex present than what is observed in the eyelash sample. Because demodicosis is not as common in the pediatric population, and mite count was not consistently correlated with the severity of chalazia [2,6,20,21], presence of Demodex mites should be taken seriously. In our study, demodicosis with 2-3 mites/12 lashes contrived only 9.75% of chalazia patients with Demodex infestation. In pediatric chalazia, microscopic examination for Demodex mites should especially careful to avoid missing a diagnosis. In addition, if there is ≥1 mite/12 lashes, it is recommended that the patient be treated as Demodex positive and undergo intervention.
In this study, it was found that Demodex mites were more frequent in patients with recurrent chalazia and those with skin surface involvement. Moreover, ocular demodicosis was significantly more prevalent in patients with recurrent, multiple, and MGD chalazia, which matched the pathogenic role of mites in meibomian and sebaceous glands [13,17,19,22]. Our results showed that demodicosis was more prevalent in chalazia patients regardless of blepharitis, similar to previously studies suggesting that demodicosis was associated with blepharitis [3,4,18] and chalazia [2,13,18,19,22]. These results also suggest that Demodex infestation could be an independent risk factor of chalazia after adjusting for the effects of blepharitis [23].
Demodex mites often utilize host cells and their metabolites, sebaceous gland secretions, sebum, and keratin as sources of nutrients. Changes in the local microenvironment of the eyelid caused by chalazia are conducive to Demodex parasitism, and demodicosis could worsen the manifestation of chalazia and cause its recurrence [8,13,15,23]. This highlights the importance of mite treatment.
Recurrent chalazia is reported in 17-25% of affected children, which is more common than in the adult population [8,24]. We observed that only 0.9% of pediatric patients with chalazia required re-operation for recurrence after comprehensive treatment for Demodex infestation. These results suggest that eradication of Demodex may be an effective method for preventing recurrence. Both 2% metronidazole ointment and Tea tree oil are reported as effective alternatives for treatment of Demodex infestation [25]. Terpinen-4-ol, which is the most active ingredient of tea tree oil, is currently the treatment of choice for pediatric demodicosis because it has fewer side effects [26,27].
Demodex mites typically complete one generation of their life cycle in 14-15 days [8]. We recommend comprehensive treatment [28] for mites because children are often reluctant to cooperate with treatment. The course of treatment is generally 1 to 3 months, encompassing several Demodex life cycles [29].
In addition, ocular discomfort was often difficult to interpret due to difficulty for children to describe the sensation, causing complaints to be overlooked [30]. In order to prevent development of demodicosis, it is important for children and their parents to maintain good ocular hygiene, including applying warm compresses and contact-isolation of demodicosis to control ocular Demodex infection [23,31].
This study had several associated limitations. Although adult or larvae Demodex were observed, Demodex folliculorum and Demodex brevis were not recorded separately for further study of which species was more prevalent in pediatric demodicosis with chalazia. Sampling the eyelash in the correct places is essential for microscopic Demodex detection, and there may be a non-invasive alternative, such as in vivo confocal microscopy, to sample eyelashes for Demodex detection, which could potentially avoid the failure to remove Demodex completely during epilation. This would also be more conducive to the review of pediatric patients. Since metronidazole gel has been reported to be both effective and safe in the treatment of demodicosis, comparing the effectiveness of different anti-mite treatments may be beneficial.
The majority of chalazia patients, especially those with recurrent and multiple chalazia, suffered more from demodicosis than healthy children. Demodicosis should be considered as a risk factor of chalazia. In children with chalazia, Demodex examination and comprehensive treatment of Demodex mites should be applied to potentially prevent recurrence.