Concurrence of ocular infection with Demodex folliculorum

Background The ectoparasite Demodex spp. is the most common human parasite detected in skin lesions such as rosacea, lichen, and keratosis. It is also an etiological factor in blepharitis. As Demodex spp. are involved in the transmission of pathogens that can play a key role in the pathogenesis of demodecosis, the aim was to assess the concurrence of Demodex folliculorum and bacterial infections. Methods The study involved 232 patients, including 128 patients infected with Demodex folliculorum and 104 non-infected patients. All patients underwent ophthalmological examination. The material for microbiological tests was collected from the conjunctival sac. Samples were plated on basic microbiological media and then incubated. Strains were identified based on morphological evaluation of the colonies on the media and preparations stained by the Gram method. Results Physiological flora was found in all patients infected with D. folliculorum and 9 (8.7%) participants from the control group. Only in patients infected with D. folliculorum we isolated Staphylococcus aureus (9 patients, 7%), Acinetobacter baumannii (one patient, 0.8%), Streptococcus pneumoniae (one patient, 0.8%), Gram-negative bacteria (one patient, 0.8%), and Bacillus spp. (one patient, 0.8%) in the conjunctival sac. Conclusions Patients infected with Demodex spp. should also undergo microbiological examination of conjunctival swabs. The treatment of each patient should be individualized, adapted to the clinical condition, and in cases of bacterial co-infection, an antibiotic and/or a topical steroid drug should be additionally prescribed. Furthermore, of the eyelid margins should be recommended.

Background The ectoparasite Demodex spp. is the most common human parasite detected in skin lesions such as rosacea, lichen, and keratosis. It is also an etiological factor in blepharitis. As Demodex spp. are involved in the transmission of pathogens that can play a key role in the pathogenesis of demodecosis, the aim was to assess the concurrence of

Background
Demodex mites are obligatory ectoparasites found in the skin, hair follicles, and outer layers of the epidermis of many mammalian species, showing a high species specificity.
Two species are found in humans-Demodex folliculorum (Simon, 1842) and Demodex brevis (Akbulatova, 1963). Considered commensals for a long time, they are now classified as parasites by most researchers. These cosmopolitan mites live in humans of all races [1].
The transmission routes of Demodex spp. have not yet been fully investigated. It is likely that infection with Demodex spp. occurs through direct contact, use of common toiletries or towels, or by airborne eggs and dust [2,3]. Skin colonization occurs during childhood or adolescence; no mites are found in the skin of newborns [4,5].
The Demodex species found in humans vary in their habitat and morphological structure.
Demodex folliculorum occurs in the hair follicle and Zeiss glands, usually forming clusters of several individuals. Demodex brevis is most often isolated as separate specimens in the depths of sebaceous glands in the skin of the face, in the Meibomian glands, and in the eyelids [5]. Both Demodex species are present in the face skin, mainly around the nose, around the eyes, on the forehead and chin [6]. In addition to facial skin, these mites can be found in other parts of the body, including the hairy part of the head, auditory canals, skin in the neckline area, genitals, hands and feet, and nipples [7][8][9]. It has been noted that D. folliculorum is more numerous, but D. brevis occupies larger areas of the skin.
Demodecosis usually causes symptoms in the skin and eyes, but it can also be asymptomatic. The symptoms of ocular demodecosis are non-specific. Patients infected with Demodex spp. have reported tearing, burning, foreign body sensations, eyelid margin hyperemia, eyelash loss, itching, eye redness, blurred vision, and conjunctivitis with excessive sensitivity to light [2,3,10,11]. Burning and itching of eyelid margins are more frequently reported on warmer days [12].
It was found that Demodex mites participate in the transmission of pathogens, which may play a key role in the pathogenesis of demodecosis [13]. Symptomatic demodecosis often occurs with simultaneous bacterial infection, which is confirmed by a decrease in the number of Demodex spp. after administration of tetracycline to people with acne [14].
Demodex folliculorum contributes to the development of rosacea by transmitting bacteria from insensitive areas to sensitive areas where inflammation may occur [15]. Together with their food, which is mainly epidermal cells and the secretions of sebaceous glands, Demodex mites can take up microorganisms from the surface of the skin. Then, through their digestive tract, microbes are transported to the hair follicles of the host [5,16]. In addition, the disintegration of Demodex spp. inside the hair follicle can lead to release of the transmitted bacteria and the development of local immune response [17]. The composition of the bacterial flora of the conjunctival sac depends on many factors, including patient age and the presence of chronic disease. For example, Propioniobacterium spp. is more common in adults, whereas Streptococcus spp. is more common in children [19]. Chronic diseases, including diabetes, may increase the number of coagulase-negative staphylococci compared to healthy patients. These bacteria produce substances inhibiting the development of pathogenic bacteria, stimulating local immunological processes and the exfoliation and regeneration of epithelial cells of the eye [20]. The aim of the study was to assess the concurrence of Demodex folliculorum and bacterial infections.

Methods
The study was carried out between October 2015 and May 2018, and was approved by the

Microbiological examination
The material for microbiological tests was collected from the conjunctival sac with a sterile swab and AMIES transport medium. Samples were plated on basic microbiological media: Columbia agar with 5% sheep blood (Chapman, MacConkey, and Sabouraud) and MacConkey medium was used to isolate and identify strains of Gram-negative bacteria.
Due to the lack of pathogenicity of this group of microorganisms in conjunctivitis, only growth morphology on the medium was evaluated, dividing bacteria into lactose-positive and lactose-negative strains.
All the microorganisms showing growth characteristic of Corynebacteria on Columbia agar with 5% sheep's blood were analyzed by Gram staining. Gram-positive cells with a characteristic club-like shape were considered to be Corynebacterium spp.
Using the disk diffusion test, the drug susceptibility of isolated strains was determined.
Methicillin resistance of S. aureus was determined using a cefoxytin disk (30 µg).
Assessment of the growth inhibition zone around the discs and analysis of the results were performed according to the guidelines of the National Reference Centre for Microbial Susceptibility (www.antybiotyki.pl).

Results
Among control subjects and patients with blepharitis symptoms, hyperopia was observed in 66 and 32 patients, myopia in 14 and 9 patients, and astigmatism in 3 control patients.  (Fig. 1A). Ophthalmic examination showed that the visual acuity (VA) of the right eye and of the left eye were 1.0 and intraocular pressure (IOP) of the right eye was 11.7 mmHg and 10.7 mmHg, respectively.
Anterior segment examination using a slit-lamp showed cylindrical dandruff on the upper eyelid and blockage of the Meibomian glands. Staphylococcus aureus was also found in a adult female patient with arterial hypertension and diabetes mellitus. Microscopic examination of her eyelashes revealed numerous larval and mature forms of D.
folliculorum. Ophthalmic examination showed that VA of the right eye was 0.9 and in the left eye was 1.0. Intraocular pressure was 12.0 mmHg, and 13.0 mmHg, respectively. The patient suffered from irritation of the eye and conjunctiva. Staphylococcus aureus sensitive to erythromycin, clindamycin, gentamicin, neomycin, tetracycline, and trimethoprim/sulfamethoxazole were observed in two patients with mature forms of D.
folliculorum. An adult female patient with thrombocytopenia without ophthalmic symptoms had hyperopia corrected by glasses. Ophthalmic examination showed that VA to be 0.

Discussion
Demodecosis is a problem in both dermatology and ophthalmology due to the chronic nature of the infection. Most studies concern the occurrence of Demodex spp. infections in the skin of the face, while research on their incidence in the eyelids is scarce. The effect of Demodex spp. infection on the visual system is a contentious issue. Some researchers indicate that these mites participate in the etiopathogenesis of eye diseases, while others disagree [21,22]. However, there are studies which offer evidence that Demodex spp.
infection may cause changes in the cornea and conjunctiva of the eye [23,24].
The incidence of Demodex spp. infection depends on climate, socioeconomic and sanitary conditions, as well as access to medical care and effectiveness of treatment [25]. In human skin, the extensiveness of Demodex spp. infection may range from 20% to 80% patients [26]. Studies on potentially healthy subjects showed that 10%-60% of respondents had Demodex spp. in eyelash follicles. A study by Kuźna-Grygiel et al. [27] showed that, in Szczecin (i.e. the capital of the region of West Pomerania, the area covered in our research), the frequency of infection with Demodex spp. in eyelashes was 61%. A similar prevalence of infection with Demodex spp. (59%) was reported by Czepita et al. [10] in their study also performed in Szczecin. Lower incidence of Demodex spp. infection was found by Sędzikowska et al. [28] in patients of hospitals in Warsaw (47%).
All surveyed residents of the Social Welfare Home in Jaromin (n = 28) were infected with Demodex spp. In a study conducted at a similar institution, the Veteran's House in Szczecin [10], infection with Demodex spp. was found in 80% of residents. The very high infection rate at the Social Welfare Home in our study may have been caused by the fact that adults with psychiatric illness can restore their mental health. In this group of patients, infection with Demodex spp. is generally higher than in control subjects. For example, in a study by Kokaçya et al. [30], patients with schizophrenia had a higher incidence of Demodex spp. (29.03%) than control group (6.7%). The same team of researchers found that in depressed patients, Demodex spp. infection was also higher than control group (23.8% vs. 9.5%) [31].
In our study, physiological flora was found in conjunctival sac swab in all patients with symptoms of blepharitis, all residents of the Welfare Center infected with Demodex spp., and in 9% of those not infected with D. folliculorum (control group). This may indicate that Demodex spp. promotes colonization of the conjunctival sac with physiological flora.
Spickett [32] showed that D. folliculorum may be a vector organism for leprosy mycobacteria (Mycobacterium leprae). Demodex mites may also transmit Staphylococcus spp. and Streptococcus spp. on its surface [17]. In a study conducted on patients, staff, and visitors of the Optometry Clinic in Oklahoma [33], S. aureus and S. epidermidis were found in 16.8% and 75.8% of subjects. In the study, two or more mites (11.6% and 5.2%, respectively) were reported more frequently in patients infected with Staphylococcus aureus than in uninfected patients. Staphylococcus aureus was found in 21.9% of patients aged 1-29 years, 13.1% aged 30-59, and 15.1% aged 60-89. In another study, Türk et al. [34] found S. aureus in two patients with blepharitis and infected with D. folliculorum. In our study, S. aureus was isolated from 7% of patients with symptoms of blepharitis, including 14.3% of residents of the Social Welfare House infected with D. folliculorum. We did not find the bacteria in the control group. In contrast, Lee et al. [35] found no differences in the incidence of bacteria between people infected and not infected with Demodex spp. Coagulase-negative Staphylococcus spp., Corynebacterium diphtheriae, and S. aureus were found in patients of both groups. There were no differences in the occurrence of MRSA between those infected and not infected with Demodex spp. In our study, MRSA was found only in patients infected with D. folliculorum, including one resident of the Social Welfare House in Jaromin.
Acinetobacter baumannii is one of the most common etiological factors of hospitalacquired infections. It shows natural mechanisms of resistance to antibiotics and chemotherapy. In the present study, A. baumannii was isolated from the conjunctival sac of a patient infected with D. folliculorum with symptoms of blepharitis. patients with papulopustular rosacea, and found specific antigens against B. oleronius in the serum.
Li et al. [37] on sera from 59 patients with diagnosed rosacea showed a statistically significant correlation between a positive serum reaction with B. oleronius antigens and the presence of Demodex spp. on eyelashes and face skin lesions. Results of a study by O'Reilly et al. [38] showed that proteins derived from B. oleronius may be a neutrophilactivating factor. Such activation of neutrophils could take place if B. oleronius proteins released from mites entered the tissues surrounding the hair follicle. This, in turn, could result in the development of local inflammation in the perifollicular tissue [38]. In our study, Bacillus spp. were isolated from the conjunctival sac of a patient with D.
folliculorum infection. In a study by Szkaradkiewicz et al. [39], 23 strains of B. oleronius were isolated from eyelashes collected from 18 patients infected with Demodex spp. and with symptoms of blepharitis; the authors observed more severe symptoms of blepharitis in patients in whom B. oleronius was found. However, B. oleronius was also found in five healthy subjects, which may undermine its role in the development of blepharitis. The authors concluded that these bacteria, living inside the intestines of the Demodex mites as symbionts, can be excreted by these mites onto the surface of human skin. Due to the fact that B. oleronius plays a significant role in the process of digestion in termites, it seems that these bacteria may play a similar role in Demodex spp. [36,40]. Lacey et al. [36] stated that two specific antigenic proteins (62 and 83 kDa) produced by B. oleronius can stimulate and be responsible for inflammation of the hair follicle. Moreover, Li et al. [37] noted a correlation between Demodex spp. infection and serum immunoreactivity to and conjunctiva and cornea of the eye, as well as pneumonia. S. pneumoniae infection can cause severe or chronic complications [41,42]. In the presented study S. pneumoniae was reported in a resident of a Social Welfare Home infected with D. folliculorum.

Conclusions
Demodex folliculorum can collect microorganisms found on the surface of the skin and transport them to the host's hair follicles. Transmission of bacteria from non-susceptible sites to sensitive areas can contribute to the development of inflammatory reactions.