The study was carried out between October 2015 and May 2018, and was approved by the Bioethics Committee of the Pomeranian Medical University in Szczecin (KB-0012/82/15). It conformed to the principles outlined in The Declaration of Helsinki as revised in 2008.
2.1. Characteristics of groups
The study involved 231 patients from Poland. Patients classified in the studied group were selected from the Ophthalmology Department (n=5) and the Ophthalmology Department of the Regional Hospital (n=45) in Kołobrzeg, the Ophthalmology Department of the Independent Public Complex of Health Care Centres in Gryfice (n=49) and residents of the Social Welfare Home (SWH) in Jaromin (29 male aged 41 to 80; mean age 59.0). In total, 128 participants diagnosed with blepharitis and infected with D. folliculorum (23-85 years of age) were chosen. The patients with blepharitis included those with at least two symptoms of blepharitis, such as burning sensation in the eyes, tearing, eyelid hyperemia, foreign body sensation, and excessive loss of eyelashes. The healthy control group included 103 non-infected patients (24-81 years of age). Exclusion criteria were as follows: using topical ophthalmic medications (except artificial tears) over the previous 3 months before the study started, a history of ocular or eyelid trauma and surgery in the last 6 months, previous diagnosis of chemical burns, Stevens-Johnson syndrome, ocular cicatricial pemphigoid, with eyelid malpositions such as entropion, ectropion, and distichiasis, signs of active ocular infection, or inflammation other than blepharitis, and general treatment with oral antibiotics.
Both groups were asked to fill the informed consent to participate in this protocol, which was followed by an interrogation carried out to capture information and slit-lamp evaluation with a magnification of ×25.
2.2. Demodex spp. examination
A total of eight eyelashes were excised per patient, four eyelashes per eye. They were extracted with fine forceps and placed separately on each end of a slide. A coverslip was placed on the top of the eyelashes after coating them with Hoyer medium. The presence and counting of Demodex were performed in the samples by light microscopy with a magnification of 4x, 10x and 40x. Infection was defined as the presence of eggs, larvae, or mature forms of
D. folliculorum on the eyelashes.
2.3. Clinical examination
Participants took part in an ophthalmic interview regarding eye problems and their personal and familial history of eye diseases. The residents from the Social Welfare Centre were not examined in detail due to their limited cooperation during the examination.
The ophthalmological examination consisted of testing of uncorrected and best corrected distance visual acuity (VA) using Snellen charts. The examination was performed at a distance of 4 m in a room providing the same lighting for all examinations. The result of the best corrected visual acuity was recorded and conversed to the LogMAR scale (decimal logarithm from the minimum angle of resolution). Intraocular pressure (IOP) was measured with Mackay-Marg Tono-Pen AVIA applanation tonometer (Reichert, USA). The measurement was performed three times and the average used for the analysis. Anterior segment examination was performed using a Haag-Streit L0185 slit lamp (Nikon, Japan).
2.4. Microbiological examination
The samples for microbiological examination were obtained from the conjunctival sac using a sterile swab and AMIES transport medium. The identification methods used in this paper correspond to those used in routine bacteriological diagnostics. Samples were plated on basic microbiological media: Columbia agar with 5% sheep blood, Chapman, MacConkey, and Sabouraud and then incubated at 37 °C for 24-48 hours. Strains were identified based on morphological evaluation of the colonies on the media and preparations stained by the Gram method.
The identification of Staphylococcus spp. was performed by determination of hemolytic capacity of colonies on Columbia agar medium with 5% sheep's blood and by evaluation of growth on Chapman medium, allowing for differentiation of staphylococci into mannitol-positive and mannitol-negative strains. All strains showing the ability to ferment mannitol were analyzed for the presence of clumping factor A, protein A, and coagulase. The presence of all three factors indicated Staphylococcus aureus.
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. Species identification was performed by VITEK Compact (bioMerieux, Poland).
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. Antybiogram was perfomed for Staphylococcus aureus strains as the pathogen caused conjunctivitis. From single colonies grown after 18-24 h, a suspension of density 0.5 according to McFarland scale (1x108 cfu/ml) was prepared and inoculated into Mueller-Hinton agar medium (bioMerieux, Poland). Subsequently, antibiotic discs with erythromycin (15 µl), clindamycin (2 µl), gentamicin (10 µl), neomycin (10 µl), tetracycline (10 µl), and trimethoprim/sulfamethoxazole (1.25/23.75 µl) were placed onto the culture medium. Methicillin-resistant Staphylococcus aureus (MRSA) was determined using cefoxitin 30-μg disks. 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).
2.5. Statistical analysis
Statistical studies were performed using Stat Soft Statistica 10.0 PL. The nonparametric Mann–Whitney test was used to evaluate the differences between Demodex spp. infection and mean IOP and VA in the analyzed groups. In order to establish possible relationships between D. folliculorum infection and the occurrence of eye diseases and symptoms in patients from the two groups, the Chi2 independence test was used. Differences were deemed statistically significant at p<0.05.