ABSTRACT
Demodex species are frequently found in blepharitis cases. This study aimed to compare the conjunctival flora of eyes with Demodex-positive blepharitis and Demodex-negative blepharitis with healthy individuals.
Eyelash epilation was performed to detect Demodex from 44 eyes of 44 patients with chronic blepharitis and 44 eyes of 44 healthy controls and examined under a microscope. A conjunctival swab was taken from the same eye and inoculated on eosin methylene blue agar, Sabouraud dextrose agar, chocolate agar, and 5% sheep blood agar. Aerobic conjunctival flora was evaluated among Demodex-positive blepharitis, Demodex-negative blepharitis and healthy eyes.
Demodex spp. was detected in 3 (6.8%) of 44 healthy controls and 24 (54.5%) of 44 patients with blepharitis. The most frequently isolated bacteria in healthy controls were coagulase-negative Staphylococci (CNS) spp. (n=32, 72.7%), Streptococcus spp. (n=16, 36.4%), Corynebacterium spp. (n=13, 29.5%). The most frequently isolated bacteria in Demodex-positive blepharitis were CNS spp. (n=14, 58.3%), Staphylococcus aureus (n=11, 45.8%), Corynebacterium spp. (n=7, 29.2%). In Demodex-negative blepharitis, CNS (n=10, 50.0%), S. aureus (n=10, 50.0%), Corynebacterium spp. (n=5, 25.0%) were most commonly isolated. S. aureus growth was significantly increased in the Demodex negative and positive blepharitis groups compared with the healthy group (p=0.001 and p=0.002, respectively). Although CNS spp. growth decreased in both groups with Demodex-negative and positive blepharitis compared with the healthy group; the decrease was significant only in those with Demodex-negative blepharitis (p=0.045). In terms of other bacterial growth, there was no significant difference between healthy eyes and Demodex positive and negative eyes with blepharitis.
We found that Demodex blepharitis has no significant effect on conjunctival flora. Blepharitis itself may be the main factor in changes in the conjunctival flora.
Keywords: Demodex, blepharitis, conjunctiva, flora
INTRODUCTION
Blepharitis is a condition characterized by inflammation of the eyelid margin, which is very common in ophthalmology practice. The effects of bacteria on the pathogenesis of blepharitis have been demonstrated frequently (1,2). Determining the types of bacteria is important for an effective treatment. Demodex spp. infestation is also common in blepharitis (3). However, Demodex blepharitis has its own characteristics (4). It is stated that Demodex spp. acts as a vector for some microorganisms in blepharitis and may cause changes in the ocular flora (3,5). Especially the symbiotic relationship with Bacillus oleronius and its effects on blepharitis has been reported (5).
The possible effects of Demodex spp. on other microorganisms are essential for determining the treatment strategy, given the importance of bacteria in blepharitis treatment. We conducted this study to compare the conjunctival flora of Demodex blepharitis, blepharitis without Demodex spp. and healthy controls to determine whether there is any difference.
METHODS
This study was conducted with 44 patients with chronic blepharitis who admitted to ophthalmology outpatient clinic and 44 age- and sex-matched healthy controls. There were 14 (31.8%) women and 30 (68.2%) men in both groups with and without blepharitis. The mean age of the group with and without blepharitis was 49.5±17.4 (20-80) and 49.1±16.9 (21-78), respectively (p=0.916). Microbiological sample analysis of the patients was performed in the microbiology and parasitology laboratory. Biomicroscopic examination was performed for the diagnosis of blepharitis in the patient and control group participating in the study. Blepharitis was accepted as the presence of erythema, telangiectasia, thickening, dandruff and choleret on the eyelid margin or tarsal conjunctiva. Severity of blepharitis was roughly equal in both eyes in all patients. Those who used topical or systemic antibiotics in the last month, those who had eye surgery, and those with other infectious or inflammatory diseases on the eyelid or eye surface were excluded from the study. The control group was selected from individuals who applied to the ophthalmology clinic only for refractive reasons and were otherwise healthy. The principles of the Declaration of Helsinki were adhered to in all steps of the study. Written and verbal consent was obtained from all patients before the study. Ethical approval was obtained from the Malatya Turgut Özal University Clinical Research Ethics Committee (decision no: 2020/206).
A minimum sample size of 20 individuals in each group was calculated for 0.80 power, 0.05 type-1 error and 0.84 effect size to identify a 40% difference in bacterial growth between controls and Demodex blepharitis.
Firstly, flora sample was taken from right eye of the patients and controls, and then eyelash epilation was performed for Demodex spp. detection from upper eyelid of the same eye.
Sampling and Examination for Demodex
The eyelash samples were examined by a specialist medical microbiologist (A.G). Two or three eyelashes (especially those with scale) taken from all patients with sterile forceps were quickly delivered to the laboratory in sterile sponges. Eyelashes brought to the laboratory were placed on the slide and then 2-3 drops of immersion were dropped on them and covered with a lamella. The area was scanned with x4 magnification of the microscope, and then the area was scanned with the diaphragm slightly closed at x10 and x40 magnification and the findings were noted. If one or more parasites are detected in this examination, Demodex spp. evaluated as positive. This review was done within an hour.
Sampling and Evaluation for Flora
The lower eyelid of the patients was pulled down slightly and flora sample was taken from the lower fornix conjunctiva with sterile swab. While taking the swab sample, care was taken to avoid touching the eyelids and eyelashes. Topical anesthetic medication was not applied to the patient for this procedure. The samples were planted in the thioglycollate broth in a short time, avoiding contamination. Then, in the microbiology laboratory, eosin methylene blue agar, sabouraud dextrose agar, chocolate agar and 5% sheep blood agar were inoculated. Media were incubated at 37 °C for 24 hours. Non-growth plates were incubated for an additional 24 hours. The incubation period of the samples planted on Sabouraud dextrose agar was extended to three weeks. Growing microorganisms were identified by standard microbiological methods.
Statistical Analysis
Statistical analysis was done with SPSS 25.0 program (Chicago, IL, USA). Results are presented as mean ± standard deviation (minimum-maximum) in measurable data. Nominal data are presented as frequency and percent. Normal distribution was evaluated with the Kolmogorov-Smirnov test. Normally distributed data were evaluated using the unpaired t-test, and non-normally distributed data were evaluated using the Mann-Whitney U test. Nominal data were evaluated using Pearson’s chi-square test or Fisher’s Exact test. A p-value of less than 0.05 was considered significant.
RESULTS
Demodex was observed in 3 (6.8%) samples in the group without blepharitis and in 24 (54.5%) samples in the group with blepharitis (p<0.001). Bacterial growth occurred in 36 (81.8%) and 38 (86.3%) samples in the group with and without blepharitis, respectively (p=0.560). The mean growth was 1.77±1.31 (0-5) and 2.00±1.29 (0-6) from each sample in the group with and without blepharitis, respectively (p=0.411). Staphylococcus aureus growth was significantly less in the group without blepharitis than with blepharitis (p<0.001). In other bacteria, growth numbers were similar between groups with and without blepharitis (p>0.05) (Table 1).
There were 61 (69.3%) samples with no Demodex spp. and 27 (30.7%) samples with Demodex spp. One to three Demodex parasites were detected in all patients except one patient with four Demodex parasites. There were 18 (29.5%) females and 43 (70.5%) males in the non-Demodex group, while there were 10 (37.0%) females and 17 (63.0%) males in the Demodex group (p=0.484). The mean age of the non-Demodex and Demodex groups was 48.3±17.7 (21-80) and 51.5±15.9 (20-71), respectively (p=0.419). The mean growth was 1.86±1.31 (0-6) and 1.93±1.29 (0-5) from each sample in the non-Demodex and Demodex groups, respectively (p=0.787). In the non-Demodex group, there were 51 (83.6%) growths in the conjunctiva samples, while in the Demodex group, there were growth in 23 (85.2%) samples (p=0.560). There was no significant difference in the number of growth of any bacteria in conjunctival flora between the Demodex and non-Demodex groups (p>0.05) (Table 2).
The mean age was 46.7±19.4 (21-80) and 51.7±15.6 (20-71) in non-Demodex blepharitis and Demodex blepharitis groups, respectively (p=0.341). There were 5 (25.0%) women and 15 (75.0%) men in the group with non-Demodex blepharitis, while there were 9 (37.5%) women and 15 (62.5%) men in the group with Demodex blepharitis (p=0.375). There was growth in 16 (80.0%) and 20 (83.3%) samples in non-Demodex and Demodex blepharitis groups (p=1.000). The mean growth number from each sample was 1.60±1.23 (0-4) and 1.91±1.38 (0-5) in non-Demodex and Demodex blepharitis groups, respectively (p=0.437). Yeast growth occurred in two patients with Demodex blepharitis and two without blepharitis. There was no yeast growth from other samples.
The four most frequently bacteria isolated in the conjunctiva of healthy eyes were coagulase-negative Staphylococci (CNS) (n=32, 72.7%), Streptococcus spp. (n=16, 36.4%), Corynebacterium spp. (n=13, 29.5%), Bacillus spp. (n=7, 15.9%). The four most frequently isolated bacteria in the conjunctiva of eyes with non-Demodex blepharitis are CNS (n=10, 50.0%), S. aureus (n=10, 50.0%), Corynebacterium spp. (n=5, 25.0%), Streptococcus spp. (n=3, 15.0%). The four most frequently isolated bacteria in the conjunctiva of eyes with Demodex blepharitis are CNS (n=14, 58.3%), S. aureus (n=11, 45.8%), Corynebacterium spp. (n=7, 29.2%), Streptococcus spp. (n=6, 25.0%), respectively (Figure 1).
S. aureus growth was significantly increased in Demodex and non-Demodex blepharitis groups compared to healthy group (p=0.001 and p=0.002, respectively), but there was no significant difference between Demodex and non-Demodex blepharitis groups (p=0.783). Although CNS growth decreased in both groups with Demodex and non-Demodex blepharitis compared to the healthy group, the decrease was significant only in the group with non-Demodex blepharitis (p=0.045). There was no significant difference between the groups with Demodex and non-Demodex blepharitis groups in CNS in conjunctival flora (p=0.580). In terms of other bacteria, there was no significant difference between healthy eyes and the groups with blepharitis with and without Demodex spp. (p>0.05) (Table 3).
DISCUSSION
Since the etiopathogenesis of blepharitis is not fully clarified, the relationship between blepharitis and microorganisms continues to be investigated. Various forms of bacteria, fungi and parasites have been isolated in blepharitis (3,6,7). The frequency and types of isolated organisms vary even in the same type of blepharitis (1,2,8). Although the increase in S. aureus in our study is consistent with the literature, the CNS decrease differs in general from the literature (1,2,8). The more frequent reporting of bacteria found in the skin flora in blepharitis indicates that organisms such as Staphylococci and Demodex spp. may invade the area (2). On the other hand, it is reported that environmental bacteria are frequently isolated in flora studies performed with blepharitis (3,8). Our results show that S. aureus and Demodex spp. are the most frequent organisms in blepharitis. This finding is consistent with the result often found in blepharitis (2,8,9). This situation indicates that no matter what mechanism is dominant, the microenvironment in blepharitis is most advantageous for Demodex spp. and S. aureus.
Demirkazık and Koltaş (10) detected Demodex in 143 (42.6%) of 335 patients with a preliminary diagnosis of blepharitis, conjunctivitis, and visual impairment. In addition, it was reported in this study that the incidence of Demodex increased significantly with increasing age. Tanrıverdi et al. (11) detected Demodex in 69 (45.1%) of 153 chronic blepharitis cases, but they did not find an age-related Demodex increase. In our study, Demodex was found in 54.5% of blepharitis patients and 6.8% of healthy controls. Although the mean age of cases with Demodex blepharitis was higher than that of cases with non-Demodex blepharitis, the difference was not significant.
The fact that Demodex spp. may cause inflammatory effects in blepharitis with Demodex (12,13) and carry bacteria with it (5,14) brings to mind its effects on other microbiological species. Zhu et al. (8) reported that the number of Propionibacterium acnes colonies on the lid margin and eyelashes increased significantly in patients with blepharitis and those with Demodex in the control group compared to those without Demodex. Yan et al. (3), in their study comparing the flora of patients with Demodex blepharitis and healthy controls through bacterial 16S RNA, identified potential bacterial genera in Demodex blepharitis as Bacilli, Firmicutes, Cyanobacteria, Lactobacillus and Streptophyta Lee et al. (15) could not find a correlation between Demodex and ocular microbiota, but emphasized that the number of cases with Demodex was low. In our study, the growth rate of S. aureus was significantly increased among the Demodex group with blepharitis compared to the healthy group. Similarly, S. aureus was increased in the group with non-Demodex blepharitis compared to the healthy group, but the CNS were decreased. These results may indicate that the presence of Demodex spp. in eyes with blepharitis has no effect on bacterial flora and that the bacterial distribution is more related to blepharitis than the presence of Demodex spp.
In our study, S. aureus was found at a rate of 12% in the conjunctiva of healthy eyes, while it was detected in approximately 50% of eyes with blepharitis regardless of the status of Demodex. Previously, S. aureus was isolated as a floral element of healthy conjunctivae (16,17), while this bacterium was reported to appear at higher rates in eyes with blepharitis (8,9,15). The presence of this bacterium was not affected by Demodex status in our study and was isolated at a significantly increased rate in eyes with blepharitis.
The CNS is often isolated in the normal conjunctival flora (17,18). Many studies report increased CNS isolation at the conjunctiva and lid margin in blepharitis with or without Demodex compared to healthy controls (18). In our study, CNS isolation was found significantly higher in the conjunctiva of healthy individuals compared to those with blepharitis without Demodex. However, the number of CNS isolations in the conjunctiva of eyes with blepharitis with and without Demodex was similar. This result, which differs from other studies (9,19), indicates that the CNS, which is a normal flora component, decrease in case of blepharitis in our study, and it may indicate that the CNS as a normal flora component may be negatively affected in blepharitis. However, the finding of more pathogenic CNS in other studies may have caused this difference.
Although Corynebacterium spp. can be found in soil, water and plants in nature, non-pathogenic species can be found in skin and mucosa (15). Most of those found in normal conjunctiva and valve flora are lipophilic species (20). It has been isolated on the conjunctiva and eyelid margin in eyes with blepharitis (1,8,15). Bezza Benkaounha et al. (1) reported that Corynebacterium spp. increased significantly in eye conjunctiva with blepharitis compared to healthy controls, but stated that this increase may not necessarily be related to pathogenesis. In our study, it was the third most common bacterial in normal flora and was the least affected by blepharitis regardless of Demodex status. Lee et al. (15) suggested that in addition to the association of blepharitis with bacteria in the skin flora, an increased proportion of bacteria such as Corynebacterium species, which can be found in pollen, dust and soil particles in cases with blepharitis, may be associated with pollen, dust and soil contamination.
Although Streptococcus spp. are frequently isolated from the skin flora, no significant increase was found in our study. Although Zhu et al. (8) isolated it more frequently in eyes with blepharitis, they did not consider it as a pathogenic organism in blepharitis because the bacterial load of Streptococcus spp. did not increase significantly. Streptococcus spp. appear to be more associated with corneal infections and dacryocystitis than blepharitis (21).
Bacillus spp. was a relatively less common type of bacteria in our study. It has been previously reported that Bacillus oleronius may be in a symbiotic relationship with the Demodex parasite and can cause infection by being transported by the parasite and multiplying under suitable conditions (5). In other studies, it has been reported that Bacillus spp. associated with Demodex may have an effect on the development of blepharitis and facial rosacea (22). Kıvanç et al. (23) reported that Bacillus spp. isolated from the conjunctiva showed antimicrobial activity against methicillin-resistant Staphylococcus species. In our study, the frequency of Bacillus spp. isolation from samples with Demodex blepharitis was similar to that of non-Demodex blepharitis and healthy controls. In addition, the presence of Bacillus spp. was not affected by the presence of Demodex, and no change was observed in the isolation frequency of other organisms in the presence of Bacillus species.
Detection and treatment of Demodex spp. has an important place in the treatment of blepharitis. In our study, frequent bacterial growth in Demodex blepharitis requires antibiotic use in addition to Demodex treatment. However, since the presence of Demodex spp. does not make a difference in bacterial distribution with non- Demodex blepharitis, it may not be necessary to take additional measures in antibiotic treatment for blepharitis when planning antibiotic treatment.
Study Limitations
The limitation of our study is that the ocular findings of Demodex blepharitis were not evaluated in relation to the presence of Demodex spp. and flora.
CONCLUSION
We think that Demodex blepharitis has no significant effect on conjunctival flora and that blepharitis itself is the main factor in determining microbial diversity.
*Ethics
Ethics Committee Approval: Ethical approval was obtained from the Malatya Turgut Özal University Clinical Research Ethics committee (decision no: 2020/206).
Informed Consent: Written informed consent was taken from all the participants.
Peer-review: Internally peer-reviewed.
*Authorship Contributions
Surgical and Medical Practices: A.G., E.E.D., Concept: A.G., E.E.D., M.F., Design: A.G., E.E.D., M.F., Data Collection or Processing: E.E.D., M.F., Analysis or Interpretation: A.G., E.E.D., M.F., Literature Search: A.G., E.E.D., M.F., Writing: A.G., M.F.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.
