Quantitative assessment of chitin synthase gene of demodex mite on eyelashes and correlation to symptomatology of blepharitis

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Aims/Purpose: to quantify the presence and activity of Demodex mites and correlate it with patient symptoms, using quantitative determination of gene copy number for the chitin synthase gene. Methods: 61 patients meeting the inclusion criteria were enrolled in the study. 8 eyelashes from each patient were aseptically placed in sterile microtubes. After DNA isolation, quantitative real‐time PCR reactions were performed with specific primers for the chitin synthase gene (DEM) (for Demodex) and beta‐actin (ACTNB) and Factor V (FV) (for human), to amplify the corresponding genes. The quantitative differences for Demodex DNA copies were relative to the simultaneous amplification of the reference human genes (CtDEM‐CtFV/ΔCt1 or CtDEM‐CtACTNB/ΔCt2). Results: 51 subjects completed the protocol. Mean age was 57.08 ± 17.35 years. Mean Ocular Surface Disease Index (OSDI) was 41.79 ± 20.05, while mean Total Ocular Symptom Score (TOSS) was 42.25 ± 23.90. Mean conjunctival hyperemia was 1.43 ± 0.48 (0‐3). Subjects had a mean Non‐Invasive Tear Break‐up Time (NIBUT) of 6.86 ± 4.32 sec. Meibography showed a mean of 1.66 ± 0.51 (0‐3), while Marx Line values were 1.56 ± 0.825 (0‐3). Pearson's correlation coefficient indicated a positive correlation of age and TOSS score (p = 0.035) as well as conjunctival hyperemia (p < 0.001) values. Higher OSDI scores were correlated to higher TOSS scores (p < 0.001), lower NIBUT values (p = 0.001) and worse meibography grade (p = 0.038). Conjunctival hyperemia showed a linear relationship with age (p < 0.001). ΔCt1 (CtDEM‐CtFV) showed a positive correlation with ΔCt2 (CtDEM‐CtACTNB) (p < 0.001). Conclusions: The present study is the first to test the number of Demodex‐severity of symptoms hypothesis, using specific genes and Real‐time PCR. Although no statistically significant correlation was revealed for the specific genes, the clinical data analysis confirmed a correct methodology and sampling, creating a frame for future research on the hypothesis.

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