Subjects

This prospective, 2-week, randomised, paired eye, investigator-masked trial, followed the tenets of the Declaration of Helsinki, was approved by the institutional ethics committee and was registered as a clinical trial (ACTRN12616000540415). Participants were required to be 18 years or older and non-contact lens wearers, with no history of major systemic, dermatological or ocular conditions, non-pregnant, no previous ocular surgery, no use of topical or systemic medications known to affect the eye and no allergies or hypersensitivity to topical medications or bee products. Eligible participants were enrolled after providing written informed consent and were required to attend three visits over the 2-week period at baseline, day 7 and day 14.

A total of 25 eligible participants were recruited, exceeding the sample size requirements for the desired study power. The designated outcome measure for determining sample size was tear film lipid layer grade. Power calculations showed that a minimum of 15 subjects was required to detect a clinically significant difference of one lipid layer grade in any of the pairwise comparisons, with 80% power (β=0.2), at a two-sided statistical significance level of 5% (α=0.05). The standard deviation (SD) of normal values was estimated at one lipid layer grade.15 Sample size estimates were determined using a uniform non-parametric adjustment, with PASS 2002 (NCSS Statistical Software, Kaysville, Utah, USA).

Treatments

Participants were randomly assigned to apply the MHME eye cream (figure 1), which was manufactured according to Good Manufacturing Practice (GMP) standards (Manuka Health New Zealand) to the left or right eye (treated eye) once a day, at night, for a period of 14 days. The fellow eye received no intervention (control eye). Two 20 mL tubes of eye cream were provided to each participant, and product application was demonstrated during the enrolment visit. Instructions were given to participants to squeeze approximately a 0.5–1 cm strip of the product (equivalent to a minimum of approximately 0.034 g±0.001 g) onto clean fingertips and to spread the cream thinly over the periocular skin of the closed upper and lower eyelids of one eye, in order to avoid direct contact with the ocular surface. Participants were then instructed to wash their hands immediately to prevent cross-contamination of the fellow eye. Participants were also advised against facial cleansing until the following morning and to exercise care to avoid the transfer of residual products to the fellow eye during facial cleansing and drying. Unused product was returned to the investigators at the end of the 14-day trial period and weighed as a measure of patient compliance.

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Figure 1

The Manuka honey microemulsion cream as it appears on extrusion from the tube (left arrow) and as it would following application thinly to the periocular area (right arrow).

Clinical measurements

McMonnies Dry Eye Questionnaire was administered to grade the severity of dry eye symptomatology at baseline. A telephone interview was conducted following the first day of eye cream application to check for immediate tolerability issues or adverse events. Participants were advised to contact the investigators during the treatment period to report adverse events at any other time.

The investigators conducting clinical measurements were masked to treatment randomisation. Participants were assessed at a single site, with a mean±SD room temperature of 20.2°C±1.4°C and relative humidity of 69.1%±4.6%. Clinical assessments were performed at baseline, day 7 and day 14 of the treatment period. Six-metre best spectacle-corrected LogMAR visual acuity (VA) was recorded. Participants were asked to rate symptoms of eyelid itching and pain on a four-point grading scale: grade 0, absent; grade 1, mild; grade 2, moderate; grade 3, severe. Bulbar conjunctival hyperaemia and palpebral erythema was assessed using the Oculus Keratograph 5M, on a grading scale from 0 to 4, using automated objective evaluation and estimation to the nearest 0.5 grade from high magnification digital imaging, respectively.

Tear film lipid layer grade and non-invasive tear film break-up time were assessed using the Oculus Keratograph 5M. Tear film lipid layer grading was based on the Guillon-Keeler grading system: grade 1, open meshwork; grade 2, closed meshwork; grade 3, wave or flow; grade 4, amorphous; grade 5, coloured fringes; grade 0, non-continuous layer (non-visible or abnormal coloured fringes).16 Break-up time was recorded as the time taken following a blink for the grid reflection to first show distortion, while the subject maintained fixation and was requested to refrain from blinking. Three measurements were averaged in each case.

The lower tear meniscus height was assessed using high magnification digital imaging captured by the Oculus Keratograph 5M. Three measurements near the centre of the lower meniscus were averaged. Tear evaporation rate was measured using a Vapometer (Delfin, Kuopio, Finland) within a swimming goggle housing.17 Differences between the evaporation rates in the open and closed eye state were recorded to factor out skin evaporation and allow quantification of evaporation from the tear film of the exposed ocular surface only. Tear film osmolarity was evaluated with a clinical osmometer (TearLab, San Diego, California, USA), using 50 nL tear samples collected from the lower lid tear meniscus. Two measurements were taken from each eye, and the higher reading for each eye was recorded.

Sodium fluorescein and lissamine green dyes were applied, in turn, to the bulbar conjunctiva in order to evaluate localised corneal and conjunctival epithelial desiccation. Staining was recorded using the modified Oxford grading scheme, where the nasal and temporal conjunctiva were divided into three areas each, and the cornea into five areas.18 Staining was graded from 0 to 5 with increasing confluence in each area and summed to provide a maximum score of 55. Lid wiper epitheliopathy (LWE) grade was assessed using lissamine green dye.19

Quantitative real-time PCR

Conjunctival impression cytology was conducted at baseline and day 14 of the treatment period, following topical anaesthetic application of one drop of Oxybuprocaine hydrochloride 0.4% (minims; Bausch & Lomb (NZ), Auckland, New Zealand) at the ocular surface. Bulbar conjunctival cells from the superior temporal ocular surface were collected using the EYEPRIM conjunctival impression device (OPIA Technologies, Paris, France). RNA extraction and purification from the conjunctival cell samples was conducted using the PureLink RNA Mini Kit (Thermo Fisher Scientific). Extracted RNA samples were tested for the presence of inhibitors before undergoing complementary DNA (cDNA) synthesis. Synthesis was performed using the SuperScript VILO cDNA Synthesis Kit and Master Mix (Invitrogen by Life Technologies). A standard beta-actin PCR and subsequent gel electrophoresis was run on a representative selection of the synthesised cDNA samples to ensure cDNA synthesis was successful. Six reference genes were tested among the sample population for stability with beta-actin and B2M being established as the most stable genes according to the NormFinder algorithm (MOMA, Aarhus, Denmark). A normalisation factor was calculated as the geometric mean of the expression of these two most stable genes and, in accordance with currently accepted practice,20 the genes of interest were quantified relatively against this factor. Quantitative PCR (qPCR) runs for quantifying the three genes of interest; matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6) and mucin-5AC (MUC5AC) were set up using the QiAgility PCR robot (Qiagen, Valencia, California, USA) with PrimeTime Assays (Integrated DNA Technologies). Internal calibrators were used in all qPCR to compensate for inter-run variations. Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were followed to ensure validity of the qPCR experiments.21

Statistics

Statistical analyses were performed with GraphPad Prism V.6.02 (http://www.graphpad.com). Repeated measures two-way analysis of variance (ANOVA) testing was performed to test the significance of treatment, time and interaction (treatment-by-time) effects on measurements over the 2-week period, where continuous variables with a normal distribution had been confirmed by the Kolmogorov-Smirnov test (p>0.05). Non-normally distributed continuous measures (non-invasive tear break-up time (NIBUT) and impression cytology measurements) were logarithmically transformed before being assessed. Ordinal data were converted to rank-values prior to undergoing analysis. Post hoc analysis for the significance of treatment effects at each time point was conducted using multiplicity adjusted Sidak's test. All tests were two tailed, and p<0.05 was considered significant. Data are presented as mean±SD, or median (IQR) unless otherwise stated.