This prospective study was approved by the research ethics committee UZ/KU Leuven, Belgium, in accordance with the tenets of the Declaration of Helsinki. This project is registered on ClinicalTrials.gov (NCT04193774). The study was performed in adult patients (minimum age 18 years).
Patients undergoing strabismus, ear, or throat surgery under general anesthesia at the University Hospitals Leuven, Belgium, were informed about the study and consent of participating volunteers was obtained before enrollment. Aforementioned indications for surgery were chosen based on no need for pretreatment with antibiotics or antiseptics before surgery. Exclusion criteria were allergy to oxybuprocaine hydrochloride, the use of topical antibiotics in the month before sampling, usage of eye drops in one eye only, and ocular surface disease.
Eligible participants filled out a short questionnaire concerning the most obvious confounding factors (age, gender, ethnical background, medication usage, ophthalmic history, sleeping side, eye rubbing). After patients were put under general anesthesia, a drop of artificial tears (Thealoz Duo; Théa Pharma, Clermont-Ferrand, France) in one eye and a drop of topical anesthetics (Minims Oxybuprocaine Hydrochloride 0.4%; Bausch + Lomb, Aubenas, France) in the other eye were applied to each subject in a randomized way by a pre-informed nurse. A timed 2 to 3 minutes passed between application of the drops and the sampling. A drop of artificial tears was applied to prevent unblinding of the executer and to mimic any diluting effect of the anesthetic drops in the control eye. Both types of drops were preservative free. By using volunteers under general anesthesia, we assured similar pressure with or without topical anesthetic because the executer was not influenced by the patient's reaction. The left and the right eye were sampled in a standardized way by a blinded medical student or resident. The student was trained before sampling and supervised by an author (HD) during the first execution. A step-by-step protocol was available as a mnemonic device for subsequent sampling moments. Sampling was performed before any presurgical disinfection of the skin and conjunctival sac. A single, sterile, nylon, flocked swab (FLOQSwabs; Copan, Brescia, Italy) was rubbed from the nasal to temporal inferior conjunctival sac and simultaneously swirled in the opposite direction of the sampling itself. The swab was placed in an Eppendorf tube and stored for less than 2 weeks at –18°C before being transferred to a −80°C freezer until further processing.