2.1 Tissue sampling
Twenty-two VKC patients (6 females, 16 males; mean age 10+3.5years) and 20 age, sex and ethnicity-matched healthy controls (HC) (7 females, 13 males; mean age 10+3.2 years) were recruited from the Ocular Allergy and Pediatric Ophthalmology services of the University of Padua between April 2019 and October 2019. Neither VKC patients nor healthy controls had a history of ocular diseases (other than VKC), infection, surgery or contact lens use. Diagnosis of VKC was based on the patient’s history and presence of typical signs and symptoms (21). Type of VKC (tarsal, limbal or mixed), positive skin prick test and/or presence of serum allergen-specific IgE (IgE positive sensitization), and disease-specific ongoing treatment (topical 1% cyclosporine A, antihistamines and/or mast cell stabilizers) were recorded. All the healthy controls were free from any topical and systemic treatment. None of subjects enrolled received antibiotics, corticosteroids, or non-steroidal anti-inflammatory drugs within 6 months (Table 1).
Each patient was swabbed at the inferior fornix of the right eye with a single-use, dry, sterile nylon swab (eSwab, Copan diagnostics Inc., Murrieta, CA, USA), placed in 1 mL sterile liquid amies medium after use and stored at -20°C until processing. All samples were collected by the same expert operator (A.L.) by passing twice in the inferior fornix avoiding touching the lid margin and without using topical anesthetic to prevent any potential contamination. Each patient/parent was also provided with a simple 10-items non-validated questionnaire investigating the presence of some of the principal factors related to allergy development including allergy family history, vaginal or cesarean delivery, maternal breastfeeding and/or formula feeding, weaning age, residence in an urban or rural area, varied or restricted diet, atopic dermatitis within the first year of life, repeated and prolonged contacts with pets, and family’s socioeconomic status. A validated, disease-specific questionnaire, the Quality of Life in Children with Vernal Keratoconjunctivitis (QUICK) questionnaire, was administered only to VKC (25). The study was approved by the Institutional Review Board and Local Ethical Committee, and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from a parent of all subjects enrolled.