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Aim: Our aim was to study prospectively the aetiology of neonatal conjunctivitis in a population-based setting.Methods: Altogether 173 neonates with clinical conjunctivitis aged on average 20 (SD 10) days were recruited from child welfare clinics in Oulu, Finland, in 2010-2015. Conjunctival specimens were collected from 167 neonates for multiplex polymerase chain reaction to detect 16 respiratory viruses, from 163 for polymerase chain reaction to detect Chlamydia trachomatis and Neisseria gonorrhoeae and from 160 for bacterial culture studies. The cases were followed up until the age of 18 months.Results: Viral conjunctivitis was diagnosed in 8/167 (4.8%; 95% CI 2.1-9.2%), chlamydial or gonococcal conjunctivitis in 0/163 cases (0%; 95% CI 0-2.2%) and other bacterial conjunctivitis in 58/160 (36%; 95% CI 29-44%). Rhinovirus was found at the ocular site in 4/167 (2.4%) neonates, adenovirus in 3/167 (1.8%) and bocavirus in 1/167 (0.6%). The most commonly isolated bacteria included Staphylococcus aureus (16%), Moraxella catarrhalis (9.4%) and Streptococcus pneumoniae (3.1%). None of these pathogens was associated with the 4/173 (2.3%) cases later operated on for persistent nasolacrimal duct obstruction.Conclusion: Chlamydia trachomatis was a rare pathogen in neonatal conjunctivitis in a population-based setting, but respiratory viruses were detected more frequently than indicated earlier. [ABSTRACT FROM AUTHOR]