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Background: Bronchiolitis is a major cause for hospitalisation in young children during the winter season, with respiratory syncytial virus (RSV) as the main causative virus. Apart from standard hygiene measures, cohorting of RSV-infected patients separately from RSV-negative patients is frequently applied to prevent crossinfection, although evidence to support this practice is lacking. The objective is to evaluate the risk of room sharing between RSV-positive and RSV-negative patients. Methods: We performed a prospective observational cohort study in children < 2 years hospitalised with acute bronchiolitis. During the first day of admission, patients shared one room, pending results of virological diagnosis (PCR). When diagnostic results were available, RSV-positive and RSV-negative patients were separated. Standard hygienic measures (gowns, gloves, masks, hand washing) were used in all patients. Results: We included 48 patients (83% RSV-positive). Co-infection was found in nine patients at admission, and two during hospitalisation (23%). The two patients with acquired co-infection had been nursed in a single room during the entire admission. None of 37 patients sharing a room with other bronchiolitis patients (20 with patients with a different virus) were co-infected during admission. Disease severity in co-infection was not worse than in monoinfection. Conclusion: One in five patients with bronchiolitis was co-infect ed, but co-infection acquired during admission was rare and was not associated with more severe disease. Room sharing between RSV-positive and RSV-negative patients (on the first day of admission) did not influence the risk of co-infection, suggesting that cohorting of RSV-infected patients separate from non-RSV-infected patients may not be indicated.