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Background: Chronic adenoid infection by β-lactam-resistant Haemophilus influenzae type b (Hib) and biofilm formation contribute to adenoid hyperplasia. Middle ear disease consequently remains a critical issue in the pediatric population. The aim of this study was to investigate the correlation of Hib biofilm formation with middle ear effusion with adenoid hyperplasia (MEE-AH) and with pediatric obstructive sleep apnea (OSA).Methods: A total of 384 patients with adenoidectomy from January 2008 to December 2012 were recruited in this investigation. Thirty-two patients (14 female and 18 male; age 4-13 years) who obtained routine adenoidectomy surgery had Hib-positive cultures were enrolled in a retrospective manner. By using polysomnography, 18 patients were diagnosed as having MEE-AH with chronic adenotonsillitis, and 14 patients were diagnosed as having pediatric OSA. The results of the Hib biofilm, antibiotic resistance profiles, and scanning electron microscopy observation, which correlated with the clinical diagnosis, were analyzed by the chi-square test and Fisher exact test.Results: Biofilm formation by Hib was significantly present in the patients with MEE-AH rather than patients with OSA. β-lactam-sensitive Hib were resistant to augmentin because of the adenoid biofilm formation. However, this finding was uncommon in the pediatric OSA group.Conclusions: Properly treating β-lactam-sensitive Hib infection may be an important issue in reducing MEE-AH and adenoid vegetation in the pediatric population. Further research is warranted to elucidate the association of Hib-related biofilm formation with treatment failure and the need to consider earlier surgical intervention. [ABSTRACT FROM AUTHOR]