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In this three-year prospective study, 137 children with acute otitis media (AOM) that had not responded after one or two empiric antimicrobial treatment courses (termed persistent AOM) underwent tympanocentesis to determine additional antimicrobial therapy based on in vitro susceptibility testing of the bacterial isolate(s). One hundred eleven children with AOM not previously treated are described for comparison. In the persistent AOM group middle ear aspirates grew Streptococcus pneumoniae (24%), Haemophilus influenzae (7%), Brahamella catarrhalis (7%), Streptococcus pyogenes (6%), Staphylo-coccus aureus (5%), two pathogens (3%) or no bacterial growth (49%); pathogens in previously untreated AOM were similar but fewer patients (30%) had no bacterial growth. After tympanocentesis additional antimicrobial therapy for persistent AOM patients utilizing drugs shown to be effective in vitro against the isolated pathogen failed to produce clinical resolution of infection in 27 (28%) of ears. Differing clinical efficacy was observed with various antimicrobials: amoxicillin (57% failure); trimethoprim/sulfamethoxazole (75% failure); cefaclor (37% failure); cefixime (23% failure); amoxicillin/clavulanate (12% failure); and cefuroxime axetil (13% failure). Presumptive clinical cure for previously untreated AOM patients was similar to that for untreated AOM except for fewer amoxicillin failures (30%). We conclude that clinical failure in persistent AOM occurs (1) even when no pathogen is isolated from tympanocentesis (50% of patients) and (2) despite demonstrated in vitro activity against culture-proved pathogens.