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Microbiologic diagnosis of septic arthritis and osteomyelitis in children is hindered by the less than optimal yield of blood and osteoarticular fluid cultures.All patients admitted to a pediatric unit for osteoarticular infections (OAI) between January 2001 and February 2004 were enrolled in this prospective study. Osteoarticular fluid and biopsy samples that were negative by conventional culture were tested by polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers.We enrolled 171 children. Culture was positive in 64 cases (37.4%), yielding Kingella kingae in 9 cases. The 107 culture-negative specimens were tested by 16S ribosomal DNA PCR. Fifteen samples (14%) were positive, all for Kingella DNA sequences. K. kingae was the second cause of OAI in this population (30.4%), after Staphylococcus aureus (38%). Patients with Kingella infection diagnosed by culture (9 cases) did not differ from those diagnosed by PCR (15 cases) in terms of their clinical characteristics (including prior antibiotic therapy). The characteristics of the 24 children with arthritis (n = 17) or osteomyelitis (n = 7) were similar to those reported elsewhere. Fever (>38°C) and symptom onset shortly before hospitalization (median, 4.5 days) were significantly associated with arthritis.Use of molecular diagnostic methods increases the identification of K. kingae in osteoarticular infections.