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Diagnosis of sexually transmitted infections in children suspected of sexual abuse is challenging due to the medico-legal implications of test results. Currently, the forensic standard for diagnosis of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections is culture. In adults, nucleic acid amplification tests (NAATs) are superior to culture for CT, but these tests have been insufficiently evaluated in pediatric populations for forensic purposes.We evaluated the use of NAATs, using urine and genital swabs versus culture for diagnosis of CT and NG in children evaluated for sexual abuse in 4 US cities. Urine and a genital swab were collected for CT and NG NAATs along with routine cultures. NAAT positives were confirmed by PCR, using an alternate target.Prevalence of infection among 485 female children were 2.7% for CT and 3.3% for NG by NAAT. The sensitivity of urine NAATs for CT and NG relative to vaginal culture was 100%. Eight participants with CT-positive and 4 with NG-positive NAATs had negative culture results (P = 0.018 for CT urine NAATs vs. culture). There were 24 of 485 (4.9%) female participants with a positive NAAT for CT or NG or both versus 16 of 485 (3.3%) with a positive culture for either, resulting in a 33% increase in children with a positive diagnosis.These results suggest that NAATs on urine, with confirmation, are adequate for use as a new forensic standard for diagnosis of CT and NG in children suspected of sexual abuse. Urine NAATs offer a clear advantage over culture in sensitivity and are less invasive than swabs, reducing patient trauma and discomfort.