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Objectives: This study compared the sensitivity and specificity of culture and two nucleic acid amplification tests (NAATs): the BD Probetec ET system (PT) and the Aptima Combo 2 (AC2) in detecting Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) in pharyngeal and rectal specimens. Methods: Male subjects were prospectively recruited at an MSM clinic in Toronto, Canada. Pharyngeal and rectal specimens were obtained for GC and CT culture, PT and AC2. Urine was also obtained for PT. A true positive was defined as: (1) positive culture, (2) positive PT and AC2 at the same site or (3) a single positive NAAT and detection of the same organism by any method at another site. Results: 248 subjects were recruited. The prevalence of pharyngeal GC was 8.1%, rectal GC 11.7%, pharyngeal CT 2.0% and rectal CT 7.7%. The sensitivity of culture for pharyngeal GC and CT was 0%; 41.4% for rectal GC and 21.1% for rectal CT. The sensitivity of PT for pharyngeal GC, rectal GC, pharyngeal CT and rectal CT was 95.0%, 93.1%, 80.0% and 94.7%, respectively. The sensitivity of AC2 was 95.0% for pharyngeal GC and 100% at all other sites. Specificity was consistently above 98%. Conclusions: PT and AC2 detected GC and CT with superior sensitivity compared to culture. They detected 73 pharyngeal or rectal GC and CT infections compared to 16 by culture, using a rigorous gold standard. NAATs should be the method of choice for the detection of GC and CT in extragenital sites in men who have sex with men. [ABSTRACT FROM AUTHOR]