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A total of 96 non-duplicate male patients with urogenital infections from 2015 to 2016 were included,they were randomly divided into urine group (n=60) and urethral swabs group (n=36).Urine specimens were split,not duplicated,and parallel aliquots were tested by culture,culture and sequencing,and Isothermal RNA Amplification Assay (IRAA) (16S rRNA).The urethral swabs were detected by culture and culture together with sequencing,respectively.The positive rate (%) of Ureaplasma in the two groups were 21.7% (13/60) and 19.4% (7/36) (p=1.000),respectively.In urine group,compared with culture and sequencing,sensitivity and specificity of IRAA were 91.7% (11/12) and 93.8% (45/48),respectively,and those of culture were 91.7% (11/12) and 100% (48/48),respectively.In urethral swabs group,compared with culture and sequencing,sensitivity and specificity of culture were 100% (7/7) and 100% (29/29),respectively.Of the 19 Ureaplasma positive specimens,68.4% (13/19) contained U.parvum only,31.6% (6/19) contained U.urealyticum only,and 0.0% (0/19) contained both.Furthermore,among the 19 Ureaplasma positive specimens,ciprofloxacin and ofloxacin showed the lowest in vitro activity.Our data suggested that urine was highly consistent with urethral swabs for detection of Ureaplasma in male patients with urogenital infections.And culture was a reliable method,with higher specificity,and even better than IRAA.