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The aim of this study was to evaluate if screening Group B Streptococcus colonization by intrapartum polymerase chain reaction could improve intrapartum administration of antibiotic prophylaxis, compared with antepartum culture screening and analyze the sensitivity and specificity of polymerase chain reaction test. 198 pregnant women with Group B Streptococcus colonization antepartum culture screening were included. When they arrived at hospital for delivery, two rectovaginal swabs were collected: for culture and polymerase chain reaction method. The rate of Group B Streptococcus colonization antepartum detected by culture was 16.7%; at delivery was 17.2% when detected by culture and 19.7% using polymerase chain reaction method. The rate of inconclusive polymerase chain reaction tests was 0.5%. Considering intrapartum culture screening as gold standard, sensitivity and specificity of polymerase chain reaction test for intrapartum Group B Streptococcus colonization was 97.1% and 95.7%, respectively. The global rate of discordance between antepartum and intrapartum Group B Streptococcus colonization was 6.6%. The rate of women not treated with intrapartum antibiotic prophylaxis in the setting of positive intrapartum culture was significantly lower using intrapartum polymerase chain reaction test (0.5%) than with antepartum culture method (3.5%, p = 0.035). The use of intrapartum antibiotic prophylaxis can be more efficient when screening Group B Streptococcus colonization intrapartum by polymerase chain reaction test. Polymerase chain reaction method had a good performance in our study, with high sensitivity and specificity. [ABSTRACT FROM AUTHOR]