Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides

Abstract

Background: Streptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening. Methods: One hundred two pregnant women who were at 35-37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses. Results: The real-time PCR assay showed 100% specificity and a limit of detection of 10 4 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7-99.6%), a specificity of 95.5% (86/90, CI 89.8-98.7%), a positive predictive value of 73.3% (11/15, CI 44.8-91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9-99.9%). Conclusion: The multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.