Evaluation of a novel real-time PCR test based on the ssrAgene for the identification of group B streptococci in vaginal swabs

Abstract

BackgroundDespite the implementation of prevention guidelines, early-onset group B streptococci (GBS) disease remains a cause of neonatal morbidity and mortality worldwide. Strategies to identify women who are at risk of transmitting GBS to their infant and the administration of intrapartum antibiotics have greatly reduced the incidence of neonatal GBS disease. However, there is a requirement for a rapid diagnostic test for GBS that can be carried out in a labour ward setting especially for women whose GBS colonisation status is unknown at the time of delivery. We report the design and evaluation of a real-time PCR test (RiboSEQ GBS test) for the identification of GBS in vaginal swabs from pregnant women.MethodsThe qualitative real-time PCR RiboSEQ GBS test was designed based on the bacterial ssrA gene and incorporates a competitive internal standard control. The analytical sensitivity of the test was established using crude lysate extracted from serial dilutions of overnight GBS culture using the IDI Lysis kit. Specificity studies were performed using DNA prepared from a panel of GBS strains, related streptococci and other species found in the genital tract environment. The RiboSEQ GBS test was evaluated on 159 vaginal swabs from pregnant women and compared with the GeneOhm™ StrepB Assay and culture for the identification of GBS.ResultsThe RiboSEQ GBS test is specific and has an analytical sensitivity of 1-10 cell equivalents. The RiboSEQ GBS test was 96.4% sensitive and 95.8% specific compared to "gold standard" culture for the identification of GBS in vaginal swabs from pregnant women. In this study, the RiboSEQ GBS test performed slightly better than the commercial BD GeneOhm™ StrepB Assay which gave a sensitivity of 94.6% and a specificity of 89.6% compared to culture.ConclusionThe RiboSEQ GBS test is a valuable method for the rapid, sensitive and specific detection of GBS in pregnant women. This study also validates the ssrA gene as a suitable and versatile target for nucleic acid-based diagnostic tests for bacterial pathogens.

Evaluation of Culture, Antigen Detection and Polymerase Chain Reaction for Detection of Vaginal Colonization of Group B Streptococcus (GBS) in Pregnant Women

“…Although we cannot rule out the possibility of detection of nonviable GBS by antigen detection and PCR assays, several conditions can explain the false negative culture results. These include antibiotics, feminine hygiene products and scanty colonization which would be difficult to obtain in culture [14,15]. We excluded the patients with prior antibiotic treatment in the third trimester of pregnancy.…”

Rapid Detection of Streptococcus agalactiae Infection Using a Loop-Mediated Isothermal Amplification Method

“…Using the selected primer set, the species-specific cAMP gene of S. agalactiae was amplified and detected without magnification in 40 minutes. While culture detection requires 36 -72 hours (6,15) and antigen detection or PCR-based assay need over two hours (7,(16)(17)(18). Secondly, LAMP method has high sensitivity for detection of S. agalactiae.…”

La synthèse des protéines par le ribosome

“…L'ARNtm peut alors représenter une alternative efficace. En effet, les séquences hautement conservées des deux extrémités 5' et 3' de l'ARNtm, couplées à sa petite taille, permettent la conception d'amorces universelles qui permettent d'amplifier aisément la partie centrale des gènes codant pour l'ARNtm, dont la divergence est a contrario considérable entre les espèces [41,42]. La méthode a été employée avec succès pour discriminer de nombreuses espèces bactériennes, que ce soit dans des échantillons biologiques humains ou divers aliments [43].…”