Development and performance evaluation of a recombinase polymerase amplification assay for the rapid detection of group B streptococcus.

Christina Clarke,Heather Carré-Skinner,Olaf Piepenburg,Louise O’Connor,Terry J Smith

doi:10.1186/s12866-016-0836-y

Abstract

BackgroundDespite the implementation of prevention guidelines, group B Streptococcal (GBS) infection remains a leading cause of sepsis, pneumonia, and meningitis, resulting in significant neonatal morbidity and mortality. Preventive approaches that identify women at risk of transmitting GBS have reduced the incidence of neonatal GBS disease, and dramatically decreased the associated mortality rates. However, there is an on-going requirement for a near-patient diagnostic test for GBS that can be carried out at the time of delivery, ideally in the labour ward setting, particularly for women of unknown GBS colonisation status at the time of delivery.MethodsIn this study, a Recombinase Polymerase Amplification (RPA) assay was developed and performance evaluated for the detection of group B Streptococcus in vaginal swabs. The assay uses the cAMP factor (cfb) gene of GBS as the target gene. The analytical performance of the assay was evaluated by testing a panel of GBS reference strains and clinical isolates, and non-GBS organisms. The limit of detection was determined and the clinical performance was evaluated by testing 124 vaginal swabs from women with both GBS positive and negative status.ResultsBased on specificity testing carried out the assay was shown to be specific for the target of interest. The limit of detection of the assay was shown to be between six and 12 genome copies and was comparable to that of a real-time PCR assay, both achieving a limit of detection below 12.5 genome copies. The performance of both assays when applied to clinical samples was identical.ConclusionA specific, sensitive RPA assay for GBS was developed. The performance of the assay for testing of clinical samples is within the acceptable range.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0836-y) contains supplementary material, which is available to authorized users.

Highlights

Despite the implementation of prevention guidelines, group B Streptococcal (GBS) infection remains a leading cause of sepsis, pneumonia, and meningitis, resulting in significant neonatal morbidity and mortality
Analytical specificity of the Recombinase Polymerase Amplification (RPA)-GBS assay The inclusivity of the assay was determined by testing a panel of GBS isolates
Limit of detection of the assay The limit of detection of the assay was determined by preparing known concentrations of GBS (S. agalactiae BCCM 15081) DNA and testing in the RPA assay

Summary

Introduction

Despite the implementation of prevention guidelines, group B Streptococcal (GBS) infection remains a leading cause of sepsis, pneumonia, and meningitis, resulting in significant neonatal morbidity and mortality. Onset GBS infection can be transmitted to the neonate during birth as it travels through the birth canal of a colonised woman and can subsequently cause infection in the neonate [8]. In some cases it can be transmitted by ascending infection to the foetus if there is premature rupture of the membranes, puerperal endometritis or if the woman develops chorioamnionitis [8], though, these conditions are relatively rare. Intrapartum chemoprophylaxis seems to have little effect on late onset disease attack rates

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