O3-S6.01 Improved diagnostics of bacterial vaginosis with molecular techniques

Abstract

Background: Bacterial vaginosis (BV) is a disturbance of the vaginal microflora. BV can cause discharge complaints and lead to pelvic inflammatory disease, ectopic pregnancy and premature birth. We evaluated a combination of PCR assay's and whole bacterial community analysis with the standard diagnostic algorithm to validate a molecular assay for the determination of BV. Methods: 160 women with vaginal discharge complaints were included. 80 women were classified as BV and 80 as non-BV according to Amsel criteria. Gram stains from vaginal smears were made for Nugent scoring. Vaginal swabs were tested with PCR assays for Gardnerella vaginalis, Atopobium vaginae, BV associated bacterium type 2 (BVAB2) and Megaspheara type 1 (MS1). Whole bacterial community analysis was performed by fluorescent Terminal Restriction Fragment Length polymorphism (TRFLP) of 16S-rDNA. TRFLP patterns and predictive fragments of a number of BV associated bacteria were analysed with Bionumerics software (Applied Maths, Belgium). Results: Compared to Amsel criteria, the highest sensitivity of 100% was achieved with a duplex PCR for G. vaginalis and/or A vaginae and the highest specificity of 86% was found with a singleplex BVAB2 specific PCR. Best overall performance was shown using a duplex real time PCR for BVAB2 and/or MS1 with a sensitivity of 90% and a specificity of 78% with respect to Amsel criteria. Using Nugent criteria as a standard, this duplex PCR has a sensitivity of 84% and specificity of 86%. From TRFLP results, the presence of predictive fragments of Prevotella, Aerococcus, Megaspheara, Mycoplasma, Peptostreptococcus, Leptotrichia, Eggerthella, Gardnerella, Atopobium and Dialister was most associated with BV positive samples. Cluster analysis of microbial profiles revealed clear differences between BVand non-BVand indicated possible intermediate or transition stages. Conclusions: A combination of bacterial species are involved in BV. For molecular diagnostics a duplex PCR of Gardnerella en/of Atopobium can be used for initial screening confirmed by a BVAB2 specific PCR. A more effective alternative is a real time duplex PCR targeting BVAB2 and/or MS1. Microbial profiling supports most targets used in the PCR assays. Cluster analysis of microbial profiles can be used to interpret discordant validation results and possibly for diagnosis.