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Evaluation and subsequent optimizations of the quantitative AmpliSens Florocenosis/Bacterial vaginosis‐FRT multiplex real‐time PCR assay for diagnosis of bacterial vaginosis

Traditional microscopy‐based methods for diagnosis of bacterial vaginosis (BV) are underutilized in many settings, and molecular techniques may provide opportunities for rapid, objective, and accurate BV diagnosis. This study evaluated the quantitative AmpliSens Florocenosis/Bacterial vaginosis‐FRT multiplex real‐time PCR (Florocenosis–BV) assay. Vaginal samples from a previous study including unselected female subjects (n = 163) and using Amsel criteria and 454 pyrosequencing for BV diagnosis were examined with the Florocenosis–BV test and additionally tested for the presence and quantity of Gardnerella vaginalis clades 3 and 4. The Florocenosis–BV assay demonstrated 100% and 98% sensitivity compared with the Amsel criteria and 454 pyrosequencing, respectively, with 91% specificity. The modified Florocenosis–BV assay (detecting also G. vaginalis clades 3 and 4) resulted in 100% sensitivity vs the Amsel criteria and 454 pyrosequencing with specificity of 86% and 88%, respectively. Further optimizations of thresholds for the quantitative parameters used in the kit resulted in 99–100% accuracy vs Amsel criteria and 454 pyrosequencing for selected parameters. The Florocenosis–BV assay is an objective, accurate, sensitive, and specific method for BV diagnosis; however, the performance of the test can be further improved with some minor optimizations.