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Background  Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by  Lactobacillus  species together with an increase of  Gardnerella vaginalis  and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence  in situ  Hybridization assay (PNA FISH) for the detection of  Lactobacillus  spp. and  G. vaginalis  in mixed samples.    Results  Therefore, we evaluated and validated two specific PNA probes by using 36 representative  Lactobacillus  strains, 22 representative  G. vaginalis  strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of  G. vaginalis  and  Lactobacillus  species  in vitro,  in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for  Lactobacillus  spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for  G. vaginalis . Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH.    Conclusions  This quick method accurately detects  Lactobacillus  spp. and  G. vaginalis  species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.

Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.