title: Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.
abstract: 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.
authors.affiliation: IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, Braga 4710-057, Portugal.
authors.forename: António
authors.initials: A
authors.lastname: Machado
authors.forename: Carina
authors.initials: C
authors.lastname: Almeida
authors.forename: Débora
authors.initials: D
authors.lastname: Salgueiro
authors.forename: Ana
authors.initials: A
authors.lastname: Henriques
authors.forename: Mario
authors.initials: M
authors.lastname: Vaneechoutte
authors.forename: Freddy
authors.initials: F
authors.lastname: Haesebrouck
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Vieira
authors.forename: Ligia
authors.initials: L
authors.lastname: Rodrigues
authors.forename: Nuno Filipe
authors.initials: NF
authors.lastname: Azevedo
authors.forename: Nuno
authors.initials: N
authors.lastname: Cerca
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D005260:Female; D006194:Gardnerella vaginalis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007778:Lactobacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D012680:Sensitivity and Specificity; D016585:Vaginosis, Bacterial / Q000175:diagnosis
chemical_list: D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids