iners (in conjugation with high loads of G. vaginalis) are commonly associated to the microflora of BV diagnosed women [4, 7,

51]. The efficiency of our multiplex PNA-FISH methodology was demonstrated by ability of the PNA probes to hybridize in a large range of Lactobacillus spp. and G. vaginalis concentrations, even in the presence of epithelial cells (see Table 4). Swidsinski and colleagues [10, 47] used a multiplex FISH methodology to study BV biofilms. A drawback of their approach is that it requires pre-treatment with lysozyme before fixation and the use of urine or paraffin-embedded samples, in opposition of our methodology that do not require a pre-treatment for FISH analysis. These experimental steps increase analysis time and decrease FISH efficiency for Lactobacillus spp. and G. vaginalis strains detection, due to the lower PD0332991 concentration number of cells available for hybridization. Another DNA hybridization test for vaginal infection was studied by Witt and colleagues that evaluated the Affirm VPIII Kit [59], which detected LY2109761 datasheet G. vaginalis, Candida spp. and Trichomonas vaginalis in clinical samples, using two distinct single-stranded nucleic acid probes for each organism, which makes the analysis more complex and vulnerable to experimental pitfalls. This validated method showed sensitivity and specificity values for G. vaginalis of 89.5% and 97.1%, respectively, both lower than our Gard162

experimental values (95.0% and 100%, respectively). Furthermore, Fredricks and colleagues developed a FISH methodology for molecular identification of unknown bacteria associated with BV [6], using DNA probes Eub338-Cy5 and G.vag198-Cy3. However, the Eub338 is an unspecific probe used to detect Lactobacillus spp., detecting all species of the order Bacillales, and G.vag198 corresponds to a twenty five oligonucleotide probe with high specificity (100%) but with low sensitivity (85.0%) when compared to our probe (see Table 2). Both these probes worked together at a hybridization temperature of 45°C, which may easily lead to the occurrence of false positive results. Moreover, previous studies

have shown that probes with Cy fluorochromes present a lower fluorescence signal than those with the corresponding Alexa Fluor [60]. selleck chemical To conclude, our main purpose was achieved by demonstrating the in vitro applicability of the PNA multiplex methodology for detection of Lactobacillus species and G. vaginalis in the presence of the HeLa epithelial cell line and other taxonomically related or pathogenic Selleck CUDC-907 bacterial strains commonly found in vaginal samples. These in vitro results confirmed the previous in silico analysis from Lac663 and Gard162 probes. Conclusions In summary, the use of the PNA multiplex FISH assay described here significantly increases the specificity and sensitivity of the detection of Lactobacillus spp. and G. vaginalis strains in mixed samples and no interference was observed in the presence of human epithelial cells.