Longitudinal qPCR Study of 5 Key Vaginal Species Figure 2. The presence and average concentrations of the 5 species during the study in group D. Legend: The chart above represents the total presence (%) of the 5 species during the days that were analysed. In addition, the actual days present versus the total days analysed are also given. The chart below represents the average concentrations (cells/ml) of the 5 species during the days that were analysed. doi:10.1371/journal.pone.0045281.g002 (#13 and #19) with a VMF shifting between grades Ib, II, I-like and IV VMF, were sporadically colonized by L. crispatus with an average bacterial concentration of log6 cells/ml. The two subjects (#18 and #25), with predominantly grade II VMF were characterized by high concentrations of L. crispatus (log5-log9 cells/ml), and – as for group N subjects with high a L. crispatus concentration, there was a 100-fold decrease during the menses. The two subjects with grade III, i.e. BV-associated VMF (#9 and #24), had no or very low concentrations of L. crispatus. Lactobacillus iners. For five out of 9 group N women, L. iners was found only around and during the menses, and only in low concentrations (log4-log6 cells/ml). In 2 women (#15 and # 22), L. iners and L. crispatus were present as a mixture during all days that were assessed. The women (#4 and # 20) with primarily grade Ib VMF carried high concentrations of L. iners (log10 cells/ ml on average). In Group D, the women (#9, #24) with predominantly grade III VMF and the women that shifted regularly between the grades Ib, II, III and IV (#13, #21, #25 ) had high concentrations of L. iners (log10 cells/ml), throughout the MCs. For 2 women (#18, #19) with constantly grade II and grade I-like/IV, respectively, L. iners was sporadically present, particularly just before, during and after the menses with concentrations varying between log5 and log6 cells/ml. Gardnerella vaginalis. In general, there was an increase in G. vaginalis concentrations when the VMF changed from normal to disturbed, and vice versa a decrease, when the disturbed VMF shifted to normal VMF (e.g. before, during and after the menses). More specifically, in Group N, all women carried G. vaginalis at a certain point in time (concentrations #log5 cells/ml ). G. vaginalis sialidase positive strains were virtually absent in Group N. G. vaginalis was present in 7 of the 8 women of group D and was only absent in one of the two subjects that used antibiotics. The two women (#13, #19) with a VMF shifting between grades Ib/ II/IV and grade I-like/IV VMF respectively, had G. vaginalis (sialidase negative strains only) concentrations lower than log6 cells/ml. Grade II and III VMF were characterized by G. vaginalis sialidase positive strains, mostly as a mixture together (minimum log8 cells/ml) with sialidase negative strains. Atopobium vaginae. A. vaginae was present in 6 of 9 women in Group N, but in 4 women only sporadically (log5 cells/ml on average) and mostly around the menses and/or when the VMF was disturbed (grade II). A. vaginae colonized two subjects during the entire study (average log9 cells/ml and log6 cells/ml respectively), with a 10 to 100-fold increase in bacterial concentrations during the menses. In Group D, half of the women, i.e. the women shifting between grades Ib/II/III or with continuous grade III VMF, had high concentrations of A. vaginae (log9 cells/ml average). The two PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45281