because it detects the most abundant species, associated with normal or disturbed VMF, but it misses A. vaginae, which produces small, slowly growing colonies, which are difficult to detect among the other BV-associated species, and also misses largely P. bivia. The effect of antibiotic therapy as determined by qPCR and culture Two women (group D) used antimicrobial agents (not specified) during the course of this study. Subject #16 followed an antimicrobial cure. Before the cure, L. iners was present at log10–log11 cells/ml, culture revealed also the presence of L. vaginalis and yeast was detected with Gram stain. During the cure, L. iners, L. vaginalis and the yeast were eradicated and instead Finegoldia magna, Atopobium parvulum, Enterococcus faecalis and Staphylococcus epidermidis were detected with culture and A. vaginae on one day with qPCR. These species possibly colonized the vagina from the rectum [55,56] during the cure. F. magna, A. parvulum, E. faecalis and S. epidermidis persisted after treatment, for the rest of the study period as determined by culture. According to qPCR results, A. vaginae, P. bivia and L. iners were detected after the cure and the yeast re-emerged as detected with Gram stain. Subject #21 followed a 9-day antibiotics cure. Before the antibiotics cure L. crispatus, L. iners, A. vaginae, G. vaginalis and P. bivia were present as determined by qPCR and L. gasseri was also present as determined by culture. During the antibiotics treatment, L. crispatus and L. gasseri disappeared completely and Escherichia coli and F. magna appeared, as determined by culture. After the cure, yeast appeared temporarily on Gram stain, and L. gasseri and L. crispatus only re-emerged post treatment at 2 and 3 weeks, respectively. These results suggest that antibiotic treatment severely affects the VMF and that (vaginal) probiotics may be recommendable to help restore the VMF after any antimicrobial treatment. Comparison of qPCR/culture and pyrosequencing Recently, Gajer et al. [12] published the first longitudinal study by pyrosequencing [12]. Although it is not easy to compare their results to those previously published, because of their use of different categorical names (community state types instead of grades), it is possible to point to several similar findings. The study of Gajer et al. [12] and this one could both establish i) strong interindividual variability of the VMF of different women, ii) similar declines of L. crispatus during the menses for the women with predominantly L. crispatus, iii) mostly in combination with replacement by L. iners or by Gram positive cocci, iv) long term stability of the VMF in most women, v) highest stability for women with grade III VMF (when assuming that their community state type (CST) IV-B largely corresponds to grade III VMF or BV- associated VMF). Gajer et al. [12] considered long term stability as another hallmark of normal VMF. However, one should be cautious, because stability does not necessarily equals eubiosis. In our longitudinal study ([10] and this study), we found that women with grade III VMF (i.e. BV associated VMF) had even more stable VMF than women with predominantly grade I VMF, and that most of these women were asymptomatic. This should not be considered as normal VMF, because also chronic BV is characterized by a very stable VMF, but it is associated with G. vaginalis and A. vaginae biofilm formation [57]. In conclusion, the more recent pyrosequencing based micro- biome studies [2,12,19] have expanded our view on the diversity of the vaginal bacterial population, but is too precocious to state that they come to opposite conclusions with respect to past studies with culture-dependent and culture-independent techniques. Longitudinal qPCR Study of 5 Key Vaginal Species Conclusions Despite the temporal, short term, dynamic changes, the VMF of most women in this study displayed long term overall stability. The principle cause of disturbance of the VMF in this study were the menses, in accordance with findings of previous studies. The use of antibiotics was also a major disturbing factor. The number of assessable incidences of sexual intercourse, although showing some influence on the presence of A. vaginae and G. vaginalis, P. bivia, was too low to draw valid conclusions. Women with grade Ia VMF, i.e. L. crispatus dominated VMF, combined long term stability with short term menses-associated fluctuations of the VMF, which entailed the reduction of L. crispatus and the strong increase of the BV-associated bacteria (A. vaginae, G. vaginalis and P. bivia) and of L. iners, which we previously also found to be strongly associated with BV-associated VMF. The VMF of women with grade III VMF showed even higher stability than grade I VMF, with constant high concentrations (log8 cells/ml average) of L. iners, A. vaginae, G. vaginalis (predominantly sialidase positive) and P. bivia, not changing during menses, and the virtual absence of L. crispatus. Only for a limited number of women, frequent short term fluctuations, i.e. shifts between grades Ib, II, III and IV VMF, were observed, with high concentrations of both L. crispatus and the BV-associated bacteria L. iners, A. vaginae, G. vaginalis and P. bivia, possibly indicating continued competition between L. crispatus and the BV-associated anaerobes. The presence of stability in grade I, and possibly even more in grade III VMF was reported in our previous study [10] and this finding was recently reconfirmed in the longitudinal pyrosequenc- ing study by Gajer et al. [12]. Limitations of the study A qPCR study is limited to the species that are analyzed, which implies that most of the vaginal microflora is not analyzed. However, due to the combination of culture [10] and qPCR, we obtained complementary information, i.e. A. vaginae and P. bivia that remained undetected in the culture study were detected by qPCR and more women appeared to be colonized by A. vaginae, G. vaginalis, L. iners and P. bivia with qPCR than could be assessed with culture. Jellyfish DNA was used as an internal inhibition control, indicating that drops in bacterial concentrations during the menses, as established with five qPCR formats were not due to PCR inhibition, i.e. not caused by blood present in the swab and/ or DNA-extract. Moreover, according to qPCR, the concentra- tions of several species increased during or around menses. No human 18S rRNA gene qPCR was used to measure human DNA levels, verifying that the swab contacted a human tissue surface and that the extraction was successful. However, we mostly analyzed DNA extracts from subsequent days, so these samples could serve as controls for each other and any aberration would not have gone unnoticed. Not all days of the 2 menstrual cycles were analyzed by qPCR for all women. Although the analyzed days were well chosen, we cannot exclude that interesting observations could have been missed. Other possible limitations to this paper were reported previously [10]. Supporting Information File S1 Bacterial concentrations of Lactobacillus cris- patus, L. iners, Atopobium vaginae, Prevotella bivia and sialidase positive Gardnerella vaginalis during the PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45281