vaginalis, P. bivia and A. vaginae appeared to be constituents of the normal vaginal community in our study population, but remained suppressed, increasing in numbers predominantly around menses or because of other disturbances, in association with a decline of L. crispatus. Indeed, around and during the menses, when the L. crispatus concentrations decreased, G. vaginalis (9 of 9 women), A. vaginae (6/9), P. bivia (8/9) and L. iners (6/9) appeared or their levels increased several logs. In general, the presence of L. crispatus on all analyzed days, in combination with its numerical dominance, appeared to be protective against disturbance of grade I VMF and the two women with the most stable grade I VMF appeared to have a lower species diversity than the other subjects (as determined by culture [10] and qPCR (this study)). In the present study, concentrations of L. crispatus below log7 cells/ml corresponded in general with poor H2O2-production. One exception was the subject that was colonized by L. jensenii (a strong H2O2-producer [43]) throughout the follow up period, as determined in our previous culture-based study [10]. It has been hypothesized that H2O2 production by L. crispatus and L. jensenii strongly contributes to colonisation resistance [44,45]. However, recent studies by O’ Hanlon et al. [46,47] have shown that cervicovaginal fluid and semen have a significant H2O2-blocking activity and that physiological concentrations of H2O2 below 100 mM did not kill any of the tested BV-associated bacteria, e.g. A. vaginae, G. vaginalis, P. bivia [46,47]. An observation supporting the findings by O’ Hanlon et al. [46,47], is the fact that in this study, in the two subjects with predominant grade II VMF and with L. crispatus concentrations above log7 cells/ml, the concomitant high H2O2 production (score 2–3) did not seem to be protective against the presence of high concentrations of BV-associated bacteria. L. iners, present in all 17 women, had an almost inverse pattern compared to L. crispatus: in women with normal VMF, it was largely absent, or barely (only around the menses) present. Interestingly, grade Ib, thus far considered as normal VMF, was characterized by high concentrations of L. iners (average log10 cells/ml). In general, L. iners appeared to be an important constituent of grade Ib, II and III VMF, confirming earlier culture results [10] and qPCR results [17] of our group. This is not in contradiction with other studies, claiming that L. iners is a predominant constituent of the VMF [2,12,16,48], with the remark that all of our studies [10,14,15,17,21] indicate that L. iners is associated with disturbed VMF, whereas others consider this species as part of the normal VMF. Compared to L. crispatus or L. jensenii, L. iners produces less H2O2 [49,50] and provides relatively lower resistance to colonization to pathogens [15,51]. In addition, Hummelen et al. [19] found that, when L. crispatus is not present in the vaginal econiche, high concentrations of L. iners are required to ensure a low pH. Growth assays showed that L. iners’ preferred carbon sources are mucins, and glycosidase activity has been predicted [20]. These metabolic properties, i.e. haemolytic and mucinolytic activity, are indicative for a more pathogenic role of L. iners, enabling it to thrive in disturbed vaginal conditions, i.e. at high pH and in a VMF dominated by anaerobes, and confirming our repeated findings of an association of this Lactobacillus species with disturbed VMF. Whether or not L.iners can play a role in initial restoration of the VMF remains to be elucidated. G. vaginalis was detected in all but one woman, but concentra- tions were very variable. G. vaginalis has been found to be present in normal VMF in several studies [10,13,16,17,19], especially around the menses. However, Roy et al. [52] found that symptoms that involve G. vaginalis were associated with .107 cfu of this species per gram of vaginal fluid [52], suggesting that the presence of G. vaginalis below this threshold is not problematic. Longitudinal qPCR Study of 5 Key Vaginal Species In general, we found that there is a tendency for the presence of sialidase producing G. vaginalis strains in disturbed VMF conditions (i.e. grade II and III, but not grades I-like and IV), although the association is not absolute. Sialidase production has been linked to biofilm production, e.g. for Streptococcus pneumoniae and Pseudomonas aeruginosa (discussed in Lopes et al. [29]), and it has been shown that there are aggregating, biofilm producing G. vaginalis strains, more associated with BV, and non-aggregative, commensal G. vaginalis strains [53]. Whether there is a link between sialidase positive G. vaginalis and biofilm formation, or between sialidase producing G. vaginalis and (recurrent) BV or preterm birth remains to be studied. P. bivia was present in 13 of the 17 women of which unexpectedly 8 out of 9 group N women, although with low concentrations (log4–log6 cells/ml), and predominantly situated around the menses or when the VMF shifted to grade I-like or grade II. In Group D, P. bivia was related to grade III BV- associated VMF, with concentrations of 2 to 3 logs higher in comparison to Group N. The presence of P. bivia was also higher around the menses. Overall, the genus Prevotella has been found to be present in normal and intermediate VMF, but predominantly and in high numbers in BV [2,16,19]. A. vaginae was present in 14 of the 17 women, i.e. also in 6 of the 9 women from Group N, but in the latter group only occasionally, and predominantly around menses and at concentrations below log6 cells/ml. In our previous qPCR based study [17] and in other studies [16,22,54], A. vaginae was also occasionally detected in normal VMF, at low numbers, and also more prominently during BV episodes. In Group D, the presence of A. vaginae coincided primarily with grade III VMF. This and previous studies indicate that A. vaginae might be a more reliable marker for BV than G. vaginalis [17,21], also because it is less frequently present than G. vaginalis in high concentrations in non grade III VMF, but it may be on the safe side to test for both A. vaginae and G. vaginalis to diagnose BV, as was suggested by Menard et al. [22]. Comparison of qPCR and culture results We compared our qPCR data (this study) with those obtained by culture for the same group of subjects [10]. When qPCR and culture results were combined, 3 to 4 Lactobacillus spp. were detected per woman in group N and 2 to 4 Lactobacillus spp. were detected per woman in group D. qPCR detected L. iners in all 9 women of group N, but this species was cultured only in 4 women, of which the 2 subjects with grade Ib VMF. L. crispatus was detected in all group N women with qPCR and in all, but the two with grade Ib with culture; A. vaginae was present in 6 women according to qPCR but in none according to culture; G. vaginalis was detected in all 9 women with qPCR, but in only 3 women with culture, and P. bivia was detected in 8 women with qPCR, but in none with culture. In group D, L. iners was detected in all 8 women with qPCR and in 5 with culture; L. crispatus was detected in all women with qPCR, but in only 2 women ( grade II and shifting grade II, Ib, IV, III) with culture; A. vaginae was detected in all women with qPCR, but in none with culture; G. vaginalis was detected in all 8 women with qPCR and in 6 women with culture; and P. bivia was detected in 5 women with qPCR and in only 2 women with culture. In summary, qPCR was more sensitive than culture, whereas culture detected only high concentrations of bacteria or failed to detect fastidious bacteria, such as A. vaginae and P. bivia. For research purposes, the qPCR results are of interest to show that BV associated species are present in low numbers in normal VMF. Culture remains useful for diagnostic purposes to some degree, PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45281