according to other studies [18–20]. A. vaginae and G. vaginalis being important markers for BV [17,21–24] were also assessed. qPCR was also performed for the G. vaginalis sialidase gene, since the presence of sialidase is considered as an indicator for BV and preterm birth (PTB) [25–28]. Moreover, we found that there is a clear genotypic distinction between sialidase producing and sialidase negative G. vaginalis strains [29]. Finally, qPCR for the P. bivia mucin-desulfating sulfatase gene (mdsC) was developed, because the activities of sialidase and sulfatase, both produced by P. bivia [30], have been suggested to be rate-limiting in the breakdown of the mucin layer and thus to be important factors in the pathogenesis of BV [30–33]. Moreover, some studies have correlated the presence of P. bivia in vaginal fluid with an important increase in PTB [34,35] and P. bivia has also been shown to enhance the growth of G. vaginalis through ammonia production [36]. In addition, we also assessed the presence of vaginal microor- ganisms producing hydrogen peroxide by culture. Some Lactoba- cillus strains have been shown to produce H2O2, which has been generally accepted as an important defense mechanism against vaginal colonisation by pathogens and a lack of vaginal H2O2- producing lactobacilli has been associated with the acquisition of BV [37–39]. Briefly, the general objectives of this study were to assess the presence and to quantify L. crispatus and L. iners during the menstrual cycle, also in relation to the BV-associated species, to assess the presence and concentrations of sialidase positive strains of G. vaginalis in normal and disturbed VMF, to assess the influence of the menstrual cycle and sexual intercourse on the presence and concentrations of the 5 species, and finally to add complementary information to the culture study previously performed [10] on this study population. Materials and Methods Ethics Statement This study was approved by the research ethics committee (EC UZG 2008/439) of the Ghent University Hospital (GUH), Belgium. Subjects Twenty-five female volunteers, aged between 18 and 35 years, were recruited after oral and written informed consent. The inclusion criteria were a regular MC and no use of contraception, except condoms. The exclusion criteria were pregnancy, com- plaints about malodorous vaginal discharge, recent history of vulval irritation, chronic use of medication, the use of antibiotics, antimycotics and antiprotozoals during the past two months, a history of vaginal surgery or hysterectomy, pelvic inflammatory disease, recurrent vaginal infections, an active vulval or vaginal dermatological aberration, vaginal douching within the last week before the study and symptomatic candidiasis or a positive Chlamydia trachomatis PCR result. After screening, 22 women were included, of which 17 completed the study. Study design The participants were asked to take vaginal swabs (ESwab, Copan, Brescia, Italy) each day of the study. The swabs were stored at 4uC and once a week all swabs were transported to the GUH. A Gram stain was made from a smear of each vaginal swab. Only the last swab of each week, taken on the day of transport to the GUH, was cultured anaerobically on Schaedler agar, Colombia agar and TMBplus agar [10]. From a selection of swabs Longitudinal qPCR Study of 5 Key Vaginal Species (see below), 200 ml (per swab) was used for DNA-extraction with the EasyMag platform (bioMe´rieux, Marcy l’Etoile, France). To limit the workload and cost of the study, DNA-extraction and qPCR were carried out only on a selection of the samples, according to the following criteria: each day of the menses, two days before and two days after the menses, two days before and after a change of the grade Ia VMF (see below), as established based on Gram stain of the vaginal swab, every other day during the change in VMF, on the day of sexual intercourse, each day that the VMF remained changed after the sexual intercourse, and until two days after the VMF restored its previous status. The number of days that were assessed per volunteer varied from 14 days (#15) to 56 days (#9) (Figures 1 and 2). Grading of Gram-stained vaginal smears The Gram stained vaginal smears were scored by four independent assessors according to previously described modified Ison and Hay criteria [10,14]. For reference, grades Ia, Iab, Ib and I-like correspond with Nugent score 0–3, grade II and IV with Nugent score 4–6 and grade III with Nugent score 7–10. However, in the modified Ison and Hay criteria, grade I-like is not considered as normal VMF [14] and the position of grade Ib as normal VMF is questionable [10]. DNA-extraction from swab medium For DNA-extraction, the ESwab medium obtained from each vaginal swab was pretreated. A total volume of 200 ml of the E- swab medium was transferred to a 2 ml tube, to which 200 ml of buffer (20 mM Tris-HCl, pH 8.0, 0.5% SDS) was added. Subsequently, 2 ml of mutanolysin (25 U/ml) was added and the mixture was incubated for 15 min at 37uC. Next, 10 ml of a 25 mg/ml proteinase K solution was added and the mixture was incubated for 15 min at 55uC. Finally, NucliSENS EasyMAG lysis buffer was added to a final volume of 2 ml, and incubated for 10 min at room temperature. Pretreated swab medium was stored at 280uC until DNA extraction. DNA extraction was performed on the NucliSENS EasyMAG (BioMe´rieux) platform, according to the manufacturer’s instructions. qPCR qPCR assays for Lactobacillus crispatus, L. iners, Atopobium vaginae, Gardnerella vaginalis, and G. vaginalis sialidase gene were carried out as described previously [17,29]. The sequence of the mucin-desulfating sulfatase gene (mdsC, NZ_ADFO01000048) from the partially sequenced P. bivia strain JCVIHMP010 (reference genome for the Human Microbiome Project, J. Craig Venter Institute, San Diego, CA) served as the template for designing the primer set for amplification of the mdsC gene from P. bivia. Primer Blast (NCBI) was used to design the following primer set: PBsulF (59 ACGTTTGGG- CAAAGCTCCTTGTCT) and PBsulR (59GCGTGTACGC- CAGTTGCAAGA). Annealing of primers on secondary struc- tures was analysed using mFOLD (http://mfold.rna.albany.edu/ ?q = mfold). Quantification of the P. bivia mucin-desulfating sulfatase gene was carried out after amplification with the LC480 SYBR GreenH I master kit (Roche, Basel, Switzerland) on the Light Cycler 480 real-time PCR system (Roche, Basel, Switzerland). Jellyfish DNA was used as an internal inhibition control, whereby a difference of 2 Cq or more was used as a cut-off to classify a sample as having significant qPCR inhibition [40]. PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45281