Longitudinal qPCR Study of the Dynamics of L. crispatus , L. iners , A. vaginae , (Sialidase Positive) G. vaginalis , and P. bivia in the Vagina Guido Lopes dos Santos Santiago1*, Inge Tency2, Hans Verstraelen 2 , Rita Verhelst2, Marijke Trog 2 , Marleen Temmerman2, Leen Vancoillie3, Ellen Decat1,4, Piet Cools1, Mario Vaneechoutte1 1 Laboratory for Bacteriology Research, Department of Clinical Chemistry, Microbiology, and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium, 2 Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium, 3 AIDS Reference Laboratory, Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University/Ghent University Hospital, Ghent, Belgium, 4 Faculty of Health Care, University College Ghent, Ghent, Belgium Abstract Background: To obtain more detailed understanding of the causes of disturbance of the vaginal microflora (VMF), a longitudinal study was carried out for 17 women during two menstrual cycles. Methods: Vaginal swabs were obtained daily from 17 non-pregnant, menarchal volunteers. For each woman, Gram stains were scored, the quantitative changes of 5 key vaginal species, i.e. Atopobium vaginae, Lactobacillus crispatus, L. iners, (sialidase positive) Gardnerella vaginalis and Prevotella bivia were quantified with qPCR and hydrogen-peroxide production was assessed on TMB+ agar. Results: Women could be divided in 9 subjects with predominantly normal VMF (grades Ia, Ib and Iab, group N) and 8 with predominantly disturbed VMF (grades I-like, II, III and IV, group D). VMF was variable between women, but overall stable for most of the women. Menses were the strongest disturbing factor of the VMF. L. crispatus was present at log7–9 cells/ ml in grade Ia, Iab and II VMF, but concentrations declined 100-fold during menses. L. crispatus below log7 cells/ml corresponded with poor H2O2-production. L. iners was present at log 10 cells/ml in grade Ib, II and III VMF. Sialidase negative G. vaginalis strains (average log5 cells/ml) were detected in grade I, I-like and IV VMF. In grade II VMF, predominantly a mixture of both sialidase negative and positive G. vaginalis strains (average log9 cells/ml) were present, and predominantly sialidase positive strains in grade III VMF. The presence of A. vaginae (average log9 cells/ml) coincided with grade II and III VMF. P. bivia (log4–8 cells/ml) was mostly present in grade III vaginal microflora. L. iners, G. vaginalis, A. vaginae and P. bivia all increased around menses for group N women, and as such L. iners was considered a member of disturbed VMF. Conclusions: This qPCR-based study confirms largely the results of previous culture-based, microscopy-based and pyrosequencing-based studies. Citation: Lopes dos Santos Santiago G, Tency I, Verstraelen H, Verhelst R, Trog M, et al. (2012) Longitudinal qPCR Study of the Dynamics of L. crispatus, L. iners, A. vaginae, (Sialidase Positive) G. vaginalis, and P. bivia in the Vagina. PLoS ONE 7(9): e45281. doi:10.1371/journal.pone.0045281 Editor: Patrick M. Schlievert, University of Iowa Carver College of Medicine, United States of America Received May 28, 2012; Accepted August 14, 2012; Published September 21, 2012 Copyright: 2012 Lopes dos Santos Santiago et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge the Geconcerteerde OnderzoeksActie fund of the University of Ghent. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Guido.LopesdosSantosSantiago@ugent.be Introduction The vaginal microflora (VMF) has been studied extensively and has been shown to be easily disturbed by exogenous and endogenous factors [1–3]. Still, the etiology of bacterial vaginosis, a polymicrobial condition whereby the lactobacilli-dominated VMF is replaced largely by anaerobes, remains to be elucidated [4] and the general dynamics of the VMF remain to be understood. Several longitudinal studies of the VMF throughout the menstrual cycle (MC), as a tool for understanding the dynamics of the VMF, have been undertaken. Most of these studies relied on analysis of Gram stained smears using the Nugent score [5–8], and some studies combined this with culture of vaginal swabs [9–11], and very recently Gajer et al. [12] combined pyrosequencing with Gram stained smears, but to our knowledge only one group [13] previously combined Gram stained smears with qPCR for analysis. The present study builds upon previous work of our group, where we reported the results of Gram stain and culture for 17 menarchal women, who self-swabbed the vagina daily during two MCs [10]. Here, we add concentrations obtained by quantative PCR for five vaginal key species, i.e. Atopobium vaginae, Lactobacillus crispatus, L. iners, Gardnerella vaginalis and Prevotella bivia, with L. crispatus being the predominant Lactobacillus in normal VMF [14– 16], L. iners being the single most predominant Lactobacillus species in bacterial vaginosis associated VMF in all of our previous studies [10,14,17], but also an important species in normal VMF, PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45281