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Quantifying Vaginal Flora Species with Real Time PCR for HIV prevention trials. Vicky Jespers 1 , Joris Menten 1 , Rita Verhelst * , Hilde Smet 1 , Sabrina Poradosú 1 , Anne Buvé 1 ,Liselotte Hardy 1 , Tania Crucitti 1 1 Institute of Tropical Medicine and * University of Ghent. Results.

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QuantifyingVaginal Flora Species with Real Time PCR for HIV prevention trials

Vicky Jespers1, Joris Menten1, Rita Verhelst*, Hilde Smet1, Sabrina Poradosú1, Anne Buvé1,Liselotte Hardy1, Tania Crucitti1

1 Institute of TropicalMedicineand* University of Ghent

Results

Introduction & Methods

BASELINE CLINICAL CHARACTERISTICS

-Presented in table 1. Twenty-one women were sexually active of whom 4 had a sexual preference for the same gender. Of the remaining 9 women, one had a sexual preference for the same gender. Prostate specific antigen (PSA) tested positive on 12 occasions over 7 women.

INTRODUCTION

-A healthy vaginal environment is protective against STIs

-Vaginal prevention products should not disturb this balance

OBJECTIVES

-To design reliable PCRs for quantification of specific vaginal flora species

-To define baseline ranges in a healthy phase I population and compare with a high risk population

METHODS: CLINICAL SET UP

-LOW RISK population: healthy European phase I women, 18-35 year, no hormones, N=30, 5 visits

  • Table 1:Baseline Clinical Characteristics

Figure 2: Log counts of bacterial cells per ml by day

in the menstrual cycle.

BASELINE VAGINAL FLORA – LOW AND HIGH RISK GROUP

-Data are presented in table 2 and figure 1.

-Extraction step (extra lysing step of the Easymag) resulted in a higher yield of DNA of 1 log difference in low risk women. PCR for human ERV was in agreement (results not shown). As a result we omitted the statistical comparison of the counts between the low and high risk women and we only present the comparison of counts within the high risk group.

¹ 5 missing values ² 4 missing values ³ 2 missing values

  • Table 2: Presence and counts of vaginal flora species for a low risk and high risk population in Antwerp, Belgium

Figure 1: Presence at baseline of species in counts/ml

-HIGH RISK population: STI clinic attenders, 16-35 year, N=41,

2 visits

-2 high vaginal flocked swabs (COPAN innovation, Italy)

Wilcoxon-rank-sum-test-result: NS:p≤0.100,+:p<0.100,*:p<0.050,**:p<0.010,***:p<0.001.

Table 3: Evolution of species presence and species counts over time – Low risk population

EVOLUTION OVER TIME – LOW RISK GROUP

-The presence or absence of a particular Lactobacillus species appeared to remain constant throughout the study (Figure 2).No predictors (partner preference, being sexually active, PSA presence, time of cycle) of being "Lactobacillus consistently present (meaning at most one visit absent)" for any of the studied Lactobacillae were identified. Longitudinal analysis of women in this group, showed that L. crispatus counts were 0.22 log higher (p<0.001) and L. iners counts were 0.83 log lower (p<0.001) at the end of the menstrual cycle. L. crispatus counts were decreased by 0.42 log after intercourse (PSA present) (p=0.002), while those of L. iners (+0.73 log, p=0.033) and of L. gasseri (+0.59 log, p= 0.058) were increased. With latent class analysis (LCA) we identified two subsets of women based on the consistent presence of the Lactobacillus species (table 3). In the first subset of women, comprising 81% of the studied low risk population, L. crispatus, L. iners, L. jensenii, and L. gasseriare consistently present in, 66%, 82%, 70%, and 67% of women, respectively. The second subset comprising 19% of the low risk population is characterized by a prevalence of G. vaginalis and A. vaginae of 100% and 88%, respectively.

† Wilcoxon rank-sum test 1 Fisher exact test 2counts in bacterial cells per ml

METHODS: LABORATORY SET UP

-Extraction: Low risk - easyMag, BioMérieux. High risk – miniMag, BioMérieux.

-Real time PCR: L. crispatus, L. iners, L. jensenii, L. gasseri, Lactobacillus sp., G. vaginalis, A. vaginae

-2 swabs pooled in PBS

‡ At most 1 visit absent

1With latent class analysis (LCA) we identified two subsets of women based on the consistent presence of the Lactobacillus species.

A combination of a quick microscopic evaluation with Nugent score together with real time PCR defining exact presence and counts up to the species level will

fine-tune the safety evaluation of vaginally applied products and this strategy should therefore be considered in future vaginal product development.

Conclusions

Institute of Tropical Medicine, Antwerp

Nationalestraat 155, B-2000 Antwerp, BelgiumTel. +32-3-247.65.52 E-mail: tcrucitti@itg.be Fax. +32-3-247.63.33 www.itg.be

2011