Quantifying Vaginal Flora Species with Real Time PCR for HIV prevention trials
Vicky
1
Jespers ,
Joris
1
Menten ,
Rita Verhelst*, Hilde
1
1
Smet
, Sabrina
1
Poradosú
, Anne
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
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.
Table 1:Baseline Clinical Characteristics
Low risk
(N=30)
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.
Table 2: Presence and counts of vaginal flora species for a low risk and high risk
population in Antwerp, Belgium
Age (years)
Mean (range)
27 (19-38)
Race N (%)
African/Caribbean
Caucasian
Hispanic
Asian
Mediterranean
0 (0)
28 (93)
0 (0)
0 (0)
2 (7)
High risk
(N=41)
¹
27 (15-47)
²
13 (34)
11 (29)
6 (16)
5 (13)
3 (8)
12 (40)
0 (0)
1 (3)
0 (0)
17 (57)
³
18 (46)
9 (23)
8 (21)
2 (5)
2 (5)
0 (0%)
12 (29%)
Contraception
N (%)
Lactobacillus total
Low Risk
High Risk: BV = 0
BV = 1
L. crispatus
Low Risk
High Risk: BV = 0
BV = 1
L. iners
Low Risk
High Risk: BV = 0
BV = 1
L. jensenii
Low Risk
High Risk: BV = 0
BV = 1
L. gasseri
Low Risk
High Risk: BV = 0
BV = 1
G. vaginalis
Low Risk
High Risk: BV = 0
BV = 1
A. vaginae
Low Risk
High Risk: BV = 0
BV = 1
† Wilcoxon rank-sum test
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
Species Present
n/N (%)
Vs Low risk
(p-value1)
30/30 (100)
29/29 (100)
12/12 (100)
-1.0
1.0
23/30 (77)
23/29 (79)
5/12 (42)
-1.0
0.066
20/30 (67)
25/29 (86)
10/12 (83)
-0.125
0.453
17/30 (57)
15/29 (52)
3/12 (25)
-0.796
0.316
19/30 (63)
7/29 (24)
1/12 (8)
-0.004
0.002
10/30 (33)
20/29 (69)
12/12 (100)
-0.006
<0.001
4/30 (13)
8/29 (28)
11/12 (92)
-0.209
<0.001
1
Fisher exact test
Vs High Risk No BV
(p-value1)
Geometric Mean
Count
(when present)
Vs High Risk No BV
(p-value†)
-1.0
937 x 106
97 x 106
11 x 106
8,97
7,99
7,041
-<0.001
-0.029
602 x 106
39 x 106
0.17 x 106
8,78
7,59
5,23
-<0.001
-1.0
545 x 106
22 x 106
38 x 106
8,74
7,34
7,58
-0.840
-0.171
40 x 106
0.55 x 106
0.25 x 106
7,60
5,74
5,40
-0.130
-0.398
977 x 103
56 x 103
(Single obs: 19 x 106)
5,99
4,75
7,28
-0.301
-0.039
1.3 x 106
0.62 x 106
42 x 106
6,11
5,79
7,62
-<0.001
-<0.001
8.6 x 106
0.12 x 106
13 x 106
6,93
5,08
7,11
2counts
None
Combined pill
Intrauterine device
Implant
Condoms
Bacterial vaginosis
² 4 missing values
³ 2 missing values
Wilcoxon-rank-sum-test-result: NS:p≤0.100,+:p<0.100,*:p<0.050,**:p<0.010,***:p<0.001.
-<0.001
Table 3: Evolution of species presence and species counts over time – Low risk
population
in bacterial cells per ml
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. gasseri are 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.
Present at least at
one visit
N=30
Lactobacillus sp.
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.
Institute of Tropical Medicine, Antwerp
2011
Nationalestraat 155, B-2000 Antwerp, Belgium
Tel. +32-3-247.65.52
E-mail: tcrucitti@itg.be
Fax. +32-3-247.63.33
www.itg.be
Consistently present‡
N=30
LCA1
Subgroup1
81%
Subgroup2
19%
30 (100%)
30 (100%)
100%
100%
L. crispatus
28 (90%)
18 (60%)
66%
36%
L. iners
23 (77%)
20 (67%)
82%
0%
L. jensenii
22 (73%)
19 (63%)
70%
18%
L. gasseri
21 (70%)
20 (67%)
67%
47%
G. vaginalis
14 (47%)
7 (23%)
34%
100%
A. vaginae
6 (20%)
2 (7%)
4%
88%
‡
At most 1 visit absent
latent class analysis (LCA) we identified two subsets of women based on the consistent presence of the Lactobacillus species.
1With
Conclusions
Figure 2: Log counts of bacterial cells per ml by day
in the menstrual cycle.
Figure 1: Presence at baseline of species in counts/ml
Counts2
Log 10
Geometric mean
(when present)
Presence
-2 high vaginal flocked swabs (COPAN innovation, Italy)
Tania
1
Crucitti
Results
¹ 5 missing values
-HIGH RISK population: STI clinic attenders, 16-35 year, N=41,
2 visits
1
Hardy ,
Institute of Tropical Medicine and * University of Ghent
Introduction & Methods
INTRODUCTION
-A healthy vaginal environment is protective against STIs
-Vaginal prevention products should not disturb this balance
1
Buvé ,Liselotte