Efficacy of orally applied probiotic capsules for bacterial vaginosis

European Journal of Obstetrics & Gynecology and Reproductive Biology 168 (2013) 75–79
Contents lists available at SciVerse ScienceDirect
European Journal of Obstetrics & Gynecology and
Reproductive Biology
journal homepage: www.elsevier.com/locate/ejogrb
Efﬁcacy of orally applied probiotic capsules for bacterial vaginosis and other
vaginal infections: a double-blind, randomized, placebo-controlled study
Goran Vujic a,*, Alenka Jajac Knez b, Vedrana Despot Stefanovic b, Vedrana Kuzmic Vrbanovic b
a
b
Department of Gynecology and Obstetrics, Medical School, University of Zagreb, Zagreb, Croatia
Jadran Galenic Laboratory, 51000 Rijeka, Croatia
A B S T R A C T
Article history:
Received 16 February 2012
Received in revised form 30 September 2012
Accepted 31 December 2012
Objective: To assess the efﬁcacy of orally administered capsules containing the probiotics Lactobacillus
rhamnosus GR-1 and Lactobacillus reuteri RC-14 (Lactogyn, JGL, Rijeka, Croatia) compared to placebo in
otherwise healthy women diagnosed with bacterial vaginosis.
Study design: Randomized, double-blind, multicentric, placebo-controlled trial, including a total of 544
subjects. Included were women older than 18 years of age, diagnosed with vaginal infection. Subjects
received either probiotic (395 subjects or 72.6%) or identical-looking placebo capsules (149 subjects or
27.4%,) per day over a period of 6 weeks. Six and 12 weeks after the beginning of the study, subjects
underwent two additional gynecological examinations and their vaginal swabs were evaluated by a
clinical cytologist.
Results: Mean follow-up period after the baseline visit was 44 days. After this period, restitution to
balanced vaginal microbiota was reported in 40 subjects (26.9%) in the placebo group, compared to 243
subjects (61.5%) in the probiotic group. Differences between groups were statistically signiﬁcant at
p < 0.001. After the additional 6 weeks of follow up, normal vaginal microbiota were still present in more
than half (51.1%) of subjects in the probiotic group, but only in around one-ﬁfth (20.8%) of subjects who
were taking placebo (p < 0.001).
Conclusion: Oral probiotics could be an alternative, side effect-free treatment for one of the most
common indications in gynecology, combining the good aspects of both metronidazole and vaginal
capsules.
ß 2013 Elsevier Ireland Ltd. All rights reserved.
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A R T I C L E I N F O
1. Introduction
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Keywords:
Bacterial vaginosis
Oral probiotics
Placebo-controlled study
Bacterial vaginosis is a very common vaginal infection, with a
prevalence of 20% in women of reproductive age [1]. Although
bacterial vaginosis is mostly a mild and in some cases even
asymptomatic disease, it can nevertheless be a cause of
endometritis and pelvic inﬂammatory disease [2,3]. Complications
in pregnancy, such as miscarriage, chorioamnionitis, premature
rupture of membranes, preterm labor and postpartal endometritis,
can all result from bacterial vaginosis [4,5]. In women undergoing
in vitro fertilization, bacterial vaginosis reduces the percentage of
implanted gametes and can also be the cause of early pregnancy
loss [6,7]. The basic pathological process in this disease is the
overgrowth of anaerobic microorganisms over the normal vaginal
microbiota. These pathogens include Gardnerella vaginalis and
sometimes Mycoplasma hominis and Mobyluncus species. It leads to
an increase of vaginal pH value and the onset of unpleasantly
? Corresponding author. Tel.: +385 1 460 46 46.
E-mail address: [email protected] (G. Vujic).
smelling vaginal discharge. Healthy vaginal microbiota include
mostly microorganisms from Lactobacillus species, whose role is to
prevent colonization by the aforementioned pathogens. Lactobacilli produce lactic acid, thus decreasing the vaginal pH [8] and
hydrogen peroxide, which is toxic to other microorganisms and
prevents their colonization in the vagina [9,10].
Bacterial vaginosis is most often treated with metronidazole,
which has no effect on lactobacilli but acts against anaerobic
microorganisms. Clindamycin is also routinely used [11,12].
Although these are considered to be safe drugs, there are a
number of side-effects noted with their use. With metronidazole,
gastrointestinal disturbances such as nausea and vomiting, and
less frequently, metallic taste in mouth, paresthesias and
leucopenia have been reported [13]. The most common sideeffects of clindamycin use are sensitivity reactions manifesting as
rash, and gastrointestinal symptoms such as anorexia, abdominal
pain, nausea, vomiting and diarrhea. The latter can also be a
symptom of pseudomembraneous enterocolitis. Less frequent
side-effects include neutropenia, leucopenia, jaundice and skeletal
muscle paralysis [14]. Both drugs can be used orally as well as
vaginally. Ten to 15% of patients do not respond to the initial
0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejogrb.2012.12.031
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2. Materials and methods
The study was designed as a randomized, double-blind,
multicentric, placebo-controlled trial. The protocol of the study
was approved by the Ethical Committee of the School of Medicine,
University of Zagreb. Recruitment was done by 55 primary care
gynecologic practitioners in central and northwestern Croatia.
Included were women older than 18 years of age, diagnosed with
vaginal infection (bacterial vaginosis, candidiasis, trihomoniasis or
combination of these conditions). Exclusion criteria were pregnancy, lactation, menstruation and a subject’s unwillingness to
provide informed consent to participate in the trial.
The initial (baseline) diagnosis of bacterial vaginosis was done
by following either the Amsel criteria or the Nugent scoring
system. In routine clinical practice in Croatia, bacterial vaginosis is
most commonly diagnosed based on the Amsel criteria [21], i.e.
thick, grayish vaginal discharge, pH of the discharge higher than
4.5, speciﬁc smell of the discharge after the addition of KOH and
presence of so-called ‘‘clue cells’’ in a saline wet mount. At least
three of these four criteria must be present for the diagnosis of
bacterial vaginosis to be conﬁrmed.
Some practitioners, however, routinely use a diagnostic method
based on the Nugent scoring system [22]. In such cases, vaginal
smears were Gram stained and evaluated using a 10-point scale
based on scoring a total number of Lactobacilli, G. vaginalis and
Mobiluncus species in a ﬁeld of view during microscopic
examination. Baseline diagnosis of vulvovaginal candidiasis and
trichomoniasis were made by routinely used diagnostic procedures. After any of the previously mentioned diagnoses was made
during the initial screening process, additional swabs for cytology
were taken to conﬁrm the diagnosis in the central laboratory.
After providing informed consent, subjects were included in the
study and assigned to either the placebo or the probiotic arm of the
trial. The randomization process was done by means of a
computer-generated randomization list, assigning each subject
to either ‘‘group A’’ or ‘‘group B’’. Randomization lists were created
using software and methodology described by Saghaei [23].
Subjects then received their supply of capsules, taking either
two ‘‘Lactogyn’’ (each capsule containing >109 CFU Lactobacillus
rhamnosus GR-1 and L. reuteri RC-14) or two identical-looking
placebo capsules per day over the period of 6 weeks.
Six and 12 weeks after the beginning of the study, all subjects
underwent two additional gynecological examinations. In subjects
with bacterial vaginosis their vaginal smears were evaluated
following the procedure used at inclusion. In cases of vulvovaginal
candidiasis, evaluation was done by determination of vaginal pH
and vaginal discharge wet mount analysis with 10% KOH, while all
subjects with trichomoniasis received a single oral dose of 2 grams
of metronidazole. Additionally, vaginal swabs from all subjects
were taken for evaluation by a clinical cytologist as well as
Lactobacillus cultivation (recovery) on agar plates.
Lactobacillus recovery from the vagina was done following the
method described by Anukam et al. [24]. After dislodging the
individual cells in sterile PBS, dilutions were plated on agar plates
with or without tetracycline (50 mg/ml). After incubation, plates
were analyzed visually, Gram stained and tested for catalase
reaction. Following such procedure, pale-yellow, tetracyclineresistant, Gram-positive and catalase-negative colonies were
considered to belong to Lactobacillus sp.
The primary outcome of the study was deﬁned as the rate of
restitution of the normal vaginal microbiota after the follow-up
period of 6 weeks. Furthermore, we observed the rate of recurrence
of vaginosis after the additional 6 weeks during which period
subjects did not take the study medication. Sample size was
calculated so that sufﬁcient statistical power (b 0.90) with
appropriately low probability of type-1 error (a < 0.05) could be
achieved. We used NCSS PASS software [25], with the null
hypothesis of achieving a 40% cure rate in placebo group vs. the
65% cure rate in the probiotic group. The null hypothesis was based
on our previous experience with this probiotic preparation
(personal communication). Such assumption resulted in a sample
size of 120 subjects per study group. In the end, however, a much
larger sample of 544 women was included in the study.
All statistical analyses were performed with Statistica software
(version 9, StatSoft, Inc, Tulsa, OK, USA). Rates and proportions
were calculated for categorical data and medians and ranges for
continuous data. Differences in continuous variables were analyzed by means of Student’s t-test (normally distributed values) or
Mann–Whitney U test (non-normally distributed data). Normality
of distribution was tested by the Shapiro–Wilk test. For categorical
variables, differences were analyzed by means of the chi-square
test. All reported p values are two sided. A p value of <0.05 was
considered to indicate statistical signiﬁcance.
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antimicrobial therapy. Among the patients who respond well
to treatment, 69% will have developed a recurrence of bacterial
vaginosis within a year, thus prompting repeated use of
antibiotics [15].
The high percentage of repeated infection in spite of
antimicrobial therapy has prompted a search for an alternative
method of treatment which could be used with the existing
regimens, or independently. Domination of lactobacilli among a
healthy vaginal microbiota and their absence in bacterial vaginosis
has resulted in the use of various species of lactobacilli orally as
well as vaginally in order to renew the vaginal microbiota [16].
These drugs, also known as probiotics, are deﬁned as living
microorganisms that, when applied in a correct dosage, can
improve health status of the host [17]. It is known that certain
strains of lactobacilli can colonize the vagina after oral or vaginal
use [18]. In addition, lactobacilli can be used for a long time
without the harmful side-effects, thus representing an appealing
alternative to antimicrobial treatment, especially in the context of
recurrent vaginal infection [19,20].
The purpose of the present study was to assess the efﬁcacy of
orally administered probiotics (Lactogyn, JGL, Rijeka, Croatia)
compared to placebo in otherwise healthy women diagnosed with
bacterial vaginosis. Our hypothesis was that the proportion of
restitution of normal vaginal microbiota will be signiﬁcantly
higher in women receiving probiotics, compared to those receiving
placebo.
3. Results
During the 12 month period from March 2008 to March 2009, a
total number of 544 subjects were included in the study. For a
detailed inclusion ﬂow chart please see Fig. 1. Study subjects were
randomized to two study groups: placebo (149 subjects or 27.39%)
and probiotic (395 subjects or 72.61%). After securing that the
necessary minimum of subjects was included in the placebo group,
increasing the number of subjects in the probiotic group was the
most efﬁcient way to achieve desired statistical power, without
denying treatment to subjects affected by bacterial vaginosis. At
the baseline visit, subjects in the two study groups were
comparable in terms of demographic data – age, number of births,
number of sexual partners, age at the ﬁrst sexual intercourse,
habits (smoking and alcohol consumption), marital status and
contraception method used (Table 1). Additionally, cytologic
smear ﬁndings were comparable in both study groups at the
baseline visit (p = 0.149; Table 2).
The mean follow-up period after the baseline visit was 44.16
days, i.e. approximately 6 weeks and 2 days, and was not
signiﬁcantly different between the two groups (p = 0.733). During
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Enrollment
77
Assessed for eligibility (n=743)
Excluded (n=92)
Not meeting inclusion criteria (n=27)
Declined to participate (n=63)
Other reasons (n=2)
Randomized (n=651)
Allocaon
Allocated to intervention (n=466)
Allocated to placebo (n=185)
Follow -Up
Lost to follow-up (n=15)
Non-compliant or stopped treatment
completely (n=9)
Not meeting inclusion criteria (12)
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Lost to follow-up (n=22)
Non-compliant or stopped treatment
completely (n=11)
Not meeting inclusion criteria (38)
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Analysis
Analysed (n=149)
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Analysed (n=395)
Table 1
Baseline characteristics of patients.
Characteristic
Placebo group
(N = 149)
Age (range)
Births
Number of sexual partners
1–3
4–6
>6
Age at the ﬁrst intercourse
<15 years
15–20 years
>20 years
Smoking (cigarettes/day)
None
<10
10–20
>20
Alcohol consumption
None
Occasionally
Daily
Marital status
Married
Never married
Separated or divorced
Contraception method
Condom
Oral contraceptives
IUD
Natural/other
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Fig. 1. Flow diagram.
p value
32.19 (18–54)
1.81
33.26 (18–58)
1.73
0.234
0.463
74.50%
18.79%
6.71%
70.89%
18.73%
10.38%
0.438
6.04%
79.87%
14.09%
4.56%
74.43%
21.01%
0.162
68.46%
12.75%
14.77%
4.03%
64.56%
16.46%
13.92%
5.06%
0.680
56.38%
40.94%
2.68%
50.07%
47.65%
2.28%
0.583
63.09%
32.89%
4.04%
56.20%
36.96%
6.84%
0.248
16.78%
24.83%
8.05%
50.33%
24.81%
20.51%
8.10%
46.48%
0.224
Co
Probiotic group
(N = 395)
the follow-up period, subjects were taking either placebo or the
probiotic capsules twice a day, as described earlier. Compliance
was comparable between the two study groups (97.64% in the
placebo vs. 91.67% in the probiotic group, p = 0.128).
After the above-mentioned follow-up period, restitution to
balanced vaginal microbiota was reported in 40 subjects (26.85%)
in the placebo group, compared to 243 subjects (61.52%) in the
probiotic group. Differences between groups were statistically
signiﬁcant at p < 0.001. At this time point, the number needed to
treat (NNT) was 2.9 with a relative risk reduction (RRR) of 47.4%.
After the additional 6 weeks of follow-up, normal vaginal
microbiota were still present in more than half (51.14%) of subjects
in the probiotic group, but only in around one-ﬁfth (20.81%) of
subjects who were taking placebo (p < 0.001). At this later time
point the NNT was 3.3, while the RRR was 38.3%. Detailed results of
cytologic smear ﬁndings are laid out in Table 3. All subjects with
Table 2
Cytologic smear results at baseline.
Finding
Gardnerella vag.
Fungi
Mixed ﬂora
Trichomonas vag.
Gardnerella vag. + mycosis
Total
Placebo group
Probiotic group
N
%
N
%
31
50
49
2
17
149
20.81
33.56
32.89
1.34
11.41
100.00
78
107
172
8
30
395
19.75
27.09
43.54
2.03
7.59
100.00
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Table 3
Cytologic smear results after 6 and 12 weeks of follow-up.
Baseline + 6 weeks
Baseline + 12 weeks
Placebo group
Placebo group
Probiotic group
%
N
%
N
%
N
%
11
14
83
0
40
1
149
7.38
9.40
55.70
0.00
26.85
0.67
100.00
12
27
109
1
243
3
395
3.04
6.84
27.59
0.25
61.52
0.76
100.00
13
19
84
0
31
2
149
8.72
12.75
56.38
0.00
20.81
1.34
100.00
11
24
155
0
202
3
395
2.78
6.08
39.24
0.00
51.14
0.76
100.00
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metronidazole gel in treatment of bacterial vaginosis [30], with
comparable results. Similar results were published by Martinez
et al. [31] (combination of tinidazole and probiotic in comparison
with tinidazole and placebo), and by Ya et al. [28] who recently
conducted a randomized, double-blind, placebo-controlled study
testing the effectiveness of probiotic vaginal capsules in preventing
recurrences of bacterial vaginosis.
Although not without certain shortcomings in terms of
population size, inclusion conditions or follow up methodology,
the above-mentioned studies established a strong case for the
existence of beneﬁciary effect of Lactobacilli in subjects with
bacterial vaginosis. With that in mind, we designed the present
study to include a relatively large number of subjects from
numerous primary care gynecological practitioners. Additionally,
we established a scrupulous procedure for both baseline and
follow-up diagnostic procedures and closely monitored subject
compliance. By applying such a study design we were able to
accrue a sufﬁcient number of subjects within a reasonable time
frame and avoid errors commonly seen in single-centre trials [32].
Finally, recruiting subjects from a wider population and testing the
effects of the treatment in a broader range of clinical settings
presented an experimental situation more typical of future use.
The main limitation of our study was a relatively short follow-up
period of 6 and 12 weeks post-baseline, which was necessary to
avoid dispersal of study subjects. Additionally, DNA probes speciﬁc
for Lactobacillus GR-1 and RC-14 would facilitate identiﬁcation of
strains in the follow-up vaginal swabs, but were unavailable at the
time the study was performed.
Our ﬁndings conﬁrm the positive effects of probiotics described
in previously mentioned studies. The results also support the
association between the proportion of subjects with positive posttreatment Lactobacillus sp. culture and the proportion of subjects
achieving restitution to balanced vaginal microbiota, which was
the main outcome of the study. In our opinion, the most promising
result of the probiotic therapy as a whole (either as vaginal
capsules or as oral therapy) is the potential to reduce or make
unnecessary the use of metronidazole in treatment of bacterial
vaginosis. Probiotic vaginal capsules have already been described
to be more effective than metronidazole for this condition [33],
while our results conﬁrm that this is true for the oral probiotic
therapy as well. We feel that oral probiotics could be an alternative,
side effect-free treatment for a very common disease in
gynecological practice, combining the good aspects of both
metronidazole and vaginal capsules. We agree with Ya et al.
[28] that potential beneﬁt of probiotics versus antibiotics can be
conﬁrmed only by means of further randomized clinical trials.
4. Comments
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unfavorable results at the second (12 week) follow-up visit
received appropriate treatment after their participation in the
study was completed.
Additional cultivation on agar plates was performed to recover
and identify Lactobacilli from the vaginal swabs. High counts
(>105 CFU/ml) of lactobacilli were recovered from 81.5% of
subjects who received probiotic, compared to only 28.9% of
subjects receiving placebo. Thus, restitution to normal (balanced)
vaginal microbiota coincided highly (p < 0.001) with presence of
lactobacilli in the vaginal swabs on both post-baseline visits.
None of the demographic variables described in Table 1 was
related signiﬁcantly to the outcome – the proportion of subjects
with the restituted vaginal microbiota after either 6 or 12 weeks
post-baseline. Borderline signiﬁcance (p = 0.082) was found,
however, when number of children was compared to the outcome.
Women with 3 or more deliveries were less likely to see restitution
to normal microbiota, compared to women with 2, 1 or no births.
This ﬁnding is clearly not due to signiﬁcantly higher mean age of
women with more children (40.1 years for women with 3 or more
children as opposed to 29.9 years for women with 2 children or
less), because age had no effect on the outcome, as mentioned
before. One possible explanation could be that both occurrence and
recurrence of bacterial vaginosis (and other female reproductive
tract infections) are more common in women with more deliveries,
a fact already shown by other researchers [26]. A much larger study
with longer follow-up period would be needed, however, to
conﬁrm or reject this hypothesis.
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Gardnerella vag.
Fungi
Mixed ﬂora
Trichomonas vag.
Bacillus vag.
Gardnerella vag. + mycosis
Total
Probiotic group
N
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Finding
Bacterial vaginosis is a condition known to be associated with
low numbers of Lactobacillus sp. bacteria in vagina [27]. Although
the source of vaginosis (reinfection or relapse) has not been fully
elucidated, it is thought that lactobacilli, by means of their
production of lactic acid, inhibit growth of other microorganisms,
thus inhibiting their overgrowth [20,28]. The same mechanism is
responsible for maintaining low vaginal pH values. The most
frequently used strains of lactobacilli in therapy are a combination
of L. rhamnosus GR-1 and Lactobacillus fermentum RC-14, shown by
in vitro studies to posses the ability of adhesion to the cells of
vaginal epithelium [29]. Because of this they are able to colonize
the vagina after oral application [13].
Studies of the effects of probiotics on bacterial vaginosis are
relatively scarce. Anukam et al. [24] conducted a randomized,
controlled, double-blind study which conﬁrmed the positive
effects of combined use of metronidazole with capsules containing
probiotic in comparison to placebo in treatment of bacterial
vaginosis. After the 30-day period, 88% of patients who received
probiotic had no signs of bacterial vaginosis, in comparison to 40%
vaginosis-free patients in placebo group (p < 0.001). The same
authors also conducted another randomized clinical trial which
compared the efﬁcacy of probiotic vaginal capsules used with the
Condensation
Oral probiotics could be an alternative, side effect-free
treatment for bacterial vaginosis, combining the good aspects of
both metronidazole and vaginal capsules.
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[19]
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[29]
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[30]
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[18]
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