Gynecol Obstet Invest 2010;70:264272 DOI: 10.

1159/000314016

Received: February 10, 2010 Accepted: February 10, 2010 Published online: October 16, 2010

Effect of Lyophilized Lactobacilli and 0.03 mg Estriol (Gynoflor) on Vaginitis and Vaginosis with Disrupted Vaginal Microflora: A Multicenter, Randomized, Single-Blind, Active-Controlled Pilot Study
G.G.G.Donders a B.VanBulck b P.VandeWalle c R.R.Kaiser d G.Pohlig d S.Gonser d F.Graf d

Infectious Diseases in Obstetrics and Gynecology, Gasthuisberg University Hospital, Leuven and General Hospital Heilig Hart, Tienen, b Departments of Obstetrics and Gynecology, A.Z. Sint Erasmus, Borgerhout-Antwerpen, and c Jan Palfijn Ziekenhuis, Merksem, Belgium; d Medinova Ltd., Zrich, Switzerland

Key Words Bacterial vaginal infections Metronidazole Gynoflor

Abstract Objectives: To evaluate the efficacy of lyophilized lactobacilli in combination with 0.03 mg estriol when compared to metronidazole in the treatment of bacterial vaginal infections. Setting: Multicenter, randomized, single-blind, activecontrolled pilot study in 3 independent gynecological practices in Belgium. Methods: Forty-six, 18- to 50-year-old premenopausal women with a disrupted vaginal flora due to a bacterial vaginal infection (bacterial vaginosis, aerobic vaginitis) were included, provided that fresh phase-contrast microscopy of the vaginal fluid showed lactobacillary flora grade 2B or 3. Patients were given a blinded box with either 12 vaginal tablets of Gynoflor (study medication) or 6 vaginal suppositories containing 500 mg metronidazole (control medication). Eight efficacy variables were studied to assess the status of the vaginal flora at entry, 37 days (control 1), 46 (control 2) weeks and 4 months after the end of therapy. Results: At control 1, the combined variables equally im-

proved in the lactobacilli group as in the metronidazole group. At control 2, the lactobacillus preparation showed slightly inferior results when compared to metronidazole. At 4 months, this analysis could not be performed due to low numbers, but analysis of recurrence rate and extra medication needed was not different between both groups. Conclusion: Lyophilized lactobacilli in combination with lowdose estriol are equivalent to metronidazole in the shortterm treatment of bacterial vaginal infections, but have less effect after 1 month. Further studies are required to evaluate the long-term efficacy of lactobacilli when applied repeatedly. Copyright 2010 S. Karger AG, Basel

Introduction

Lactobacilli are of crucial importance for the maintenance of the vaginal environment, since they resist colonization of pathogens through the production of bacteriocins, lactic acid, and hydrogen peroxide as well as through competitive adherence to the vaginal epithelium [13].
G. Donders, MD, PhD Infectious Diseases in Obstetrics and Gynecology, Gasthuisberg University Hospital Herestraat 49 BE3000 Leuven (Belgium) Tel. +32 16 34 42 04, Fax +32 16 82 27 14, E-Mail gilbert.donders@femicare.net

2010 S. Karger AG, Basel 03787346/10/07040264$26.00/0 Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Accessible online at: www.karger.com/goi

An extremely small amount of estriol, a weak human estrogen derived from estradiol in the placenta, was added in order to help proliferate the vaginal epithelium, making enough glycogen available from desquamated superficial cells to act as a nutritional substrate for the lactobacilli [4]. Exogenous vaginal lactobacilli can produce lactate and lower the vaginal pH, inhibiting the growth of pathogens, while concomitantly favoring the development of the lactobacilli. Disturbance of this delicate ecosystem by for example hormonal changes, sexual behavior, pregnancy or antibiotic therapy can diminish the levels and activity of the lactobacilli, rendering the vagina more susceptible to infection by pathogens. Vaginal application of exogenous lactobacilli with a low dose of estriol has been shown beneficial in several clinical indications, such as (recurrent) bacterial vaginosis, abnormal vaginal discharge of unknown origin, atrophic vaginitis and as a reconstitution therapy after anti-infective treatment [510]. At present, metronidazole or clindamycin are the standard treatments of bacterial vaginosis (BV) and abnormal vaginal flora (AVF). However, in gynecological practices treatment failures occur and recurrent infections are frequent. Also, neither of both antibiotics is free of complications, and for recurrent disease, many women are reluctant to take them repetitively. Amongst the most frequent non-trichomonal pathological conditions linked to AVF are bacterial vaginosis (BV) and, less frequently, aerobic vaginitis (AV) [11]. BV is characterized by a fishy smelling, grayish-white vaginal discharge and vaginal pH above 4.5, the presence of typical granular flora with clue cells on microscopy [12]. Lactobacilli are decreased or absent and replaced by high numbers of facultative anaerobes. The condition is typically not accompanied by vaginal leukocytosis and causes no inflammation. AV also causes an increased vaginal pH and increased discharge, but is associated with overgrowth of enteric aerobes like group B streptococci, Escherichia coli and Staphylococcus aureus, and is often accompanied by the presence of increased vaginal leukocytosis and parabasal epitheliocytes [11]. AV elicits an intense immune response with high levels of interleukin-6, interleukin-1 and leukemia inhibitory factor in the vaginal fluid [11, 13], and may be a potential cofactor in the pathogenesis of infection-related complications in pregnancy such as cervix shortening, chorioamnionitis, preterm rupture of the membranes and preterm delivery [1517]. Although some treatment trials have shown some success with topical antibiotics like kanamycin, standard therapy is not yet available for AV [18, 19].
Lyophilized Lactobacilli and Gynoflor in Disrupted Vaginal Microflora

The aim of the present study was to assess the potential value of vaginal tablets containing viable lactobacilli and a low dose of estriol (0.03 mg) as compared to metronidazole (500 mg vaginal suppositories) in the treatment of bacterial vaginitis or vaginosis. Others authors have used similar vaginal applications of high doses of metronidazole demonstrating superior short- and long-term cure rates in patients with AVF [20].
Patients and Methods
Study Design This multicenter, randomized, active-controlled, singleblinded clinical trial was designed as a pilot study to evaluate the feasibility of a future large-scale study for short- and long-term follow-up. The study protocol was approved by the responsible Ethics Committee before the study was started (Ethical Committee, Heilig Hart Kliniek, Tienen, Belgium). The study was performed according to the Declaration of Helsinki and the applicable cGCP guidelines in 3 independent gynecological practices in Belgium. Patient Selection Forty-six, 18- to 50-year-old premenopausal women with signs or symptoms of a bacterial vaginal infection and a disrupted vaginal microflora showing a lactobacillary grade IIbIII [21] were included in the study after they had given their written informed consent. The inclusion criterion had to be validated by (1) a blinded confirmatory second reading of the microscopy slide, (2) a vaginal lactate concentration less than 30 mg/100 ml, and (3) a vaginal pH above 4.5. Detailed history of vaginal infections was recorded for all patients (table1). Exclusion criteria were pregnancy, ovariectomy, tumors in the genital tract or breast, hypersensitivity to the study medication, uterine or vaginal bleeding of unknown origin, use of unopposed progestins, use of anti-infective (local or systemic) therapy in the previous 14 days, use of any vaginal medication in the previous 7 days, vaginal douching during the previous 48 h, or nonprotected sexual contact during the last 24 h. Women with sexually transmitted infections, such as Neisseria gonorrhoea, Chlamydia trachomatis, Treponema pallidum, herpes simplex, Trichomonas vaginalis, human papilloma virus, or HIV were also excluded. A flow diagram depicting information about the numbers of participants at the different stages of the trial is shown in figure 1. BV was diagnosed when 3 of 4 of the parameters described by Amsel et al. [12] were present. Diagnosis of AV was based on the lactobacillary grades, the presence of parabasal cells, the proportional numbers of leukocytes, the toxic appearance of the leukocytes and the presence of cocci in chains of small rods in the wet mount examination, as described elsewhere [11]. Patients could only be included if the lactobacillary flora was moderately to severely disturbed, i.e. lactobacillary grade IIb or III (see Lactobacillary Grade). Medication Patients received either test or control medication according to the randomization list. Each vaginal tablet of the test preparation (Gynoflor, Medinova Ltd., Switzerland) contains at least 1 !

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107 colony-forming units of live Lactobacillus acidophilus, 0.03 mg estriol and approximately 600 mg lactose. The test medication was stored at 28 C. Suppositories containing 500 mg metronidazole (Flagyl, Rhone-Poulenc Rorer) were used as active control. Patients applied either one vaginal tablet or suppository daily for 12 or 6 days, respectively. Due to the different treatment periods (lactobacilli: 12 days; metronidazole: 6 days), only patient blinding was possible. Allocation to either group was performed using a random allocator (Rancode, Data analysis and Study planning, Munich/ Gauting, Germany). The weighting of the two groups was roughly 1:1 (lactobacilli: 22; metronidazole: 24).

plasma hominis and Ureaplasma urealyticum were tested on a vaginal swab by the Mycoscreen test. A Dacron swab was first rotated in the cervix and than soaked in the vaginal fluid, to be transported to the lab in an A3 transport medium. The swab was incubated for 48 h in lyophilized urea-arginine broth at 37 C. Positive (phenol red) samples were for 48 h cultured on A7 agar at 37 C to identify and quantify the mycoplasmata and Mycofast was used for the antibiogram.

Follow-Up The first control examination (C1) was performed at 37 days, the second examination (C2) at 46 weeks and the third (C3) at 4 months after the end of therapy. Primary Efficacy Parameters The following 8 efficacy variables were considered in the confirmatory statistical analysis as described below. Lactobacillary Grade Vaginal secretion was examined immediately using phasecontrast microscopy at !400 magnification. The lactobacillary grade was assessed as described by Donders [22]. Briefly, grade I corresponds to a predominance of lactobacillary morphotypes, with very few coccoid bacteria present, grade IIa to a mixed flora with lactobacilli and other germs, but lactobacilli outnumbering other germs, grade IIb to a mixed flora with lactobacilli and other germs, but fewer lactobacilli than others, and grade III to a grossly abnormal flora with numerous other bacteria and virtually no lactobacilli present. Lactate Concentration A standardized vaginal washing with 2 ml 0.9% NaCl was performed by flushing and reaspirating 2 ml of saline through a 21/4 gauge needle in the left, central and right upper vaginal vaults. The lavage fluid was shipped for analysis of lactate levels to the central laboratory within 6 h after sampling and stored for later analysis at 18 C. The lactate concentration in the vaginal rinsing fluid was measured by a validated gas-liquid chromatography [22, 23].

Patients Complaints At entry and at each control examination, the patients were asked whether they had suffered from vaginal discharge, bad smell, vulvo-vaginal itching, burning sensation, painful intercourse, painful micturation and abdominal discomfort during the last 4 weeks before the entry examination or since the last control, respectively. For the presence of each symptom, 1 point was added and the total patients complaints score could thus have values from 0 to 7. Objective Signs and Symptoms Appearance of the vaginal discharge (thin white, sticky yellow or other) and smell (fishy or rotten) were evaluated as present (adding 1 point) or absent (0). Redness of the vulva and vagina, ulceration of the vagina and cervix, and painful cervix and uterus/adnexae due to inflammation were reported as none (0), moderate (1) or severe (2). The total symptoms score was calculated by adding up the respective figures, leading to values between 0 and 14. Maturation Index The various cell types of the vaginal epithelium (parabasal, small intermediate, large intermediate and superficial cells) were determined and the Maturation Index was calculated according to the following formula: MI = 0!(1) + 0.33!(2) + 0.67!(3) + 1!(4), where 1 = % parabasal cells, 2 = % small intermediate cells, 3 = % large intermediate cells, and 4 = % superficial cells. The Maturation Index can take figures between 0 and 100. Vaginal Ecology Score (VECO) [31] The VECO score constitutes a surrogate marker comprising relevant parameters for the characterization of the vaginal milieu. Using phase-contrast microscopy (magnification !400), the vaginal smear was analyzed for lactobacillary grade, presence of leukocytes and red blood cells (background activity), and lysis of the epithelial cells. Lactobacillary morphotypes were evaluated as normal, leptosomic, coarse or mixed types. Each parameter can get a score ranging from 1 to +3. A total score could thus have values from 0 to 11. The higher the score, the more disturbed is the vaginal ecosystem. It should be noted that in this study the original VECO score was used with two modifications: (1) epithelial cell abnormality was not evaluated, which decreased the possible maximum score by 2, and (2) pathogens were assessed using Grams staining, instead of Papanicolaous method. Tolerability The tolerability of the treatment was assessed using a 4-point estimation scale (very good, good, moderate, poor) by both the investigators and the patients. The type, time of onset, intensity, duration and causality of adverse events were recorded during the study.

Vaginal pH Vaginal pH was measured using pH strips (Merck, color scale 4.07.0) at the lateral vaginal wall of the upper third of the vagina. Microbial Pathogens Gram-positive, Gram-negative, and Gram-variable microorganisms were classified in a semiquantitative manner (none, few, moderate, many) [24]. In case more than one pathogen was evident, the score of the most abundant pathogen was used. Microbiological cultures were processed from a cotton-tipped swab from the posterior vault and immediately placed in Amies-modified Stuart medium for transport to the central laboratory (Femicare, Tienen, Belgium) within 6 h. Cultures were performed for microorganisms such as Escherichia coli, Enterococci (E. faecalis), Gardnerella vaginalis, group B streptococci (GBS), Staphylococcus aureus, Prevotella (former Bacteroides), and Candida sp. Myco-

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Table 1. Demographics for all patients enrolled

Criteria Number of patients Diagnosis Bacterial vaginosis Aerobic vaginitis Mean age, years 8 SD Entire population Patients with BV Patients with AV History of pregnancy Parity, range Miscarriage, abortion, extrauterine pregnancy Preterm delivery (<37 weeks) Sexual history Use of oral contraceptives Regular use of condoms Change of sex partner in the last 2 months More than 1 lifetime sex partner History of vaginal infections in the last 12 months Duration of vaginal infection(s): more than a year Frequency of vaginal infection(s): more than 3 per year Previous/current medical disorders Diabetes Intake of corticosteroids (rheuma, autoimmunity) Hysterectomy (with preservation of adnexae) Prior surgery on the cervix (LLETZ, laser, operative)

Lactobacilli group 22 14 8 36.089.2 35.1810.0 37.588.0 03 3/22 1/22 10/22 2/22 4/22 9/22 9/22 10/22 0/22 0/22 2/22 2/22

Metronidazole group 24 14 10 31.688.2 30.888.4 32.888.2 02 4/24 0/24 11/24 5/24 1/24 11/24 7/24 8/24 0/24 1/24 0/24 1/24

Entire population 46 28 18 33.788.9 32.188.4 36.189.3 03 7/46 1/46 21/46 7/46 5/46 20/46 16/46 18/46 0/46 1/46 2/46 3/46

Statistical Analysis An Intent-To-Treat anaysis (ITT) was performed with all 44 patients. The group comparison was done using a test for noninferiority of the test compared to the standard treatment. Sample Size In order to obtain a 33% difference in a group of women that have 70% chance of suffering from relapses during 4 months after therapy, it was calculated that a test group of at least 40 women (1:1) had to be examined in order to obtain a single-sided type I error of 0.05 with a power of 80%. Confirmatory Analysis The eight efficacy variables were combined in the confirmatory analysis using the Wei-Lachin procedure, a generalized multivariate Wilcoxon-Mann-Whitney test [2528]. The confirmatory analysis of the combined 8 variables was performed with the ITT population for C1 (difference between C1 and Entry (E)) and at C2 (difference between C2 and E), tested in a confirmatory manner as described above and interpreted in an exploratory manner according to the principle of an a priori ordered hypothesis [29]. The point estimators and their confidence intervals were calculated for both the combined test and the single tests, so that conclusions concerning noninferiority could be drawn based on confidence intervals [30].

Group Comparison The single efficacy variables were analyzed individually in an exploratory manner using the Wilcoxon-Mann-Whitney U test at C1 and C2. The confirmatory analysis at C1 was done with the combined variables using the Wei-Lachin procedure. The nonparametric, robust Wilcoxon-Mann-Whitney U test, one-sided, was used as the statistical test procedure. The MannWhitney estimator P(X ! Y) was taken as effect measure for stochastic superiority. The alpha of the study was stipulated as 0.025, one-sided, the associated confidence interval being 97.5%, onesided. The test for noninferiority is performed using the confidence interval of the Mann-Whitney estimator P(X ! Y) + 0.5P(X = Y), whereby noninferiority is proven at the 2.5% level if the calculated one-sided confidence interval lies above 0.36. The value of 0.36 corresponds to a medium-sized or relevant inferiority [29, 30].

Results

The demographic characteristics are summarized in table1. No difference in characteristics was observed between the treatment groups. Among the enrolled women
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267

n = 46 Patients randomized and treated

n = 22 Patients receiving Gynoflor

n = 24 Patients receiving Flagyl

n = 21 present at C1

n=1 missing at C1

n = 20 present at C1

n=3 missing at C1 n=1 withdrawn

n = 15 present at C2

n=7 withdrawn

n = 17 present at C2

n=1 missing at C2 n=5 withdrawn

n=7 present at C3

n=8 withdrawn

n=8 present at C3

n = 10 withdrawn

Fig. 1. Flow chart of study patients.

(n = 46) BV was diagnosed in 28 (60.9%) and AV or mixed AV/BV in 18 cases (39.1%). 32% (n = 7) of the patients treated with lactobacilli and 25% (n = 6) of the patients treated with metronidazole dropped out after C1 (difference not significant, fig.1). By the time of C3, 15 patients treated with lactobacilli and 16 treated with metronidazole had dropped out of the study (difference not significant). Except for 2 patients who discontinued due to an adverse event (under metronidazole-treatment 1 before C1 and 1 after C2), the reasons of the dropouts after C1 and C2 were due to relapses. The Kaplan-Meier-like graph shows the number of patients that did not require additional medication at any given time of the study (fig.2). There was no difference in the course of both graphs of patients using lactobacilli or metronidazole. The confirmative analysis was done with eight variables at C1 within the ITT population (table 2). The Mann-Whitney (MW) estimator for the combined variables is 0.46 with a lower bound 97.5% confidence interval
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of 0.36. The data indicate one-sided equivalence (at least as good) at the 2.5% level for Gynoflor, when compared with metronidazole (fig. 3, upper panel). At C2 the MW = 0.43 with a confidence interval of 0.32, indicating a slight inferiority of the test preparation when compared to control medication (fig.3, lower panel). At C1, the MW values of all the single variables are within 0.36 and 0.5, except for the variable patients complaints (MW = 0.32). Remarkably, lactobacillary grades improved and lactic acid concentration was higher in the test group than in the control group (MW = 0.53 and 0.52, respectively). Also at C2, the MW values for most single variables are 10.36 except for the pH (MW = 0.35). Compared with the test group, the markers of lactobacillary colonization were now better in the control group (both MW = 0.39). On the other hand, the Maturation Index, a marker for proliferation of the epithelium, was more improved in the test medication than in the control group (MW = 0.67).

Donders /VanBulck /VandeWalle / Kaiser /Pohlig /Gonser /Graf

Table 2. Baseline values and outcomes at the first and second follow-up of the ITT population
Entry (E)
lactobacilli (n = 22) metronidazole (n = 22)

Control 1 (C1)
lactobacilli (n = 20) metronidazole (n = 20) MannWhitney estimator P(X < Y + 0.5P (X = Y) MannWhitney 97.5% CI onesided

Control 2 (C2)
lactobacilli (n = 19) metronidazole (n = 21) MannWhitney estimator P(X < Y) + 0.5P (X = Y) MannWhitney 97.5% CI onesided

Primary efficacy variable, mean (SD) Lactobacillary grade 3.86 (0.35) Lactate concentration 43.09 (41.06) Vaginal pH 5.88 (0.75) Micropathology 2.09 (0.92) Patients complaints 3.14 (1.25) Objective signs and symptoms 3.23 (1.19) Maturation Index per field of view (!1,000) 64.68 (27.05) VECO score 5.00 (0.82) Combined results Microbiological culture, n (%) 14 (63.6%) 6 (27.3%)

3.95 (0.21) 45.8 (58.46) 5.91 (0.89) 2.27 (0.77) 3.77 (1.45) 3.00 (1.27) 73.71 (17.97) 4.91 (0.97)

2.45 (1.15) 91.16 (97.99) 5.16 (0.90) 1.79 (0.71) 1.38 (1.40) 1.70 (1.69) 73.07 (15.53) 4.80 (1.61)

2.60 (1.14) 77.74 (43.92) 4.96 (0.60) 1.89 (0.74) 1.15 (1.46) 1.05 (1.61) 79.30 (14.50) 4.25 (1.12)

0.53 0.52 0.46 0.46 0.32 0.44 0.54 0.41 0.46

0.36 0.35 0.28 0.28 0.16 0.27 0.36 0.24 0.36

3.00 (1.20) 78.19 (116.30) 5.46 (0.94) 1.94 (0.80) 2.11 (1.52) 2.05 (1.54) 75.93 (18.38) 5.00 (1.56)

2.52 (1.36) 95.43 (91.80) 4.93 (0.75) 1.86 (0.79) 2.05 (2.48) 1.57 (1.69) 76.41 (16.12) 4.43 (1.60)

0.39 0.39 0.35 0.42 0.37 0.45 0.67 0.37 0.43

0.23 0.22 0.16 0.25 0.21 0.28 0.50 0.20 0.32

Gardnerella vaginalis Streptococcus sp.

15 (68.2) 6 (27.3)

7/19 (36.8) 3/18 (15.8) 13/20 (65) 16/20 (80)

6/19 (31.6) 8/19 (42.1) 11/20 (55) 16/21 (76.2)

7/18 (38.9) 3/18 (16.7)

7/21 (33.3) 10/21 (47.6)

Good to very good tolerability of the treatment, n (%) Assessed by investigator Assessed by patients

9/19 (47.4) 9/19 (47.4)

11/20 (55) 11/19 (57.9)

Numbers of patients with positive culture of G. vaginalis, indicative for BV, decreased by half after both treatments. This remained stable for 1 month after treatment. Numbers of patients with positive culture of streptococci, primarily involved in the pathogenesis of AV, decreased to the half in the lactobacilli group, but increased from 27 to 48% in the metronidazole group (42% after 1 week, 48% after 1 month). At C1, the tolerability of the lactobacilli treatment was rated as good to very good in 65 and 80% of the cases by the investigators and patients, respectively. Corresponding values for the metronidazole treatment were 55 and 76%, respectively. At C2, about half of the patients were dissatisfied in both treatment groups, most likely due to relapses. Two adverse events were observed in the lactobacilli group, one of which was mild nausea and headache. The other, vulvo-vaginal itching, was related to upcoming candidiasis and most likely not causally linked to treatment with lactobacilli. In the metronidazole group two adverse events were reported, one (abdominal cramps) with definite causality as stated by the investigator. The other adverse event (gastritis, hemoptosis) was reported as not being linked to the metronidazole treatment. One other minor adverse event was not specified further.
Lyophilized Lactobacilli and Gynoflor in Disrupted Vaginal Microflora

20 Number of patients

10 Metronidazole Lactobacilli 0 Start 1 week 1 month 4 months

Fig. 2. Kaplan-Meier-like graph showing patients not requiring additional therapy for bacterial vaginitis and vaginosis.

Discussion

This study was designed as a pilot study with a small population of premenopausal women with the intention to evaluate the feasibility of a possible large-scale future trial comparing the efficacy of probiotics (exogenous lactobacilli) to vaginally applied metronidazole for the treatGynecol Obstet Invest 2010;70:264272

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1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Superiority Gynoflor

Superiority Flagyl Lacto Lacta pH MicPa PatCo ObSig MatIn VECO E-C1 C1-E E-C1 E-C1 E-C1 E-C1 C1-E E-C1 Combined result

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Mann-Whitney statistic

1.0 0.9 Mann-Whitney statistic 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Superiority Gynoflor

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

Fig. 3. ITT population using Mann-Whit-

ney statistic and confidence interval for non-inferiority (97.5% CI, one-sided). Univariate results (Wilcoxon tests) and combined result (Wei-Lachin procedure) are shown for the first control visit after 1 week (C1, upper panel) and the second control visit after 1 month (C2, lower panel). Lacto = Lactobacillary grade; Lacta = lactate; MicPa = micro-pathology; PatCo = patients complaints; MatIn = Maturation Index; VECO = VECO score.

Superiority Flagyl Lacto Lacta pH MicPa PatCo ObSig MatIn VECO E-C2 C2-E E-C2 E-C2 E-C2 E-C2 C2-E E-C2 Combined result

ment of bacterial vaginal infections. Important clues are provided for a confirmatory study. Confirmatory analysis was done with the combined eight variables which reflects the status of the vaginal milieu as well as the disease symptoms. The results demonstrate one-sided equivalence at the 2.5% level of significance, indicating that lactobacilli are at least as good as metronidazole at 1 week after the stop of treatment. The data for lactobacilli at 46 weeks after the end of therapy, however, are lower and suggest inferiority of lactobacilli as compared to metronidazole. Originally, the analysis at visit 3 (4 months after the end of therapy) was also planned but turned out to be too long to perpetuate a sufficient number of patients. Two thirds had dropped out of the study in both groups, which was mainly due to relapses or use of medication that was not allowed in the study (protocol violation). In some studies, symptomatic candidiasis had developed in 14 30% of women treated with metronidazole [1]. Other au270
Gynecol Obstet Invest 2010;70:264272

thors reported relapse rates of BV between 14 und 39% within 1 month after metronidazole treatment and in at least 30% within 3 months [3135]. Both factors could have played a role in the high dropout rate and use of medication in this study. Therefore, since both treatments had similar high dropout rates, it may be necessary to change the prevailing idea of single episode treatment for a long-standing, chronically recurring disease, and to see treatment more in the strategy of intermittently sustaining the defensive flora either by application of chemicals such as metronidazole or clindamycin, or by application of exogenous lactobacilli at regular intervals, e.g. monthly. While frequent and prolonged use of antibiotics like metronidazole is afflicted with side effects [36], the application of exogenous lactobacilli may be a much safer alternative treatment for that purpose. Lactobacilli offered a longer protection against the regrowth of pathogens such as group B streptococci.
Donders /VanBulck /VandeWalle / Kaiser /Pohlig /Gonser /Graf

In a study by Ozkinay et al. [10], the status of the vaginal microflora was assessed after anti-infective treatment of vaginal infections using standard therapy like metronidazole, and the patients were then treated with Gynoflor in a randomized double-blind placebo-controlled trial. The results showed that the vaginal microflora improved significantly more under Gynoflor therapy compared to placebo. Thus, active restoration of a disrupted microflora after anti-infective therapy may be important to reduce recurrences. Due to the low numbers of patients in the subgroups, discussion at length about the specific BV and AV outcomes was not possible. However, the beneficial effect of Gynoflor on the recovery of G. vaginalis and group B streptococci plead for further studies in the treatment and long-term prevention of recurrences of both condi-

tions, even more so as this as well as former studies showed only sporadic adverse events and no serious side effects [6, 10]. We conclude that in symptomatic premenopausal women with disrupted vaginal flora due to bacterial vaginal infections like BV and AV, a physiological treatment such Gynoflor enables a valuable and safe alternative to therapy with an anti-infective agent like metronidazole. However, after a single treatment episode, metronidazole was more effective than exogenous lactobacilli after 1 month, indicating that a long-lasting effect with Gynoflor or other probiotics might only be obtained when applied repetitively. Therefore, future studies should address the possible benefits of repetitive application of these therapies.

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