BACTERIAL VAGINOSIS - LOCAL LACTOBACILLUS CASEI VAR RHAMNOSUS DÖDERLEIN MONOTHERAPY
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1 International Journal of Probiotics and Prebiotics Vol. 9, No. 4, pp , 2014 ISSN print, Copyright 2014 by New Century Health Publishers, LLC All rights of reproduction in any form reserved BACTERIAL VAGINOSIS - LOCAL LACTOBACILLUS CASEI VAR RHAMNOSUS DÖDERLEIN MONOTHERAPY 1 Stefan Kovachev and 2 Rossitza Vatcheva-Dobrevska 1 Department of General and Oncogynecology Military Medical Academy Sofia, Bulgaria; and 2 Department of Microbiology and Virology, University Hospital Queen Joanna - ISUL Sofia, Bulgaria [Received June 18, 2014; Accepted August 26, 2014] [Communicated by Prof. Francesco Marotta] ABSTRACT: The objective of the current research is to establish the efficacy of the local probiotic monotherapy in the treatment of bacterial vaginosis. 139 (100%) women with bacterial vaginosis, randomized into two groups are included in the research. In the first group 85 women were treated with local (10 applications) probiotic medicine, containing lactobacillus type Lactobacillus casei var rhamnosus Döderlein Lcr35. In the second group 54 patients were treated with five days, oral administration of Clindamycin (600mg) BID and local therapy of two vaginal ovules containing 1000mg Metronidazole each, which were administered locally every other day. Additionally in this group was administered again the same local probiotic. The efficacy of the therapeutic scheme was evaluated via comparison of patients clinical complains in the different groups, of data from clinical examinations and microbiological tests for each patient. One month after the probiotic monotherapy, the clinical efficacy in this group is 42.7% and microbiological efficacy 41.3%. They are lower than in the second group with combine treatment: clinical efficacy 87.5%, microbiological efficacy 80.3 %. Our results show that the local probiotic monotherapy has fewer efficacies in comparison to the nitroimidazole/lincozamide/ probiotic scheme for treatment of bacterial vaginosis. KEY WORDS: Bacterial vaginosis, Lactobacilli, Monotherapy, Probiotic, Vaginal flora Corresponding Author: Stefan Kovachev, MD, PhD, P.U.Todorov Blvd, Bl. No.5, entr.b, fl. No. 25, Sofia, Bulgaria-1404; Telephone: (+) ; stkovachev@abv.bg INTRODUCTION The use of probiotics to restore and maintain normal vaginal flora represents a promising alternative or addition to conventional therapy for the treatment of bacterial vaginosis (Fredricsson et al., 1989; Chimura et al., 1995; Hemalatha et al., 2012). Probiotics can be used independently or in addition to the primary therapy of bacterial vaginosis (Fredricsson et al., 1989; Chimura et al., 1995). Are conducted clinical trials for their action in both cases of application (Fredricsson et al., 1989; Chimura et al., 1995; Hemalatha et al., 2012). These studies apply specific strains of Lactobacilli orally or locally and tracked their ability to colonize the vagina of patients with symptomatic and asymptomatic bacterial vaginosis, to reduce colonization of pathogens and to remove the symptoms of it, when they are presented. Therefore, the use of probiotics in the treatment of bacterial vaginosis, to restore and maintain normal vaginal flora represents a promising alternative to conventional therapy (Fredricsson et al., 1989; Chimura et al., 1995; Hemalatha et al., 2012). The aim of our study was to establish the clinical and microbiological efficacy of local probiotic Lactobacillus casei var rhamnosus Döderlein monotherapy of bacterial vaginosis and effect on the vaginal flora. MATERIALS AND METHODS The survey is open, single-site and was conducted in our, gynecological outpatient clinic Military Medical Academy Sofia, Bulgaria, between year. To patients was provided information for the purposes of the investigation and the conditions of inclusion. We have the informed consent of each patient. The study included a total of 206 women aged between 17 and 50 years with clinical and/or microbiological established bacterial vaginosis. Of the inspection review days after incorporation came forward 139 women. Only their data and results are handled in the survey. Excluded are patients with established Neisseria Gonorrhoae, HSV, HPV, Chlamydia Trachomatis, HIV and other vaginal infections. Not included pregnant women and those taking corticosteroids, antibiotics,
2 130 imidazoles, probiotics or used vaginal preparations in the last month. Are not included in the study immune compromised patients, women with autoimmune diseases with endocrine diseases or diabetes. Excluded are patients with cancer disease. Of all the women in the inclusion in the study has drawn a medical history, performed gynecological examination and conducted microbiological tests. Upon examination of the vagina to make evaluation of vaginal secretion (determined often by patients like - fluor) according to the following specifications: quantity; consistence; color; the odor, which is reflected in the data sheet. The amount of vaginal discharge (flow), is estimated at 4 degrees from 0 (+) (+) to (+++). Upon examination of the vagina from the back vaginal vault with a dry sterile swab is taken vaginal secret for microbiological research. For the diagnosis of bacterial vaginosis, were used following clinical criteria introduced in gynecology practice from Amsel et al., 1983: - Homogeneous vaginal discharge (color and amount may vary) - Amine (fishy) odor when potassium hydroxide solution is added to vaginal secretions (commonly called the "whiff test") - Presence of clue cells (greater than 20%) on microscopy - Vaginal ph greater than 4.5 (Amsel et al., 1983). The establishment of three of the following four criteria is considered sufficient for diagnosis. For ph-metry indicator (Merck) strips are used in the range 4-7 as the pathological results are accepted on a ph above 4.5. Whiff test of vaginal secretion is reported as positive in separating of unpleasant smell. For the preparation of the wet mount, to the vaginal secretion affixed to a slide we add saline (0.9% NaCl). Wet mount was observed with a light microscope. The diagnosis of BV is placed in the presence of the "clue cells". Clue cells > 20% of the light field is considered a positive sign for BV. Not all patients included in the study, show a pronounced clinical symptoms. For many of them it was the diagnosis non-symptomatic and put through a microscopic examination of a Gram stained preparation of a vaginal secretion. Preparations in Gram were evaluated for the following morfotypes in surveillance with immersion (x 1000): large, Gram-positive rods (morfotype- laktobacilli); petty Gram-variabiles rods (G. vaginalis morfotype); small gram-negative rods (Bacteroides spp., morfotype); curved gram-variabilni rods (Mobiluncus morfotype) and Grampositive cocci. We used Nugent's criteria for the evaluation of microscopic Gram stained preparations introduced by Nugent et al., This method separates those with normal flora dominated by lactobacilli and smears with mixed flora characteristic of bacterial vaginosis (Nugent et al., 1991). The method is modified by other authors in 1991, to include intermediate categories, demonstrating the presence of mixed microbial flora with a significantly amount of laktobacilli strains (Nugent et al., 1991). Studies conducted in years 2002 and 2006 were evaluated and smears of vaginal flora were arranged in the following groups (grades): Grade 0, epithelial cells with no bacteria Grade 1, normal vaginal flora (lactobacillus morphotypes alone) Grade II, reduced numbers of lactobacillus morphotypes with a mixed bacterial flora Grade III, mixed bacterial flora only, few or absent lactobacillus morphotypes Grade IV, Gram-positive cocci only (Nugent et al., 1991; Ison et Hay, 2002). Grades 0, I, and IV are found in women without BV. Grade II is intermediate and not found in women with BV as defined by Amsel's criteria. Grade III is consistent with BV as diagnosed by Amsel's criteria (Ison et Hay, 2002). Randomization. Patients with bacterial vaginosis were randomized into two groups in 1:1 correlation. In the first clinical group fell 103 (85/82.5% on control review) women who held the first therapeutic treatment scheme. In the second group there was 103 (54/52.4% on control review) patients who were treated with a second therapeutic scheme. Therapeutic scheme. Patients in the first group held therapy with local application (10 days) of probiotic vaginal ovules containing live laktobacilli species: Lactobacillus casei var. rhamnosus Döderlein Lcr35 - Gynophilus; Laboratoires Lyocentre, France. In the second group (second therapeutic scheme) there was therapy with five daily oral dose of clindamycin, divided into two take 600 mg in 12 hours and local therapy of two vaginal ovules containing 1000 mg metronidazole which was administered on day first and third. By the fifth day after this combined therapy, additionally was administered again local probiotic Lactobacillus casei var. rhamnosus Döderlein Lcr35. Recommended sexual abstinence during the treatment to the control examination after him. Of sex partners was hired two days of oral therapy with tinidazol. Tracing was performed with a control review of day after the therapy, which involved a gynecological examination and microbiological examination with direct microscopy of wet and Gram stained mount of vaginal samples. The effectiveness of the therapeutic scheme was estimated through comparison of clinical complaints of the patients groups, data from the clinical examination and microbiological tests for each of them. Statistical analyses. Clinical and microbiological data were analysed using the test c2 for evaluation of the results obtained during the review. Statistical significance is taken in P < For each monitored parameter we reports the proportion (in percent) of patients with improvement in each treatment scheme individually. We compared how many times there was a decrease in the number of patients with the specific complaint (positive/negative, laboratory or
3 131 microbiological examination) as a result of the treatment. Two factor dispersion analysis with repeated observations was used (2-way ANOVA with repeated measures) with subsequent (post hoc) comparisons of the effect of therapy by a method of Tukey (Tukey's HSD). RESULTS A total of 206 subjects were recruited for this study. At the end of the study, data from 67 (32.5%) subjects were dropped from analysis due to non-compliance. TABLE 1. Clinical signs of bacterial vaginosis (BV) before and after therapy in the two groups. Abbreviations: Th, therapy GROUP I GROUP II CLINICAL INDICATORS Before Th After Th Before Th After Th N % N % N % N % Clinical complaints Vaginal fluorine (++; +++) ph-alkaline Whiff test (+) Clue cells Amsel s criteria (+) TABLE 2. Basic microbiological indicators of bacterial vaginosis (BV) before and after therapy in the two groups. Abbreviations: Th, therapy MICROBIOLOGICAL INDICATORS GROUP I GROUP II Before Th After Th Before Th After Th N % N % N % N % Gram-stained vaginal samples Anaerobe mixed flora - BV Absence of lactobacilli Nugent s grades of vaginal flora: II/III
4 132 TABLE 3. Statistical analysis of the results by two-factor variance analysis with repeated observations (2-way ANOVA with repeated measures) with subsequent (post hoc) comparisons of the effect of therapy by Tukey s HSD method. THERAPEUTIC SCHEME COMPARISON OF THE EFFECTS OF THE TWO THERAPEUTIC SCHEMES (I AND II) DIFFERENCE IN AVERAGE STANDARD ERROR P-VALUE (RELEVANCE VALUE) II Therapeutic scheme I * I Therapeutic scheme II (0,25) TABLE 4. Improvement of clinical and microbiological indicators after therapy in the two groups. MICROBIOLOGICAL AND CLINICAL INDICATORS IMPROVEMENT AFTER THERAPY Gram-stained vaginal samples Clinical efficacy of the therapy From Table 1, it is clear that in a smaller percentage of clinical complaints of patients after therapy (a basic indicator Amsel's criteria -11.1%) There are in the second group. In the first group, Amsel's criteria (+) patients after probiotic monotherapy were 53.0%. The microbiological efficacy of the therapy The results in the Table 2 indicate more serious reduction of microbiological indicators in a second clinical group from 3.7% to 18.5%. After her follow the results in the first group, from 43.5 percent to 56.5%. In Table 3, statistical significance was found in a second therapeutic group, compared with the first. From Table 4 shows that the improvement of the clinical and microbiological indicators after the treatment is greater in the group with the combination therapy compared with group held monotherapy. DISCUSSION Many studies apply specific strains of Lactobacilli orally or locally and tracked their ability to colonize the vagina of patients with symptomatic and asymptomatic bacterial vaginosis, to reduce colonization of pathogens and to remove the symptoms of it, when you have them. Hallen et al., 1992, discovering that in the group of patients with dysbacteriosis with dominant anaerobic strains which applied 7-10 days Groups (therapeutic schemes) I II % % Anaerobe mixed flora - BV Lactobacilli Nugent s grades of vaginal flora Grades: II/III Clinical criteria Amsel s criteria (+) Lactobacilli has more healed women compared with that using a placebo. Restoration of normal vaginal microflora settled in 57% (16 out of 28 women) of patients treated with Lactobacilli and at 0% (none of the 29 women) received placebo (Hallen et al., 1992). Rossi et al., 2010 explore long-term effect of the treatment with local probiotic of 40 women with bacterial vaginosis. The authors identify 80% of clinical efficacy of probiotic therapy at the end of the study (Rossi et al., 2010). Hemalatha et al., 2012, using probiotic monotherapy in the treatment of 67 women with bacterial vaginosis. Clinical efficacy of therapy (Amsel) established by the authors is 47% (Hemalatha et al., 2012). A prospective study of Chimura et al., 1995 confirmed the effect of local application of Lactobacilli in tracking the 11 women with bacterial vaginosis. The authors establish statistically significant reduction of vaginal inflammation, and ph and disappearance of all 14 gramnegative strains of microorganisms isolated in the first 3 days after the treatment (Chimura et al., 1995). Microbiologically, it is cured completely in 6 (54.5%) of the 11 women and was partially healed in three of them (27.3%), (Chimura et al., 1995). Mastromarino et al., 2009, conducted a placebo controlled study of the effectiveness of vaginal probiotics in the treatment of 39 women with bacterial vaginosis. Two weeks after the treatment, the clinical and microbiological efficacy is 61% in women using probiotics and 19% in the placebo group (Mastromarino et al., 2009). Bulgarian, placebo controlled study of Sigridov et al., 2007, lays down that the local application of Lactobacilli for one month in 20 patients
5 133 with bacterial vaginosis leads to a cure in 90% of cases, while in the placebo group healing is 67%. Follow-up of patients after three months showed 90% healed in the group treated with Lactobacilli and 50% for those treated with placebo (Sigridov et al., 2007). In contrast, several other randomized clinical trials are not find significant difference between the therapeutic outcomes of women with BV, applied locally specific strains of Lactobacilli, and those treated with vaginal lincozamides/nitroimidazoles, even on the contrary (Fredricsson et al., 1989; Erikson et al., 2005). Fredricsson et al treated 14 women with bacterial vaginosis with local L. acidophilus and achieve therapeutic effect only in one of them, whereas in the group with vaginal metronidazole it is 92.9%. In addition, the authors do not detect the increasing number of lactic acid bacteria in the vagina of women treated with L. acidophilus (Fredricsson et al., 1989). Erikson et al., 2005 used vaginal clindamycin for treatment of 187 women with BV and continue then with a local L. gasseri, L. casei, L. rhamnosus, L. fermentum or placebo within one menstrual cycle. Therapeutic results after the second menstrual cycle defined by Amsel criteria or by Nugent-Ison- Hay score of microscopic preparation of vaginal contents do not differ statistically significant in patients treated with Lactobacilli by those with placebo (Erikson et al., 2005). Our results shows clinical efficacy (Amsel's clinical criteria) of the probiotic monotherapy 47%. The therapy was suffered by patients without serious side effects. The microbiological efficacy (Gram stain of vaginal samples Nugent's criteria) was 41.1%. These results are lower than those established in clinical studies that we have quoted. They are lower than results that we received in the group, with a combined nitroimidazole/lincozamide/probiotic therapy with. In this group we received clinical efficacy 88.9% and microbiological efficacy 79.6%. Our results obtained in this study show that for the moment the local probiotic monotherapy have lesser efficacy in comparison with the combined therapeutic nitroimidazole/lincozamide/probiotic scheme for treatment of bacterial vaginosis. CONFLICT OF INTEREST DISCLOSURE I declare that there is no conflict of interest. REFERENCES Amsel R, Totten PA, Spiegel CA, Chen KCS, Eschenbach D, Holmes KK. (1983). Non- specific vaginitis: diagnostic and microbial and epidemiological associations. American Journal of Medicine 74: vaginal clindamycin ovules. Acta Dermato-Venereologica 85: Fredricsson B, Englund K, Weintraub L, Olund A, Nord CE. (1989). Bacterial vaginosis is not a simple ecological disorder. Gynecologic and Obstetric Investigation 28: Hallen A, Jarstrand C, Pahlson C. (1992). Treatment of bacterial vaginosis with lactobacilli. Sexually Transmitted Diseases 19: Hemalatha R, Mastromarino P, Ramalaxmi BA, Balakrishna NV, Sesikeran B. (2012). Effectiveness of vaginal tablets containing lactobacilli versus ph tablets on vaginal health and inflammatory cytokines: a randomized, double-blind study. European Journal of Clinical Microbiology and Infectious Diseases 31: Ison CA, Hay PE. (2002). Validation of a simplified grading of Gram stained vaginal smears for use in genitourinary medicine clinics. Sexually Transmitted Infections 78: Mastromarino P, Macchia S, Meggiorini L, Trinchieri V, Mosca L, Perluidgi M, Midulla C. (2009). Effectiveness of Lactobacillus-containing vaginal tablets in the treatment of symptomatic bacterial vaginosis. Clinical Microbiology and Infection 15: Nugent RP, Krohn MA, Hillier SL. (1991). Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. Journal of Clinical Microbiology 29: Rossi A, Rossi T, Bertini M, Caccia G. (2010). The use of Lactobacillus rhamnosus in the therapy of bacterial vaginosis. Evaluation of clinical efficacy in a population of 40 women treated for 24 months. Archives of Gynecology and Obstetrics 281: Sigridov I, Ivanov S, Batashki I. (2007). Preliminary results of Lactofem trial-first impressions. Akush Ginekol (Sofiia) 46 Suppl 2: Chimura T, Funayama T, Murayama K, Numazaki M. (1995). Ecological treatment of bacterial vaginosis. Japanese Journal of Antibiotics 48: Eriksson K, Carlsson B, Forsum U, Larsson PG. (2005). A double-blind treatment study of bacterial vaginosis with normal vaginal lactobacilli after an open treatment with
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