THE EFFICACY OF PROBIOTIC B. COAGULANS (SNZ-1969) TABLETS IN THE TREATMENT OF RECURRENT BACTERIAL VAGINOSIS

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1 International Journal of Probiotics and Prebiotics Vol. 12, No. 4, pp , 2017 ISSN print, Copyright 2017 by New Century Health Publishers, LLC All rights of reproduction in any form reserved Research Article THE EFFICACY OF PROBIOTIC B. COAGULANS (SNZ-1969) TABLETS IN THE TREATMENT OF RECURRENT BACTERIAL VAGINOSIS 1 V. Usha Rani, 2 S.R. Rao, 3 P. Grisha, 4 A.V. Sharma and 5 M.S. Usha 1,2,3,5 Osmania Medical College & Osmania General Hospital, Hyderabad; and 4 Regulatory Affairs and R&D Sanzyme Limited, Hyderabad, India [Received June 25, 2017; Accepted November 10, 2017] [Communicated by Prof. Francesco Marotta] ABSTRACT: Bacterial vaginosis (BV) is a common condition affecting 10 30% of women in developed nations. The current recommended treatment for BV is oral or vaginal antibiotics; however, recurrence rates of up to 60% within 12 months of treatment have been reported previously. The present study aimed to evaluate the efficacy of Bacillus coagulans (SNZ- 1969) against BV and the ability of this bacterial species to prevent BV recurrence in women aged years. The study assessed 173 women, and of these women, 120 showed recurrent BV according to Nugent s and Amsel s criteria. These 120 women were randomly assigned to the following study arms: metronidazole arm and metronidazole + B. coagulans arm. The metronidazole + B. coagulans arm showed better success (86.6%) in treating and minimizing recurrence of vaginal infections and consequently symptoms associated with BV, owing to the large number of lactobacilli administered in the B. coagulans arm. Oral supplementation with probiotics can be an efficient approach for the treatment of BV. Oral therapy for a longer duration would be more effective despite intestinal passage, vaginal ascension, and growth of lactobacilli requiring a long time. Long-term administration could probably allow controlled release of lactobacilli for preventing recurrence of BV. KEY WORDS: Bacillus coagulans, SNZ-1969, Vaginosis Corresponding Author: Dr. AV Sharma, Regulatory Affairs and R&D, Sanzyme (P) Limited, Hyderabad, India ; aries2360@gmail.com INTRODUCTION Bacterial vaginosis (BV) is a poly-microbial synergistic infection characterized by complex changes in the normal vaginal flora attributed to a reduction in the prevalence of lactobacilli and an increase in the concentration of pathogenic organisms (Vigneshwari et al., 2014; Udaylaxmi et al., 2011). In the healthy vagina, lactobacilli inhibit the growth of other microorganisms through certain properties, such as their adhesive ability, production of acids, bacteriocins, hydrogen peroxide, and bio-surfactants, and their ability to compete for mannose and glycoprotein receptors. BV is the most common cause of vaginitis in women of reproductive age, and it is a commonly encountered infection in the gynecology outpatient setting. More than 90 million cases of BV have been reported worldwide (Vigneshwari et al., 2014). According to a mid-term review of National AIDS Control Program III, the prevalence of BV in the adult population in India ranges from 17.8% to 63.7% (NHFS ). The most common pathogenic organisms causing vaginosis are facultative anaerobic bacteria, such as Gardnerella vaginalis and Mycoplasma, anaerobic gram-negative rods, such as Prevotellabivia, Prevotella intermedia, and Bacteroides spp., anaerobic gram-positive rods, such as Mobiluncus morphotypes, and anaerobic gram-positive cocci, such as Peptostreptococcus spp. (Vigneshwari et al., 2014). The etiology of BV is probably multi-factorial, and the factors initiating the shift are unclear. The local ph of the vagina can increase mainly because of a reduction in the number of hydrogen peroxide-producing lactobacilli, making it more susceptible to the growth of pathogenic organisms.bv may be symptomatic or asymptomatic. Symptomatic women commonly present with increased vaginal discharge, which is characteristically foul smelling, thin, gray, and homogeneous (Udaylaxmi et al., 2011). BV can lead to a variety of obstetric and

2 176 B. coagulans for BV treatment gynecological complications, such as endometritis, salpingitis, and pelvic inflammatory disease (PID), as well as pregnancy complications, such as premature rupture of membranes, preterm labor, spontaneous abortion, chorioamnionitis, postpartum endometritis, cuff cellulitis, and vault infections after hysterectomy (Scoper, 1999). Multiple criteria are used for the diagnosis of BV. One of the approaches for diagnosis is Amsel s composite criteria, which includes clinical diagnosis and a few simple laboratory tests. BV can also be diagnosed using Spiegel s and Nugent s criteria (Spiegel, 1991; Nugent et al., 1991). Both these criteria are based on the evaluation of normal flora in gram-stained smears of vaginal discharge. Metronidazole has been suggested as the most effective drug for treating BV. No antibiotic has been found to be completely effective in the treatment and prevention of BV (Hill, 1993; Majeroni, 1998). Recurrent infections are common, and efforts to reduce the prevalence of BV have been unsuccessful. Apart from the complications related to the use of antibiotics, the drug resistance of microorganisms is increasing (Beigi et al., 2004). Therefore, there is a need to develop new treatment approaches for BV in order to improve the low efficiency of existing treatment regimens. Many researchers have studied the effects of probiotics as an adjunct for BV (Reid and Bruce, 2003; Parma et al., 2014). Probiotics have been defined as live microorganisms which, when administered in adequate amounts, confer a health benefit to the host. Probiotic organisms are believed to have the potential to prevent diseases because of their antagonistic activities against pathogens in vivo. The colonization of the sterile infant vagina with commensal organisms marks the beginning of the protective mechanism against pathogenic organisms. They might fall prey to the insults caused due to various etiologies. The introduction of lactobacilli restores the ecological balance of the vagina, resulting in more disease-free reproductive years in a woman. Probiotics will restore normal vaginal flora, and normal ph will be maintained. Efforts to artificially restore normal vaginal flora with the use of probiotics could provide a reliable alternative treatment to antibiotics and a preventive regimen. The present study was performed to evaluate the efficacy of Bacillus coagulans (SNZ-1969) against BV and the ability of this bacterial species to prevent BV recurrence in women aged years. This study had two objectives. First, to assess the efficacy of the administration of 120 million colony forming units (CFU) of a probiotic (B. coagulans, SNZ-1969) and metronidazole for the treatment, prevention and recurrence of BV was evaluated. Second, to determine the prevalence of BV among women of reproductive age. MATERIALS AND METHODS Specimen collection was performed in a well-lit room. Patients were asked to lie on the examination table in the lithotomy position. An un-lubricated Cusco vaginal speculum was introduced into each participant s vagina under aseptic conditions, and the nature of the discharge and the condition of the vagina and cervix were noted. Diagnosis of BV was made according to Nugent s scoring (Nugent et al., 1991) and Amsel s criteria (Amsel et al., 1983). Study design and duration This was a two-arm, cross-sectional study performed for 10 months (February 2015 to November 2015) (Figure 1). The study was conducted at the Department of Obstetrics &Gynecology, Modern Government Maternity Hospital (MGMH) (Petlaburz, Hyderabad, India). Prior to performing the study, approval was obtained from the Institutional Ethics Committee (IEC) (Registration No. ECR/300/Inst/AP/2013) of Osmania Medical College (Hyderabad, India) (Figure 1). FIGURE 1. Flow Chart for Subject selection and Treatment. Detailed History and Clinical Examination Arm I (n=60) Treated with metronidazole 400mg BID x7 days 362 Women with Vaginal Discharge attended the Gynaecology OPD 120 had recurrent BV 173 diagnosed to have BV based on Amsel s criteria & Nugent s scoring Arm II (n=60) Treated with metronidazole 400 mg BID x 7days + BC- SNZ million CFU) TID X 21 days Inclusion criteria Women of reproductive age who were over 18 years but less than 41 years (either married or unmarried) with clinical symptoms of BV were included in this study. All participants were required to sign an informed consent form in their local language (Telugu/Urdu), Hindi, or English. Women were included if they did not have vaginal bleeding and if they did not have mixed vaginal infections (sexually transmitted diseases and HPV infection). The investigators ensured that the women participating in this study were not receiving anticoagulants or antibiotics. Women were included if they were not receiving vaginal antifungal agents or antibiotics 10 days prior to their scheduled enrolment in the study. Additionally, women were included if they were willing to restrain from using douches, spermicides, or other vaginal medications, except concomitant medications under the supervision and advice of the investigators. Moreover, the participants were asked to abstain from sexual intercourse at least 48 hours prior to the final study visit.

3 B. coagulans for BV treatment 177 Exclusion criteria Stringent exclusion criteria were followed, and normal female individuals were not allowed to participate in this study. Female individuals with abnormal Pap smears, genital carcinomas, or other acute/serious gynecological complications were excluded. Women were excluded if they refused to sign the informed consent form. Diagnosis according to Amsel s criteria Amsel s composite criteria includes presence of homogeneous vaginal discharge, vaginal ph of > 4.5, presence of clue cells in wet mounts of vaginal discharge, and a positive whiff test. dried, heat fixed, and stained with Gram stain. 3 Each bacterial morphotype was quantified under an oil immersion objective (l000 ) by using the following scheme: 1+, <1 per field; 2+, 1 5 per field; 3+, 6 30 per field; 4+, >30 per field. Large grampositive rods were considered as Lactobacillus morphotypes; small gram-negative and gram-variable rods were considered as G. vaginalis and Bacteroides spp. morphotypes; curved gramvariable rods were considered as Mobiluncus spp. morphotypes. Scoring was performed as shown in Table 1. The scores were added to yield a final score. The criterion for BV was a score of 7 or higher. A score of 4 6 was considered as intermediate, and a score of 0 3 was considered as normal. TABLE 1. Interpretation of Nugent scores. 1-3, Normal Vaginal Flora; 4-6, Intermediate and 7-10, Indicative of Bacterial Vaginosis Lactobacilli Morphotype Score Gardnerella & Bacteroides spp. Morphotype Score Curved Gram variable Rods Score < 1 1 < < Total According to Amsel, if at least 3 of the 4 criteria are positive, the patient is considered to have BV. Vaginal ph The vaginal ph was tested using a ph paper (Qualigens Fine Chemicals, India) by dipping the paper in secretions pooled at the posterior region. This was compared with a standardized colorimetric reference chart to estimate the actual ph. Whiff test A drop of vaginal fluid was taken on a grease-free glass slide, and a drop of 10% KOH was added. An intense putrid fishy odor was indicative of a positive reaction. Presence of clue cells A drop of vaginal fluid was mixed with a drop of normal saline on a clean grease-free glass slide, and a cover slip was placed over the drop. The slide was observed under 10 and 40 magnifications within 10 minutes. Vaginal epithelial cells, which were coated with coccobacilli resulting in a cell border that was indistinct or stippled instead of sharply defined, were considered as clue cells. Clue cells are a characteristic feature of BV. If clue cells represent 20% or more of the epithelial cells in a high-power field, the result is considered positive. The presence of any motile trichomonads, budding yeast cells, and pseudohyphae were also noted. Diagnosis according to Nugent s criteria Vaginal discharge was smeared on clean glass slides, air Evaluation of the efficacy of probiotics with regard to BV recurrence A total of 120 participants who had BV recurrence (3 or more episodes in the past 1 year) were included in the next step of the study. This study was designed as a prospective, non-blinded, two-arm, cross-sectional study. These 120 patients were randomly assigned to the following arms: 1) metronidazole arm (metronidazole 400 mg BID 7 days, n = 60) and 2) metronidazole + B. coagulans arm (B. coagulans [SNZ-1969] [120 million CFU] 1 Tab TID 21 days + metronidazole 400 mg BID 7 days, n = 60). Study participants were assessed 28 days after the initiation of therapy, according to Amsel s criteria and Nugent s score. The primary end point of the study was considered when patients had symptomatic alleviation from the signs and symptoms of BV. The secondary end point was considered as the absence of Amsel s criteria or the existence of only 1 criterion along with a Nugent s score of < 3. The absence of Amsel s criteria or the existence of only 1 criterion was used to determine treatment success. TABLE 2. Incidence of BV in various age groups. Age group n (%) (16.1%) (46.9%) (25.4%) (6.9%) 5 > 41 3 (1.73%) Total 173

4 178 B. coagulans for BV treatment TABLE 3. Effect of marital status on vaginal discharge Marital status Vaginal discharge (n=) 1 Married 248 (68.5%) 2 Unmarried 114 (41.49%) Total 362 TABLE 4. Symptoms and Rates of recurrence of BV BV clinical symptoms Rate of recurrence Observations n No. of Episodes the Previous year n 1 Vaginal Discharge 173 (100%) 0 33 (%19.05) 2 Malodour 173 (100%) (11.56%) 3 Itching 83 (47.9%) (51.44%) 4 Dysuria 52 (30.05%) > 6 31 (17.91%) TABLE 5. Effect of treatment with metronidazole or metronidazole + BC SNZ-1969 on symptomatic relief Treatment regimen Metronidazole Metronidazole + BC SNZ-1969 Before (n) After Before (n=) After 1 Vaginal Discharge (56.6%) 60 8 (13.3%) 2 Malodour (50%) 60 7 (11.6%) 3 Itching (18.3%) 26 3 (5%) 4 Dysuria 17 9(15%) 22 6 (10%) TABLE 6. Effect of treatment with metronidazole or metronidazole + BC SNZ-1969 on Amsels criteria changes Variable Metronidazole Metronidazole + BC SNZ-1969 Before After Before After 1 Vaginal Discharge 60 (100%) 34 (56.6%) 60 (100%) 8 (13.3%) 2 Clue cells 42 (70%) 12 (20%) 43 (71.6%) 12 (20%) 3 Whiff test 60 (100%) 34 (56.6%) 60 (100%) 8 (13.3%) 4 ph > (68.3%) 25 (41.6%) 39 (65%) 7 (11.6%) RESULTS A total of 362 patients of reproductive age with complaints of vaginal discharge were examined for BV. Among these patients, 173 were diagnosed with BV. The primary symptom observed in the 173 women was vaginal discharge with malodour. On further assessment of patient history, 120 patients were found to have recurring episode (Figure 1). Only women with BV were allowed to participate in the study, provided they met the inclusion criteria. The incidences of BV in women of various age groups are shown in Table 2. In our study, the incidence of BV was the maximum in the age group of years (n = 86, 49.7%) (mean age in this group was 28 years), followed by the age group of years (n = 44, 25.4%), implying that the incidence was highest in women of reproductive age (Table 4) The incidence of vaginal discharge according to marital status is presented in Table 3. Vaginal discharge was more commonly noted in married women than in unmarried women (n=248 [68.5%] vs. n=114 [49.4%]). The symptoms and rate of BV recurrence are presented in Table 3. Symptomatic relief and classification according to Amsel s criteria after treatment on the basis of the treatment regimen and characteristics are shown in Tables 5 and 6, respectively. A significant reduction in symptoms and scoring was observed in the metronidazole + B. coagulans arm, when compared to the findings in the metronidazole arm. Additionally, the cure rate was higher in the metronidazole + B. coagulans arm than in the metronidazole arm (86.6% vs. 41.6%; Tables 5 and 6). DISCUSSION BV is the most common cause of vaginal discharge among women of reproductive age, with a prevalence of 16-69%, depending on the population studied. Culture is the gold standard method for the diagnosis of most bacterial diseases; however, culture is not the gold standard for the diagnosis of BV because organisms that are involved in BV cannot be easily isolated in the laboratory and normal women have these organisms in their vagina in small numbers. Multiple criteria are often used for the diagnosis of BV. One of the approaches for diagnosis is Amsel s composite criteria, which includes clinical diagnosis and a few simple laboratory tests. BV can also be diagnosed according to Spiegel s and Nugent s criteria. Both these criteria are based on the evaluation of normal flora in gram-stained smears of vaginal discharge. We screened 362 individuals who visited our out-patient department complaining of vaginal discharge, and of these,

5 B. coagulans for BV treatment (48%) were diagnosed as having BV according to Amsel s and Nugent s criteria. Similar prevalence rates have been reported in other studies (Figure 2 and Tables 7). FIGURE 2. Effect of treatment on pre-treatment (Black bar) vs. post-treatment (Grey bar) Nugent Scores. BC= Bacillus coagulans and met= Metronidazole Nugent Score (Pre- and Post -Treatment) met met + BC- SNZ TABLE 7. Prevalence of BV as reported in different populations Study Prevalence rate of BV 1 Nawani et al., % 2 Tiyyagura et al., % 3 Present study (Usha Rani et al.) 48.0% Affected age group: BV was most common in the age group of years. The disease occurred mainly in young women of reproductive age, and this finding is consistent with the findings of previous studies. Changes in the structure and composition of the vaginal ecosystem maybe influenced by age, infection, method of birth control (contraceptives), frequency of sexual activity, and number of sexual partners. These features are mainly noted in women of reproductive age. Symptoms: The most common symptoms of BV were vaginal discharge and malodor, which were noted in 173 cases, followed by itching and dysuria. These findings were consistent with the findings of a previous study (Falagasetal, 2007). Recurrent BV is defined as having more than 3 episodes of BV in the past 1 year. In our study, 120 participants had recurrent BV. Regardless of the significant progress reported on the understanding of the epidemiology, pathophysiology, and treatment of BV, scant information is available regarding recurrent BV. Treatment with probiotics: The loss of vaginal lactobacilli appears to be the major factor in the cascade of changes leading to BV, and relapse is associated with failure to establish healthy lactobacilli-dominated vaginal flora. The effectiveness of oral probiotics is associated with the following conditions: increased movement of probiotic and/or indigenous lactobacilli from rectal skin to the vagina; reduced movement of pathogens from rectal skin to the vagina; and enhancement of intestinal mucosal immunity, which affects vaginal immunity, rendering the environment less receptive to organisms responsible for BV. In a previous study, on treating vaginal dysbiosis with dominant anaerobic species, local monotherapy with probiotics was less effective than combined therapeutic schemes (Kovachev et al, 2013). The results of another study on lactobacilli involving 399 vaginal samples (Ehrstrom et al, 2010) showed that short-term probiotic supplementation can result in vaginal colonization of exogenous lactobacilli for more than 6 months. In the present study, there was a significant difference between the 2 study groups, with better cure rates in the metronidazole + B. coagulans arm than in the metronidazole arm (86.6% vs. 41.6%) (Table 8). Many other studies have also shown that patients receiving metronidazole + B. coagulans had better treatment efficiency (Table 9). TABLE 8. Recurrence rates of BV as reported in different populations Study Rate 1 Bradshaw et al., % 2 Wilson, % 3 Present study (Usha Rani et al.) 69% TABLE 9. Cure rates of BV: metronidazole vs. metronidazole + BC SNZ-1969 treatment Drugs administered BV Cure rate 1 Metronidazole 41.6% 2 metronidazole + BC-SNZ % TABLE 10. Cure rates of BV following metronidazole or metronidazole + BC SNZ-1969 treatments Study metronidazole (n=) metronidazole + probiotic (n=) 1 Anukam et al, Larson et al., Petricevic et al., Sudha et al., Tafazzoli et al., Present study (Usha Rani et al) 41.6% 86.6%

6 180 B. coagulans for BV treatment TABLE 11. Metronidazole vs. metronidazole + BC SNZ-1969 treatments of BV: Analysis of outcome and Interpretation of data. Parameters metronidazole + BC SNZ-1969 (n=) Metronidazole (n=) p-value Vaginal discharge Relief / Cured 52 8 p < 0.05 Persistent / Not Cured Malodour/Whiff test Relief 53 7 Persistent p < 0.05 Itching Reduced/Cured 23 3 Persistent NS Dysuria Reduced/Cured 16 6 Persistent 8 9 NS Nugent s criteria** (Figure1) BV Cured 52 8 p < 0.05 BV Not Cured Recurrent BV among the Study subjects BV Curative Rates 52 8 p < 0.05 Not Cured Thus, the findings in our study are consistent with the findings in the majority of previous studies (Eriksson et al., 2005; Falagas et al., 2007; Bodean et al., 2013; Ling et al., 2013); however, there were some differences, possibly because of factors, including the use of B. coagulans instead of Lactobacillus spp., use of Lactobacillus alone in some studies, differences in the Lactobacillus colony count, vaginal use of Lactobacillus, and use of clindamycin instead of metronidazole. The present study had some limitations. This was a preliminary study with a small number of patients and was an open trial. Further studies are needed to confirm the effects of probiotics in a large number of patients before probiotics can be widely accepted as an alternative mode of treatment in patients with symptoms of BV. Significant reductions in the symptoms and scoring are observed in the treated cases CONCLUSION The prevalence of BV was 48%, and the rate of recurrence was 69% (Table 10). The present study concluded that the number of Lactobacilli was higher among patients treated with a probiotic and metronidazole than among patients treated with metronidazole alone. Treatment using a probiotic along with metronidazole for BV showed better success (86.6%) in treating and minimizing recurrence of vaginal infections and consequent symptoms, such as vaginal discharge, malodor, burning micturition, and itching. Hence, oral supplementation with a probiotic can help improve the treatment of BV (Table 11). FUTURE IMPLICATIONS When managing women with recurrent BV, it is important to recognize not only the physical symptoms or discomfort of BV but also the significant and distressing psychosocial sequel experienced by women. While BV is often considered a minor and common vaginal condition by clinicians, its recurrent nature and the substantial impact it can have on a woman s emotional, sexual, and social lives render it an important infection. Therefore, treatment is imperative to prevent recurrence. REFERENCES Amsel, R., Totten, P. A., Spiegel, C. A., Chen, K. C. S., Eschenbach, D. A. and K. K. Holmes. (1983). Nonspecific vaginitis: diagnostic criteria and epidemiologic associations. American Journal of Medicine 74: Anukam, K.C., Osazuwa, E., Osemene, G.I., Ehigiagbe, F., Bruce, A.W. and Reid, G. (2006). Clinical study comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat symptomatic bacterial vaginosis. Microbes and Infections 8: Beigi, R. H., Austin, M. N., Meyn, L. A., Krohn, M. A. and Hillier S. L. (2004). Antimicrobial resistance associated with the treatment of bacterial vaginosis. American Journal of Obstetrics and Gynecology 191: Bodean, O., Munteanu, O., Cirstoiu, C., Secara, D. and Cirstoiu, M. (2013). Probiotics a helpful additional therapy

7 B. coagulans for BV treatment 181 for bacterial vaginosis. Journal of Medicine and Life 6: Bradshaw, C.S., Morton, A.N., Hocking, J., Garland, S.M., Morris, M.B., Moss, L.M., Horvath, L.B., Kuzevska, I. and Fairley, C.K. (2006). High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. Journal of Infectious Disease. 193: Ehrström, S., Daroczy, K., Rylander, E., Samuelsson, C., Johannesson, U., Anzén, B. and Påhlson, C. (2010). Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvo-vaginal candidiasis. Microbes and Infection 12: Eriksson, K., Carlsson, B., Forsum, U. and Larsson, P-G. (2005). A double-blind treatment study of bacterial vaginosis with normal vaginal lactobacilli after an open treatment with vaginal clindamycin ovules. Acta dermatovenereologica 85: Falagas, M. E., Betsi, G.I. and Athanasiou, S. (2007). Probiotics for the treatment of women with bacterial vaginosis. Clinical Microbiology and Infection 13: Hill, G.B. (1993). The microbiology of bacterial vaginosis. American Journal of Obstetrics and Gynecology, 169: Kovachev, S. and Dobrevski-Vacheva, R. (2013). Probiotic monotherapy of bacterial vaginosis: An open, randomized trial. Akush Ginekol (Sofiia) 52: Larsson, P.G., Stray Pederson, B., Ryttig, K.R. and Larsen, S. (2008). Human lactobacilli as supplementation of clindamycin to patients with bacterial vaginosis reduce the recurrence rate; a 6-month, double blind, randomized, placebo-controlled study. BMC Women s Health 8: 3. Ling, Z., Liu, X., Chen, W., Luo, Y., Yuan, L., Xia, Y., Nelson, K.E., Huang, S., Zhang, S., Wang, Y., Yuan, J., Li, L. and Xiang, C. (2013). The restoration of the vaginal microbiota after treatment for bacterial vaginosis with metronidazole or probiotics. Microbial Ecology 65: Majeroni, B.A. (1998). Bacterial vaginosis: An update. American Family Physician 57: Nawani, M. and Sujatha, R. (2013). Diagnosis and prevalence of bacterial vaginosis in a tertiary care centre at Kanpur. Journal of Evolution of Medical and Dental Sciences 2: Nugent, R. P., Krohn, M. A. and Hillier, S. L. (1991). Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. Journal of Clinical Microbiology 29: Parma, M., Stella, V.V., Bertini, M. and Candiani, M. (2014). Probiotics in the prevention of recurrences of bacterial vaginosis. Alternative Therapies in Health and Medicine. 20 Suppl 1: Petricevic, L. Witt, A. (2008). The role of Lactobacillus casei rhamnosus Lcr35 in restoring the normal vaginal flora after antibiotic treatment of bacterial vaginosis. BJOG-An International Journal of Obstetrics & Gynaecology 115: Reid, G. and Bruce, A.W. (2003). Urogenital infections in women: can probiotics help? Postgraduate Medical Journal 79: Spiegel, C.A. (1991). Bacterial vaginosis. Clinical Microbiological Review 4: Scoper, D.E. (1999). Gynecologic Complications of Bacterial Vaginosis: Fact or Fiction? Current infectious disease reports 1: Sudha, R.M., Yelikar, K.A., and Deshpande, S. (2012). Clinical study of Bacillus coagulans unique IS-2 (ATCC PTA ) in the treatment of patients with bacterial vaginosis. Indian Journal of Microbiology 52: Tafazzoli, H.H., Amiraliakbari, S., Afrakhteh, M., Alavi Majd, H. and Nouraei, S. (2014). Comparison of Metronidazole versus a Combination of Metronidazole plus Probiotics in the Treatment of Bacterial Vaginosis. Journal of Womens Health, Issues and Care 3:3. doi: / Tiyyagura, S., Taranikanti, M., Ala, S. and Mathur, D.R. (2012). Bacterial Vaginosis in Indian Women in the Reproductive Age Group. International Journal of Biomedical Research 3: Udayalaxmi, G.B., Subbannayya, K. and Shalini, S. (2011). Comparison of the Methods of Diagnosis of Bacterial Vaginosis. Journal of Clinical and Diagnostic Research 5: Vigneshwari, R.S., Rambabu, T., Jeyaseelan, T.S., Revathi, P., Deivam, S. and Uma, A. (2014). Role of probiotics in the treatment of bacterial vaginosis. International Journal of Scientific and Technology Research 3:1-4 Wilson, J. (2004). Managing recurrent bacterial vaginosis. Sexually Transmitted Infections 80: 8-11.

8 182 B. coagulans for BV treatment