Is it possible to prevent recurrent vulvovaginitis? The role of Lactobacillus plantarum I1001 (CECT7504)

ORIGINAL ARTICLE Is it possible to prevent recurrent vulvovaginitis? The role of Lactobacillus plantarum I1001 (CECT7504) S. Palacios1 & J. Espadaler2 & J. M. Fernández-Moya3 & C. Prieto4 & N. Salas1 Received: 16 March 2016 /Accepted: 20 June 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The purpose of this study was to prospectively evaluate the impact of the use of L. plantarum I1001 applied vaginally on Vulvovaginal Candidiasis (VVC) time-until- recurrence after treatment with single-dose vaginal clotrima- zole. This was a clinical open-label, prospective study of two non-randomized parallel cohorts with symptomatic acute VVC: (1) 33 sexually active women 18–50 years old, pre- scribed a standard single-dose 500 mg vaginal tablet of clotri- mazole followed by vaginal tablets with L. plantarum I1001 as adjuvant therapy, and (2) 22 women of similar characteris- tics but prescribed single-dose clotrimazole only. Use of the probiotic and factors that might influence recurrence risk (age, recurrent VVC within previous year, antibiotic prior to study enrolment, diaphragm or IUD contraception, among others) were included in a multivariate Cox regression model to adjust for potential between-cohort differences. Probiotic use was associated with a three-fold reduction in the adjusted risk of recurrence (HR [95 %CI]: 0.30 [0.10–0.91]; P = 0.033). Adjusted free-survival recurrence was 72.83 % and 34.88 % for the probiotic and control groups, respectively. A higher cumulative recurrence was also observed in cases with use of antibiotics prior to enrolment (HR [95 %CI]: 10.46 [2.18– 50.12]; P = 0.003). Similar findings were found at six months after azole treatment in women with RVVC. Overall, good compliance with the probiotic was reported for 91.3 % of women. The study suggests that follow-up therapy with vag- inal tablets with L. plantarum I1001 could increase the effec- tiveness of single-dose 500 mg clotrimazole at preventing recurrence of VVC, an effect that was also observed in women with recurrent vulvovaginal candidiasis (RVVC) after six months of azole treatment. Introduction Vulvovaginal candidiasis (VVC) is one of the most frequent infections of the female genital tract and is mainly associated with Candida albicans (followed by Candida glabrata) [1]. Candida species are normally found in the lower genital tract of 10–20 % of healthy women of childbearing age [2]. Evolution from colonization to symptomatic infection involves different host factors such as susceptibility and inflammatory responses and/or the imbalance of vaginal microbiota [3]. Available literature on VVC suggests that 75 % of women will show a VVC episode [4, 5] through their lifetime, and that 5–10 % of all women will experience recurrent vulvovaginal candidiasis (RVVC), i.e ≥4 episodes/year [6]. Short-term and single-dose vaginal antifungals are success- ful in 80–90 % of acute uncomplicated cases [7, 8]. However, azole resistance rates are above 15 % in women with RVVC [9, 10]. Antifungal resistance, infection recurrence and side effects of pharmacological treatments are key issues for patients and their physicians seeking alternative interventions for VVC. Electronic supplementary material The online version of this article (doi:10.1007/s10096-016-2715-8) contains supplementary material, which is available to authorized users. * C. Prieto cprieto@gynea.com 1 Instituto Palacios de Salud y Medicina de la Mujer, Antonio Acuña 9CP, 28009 Madrid, Spain 2 Autonomous University of Barcelona, AB-Biotics S.A. Eureka building, 08193 Bellaterra, Barcelona, Spain 3 Instituto de Medicina EGR, Camino de la Zarzuela, 19, 28023 Aravaca, Madrid, Spain 4 Gynea Laboratorios by Kern Pharma, Pol. Ind. Colom II, C/Venus, 72, 08228 Terrasa, Barcelona, Spain Eur J Clin Microbiol Infect Dis DOI 10.1007/s10096-016-2715-8.

Probiotics are defined as Blive microorganisms, which when administered in adequate amounts, confer a health ben- efit on the host^ [11]. Probiotic properties are strain-specific so their positive health effects cannot be extrapolated to other strains of the same species or genus [12]. Lactobacilli are the predominant bacteria in a healthy vaginal ecosystem, and have been proposed for the treatment and prevention of genitouri- nary infections including candidiasis [4, 6]. Local use of these microorganisms results in immunomodulation responses and the restoration of vaginal microbiota, interfering with the col- onization and growth of potential pathogens such as Candida [13]. A Lactobacilli-dominated vaginal microbiota produces significant levels of lactic acid with strong microbicidal prop- erties [14]. Evidence of synergy between adjuvant local Lactobacilli and azole treatment for VVC has been reported [9, 15]. Nevertheless, evidence of the benefit of adding probiotics to the standard azole on the risk of symptomatic recurrence is lacking. Recently, a pilot study for the evaluation of colonization and tolerability of a vaginal tablet of L. plantarum strain I1001 (deposit code CECT7504) in healthy women, reported that use of this formulation, three times a week on alternate days, achieves an adequate vaginal lactobacilli concentration [16]. These positive results led us to prospectively evaluate the impact of the use of L. plantarum I1001, applied vaginally, on VVC recurrence after a single-dose vaginal clotrimazole. Patients and methods A clinical open-label, prospective study of two non- randomized parallel cohorts was conducted in the out-patient gynaecological departments of the Instituto Palacios de Salud y Medicina de la Mujer and the Instituto de Medicina EGR (Madrid) from June 2013 until February 2015. The first cohort (n = 33) corresponded to sexually active women between 18 and 50 years old with symptomatic acute VVC, who were prescribed with a standard single-dose 500 mg vaginal tablet of clotrimazole, followed by the L. plantarum I1001 vaginal probiotic tablet (Melagyn® Probiotico Vaginal, Gynea by Kern Pharma, Spain; each tablet contains L. plantarum I1001 at minimum 1 × 108 colony forming units) as adjuvant therapy (one tablet, three times a week on alternate days, for two consecutive months, exclud- ing days with menstruation). The second cohort (n = 22) com- promised women of similar characteristics but prescribed the single-dose clotrimazole treatment only. Exclusion criteria (both cohorts) were pregnancy/breast feeding, delivery or abortion within the previous trimester, signs of other vaginal infections, abnormal genital bleeding within the previous semester, or any concomitant medication during follow-up that might significantly influence the evaluation and/or study results (including beta-lactams, clindamycin and tetracycline). Informed written consent was obtained from all participants prior to enrolment. All the study materials were approved by the Ethical Committee of the Hospital Universitario de la Princesa (Madrid, Spain). Vaginal probiotic and dose administration L. plantarum I1001 (CECT7504) is a selected, patented strain which has been tested in vitro, showing good adherence to vaginal epithelial cells (VEC), high acidification of simulated vaginal media, high tolerance to antimicrobial factors of inflamed vaginal fluid, and intrinsically resistant to high con- centrations of some typical antibiotics (ATB) and antifungals used for vaginal affections. This L. plantarum I1001 formula- tion has been previously tested in healthy women during a pilot open label clinical trial at Hospital Vall d’Hebron (Barcelona, Spain) [16], showing successful colonization of the vagina for at least 48 hours, therefore, allowing its appli- cation on alternate days. Data collection and follow-up The primary outcome was the recurrence-free survival of signs/symptoms of VVC, as collected in the follow-up visits at two and three months after the azole treatment, within the possibility of a six-month visit for women with history of RVVC. Specific risk factors (age, diabetes mellitus, RVVC within the last year, ATB prior to enrolment, diaphragm/intrauterine device or oral contraception, immunosuppression and history of other non-candida vulvovaginitis [VV]) were recorded at baseline. Self-reported presence of pruritus, vulvar soreness/ irritation and burning vulvar pain, and signs of vaginal dis- charge, vulvar erythema and edema, and malodorousness, were assessed using a semi-quantitative scale (0 = absent, 1 = light, 2 = moderate and 3 = severe). In the L. plantarum I1001 cohort, physicians rated the effectiveness and tolerabil- ity of the product, using a semi-quantitative scale (0 = inade- quate, 1 = fair, 2 = good, 3 = very good), while women also reported their opinion on the product and evaluated its tolera- bility and their satisfaction using the Spanish version of the Treatment Satisfaction Questionnaire for Medication (TSQM version 1.4) [17]. Statistical analysis Baseline characteristics (including known risk factors for VVC) of both cohorts were compared with Fisher’s exact test (categorical variables) and Mann-Whitney’s U test (quantita- tive variables). Values were summarized and presented as per- centages or mean ± standard deviation (SD). For the main clinical outcome (VVC recurrence-free survival), use of the Eur J Clin Microbiol Infect Dis.

vaginal probiotic and additional factors that might influ- ence the risk of recurrence (see above) were included in a multivariate Cox (proportional-hazards) regression model; these results are presented as Hazard ratios (HR) and their 95 % confidence intervals (95 %CI). Drop-outs were cen- sored at the time of last follow-up. In an attempt to assess the change in recurrence within cohorts, we compared the average number of VVC episodes per trimester within 12 months prior to enrolment including the baseline episode of women with three-months follow-up (obtained from clini- cal records) to those observed during the follow-up at three months, by using the non-parametric Wilcoxon signed-rank test. Only subjects with complete clinical record for the past 12 months and complete follow-up at three months were considered for this later analysis. Results on the TSQM are presented as median and interquartile range (IQR), because of their non-normality. No imputation was performed for missing values, and significance was set at a level of 5 %, two-tailed. All statistical tests were performed with SPSS Statistics for Windows v22.0 (IBM Corp. 2013. Armonk, NY). Results Baseline data A general description of the patients and current VVC episode is presented in Table 1. Flow of patients throughout the study is presented in Fig. 1. Drop-out rate was slightly higher in the clotrimazole-only cohort (18.5 % vs. 5.7 % at 2 months, and 24.1 % vs. 15.4 % at 3 months), although the difference did not reach statistical significance. Among the 55 women from both cohorts, 50 (91 %) had a history of previous VVC anytime during their lifespan (Table 1), from which 33 (63.5 %) met criteria for VVC within the 12 months prior to enrolment. The number of VVC epi- sodes during the previous 12 months was higher in the L. plantarum I1001 cohort (4.55 ± 2.58 vs. 3.53 ± 1.88), al- though this difference did not reach statistical signifi- cance. Conversely, previous non-Candida VVs were re- ported exclusively in the antifungal-only cohort (n = 3, P = 0.059, statistical trend). Current VVC was generally characterized by the specialist with vulvar erythema, vaginal discharge, edema and burning/ soreness, without differences between cohorts regarding the number of patients with each symptom. Patients’ and gynaecologists’ evaluation of the vaginal probiotic Overall, patients treated with the vaginal probiotic reported a compliance of 91.3 %. The TSQM (n = 29) results showed that patients reported high ratings of efficacy, lack of side effects, convenience and overall satisfaction (Table 2). Only one patient reported a side effect (edema and erythema) of moderate intensity, and rated it as Bsomewhat affecting her satisfaction with the treatment^ in the TSQM questionnaire (i.e. a score of 3 in a Likert 1–5 scale). Moreover, gynecolo- gists considered efficacy of the test product in their patients (n = 31) as Bgood^/Bvery good^ in 87 % of cases, and tolera- bility as Bgood^/Bvery good^ in 100 % of them. Time until symptomatic recurrence Overall, 10/31 (32.2 %) of women in the probiotic group and 10/17 (58.8 %) of women in the control group had experienced one or more symptomatic recurrences during the 3–month follow-up (P = 0.125). The Cox regression model on the combined cohorts (Table 3) showed that adjusted HR for developing a new VVC within three months of the single-dose clotrimazole, depending on probiotic use, was 0.30 (95 %CI: 0.10–0.91) and that this effect was statistically significant (P = 0.033, Fig. 2). Among other risk factors, ATB prior to enrolment also showed a significant effect, with an adjusted HR of 10.46 (95 %CI: 2.18–50.12); (P = 0.003, Fig. 3). Thus, comparison of the recurrence-free survival shows a clear increase in ad- justed survival at 3 months (73 % vs. 35 %, i.e. a +108.6 % increase) in women receiving the probiotic, while the ‘previ- ous ATB’ factor shows marked effect in the opposite direction (6 % vs. 68 %). Analysis of the subgroup with history of RVVC (n = 23 of the clotrimazole + probiotic cohort, and n = 12 from the clo- trimazole cohort) at 6-months of follow-up, showed a similar difference in adjusted survival among treatment cohorts (63 % vs. 22 %, Supplementary material). The adjusted HR for symptomatic recurrence depending on probiotic use was 0.30 (95 %CI: 0.10–0.89; P = 0.03), likewise for the effect of ‘previous ATB’ (Supplementary material). Differences in the number of episodes In the L. plantarum I1001 cohort the average number of VVC episodes per trimester during the 3-month follow-up was 0.45 ± 0.72, down from 1.14 ± 0.65 episodes/trimester during the 12 months prior to enrolment, thus representing a reduction of 59 % (P = 0.001). Conversely, such difference was not ob- served in the clotrimazole-only cohort (0.67 ± 0.62 vs. 0.88 ± 0.47 episodes/trimester; P > 0.1). Discussion In the present study, follow-up therapy with vaginal tab- lets with L. plantarum I1001 after a single-dose 500 mg clotrimazole was found to significantly reduce the risk of Eur J Clin Microbiol Infect Dis.

symptomatic recurrence within 3 months after an acute VVC, with similar findings in the subset of patients with RVVC within 6 months of follow-up. Although most baseline characteristics were comparable, patient distribution among cohorts was not randomized and resulted in a larger number of the VVC episodes of women Table 1 Demographic, reproductive and gynaecological characteristics Characteristic Total sample Clotrimazole + L. plantarum I1001 Clotrimazole P-value Total cases 55 33 22 Age (years) 33.36 ± 8.61 33.58 ± 8.23 33.05 ± 9.34 0.830 Marital status Single 27 (49.1 %) 16 (48.5 %) 11 (50.0 %) 0.190 Divorced 2 (3.6 %) 0 (0.0 %) 2 (9.1 %) Married/living together 26 (47.3 %) 17 (51.5 %) 9 (40.9 %) Gynaecological history and reproductive state Child bearing age 51 (92.7 %) 32 (97.0 %) 19 (86.4 %) 0.290 Perimenopause 4 (7.3 %) 1 (3.0 %) 3 (13.6 %) Children (yes) 19 (34.5 %) 10 (30.3 %) 9 (40.9 %) 0.564 Relevant medical and/or surgical history 11 (20.4 %) 9 (28.1 %) 2 (9.1 %) 0.167 Risk factors for VV Antibiotics prior to study enrolmenta 6 (11.5 %) 4 (12.9 %) 2 (9.5 %) 1.000 Diaphragm or IUD contraceptiona 3 (5.8 %) 2 (6.5 %) 1 (4.8 %) 1.000 Oral contraceptiona 11 (21.2 %) 6 (19.4 %) 5 (23.8 %) 0.739 Diabetes mellitusa 0 (0 %) - - - Immunosuppressiona 1 (1.9 %) 1 (3.2 %) 0 (0.0 %) 1.000 Othersa 4 (7.7 %) 2 (6.5 %) 2 (9.5 %) 1.000 Vulvovaginitis history Other non-candida VV 3 (5.6 %) 0 (0.0 %) 3 (13.6 %) 0.059 Any previous VVC 50 (90.9 %) 29 (87.9 %) 21 (95.5 %) 0.638 Age at first VVC episodeb 25.15 ± 7.73 25.14 ± 8.07 25.17 ± 7.37 0.742 Total VVC episodes everb 9.54 ± 7.96 9.66 ± 6.04 9.38 ± 10.20 0.242 RVVC evera 35 (67.3 %) 23 (69.7 %) 12 (63.2 %) 0.761 Total VVC in the last 12 monthsc 4.22 ± 2.40 4.55 ± 2.58 3.53 ± 1.88 0.142 Any previous VVC in the last 12 monthsa 33 (63.5 %) 23 (69.7 %) 10 (52.6 %) 0.246 Last VVC episode Months since last VVCd 4.06 ± 3.73 3.72 ± 3.49 4.62 ± 4.13 0.413 Symptomatic days during the last VVCe 4.36 ± 2.55 4.31 ± 2.71 4.43 ± 2.36 0.719 VVC signs and symptoms Pruritus 25 (75.8 %) 19 (86.4 %) 0.495 Burning pain/soreness 25 (75.8 %) 20 (90.9 %) 0.284 Vulvar erythema 29 (87.9 %) 21 (95.5 %) 0.638 Vaginal discharge 28 (84.8 %) 20 (90.9 %) 0.689 Vulvar pain/dyspareunia 17 (51.5 %) 12 (54.5 %) 1.000 Edema 27 (81.8 %) 19 (86.4 %) 0.727 Malodourness 11 (33.3 %) 7 (31.8 %) 1.000 VV vulvovaginitis, VVC vulvovaginal candidiasis, IUD intrauterine device, RVVC recurrent vulvovaginal candidiasis Values are presented as percentages or mean ± standard deviation (SD). Percentages are calculated based on the total number of patients available for each item. Comparison among the two intervention groups (P value) was performed with Fisher's test (categorical variables) or Mann Whitney test (quantitative variables) a Data available for 52 patientsb Data available for 47 patients c Data available for 46 patients d Data available for 45 patients e Data available for 50 patients Eur J Clin Microbiol Infect Dis.

with any previous VVC in the last 12 months and a dif- ferent history of non-Candida VV in the probiotic cohort. Taking into account the lack of randomization and those differences between cohorts, we considered appropriate to use a multivariate survival test to study the primary out- come (the recurrence-free survival). After adjustment for potential confounding factors by Cox multivariate analy- sis, the effect of adjuvant probiotic was significant in the full study population (at 3-months) and in women with RVVC within a 6-month follow-up. Nevertheless, we were surprised to observe that the hazard ratio was virtu- ally the same in the full population at 3 months and in the subpopulation with RVVC at 6 months. However, this lack of difference may be due to the small study sample. In future studies, it would be interesting to reproduce the same analysis using a larger sample in order to clear up whether there are any differences between the evaluation at 3- and 6-months of follow-up regarding the use of probiotics. Also, the difference in the fraction of women experiencing at least one symptomatic recurrence between the two study cohorts (32.2 % vs 58.8 %) did not reach statistical significance. However, this analysis at study endpoint does not take into account the baseline differences between the two cohorts, nor captures the time-to-event effect as the Cox analysis does. The latter may be of special relevance in the study population, as it was particularly prone to recurrence (67.3 % had a history of RVVC at any time of their lives and 63.5 % reported at least one previous VVC episode within the 12 months prior to enrolment). Variability among candidiasis and the selection of treat- ment are issues that have aroused considerable discussion. Recent reports suggest the possibility of two types of VVC: (a) the typical, characterized by dense vaginal discharge with occasional recurrences, and (b) the cyclic-recurrent, with hy- persensitive reactions even in presence of small quantities of Candida [4]. In both cases, a strong vaginal ecosystem is de- sirable to fend off Candida strains and reduce the risk of VVC recurrence. Several authors suggest that maintenance ther- apy needs to be given frequently enough to prevent vag- inal regrowth or recolonization, and transformation to a symptomatic state in cases with Bhost intolerance^; how- ever, once the azole is suspended, the risk of a new epi- sode is particularly high [8]. Witt et al. [18] found no advantages in treating RVVC with monthly 200 mg itraconazole, despite the adjuvant use of local L. gasseri lyophilisates for 6 days. Thus, in our case, the pro- biotic intervention was maintained over 2 months to ensure proper concentrations of lactobacilli. Probiotics are being used in the gynaecological field as an alternative intervention for VVC and its prevention after ATB therapy, and so for bacterial vaginosis. Evidence suggests that the coaggregation of lactobacilli prevents Candida from bind- ing to VEC [19], therefore improving the efficacy of antifun- gals [20, 21]. A retrospective comparative study by De Seta et al. [21] showed a better subjective resolution of typical VVC symptoms after treatment with clotrimazole 2 % vaginal cream for 3 days followed by vaginal application of Fig. 1 Patients flow-chart across study visits Table 2 Patients satisfaction with L. plantarum I1001 vaginal tablets (TSQM questionnaire) Parameter Total number of patients Median IQR Efficacy 29 83.3 66.7–83.3 Side effects (lack of) 29 100 100–100 Convenience 29 83.3 66.7–83.3 Overall satisfaction 29 76.4 52.8–76.4 IQR interquartile range Table 3 Cox proportional-hazards regression model for recurrence of VVC at 3 months Variable Hazard ratio 95 % CI P-value Lower Upper Age 0.96 0.90 1.03 0.228 Any previous VVC within the last 12 months 1.69 0.49 5.79 0.406 Antibiotics prior to enrolment 10.46 2.18 50.12 0.003 Diaphragm or IUD contraception 1.55 0.30 7.91 0.600 Immunosuppression 0.42 0.03 5.15 0.493 Oral contraception 0.97 0.22 4.32 0.965 History of other non-candida VV 0.34 0.03 3.52 0.365 Use of L. plantarum I1001 0.30 0.10 0.91 0.033 VVC vulvovaginal candidiasis, RVVC recurrent VVC, VV vulvovaginitis, IUD intrauterine device, CI confidence intervals Eur J Clin Microbiol Infect Dis.

L. plantarum P17630 daily for 6 days, plus a weekly applica- tion for 4 weeks, all compared with azole cream alone. However, they excluded patients with known risk factors of VVC (RVVC, prior ATB, etc.). In our study, on the contrary, no exclusion of them was considered since these women are more likely to require supplementary therapies to standard treatments. Despite the similarities between the published evidence and our results, it should be stressed that most reports have assessed multidose azole treatment [20, 21]; hence, the effects of the L. plantarum I1001 strain may be of particular interest since use of these adjuvant vaginal tablets was associated with a significant 3-fold reduction in the HR of recurrence, during a 3-month follow-up. To our knowledge, this is the first study to report a significant effect of a probiotic in the risk of symp- toms recurrence in women with high prevalence of RVVC. It is noteworthy that ATB prior to enrolment significantly in- creased the VVC recurrence risk, consistent with previous reports such as those from Pirotta et al. [22] and Spinillo et al. [23], who also concluded that this factor could entail even a first-episode of VVC. Their findings and those present- ed herein enhance the role of vaginal probiotics as a preven- tive strategy (complementing pharmacological treatment), and a primary intervention for reducing the risk of vaginal infec- tions. Since ‘Prior ATB’ per se seems to significantly increase probabilities of VVC and of recurrences, L. plantarum I1001 Fig. 2 Recurrence-free survival curve 3 months after single-dose clotrimazole treatment. Comparison of the overall recurrence-free survival between patients treated with clotrimazole 500 mg single dose-only vs. clo- trimazole + L. plantarum I1001. The difference on adjusted sur- vival between the two cohorts is significant, with a higher recurrence-free survival in pa- tients receiving adjuvant vaginal probiotic (HR [0.30 [95 %CI: 0.10–0.91]; P = 0.033, multivari- ate Cox regression model) Fig. 3 Recurrence-free survival curve 3 months after single-dose clotrimazole treatment. Patients with history of antibiotic treat- ment (ATB) prior to enrolment. Comparison of the overall recurrence-free survival depend- ing on the use of antibiotic treat- ment prior to enrolment. The dif- ference on adjusted survival be- tween the two cohorts achieves significance with a markedly lower recurrence-free survival in patients with such risk factor (HR: 10.46 [95 %CI: 2.18–50.12]; P = 0.003, multivariate Cox regres- sion model) Eur J Clin Microbiol Infect Dis.

could be of relevance as a resource for women needing to replenish their vaginal microbiota after taking ATB. It has to be noted that, in our study, VVC relapse was considered on the basis of any self-reported sign/symptom suggesting a new episode. Despite the subjective nature of these criteria, it could probably represent a more realistic and practical criteria in daily practice, considering that the pure presence of symptoms can entail a medical consultation and/ or use of over-the-counter medication. Beyond the clinical impact of the probiotic, high rates of satisfaction, tolerability and effectiveness were reported by both patients and gynaecologists. Our study has certain limitations that must be pointed out, such as the previously commented lack of randomization and the open-label nature of the trial, thus a placebo effect cannot be ruled out. However, it is worth pointing out that the high number of women fulfilling RVVC criteria suggests they may be well aware of the symptoms of a true VVC episode. And finally, the sample size available was limited, and drop-outs during the study follow-up were not uncommon. Thus, addi- tional studies should be conducted to evaluate the effective- ness of L. plantarum I1001, using double blind randomized designs and larger sample sizes. Also, it would be interesting to evaluate the efficacy of L. plantarum I1001 as add-on or follow-up therapy of other antifungal regimes, as well as for preventing the recurrence of bacterial vaginosis. Acknowledgments The authors thank all the gynaecologists of the Instituto Palacios de Salud y Medicina de la Mujer and the Instituto de Medicina EGR Madrid who actively participated in the study and to Dr. Cindy L. Larios from the Medical Department of Clever Instruments S.L. (Barcelona) for the statistical analysis and editorial assistance for the manuscript. Compliance with ethical standards Funding Gynea by Kern Pharma funded the study. Conflict of interest J. Espadaler is a full-time employee of AB-Biotics S.A; C. Prieto is a full-time employee of Gynea by Kern Pharma. Ethical approval All the study materials were reviewed and approved by the Ethical Committee of the Hospital Universitario de la Princesa (Madrid, Spain). Informed consent Informed written consent was obtained from all participants prior to enrolment. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. References 1. Richter SS, Galask RP, Messer SA, Hollis RJ, Diekema DJ, Pfaller MA (2005) Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases. J Clin Microbiol 43:2155–2162 2. Tibaldi C, Cappello N, Latino MA et al (2009) Vaginal and endocervical microorganisms in symptomatic and asymptomatic non-pregnant females: risk factors and rates of occurrence. Clin Microbiol Infect 15:670–679 3. Peters BM, Yano J, Noverr MC, Fidel PL (2014) Candida vaginitis: when opportunism knocks, the host responds. PLoS Pathog 10: e1003965 4. Murina F, Graziottin A, Felice R et al (2011) The recurrent vulvovaginal candidiasis: proposal of a personalized therapeutic protocol. ISRN Obstet Gynecol 2011:806065 5. Marchaim D, Lemanek L, Bheemreddy S et al (2012) Fluconazole- resistant Candida albicans vulvovaginitis. Obstet Gynecol 120: 1407–1414 6. Sobel JD (2016) Recurrent vulvovaginal candidiasis. Am J Obstet Gynecol 214:15–21 7. Mendling W, Brasch J, Cornely OA, Effendy I, Friese K, Ginter- Hanselmayer G et al (2015) Guideline: vulvovaginal candidosis (AWMF 015/072). S2k (excluding chronic mucocutaneous candidosis). Mycoses 58:1–15 8. Dovnik A, Golle A, Novak D, Arko D, Takac I (2015) Treatment of vulvovaginal candidiasis: a review of literature. Acta Dermatovenerol Alp Pannonica Adriat 24:5–7 9. Kovachev SM (2015) Local probiotic therapy for vaginal Candida albicans infections. Probiotics Antimicrob Proteins 7:38–44 10. Sobel JD, Zervos M, Reed BD, Hooton T, Soper D, Nyirjesy P et al (2003) Fluconazole susceptibility of vaginal isolates obtained from women with complicated Candida vaginitis: clinical implications. Antimicrob Agents Chemother 47:34–38 11. Food and Agriculture Organization of the United Nations and World Health Organization. 2001, posting date. Regulatory and clinical aspects of dairy probiotics. Food and Agriculture Organization of the United Nations and World Health Organization Expert Consultation Report. Food and Agriculture Organization of the United Nations and World Health Organization Working Group Report. http://www.who. int/foodsafety/fs_management/en/probiotic_guidelines.pdf. Accessed 30 June 2016 12. Hill C, Guarner F, Reid G et al (2014) Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11:506–514 13. Borges S, Silva J, Teixeira P (2014) The role of lactobacilli and probiotics in maintaining vaginal health. Arch Gynecol Obstet 289:479–489 14. O’Hanlon DE, Moench TR, Cone RA (2013) Vaginal pH and microbiocidal lactic acid when lactobacilli dominate the microbio- ta. PLoS One 8:e80074 15. Martinez RC, Franceschini SA, Patta MC, Quintana SM, Candido RC, Ferreira JC et al (2009) Improved treatment of vulvovaginal candidiasis with fluconazole plus probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14. Lett Appl Microbiol 48:269–274 16. Prieto C (2016) Lactobacillus plantarum I1001 en la prevención de la candidiasis vulvovaginal recurrente. Oral communication at the Workshop SEPYP (Sociedad Española de Probióticos y Prebióticos). Seville, Spain 17. Atkinson MJ, Sinha A, Hass SL et al (2004) Validation of a general measure of treatment satisfaction, the Treatment Satisfaction Eur J Clin Microbiol Infect Dis.

Questionnaire for Medication (TSQM), using a national panel study of chronic disease. Health Qual Life Outcomes 2:12 18. Witt A, Kaufmann U, Bitschnau M, Tempfer C, Ozbal A, Haytouglu E et al (2009) Monthly itraconazole versus classic ho- meopathy for the treatment of recurrent vulvovaginal candidiasis: a randomised trial. BJOG 116:1499–1505 19. Falagas ME, Betsi GI, Athanasiou S (2006) Probiotics for preven- tion of recurrent vulvovaginal candidiasis: a review. J Antimicrob Chemother 58:266–272 20. Ehrstrom S, Daraczy K, Rylander E et al (2010) Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal can- didiasis. Microbes Infect 12:691–699 21. De Seta F, Parazzini F, De Leo R, Banco R, Maso GP, De Santo D et al (2014) Lactobacillus plantarum P17630 for preventing Candida vaginitis recurrence: a retrospective com- parative study. Eur J Obstet Gynecol Reprod Biol 182:136– 139 22. Pirotta M, Gunn J, Chondros P, Grover S, O'Malley P, Hurley S, Garland S (2004) Effect of lactobacillus in preventing post- antibiotic vulvovaginal candidiasis: a randomised controlled trial. BMJ 329:548 23. Spinillo A, Capuzzo E, Acciano S, De Santolo A, Zara F (1999) Effect of antibiotic use on the prevalence of symp- tomatic vulvovaginal candidiasis. Am J Obstet Gynecol 180:14–17 Eur J Clin Microbiol Infect Dis.