Efficacy and safety of vaginal suppositories containing combination of Natamycin and Lactulose in treatment of vulvovaginal candidiasis: international, randomized, controlled, superiority clinical trial (combination of Natamycin and Lactulose for treatment of vulvovaginal candidiasis)

Abstract

Background

The study aimed to assess the efficacy and safety of Natamycin + Lactulose vaginal suppositories (100 mg natamycin and 300 mg lactulose) (AVVA RUS JSC, Russia) in adult females with vulvovaginal candidiasis.

Methods and Results

An international, randomized, controlled, assessor-blinded clinical trial enrolled 218 females randomly distributed into three groups: Natamycin + Lactulose (92 patients), Lactulose (36 patients), and Pimafucin® (90 patients). The study drug and comparator drugs had an identical dosing regimen (one suppository intravaginally once a day at bedtime for six days). The study involved four visits to the study site with examination at Visits 2 and 3.

The fixed-dose combination of Natamycin + Lactulose was superior to both comparator drugs in terms of the primary efficacy endpoint defined as the percentage of patients achieving a clinical recovery: the absence of symptoms of vulvovaginal candidiasis. At Visit 2, clinical recovery was reported in 81.6% of females in the Natamycin + Lactulose group compared to 42.9% and 62.3% of patients in the Lactulose and Pimafucin groups, respectively. The difference in proportions was 38.8% and 18.4%. In the Natamycin + Lactulose group, microscopic recovery was observed in 75.9% of patients at Visit 2 and in 90.8% of patients at Visit 3. In the Lactulose group, 45.7% and 74.3% subjects responded positively at Visits 2 and 3. In Pimafucin group, microscopic recovery was reported in 71.3% and 88.5% of patients at Visits 2 and 3, respectively, while no differences were observed between the Natamycin + Lactulose and Pimafucin groups at both visits. At Visit 3, the number of vaginal lactobacilli was significantly higher in the Natamycin + Lactulose group. In females with the low baseline content of vaginal lactobacilli, the combination drug under investigation increased the vaginal lactobacilli content to the reference values in 15.4% and 20.9% of patients at Visit 2 and Visit 3, respectively.

Conclusions

The fixed-dose combination Natamycin + Lactulose 100 mg + 300 mg vaginal suppositories (AVVA RUS JSC, Russia) demonstrated superior efficacy compared to 1) Pimafucin 100 mg and 2) Lactulose 300 mg vaginal suppositories in adult females with vulvovaginal candidiasis.

Trial registration

NCT06411314, retrospectively registered on May, the 13th, 2024.

Introduction

Although the pharmaceutical industry has experienced rapid growth introducing numerous antimycotic drugs, treatment of vulvovaginal candidiasis (VVC) remains a significant concern. High incidence of the disease, long-term clinical course [1], frequent recurrence [2, 3], and increasing azole resistance [4] require searching for new treatment options for VVC patients. Currently, restoring the normal vaginal microbiota is the mainstream concept for VVC treatment. Physiological mechanisms of normal vaginal microbiota colonization play a crucial role in suppressing the morphological proliferation of Candida yeasts into their hyphal form, which ultimately determines the degree of fungal pathogenicity [5,6,7,8]. An effective treatment strategy involves combining antimycotic drugs with a known mechanism of action and drugs that help to restore the normal vaginal microbiota [5]. The efficacy of various dosage forms of probiotics has been studied for decades; however, the results remain contradictory [9,10,11,12].

The aim of this study is to evaluate the efficiency of a combination drug containing a prebiotic lactulose and an antimycotic component. Despite limited data on using lactulose in treating VVC, numerous studies investigated its effect on intestinal microbiota, and specifically, bifidobacteria growth which implies its potential benefits for restoring vaginal microbiota [13].

The study drug, Natamycin + Lactulose, is a combination of the antimycotic natamycin and the prebiotic lactulose. Natamycin is a broad-spectrum polyene antibiotic. Its fungicidal activity is mediated by its binding to fungal cell membrane sterols, which increases the permeability of the cell membrane and leads to the loss of essential cellular constituents causing cell lysis. Natamycin is active against most yeast-like fungi, dermatophytes, yeasts, and other fungi, as well as against certain protozoa like trichomonas; however, it is inefficient against Gram-positive and Gram-negative bacteria. Since natamycin is poorly absorbed with less than 3% of the drug penetrating the skin and intact mucous membranes, it is used as a local action drug and has practically no systemic disorders or side effects [14].

Lactulose is a synthetic disaccharide consisting of the galactose and fructose residues linked by a ß-glycosidic bond [15]. It is not digested by mammalian enzymes; however, it can be fermented by the normal microflora [16], promoting the growth of bifidobacteria and lactobacilli. Lactulose fermentation produces small chain fatty acids (lactic, acetic, and formic) which inhibit the growth of Candida fungi and other pathogens [17]. Animal studies have demonstrated that oral lactulose is poorly absorbed and is further metabolized by the intestinal microbiota [18]. Therefore, we do not expect the systemic absorption of lactulose upon intravaginal administration.

Since natamycin does not inhibit the growth and reproduction of lactobacilli, we suggested that combining antimycotic and prebiotic therapies for vulvovaginal candidiasis might have a synergistic effect, thus promoting the growth of normal vaginal microflora while inhibiting the growth of Candida.

The first objective of this clinical trial was to estimate the efficiency of combining Natamycin and Lactulose (Natamycin 100 mg + Lactulose 300 mg vaginal suppositories) compared to Pimafucin (Natamycin 100 mg vaginal suppositories) or Lactulose 300 mg vaginal suppositories. The second objective of the study was to scrutinize the safety of the combination suppositories for treating VVC in non-pregnant adult females. The study was designed following the U.S. Food and Drug Administration (FDA) guidelines on developing drugs for VVC treatment [19]. Since the effects of lactulose on fungal infection are currently unknown, we chose an adaptive trial design for this study.

Methods

The Natamycine + Lactulose-supp-12/21 study (Permission # #482 from 11.08.2022) was conducted from December 2022 to July 2023 in two countries: the Russian Federation (one study site) and the Republic of Belarus (five study sites). Prior to subjects` enrollment, the ethical approval was obtained from the Ethics Review Board of the Russian Ministry of Health (Abstract of Record No. 305 of March 22, 2022) and local ethics committees (Statement w/n of December 05, 2022 issued by the Institutional Review Board of Minsk Municipal Polyclinic No.4; Statement w/n of November 28, 2022 issued by the Institutional Review Board of Minsk Municipal Outpatients Clinic No.5; Statement w/n of December 05, 2022 issued by the Ethics Committee of Minsk Municipal Emergency Care Hospital; Statement w/n of December 02, 2022 issued by the Institutional Review Board of Minsk Frunzenskiy District Central Polyclinic No.2; Statement w/n of November 29, 2022 issued by the Institutional Review Board of Minsk Municipal Hospital No.1). The study was coordinated by the Department of Reproductive Medicine and Surgery at the A.I. Evdokimov Moscow State Medical and Dental University and performed in compliance with the World Medical Association Declaration of Helsinki and the International Conference on Harmonization guidelines (Good Clinical Practice). Prior to enrollment, participants received written and verbal explanations of the study's aims, objectives, and methods, as well as the potential benefits and risks associated with participation. The subject and investigator completed the informed consent form in duplicate, with dated handwritten signatures. The investigator kept the first copy of the signed informed consent forms in the Investigator File, while the second copy was given to the subject. The study was reported in accordance with the Consolidated Standards for Reporting Trials (CONSORT) guidelines (Additional file 1). The study reached a pre-specified level of significance for the primary efficacy endpoint (P < 0.01556) and was terminated at the interim analysis.

Patients

The study enrolled females aged 18 to 60 with a clinical and microscopic diagnosis of vulvovaginal candidiasis (ICD-10 B37.3) confirmed by meeting all of the following criteria: 1) two or more of the following signs and symptoms of vulvovaginal candidiasis: white or yellowish-white curd-like, thick, or creamy vaginal discharge; vulvar itching, burning, and pain; anogenital itching and burning; discomfort in the vulva; itching, burning, painful urination (dysuria); 2) yeast cells in the vaginal swab specimen; 3) vaginal pH ≤ 4.5. The diagnostic criteria were standardized across the sites and were consistent with the FDA guidelines for developing drugs for treating VVC [1] as well as Appendix 100 [Additional file 2]. Pregnant and lactating women were excluded from the study. All patients agreed to use the effective contraceptive methods during the study and for 3 weeks after its completion. The acceptable methods of contraception included low- or microdose combined oral contraceptives, barrier, or dual barrier methods. Patients were excluded from the study if they met any of the following criteria: a clinical and laboratory diagnosis of bacterial vaginosis; vulvovaginitis caused by specific pathogens such as Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae; cancer, chronic inflammatory, or atrophic diseases of the female genital tract; previous surgeries on external or internal genitalia within 6 months; childbirth and abortion within 6 months. Patients must have had a stable menstrual cycle of 35 days or less. To be eligible for the study, participants were required to refrain from taking any prebiotics or probiotics as well as systemic or topical antifungal, antibacterial, or antiprotozoal medications for at least 2 weeks prior to the screening and throughout the study. The study subjects were not allowed to use intrauterine devices (hormonal or non-hormonal), topical vaginal antifungal or antibacterial medications, or vaginal antiseptics within 7 days prior to the screening visit. Women with severe disease or chronic disease exacerbation were also excluded from the screening. The study cohort was selected from female patients who visited the specialized departments of medical care facilities and was therefore representative of the general population.

Study design

This study was an international, randomized, comparative, open-label, assessor-blinded phase III clinical trial. The statistical objective of the study was to demonstrate that the original fixed-dosed combination Natamycin + Lactulose 100 mg + 300 mg vaginal suppositories (AVVA RUS JSC, Russia) was superior to Pimafucin® (Natamycin) 100 mg (Temmler Italia S.r.L., Italy) and Lactulose 300 mg vaginal suppositories. The study involved four in-person visits to the study site, i.e. Visit 0 (Screening, Days -7 to -1), Visit 1 (Randomization, Day 1), Visit 2 (Treatment Assessment, Day 7 ± 1), and Visit 3 (Treatment Assessment, Day 20–27 ± 1). The study duration was 34 ± 2 days.

Randomization and treatment

The female subjects were registered at the study sites using OpenClinica 3.14 (OpenClinica, LLC), a centralized web-based system. The study subjects were randomly assigned to treatment groups using a block randomization method with varying block sizes by the Interactive Web Response System. The randomization code assigned to a patient was documented in the source documents, electronic case report forms, adverse event (AE) reports, etc. The assigned randomization code remained unchanged throughout the study. The subjects were randomly distributed into three groups (1:0.5:1): Group 1, Natamycin + Lactulose 100 mg + 300 mg vaginal suppositories (AVVA RUS JSC, Russia); Group 2, Lactulose 300 mg vaginal suppositories; and Group 3, Pimafucin® (natamycin) 100 mg vaginal suppositories (Temmler Italia S.r.l., Italy). The study drug (Natamycin 100 mg + Lactulose 300 mg) and comparator drugs (Pimafucin® 100 mg and Lactulose 300 mg) had an identical dosing regimen, i.e. one suppository was administered intravaginally once a day at bedtime for six consecutive days. The suppository was inserted as far as possible into the vagina in the patient’s supine position.

The differences between visual characteristics of the study drug Natamycin + Lactulose and the comparator drug Pimafucin® made complete blinding of the investigator and patient impossible. Therefore, we employed an open-label design. To account for the potential impact of the open-label design on the efficacy endpoint assessments, patients were examined by an assessing physician who was blinded to the treatment allocation.

Study procedures

At the screening visit, the volunteers met with a physician and received complete written and verbal information about all study procedures and risks. After signing and dating the Informed Consent Form, a physician assessed whether the potential participant met the inclusion/exclusion criteria at a screening visit. The screening visit comprised a comprehensive physical examination (including pelvic examination), an assessment of demographic characteristics, body size, and medical and medication history. An amine test was performed to verify the diagnosis of bacterial vaginosis. At the Screening Visit, vaginal specimens were tested for urogenital infections (Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae) by polymerase chain reaction. Blood samples were collected for HIV, hepatitis B (HBs-Ag), hepatitis C (anti-HCV), syphilis, and hematology. The patients also had rapid urine β-hCG tests to rule out pregnancy and urinalysis.

At Visit 1, patients were reassessed for compliance with inclusion and non-inclusion criteria, randomized, and administered the assigned drug. The patient received training on suppository administration and was advised to make lifestyle changes to avoid using any vaginal insertion devices, such as spermicides, swabs, tampons, sprays, diaphragms, or condoms. Additionally, the patient was instructed to refrain from sexual intercourse for 48 h prior to her next visit.

At the follow-up Visits 2 and 3, patient’s complaints were recorded, and a complete physical examination was performed. A blinded assessing physician conducted a pelvic examination and obtained a vaginal swab specimen. At Visit 2, patients presented empty suppository packs for compliance assessment. During Visit 3, blood and urine samples were collected for hematology and urinalysis, respectively.

Safety was assessed throughout the study by patient interviews, physical examinations, laboratory tests, and instrumental methods.

Pelvic examination

A standard pelvic examination was performed. During the pelvic examination at all study sites, an investigating physician (non-blinded, Visit 0) and an assessing physician (blinded, Visit 2, 3) assessed the severity of VVC symptoms using a 4-point scale (Additional file 3).

Microscopic examination (Native Vaginal Specimen With and Without 10% Potassium Hydroxide (КOH))

At Visits 0, 2, and 3, a biological sample for microscopic examination (native vaginal specimen with and without 10% potassium hydroxide) was taken with a Folkman spoon from the posterolateral vaginal vault. The native specimens were microscopically evaluated for the presence and shape of epithelial cells, presence, and number of polymorphonuclear leukocytes, clue cells, lactobacilli and other microflora, pseudomycelium of yeast-like fungi, and trichomonas identified by their characteristic motility. For the native specimen, the microscopy results were reported in compliance with the Guidance for biological sample collection and laboratory testing at the study site, version 1.0 of December 14, 2022 (Additional file 4).

At Visits 0, 2, and 3, a biological sample for microscopic examination (native vaginal specimen with 10% potassium hydroxide) was taken with a Folkman spoon from the posterolateral vaginal vault. One or two drops of warm (preferably 37°C) 10% potassium hydroxide and a biological sample were placed on the slide. The biological sample was mixed with one or two drops of warm 10% potassium hydroxide, covered with a cover glass and immediately viewed using a light microscope (MIKMED-5 Medical Microscope; LOMO JSC, Russia) with increasing magnification (× 5, × 10, and × 40). For the native specimen with 10% potassium hydroxide, the microscopy results were reported in compliance with the Guidance for biological sample collection and laboratory testing at the study site, version 1.0 of December 14, 2022 (Additional file 5).

Culture test (Vaginal Swab Culture) for Candida spp. and Lactobacillus spp

A culture test was performed at Visits 0, 2, and 3 to determine the endogenous vaginal microflora. The test provided information on the presence or absence of growth, the number of Lactobacilli, opportunistic microorganisms, and fungi grown in the culture, as well as the genus and species of all representatives, including fungi. Additionally, a descriptive picture of swab microscopy (bacterioscopy) was included.

Evaluation of vaginal pH

Vaginal pH was measured at Visits 0, 2, 3 by semi-quantitative method using Colpo-Test pH Indicator Strips (BIOSENSOR AN LLC, Russia). pH of a test sample was determined semi-quantitatively by visually comparing the colors and color intensities of the test strip with the reference color scale.

Patient’s efficacy assessment by Four-Point Scale

The patient completed a questionnaire in the presence of the investigating physician as a part of the efficacy assessment. The questionnaire contained closed-ended responses ranging from 0 point (no clinical symptoms) to 3 points (severe clinical symptoms) (Additional file 3).

Vital signs

Heart rate, respiratory rate, systolic and diastolic blood pressure levels, and body temperature were measured during all visits. The patient's blood pressure was measured at the brachial artery in a seated position, following the standard guidelines.

Efficacy and safety

The primary efficacy endpoint in this study was defined as the proportion (%) of patients who achieved a clinical response (recovery) at Visit 2. The clinical response was considered as the absence of significant signs and symptoms of VVC (white or yellowish-white curd-like, thick, or creamy vaginal discharge; vulvar itching, burning and pain; anogenital itching and burning; discomfort in the vulva; itching, burning, painful urination [dysuria]). Secondary efficacy endpoints were as follows: the proportion (%) of patients with the clinical response at Visit 3; the proportion (%) of patients with microscopic recovery (the absence of Candida spp. at Visits 2 and 3; the proportion of patients with overall (clinical and microscopic) recovery at Visits 2 and 3; the patient's efficacy assessment by the 4-point scale at Visits 2, 3; the microscopic change in the lactobacilli count in vaginal specimens at Visits 2 and 3 compared to a baseline (Visit 0).

The safety endpoints assessed in this study included an incidence of any adverse events, an incidence of serious adverse events (SAEs), an incidence of AEs and SAEs probably related (in the investigator's opinion) with the study drug at the study dose, and an incidence of AEs and SAEs that led to study drug discontinuation.

The dynamics of clinical symptoms, laboratory values and pH test are provided in Additional files 6–12.

Statistical analysis

Primary efficacy endpoint

The study tested a hypothesis, whether the study drug Natamycin + Lactulose was superior to each of the comparator drugs. The following statistical hypotheses were suggested: H₀: p_A – p_B ≤ 0.05; H_A: p_A – p_B > 0.05, where p_A is the proportion of patients with the clinical response (recovery) at Visit 2 in the Natamycin + Lactulose group; p_B is the proportion of patients with the clinical response (recovery) at Visit 2 in the Lactulose or Pimafucin groups. Hierarchical testing was performed to control the overall type I error throughout the study. First, Natamycin + Lactulose and Lactulose were compared. If Natamycin + Lactulose was statistically significant superior, it was compared with Pimafucin. To confirm the study hypothesis, the combination drug had to demonstrate superiority to both comparator drugs.

The one-tailed Fisher's exact test was used because of the nature of the superiority hypothesis. Considering the selected method of analysis for evaluating the statistical hypotheses, we calculated the proportions (expressed in %), the difference in proportions between the groups and confidence intervals for the proportions and difference in proportions between the groups. The confidence interval for the proportions was calculated using the Clopper-Pearson method for binomial proportions, while the confidence interval for the difference in proportions was calculated using the Newcombe-Wilson method. To demonstrate the superiority of the study drug over the comparator, we assumed that the confidence interval for the between-group difference in proportions (p_A-p_B) of patients who achieved a clinical response (recovery) at Visit 2 would not cross the lower limit of 0.05 (5%). The P-value obtained for the one-tailed Fisher's exact test to demonstrate efficacy was P < 0.01556 for the interim analysis and P < 0.013812 for the final analysis while maintaining the underlying assumptions. The P-values for the comparisons of Natamycin + Lactulose to Lactulose and Natamycin + Lactulose to Pimafucin were identical.

The study had an adaptive design, allowing for the sample size to be recalculated based on the results of the interim analysis. The sample size was calculated using an adaptive approach based on a group sequential design. The interim analysis was assumed to be conducted after 50% enrollment was achieved. For the interim analysis, it was determined that Group 1 and Group 3 would each require the recruitment of 88 patients (including dropouts during the study). If the interim analysis confirmed the underlying assumptions, 88 more patients would be recruited into each group (1 and 3). Group 2 (Lactulose) should include 34 patients before the interim analysis and another 34 patients after the interim analysis.

The main analysis was performed in the per-protocol (PP) population.

Secondary efficacy endpoints

Secondary efficacy endpoints were analyzed using the Fisher's exact test or the χ2 (‘chi-square’) test (depending on an expected value in the contingency tables). The Clopper-Pearson binomial method was used to calculate the 95% confidence interval for the proportions.

To analyze the microscopic change in the lactobacilli count in vaginal specimens at Visits 2 and 3 compared to baseline, we used the Mann–Whitney test for a between-group comparison and the paired Wilcoxon test for a within-group comparison.

The main analysis of the secondary efficacy endpoints was performed using the PP population.

Statistical analysis was conducted using Statistica version 10.0. Analysis of demographic and other baseline characteristics was applied to the full analysis set and the safety analysis set. Baseline characteristics were evaluated only in the safety analysis set, since there were no differences between the sets. Descriptive statistics were used to present all group data, including demographics, laboratory results, instrumental and physical examination findings, and vital signs, for each treatment group. Comorbidities and adverse events were coded using MedDRA. Concomitant medications were coded using the ATX classification.

Results

Patients

In the study, 219 patients were screened, and 218 of them were randomized, 4.13% (9/218) of the randomized women withdrew from the study prematurely, i.e. 5.43% (5/92) of patients withdrew from the Natamycin + Lactulose group, and 3.33% (3/90) of patients withdrew from the Pimafucin group (between-group differences were not statistically significant, P = 0.721). In the Lactulose group, 2.78% (1/36) of women were excluded, which was not statistically significant (P = 1.000) when compared to the main group (Fig. 1). Thus, the PP population included 209 patients: 87 patients in the main group, 87 patients in the Pimafucin group, and 35 patients in the Lactulose group. The safety analysis set included 217 patients, as one female did not receive any dose of the study drug.

Fig. 1

CONSORT 2010 Flow Diagram. The distribution of patients in the study of Natamycin + Lactulose combination for treatment of vulvovaginal candidiasis

Erroneous inclusion: patients who met the non-inclusion criteria; Non-compliance: patients who did not complete the treatment; Protocol non-compliance: patients who had serious violations of the protocol; Withdrawal of informed consent: patients who did not wish to continue participation in the study; AE: occurrence of an adverse event that caused a patient to withdraw from the study.

Patients in the study and comparison groups had comparable demographic characteristics, including age and body mass index. The differences in body weight between the Natamycin + Lactulose and Lactulose groups were not statistically significant, as the body mass index did not differ between the groups (Table 1). As the study only included females, comparing and describing groups by sex is not applicable.

Table 1 Demographics and baseline characteristic of Patients from Three Groups (Safety Analysis Set)

Primary efficacy endpoint

During the study, we obtained the following results. At Visit 2, clinical recovery in the PP population was reported in 81.6% (71/87) of females in the Natamycin + Lactulose group compared to 42.9% (15/35) in the Lactulose group. The difference in the proportions of recovered patients was 38.8% (hereafter, ‘study drug’ minus ‘comparator’) with a 95% CI of 19.9%-55.1%. The statistical analysis demonstrated that Natamycin + Lactulose was superior to Lactulose. The lower limit of 95% confidence interval did not cross the value of 0.05 (5%), while the P value of the one-sided Fisher's exact test was less than 0.001 (Table 2).

Table 2 The percentage of Patients With Clinical Recovery (Visit 2). A comparative analysis of the Natamycin + Lactulose and Lactulose Groups

Since the combination drug Natamycin + Lactulose showed greater efficacy than Lactulose, we compared the study drug with the second comparator. In the Pimafucin group of the PP population, 62.3% (55/87) of patients achieved clinical recovery by Visit 2. The difference in proportions was 18.4% (95% CI, 5.11%-30.9%). The statistical analysis demonstrated that similarly, Natamycin + Lactulose was superior to Pimafucin. The lower limit of 95% confidence interval did not cross the value of 0.05 (5%) and the P value of the one-sided Fisher's exact test is 0.005 (Table 3).

Table 3 The percentage of Patients With Clinical Recovery (Visit 2). A comparative analysis of the Natamycin + Lactulose and Pimafucin Groups

Secondary efficacy endpoints

At Visit 3, clinical recovery in the PP population was reported in 90.8% (79/87) of females in the Natamycin + Lactulose group compared to 62.9% (22/35) in the Lactulose group. The between-group differences were statistically significant (P < 0.001)(Fig. 2). In the PP population, a comparative analysis between the Natamycin + Lactulose and Pimafucin groups revealed a statistically significant difference in the proportions of recovered patients at Visit 3, demonstrating the superiority of the study drug (P = 0.021). Thus, clinical recovery occurred in 90.8% (79/87) of females in the main group and in 78.2% (68/87) of females in the comparison group (Fig. 2).

Fig. 2

The number and proportion of patients with the clinical response (recovery) at Visit 3. The horizontal axis shows the number of patients with clinical recovery in each group (Lactulose group, Natamycin + Lactulose group and Pimafucin group), the vertical axis shows and the proportion (%) of patients

In the Natamycin + Lactulose group, microscopic recovery (Visit 2) was observed in 75.9% (69/87) of patients at Visit 2 (Fig. 3) and in 90.8% (79/87) of patients at Visit 3 (Fig. 4). In the Lactulose group, 45.7% (16/35) and 74.3% (26/35) subjects responded positively at Visits 2 and 3, respectively. Statistically significant differences were observed between the groups at both time points (Visit 2, P = 0.001, Visit 3, P = 0.017). In the Pimafucin group, 71.3% (65/87) and 88.5% (77/87) of patients showed microscopic recovery at Visits 2 and 3, respectively. For microscopic recovery, no statistically significant differences were found between the Natamycin + Lactulose and Pimafucin groups.

Fig. 3

The number and proportion of patients with microscopic recovery at Visit 2. The horizontal axis shows the number of patients with microscopic recovery in each group (Lactulose group, Natamycin + Lactulose group and Pimafucin group), the vertical axis shows the proportion (%) of patients

Fig. 4

The number and proportion of patients with microscopic recovery at Visit 3. The horizontal axis shows the number of patients with microscopic recovery in each group (Lactulose group, Natamycin + Lactulose group and Pimafucin group), the vertical axis shows the proportion (%) of patients

At Visit 2, overall (clinical and microscopic) recovery in the PP population was reported in 71.1% (61/87) of females in the Natamycin + Lactulose group compared to 22.9% (8/35) in the Lactulose group (Fig. 5). The between-group differences were statistically significant, demonstrating the superiority of the study drug (P < 0.001).

Fig. 5

The number and proportion of patients with the overall recovery at Visit 2. The horizontal axis shows the number of patients with clinical and microscopic recovery in each group (Lactulose group, Natamycin + Lactulose group and Pimafucin group), the vertical axis shows the proportion (%) of patients

At Visit 3, Natamycin + Lactulose also showed greater efficacy compared to Lactulose (P < 0.001) (Fig. 6). At this time point, 85.1% (74/87) of the subjects treated with the combination drug showed clinical and microscopic recovery, compared to 51.4% (18/35) of those who received Lactulose monotherapy. At Visit 2, we observed no statistically significant difference in clinical and microscopic efficacy in the PP population between Natamycin + Lactulose and Pimafucin (P = 0.083). However, the fixed combination was more effective compared to Pimafucin monotherapy at Visit 3 (P = 0.028).

Fig. 6

The number and proportion of patients with the overall recovery at Visit 3. The horizontal axis shows the number of patients with clinical and microscopic recovery in each group (Lactulose group, Natamycin + Lactulose group and Pimafucin group), the vertical axis shows the proportion (%) of patients

At Visits 2 and 3, the study subjects rated treatment efficacy using a 4-point scale. At Visit 2, in the PP population, 2.30% (2/87), 54.0% (47/87), and 43.7% (38/87) of the women in the Natamycin + Lactulose group, and 10.3% (9/87), 44.8% (39/87), and 44.8% (39/87) of the patients in the Pimafucin group rated the therapy as satisfactory, good, and excellent, respectively (no between-group difference, P = 0.700). At the same visit, the treatment was rated as satisfactory, good, and excellent 17.1% (6/35), 48.6% (17/35), and 31.4% (11/35) of women in the Lactulose group rated, respectively (between-group differences were not statistically significant compared to the Natamycin + Lactulose group, P = 0.058) (Fig. 7).

Fig. 7

The distribution of patients by subjective assessment of efficacy at Visit 2. The horizontal axis shows the Lactulose, Natamycin + Lactulose and Pimafucin group, the vertical axis shows the number of patients in each group who evaluated the treatment as excellent, good, satisfactory, or worse

At visit 3, in the PP population, 2.30% (2/87), 29.9% (26/87), and 66.7% (58/87) of the subjects from the Natamycin + Lactulose group, and 1.15% (1/87), 41.4% (36/87) and 57.5% (50/87) of the patients in the Pimafucin group rated the therapy as satisfactory, good, and excellent, respectively (no between-group difference, P = 0.357). At the same visit, the treatment was evaluated as satisfactory, good, and excellent by 2.86% (1/35), 51.4% (18/35) and 37,1% (13/35) of women in the Lactulose group, respectively (between-group differences were statistically significant compared to the Natamycin + Lactulose group, P = 0.007) (Fig. 8).

Fig. 8

The distribution of patients by subjective assessment of efficacy at Visit 3. The horizontal axis shows the Lactulose, Natamycin + Lactulose, and Pimafucin groups, the vertical axis shows the number of patients in each group who evaluated the treatment as excellent, good, satisfactory, or worse

At Visit 3, the number of vaginal lactobacilli (log scale) was significantly higher in the Natamycin + Lactulose group. Thus, at Visit 0, the average lactobacilli count (Me (1Pr; 3Pr) in main study group was 4.00 (0.00; 5.00) lg CFU/swab. At Visit 3, we observed a statistically significant growth to 5.00 (3.00; 6.00) lg CFU/swab (the within-group difference was statistically significant, P < 0.001). The comparison groups showed less pronounced improvement over time. At Visit 3, we also found statistically significant differences between the combination group and both the Lactulose group (P = 0.028) and the Pimafucin group (P < 0.001).

Using microscopic examination, we determined the number of patients with normal or low levels of vaginal lactobacilli. Among the VVC females with low baseline values of vaginal lactobacilli, the combination of Natamycin + Lactulose increased the vaginal levels of lactobacilli to the reference values in 15.4% of patients (95% CI, 2.03–28.7) at Visit 2 (statistically significant [95% CI does not include zero]). At Visit 3, Natamycin + Lactulose increased the vaginal levels of lactobacilli to the reference values in 20.9% (95% CI: 5.91—35.85) of patients with low baseline values of vaginal lactobacilli (statistically significant [95% CI of the difference does not include zero]). Monotherapy with Pimafucin and Lactulose had no effect on the number of patients with normal or low lactobacilli levels by Visits 2 and 3 as compared to Visit 0. At Visit 3, the proportion of women with normal lactobacilli levels in the Natamycin + Lactulose group was statistically significantly higher than in the Pimafucin group (P = 0.007), given the similar baseline values at Visit 0 (P = 0.675) (Table 4, Additional file 5).

Table 4 The Lactobacilli Count in Vaginal Specimens (Safety Analysis Set)

Safety

During the study, adverse events (AE) were reported in 6.59% (6/91) of patients from the Natamycin + Lactulose group and in 11.1% (10/90) of patients from the Pimafucin group. However, we did not observe any statistically significant differences between the groups (P = 0.284). No AEs were reported in the Lactulose group. No SAEs occurred in any patient from any group.

The most frequent AE was mild anemia (2 and 8 cases in the study drug group and 5 of 11 cases in the Pimafucin group). Genitourinary AEs were reported in 2 of 8 cases in the Natamycin + Lactulose group and in 5 of 11 cases in the Pimafucin group.

Two of 8 mild AEs in the Natamycin + Lactulose group were related to the study drug, i.e. loose stools (n = 1) and vulvovaginal itching (n = 1). In the Pimafucin group, the same number of mild AEs were associated with the treatment, i.e. irritation at the administration site (n = 1) and premature menstruation (n = 1). Thus, we observed a total of four adverse reactions during the study.

For moderate AEs, we did not detect any association with the treatment in all groups.

We found clear association with treatment for one event in the Pimafucin group, while no association for any event in the Natamycin + Lactulose group (no between-group differences, P = 1.000). We determined a probable association for one event in the main group and didn’t observed it in the Pimafucin group (P = 1.000). One AE in the study drug group, and one AE in the comparison group could be related to the treatment (P = 1.000). 6 observed AEs in the Natamycin + Lactulose group and 8 AEs in the comparison group (no statistically significant between-group differences, P = 1.000) were unlikely to be associated with the treatment. We did not detect any conditional association for any of the AEs in both groups and an unclear relationship of one AE to Pimafucin (no statistically significant between-group differences, P = 1.000).

Thus, we did not observe any statistically significant differences in the AE association with the treatment: P = 0.577 between the Natamycin + Lactulose/Pimafucin study groups.

Three of all 19 events in both groups were of moderate severity, while 16 AEs were mild; no severe events were reported (Table 5). We observed one moderate event in the Natamycin + Lactulose group compared to two events in the comparison group and detected no statistically significant differences between the groups (P = 0.737).

Table 5 Adverse Events Reported in Study Subjects (Safety Analysis Set)

Discussion

The treatment of vaginal infections with combination of antimycotic drug with prebiotic for simultaneous treatment of pathogenic microflora by Natamycin and re-colonization stimulation of favorable microorganisms presents the novel approach to therapy with few analogues overall in the world. In our study, we demonstrated the greater efficacy of the original fixed-dose combination of antimycotic Natamycin with the prebiotic Lactulose vaginal suppositories compared to the Lactulose in adult female patients with vulvovaginal candidiasis. We observed these effects at all time points (at Visits 2 and 3) for both clinical and microscopic recovery. According to the questionnaires completed by the patients at the final visit, they were more satisfied with the treatment using Natamycin + Lactulose than with Lactulose monotherapy. In the Lactulose group, the vaginal culture showed no change in the lactobacilli counts at Visits 2 and 3 compared to the baseline. At the last visit, we observed a statistically and clinically significant difference between the groups. The number of colonies was higher in the Natamycin + Lactulose group.

We evaluated the efficacy of the drugs based on the primary efficacy endpoint, i.e. clinical recovery at Visit 2. The combination drug was found to be more effective than Natamycin monotherapy in terms of clinical recovery achieved at the final visit. We detected no differences in the microscopic recovery between both treatments. However, number of patients with the overall recovery at the final visit was higher in the main study group as compared to the comparator group.

Subjective assessment of treatment efficacy showed no differences between the Natamycin + Lactulose and Pimafucin groups. In the Pimafucin group, no significant difference in the vaginal lactobacilli counts was observed between the baseline and assessment visits. At the final visit, the proportion of women with normal lactobacilli levels in the Natamycin + Lactulose group was statistically significantly higher than in the Pimafucin group, given the similar baseline values.

The safety assessment revealed no significant differences between Natamycin + Lactulose and the comparator drugs. The safety profile of Natamycin + Lactulose was comparable to that of Pimafucin.

Rapid development of research on microbiota has drawn much attention to prebiotics. The study demonstrated the beneficial effect of combining drug Natamycin + Lactulose on the lactobacilli counts in patients with vulvovaginal candidiasis. In the Natamycin + Lactulose group, we observed an increase in the lactobacilli counts at the final visit compared to the baseline values and a higher percentage of patients who achieved the reference levels of lactobacilli.

Previous data has shown that a reduction in Lactobacillus concentration is not typical of VVC [20, 21]. At the baseline visit, 35.9% (78/217) of the total samples had reference levels of this saprophyte in their vaginal discharge. However, a next-generation sequencing study has shown VVC-related changes in the vaginal microbiota to be more intricate and diverse than previously thought [22]. As the group with higher vaginal lactobacilli counts showed better clinical and microscopic outcomes, we suggested that the patients required correction of the vaginal biocenosis. Overall, positive effect of Lactulose and other prebiotics on vaginal microbiome (especially Lactobacilli) was described earlier and could explain both high recovery rate in patients received Lactulose in monotherapy regimen and in combination with Natamycin [23].

Normalization of flora in the presence of lactulose support a faster recovery of VVC which is an important finding and the novelty in this study. This result is in line with the importance of a eubiotic state in health in general, as well as in gynecological conditions. VVC may manifest as a result of vaginal dysbiosis, primarily caused by a reduction of the vaginal dominant Lactobacilli. Lactobacilli are the dominant inhabitants of the vaginal microbiome, ensuring that other anaerobes are kept in check. A shift in the vaginal flora from the dominant singular Lactobacillus to a polymicrobial state (dysbiosis) may result in vaginal infections including VVC and other non-infectious gynecological diseases [24,25,26].

The faster efficacy of the combination therapy as well as the increase of lactobacilli has an important implication. In VVC during treatment with an antimycotic, symptoms disappear, however vaginal normal flora is not restored, increasing the chances of re-infection. Lactulose in the Natamycin + Lactulose fixed dose combination, as a prebiotic, can restore vaginal normal flora by increasing the number of Lactobacilli, thus shortening the time for clinical remission, restore normal flora and possibly prevent re-infections. As lactulose promotes growth of Lactobacilli which has been shown in this study, and Natamycin eradicates Candida spp, the causative agent of VVC, the fixed dose combination restores normal vaginal microbiome.

Lactobacilli (increased by the fixed dose combination) can produce metabolites including lactate, antimicrobials, and lactic acid to inhibit the proliferation of pathogens. The lactic acid produced acidifies the vaginal pH (3.5–4.5) and such low pH offers protection against viral, bacterial, and parasitic infections. Lactobacilli also produce bacteriocins and hydrogen peroxide which have bactericidal and antimicrobial activity respectively, thus discouraging colonization of pathogenic organisms. They can also competitively exclude pathogens from adhering to epithelium and trap pathogens by direct physical contact to prevent the colonization of pathogens and can regulate the immune or inflammatory response, particularly relieving the inflammation by decreasing cytokines like IL-1β. Finally, Lactobacilli can improve the barrier function by producing lactate, which can increase the mucus viscosity to facilitate viral trapping, and inhibiting pathogens from damaging the DNA of epithelial cells [25]. Therefore the benefits of Lactobacilli are apparent and in this study the superior efficacy of the fixed dose combination can naturally be attributed to the effect of lactulose on restoring normal flora.

Study limitations

• 1. The study subjects were non-pregnant females at the reproductive age, so the results may be different in other age groups and in pregnant women.

• 2. The vaginal culture test for Candida spp. and Lactobacillus spp. that was used for the microscopic assessment has certain sensitivity limitations.

• 3. The study did not include females with recurrent vulvovaginal candidiasis. Additionally, the follow-up period was limited to one month, which prevents drawing conclusions about the long-term effects of the study combination.

Conclusions

In the present international, randomized, controlled, assessor-blinded clinical trial, we demonstrated that the efficacy of the original fixed-dose combination Natamycin + Lactulose 100 mg + 300 mg vaginal suppositories (AVVA RUS JSC, Russia) was superior to the Pimafucin® (Natamycin) 100 mg vaginal suppositories (Temmler Italia S.r.L., Italy) and the Lactulose 300 mg vaginal suppositories in adult female patients with vulvovaginal candidiasis and had a comparable safety profile.