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)

doi:10.21203/rs.3.rs-4963521/v1

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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)

https://doi.org/10.21203/rs.3.rs-4963521/v1

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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 who were randomized 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 daily at bedtime for six days). The study involved four visits to the study site with the examination at Visits 2 and 3.

Fixed combination of Natamycin + Lactulose was superior to both comparator drugs for the primary efficacy endpoint defined as percentage of patients who achieved a clinical recovery: the absence of symptoms of vulvovaginal candidiasis. At Visit 2, clinical recovery was reported in 81.6% of females in Natamycin + Lactulose group compared to 42.9% in Lactulose group and 62.3% of patients in Pimafucin group. The difference in proportions was 38.8% and 18.4%. In Natamycin + Lactulose group, microscopic recovery (Visit 2) was observed in 75.9% of patients at Visit 2 and in 90.8% of patients at Visit 3. In Lactulose group, 45.7% and 74.3% subjects responded positively at Visits 2 and 3. In Pimafucin group, 71.3% and 88.5% of patients showed microscopic recovery at Visits 2 and 3. For the microscopic recovery, no differences were reported between Natamycin + Lactulose and Pimafucin groups at both visits. At Visit 3, the number of vaginal lactobacilli was significantly higher in Natamycin + Lactulose group. In females with low baseline values of vaginal lactobacilli, the study combination increased the vaginal levels of lactobacilli to the reference values in 15.4% of patients at Visit 2, and in 20.9% of patients at Visit 3.

Conclusions: The fixed 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 13^th, 2024.

vulvovaginal candidiasis

natamycin

lactulose

prebiotic

lactobacilli

Although the pharmaceutical industry has experienced rapid growth and there are many antimycotic drugs available, the treatment of vulvovaginal candidiasis (VVC) continues to be a significant concern. Due to the high incidence of the disease, long-term clinical course (1), frequent recurrence (2, 3), and increasing azole resistance (4), there is an unmet need for new treatment options for patients with VVC. Currently, restoring 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). Combining antimycotic drugs with known mechanism of action with drugs that promote the restoration of normal vaginal microbiota is a highly effective strategy for improving treatment (5). The efficacy of various dosage forms of probiotics has been studied for decades, however the results remain contradictory (9, 10, 11, 12). Thus, our aim is to study a combination drug that contains a prebiotic, lactulose, and an antimycotic. Although there is limited data on the use of lactulose in treating VVC, it is important to note that numerous studies investigating its effect on intestinal microbiota, and specifically bifidobacteria growth suggesting its potential benefits (13).

The study drug, Natamycin + Lactulose, is a combination of antimycotic natamycin and the prebiotic lactulose. Natamycin is a broad-spectrum polyene antibiotic. Its fungicidal activity is due to its binding to fungal cell membrane sterols, thereby increasing permeability of the cell membrane, leading to a loss of essential cellular constituents ultimately causing cell lysis. Natamycin is active against most yeast-like fungi, dermatophytes, yeasts, and other fungi, as well as against some protozoa like trichomonas, but is not effective against Gram-positive and Gram-negative bacteria. As natamycin has a local action and is poorly absorbed with less than 3% penetrating through the skin and intact mucous membranes, its topical use is associated with practically no systemic disorders or side effects (14).

Lactulose is a synthetic disaccharide consisting of 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. Fermentation of Lactulose yields 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 slightly absorbed, and is metabolized by the intestinal microbiota (18). Systemic absorption of lactulose is thus not expected with intravaginal administration.

Since natamycin does not inhibit the growth and reproduction of lactobacilli, we hypothesize that combining antimycotic and prebiotic therapies for vulvovaginal candidiasis will have a synergistic effect. This combination will stimulate the growth of normal vaginal microflora while inhibiting the growth of Candida.

Therefore, the clinical trial aimed to test whether at the combination of Natamycin and Lactulose (Natamycin 100 mg + Lactulose 300 mg vaginal suppositories) has superior efficacy compared to Pimafucin (Natamycin 100 mg vaginal suppositories), or Lactulose 300 mg vaginal suppositories. The second objective of the study was to investigate the safety of the combination suppositories in the treatment of vulvovaginal candidiasis in non-pregnant adult females. The study was designed based on the U.S. Food and Drug Administration (FDA) guidelines for developing drugs for treating VVC (19). An adaptive trial design was proposed for this study as the effects of lactulose on fungal infection are currently unknown.

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 three 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 effective contraceptive methods throughout 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; chronic inflammatory, atrophic, or oncologic diseases of the female genital tract; previous surgery 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 screening and throughout the study. 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 or chronic disease in the period of exacerbation diseases were also excluded from the screening. The study population was selected from female patients who visited specialized departments of medical care facilities and is therefore representative of the general population.

Study Design

This 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 combination Natamycin + Lactulose 100 mg + 300 mg vaginal suppositories (AVVA RUS JSC, Russia) was superior to Pimafucin® (Natamycin) 100 mg vaginal suppositories (Temmler Italia S.r.L., Italy) and Lactulose 300 mg vaginal suppositories. To achieve the study objective, 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 through the Interactive Web Response System. The randomization code assigned to the 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. To achieve the study objective, three groups were formed after randomization (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 daily at bedtime for six consecutive days. The suppository was inserted as far as possible into the vagina into the vagina in the patient’s supine position.

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

Study Procedures

At the screening visit, women who wished to participate 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 conducted a screening visit to assess whether the participant met the inclusion/exclusion criteria. 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 how to administer suppositories 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 hours prior to her next visit.

At follow-up Visits 2 and 3, patient’s complaints were reported, and a complete physical examination was performed. A blinded assessing physician conducted a pelvic examination and obtained a vaginal swab specimen. At Visit 2, the 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 (КОН))

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 (x5, x10, and x40). 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 4).

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). The pH of the test sample was determined semi-quantitatively by visually comparing the colors and color intensities of the test strip with those of the reference color scale.

Patient’s Efficacy Assessment by Five-Point Scale

The patient completed a questionnaire in the presence of the investigating physician as part of the efficacy assessment. The questionnaire contained closed-ended responses ranging from 1 point (no clinical symptoms) to 5 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 while in a seated position, following 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: proportion (%) of patients with the clinical response at Visit 3; proportion (%) of patients with microscopic recovery (absence of Candida spp. at Visits 2 and 3; proportion of patients with overall (clinical and microscopic) recovery at Visits 2 and 3; patient's efficacy assessment by the 5-point scale at Visits 2, 3; microscopic change in lactobacilli count in vaginal specimens at Visits 2 and 3 compared to 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.

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: р_А – р_В ≤ 0.05; H_A: р_А – р_В > 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 group or Pimafucin group. To control the overall type I error throughout the study, hierarchical testing was conducted. Firstly, a comparison was made between Natamycin + Lactulose and Lactulose. If statistically significant superiority was evident, then a comparison was made between Natamycin + Lactulose and Pimafucin. To confirm the study hypothesis, the combination 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 statistical hypotheses, we calculated the proportions (expressed in %), the difference in proportions between groups and confidence intervals for the proportions and difference in proportions between 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 to 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, another 88 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 analyse the microscopic change in lactobacilli count in vaginal specimens at Visits 2 and 3 compared to baseline, we used the Mann-Whitney test for between-group comparison and the paired Wilcoxon test for 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 planned for the full analysis set and the safety analysis set. Baseline characteristics were evaluated only in the safety analysis set as the sets did not differ. 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.

Patients

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 the patient to withdraw from the study.

Figure 1. CONSORT 2010 Flow Diagram. Distribution of patients in study of Natamycin + Lactulose combination for treatment of vulvovaginal candidiasis.

The demographic characteristics of the patients in the study group and comparison group were comparable, 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)
Characteristics	Natamycin + Lactulose (n = 91)	Lactulose (n = 36)	Pimafucin (n = 90)
White; n (%, 95% CI)	91 (100.0, 95.9–100.0)	36 (100.0, 90.4–100.0)	90 (100.0, 95.9–100.0)
Age, years; M (SD, min-max)	34.5 (8,31, 19–54)	34.3 (7.53, 20–48)	34.8 (8.73, 19–54)
Weight, kg; M (SD, min-max)	64.6 (11.3, 42.0-100.0)	59.9 (11.0, 41.0-100.0)	63.6 (12.4, 42.0-100.0)
BMI, kg/m²; M (SD, min-max)	23.1 (4.15, 15.0-35.4)	21.9 (4.03, 17.4–38.1)	22.8 (4.17, 16.6–38.1)
Medical History, n (%, 95% CI)
- Comorbidities	22 (24.2, 16.5–33.9)	6 (16.7, 7.87–31.9)	18 (20.0, 13,0-29.4)
- Concomitant treatment	8 (8.79, 4.52–16.4)	2 (5.56, 1.54–18.1)	8 (8.89, 4.57–16.6)
Symptomatic VVC, (%, 95% CI)
- White or yellowish-white curd-like, thick or creamy vaginal discharge	0 points: 0 (0.0, 0.0-0.45) 1 point: 3 (3.30, 1.13–9.25) 2 points: 71 (78.0, 68.5–85.3) 3 points: 17 (18.7, 12.0-27.9)	0 points: 0 (0.0, 0.0-9.64) 1 point: 1 (2.78, 0.49–14.2) 2 points: 28 (77.8, 61.9–88.3) 3 points: 7 (19.4, 9.75-35.0)	0 points: 0 (0.0, 0.0-4.09) 1 point: 3 (3.33, 1.14–9.35) 2 points: 70 (77.8, 68.2–85.1) 3 points: 17 (18.9, 12.1–28.2)
- Vulvar itching, burning and pain	0 points: 0 (0.0, 0.0-0.45) 1 point: 4 (4.40, 1.72–10.8) 2 points: 70 (76.9, 67.3–84.4) 3 points: 17 (18.7, 12.0-27.9)	0 points: 0 (0.0, 0.0-9.64) 1 point: 2 (5.56, 1.54–18.1) 2 points: 29 (80.6, 56.0-84.2) 3 points: 5 (13.9, 6.08–28.7)	0 points: 0 (0.0, 0.0-4.09) 1 point: 4 (4.44, 1.74–10.9) 2 points: 71 (78.9, 69.4–86.1) 3 points: 15 (16.7, 10.04–25.7)
- Anogenital itching and burning	0 points: 60 (65.9, 55.3–74.9) 1 point: 20 (22.0, 14.7–31.5) 2 points: 11 (12.1, 6.89–20.4) 3 points: 0 (0.0, 0.0-0.45)	0 points:20 (55.6, 39.6–70.5) 1 point: 10 (27.8, 15.9–44.0) 2 points: 6 (16.7, 7.87–31.9) 3 points: 0 (0.0, 0.0-9.64)	0 points: 66 (73.3, 63.9–81.4) 1 point: 18 (20.0, 13.0-29.4) 2 points: 5 (5.56, 2.40–12.4) 3 points: 1 (1.11, 0.20–6.03)
- Discomfort in the vulva	0 points:24 (26.4, 18.4–36.3) 1 point: 8 (8.79, 4.52–16.4) 2 points: 47 (51.7, 41.5–61.6) 3 points: 2 (13.2, 7.71–21.7)	0 points: 9 (25.0, 13.8–41.1) 1 point: 3 (8.33, 2.89–31.8) 2 points: 20 (55.6, 39.6–70.5) 3 points: 4 (11.1, 4.41–25.3)	0 points: 35 (38.9, 29.5–49.2) 1 point: 11 (12.2, 6.96–20.6) 2 points: 34 (37.8, 28.5–48.1) 3 points: 10 (11.1, 6.15–90.3)
- Itching, burning, painful urination (dysuria)	0 points: 52 (57.1, 46.9–66.8) 1 point: 29 (31.9, 23.2–42.0) 2 points: 9 (9.89, 5.29–17.7) 3 points: 1 (1.10, 0.19–5.97)	0 points: 21 (58.3, 42.2–72.9) 1 point: 11 (30.6, 18.0-46.9) 2 points: 3 (8.33, 2.89–31.8) 3 points: 1 (2.78, 0.49–14.2)	0 points: 64 (71.1, 61.0-79.5) 1 point: 19 (21.1, 14.0-30.6) 2 points: 5 (5.56, 2.40–12.4) 3 points: 2 (2.22, 0.61–7.74)

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

(It is situated in the list of tables at the end of the document text file)

Primary Efficacy Endpoint

The study yielded 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 proportions 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. This was supported by the fact that 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 was less than 0.001 (Table 2).

Table 2

Percentage of Patients With Clinical Recovery (Visit 2). Comparative analysis of Natamycin + Lactulose and Lactulose Groups
Variable	Value
Natamycin + Lactulose (n = 87)	71 (81.6%); 95% CI, 72.2–88.4
Lactulose (n = 35)	15 (42.9%); 95% CI, 28.0-59.1
Difference in Proportions, %	38.8
95% CI, %	19.9–55.1
P-value for the one-tailed Fisher’s exact test	< 0.001

The study drug was compared with a second comparator based on the evidence of the superiority of Natamycin + Lactulose to Lactulose. In the Pimafucin group of the PP population, 62.3% (55/87) of patients had clinical recovery by Visit 2. The difference in proportions was 18.4% (95% CI, 5.11%-30.9%). The statistical analysis demonstrated that Natamycin + Lactulose was superior to Pimafucin. This was supported by the fact that 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

Percentage of Patients With Clinical Recovery (Visit 2). Comparative analysis of Natamycin + Lactulose and Pimafucin Groups
Variable	Value
Natamycin + Lactulose (n = 87)	71 (81.6%); 95% CI, 72.2–88.4
Pimafucin (n = 87)	55 (62.3%); 95% CI, 52.7–72.6
Difference in Proportions, %	18.4
95% CI, %	5.11–30.9
P-value for the one-tailed Fisher’s exact test	0.005
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. Between-group differences were statistically significant (P < 0.001) in favor of Natamycin + Lactulose (Fig. 2). In the PP population, a comparative analysis between the Natamycin + Lactulose and Pimafucin groups revealed a statistically significant difference in the proportion of recovered patients by Visit 3, favoring 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).

Figure 2. Number and proportion of patients with the clinical response (recovery) at Visit 3.

Number of patients with clinical recovery in each group (Lactulose group, Natamycin + Lactulose group and Pimafucin group) are on the horizontal axis and the proportion (%) of patients is on the vertical axis.

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.

Figure 3. Number and proportion of patients with microscopic recovery at Visit 2.

Number of patients with microscopic recovery in each group (Lactulose group, Natamycin + Lactulose group and Pimafucin group) are on the horizontal axis and the proportion (%) of patients is on the vertical axis.

Figure 4. Number and proportion of patients with microscopic recovery at Visit 3.

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). Between-group differences were statistically significant, favoring the study drug (P < 0.001).

Figure 5. Number and proportion of patients with the overall recovery at Visit 2.

Number of patients with clinical and microscopic recovery in each group (Lactulose group, Natamycin + Lactulose group and Pimafucin group) are on the horizontal axis and the proportion (%) of patients is on the vertical axis.

At Visit 3, Natamycin + Lactulose also showed better efficacy compared to Lactulose (P < 0.001) (Fig. 6). At this time point, 85.1% (74/87) of the subjects treated with the combination showed clinical and microscopic recovery, compared to 51.4% (18/35) of those who received Lactulose monotherapy. At Visit 2, there was 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 superior to Pimafucin monotherapy at Visit 3 (P = 0.028).

Figure 6. Number and proportion of patients with the overall recovery at Visit 3.

At Visits 2 and 3, the study subjects rated the efficacy by a 5-point scale. At Visit 2, in the PP population, 2.30% (2/87), 54.0% (47/87), and 43.7% (38/87) of the Natamycin + Lactulose group, and 10.3% (9/87), 44.8% (39/87), and 44.8% (39/87) of the Pimafucin group rated the therapy as satisfactory, good, and excellent, respectively (no between-group difference, P = 0.700). At the same visit, 17.1% (6/35), 48.6% (17/35), and 31.4% (11/35) of women in the Lactulose group rated the treatment as satisfactory, good, and excellent, respectively (between-group differences were not statistically significant compared to the Natamycin + Lactulose group, P = 0.058) (Fig. 7).

Figure 7. Distribution of patients by subjective assessment of efficacy at Visit 2.

Lactulose group, Natamycin + Lactulose group and Pimafucin group are on the horizontal axis and the number of patients in each group who evaluated the treatment as excellent, good, satisfactory, or worse is on the vertical axis.

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

Figure 8. Distribution of patients by subjective assessment of efficacy at Visit 3.

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

The microscopic examination examined 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]). The use of Pimafucin and Lactulose had no effect on the number of patients with normal or low lactobacilli levels by Visits 2 and 3 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), with similar baseline values at Visit 0 (P = 0.675) (Table 4, Additional file 5).

Table 4

Lactobacilli Count in Vaginal Specimens (Safety Analysis Set)
Characteristics	Natamycin + Lactulose (n = 91)	Lactulose (n = 36)	Pimafucin (n = 90)
Lactobacilli count at Visit 0, lg CFU/swab, Me (1Pr; 3Pr)	4.00 (0.0; 5.00)	4.50 (0.0; 5.00)	4.00 (0.0; 5.00)
Lactobacilli count at Visit 2, lg CFU/swab, Me (1Pr; 3Pr)	4.00 (0.0; 5.00)	4.50 (0.0; 5.00)	4.00 (0.0; 5.00)
Lactobacilli count at Visit 3, lg CFU/swab, Me (1Pr; 3Pr)	5.00 (3.00; 6.00)	4.50 (0.0; 5.00)	4.00 (0.0; 5.00)
Number (%) of patients with normal lactobacilli counts at Visit 0, n (%)	29 (31.5)	18 (50.0)	31 (34.4)
Number (%) of patients with normal lactobacilli counts at Visit 2, n (%)	43 (46.7)	12 (33.3)	40 (44.4)
Number (%) of patients with normal lactobacilli counts at Visit 3, n (%)	48 (52.2)	12 (33.3)	29 (32.2)

Safety

During the study, adverse events were reported in 6.59% (6/91) of patients in the Natamycin + Lactulose group and in 11.1% (10/90) of patients in the Pimafucin group, but no statistically significant differences between the groups were found (P = 0.284). No AEs were reported in the Lactulose group. No SAEs occurred in any patient of all groups.

The most frequent SAE 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, a total of four adverse reactions were identified during the study.

For moderate AEs, there was no association with the treatment in all treatment groups.

A definite association was established for one event in the Pimafucin group. In the Natamycin + Lactulose group, no association with treatment was established for any event (no between-group differences, P = 1.000). A probable association was established for one event in the main group; this type of association was not reported for the Pimafucin group (P = 1.000). One AE in the study drug group, and one AE in the comparison group were possibly related to the treatment (P = 1.000). An unlike relation was determined for 6 AEs in the Natamycin + Lactulose group and 8 AEs in the comparison group (no statistically significant between-group differences, P = 1.000). A conditional association was not established for any of the AEs in the two groups, while one AE was indefinitely related to Pimafucin (no statistically significant between-group differences, P = 1.000).

Thus, there were no statistically significant differences in the AE association with the treatment: P = 0.577 between the study groups Natamycin + Lactulose/Pimafucin.

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

Table 5

Adverse Events Reported in Study Subjects (Safety Analysis Set)
N = 8	Mild AEs	Mild AEs	Moderate AEs	Moderate AEs	Severe AEs	Severe AEs
Natamycin + Lactulose Group
System Organ Class / Event	Drug-related	Non-drug-related	Drug-related	Non-drug-related	Drug-related	Non-drug-related
Anemia	0	2	0	0	0	0
Gastrointestinal disorders	1	3	0	0	0	0
Infections and infestations	0	0	0	1	0	0
Reproductive system and breast disorders	1	0	0	0	0	0
Pimafucin Group
System Organ Class / Event	Drug-related	Non-drug-related	Drug-related	Non-drug-related	Drug-related	Non-drug-related
Anemia	0	5	0	0	0	0
General disorders and administration site conditions	1	0	0	0	0	0
Infections and infestations	0	1	0	0	0	0
Renal and urinary disorders	0	0	0	1	0	0
Reproductive system and breast disorders	1	1	0	0	0	0
Musculoskeletal and connective tissue disorders	0	0	0	1	0	0

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

(It is situated in the list of tables at the end of the document text file)

The study demonstrated the superior efficacy of the original fixed combination Natamycin + Lactulose 100 mg + 300 mg vaginal suppositories (AVVA RUS JSC, Russia) compared to Lactulose 300 mg vaginal suppositories in adult female patients with vulvovaginal candidiasis. The superiority was demonstrated at all time points (at Visits 2 and 3), both for clinical and microscopic recovery and therefore for the overall (both clinical and microscopic) recovery. The analysis of the questionnaires completed by the patients at the final visit, satisfaction with treatment was higher with Natamycin + Lactulose than with Lactulose monotherapy. In the Lactulose group, the vaginal culture showed no change in lactobacilli counts at Visits 2 and 3 compared to baseline. At the last visit, there was a statistically and clinically significant difference in favor of Natamycin + Lactulose. This was demonstrated by the increase in the number of colonies.

The superior efficacy of the study drug Natamycin + Lactulose to Pimafucin was obtained for the primary efficacy endpoint, i.e. clinical recovery at Visit 2. The combination drug was found to be more effective than Natamycin monotherapy in achieving clinical recovery at the final visit. No differences were found between the two treatments for microscopic recovery. However, the main group had a higher number of patients with overall recovery at the final visit compared to the comparator group.

According to the subjective assessment of treatment efficacy, no differences between the groups of Natamycin + Lactulose and Pimafucin were found. In the Pimafucin group, no significant difference in 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, with similar baseline values.

The safety assessment revealed no significant differences between Natamycin + Lactulose. The safety profile of Natamycin + Lactulose was found to be comparable to that of Pimafucin in the treatment of patients with vulvovaginal candidiasis.

With the rapid development of microbiota science, including the vaginal microbiota, interest in prebiotics is growing rapidly. The study demonstrated a positive effect of Natamycin + Lactulose on lactobacilli counts in patients with vulvovaginal candidiasis. In the Natamycin + Lactulose group, there was an increase in lactobacilli counts at the final visit compared to baseline values. As a result, more patients achieved reference levels of lactobacilli.

Previous data has shown that a reduction in Lactobacillus concentration is not a characteristic of VVC (20, 21). At the baseline visit, 35.9% (78/217) of the total sample had reference levels of this saprophyte in their vaginal discharge. However, a next-generation sequencing study has revealed that VVC-related changes in the vaginal microbiota are more intricate and diverse than previously thought (22). Based on this hypothesis, it can be suggested that the patients required correction of the vaginal biocenosis. This is because the group with higher vaginal lactobacilli counts showed better clinical and microscopic outcomes.

Thus, the original fixed combination Natamycin + Lactulose 100 mg + 300 mg vaginal suppositories (AVVA RUS JSC, Russia) demonstrated superior efficacy compared to Pimafucin® (Natamycin) 100 mg vaginal suppositories (Temmler Italia S.r.L., Italy) and Lactulose 300 mg vaginal suppositories in adult female patients with vulvovaginal candidiasis, and comparable safety profile.

Study Limitations

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.

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

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.

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

VVC, vulvovaginal candidiasis; AE, adverse event; SAE, serious adverse event; FDA, U.S. Food and Drug Administration.

Ethics approval and consent to participate

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.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

Declare none

Funding

The study was funded by JSC "AVVA RUS"

Authors' contributions

ONV, EVA, OVM and AVD developed the study design, ONV and EVA conducted the research, ONV, EVA, OVM, SAT and EAL analyzed and interpreted the data, ONV, EVA and OVM prepared the original draft of the manuscript, SAT, EAL, LZ and AVD finalized the manuscript. All authors read and approved the final manuscript.

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Competing interest reported. OVM, SAT and EAL are employees of AVVA RUS JSC that produce the combination suppositories

Additionalfile1.docx
File name - Additional file 1 File format- .docx Title of data - Table S1. CONSORT 2010 Checklist Description of data – CONSORT 2010 Checklist
Additionalfile2.pdf
File name - Additional file 2 File format- .pdf Title of data - Text S1. Appendix100. Diagnoses Appendix Description of data - Appendix100. Diagnoses Appendix
Additionalfile3.docx
File name - Additional file 3 File format- .docx Title of data - Table S2. Assessment of the severity of the symptom of vulvovaginal candidiasis on a 4-point scale Description of data - Assessment of the severity of the symptom of vulvovaginal candidiasis on a 4-point scale
Additionalfile4.docx
File name - Additional file 4 File format- .docx Title of data - Table S3. Registration of native smear assessment results Description of data - Registration of native smear assessment results
Additionalfile5.docx
File name - Additional file 5 File format- .docx Title of data - Table S4. Registration of the evaluation results with the addition of a 10% KOH solution Description of data - Registration of the evaluation results with the addition of a 10% KOH solution

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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)

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