Evaluating the Effectiveness in Supportive Treatment of Vaginitis of Vaginal-spraying Bacillus Probiotic Spore Suspension (LiveSpo X-Secret)

Vaginitis is a disease related to women's reproductive tract including bacterial vaginosis (Bacterial vaginosis - BV), sexually transmitted diseases (Sexually Transmitted Diseases - STDs)... This is considered is a common infection in the world, especially in developing and underdeveloped tropical countries, with up to 3/4 of the population suffering from STDs, mainly in the reproductive age group of 15-24. There are many microbial agents that cause gynecological diseases, including bacteria, viruses, fungi, flagellates... Bacterial vaginosis is an endogenous vaginal infection caused by more than 30 different species of bacteria, mainly mainly anaerobic bacteria such as Gardnerella vaginalis, Mycoplasma hominis, M. genitalium... Vaginitis is quite common in Vietnamese women. This is an issue of great concern in public health care because sexually transmitted diseases often have mild or no symptoms, so they are easy to ignore. Besides, the use of broad-spectrum antibiotics can lead to side effects such as loss of appetite, fatigue and sexual problems, while repeated treatments can lead to the appearance of antibiotic resistance, poses a significant challenge to the treatment of BV and STDs worldwide. The disease is normally caused by the replacement of Lactobacillus (a group of beneficial bacteria of the vagina) leading to an overgrowth of anaerobic bacteria. Probiotics are microorganisms that are beneficial to human health when consumed in sufficient amounts, playing an important role in maintaining the balance of the body's microflora, especially in the intestines and vagina. Studies have found that probiotics may help reduce the incidence and severity of vaginal infections, including bacterial vaginosis and yeast infections. Currently, probiotics formulated as oral capsules or gynecological suppositories containing Bifidobacterium or Lactobacillus strains are being studied as a potential adjunctive therapy for the treatment of vaginal infections. However, these strains have low viability and are sensitive to temperature, humidity and pH, making preservation difficult. Their effectiveness therefore depends on the specific strains used and the dosage required to achieve high concentrations in the vaginal tract. Beneficial bacteria of the Bacillus genus have been studied for a long time for their safety. For example, Bacillus subtilis, B. clausii and B. coagulans are often aerobic or facultative anaerobic bacteria, capable of growing rapidly and forming spores that are resistant to harsh conditions such as lack of nutrition, lack of oxygen, high temperatures and even antibiotics. In addition, these bacteria are particularly well-suited for usage as direct and in-situ probiotic vaginal sprays since they can also secrete antibacterial compounds, lactic acid, H2O2, and activate the mucosal layer of intestinal immune system. The vaginal-spraying probiotic therapy helps support the treatment and prevention of sexually transmitted pathogens, providing a promising support/alternative solution to traditional antibiotic therapy.

The aim of this study is to assess if vaginal-spraying Bacillus probiotic spores can can reduce clinical symptoms and improve various sub-clinical markers of vaginitis patients.

Methods: Prospective design of randomized, double-blind, and controlled clinical trial. The experiment was conducted on women with vaginitis and the obtained data was compared either between 2 groups or among two-to-four time points of treatment period of the same group. The control group was applied the routine treatment at the hospital combined with spraying 0.9% NaCl physiological saline into the vagina 3 times/time x 2 times/day continuously for 28 days. The test group was applied routine treatment at the hospital combined with spraying LiveSpo X-SECRET into the vagina 3 times/time x 2 times/day continuously for 28 days.

Samples are taken at day 0 and one or more of the following time points: day 3, 7, 14, and/or 28, depending on each research unit's ability to control sampling at certain intervals during the treatment procedure. In details, samples were taken at day 0, 7, and 28 at Hanoi OGH; and at day 0, 3, 7, 14, 28 at Bac Ninh CDC. Sub-clinical indicators related to vaginitis was monitored at different time points, including:

• Bacterial staining tests: These tests were conducted routinely at the Departments of Microbiology, both at Hanoi OGH and Bac Ninh CDC, at day 0 and day 28.
• Real time PCR assay to detect 9 main microorganisms that cause inflammation in vaginal swab samples: Gardnerella vaginalis (GV); Neisseria gonorrhoeae (NG); Chlamydia trachomatis (CT); Candida albicans (CA); Trichomonas vaginalis (TV); Mycoplasma hominis (MH); Mycoplasma genitalium (MG); Human alphaherpesvirus type 1 (HSV-1); Human alphaherpesvirus type 2 (HSV-2). This assay was performed at the Department of Microbiology, Fungi, and Parasitology Testing, Central Dermatology Hospital, using commercial kits or an "in-house" kit that was recently developed and published internationally by the research team (Bui et al. ., 2023). The assay was carried out at day 0, 7, and 28 for samples collected at Hanoi OGH; and at day 0, 3, 7, 14, 28 for samples collected at Bac Ninh CDC.
• Real time PCR assay to detect Bacillus sp. including B. subtilis, B. clausii and B. coagulans in vaginal swab samples. This experiment was performed at the Spobio Research Center, Anabio R&D. The assay was carried out at day 0, 7, and 28 for samples collected at Hanoi OGH; and at day 0, 3, 7, 14, 28 for samples collected at Bac Ninh CDC.
• Analysis of immune index including 5 types of cytokines IL-1β, IL-6, IL-8, IL-10, TNF-α and IgA was performed using ELISA technique from vaginal swab samples collected at days 0, 7, and 28 at Hanoi OGH only. The tests are conducted at the Department of Microbiology, Fungi, and Parasitology Testing, Central Dermatology Hospital or Spobio Research Center, Anabio R&D.
• Changes in microbiome using 16S rRNA metagenome analysis: This microbiome analysis was conducted for representative vaginal swab samples from patients of each research group (expected 10-15 patients/group) using new generation sequencing (NGS) on an Illumina MiSeq system (Illumina, San Diego, CA, USA) with a 2 × 250 bp run configuration. The assay was carried out at day 0 and one or more of the following time points day 7 and 28 for samples collected at both Hanoi OGH and Bac Ninh CDC.