Department of Obstetrics and Gynecology, Peking University Shenzhen Hospital
Phase: PHASE4
Conditions: Bacterial Vaginosis
Sponsor: Peking University Shenzhen Hospital

 
Probiotics as Adjuvant Treatment for 
Bacterial Vaginosis 
 
NCT03894813 
 
2021-02-20 
 
 

Study Protocol with SAP 
This study was a single-centre, prospective, parallel-group, randomized, controlled 
study. Women who came to the gynaecological clinic of Peking University Shenzhen 
Hospital, China, between March 2019 and June 2019 with abnormal vaginal discharge 
symptoms were recruited, and eligible subjects were informed of the study protocol 
and enrolled in the study. The inclusion criteria were women aged between 18 and 65 
years old, premenopausal women, women with a history of sexual activity, and 
women with a Nugent’s Gram stain score of 7 or higher. The exclusion criteria were 
mixed vaginitis, such as vulvovaginal candidiasis (VVC), Trichomonas vaginalis 
(TV) infection, Chlamydia trachomatis (CT) infection or gonococcal vaginitis; 
planning for or being pregnant; breast-feeding; pelvic inflammatory disease; allergy to 
metronidazole; currently using antibiotics; long-term use of contraceptives or 
immunosuppressants or anaphylactic constitution; and a history of systemic organic 
diseases or psychiatric diseases. The study was conducted in accordance with the 
Declaration of Helsinki, was approved by the Medical Ethnic Committee of PKUSZH 
(with the Unique Protocol ID: PUshenzhenH2018-016) and was first posted on 
ClinicalTrials.gov (NCT03894813) on March 26, 2019. Informed consent was 
obtained from all subjects involved in the study. 
The sample size was calculated based on the non-inferiority test 
(https://www.cnstat.org/samplesize/12/), which referred to Gregor Reid et al, who 
carried out studies with the same Lactobacillus strains administered orally to women 
with bacterial vaginosis (BV). The relevant parameters were determined as follows: 
test level α=0.05, test power 1-β=0.80, Pt=0.90, Pc=0.75, Nt:Nc=1:1, boundary value 
Δ= -0.0.068 (excellent), and Fisher’s exact test. As a result, the sample size of each 
group was calculated as 50 patients, 100 patients in total. Considering a loss to 
follow-up rate of 20%, a sample size of 120 patients was finally determined, with 60 
patients in each group. 
After obtaining written informed consent, the participants were first asked to complete 
a questionnaire on demographic characteristics, reproductive health and sexual 
behaviour. They were subsequently randomly assigned to either the metronidazole 
group or the adjunctive probiotic group according to the random number table. The 
study was not blinded to the researchers or the patients. Starting from the enrolment 
day, the participants in the adjunctive probiotic group received orally administered 
probiotic drinks containing L. rhamnosus GR-1 and L. reuteri RC-14 (≥1 × 109 
CFU per day, for 30 days) and vaginally administered metronidazole suppositories 
(0.2 g per day, for 7 days), and the participants in the metronidazole group received 
metronidazole vaginal suppositories only. Clinical follow-up visits were scheduled at 
30 and 90 days after starting treatment. The intervention products were dispensed by 
the investigator at the initial visit, and the compliance of participants was assessed by 
counting returned containers and filling out a questionnaire. The feasibility, 
acceptability and adherence to the administered products; vaginal symptoms; and 

adverse events (AEs) were also assessed by self-report in medication diaries and 
questionnaires at follow-up visits. Therapy was initiated after the enrolment visit and 
suspended when menstruation began and continued immediately when it finished. 
Patients were asked to avoid sexual intercourse and vaginal douching during the first 
7 days of treatment with metronidazole suppositories. 
General information and clinical signs and symptoms of BV, including homogeneous 
and thin vaginal discharge, unpleasant odour (such as a “fishy” smell) and vulvar 
discomfort (such as itching or burning), were recorded at 0 day, 30 days and 90 days. 
The efficacy outcome was evaluated by the cure rate according to per-protocol (PP) 
analysis, which was the percentage of participants who did not present with BV 
(defined by Nugent score < 7) at any of the follow-up visits. Those with a Nugent 
score ≥ 7 in the 30-day follow-up was excluded from the trail, but when we 
evaluate the overall recurrence rate of the 90-day follow-up, these participants were 
still included in. At each follow-up visit, patients were requested to report any 
unexpected symptoms during the study period. AEs were recorded in the case report 
form (Table S1). 
The diagnosis criteria of BV were based on Nugent Gram stain scoring of vaginal 
smears. The Gram-stained smear slides were examined microscopically, and the 
number of bacterial cells with different morphotypes was counted under 1000 × 
magnification. Each slide was identified according to Nugent score. A Nugent score 
of 0-6 was considered BV negative and of 7-10 was considered BV positive. The 
Gram stains were analysed in a double-blind manner by two experienced cytology 
technicians. 
This study was a single-centre randomized controlled trial with two arms allocated at 
a ratio of 1:1. SPSS 13.0 software was applied to generate a random number table, 
and the random order was assigned depending on the order of enrolment.