INTRODUCTION

Bacterial Vaginosis (BV) is the most common cause of abnormal vaginal discharge in women of childbearing age.1 It is a polymicrobial clinical syndrome characterized

by a profound change in vaginal microbiota from a Lactobacillius- dominant state to anaerobic bacteria of high diversity including Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp, Prevotella spp, and

other BV-associated bacteria (BVAB).2,3 This change is accompanied by a rise in vaginal pH and increased amines which produce typical odor. BV increases the incidence of gynecologic and obstetric diseases, including of spontaneous abortion, premature labour, chorioamnionitis, and postpartum endometritis and pelvic inflammatory disease (PID).4,5 Also, it associates with a 2-3 fold increased risk of acquiring sexually transmitted diseases (STDs) such as chlamydial infection, gonorrhea, genital herpes and human immunodeficiency virus (HIV) infection.6-8

The recommended treatment is metronidazole or clindamycin.9 In Thailand, oral metronidazole is more commonly prescribed for BV. However, the metronidazole 400 mg oral tablet taken 3 times day commonly elicits adverse events such as a metallic taste, nausea and vomiting resulting in poor compliance.10 A single oral dose of metronidazole 2gm has comparable efficacy with a 7-day course10 and previous studies demonstrated less gastrointestinal side effects.

Therefore, the 2 g metronidazole regimen is included in the treatment guideline provided by the Australian Sexual Health Alliance.14

A novel vaginal ovule containing metronidazole 750 mg plus miconazole nitrate 200 mg (Neo-penotrans Forte®; NPF, Exeltis, Thailand) may be an alternative treatment modality. Previous studies showed that an oral metronidazole tablet can be used intravaginally for treating women with BV. The novel vaginal ovule, which dissolves more readily, had been reported to have high efficacy against BV, trichomoniasis and fungal infection: 75-96%, 100%, and 82-90%, respectively.15,16 Also, NPF can effectively cure mixed infection.15,16 A monthly 7-day course of NPF for up to 3-8 months was found to prolong remission period among women with recurrent BV.17 Another benefit of this vaginal suppository is that there have never been any serious adverse events reported.16,18 However, with different backgrounds of the users, the efficacy is yet to be validated in Thai women. The present study aims to compare the cure rate and symptomatic recurrence rate between a 7 day-course of vaginal metronidazole 750 mg plus miconazole nitrate 200 mg (NPF®) and a single oral dose of 2 g oral metronidazole in treating BV. Side effects of treatment and the women’s satisfaction were also evaluated.

MATERIALS AND METHODS

This prospective open label randomized clinical trial was carried out at the Department of Obstetrics and Gynaecology, Faculty of Medicine Siriraj Hospital,

Original Article SMJ

Mahidol University, during September 2019 – March 2020. The ethical approval was obtained from the Siriraj Institutional Review Board (Si 222/2019). The trial was registered at the Thai Clinical Trials Registry (TCTR20200902002).

Participants

All women who aged 18-45 years and were diagnosed with BV by Amsel’s criteria were invited to participate in the study. The diagnosis based on Amsel’s criteria required at least 3 of the following criteria: thin white/grey homogenous discharge, pH>4.5, fishy (amine) odor and the presence of clue cells.19 The exclusion criteria were women who: had a history of allergy to metronidazole or miconazole, were immunocompromised, had previous episodes of BV within 3 months, had taken medications that could disrupt the vaginal ecosystem e.g. anti-parasitic drugs, oral antibiotics, any vaginal medications, anti- coagulant, or disulfiram within the previous month, had co-incidental other STIs or cervical pre-cancerous or cancerous lesions, was currently pregnant or lactating, or had abnormal uterine bleeding.

Intervention

All women who presented with abnormal vaginal discharge at the Clinic were informed about the study before entering examination rooms. Then, they underwent history-taking, pelvic examination and wet preparation as a part of routine practice. The initial evaluation using a microscope took around 5 minutes for diagnosing BV based on Amsel’s criteria. The eligible participants were explained in detail about the study by a study nurse and signed the informed consent. After that, another high vaginal swab was collected for gram stain and consequently Nugent’s scoring system. Demographic data, as well as pre-treatment symptom evaluation, were then collected by the study nurse.

The randomization was computer-generated using block-of-four with a ratio 1:1. Each participant was allocated to receive either a 7-day course of vaginal ovules containing metronidazole 750 mg plus miconazole nitrate 200 mg (Neo-penotrans Forte®; NPF, Exeltis, Thailand) or a single dose of oral 2gm metronidazole (Metrolex®, Siam Bheasach Co., Ltd., Thailand).

The participants who were assigned to use vaginal tablets were trained to perform a proper self-insertion of vaginal tablets using a manikin. The single oral dose of metronidazole was given 45 minutes following the oral consumption of domperidone 10 mg at the Clinic on the recruitment day. All participants were asked to comply

with the following prohibitions: no sexual activity, no vaginal douching or cleansing, and avoiding alcoholic beverages for one week following treatment.

All participants were scheduled for a 2-week follow up visit. Clinical response, wet preparation and gram stain for Nugent scoring system were done by an investigator (Chayachinda C). Satisfaction and side effects were evaluated by a study nurse. Those who had persistent BV were treated with metronidazole 400 mg per oral thrice a day for 7 days; or who had other diagnosis were treated accordingly. At 1-month and 3-month post-treatment, all participants were telephoned asking about symptomatic recurrence and/or additional treatment.

Outcome measures

At 2-week follow-up, outcome measures were clinical response, cure rate by Amsel’s criteria, cure rate by Nugent’s scoring system, satisfaction and side effects. Five key symptoms which were evaluated by a study nurse including vaginal discharge, irritation, itching, odor and coital pain, were graded into 0 (no/absent), 1 (mild), 2 (moderate) and 3 (severe). This results in the ranging score from 0-15. At least 75% reduction of the scores was defined as ‘clinical response’. Satisfaction and side effects were also evaluated by the study nurse; and being reported using 0-10 from ‘no’ to ‘maximum’.

The diagnosis of BV based on Amsel’s criteria (Chayachinda C) required at least 3 of the following criteria: thin white/grey homogenous discharge, pH

>4.5, fishy (amine) odor and the presence of clue cells.19 The Nugent scoring was assessed by a blinded microbiologist. The gram-stained slides, two for each participant (first visit and follow-up visit), were labeled using code numbers; and were sent to the microbiologist all at once. The scoring system was done by looking for Lactobacillus spp., Gardnerella/ Bacteroides spp., and curved gram variable rods. The scores ranged from 0-10 and are categorized into 3 groups: score 0-3 (normal), score 4-6 (intermediate flora) and score 7-10 (BV).20 Cure rate was defined as the conversion of BV to non- BV, including reduction of Amsel’s criteria from ≥3 to <3 criteria or that of Nugent score from ≥7 to <7.

At 1-month and 3-month post-treatment, the telephone interview regarding current symptoms, symptomatic recurrence and additional treatment was done by a study nurse who was blinded to the allocation.

Sample size calculation and statistical analysis Data analyses were carried out with STATA (version

12.0; Stata Corp., College Station, Texas, USA). To

describe the characteristics of the participants, mean

±standard deviation, n (%), and median with range were used. For categorical variables, comparisons were performed using the chi-squared test or Fisher’s exact test. The distribution of each continuous variable was tested using the Shapiro–Wilk test. Parametric continuous variables were compared with Student’s t-test, while nonparametric continuous variables were analyzed with the Wilcoxon rank-sum test. Univariate and multivariate logistic regression were used to determine treatment efficacy. A P-value of < 0.05 was deemed statistically significant.

The sample size calculation was undertaken using a formula that compares 2 proportions. A study by Chaithongwongwatthana et al. showed that the efficacy of a single dose of 2 g metronidazole to treat BV was 78.6%12, whereas another study by Regidor showed that the cure rate in women using NPF was 98.1%.16 The required sample size was determined to be 30 per group (power, 70%; alpha, 0.05). As a lost-to-follow-up rate of 30% was expected, 40 participants needed to be recruited to each group.

RESULTS

Of 84 eligible participants, 70 came for the two- week follow-up and were included in the analysis. (NPF N=34, MET N=36) Sixty-nine and 60 were contacted at one-month and three-month respectively (Fig 1). The average age and body mass index (BMI) were 32.3±7.9 years and 21.6±3.8 kg/m2. Around half of all participants reported regular external vaginal cleansing after urination and 8.6% reported ever vaginal douching. Ten participants reported history of sexually transmitted diseases (STD), including 5 genital warts, 3 genital herpes and 2 PID. (Table 1)

All treatment outcomes at two weeks are shown in Table 2. After adjusting for age, sexual experience and number of lifetime sex partners, both NPF and MET had comparable efficacy. Improvement of each symptom is demonstrated in Table 2. Table 3 shows clinical score, Amsel’s criteria and Nugent’s scores before the intervention and 2-week post treatment. There was no difference between NPF and MET except that the total Nugent’s score in NPF group was significantly lower at 2-week follow-up (5.4±1.9 vs 6.8±1.9, p=0.004). At the two-week follow-up, four participants were diagnosed with vaginal candidiasis (NPF 1/34, 2.9% vs MET 3/36, 8.3%, p=0.331); and none had BV.

Both groups reported high satisfaction (NPF 8.5±1.4 vs MET 7.9±2.0, p=0.125). No drug allergy was reported but the side effects were as the followings: loss

Fig 1. Flow of the participants.

of appetite/metallic taste (NPF 5/34, 14.7% vs MET 6/36, 16.7%, p=0.822), nausea (NPF 3/34, 8.8% vs MET 6/36, 16.7%, p=0.327), dizziness (NPF 4/34, 11.8% vs MET 5/36, 13.9%, p=0.791), vaginal irritation (NPF 3/34, 8.8% vs MET 6/36, 16.7%, p=0.327) and pelvic pain/ diarrhea (NPF 2/34, 5.9% vs MET 1/36, 2.8%, p=0.522). No symptomatic, recurrent episode of BV was reported at one-months and three-month telephone follow-ups.

DISCUSSION

Both vaginal tablets containing metronidazole

(750 mg) and miconazole nitrate (200 mg) and a single dose of oral metronidazole (2 g) for treating

women with BV demonstrate comparable clinical cure and laboratory-based cure rate. The high efficacy demonstrated by NPF in the current investigation was consistent with a meta-analysis done by Lugo-Miro VI et al.10 Our findings support the potential of this medication as a first-line treatment for BV. Given that oral metronidazole has remarkable side effects of gastrointestinal irritation when BV is localized dysbiosis of the vaginal ecosystem, topical treatment modality appears promising. Moreover, a vaginal biofilm of G. vaginalis may limit the treatment efficacy of oral metronidazole21 whereas vaginal metronidazole may better disrupt such protective factor.

CONCLUSION

Amsel’s criteria at two-week follow-up were not different whereas Nugent’s scores were. Amsel’s criteria partially belonged to clinical-based diagnostic methods when Nugent’s scoring system was mainly a laboratory- based method. The latter one was usually used as a gold-standard diagnostic method in research study. Obviously, Nugent’s scoring system had a better reflection of vaginal ecosystem as our previous study showed that, based on this method, 20% of asymptomatic pregnant women had BV; and tended to have worse pregnancy outcomes.22 Therefore, the higher cure rate based on Nugent’s scoring system in the present study suggested the better resolution of vaginal dysbiosis following NPF treatment.

Contrasting to previous studies, none of the participants in both groups reported symptomatic recurrence. A longer course of metronidazole results in a lower incidence of recurrence at 1 month.10 Moreover, a long-term study in Australian women found that over 50% of BV-diagnosed women receiving or not receiving adjuvant treatment reported recurrent BV episodes at their 6-month follow-up.23 This may be partly explained by the fact that practice to achieve and maintain vaginal hygiene were emphasized during participant counselling,

such as avoiding excessive cleansing, vaginal douching, and the wearing of tight garments. Additionally, the follow-up period was short; and BV can also be asymptomatic.

Compatible with previous studies, the combination of metronidazole and miconazole in a vaginal tablet appear not to cause severe adverse events; and mitigate the coincidence of BV and vaginal candidiasis (VC).15,16 The coincidence of BV and VC and VC as a consequence of BV have been evident. The coincidence was reported in 15.2% of American non-pregnant women16 and 13.3% of Thai pregnant women.22 Furthermore, the administration of miconazole (200 mg) vaginal suppositories for 3 days is a recommended regimen for treating women with vaginal candidiasis (VC) treatment guidelines.9,23 As a consequence, pseudohyphae was detected around three times higher in the MET group at 2-week.

Although none of the participants required additional BV treatment within 3 months post-treatment, a quarter of the participants reported a malodorous vaginal discharge or fishy odor following sexual intercourse. This supports the dynamic and self-heal of vaginal microbiome. Despite the fact that seminal fluid can precipitate the incidence of BV, the resumption of individual participant’s normal life is the goal of BV

treatment. As such, other risk factors of BV occurrence should be serious taken into consideration such as excessive vulvar cleansing and vaginal douching.

The strength of this study is its randomized design. Although the participants could not be blinded, the investigators who evaluated the outcome measures and the statistician were. The limitations of the study were its small sample size and short follow-up period. One of the biggest concerns among women with BV is the frequent recurrence of the condition. Only symptomatic recurrence is approached at 1- and 3-month while BV can be asymptomatic. Another limitation was that the administrative route and the duration of treatment period of the two assigned treatment were different. Provide that double-dummy, placebo-controlled design and daily self-record of symptoms had been applied, the study would have had less bias in outcome measurement but probably more advantage of the novel treatment.

In conclusion, for BV treatment, both vaginal tablets containing metronidazole (750 mg) plus miconazole nitrate (200 mg) and oral metronidazole (2 g) show comparable efficacy and side effects.

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