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Prevention of recurrence of bacterial vaginosis using lactobacilli-containing vaginal tablets among women with HIV: a randomized, placebo-controlled, double-blinded phase IV trial
The prevalence of bacterial vaginosis (BV) was 28.4% among women infected with HIV.
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The use of vaginal tablets were highly acceptable in women infected with HIV (97% and 90%).
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Lactobacilli-containing vaginal tablets were found safe in women infected with HIV.
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Women with <7 years of HIV had higher odds (1.7 times) of having BV infection.
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There was no difference in the survival estimates for BV recurrence observed in the study arms.
Abstract
Objectives
The effectiveness of lactobacilli-containing vaginal tablets (VT) in bacterial vaginosis (BV) recurrence prevention among women infected with HIV treated with standard oral metronidazole in Pune, India was studied.
Methods
Women infected with HIV with confirmed BV diagnosis (Nugent score ≥7 and Amsel criteria >3) were enrolled in a 12-month, double-blind, randomized, placebo-controlled, phase IV study between 2018 and 2021. After a standard course of oral metronidazole for 7 days (400 mg three times a day), women were randomly assigned to either lactobacilli-containing or placebo VT arms to receive VTs for 4 months. BV recurrence was assessed after the initial cure from BV.
Results
Of the 464 women infected with HIV, 80 women with confirmed BV were enrolled. The retention was affected due to the COVID-19 pandemic (6-month retention rates 78%). The cure was seen in 85% and 93.5% of participants from the treatment and placebo arms, respectively, after four VT cycles. BV recurrence was seen in 41.4% and 44.8% in the treatment and placebo arm, respectively, with no significant difference in the two groups.
Conclusion
The lactobacilli-containing VT was acceptable and safe; however, the addition of VT over standard oral metronidazole did not show any additional benefit in the prevention of BV recurrence in women infected with HIV, indicating the need for long-term randomized trials among them. Registered at Clinical Trials Registry- India, (CTRI) Number: CTRI/2018/04/013298
CDC BV Working Group Indications for therapy and treatment recommendations for bacterial vaginosis in nonpregnant and pregnant women: a synthesis of data.
]. BV is characterized by either absence or depletion of lactobacilli and the proliferation of other anaerobic bacteria, such as Gardnerella vaginalis, Mobiluncus, Prevotella species, and the recently identified Atopobium vaginae [
CDC BV Working Group Indications for therapy and treatment recommendations for bacterial vaginosis in nonpregnant and pregnant women: a synthesis of data.
]. Although the treatment is effective, failure to complete the treatment due to side effects and poor recolonization by normal vaginal flora leads to recurrence [
The effectiveness of vaginal tablets (VTs) containing three lactobacilli strains for the treatment of BV and their ability to re-establish a normal flora was studied in 39 women who were HIV-negative in a double-blind, placebo-controlled clinical trial [
]. The lactobacilli treatment was successful 2 weeks after the completion of therapy, with the Nugent score (NS) <7 in 61% of patients treated with Lactobacillus compared with 19% in the placebo group (P <0.05). The BV recurrence was studied only up to 2 weeks in women who were HIV-negative, and the long-term effect was not assessed in this trial.
The prevalence of BV among women infected with HIV in India is 47.7% [
HIV Epidemiology Research Study. Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract.
] and also appears to influence the plasma VL in women infected with HIV. Hence, BV recurrence prevention remains crucial for women infected with HIV. Many strains of lactobacilli have been used in BV treatment and recurrence prevention [
Effectiveness of vaginal tablets containing lactobacilli versus pH tablets on vaginal health and inflammatory cytokines: a randomized, double-blind study.
]. However, due to the paucity of adequate data from well-designed long-duration studies, they are not recommended for the treatment and prevention in women infected with HIV. We conducted a phase IV study to determine the effect of multiple treatment cycles with lactobacilli-containing VT in addition to standard oral metronidazole therapy in the prevention of BV recurrence and assess its acceptability among women infected with HIV.
Methods
This academic trial was conducted between October 2018 and June 2021 in a clinic-based setting in Pune, India using lactobacilli containing VT in the treatment and a placebo in the second arm.
Participants
We screened women infected with HIV complaining of white vaginal discharge; the women were aged 18-40 years, were on antiretroviral therapy (ART)-naïve or on ART, had regular menstrual cycles, were willing to be abstinent from sexual activity for days as mentioned in the protocol, had the facility to store study VT at the desired temperature (between 2 and 8°C), and were able and willing to provide written informed consent. Women who received oral or vaginal antimicrobial or any probiotic therapy within 14 days before enrollment, pregnant or lactating up to 6 months, reported or known allergy/sensitivity to metronidazole or lactobacilli-containing formulations, use of any vaginal medication in the previous 7 days of enrollment, and had any condition that, in the opinion of the study staff, would make participation in the study unsafe, complicate the interpretation of study outcome data, or otherwise interfere with achieving the study objectives were excluded.
Study procedures
Women infected with HIV were screened for BV after obtaining written informed consent. After medical history and examination, genital samples were collected to ascertain the presence of BV based on the Amsel criteria (AC) [
]. BV was confirmed if three of four of AC were positive and NS was ≥7. Eligible women were enrolled within 7 days of their screening.
At enrollment, the data were collected on sociodemographic information, sexual behavior, information on HIV, and ART, followed by a clinical examination by a clinician. All enrolled women were counseled about the need for sexual abstinence for 15 days in the first month and 8 days every month for the next 3 months while using the metronidazole and VT tablets. Risk reduction and safe sex practice counseling was provided at enrollment and during follow-up visits by a trained counselor. A urine pregnancy test was performed to rule out pregnancy. Blood samples for complete blood count, cluster of differentiation (CD)4, plasma VL, storage and vaginal swabs for gram stain, pH, KOH (potassium hydroxide) test, and wet mount were collected. The participants were prescribed and dispensed metronidazole 400 mg to be taken three times a day for 7 days.
On the eighth day, metronidazole adherence assessment, dispensing of study VT as per the randomization code, and adherence counseling were done by a trained pharmacist. The participants were advised to use VT every night vaginally for the next 8 days.
For 2-4 months, the 8-days course of VT was dispensed after the clinical and adherence assessment and negative urine pregnancy test. Women were counseled that they can be sexually active except for the 8 days while using VTs. Participants were assessed for cure, which was defined as the negative AC and NS of <7 at the test-of-cure visit 21-30 days after the day of enrollment. Participants found not cured were offered an alternate treatment—clindamycin 300-mg capsule taken two times a day for 7 days.
Between 5 and 12 months, a clinical and laboratory assessment for BV recurrence was done between 7 and 14 days after their first day of menses.
Intervention
We used Flora Balance, which is an effervescent rapid-release VT containing not less than one billion colony-forming units of probiotic mix of three strains namely Lactobacillus plantarum DSM 24801, Lactobacillus salivarius DSM 24800, and Lactobacillus brevis DSM 27961 and excipients, in the treatment arm, whereas the placebo contained only excipients (no active ingredient). These strains were selected based on their specific characteristics and recorded data on vitality, good adhesion properties (anti-Gardnerella vaginalis activity and H2O2 production), and co-aggregation with BV-related microorganisms. These tablets were approved for use in the Employees’ State Insurance Corporation hospitals for the treatment of BV by the Drug Controller General of India on March 28, 2017.
Participants were instructed to store the VT in a refrigerator at 2 to 8°C, away from the direct source of light, heat, and children. Participants were counseled about inserting VT (without crushing or breaking) at bedtime inside the vagina with the middle finger while lying in a recumbent position and after washing their hands with soap. In case of expulsion, participants were instructed not to reinsert the tablet and report it to the study team.
Sample size and interim analysis
Assuming that the intervention group will show only 50% BV recurrence after 1 year of treatment as an effect of intervention compared with the placebo group, with an anticipated difference percent recurrence of at least 30% between the groups, with a 5% level of significance, the sample size was determined as 38 in each group. After considering a 5% anticipated loss to follow-up (LFU), the sample size was rounded off to 40 per group.
The interim analysis was conducted 1 year after completing four cycles of VT by the first 40 enrolled participants for safety assessment and unmet medical needs of VTs. During the interim analysis, it was observed that 15 women were LFU, despite adequate retention efforts; hence, 15 new recruitments to compensate for the LFU were recommended by the Data Safety Monitoring Board (DSMB). These new enrollments were completed as per the original randomization arm of the women LFU.
Randomization
A randomization plan was provided by the pharmaceutical company manufacturing VTs in a 1 : 1 ratio to receive either lactobacilli-containing or placebo VTs. They were labeled sequentially by the pharmaceutical company as per the randomization codes generated by them and couriered to the study team in a temperature-controlled environment. The lactobacilli-containing and placebo VT looked exactly similar in color, texture, and packaging. The prelabeled VTs were received directly by the pharmacist. This ensured the concealment of the randomization until the data analysis started. The participants and all investigators were blinded to the intervention.
Data collection
The data were collected through structured case record forms in the local language. Adherence was assessed through a questionnaire, pill count, and diary record that was given to each participant at enrollment. The expelled tablet/s were counted as missed tablets.
Adverse event monitoring
DSMB was established as recommended by the ethics committee for monitoring the safety of the participants. Participants were assessed for any adverse events (AEs) for metronidazole and VT by a clinician. The data regarding serious AEs, as per schedule Y, were collected and reported, as per the timelines. The grading of AE severity and assessment for relatedness using the Division of AIDS Toxicity Table [
National Institute of Allergy and Infectious Diseases Division of AIDS (DAIDS) Table for grading the severity of adult and pediatric adverse events, corrected. Version 2.1.
Division of AIDS National Institute of Allergy and Infectious Diseases,
Maryland2017
] were done by the clinician and the Principal Investiagtor. All AEs were sent to DSMB cumulatively and electronically during the interim analysis review.
Primary outcomes
The primary outcome was a recurrence of BV after the initial cure. The initial cure was defined as “AC-negative” and “NS <7” after the completion of oral metronidazole and four cycles of the study VT. Clinical recurrence was defined as BV-positive by AC. Confirmatory recurrence was defined as “NS ≥7” and “positive by AC”. The time to recurrence was calculated as the difference between cure and first clinical/confirmatory recurrence. The secondary outcomes included an assessment of adherence and acceptability of VT.
Statistical analysis
The data were entered in Microsoft Excel and analyzed in STATA version 16.1. The BV prevalence and factors associated with BV were calculated for initial and newly screened women. Factors showing a P-value <0.02 in the binary logistic regression were included in the multivariable logistic regression model. The recurrence, safety, adherence, and acceptability assessment included only women who were randomized to either arm from old and newly enrolled women. The cure rates were calculated for those completing the four VT cycles. The clinical and confirmatory recurrence were calculated. Kaplan-Meier methods were used to represent the survival curves for the time until the BV recurrence, and the log-rank and Tarone-Ware tests were used to compare survival curves between the two arms. The information loss during the COVID-19 pandemic was explained using the best- and worst-case scenario analysis using the Kaplan-Meier method.
Impact of the COVID-19 pandemic on the study implementation
In March 2020, a nationwide lockdown was announced. Maharashtra was the highest-hit state by the pandemic. Stringent restrictions were imposed on travel to ensure infection control. Due to their immunocompromised status, patients with HIV were considered to be at a higher risk of COVID-19 infection and its complications. Many study participants relocated to their native places and did not return. To ensure ART adherence, the National AIDS Control Organization issued guidelines on dispensing ART near patients’ homes, minimizing their exposure to infection. Considering the vulnerability to COVID-19 and the safety of women infected with HIV, DSMB recommended holding the study follow-up visits between April and June 2020, which led to the missing of initial cycles of VT for newly enrolled participants and no monitoring for BV recurrence. Due to fear of stigma and possible breach of confidentiality, home dispensing and data collection options could not be implemented. The study was prolonged due to the pandemic and was stopped in June 2021 due to funding shortage and in consultation with sponsors that led to 78% retention rates at the end of 6 months. This was a limitation.
Results
Of the 399 initially screened women infected with HIV, 112 were eligible and 80 were enrolled. One participant did not return for randomization; hence, 79 women were randomized to treatment (n = 39) and placebo (n = 40) arms. After the interim review, 65 screenings and 15 new enrollments were completed as recommended by the DSMB. Thus, a total of 464 (399 + 65) screenings were done. Figure 1 shows the participant flow from screening to enrollment.
Figure 1Participant flow of participants under the study. BV, bacterial vaginosis; TAH, total abdominal hysterectomy.
The prevalence of BV at the screening was 33.2% and 28.4% by AC and NS ≥7, respectively. There was 95.3% Kappa agreement (P <0.01) between AC and NS. More than half of the participants were asymptomatic at screening (54%). The median duration of BV symptoms reported by women was 2 months (Q1, Q3: 1, 3.5). The odds of BV among women with HIV <7 years was 1.7 times higher than that of women with HIV >7 years.
Table 1 describes the sociodemographic characteristics of the enrolled women (n = 79, one participant was not randomized). The mean age for enrolled women was 34 years (SD: 4.7) and all were on ART. The characteristics of women in both arms were comparable, except for the baseline CD4 count. The women spent a median of 537 (interquartile range: 280-619) and 540 (interquartile range: 205-618) person-days in the treatment and the placebo arm, respectively. Overall, 25% had a baseline CD4 count of <500 cells/mm3. Sexual debut aged <16 years was seen in 23%. Three (3.8%) women reported ≥2 current male partners, and 5 (6.3 %) reported ≥2 male partners in the last 12 months. Absence or inconsistent condom use in the last 12 months was reported by 37 (46%) of the women. Grade II and III anemia at enrollment as per the Division of AIDS Toxicity Table was noted in 30% [
National Institute of Allergy and Infectious Diseases Division of AIDS (DAIDS) Table for grading the severity of adult and pediatric adverse events, corrected. Version 2.1.
Division of AIDS National Institute of Allergy and Infectious Diseases,
Maryland2017
Six (15%) and nine (22.5%) women from the treatment and placebo arm, respectively, were LFU before the completion of four VT cycles and hence excluded from the cure analysis. Cure after four VT cycles was seen in 85% and 93.5% of the participants from treatment and placebo arms, respectively.
Recurrence of BV
Clinical recurrence after the cure was seen in 13/29 (44.8%); 95% confidence interval (CI; 26.4-64.3%) participants in both arms. Confirmatory recurrence was seen in 12/29 (41.4%; 95% CI 23.5-61.1%) and 13/29 (44.8%; 95% CI 26.4-64.3%) in the treatment and placebo arm, respectively. The Kaplan-Meier survival curves for treatment and placebo arms overlap each other, which indicated no significant difference in survival estimates for BV recurrence (Figures 2 and 3). Overall, the median recurrence time was lesser in the placebo arm than in the treatment arm. The median survival time was significantly higher in the treatment arm than the placebo arm (P <0.01). The survival probability was higher among the treatment (at t = 200,300,400,500 days) than the placebo arm in the actually observed BV recurrence, best-case scenario, and worst-case scenario (Figures 3 and 4). The overlapping curves indicate short-term benefits but without any evidence of long-term benefits and greater variance in the study group.
Figure 2Kaplan-Meier survival estimates with time for the first recurrence less than 1 year, in vaginal tablets study.
Figure 4Best and worst-case scenarios: Kaplan-Meier survival estimates when LFU are considered as non-recurrent BV cured cases till May 31, 2021 BV, bacterial vaginosis; LFU, loss to follow-up.
The lactobacilli-containing tablets were found safe for use. Self-limiting minor AEs, such as vaginal irritation, discomfort, and vaginal redness were reported by seven participants. Vaginal irritation was similar in both arms (approximately 20%). None of the participants discontinued the treatment due to AEs. The two serious AEs reported during the study were unrelated to the VT (death of a participant after a road traffic accident and hospitalization for COVID-19).
Adherence to metronidazole and VT
Adherence to metronidazole was 100% in 34/39 (87.2%) and 35/40 (87.5%) participants in the treatment and placebo arms, respectively. The adherence to VT was 100% in 33 (85%) and 35 (87.5%) participants from the treatment and placebo arm, respectively (P = 0.71). Two reported VT expulsions were considered as missed tablets in the adherence calculation.
Acceptability of VTs
A total of 33/34 (97%) and 28/31 (90%) women in treatment and placebo arms, respectively, used the tablets as prescribed and had no issues in storing and using them. About 70% and 64% of participants from the treatment and placebo arms expressed their preference to receive VTs at a private clinic over an ART center.
Discussion
Four cycles of lactobacilli-containing VT in combination with standard oral metronidazole therapy were safe and acceptable to women infected with HIV. The effectiveness of these VTs was established among 39 women who were HIV-negative [
]; however, the same could not be confirmed in women infected with HIV for a longer duration. Although no added therapeutic benefit in preventing BV recurrence was seen in our study, there were no side effects reported.
BV is one of the most common causes of white discharge among women of reproductive age [
CDC BV Working Group Indications for therapy and treatment recommendations for bacterial vaginosis in nonpregnant and pregnant women: a synthesis of data.
] and up to 80% in those with HIV in rural South Africa compared with 54% in women without HIV. The BV prevalence in our study was higher than in another study conducted in Maharashtra [
]. BV is predisposed by various factors, such as multiple sexual partners; hormonal fluctuations; underlying medical conditions; and the use of intravaginal and systemic antibiotics, local hygiene, and intravaginal washing practices [
]. Nearly half of the participating women reported inconsistent condom use in the last 12 months; few reported multiple sex partners. Although no associations were observed, still, a higher proportion of inconsistent condom use reported by the participating women infected with HIV indicate a need for safe sex and risk reduction counseling along with treatment of BV. The feasibility of a basket of protection and treatment against BV can be tested in the future study because BV has been associated with various gynecologic and obstetric complications, such as pelvic inflammatory disease, cervicitis, preterm labor and delivery, and postpartum endometritis [
]. BV recurrence prevention allows the prevention of these complications in women without HIV, as well as women infected with HIV. This calls for early diagnosis and treatment.
The burden of bacterial vaginosis: women's experience of the physical, emotional, sexual and social impact of living with recurrent bacterial vaginosis.
]. More than 50% of women were asymptomatic at screening in our study. The absence of symptoms makes it difficult for treatment because women are not aware of BV. Another important challenge is inability of women to understand the difference between physiological and pathological white discharge. Most women attribute white discharge to causes, such as stress, weakness, hot foods, and over exertion [
] and fail to understand when to seek care. Women essentially seek medical care when the discharge persists for more than 2-3 months and has a bad odor [
]. The health-seeking behavior reported in our study was suboptimal. Similar to other studies, the majority of our participants were experiencing white discharge for 2 months and the majority of them did not consider it abnormal [
]. This indicates an urgent need for creating awareness among women of reproductive age about the importance of early diagnosis of pathological white discharge and timely intervention for the treatment and prevention under the sexual and reproductive health care programs.
Although, BV is not a sexually transmitted disease [
]. Early diagnosis, treatment, and prevention of recurrence are important for the prevention of transmission of HIV to sex partners who are HIV-negative. Moreover, the risk perception among women infected with HIV is limited. This also indicates the need for education and counseling of women infected with HIV about abnormal white discharge and its consequences for better reproductive health and prevention of HIV transmission to their partner who is HIV-negative.
To the best of our knowledge, this is the first study to document the acceptability of lactobacilli-containing VT for the prevention of the recurrence of BV among women infected with HIV. The majority of the women infected with HIV enrolled in the study reported tablets as acceptable and easy to use. Most women reported their preference to receive these tablets in private clinics over ART centers or government hospitals. This highlights the confidentiality issues.
One of the strengths of our study is the use of both AC and NS for confirmation of the diagnosis of BV, which made it comparable with other studies. The adherence to antibiotics and study tablets was high. The study groups were comparable in sociodemographic and behavioral characteristics at baseline, except for CD4 count, which did not affect the association between VT use and recurrence of BV in both arms.
Our limitations include the LFU during COVID-19, which limited the assessment of the study end points for 12 months for each woman. The use of NS for end point assessment limited the inclusion of women with intermediate flora as used by Joshi et al. [
]. Hence, the use of NS is likely to give an underestimation of the recurrence rate of clinically evident BV. A point-of-care test is needed for timely confirmation of BV among women infected with HIV. The variance for recurrence was found to be high hence generalizability is limited.
Although this study showed no additional benefit from combining oral metronidazole with lactobacilli-containing VT, they were found to be acceptable, with no related side effects. The COVID-19 pandemic did affect the retention of participants in the study, which could have led to an underestimation of the association between BV recurrence and VT use. Currently, internationally recommended antibiotic treatments are provided as monotherapies; however, the recurrence is high and the sustained cure rates are less. There is a need for more effective combination therapies that can restore the vaginal flora and sustain it for the BV recurrence prevention among women infected with HIV. Further large-scale studies are required to understand the effect of augmentation of antibiotic treatment with lactobacilli-containing VT on the BV recurrence in women infected with HIV and on HIV VL shedding to assess the impact on the transmission of HIV to their partners who are HIV-negative.
Funding
The study was funded by the Indian Council of Medical Research, India (Sanction no 5/7/716/2011-RHN). The study VTs were provided by NextGen Pharma Private Limited. Sponsors have no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
Ethical approval
The protocol and the written informed consent forms of the study were approved by the institutional ethics committee (NARI/EC Protocol No: 2012-02). Written informed consent was obtained from all the study participants at screening and enrollment.
Author contributions
SB has done study conceptualization, writing, securing funding, and for overall implementation SB and SS were responsible. PS, TLK, GR, and AK contributed to data collection. AM has contributed to all the laboratory tests related to eligibility and confirmation of BV. SuS contributed to data cleaning and analysis. SB, SS, SuS, PS, AK, TLK, GR, AS, and AM contributed to manuscript writing. SS and SB finalized the manuscript.
Data availability statement
Data will be available at the ICMR-National AIDS Research Institute centralized data repository system and is available on request to [email protected]
Declaration of competing interest
The authors have no competing interests to declare.
Acknowledgments
The study was led by ICMR-National AIDS Research Institute, Pune. The authors acknowledge the support received from the Director of the ICMR-National AIDS Research Institute for the entire study. The authors are thankful for the funding and continued support received by the Indian Council of Medical Research, India. The authors sincerely acknowledge the support received from the Maharashtra State AIDS Control Society and DSMB. The entire study team expresses their heartfelt thanks to the study participants, without whom this study would not have been completed. The authors thank NextGen Pharma Private Limited for providing prelabeled VTs with lactobacilli and a placebo. The authors are thankful to Dr. Leena Rawat of the ART Center, Armed Forces Medical College, Pune for her support in study recruitment.
HIV Epidemiology Research Study. Association between bacterial vaginosis and expression of human immunodeficiency virus type 1 RNA in the female genital tract.
Effectiveness of vaginal tablets containing lactobacilli versus pH tablets on vaginal health and inflammatory cytokines: a randomized, double-blind study.
The burden of bacterial vaginosis: women's experience of the physical, emotional, sexual and social impact of living with recurrent bacterial vaginosis.
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