Preparing for a clinical trial of interventions to maintain normal vaginal microbiota for preventing adverse reproductive health outcomes in Africa

Human body surfaces are covered by bacteria called the human microbiota. This microbial community is believed to contain at least 10 times the number of cells and 100 times the number of genes in the human body. Considerable progress has been made in characterising the human microbiota in recent years. Researchers have discovered that these microbial communities are needed for proper functioning of the human body, for example to digest complex carbohydrates in the gut and to prevent pathogen invasion. At the same time, imbalances of the normal microbiota have been shown to be associated with a number of diseases.

The human vagina also contains bacterial communities of mostly lactobacilli that are thought to protect women and their foetuses from pathogen invasion. When these bacterial communities are imbalanced, pathogens may enter the uterus, placenta, membranes surrounding the foetus, or abdominal cavity, causing pelvic inflammatory disease, miscarriages, pre-term births, or maternal or neonatal sepsis. These imbalances have also been associated with increased transmission of HIV and other sexually transmitted infections (STIs) between sexual partners and mother-child pairs. Some of the imbalances have been diagnosed in the past (using symptoms reported by the patient and microscopy of vaginal fluid) as bacterial vaginosis (BV). Unfortunately, BV has been notoriously difficult to treat, and even when treated successfully, recurrence rates are high. Because researchers have not been able to prevent BV recurrences during pregnancy, they have also not been able to prevent subsequent poor pregnancy outcomes.

In this development grant, we will develop interventions to maintain normal vaginal microbiota after initially having cured BV. The study participants will be Rwandan women who are at high risk of HIV and other STIs but who are not pregnant. We will ask one group of 15 women to use a vaginal gel (0.75% metronidazole gel) twice per week for 2 months, a second group of 15 women to use a vaginal probiotic tablet called Gynoflor thrice weekly for 2 months, and a third group of 15 women to use a vaginal probiotic capsule called Ecologic Femi thrice weekly for 2 months. Both probiotics contain 'healthy' bacteria called lactobacilli, and Gyneflor also contains a very low dose of oestriol. A further group of 15 women will form the control group. All women in all groups will receive safer sex and family planning counselling as well as individualised counselling on how to integrate the study interventions into their usual vaginal hygiene practices. At the end of the project, we will evaluate whether we were able to maintain healthy vaginal microbiota during the 2-month intervention period as well as the 4 months after cessation of the interventions. We will also evaluate the acceptability and feasibility of these interventions.

A novel aspect of this trial is that we will not only use standard methods to evaluate the vaginal microbiota (i.e. reporting of symptoms and microscopy of vaginal fluid) but also new molecular methods based on the sequencing of bacterial genes. This allows for a holistic and in-depth characterisation of microbial communities.

The results of this trial will be used to plan a larger randomized controlled clinical trial of the most promising intervention(s).

Women will be screened for BV by Nugent score, vaginal candidiasis, sexually transmitted infections (STIs), and urinary tract infection (UTI) and will be treated as indicated using standard antibiotic therapies. Women whose vaginal microbiota (VMB) are successfully normalised (BV negative by Amsel criteria, and no Trichomonas vaginalis or yeasts on wet mount) are then randomised into four groups (with 17 women per group): - Group 1: Behavioural 'vaginal practices cessation and safer sex' intervention only. - Group 2: Behavioural intervention plus oral metronidazole, 500 mg twice weekly for two months. - Group 3: Behavioural intervention plus Gynophilus LP vaginal tablet, once every four days for two months. - Group 4: Behavioural intervention plus Ecologic Femi vaginal capsule, daily for the first five days followed by thrice weekly for a total of two months. Gynophilus (Biose, Clermont-Ferrand, France) is a vaginal tablet containing only one Lactobacillus species and Ecologic Femi (Winclove, Amsterdam, The Netherlands) is a vaginal capsule containing multiple Lactobacillus species and Bifidobacterium bifidum. Both companies will provide the study products free of charge. All women in all groups will visit the study clinic at screening, enrolment/randomisation (=initiation of interventions), Day 7, Month 1, Month 2 (=termination of interventions) and Month 6. The Day 7 visit was added to evaluate immediate effects of the interventions. We will ask a subgroup of three women per group (N=12 women) to self-sample at home every Monday, Wednesday, and Friday for one menstrual cycle. The samples will either be collected by a study staff member or dropped off by the participant at the study clinic once per week. Nugent scores and Illumina MiSeq sequencing will be done on all approximately 600 vaginal samples to be collected. HIV, STI, UTI and pregnancy testing will be done at screening, Month 6 and when clinically indicated.

Human body surfaces are covered by bacteria called the human microbiota. This microbial community is believed to contain at least 10 times the number of cells and 100 times the number of genes in the human body. Considerable progress has been made in characterising the human microbiota in recent years. Researchers have discovered that these microbial communities are needed for proper functioning of the human body, for example to digest complex carbohydrates in the gut and to prevent pathogen invasion. At the same time, imbalances of the normal microbiota have been shown to be associated with a number of diseases. The human vagina also contains bacterial communities of mostly lactobacilli that are thought to protect women and their foetuses from pathogen invasion. When these bacterial communities are imbalanced, pathogens may enter the uterus, placenta, membranes surrounding the foetus, or abdominal cavity, causing pelvic inflammatory disease, miscarriages, pre-term births, or maternal or neonatal sepsis. These imbalances have also been associated with increased transmission of HIV and other sexually transmitted infections (STIs) between sexual partners and mother-child pairs. Some of the imbalances have been diagnosed in the past (using symptoms reported by the patient and microscopy of vaginal fluid) as bacterial vaginosis (BV). Unfortunately, BV has been notoriously difficult to treat, and even when treated successfully, recurrence rates are high. Because researchers have not been able to prevent BV recurrences during pregnancy, they have also not been able to prevent subsequent poor pregnancy outcomes. In this development grant, we will develop interventions to maintain normal vaginal microbiota after initially having cured BV. The study participants will be Rwandan women who are at high risk of HIV and other STIs but who are not pregnant. We will ask one group of 17 women to use an oral antibiotic used to treat vaginal infections (metronidazole 500 mg) twice per week for two months, a second group of 17 women to use a vaginal probiotic tablet called Gynophilus LP once every four days for two months, and a third group of 17 women to use a vaginal probiotic capsule called Ecologic Femi once per day for the first five days followed by thrice weekly for a total of two months. Both probiotics contain 'healthy' bacteria called lactobacilli. A further group of 17 women will form the control group. All women in all groups will receive safer sex and family planning counselling as well as individualised counselling on how to integrate the study interventions into their usual vaginal hygiene practices. At the end of the project, we will evaluate whether we were able to maintain healthy vaginal microbiota during the two-month intervention period as well as the four months after cessation of the interventions. We will also evaluate the acceptability and feasibility of these interventions. A novel aspect of this trial is that we will not only use standard methods to evaluate the vaginal microbiota (i.e. reporting of symptoms and microscopy of vaginal fluid) but also new molecular methods based on the sequencing of bacterial genes. This allows for a holistic and in-depth characterisation of microbial communities. The results of this trial will be used to plan a larger randomized controlled clinical trial of the most promising intervention(s).