Preparing for a Clinical Trial of Interventions to Maintain Normal Vaginal Microbiota for Preventing Adverse Reproductive Health Outcomes in Africa
Phase: NA
Conditions: Bacterial Vaginosis
Sponsor: Janneke van de Wijgert

22 April 2015 
 
1 
 
 
 
 
 
 
 
 
STUDY PROTOCOL 
 
 
Preparing for a clinical trial of interventions to maintain 
normal vaginal microbiota for preventing adverse 
reproductive health outcomes in Africa 
 
 
Sponsor 
University of Liverpool (UoL)
Study site 
Rinda Ubuzima (RU)
KN 50th Avenue (formerly Rue Akagera 716), Kiyovu, Kigali, Rwanda 
P.O. Box 4560, Kigali, Rwanda 
Tel: +250-252-503431 
Fax: +250-252-503389 
Chief investigator
Professor Janneke van de Wijgert, PhD MPH MSc
Institute of Infection and Global Health, University of Liverpool 
Ronald Ross Building, 8 West Derby Street, Liverpool L69 7BE 
Tel office: +44-151-795-9613; Tel mobile: +44-755-719-5108 
Email: j.vandewijgert@liverpool.ac.uk 
Principal Investigator
Stephen Agaba, MD
Rinda Ubuzima (see contact details above) 
Tel mobile: +250-788 357 866 
Email: agabakarema@yahoo.co.uk 
Data collection period
1 February 2015 – 31 January 2016
Protocol version, date
Version: 1.0; Date: 22 April 2015
Most recent approved 
version, date 
Not applicable
Previous approved 
versions, dates 
Not applicable
 
Protocol Approval: 
Chief Investigator
Principal Investigator
Janneke van de Wijgert
Stephen Agaba
 
 
 
Signature 
Signature
Date: 24 April 2015
Date: 24 April 2015

22 April 2015 
 
2 
STATEMENT OF PROTOCOL COMPLIANCE AND CONFIDENTIALITY  
 
By signing this statement, the Principal Investigator agrees to: 
 
• 
Conduct the present study in full accordance with the most recent approved version of the 
protocol, within applicable timelines, according to the relevant standard operating procedures 
and in full agreement with all applicable regulations and the international guidelines regarding 
the conduct of clinical research. 
• 
Permit trial-related monitoring, audits, independent ethics committee review, and regulatory 
inspections, providing direct access to source data/documents during and after the course of 
the trial. 
• 
Make the protocol and all relevant related study documentation available to all physicians, 
nurse/counsellors, laboratory staff and other personnel who participate in conducting this study. 
Ensure that the study team receives adequate training so that they are fully informed and 
qualified to conduct the study. 
• 
Maintain all study documentation in accordance with the International Conference on 
Harmonisation (ICH) Good Clinical Practice (GCP) (1) and other relevant guidelines or until the 
Chief Investigator stipulates in writing that maintenance is no longer required. 
 
The Principal Investigator also acknowledges that this protocol contains confidential information. As 
such, the information may not be disclosed to anyone except persons involved in implementation of the 
study, relevant ethics committees and relevant drug regulatory authorities, unless specific permission is 
granted in writing by the Chief Investigator, or disclosure is required by relevant laws and regulations. 
The Principal Investigator: 
• 
Authorises the Chief Investigator and her team at the University of Liverpool (UoL), Liverpool, 
UK, to have full access to all data pertinent to this study and to receive and test study 
specimens as described in detail in this protocol. 
• 
Understands that copies of all computerised databases will be kept and fully accessible by the 
study team at the UoL, the study team at Rinda Ubuzima (RU), as well as third parties who have 
been given permission by the Chief Investigator and the Principal Investigator. 
• 
Acknowledges that scientific publications and other planned uses of the study data and study 
specimens will be governed by a separate data and specimens sharing policy, and will adhere to 
the agreements set forth in that policy. 
 
Principal Investigator:
Stephen Agaba 
 
 
 
24 April 2015 
Signature 
Date
 
 
 

22 April 2015 
 
3 
TABLE OF CONTENTS 
 
Statement of protocol compliance and confidentiality .......................................................................... 2 
Table of contents .................................................................................................................................... 3 
List of acronyms ...................................................................................................................................... 5 
1. 
Protocol team roster ....................................................................................................................... 6 
2. 
Synopsis ........................................................................................................................................... 7 
3. 
Introduction .................................................................................................................................... 9 
3.1 
Background .............................................................................................................................. 9 
3.2 
Rationale ................................................................................................................................ 11 
4. 
Study design, objectives and endpoints ........................................................................................ 11 
4.1 
Study design .......................................................................................................................... 11 
4.2 
Specific objectives and endpoints ......................................................................................... 12 
4.3 
Sample size ............................................................................................................................ 13 
4.4 
Randomisation and blinding .................................................................................................. 14 
5. 
Study population ........................................................................................................................... 14 
5.1 
Target population .................................................................................................................. 14 
5.2 
Eligibility criteria .................................................................................................................... 14 
5.3 
Recruitment ........................................................................................................................... 15 
5.4 
Retention and withdrawals ................................................................................................... 15 
6. 
Study procedures - Clinical ............................................................................................................ 16 
6.1 
Visit flow chart and schedule of assessments ....................................................................... 16 
6.2 
Screening ............................................................................................................................... 16 
6.3 
Results visit ............................................................................................................................ 18 
6.4 
Enrolment visit....................................................................................................................... 18 
6.5 
Follow-up visits Day 7, Month 1 and Month 2 ...................................................................... 19 
6.6 
Final Month 6 visit ................................................................................................................. 20 
6.7 
Frequent sampling group ...................................................................................................... 20 
6.8 
Unscheduled visits ................................................................................................................. 21 
6.9 
Withdrawal procedures ......................................................................................................... 21 
7. 
Study procedures – Social science ................................................................................................ 21 
8. 
Study procedures - Laboratory...................................................................................................... 22 
8.1 
Diagnostic tests ..................................................................................................................... 22 
8.2 
Research tests ........................................................................................................................ 22 
9. 
Data management and analysis .................................................................................................... 23 
10. Ethical issues and procedures ....................................................................................................... 23 
10.1 
Ethical review ........................................................................................................................ 23 
10.2 
Risks and benefits .................................................................................................................. 24 

22 April 2015 
 
4 
10.3 
Compensation and insurance ................................................................................................ 24 
10.4 
Confidentiality ....................................................................................................................... 24 
10.5 
Informed consent procedures ............................................................................................... 25 
11. Investigational products ................................................................................................................ 25 
11.1 
Study products ....................................................................................................................... 25 
11.2 
Drug regulatory authorities ................................................................................................... 27 
11.3 
Study product accountability and packaging ........................................................................ 27 
11.4 
Adherence assessments ........................................................................................................ 27 
12. Pharmacovigilance ........................................................................................................................ 27 
12.1 
Definition of adverse events ................................................................................................. 27 
12.2 
Definition of serious adverse events ..................................................................................... 27 
12.3 
Assessing AE/SAE severity and relationship to study products ............................................. 28 
12.4 
Reporting of AEs/SAEs ........................................................................................................... 28 
13. Other ............................................................................................................................................. 29 
13.1 
Funding .................................................................................................................................. 29 
13.2 
Study management and oversight ........................................................................................ 29 
13.3 
Protocol amendments ........................................................................................................... 29 
13.4 
GC(L)P monitoring ................................................................................................................. 30 
13.5 
Study termination .................................................................................................................. 30 
13.6 
Dissemination of results ........................................................................................................ 30 
13.7 
Archiving ................................................................................................................................ 30 
14. References ..................................................................................................................................... 31 
15. Appendices .................................................................................................................................... 33 
15.1 
Visit flow chart ....................................................................................................................... 33 
15.2 
Schedule of assessments ....................................................................................................... 34 
15.3 
Package inserts for metronidazole oral tablets, metronidazole vaginal gel, Ecologic Femi, 
and Gynophilus ...................................................................................................................... 35 
 
 
 
 
 

22 April 2015 
 
5 
LIST OF ACRONYMS 
 
AE
Adverse event
BV 
Bacterial vaginosis 
CGR
Centre for Genomics Research
(e)CRF  
(electronic) Case report form 
CVL 
Cervicovaginal lavage 
CT 
Chlamydia trachomatis 
EC 
Executive committee 
FDA
Food and Drug Administration
FGD 
Focus group discussion 
GBP
Great Britain pound
GCP 
Good Clinical Practice 
GCLP 
Good Clinical Laboratory Practice 
GMP
Good Manufacturing Practice
HIV 
Human immunodeficiency virus 
HPV
Human papillomavirus
HSV-2
Herpes simplex virus type 2
ICF 
Informed consent form
ICH 
International Conference on Harmonization 
IDI 
In-depth interview 
IGH
Institute of Infection and Global Health
MRC
Medical Research Council
NG 
Neisseria gonorrhoea 
PCR 
Polymerase chain reaction 
PIS
Participant information sheet
PPM
Potentially pathogenic micro-organism
qPCR
Quantitative polymerase chain reaction
RU 
Rinda Ubuzima 
RWF
Rwandese franc
SAE 
Serious adverse event 
SOP 
Standard operating procedure 
(e)SPIR 
(electronic) Study participant identification register
STI 
Sexually transmitted infection 
SUSAR 
Suspected unexpected serious adverse event
TSC 
Trial steering committee
TV 
Trichomonas vaginalis 
UoL
University of Liverpool
UK
United Kingdom
USA
United States of America
UTI
Urinary tract infection
VMB
Vaginal microbiome
WHO
World Health Organisation
 
 

22 April 2015 
 
6 
1. PROTOCOL TEAM ROSTER 
 
Rinda Ubuzima (RU)
RU function title
Study role
E-mail address
Stephen Agaba, MD
Study Physician
Principal Investigator 
agabakarema@yahoo.co.uk
Gilles Ndayisaba, MD
Executive Committee 
(EC) member 
Co-Investigator (clinical)
ndayisaba@gmail.com
Mireille Uwineza
Research Nurse
Study coordinator
uwinezamireille@yahoo.fr
Evelyne Kestelyn, 
MA 
EC member, RU 
Director 
 
Co-investigator (ICH 
GCP, social science) 
e.kestelyn@liv.ac.uk
Jennifer Van Nuil, 
PhD 
Consultant Medical 
Anthropology 
Co-investigator (social 
science) 
ilovannuil@gmail.com
Marie Michelle 
Umulisa, MA 
Recruitment & 
Outreach Manager 
Research Nurse 
Co-investigator 
(recruitment, outreach, 
social science) 
mariemichelleu@yahoo.fr
Lambert Mwama-
rangwe, BSc 
Laboratory Manager
Laboratory coordinator
mwambara2@yahoo.fr
Vincent Karangwa
EC member, Admin 
& Finance Manager 
Administrative oversight 
at RU 
karavinc@yahoo.fr
University of 
Liverpool (UoL) 
UoL role
Study role
Email address
Janneke van de 
Wijgert, PhD MPH 
MSc 
Professor of
Infection & Global 
Health 
(also RU EC member) 
Chief Investigator 
j.vandewijgert@liv.ac.uk
Alistair Darby, PhD
Lab scientist at UoL 
Centre for Genomics 
Research (CGR) 
Co-investigator (vaginal 
microbiome (VMB) 
testing CGR) 
acdarby@liv.ac.uk
Christiane Hertz-
Fowler, PhD 
Lab scientist and 
manager at CGR 
Co-investigator (VMB
testing CGR) 
c.hertz-fowler@liv.ac.uk
 
Trial Steering Committee
Institution
Email address
Vicky Jespers, MD PhD MEpi
Tania Crucitti, PhD PharmD 
Institute of Tropical Medicine, Antwerp, 
Belgium 
vjespers@itg.be
tcrucitti@itg.be 
Jacques Ravel, PhD
Institute for Genome Sciences, 
University of Maryland School of 
Medicine, Baltimore, MD, USA 
jravel@som.umaryland.edu
Anna Maria Geretti, MD PhD
FRCPath 
UoL IGH
geretti@liv.ac.uk
Jessica Younes, PhD
Winclove Probiotics, Amsterdam, 
Netherlands 
j.younes@winclove.nl
Adrien Nivoliez, PhD
Probionov, Arpajon-sur-Cère, France
a.nivoliez@probionov.com
 
 
 

22 April 2015 
 
7 
2. SYNOPSIS 
 
Purpose 
Women who are treated with oral antibiotics for a vaginal infection (such as 
bacterial vaginosis or BV) are left with a vulnerable vaginal microbiome 
(VMB). This often leads to recurrent BV. The purpose of this study is to 
determine the feasibility, and collect pilot data, on interventions to restore 
and maintain a healthy VMB for 2-6 months after oral antibiotic treatment 
for vaginal infection. This is important because BV is associated with 
increased HIV acquisition, adverse pregnancy outcomes, and 
maternal/neonatal infections. The data will be used to choose the best 
interventions and to inform the design of a future efficacy trial. 
Study Design
Sixty HIV-negative, non-pregnant, sexually active women aged 18-45 with BV 
by Amsel criteria and/or Trichomonas vaginalis (TV) on wet mount or by 
culture will be treated using oral metronidazole for 7 days. After successful 
treatment (defined as no BV by Amsel criteria and no TV on wet mount) and 
when they are free of vaginal candidiasis, other curable sexually transmitted 
infections (STIs) and urinary tract infection (UTI)), they will be randomised to 
4 different VMB maintenance interventions (15 per group) within 3 days of 
completing oral metronidazole treatment: 1) Behavioural ‘vaginal practices 
cessation and safer sex’ counselling only (control); 2) Behavioural counselling 
plus 0.75% metronidazole vaginal gel, one applicator (5g) twice weekly for 
two months; 3) Behavioural counselling plus Ecologic Femi vaginal capsule, 
once per day for 5 days immediately after oral metronidazole treatment 
followed by thrice weekly, for two months; 4) Group 4: Behavioural 
counselling plus Gynophilus vaginal tablet, once every 4 days for two months. 
In all 3 biomedical intervention groups, vaginal product use may be ceased 
temporarily during menstruation. Participants will be asked to adhere to the 
interventions for 2 months, and VMB assessments will take place before 
(screening and enrolment visits), during (Day 7, Month 1 and Month 2 visits), 
and after the interventions (Month 6 visit). Any episode of symptomatic BV 
by Amsel criteria, symptomatic vaginal candidiasis, STI or UTI diagnosed 
during follow-up will be treated using standard oral therapies and the 
interventions will be allowed to continue during this treatment. A subgroup 
of 3 women in each study group (N=12) will be asked to self-collect vaginal 
swabs at home every Monday, Wednesday, and Friday for the first month of 
the intervention to allow for an in-depth evaluation of VMB adjustments to 
the initiation of the interventions (the ‘frequent sampling subgroup’).   
Study Site 
Rinda Ubuzima (RU), Kigali, Rwanda
Study Products
• Vaginal antibiotic: 0.75% metronidazole vaginal gel (Graceway 
Pharmaceuticals, Bristol, TN, USA) 
• Vaginal probiotic: Ecologic Femi vaginal capsule (Winclove Probiotics, 
Amsterdam, The Netherlands) 
• Vaginal probiotic: Gynophilus vaginal tablet (Probionov, Clermont-
Ferrand, France) 
Study Population
HIV-negative, non-pregnant, sexually active women between 18-45 years at 
high risk of HIV, STIs, and BV in Kigali, Rwanda. RU has worked with this 
target population for 10 years. The BV prevalence was at least 40% in 
previous studies. 
Study Duration
One year
Study Endpoints
Primary clinical endpoints will be the safety and preliminary efficacy of the 
study products, the latter defined as incidence of BV by Amsel criteria, BV by 

22 April 2015 
 
8 
Nugent score (Nugent 7-10), intermediate microbiota (Nugent 4-6), TV (by 
culture) and symptomatic vaginal candidiasis (by wet mount) during follow-
up. Secondary clinical endpoints will be membership of specific VMB clusters 
over time as determined by clustering analysis of all available Illumina MiSeq 
data, the presence over time of individual VMB bacteria (as determined by 
Illumina MiSeq), and the incidence of STIs (HIV, syphilis, herpes simplex type 
2 (HSV-2), Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)), and 
UTI (by urinalysis dipstick) during the follow-up period. In addition, we will 
conduct focus group discussions (FGDs), in-depth interviews (IDIs) and a 
quantitative survey to address specific feasibility objectives. 
 
 
 
 

22 April 2015 
 
9 
3. INTRODUCTION 
 
3.1 Background 
 
Microbiota are bacterial communities (consisting predominantly of commensal bacteria) that live in 
or on the human body, including the vagina. Under normal circumstances, these bacterial 
communities do not cause disease, and humans are dependent on them for protection against 
pathogens and for specific bodily functions (such as the processing of food in the gut). Healthy 
vaginal microbiota (VMB) consist predominantly of lactobacilli, which are thought to restrict growth 
of other bacteria by keeping the vagina acidic and via other antimicrobial mechanisms (2). Clinical 
conditions associated with an imbalanced VMB include bacterial vaginosis (BV) and vaginitis. In these 
conditions, lactobacilli are replaced by other micro-organisms that can cause symptoms, such as a 
high diversity of anaerobic bacteria in BV and potentially pathogenic micro-organisms (PPMs, such as 
streptococci, staphylococci, and Escherichia coli) in vaginitis. Like commensals, PPMs are harmless 
under normal circumstances. However, they are more likely to cause severe invasive disease under 
special circumstances such as wound infections and sepsis in vulnerable patients (3). In pregnant 
women, Group B streptococci are most feared as a frequent cause of maternal and neonatal 
infections and mortality (4). 
 
BV is a complex condition involving multiple bacteria and can therefore be defined in multiple ways. 
In clinic settings, it is often diagnosed based on patient-reported symptoms alone or using the Amsel 
criteria (5). In the case of Amsel criteria, BV is diagnosed when 3 of the following 4 criteria are 
present: 1) clue cells on wet mount microscopy (a wet mount is vaginal fluid on a microscopy slide); 
2) a ‘fishy’ odour after adding 10% KOH to the wet mount; 3) vaginal pH > 4.5; and 4) thin, 
homogenous vaginal discharge present. Both patient-reported symptoms as well as the Amsel 
criteria have poor sensitivity because many women with BV are asymptomatic. Therefore, in 
research settings, Gram stain Nugent scoring is more often used (6). Vaginal fluid is smeared on a 
microscopy slide, stained, and scored under the microscope based on the presence and relative 
quantity of three bacterial morphotypes (shapes). In this case, the diagnosis does not depend on the 
presence of signs or symptoms and is therefore more sensitive. A Nugent score of 0-3 is considered 
normal, 4-6 is referred to as intermediate microbiota, and 7-10 BV. Unfortunately, Nugent scoring is 
labour-intensive and is therefore usually not a feasible alternative to the Amsel criteria. Other micro-
organisms that are often found in the vagina include Candida yeasts, Trichomonas vaginalis (TV) and 
other sexually transmitted pathogens. Vaginal candidiasis and TV are typically diagnosed by 
visualising the organisms on the wet mount by microscopy and/or by culturing them, and other 
sexually transmitted pathogens by commercialised pathogen-specific diagnostic assays.  
 
Epidemiological studies using the Amsel criteria or Nugent scoring have shown that BV increases risk 
of preterm birth and infections in pregnant women and their neonates (7). Furthermore, BV and 
intermediate microbiota by Nugent score, TV and vaginal candidiasis, have been associated with 
increased transmission of HIV and other chronic sexually transmitted infections (STIs) (8). Studies 
have also shown that the female urinary tract has its own microbiome that resembles the VMB, and 
presence of E. coli in the VMB might therefore increase risk of urinary tract infections (UTIs) (9). 
 
In the last decade, phylogenetic analyses of vaginal samples have shown that the VMB is more 
complex than previously thought (10-14). In these studies, vaginal samples were taken, and the 
bacterial DNA in those samples was characterised using molecular methods. The molecular method 
that is currently the most commonly used is sequencing of a portion of the bacterial 16S ribosomal 
RNA gene; this gene is ubiquitously present in bacteria (not in human cells) and can be used to 
identify bacteria at the genus or even species level because of differences in the nucleotide 
sequences of the gene. Using such molecular methods, several bacteria were identified that had 

22 April 2015 
 
10 
previously been missed because they were difficult to culture (10), and important differences were 
discovered between different species of the genus Lactobacillus. In a landmark study in women in 
the U.S. without vaginal symptoms, 5 VMB clusters were identified, 4 of them dominated by L. 
crispatus, L. iners, L. gasseri, L. jensenii, respectively, and the fifth one consisting of a mixture of 
anaerobes (11, 12). A second study in the U.S., this time in women with clinical BV, identified 
multiple VMB clusters consisting of highly diverse mixtures of anaerobic bacteria (13). These U.S. 
studies identified racial differences, with the highest prevalence of non-lactobacilli clusters in black 
women (11). The Rinda Ubuzima (RU) team was the first to describe the VMB of African women in a 
comprehensive manner (15). In a cohort of Rwandan women at high risk of acquiring HIV, we 
identified 6 VMB clusters, dominated by L. crispatus, L. iners, an anaerobe mixture with intermediate 
bacterial load, and anaerobe mixtures with high bacterial loads (three distinct clusters), respectively. 
We found significant trends in prevalence of HIV/STIs from low prevalence in the L. crispatus cluster, 
to higher prevalence in the L. iners cluster, and highest prevalence in the mixed anaerobe clusters. 
Until recently, molecular methods were not incorporated into large epidemiological studies with 
clinical outcomes due to limited availability and high costs. However, the Illumina MiSeq next 
generation sequencing platform (which is available in select laboratories in Europe but not in 
Rwanda; http://www.illumina.com/systems/miseq.ilmn) now makes high throughput sequencing 
possible and affordable. High throughput sequencing currently does not yet have any routine 
diagnostic value and is only used for research. 
 
Even though evidence suggesting that not only symptomatic BV but also asymptomatic BV and 
intermediate microbiota cause long-term adverse outcomes is mounting, only symptomatic BV tends 
to be treated. BV is notoriously difficult to treat (16-18). About 60-80% of patients are cured after a 
standard course of antibiotics (typically oral or vaginal metronidazole or clindamycin) but recurrence 
rates are high: around 50% within 6 months (16). Two main reasons for this have been hypothesised: 
1) the antibiotics not only reduce the abundance of BV-associated bacteria but also of healthy 
lactobacilli, which means that the VMB continues to be fragile after treatment; and/or 2) the 
antibiotics cannot penetrate the adherent biofilm that is often formed by the BV-associated 
bacteria, such as Gardnerella vaginalis (16). Antibiotic resistance does not seem to play a major role. 
BV tends to recur around the time of menstruation, which is thought to be due to declining 
oestrogen levels and/or declining vaginal pH due to menstrual blood flow, and after triggering 
events such as unprotected sex or vaginal douching. Many antibiotic, sexual behaviour, and vaginal 
hygiene interventions to reduce BV recurrences, and subsequently reduce adverse pregnancy 
outcomes, have been tried but with limited success thus far (reviewed in 16-19). None of these 
studies incorporated longitudinal molecular VMB assessments. TV is often associated with BV and 
the treatment for the two conditions is identical.  
 
Therapies to restore and maintain a healthy VMB after antibiotic BV or TV treatment are currently 
not standard practice, but some clinicians in Europe and North America recommend twice weekly 
0.75% metronidazole vaginal gel for 4-6 months to lower the risk of BV recurrence (16). This 
recommendation was tested in a randomised controlled trial in the U.S., which showed a statistically 
significant 57% reduction in the BV recurrence rate (20). Unfortunately, the effect disappeared after 
cessation of the intervention. However, since metronidazole can safely be used during pregnancy 
and has a high resistance threshold, this intervention could potentially be used in the second and 
third trimester of pregnancy in women at high risk of adverse pregnancy outcomes associated with 
an imbalanced VMB. Another study in American women at high risk of BV and STIs showed that 
metronidazole gel twice per week for 6 months significantly reduced BV and STI incidence in the 
intervention group (21). Two recent African trials evaluating oral (2g monthly) and vaginal 
metronidazole (5 nights every 3 months) for BV prevention showed significant beneficial effects, but 
the effects were modest, most likely due to the infrequent dosing (22, 23). 
 

22 April 2015 
 
11 
The World Health Organization (WHO) 2001 definition of probiotics is ‘live micro-organisms which, 
when administered in adequate amounts, confer a health beneﬁt on the host’. Evidence is mounting 
that probiotic lactobacilli can restore and maintain lactobacilli-dominated VMB, are better able to 
disrupt BV-associated biofilms than antibiotics, and can be used safely for long periods without the 
risk of developing resistance (24). We have identified two vaginal probiotics that are licensed in 
Europe as medical devices and available over-the-counter: Ecologic Femi (Winclove Probiotics BV, 
Amsterdam, The Netherlands) and Gynophilus (Probionov, Clermont-Ferrand, France). While both 
vaginal probiotics contain lactobacilli with Qualified Presumption of Safety status in Europe (25), 
Ecologic Femi is a vaginal capsule containing multiple species of Lactobacillus and Gynophilus is a 
vaginal tablet containing one Lactobacillus species (together with its required nutrients) that was 
specifically designed to be released slowly over time. Clinical studies with Gynophilus in Austria, 
France, Russia and Poland thus far have shown beneficial VMB effects (27-30) and bothproducts are 
deemed safe (see section 11.1). 
 
To summarise: current knowledge suggests that standard antibiotic treatment of BV alone is not 
sufficient in women with recurrent BV. The antibiotics also reduce the number of lactobacilli in the 
VMB, thereby leaving the VMB fragile. We believe that standard antibiotic treatment should be 
accompanied by behavioural counselling (safer sex and cessation of traditional vaginal practices), 
and perhaps also by promising vaginal antibiotic or probiotic VMB maintenance therapies. 
 
3.2 Rationale 
 
The RU team has worked with women at high risk of HIV and other STIs in Kigali since 2004 (31). We 
believe that interventions to reduce the extremely high prevalence and incidence of BV and other 
VMB imbalances are needed in this population, not only because BV is an important health problem 
in its own right, but also to prevent more serious consequences such as increased HIV/STI 
transmission and adverse pregnancy outcomes (all of which are also common in our study 
population). Our long-term goal is to conduct a randomised controlled efficacy trial of promising 
interventions (standard antibiotic treatment, followed by novel antibiotic and/or probiotic 
maintenance therapies, accompanied by behavioural counselling) to normalise the VMB for at least 
6 months in order to reduce the incidence of BV, HIV/STIs, and adverse pregnancy outcomes. 
However, as a first step, we will conduct a pilot study to determine the feasibility of conducting such 
a trial, and to determine the longitudinal molecular effects of these promising interventions on the 
VMB of high risk women. These data are needed to choose the best interventions and to inform the 
design of the future efficacy trial.  
 
4. STUDY DESIGN, OBJECTIVES AND ENDPOINTS 
 
4.1 Study design 
 
Sixty HIV-negative, non-pregnant, sexually active women aged 18-45 with BV by Amsel criteria 
and/or TV will be treated using oral metronidazole for 7 days. After successful treatment (defined as 
no BV by Amsel criteria and no TV on wet mount) and when they are free of vaginal candidiasis, 
other curable STIs and UTI, they will be randomised to 4 different VMB maintenance interventions 
(15 per group) within 3 days after completing oral metronidazole treatment: 
− 
Group 1: Behavioural ‘vaginal practices cessation and safer sex’ counselling only (control); 
− 
Group 2: Behavioural counselling plus 0.75% metronidazole vaginal gel, one applicator (5g) 
twice weekly for two months. If 0.75% metronidazole vaginal gel with stability data up to 30 °C 
cannot be obtained, we will use oral metronidazole 400-500mg twice weekly for two months 
(see section 11.1). 
− 
Group 3: Behavioural counselling plus Ecologic Femi vaginal capsule, once per day for 5 days 
immediately after oral metronidazole treatment followed by thrice weekly, for two months 

22 April 2015 
 
12 
− 
Group 4: Behavioural counselling plus Gynophilus vaginal tablet, once every 4 days for two 
months. 
 
In all 3 biomedical intervention groups, vaginal product use may be ceased temporarily during 
menstruation. Participants will be asked to adhere to the interventions for 2 months, and VMB 
assessments will take place before (screening and enrolment visits), during (Day 7, Month 1 and 
Month 2 visits), and after the interventions (Month 6 visit). Any episode of symptomatic BV by Amsel 
criteria, symptomatic vaginal candidiasis, STI (including TV) or UTI diagnosed during follow-up will be 
treated using standard oral therapies and the vaginal interventions will be allowed to continue 
during this treatment. A subgroup of 3 women in each study group (N=12) will be asked to self-
collect vaginal swabs at home every Monday, Wednesday, and Friday for the first month of the 
intervention to allow for an in-depth evaluation of VMB adjustments to the initiation of the 
interventions (the ‘frequent sampling subgroup’). These samples will be collected by a staff member, 
or dropped of by the participant, at least once per week at a time and location that is acceptable to 
the participant.   
 
Primary clinical endpoints will be the safety and preliminary efficacy of the study products, the latter 
defined as incidence of BV by Amsel criteria, BV by Nugent score (Nugent 7-10), intermediate 
microbiota (Nugent 4-6), TV by culture and symptomatic vaginal candidiasis (by wet mount) during 
follow-up. Secondary clinical endpoints will be membership of specific VMB clusters over time as 
determined by clustering analysis of all available Illumina MiSeq data, the presence over time of 
individual VMB bacteria (as determined by Illumina MiSeq), and the incidence of STIs (HIV, syphilis, 
HSV-2, CT, and NG), and UTI (by urinalysis dipstick) during the follow-up period. In addition, we will 
conduct focus group discussions (FGDs), in-depth interviews (IDIs) and a quantitative survey to 
address specific feasibility objectives (see 4.2 below). 
 
4.2 Specific objectives and endpoints 
 
Primary clinical objectives
Endpoints
1. To determine the safety of the 
three biomedical interventions 
compared to the control group. 
− Self-reported solicited and unsolicited (serious) adverse 
events (AEs) and social harms 
− Clinician-observed speculum exam findings 
2. To determine the preliminary 
efficacy of the three biomedical 
interventions compared to the 
control group in reducing BV 
recurrence. 
− BV by Amsel criteria, Nugent score 4-10, TV by culture and 
symptomatic vaginal candidiasis at any time during the 
intervention (Day 7, Month 1, Month 2) 
− BV by Amsel criteria, Nugent score 4-10, TV by culture, and 
symptomatic vaginal candidiasis after cessation of the 
intervention (Month 6) 
− Abnormal Nugent scores will be analysed as two categories 
(4-6 and 7-10) instead of one category (4-10) if statistical 
power permits. 
Secondary clinical objectives
Endpoints
1. To determine which VMB 
changes at the molecular 
bacterial level are induced by 
initiation and cessation of the 
various interventions compared 
to the control intervention.  
 
− Membership of specific VMB clusters (as determined by 
clustering analysis of all Illumina MiSeq data) over time  
− The presence over time of individual VMB bacteria as 
determined by Illumina MiSeq (and, if funding permits, 
other molecular methods), including the PPMs 
streptococci, staphylococci, and E coli. 
− The additional data points in the frequent self-sampling 
group will be analysed separately. 
2. To determine preliminary 
− Incidence of the following STIs between screening and 

22 April 2015 
 
13 
efficacy of the three biomedical 
interventions compared to the 
control group in reducing the 
incidence of STIs and UTI. 
Month 6: HIV, HSV-2, syphilis, CT, NG, (and, if funding 
permits, human papillomavirus (HPV)) 
− Incidence of UTI between screening and Month 6 
Feasibility objectives
Endpoints
1. To determine what Rwandan 
women know about BV and its 
impact on reproductive health 
outcomes. 
− Quantitative survey with women attending recruitment 
and screening sessions, and FGD with women enrolled in 
the trial. 
2. To determine acceptability of 
and adherence to the 4 
interventions.  
[In the case of the control intervention, to 
determine if women who do not want to 
completely cease vaginal cleansing are willing 
to use clean water only in a manner that does 
not interfere with the biomedical 
interventions.]
− Quantitative survey, FGD and IDI data from women 
enrolled in the trial on acceptability and adherence. 
− Triangulation of quantitative and qualitative adherence 
data (see below). 
− The number of women withdrawing from the trial because 
of discontent with the interventions. 
3. To determine the acceptability 
and feasibility of vaginal self-
sampling at home three times 
per week for 4 weeks (with 
sample pick-up or drop-off). 
− Quantitative survey and FGD data from women enrolled in 
the frequent self-sampling group on acceptability and 
feasibility. 
− The proportion of samples with sufficient DNA yield to 
generate Illumina MiSeq results. 
4. To determine the required 
frequency of vaginal sampling in 
a future clinical trial. 
− Using the above-mentioned primary and secondary clinical 
endpoints. 
5. To determine the likely 
sustainability of the various 
interventions. 
− Using the above-mentioned clinical and feasibility 
endpoints as well as cost data from this pilot study and IDI 
data from clinicians and policymakers. 
 
If funding permits, we will also assess the following additional secondary and tertiary clinical 
endpoints using stored specimens: 1) additional VMB characterisation using other molecular 
methods such as quantitative PCR (qPCR) to quantify individual bacterial species, TV, and Candida 
albicans (secondary; addressing secondary objective 1 above); 2) HPV typing of endocervical 
specimens (secondary; addressing secondary objective 2 above); 3) urine microbiome 
characterisation by Illumina MiSeq and/or other molecular methods (tertiary), VMB biofilm 
characterisation(tertiary); and characterisation of the VMB and associated metabolites and immune 
responses in cervicovaginal lavages (CVLs; tertiary). 
 
4.3 Sample size 
 
This pilot study is descriptive and one of its aims is to inform sample size calculations of a future 
efficacy trial. Formal sample size calculations were therefore not performed. In the clinical trial, 60 
women will be randomised into 4 groups consisting of 15 women each. Based on previous 
experiences with reproductive health clinical trials in the same study population, we expect to have 
to screen at least 120 women to enrol 60.  
 
In the qualitative research, the number of FGDs or IDIs held will be governed by data saturation: no 
new discussions/interviews will be done when they no longer generate new information. The sample 
sizes listed in this study protocol are estimates based on our previous experiences with qualitative 
research. For feasibility objective 1, we expect to conduct quantitative face-to-face interviews with 
at least 30 women attending recruitment sessions who did not pass pre-screening (in addition to the 
approximately 120 women attending screening visits) and conduct 2 FGDs with women enrolled in 

22 April 2015 
 
14 
the trial. For feasibility objective 2, we expect to conduct 2 FGDs and 5 IDIs (in addition to 
quantitative face-to-face interviews with the 60 women enrolled in the trial). For feasibility objective 
3, we expect to conduct one FGD with all 12 women in the frequent sampling subgroup. For 
feasibility objective 5, we expect to conduct 5 IDIs with clinicians and policymakers. The total 
number of FGDs and IDIs will therefore be approximately 5 FGDs and 10 IDIs. 
 
4.4 Randomisation and blinding 
 
Participants will be randomised at the enrolment visit. Study group allocation will be concealed until 
the participant was confirmed to be eligible and has provided written informed consent for 
enrolment. Computer-generated group allocations will be generated prior to study initiation and 
concealed in randomisation envelopes numbered 1-60. The envelopes will be opened in consecutive 
order as new participants are randomised. The study clinicians cannot be blinded because the study 
products look different and dosing schedules are different. However, we will blind the laboratory 
technicians at RU and at UoL so that the assessment of all laboratory endpoints is blinded. This will 
be achieved by ensuring that study samples and accompanying documents that are shared with the 
laboratory technicians do not contain any information about the study arm that the participant was 
randomised to.    
 
5. STUDY POPULATION 
 
5.1 Target population 
 
Our target population is HIV-negative, non-pregnant, sexually active women between 18-45 years at 
high risk of HIV/STIs/BV in Kigali, Rwanda. RU has worked with this target population for 10 years 
(15, 31). The HIV prevalence in a previous study in this population was 24% (31), the BV prevalence 
40% (15), and the TV prevalence 17% (31). The HSV-2 and HPV status of participants will be 
determined (the latter only if funding permits; see below), and will be considered for inclusion in 
statistical models as a confounding variable, but will not be used to select women for enrolment. We 
decided to limit inclusion to women of reproductive age (18-45 years) because our long-term goal is 
to identify an efficacious intervention that can subsequently be tested in women of reproductive age 
with high risk of HIV/STIs/BV and/or pregnancy. An intervention that is efficacious in women of 
reproductive age is not necessarily efficacious in postmenopausal women due to expected VMB 
changes after menopause. 
 
5.2 Eligibility criteria 
 
Inclusion criteria: 
− 
Female, 18-45 years old 
− 
Sexually active, defined as having had sex at least twice in the two weeks prior to screening 
− 
At high risk of HIV/STIs/BV, defined as having had more than one sexual partner in the last 12 
months OR having been treated for an STI and/or BV in the last 12 months 
− 
Successfully treated for BV by Amsel criteria or TV (defined as no BV by Amsel criteria and no TV 
on wet mount at the enrolment visit) and free of HIV (at screening), pregnancy (at screening 
and the enrolment visit), symptomatic vaginal candidiasis and UTI (at the enrolment visit), and 
syphilis, NG, and CT (at the results visit). Due to the long turn-around time for NG and CT lab 
test results, women may be enrolled when their NG and CT results are not yet known, but a 
new participant will be randomised for each woman whose screening sample comes back 
positive for NG and/or CT after having been randomised. The NG/CT-positive women will be 
allowed to complete the study but their data may be removed from some analyses.  
− 
Currently in good physical and mental health as judged by a study physician 
− 
Willing and able to adhere to study procedures and provide written informed consent. 

22 April 2015 
 
15 
According to Rwandan ethics guidelines, unmarried women aged 18-20 also need the written 
informed consent of a parent or guardian.  
− 
Women are allowed to use contraception, including hormonal contraception; use of these 
methods will be taken into account in the instructions given to women about probiotic use and 
in the data analysis. 
 
Exclusion criteria: 
− 
HIV positive 
− 
Pregnant 
− 
Clinician-observed genital ulcers, condylomata, or other genital abnormalities at screening 
and/or enrolment 
− 
Underwent a gynaecological surgery/invasive procedure in the 3 months prior to screening 
− 
History of significant urogenital prolapse, undiagnosed vaginal bleeding, urine or faecal 
incontinence, or blood clotting disorders 
− 
Allergic to metronidazole or any other components of the study drugs 
− 
Not willing to stop use of other oral or vaginal probiotics from the screening visit until the end 
of study participation 
− 
Participating in another health intervention study 
− 
For any other reason potentially interfering with participant safety or protocol adherence as 
judged by the Principal Investigator (these reasons will be recorded) 
 
5.3 Recruitment 
 
RU will use its network of community mobilisers to organise recruitment meetings in the Kigali 
neighbourhoods where recruitment for previous RU studies with high risk women took place. These 
neighbourhoods are Gikondo, Kimihurura, Remera and Muhima. Recruitment will not start until 
permission has been obtained from the local authorities in those neighbourhoods. Standard RU 
procedures will be used to ensure that confidentiality is not breached. For example, the project 
vehicles do not carry a logo and outreach staff do not wear uniforms. During recruitment meetings, 
study staff will explain the study to potential participants, and there will be sufficient time for 
questions and answers. At the end of the meeting, study staff will check potential eligibility of 
individual women who are interested in participating using an anonymous checklist (referred to as 
pre-screening). No individual data will be recorded during this pre-screening process; only overall 
statistics (number of women who attended the recruitment session, were pre-screened, and were 
deemed eligible after pre-screening) will be kept for each recruitment session. Women who are 
interested and deemed potentially eligible at pre-screening will be asked for contact information 
(this information cannot be linked to the pre-screening checklist) and will be invited to attend a 
screening visit at the RU research clinic in Kiyovu, Kigali.  
 
5.4 Retention and withdrawals 
 
Study staff, with the help of community mobilisers, will make every effort to retain participants to 
minimise possible bias associated with loss-to-follow-up. All participants are required to provide 
contact information for herself and at least one other contact person at screening, and this 
information will be verified at each subsequent visit; they will consent to this during the screening 
and enrolment informed consent processes, respectively. Study staff will ask each participant for 
permission to contact her and her contact person(s) in specific ways, such as by mobile phone 
and/or home visit. If a participant misses a visit, study staff will attempt to contact her in accordance 
with what was agreed. All contact attempts will be documented in the participant’s records.  
 
Visit windows for the study visits are as follows: 
• 
Screening + results visits: the entire screening process should take no longer than 6 weeks 

22 April 2015 
 
16 
• 
Enrolment visit: maximum of 3 days after completion of oral metronidazole treatment 
• 
Day 7: Day 5 – Day 9 
• 
Month 1: ± 7 days 
• 
Month 2: ± 7 days 
• 
Month 6: - 7 days 
 
If a participant misses a visit window, this will be considered non-adherence to the visit schedule but 
not a protocol deviation. A participant will be considered lost to follow-up only at study close-out 
after multiple contacts by staff (up to a maximum of three face-to-face contacts per missed visit) to 
complete the participant’s visit schedule have been unsuccessful. 
 
Participants may voluntarily withdraw from the study for any reason at any time during the study. If 
a participant does withdraw from the study, study staff will attempt to complete any outstanding 
evaluations of the participant (see ‘withdrawal procedures’) but only if the participant agrees. The 
reason(s) for the withdrawal will be recorded in the participant’s records. The Principal Investigator 
may also discontinue participants (for example, if this is deemed in the best interest of the 
participant) but will first ask the Chief Investigator for a second opinion. Women who become 
pregnant or acquire an STI (including HIV) during the study may continue. Participants who are 
discontinued from the study cannot be rescreened. 
 
6. STUDY PROCEDURES - CLINICAL 
 
At all study visits, curable genital infections (syphilis, NG, CT, TV, symptomatic BV, and symptomatic 
vaginal candidiasis) will be treated at RU free of charge and partner notification and treatment 
services will be offered if applicable. Women will also receive reproductive health counselling and 
condoms free of charge. Any other medical conditions (including newly diagnosed HIV and 
pregnancy) and reproductive health needs (including contraception) will be actively referred to local 
clinics or referral hospitals. The RU study team is well connected with available services in Kigali.  
 
The order of procedures at study visits as described below may be modified somewhat to improve 
clinic flow but informed consent procedures will always be done before any other procedures, and 
face-to-face interviewing will be done before counselling in order not to bias the interview results. 
 
6.1 Visit flow chart and schedule of assessments 
 
See Annex 1 and 2. 
 
6.2 Screening 
 
The screening process is in at least two study visits (screening and results visit) and should take no 
longer than 6 weeks. 
 
Upon arriving at the RU research clinic all potentially eligible participants will be assigned a 
participant identification number after which the screening informed consent process will be done 
according to existing RU standard operating procedures. It will include a group educational session, 
followed by an individual session consisting of a formal assessment of literacy, a discussion about 
the study with a research nurse, a formal assessment of understanding of the study, and the actual 
signing of the consent form (see 10.5 for details). 
 
After written informed consent has been obtained, the following procedures will take place: 
1. 
Collection of contact information; 
2. 
Completion of screening eligibility checklist. Women who are menstruating at the time of this 

22 April 2015 
 
17 
visit will be asked to return for completion of screening procedures after they have finished 
menstruating. 
3. 
Face-to-face interview (sociodemographic characteristics, sexual behaviour, vaginal practices, 
circumcision status and penile hygiene of male partners, medical and reproductive history with 
an emphasis on STIs and recurrent reproductive tract infections, current medications and 
contraception, current genital symptoms, and BV knowledge); 
4. 
HIV pre-test and HIV/STI/BV risk reduction counselling; 
5. 
Collection of blood (10 ml; for HIV, HSV-2 and syphilis testing) and urine (for pregnancy and 
urinalysis dipstick testing); 
6. 
Rapid HIV, pregnancy and urinalysis dipstick tests in the onsite RU laboratory; 
7. 
Post-HIV test counselling, provision of rapid test results, and provision of treatment (for UTI on 
urinalysis dipstick) and referrals (for HIV and pregnancy) as needed. 
8. 
If HIV-positive and/or pregnant: not eligible for enrolment. A speculum examination will only be 
done when clinically indicated but women will be asked to donate vaginal swabs for molecular 
VMB testing. These swabs will be collected without speculum insertion, and can be collected by 
the clinician or by the woman herself depending on her preference.  
9. 
If HIV-negative and not pregnant: Speculum examination with visual inspection and specimen 
collection in the following order: 1) vaginal swabs for vaginal pH measurement, wet mount, 
Gram stain Nugent scoring, TV culture, and VMB assessments (see 8.2); 2) endocervical swabs 
for NG/CT PCR and HPV typing; and 3) CVL for additional VMB assessments (see 8.2). 
10. Wet mount microscopy (Amsel criteria, Candida, and TV) in the onsite RU laboratory; 
11. Provision of treatment as follows: 
− If only BV by Amsel criteria and no/minor vaginal symptoms: postpone treatment until the 
results visit 1-2 weeks later. According to local treatment guidelines and the World Health 
Organisation, asymptomatic BV does not have to be treated. However, in this study, all 
women with BV by Amsel criteria will be treated because we want to achieve a healthy VMB 
in all women. However, postponing treatment to allow for other diagnostic testing to be 
completed first will not jeopardise that goal. 
− If BV by Amsel criteria and significant vaginal symptoms: start oral metronidazole for 7 days, 
and schedule a results visit for within 3 days after completion of this treatment. This will give 
the team 10 days to obtain all other STI test results, which will determine final eligibility for 
enrolment (see 6.3.2). 
− If TV on wet mount: start oral metronidazole for 7 days, and schedule a results visit for 
within 3 days after completion of this treatment. This will give the team 10 days to obtain all 
other STI test results, which will determine final eligibility for enrolment (see 6.3.2). 
− If vaginal candidiasis and vaginal symptoms (regardless of BV or TV): start standard 
antifungal treatment and repeat screening after treatment (see 6.3.2).  
12. Reimbursement, and scheduling of the results visit in 1-2 weeks for all HIV-negative, non-
pregnant women regardless of eligibility up to that point (allowing sufficient time to complete 
all diagnostic laboratory testing); 
13. Participant leaves the RU clinic; 
14. Further local laboratory testing:  
− Blood specimens: HSV-2 and syphilis serology 
− Vaginal specimens and CVL: TV culture, and initial processing and storage of samples for 
Gram stain Nugent scoring at RU and VMB and biofilm assessments at UoL (see 8.2) 
− Endocervical specimens: NG/CT PCR, and initial processing and storage of one swab for HPV 
typing in Europe (if funding permits; see 8.2). We will only conduct HPV typing if funding 
permits as this test is not available in Rwanda and does not have any therapeutic 
consequences for women. 
 
 
 

22 April 2015 
 
18 
6.3 Results visit 
 
All HIV-negative, non-pregnant women regardless of eligibility up to that point will be asked to 
return to the study clinic 1-2 weeks after the screening visit to obtain their screening test results. 
 
The following procedures will take place at the results visit: 
1. 
Confirmation of contact information; 
2. 
Provision of HSV-2, syphilis, NG, CT, and TV culture results. 
− If syphilis, NG or CT positive or received treatment for candidiasis at screening (regardless of 
other test results): Treat STIs according to national guidelines and ask the woman to return 
to RU as soon as possible after completing her STI treatment for a repeat wet mount. 
Women who received candidiasis treatment at screening can provide a repeat wet mount at 
this visit. Vaginal swabs to measure vaginal pH and prepare a wet mount (as well as a slide 
for Gram stain Nugent scoring) will be obtained without speculum. If negative for BV by 
Amsel criteria and TV on wet mount, the woman is not eligible for enrolment. If positive for 
BV by Amsel criteria and/or TV on wet mount, treat using oral metronidazole for 7 days and 
schedule the enrolment visit within 3 days of completing treatment. 
− If TV culture positive and not yet treated for TV (based on wet mount) at screening: treat 
using oral metronidazole for 7 days and schedule the enrolment visit within 3 days of 
completing this treatment. 
− If BV by Amsel criteria at screening and not yet treated (regardless of symptoms): treat using 
oral metronidazole for 7 days and schedule the enrolment visit within 3 days of completing 
this treatment.  
− A positive HSV-2 result will not have any treatment or eligibility consequences. 
3. 
Due to the logistics of NG/CT PCR, we expect long turn-around time. For this reason, and 
because the prevalence of NG and CT is much lower than the prevalence of the other STIs, 
women may be enrolled when their NG and CT results are not yet known. However, a new 
participant will be randomised for each woman whose screening sample comes back positive 
for NG and/or CT after having been randomised. The NG/CT-positive women will be allowed to 
complete the study but their data may be removed from some analyses.   
4. 
Due to the logistics of Nugent scoring, Nugent scores may not be available at the enrolment 
visit. Results will not be used to guide enrolment and treatment decisions anyway, but will only 
be used in the data analysis. 
5. 
Confirmation of eligibility thus far using the enrolment checklist. The screening process may be 
repeated up to 3 times.  
 
6.4 Enrolment visit 
 
The enrolment visit will be scheduled as soon as possible, and within a maximum of 3 days after 
completion of oral metronidazole treatment. This window of 3 days is needed in case the last day of 
oral metronidazole treatment is in the weekend or during menses. At the enrolment visit, the 
following procedures will take place: 
1. 
Urine pregnancy and urinalysis dipstick testing. If pregnant, not eligible. If UTI, treat and do not 
yet randomise. Repeat wet mount (and collect Gram stain for Nugent scoring) and enrol based 
on new wet mount results as in 6.3.2. 
2. 
Enrolment informed consent procedures; 
3. 
Face-to-face interview (update on current medications, contraception and genital symptoms 
and baseline assessment of AEs and social harms) 
4. 
Speculum examination with collection of vaginal swabs for vaginal pH measurement, wet 
mount, Gram stain Nugent scoring, TV culture and VMB assessments (see 8.2); endocervical 
swabs to store for potential NG/CT testing (if required for clinical reasons or for VMB 
interpretation); and a CVL to store for additional VMB assessments (see 8.2). If BV by Amsel 

22 April 2015 
 
19 
criteria, TV on wet mount or symptomatic vaginal candidiasis on wet mount, treat and do not 
yet randomise. Repeat wet mount (and collect Gram stain for Nugent scoring) and enrol based 
on new wet mount results as in 6.3.2. 
5. 
Randomisation;  
6. 
HIV/STI/BV risk reduction counselling plus explanation of the intervention that the participant 
was randomised to (including detailed explanations of potential side effects, and in the case of 
metronidazole, potential side effects when metronidazole is combined with alcohol use), 
provision of study products, and application of the first dose of the relevant intervention under 
supervision of a study staff member (and practice of vaginal self-sampling if in the frequent 
sampling group). Women will be asked to stay in the study clinic for a minimum of about 30 min 
after application of the first dose to identify and treat any immediate allergic reactions (these 
are exceedingly rare). 
7. 
Reimbursement, and scheduling of the Day 7 visit; 
8. 
Participant leaves the RU clinic; 
9. 
Further laboratory testing:  
− Vaginal swabs and CVL: Initial processing and storage of a vaginal Gram stain for Nugent 
scoring at RU, and vaginal swabs and CVL for VMB and biofilm assessments at UoL (see 8.2); 
− Endocervical swabs: Initial processing and storage for potential local NG/CT testing; 
− Urine specimens: Initial processing and storage for urine microbiome analyses in Europe (if 
funding permits; see 8.2). 
 
Use of Amsel criteria:  As noted in the introduction, one of the four Amsel criteria is the presence of 
vaginal discharge, and we may therefore miss asymptomatic cases of BV if we use the Amsel criteria 
instead of Nugent scoring. This is not a problem for the women themselves because asymptomatic 
BV is normally not treated, but it is a missed opportunity for this study. We will closely monitor our 
Amsel data in the beginning of the study (compare the Amsel data with the Nugent data), and if 
warranted, we may decide to only use the three laboratory Amsel criteria to guide enrolment and 
treatment decisions. In that case, BV by Amsel criteria would be defined as 2 of the 3 laboratory 
criteria throughout this protocol.  
 
6.5 Follow-up visits Day 7, Month 1 and Month 2 
 
Follow up visits in the clinical trial will take place at Day 7 (7 days after start of the intervention), 
Month 1, Month 2 (cessation of the intervention), and Month 6 (4 months after cessation of the 
intervention).  
 
The following procedures will take place during the Day 7, Month 1 and Month 2 follow up visits: 
1. 
Confirmation of contact information. Women who are menstruating at the time of a study visit 
will be asked to return for completion of visit procedures after they have finished menstruating. 
2. 
Face-to-face interview (update since the last study visit on: current medications, contraception, 
genital symptoms, sexual behaviour, vaginal practices, penile hygiene practices of male 
partners, having been diagnosed and/or treated for STIs/BV/vaginal candidiasis or other 
medical conditions, adverse events/social harms, and acceptability of and adherence to the 
interventions). 
3. 
Speculum examination with collection of specimens: vaginal specimens for vaginal pH, wet 
mount, Gram stain Nugent scoring, TV culture and VMB and biofilm assessments at UoL, and a 
CVL to store for additional VMB assessments at UoL. If the participant has signs or symptoms of 
a reproductive tract infection, specimens will be collected for diagnostic testing as described for 
the screening visit. 
4. 
Counselling with as main aim to address any questions or concerns the participant may have 
about her intervention. At the Month 2 visit, the intervention will be terminated. 
5. 
Reimbursement, and scheduling of the next visit;  

22 April 2015 
 
20 
6. 
Participant leaves the RU clinic; 
7. 
Further laboratory testing:  
− Diagnostic specimens (only in the case of genital signs or symptoms): see screening visit 
− Initial processing and storage of vaginal Gram stains for Nugent scoring at RU, and vaginal 
swabs and CVLs for VMB and biofilm assessments at UoL. 
 
6.6 Final Month 6 visit 
 
The following procedures will take place during the Month 6 visit: 
1. 
Confirmation of contact information. Women who are menstruating at the time of this visit will 
be asked to return for completion of visit procedures after they have finished menstruating. 
2. 
Face-to-face interview (the same topics as the other follow up visit interviews but with an 
emphasis on overall evaluation of the feasibility and acceptability of the interventions) 
3. 
Pre-HIV test and HIV/STI/BV risk reduction counselling 
4. 
Collection of blood (10 ml; for HIV, syphilis and HSV-2 testing, the latter only if negative at 
baseline) and urine (for pregnancy and urinalysis dipstick testing) 
5. 
Speculum examination with specimen collection in the following order: 1) vaginal swabs for 
vaginal pH measurement, wet mount, Gram stain Nugent scoring, TV culture, and VMB and 
biofilm assessments at UoL; 2) endocervical swab for NG/CT PCR and HPV typing (if funding 
permits); 3) CVL for additional VMB assessments at UoL. 
6. 
Rapid HIV, pregnancy, urinalysis dipstick, and wet mount (Amsel criteria, Candida and TV) 
testing in the onsite RU laboratory 
7. 
Post-HIV test counselling, provision of rapid test results, and treatment and referrals as needed 
8. 
Participant leaves the RU clinic. She will be told that she will be contacted should any of the 
other diagnostic test results come back positive. 
9. 
Further local laboratory testing:  
− Blood specimen: syphilis and HSV-2 serology; 
− Vaginal specimens and CVL: Nugent scoring, TV culture, initial processing and storage for 
VMB and biofilm assessments at UoL; 
− Endocervical specimen: NG/CT PCR, HPV typing (if funding permits); 
− Urine specimen: Initial processing and storage for urine microbiome assessments in Europe 
(if funding permits). 
 
6.7 Frequent sampling group 
 
During the enrolment informed consent procedures, women will be asked if they are willing to take 
part in the frequent sampling procedures on top of regular study procedures should they be 
selected. Only 3 women per randomisation group (total N=12) will be selected. The actual selection 
of women will be guided by the overall study visit schedule to ensure that the study staff responsible 
for specimen pick-up at the participants’ homes and the laboratory teams can cope with the 
additional workload. Women in the frequent sampling group will undergo regular study procedures, 
but in addition, will be asked to collect two vaginal swabs on Monday, Wednesday and Friday 
morning at home during the first month of the intervention. Sample collection may continue during 
menses. Women will be taught how to self-sample by RU study staff at their enrolment visit. All self-
collection materials will be provided to the participant in such a manner that hygienic storage at 
home is possible. Participants will be asked to store the specimens in a refrigerator or a cool place 
away from direct sunlight. The study team assumes that most women will not have access to a 
refrigerator and will therefore provide storage media that will protect the samples from 
degradation. Specimens will be picked up by study staff, or dropped off by study participants, on a 
weekly basis (or more often if feasible) and will be processed and stored by the RU laboratory 
immediately after receipt. The exact pick-up/drop-off schedule will be negotiated with each 
participant individually to ensure that it is feasible and acceptable to them. 

22 April 2015 
 
21 
 
To preserve confidentiality and prevent social harms, the following procedures will be followed: 
• 
Only women who are enrolled in the study will be asked whether or not they are interested in 
participating in the self-sampling component of the study.  
• 
Only women who are interested in participating in the self-sampling component will be 
considered for selection. 
• 
The selected participants can freely choose their preferred collection method: home visit by 
study staff or sample drop-off. 
• 
Drop-off sites will be selected with the help of our outreach team, using stringent criteria to 
preserve the participant’s confidentiality and ensure easy access. The locations we used in 
previous studies were rooms in health centres or one stop centres close to the participants’ 
homes. 
• 
Home-visits will only be conducted with permission from the participant by our female outreach 
staff who have been thoroughly trained in how to preserve confidentiality. These female staff 
will always be accompanied by a male driver, who has also been trained in how to preserve 
confidentiality. RU cars do not display any logos or project identifiers and RU staff do not wear 
uniforms. 
 
6.8 Unscheduled visits 
 
All participants will be encouraged to visit the RU clinic in between study visits when they experience 
SAEs, AEs that they suspect are product-related, or new episodes of STIs, BV or vaginal candidiasis. 
Study staff may ask participants to return to the RU clinic in between regular study visits if diagnostic 
tests come back positive and require counselling, partner notification, treatment and/or referral. 
Other unscheduled visits may take place at the request of participants or study staff at any time 
during the study. All unscheduled visits will be documented. 
 
6.9 Withdrawal procedures 
 
Women who choose to withdraw during the first 2 months of study participation will be asked if 
they are willing to answer a few questions (focusing on reasons for withdrawal, acceptability of and 
adherence to the intervention, adverse events/social harms and current genital symptoms) and to 
provide vaginal swabs (without insertion of a speculum) for VMB assessments. Women who choose 
to withdraw between the Month 2 and Month 6 visits will first be asked if they are willing to 
complete all Month 6 procedures at the time of withdrawal, and if not, if they are willing to answer a 
few questions and provide vaginal swabs (without insertion of a speculum) for VMB assessments. 
Only the procedures that the participant agrees to will be implemented. 
 
7. STUDY PROCEDURES – SOCIAL SCIENCE 
 
As mentioned in section 4.3 (sample size), a total of about 5 FGDs and 10 IDIs will be conducted to 
address the feasibility objectives. In addition, we expect to conduct quantitative face-to-face 
interviews focusing on BV knowledge with at least 30 women attending recruitment sessions (using 
similar questions that will be included in the screening interview, which will be completed by 
approximately 120 women during screening visits).  
 
At the recruitment sessions prior to the recruitment presentation, women will be asked if they are 
willing to be interviewed for about 15 min about their BV knowledge (the recruitment presentation 
contains explanations about BV and might therefore bias the interview results). If they say yes, study 
staff will explain the interviewing procedures to them, will give them an information sheet, and will 
ask for verbal consent. No identifying or contact information will be recorded, and the interview will 

22 April 2015 
 
22 
be conducted outside of hearing distance of others present at the recruitment site. After the 
interview, the pre-screening eligibility checklist will be administered (again, anonymously) so that we 
know whether the woman is potentially eligible for the trial or not. Only the overall outcome of that 
assessment will be written on the person’s anonymous questionnaire. After the interview, 
participants will be given a small token of appreciation, such as a bar of soap or hand lotion. They 
will not be given a monetary reimbursement. Interviews will be conducted until a total N of 30 has 
been reached. 
 
At screening visits, all women will be asked if they would be willing to participate in FGDs or IDIs at a 
later date should they be selected. Only women who said yes may be approached by study staff. 
Selected women will be asked to provide separate informed consent for each FGD and/or IDI that 
they participate in. All FGD and IDI procedures with study participants will take place at the RU 
research clinic. The FGDs/IDIs on acceptability and adherence will be held with participants who 
have completed their 2-month intervention period, the FGD with the frequent self-sampling group 
after all 12 women have completed the frequent self-sampling procedures, and the other FGDs/IDIs 
at any time during the data collection period. 
 
Approximately 5 IDIs will be conducted with clinicians and health policymakers. The interviewees will 
be selected from among RU’s professional network in Kigali, and an attempt will be made to include 
professionals with different types of relevant expertise. They will be approached by the Principal 
Investigator. Selected interviewees will be asked to provide written informed consent for the IDI. 
The IDI procedures will take place in a private location chosen by the interviewee. 
 
8. STUDY PROCEDURES - LABORATORY 
 
8.1 Diagnostic tests 
 
The following diagnostic tests will be performed in the onsite RU laboratory (or at the National 
Reference Laboratory in Kigali if needed) using validated test kits and standard operating 
procedures: 
− Urine hCG pregnancy test 
− HIV by rapid test algorithm based on the most recent Rwandan HIV voluntary counselling and 
testing guidelines 
− Syphilis by RPR and Treponema pallidum-specific test 
− HSV2 by serology 
− CT and NG by PCR (at the National Reference Laboratory in Kigali) 
− TV by wet mount microscopy and InPouch culture 
− Vaginal candidiasis by wet mount microscopy 
− BV by Amsel criteria and Gram stain Nugent scoring 
− UTI by urinalysis dipstick test 
− If funding permits, the presence of HPV types (and particularly oncogenic HPV types) will be 
determined using molecular methods in a specialised laboratory in Europe. This test is not 
available in Rwanda and does not have any therapeutic consequences for Rwandan women. 
 
8.2 Research tests 
 
Most testing for research purposes will be conducted at UoL, Liverpool, UK. Specimens will be 
processed and stored (at -80°C, or -20°C if -80°C is not possible) at RU and shipped in batches from 
RU to UoL using RU standard operating procedures (which are based on local and international 
shipping regulations) and Material Transfer Agreements. They will be stored at -80°C at UoL 
immediately after arrival until testing.  

22 April 2015 
 
23 
 
- 
DNA will be extracted from vaginal swabs (and/or CVLs), bacterial DNA will be amplified using 
primers directed to highly conserved regions of the 16S rRNA gene, and bacteria will be 
characterised using Illumina MiSeq sequencing of variable regions of the 16S rRNA gene. These 
procedures were adapted from Fadrosh et al (32) and have been successfully used in previous 
studies by UoL. 
- 
If funding permits, individual VMB bacteria (commensals, PPMs, and probiotic strains), TV and 
Candida albicans may be further investigated using qPCR or other molecular methods. 
- 
If funding permits, vaginal fluid on microscopy slides may be evaluated for biofilm. 
- 
If funding permits, CVLs may be used to characterise bacterial metabolism (by assessing the 
presence of bacterial metabolites or proteins in the CVLs) and host immune responses (by 
identifying the presence of cytokines, other inflammatory markers, and human proteins in the 
CVLs).   
- 
If funding permits, urine samples will be analysed by Illumina MiSeq at UoL to characterise the 
urine microbiome and compare this to the VMB of the same women. 
 
9. DATA MANAGEMENT AND ANALYSIS 
 
We will use paper-based source documents and electronic case report forms (eCRFs). All study 
documents will be kept at RU in lockable cabinets and/or rooms for confidentiality reasons. 
Documents containing names and/or signatures will be kept separately from all other study 
documents containing identification numbers. 
 
The electronic study participant identification register (eSPIR; containing participant names, 
identification numbers, contact information, contact attempts, and visit attendance) and clinical trial 
database (containing all CRF data) will be programmed by specialised clinical database programmers. 
Data will be entered into the eSPIR by the RU receptionist when the participant first enters the RU 
research clinic at each study visit. CRF data will be double-entered into the clinical database by RU 
data entry clerks after initial monitoring for CRF completeness and accuracy has been completed. 
FGDs and IDIs will be tape-recorded, transcribed verbatim, translated into English, and analysed in 
Atlas Ti software. The SPIR, clinical database, transcripts, and Atlas Ti data will be kept on password-
protected RU computers and access will be limited to key project staff. Data will be backed-up 
regularly at RU, and will be shared with the UoL Chief Investigator at regular intervals for 
safekeeping at UoL. The RU standard operating procedures related to data entry and management, 
and the UoL data safety and privacy guidelines, will be followed. The molecular VMB data and other 
laboratory research data will be managed separately at UoL. These data will only contain participant 
identification numbers, and UoL laboratory staff will not have access to the SPIR or any other 
personal identifying information. 
 
The analysis of primary and secondary clinical outcomes will be conducted by the UoL Chief 
Investigator or designee(s) in close collaboration with the Principal Investigator. These analyses will 
be conducted in accordance with a priori developed statistical analysis plans, which will be circulated 
to all co-investigators and external advisors for review and input. The feasibility outcomes will be 
analysed by the RU social science team led by Jennifer Van Nuil.  
 
10. ETHICAL ISSUES AND PROCEDURES 
 
10.1 
Ethical review 
 
The protocol will be reviewed by the Principal Investigators, co-investigators, and trial steering 
committee (TSC) members and approved by the Chief Investigator and Principal Investigator. Prior to 

22 April 2015 
 
24 
enrolment of the first participant, the protocol will be approved by the Rwanda Biomedical Centre 
National Health Research Committee (this is an administrative approval, not an ethics approval), and 
two ethics committees: the National Ethics Committee of Rwanda and the UoL Research Ethics Sub-
committee for Physical Interventions (RESPI). 
 
10.2 
Risks and benefits 
 
Physical burdens may include discomfort in the vagina during collection of vaginal and endocervical 
swabs or use of the vaginal products and/or bruising at the site of venipuncture. Psychological 
burdens may include embarrassment during the face-to-face interview and pelvic exam procedures, 
and personal and/or interpersonal stress if HIV infection and/or an STI is diagnosed. Though unlikely, 
a breach of confidentiality is a potential risk. Furthermore, women may have to spend a significant 
amount of time at the study clinic (2-4 hours), especially at the screening and Month 6 visits. 
 
Participants will receive their HIV test results according to the national HIV voluntary counselling and 
testing guidelines. Pregnant women will be referred to an antenatal clinic where they will receive 
more comprehensive care than we can offer them. Although a positive HSV-2 or HPV status does not 
lead to medical intervention, clients who test positive for HSV-2 or high-risk HPV will be informed of 
their result. They will be educated about HSV-2, and told to seek care immediately (as well as use 
condoms) if they experience an active herpes outbreak. They will also be educated about HPV, the 
link between HPV and cervical cancer, and how best to prevent HPV infections. 
 
Benefits for all participants include free HIV counselling and testing, and free diagnosis and 
treatment of several common STIs, BV, and vaginal candidiasis. Participants who suffer from 
recurrent BV may experience clinical benefit from the study interventions. In addition to treatment 
and referral for themselves, participants will be offered partner notification services if they are 
newly diagnosed with HIV or another STI. They will also receive male condoms free of charge 
throughout the study. 
 
10.3 
Compensation and insurance 
 
Women will be compensated in the amount of 3,000 Rwandese francs (the equivalent of 3 GBP) per 
visit at the RU study clinic that is scheduled by study staff. They will not receive monetary 
compensation for unscheduled visits at their own initiative but will receive the benefits described 
under 10.2.  
 
UoL will obtain a no-fault liability insurance covering the entire conduct of the study. This insurance 
will cover the trial participants and study staff for any damage or injury that results from any study-
related activities or procedures.  
 
10.4 
Confidentiality 
 
Every attempt will be made to maintain the confidentiality of study participants. All women will be 
assigned a unique personal identification number for use on all study forms containing interview, 
clinical, and laboratory data. This identification number will be linked to women’s personal 
information in a central registry (the eSPIR). Documents containing the names and/or signatures of 
participants (such as consent forms) will be kept separately from all other study documents 
containing identification numbers only. Each specimen will also be given a unique identification 
number to link them to other laboratory results and questionnaire data of the same person at the 
same visit. In FGDs, participants will be told they can use a name other than their given name for 
confidentiality. Transcripts from the FGDs and/or IDIs will not contain participants’ given names. 
 

22 April 2015 
 
25 
All interviews, counselling procedures and clinical procedures will be conducted in private and study 
staff will be told not to share confidential information regarding study participants with anyone 
outside the study team without a participant’s consent. All completed study forms and documents 
will be kept in lockable rooms and/or cabinets at RU. Care will be taken to maintain confidentiality 
during follow-up, particularly when conducting home visits for participant tracing purposes. Study 
results will be presented as aggregated data, with no personal information. 
 
10.5 
Informed consent procedures 
 
Written informed consent will be obtained from all study participants. The informed consent form 
(ICF) has to be co-signed by a parent or guardian when the participant is aged 18-20 and unmarried 
(by Rwandan law, women aged 18-20 who are legally married are no longer considered legal minors 
and can sign for themselves). Participants with insufficient literacy (as determine by formal literacy 
assessment) can provide a thumbprint but the informed consent process should be observed by an 
independent witness who co-signs the ICF. The witness cannot be a RU staff member, but can be 
another study participant. 
 
Four separate participant information sheets (PIS) and informed consent forms (ICF) will be used: 
one for screening, one for enrolment, one for FGDs and one for IDIs. A fifth PIS will be given to 
women participating in the BV interview at recruitment sites but we will obtain verbal consent from 
them given the anonymous nature of the short interview (15 min) and lack of any other study 
procedures. In the clinical trial (screening and enrolment visits), the informed consent process will 
start with a group educational session, followed by an individual session attended by the participant 
and, if applicable, her parent/guardian and/or her witness. The individual session consists of a 
formal assessment of literacy, a discussion about the study with a research nurse counsellor, a 
formal assessment of understanding of the study, and the actual signing of the consent form. The 
FGDs will only be done with participants who have been enrolled in the trial for a while. Study staff 
will therefore know them well, and may skip the formal assessments of literacy and understanding if 
they feel confident about the participant’s literacy and understanding of the study. In the case of 
IDIs with clinicians and policymakers, the informed consent process will be individual, and will not be 
accompanied by formal assessments of literary and understanding because the interviewees are 
highly educated professionals.  
 
All consent procedures (including the literacy assessment and assessment of understanding) will be 
conducted in the language chosen by the participant and will be completed before study procedures 
take place. In all cases, two copies of the PIS and ICF documents will be provided to the participant 
(and her parent/guardian and/or witness if applicable); they will be asked to leave one fully signed 
copy at RU for the study files and to take one fully signed copy home for their own records. 
 
During the screening and enrolment consent process, participants will be asked to sign separately 
for long-term storage of specimens for the future testing that has already been specified in this 
study protocol (tertiary objectives) or for closely related additional VMB research. Any additional 
research that is not closely related to the study objectives in this protocol would be formally 
approved by the study sponsor and the ethics committees listed in secion 10.1 via a protocol 
amendment prior to initiation of testing. If a participant does not consent to long-term storage, her 
specimens will be destroyed after the primary and secondary study objectives have been achieved. 
 
11. INVESTIGATIONAL PRODUCTS 
 
11.1 Study products 
 
The active ingredient in metronidazole oral tablets and vaginal gel is the antibiotic metronidazole. 

22 April 2015 
 
26 
For the initial treatment of BV and TV, RU uses generic metronidazole oral tablets that are readily 
available in local licensed pharmacies in Kigali (for an example, see the package insert for Eflaron in 
Appendix 15.3). These generic tablets were approved by the Rwandan Government and are stable at 
temperatures up to 30 °C. The 0.75% metronidazole gel MetroGel-Vaginal (Graceway 
Pharmaceuticals, Bristol, TN, USA) is approved by the US Food and Drug Administration (FDA) and is 
also stable at temperatures up to 30 °C (see US FDA Prescribing Information in Appendix 15.3). Each 
gram of gel contains 7.5mg metronidazole, 0.8mg methylparaben, 0.2mg propylparaben, in a gel 
consisting of purified water, propylene glycol, carbomer 934P and edentate disodium. The gel is 
formulated at pH 4.0 and comes in a 70g aluminium tube with 5 disposable applicators. It is 
administered into the vagina using a disposable applicator (which has to be filled by the user), and 
5g of gel is administered with each application. MetroGel-Vaginal is not available in Rwanda and will 
be imported from the USA by RU for use in this study only. It can be used for BV treatment, but as 
described in the introduction, some physicians also use it as a maintenance therapy to prevent BV 
recurrence in the same dosing schedule as we are proposing to use in this study (16, 20). If RU is not 
able to import MetroGel-Vaginal gel, we will use metronidazole oral tablets (the same generic 
tablets as described above) 400-500mg twice per week for 2 months instead. These tablets have 
been approved for prophylaxis of anaerobic infections.   
 
The other two study products are vaginal probiotics. In Europe, vaginal probiotics are typically 
registered as medical devices; this applies to the probiotics to be used in this study as well. The 
Notified Bodies do not consider them medicines because they are applied to a body surface but not 
absorbed by the body. The Dutch Notified Body explains this as follows: ‘The device obtains its 
intended action by enhancing the natural vaginal flora by preventing or alleviating any imbalance in 
this flora. It achieves this principally by promoting the formation of lactic acid, the production of 
hydrogen peroxide, and competition with potential pathogens for space and substrate. This action is 
not considered to be falling under pharmacological, immunological or metabolic means; it does not 
directly act on the human body through the actions mentioned.’ Furthermore, the vaginal probiotics 
to be used in this study only contain bacterial strains with Qualified Presumption of Safety status 
(23), have CE marketing approval in Europe, and are produced in facilities with current Good 
Manufacturing Practice (cGMP) certificates. See Appendix 15.3 for the package inserts. 
 
Ecologic Femi (Winclove Probiotics, Amsterdam, The Netherlands) is a vaginal capsule containing 
multiple lactic acid-producing bacteria (mainly lactobacilli). These bacteria include Bifidobacterium 
bifidum W28, L. acidophilus W70, L. helveticus W74, L. brevis W63, L. plantarum W21, and L. 
salivarius W24. They were selected for their ability to produce lactic acid and hydrogen peroxide, 
adhere to epithelial cells, and inhibit growth of C. albicans. Ecologic Femi was found to be stable at 
25°C and 30°C for up to 2 years. The dosing schedule that we propose to use in this study has been 
approved by Winclove and the Dutch Notified Body. 
Gynophilus LP vaginal tablets (Probionov, Clermont-Ferrand, France) were designed for controlled 
release of lactobacilli in the vagina. Each Gynophilus tablet contains 876.9 mg of Lcr Regenerans, a 
culture of the Lactobacillus casei rhamnosus 35 (Lcr35) strain specially designed for the vaginal 
environment. It has a long history of use in gynaecology (more than 30 years) and an excellent safety 
profile (clinical trials for vaginal use of this strain have been conducted and an Investigator’s 
Brochure is available). Lcr Regenerans’ ability to ferment glycogen to lactic acid is five times superior 
to that of the native Lcr35 strain; 95% of the lactic acid produced by Lcr35 is L-lactate, corresponding 
to the lactic acid naturally present in the vaginal cavity. A tablet of Lcr Regenerans contains not only 
the probiotic strain but its culture medium as well. The tablet disintegrates in the vagina and forms a 
gel. The tablets are stable at 25°C and 30°C for up to 10 months (stability testing of the tablet is 
ongoing and the same strain in a capsule is stable up to 2 years). The dosing schedule that we 
propose is as recommended by Probionov and approved by the French Notified Body. 
 
 
 

22 April 2015 
 
27 
11.2 Drug regulatory authorities 
 
Since all study products are available on the market in Europe (vaginal probiotics), the USA 
(Metrogel-Vaginal), or Rwanda (generic metronidazole oral tablets), no regulatory approval is 
needed for this study protocol. However, the study products are currently not available in Rwanda 
(with the exception of generic metronidazole oral tablets), and we will therefore obtain import 
permits from the Rwanda Minister of Health and the Ministry of Health Pharmacy Task Force prior to 
shipment of the study products to Rwanda. Details about this procedure are available in the RU  
SOP for Investigational Medicinal Product Importation. 
 
11.3 Study product accountability and packaging 
 
Metronidazole oral tablets and vaginal gel, Ecologic Femi capsules and Gynophilus tablets can be 
kept at room temperature, away from direct sunlight exposure Study products will be shipped in 
their commercial packaging from Europe or the USA to Rwanda, and will be kept in the on-site 
pharmacy room at RU after arrival in Rwanda. The temperature of this onsite pharmacy room is 
monitored and recorded every work day. The RU study product accountability SOPs will be followed. 
Participants will receive a sufficient amount of study product from the RU pharmacy to bridge the 
time period between study visits, up to the upper visit window. The study products will be given to 
participants in their original commercial packaging. In addition, a study leaflet with product and use 
information will be provided to each study participant in a language that is understandable to her. 
Participants will be asked to bring the packaging of used products as well as unused study products 
with them to the RU clinic at each study visit. Study staff will record study product dispensed, used 
and returned unused at each study visit. All unused study products that are left-over at study close-
out, as well as the packaging of used products returned to RU, will be incinerated in a Government 
incineration facility in Kigali (see RU SOP for Investigational Medicinal Product Destruction). 
 
11.4 Adherence assessments 
 
Adherence to the study interventions will be assessed at the Day 7, Month 1 and Month 2 visits by 
structured interviewer-administered questionnaire, review of a diary card that the participant will be 
asked to complete in between study visits, review of returned used packaging and unused product, 
and by asking the participant to complete a self-rating adherence scale. These different sources of 
data will be triangulated to arrive at an overall level of adherence for each participant over time. 
 
12. PHARMACOVIGILANCE 
 
12.1 Definition of adverse events 
 
An adverse event (AE) is any untoward medical occurrence, and a social harm any untoward social 
occurrence, in a study participant during study participation, whether or not there is a causal 
relationship with the intervention that the participant received.  
An unexpected AE is an AE, the nature or severity of which is not consistent with the applicable 
product information (Summary of Product Characteristics or Investigator’s Brochure). The final 
determination of whether an AE is unexpected will be made by the Chief Investigator, but the 
Principal Investigator should also be aware of all applicable product information. 
 
12.2 Definition of serious adverse events 
 
A serious adverse event (SAE) is an AE that results in death, is life-threatening, requires or prolongs 
hospitalisation, results in a congenital anomaly/birth defect, or results in persistent or significant 
incapacity or disability.  

22 April 2015 
 
28 
 
An suspected unexpected serious adverse event (SUSAR) is an SAE, the nature or severity of which is 
not consistent with the applicable product information (Summary of Product Characteristics or 
Investigator’s Brochure). The final determination of whether an AE is unexpected will be made by 
the Chief Investigator, but the Principal Investigator should also be aware of all applicable product 
information.  
 
12.3 Assessing AE/SAE severity and relationship to study products 
 
The Principal Investigator is responsible for assessing the severity of AEs/SAEs occurring on 
study. The following criteria will be used: 
• 
Mild: Transient or mild discomfort (<48 hours), no medical intervention/therapy required; 
• 
Moderate: Mild to moderate limitation in activity, some assistance may be needed, no or 
minimal medical intervention/therapy required; 
• 
Severe: Marked limitation in activity, some assistance usually required, medical 
Intervention or therapy required.  
 
The Principal Investigator is responsible for determining the relationship of all AEs/SAEs with study 
product use based on the following criteria: 
• 
Not Related: There is no temporal or causal relationship to the study product administration, 
and the AE/SAE is clearly explained by another cause (concurrent disease, concomitant 
medication, etc.); 
• 
Probably Not Related (Unlikely): There is a temporal relationship to study product 
administration, but there is no reasonable causal relationship, and the AE/SAE is more likely 
explained by another cause (concurrent disease, concomitant medication, etc.); 
• 
Possibly Related: The AE/SAE can be explained by another cause but the possibility of a 
relationship with the study product cannot be ruled out. 
• 
Probably Related: There is a temporal and causal relationship between the study product and 
the AE/SAE, and the AE/SAE is more likely explained by the study product than by another 
cause; 
• 
Definitely Related: There is a temporal and causal relationship between the study product and 
the AE/SAE, and the AE/SAE responds to withdrawal of the study product (and recurs with 
repeat administration of the study product). 
 
12.4 Reporting of AEs/SAEs 
 
All SAEs, whether or not deemed expected or study product-related, must be reported by the 
Principal Investigator to the Chief Investigator within 24 hours using a standardised SAE Notification 
Form by email (j.vandewijgert@liv.ac.uk). The SAE Notification Form should be completed with all 
available information at the time of reporting. The Principal Investigator is required to write a 
second detailed written report after SAE follow-up has been completed. SAE follow-up will be 
considered completed when the participant returns to her usual health or until the Principal 
Investigator does not expect further improvement or worsening of the event. 
 
At study visits, some AEs/SAEs will be reported spontaneously when asking if any medical or social 
problems occurred (unsolicited) and some will be reported only after specific questioning (solicited). 
Participants will be encouraged to contact study staff in between study visits to report SAEs, AEs that 
they suspect are product-related, and new episodes of STIs, BV or vaginal candidiasis. They will be 
allowed to contact study staff for any other AEs in between study visits as well. Treatment, 
counselling, or referral for SAEs, AEs or social harms that are judged possibly/probably/definitely 
related to study participation will be provided by RU staff, to the extent possible, at no cost to the 

22 April 2015 
 
29 
participant. All SAEs and AEs will be followed up until resolution or until the Principal Investigator 
does not expect further improvement or worsening of the event. Should a participant become 
pregnant during the study, she will be followed up by the study team until delivery, and the 
pregnancy outcome will be documented. Any pregnancy outcome that meets the criteria for SAE 
reporting (e.g. congenital anomalies) will be reported as an SAE. 
SAEs and AEs will be reported to the ethics committees listed in section 10.1 in accordance with 
their guidelines. 
 
13. OTHER 
 
13.1 
Funding 
 
This study is funded by the UK Medical Research Council (MRC). The main funding contract is 
between the UK MRC and the UoL IGH (grant number MR/M017443/1) and by subcontract between 
UoL IGH and RU. 
 
13.2 
Study management and oversight 
 
The trial sponsor is the University of Liverpool (UoL) and this protocol and other trial documentation 
was therefore reviewed and approved by the UoL Sponsorship Committee. Overall trial management 
at UoL will be coordinated by the Chief Investigator Prof. van de Wijgert in the Institute of Infection 
and Global Health. She will make use of resources available through the UoL Research Support 
Office. VMB testing will take place at the UoL Centre for Genomics Research. 
 
Trial implementation in Rwanda will be managed by the RU team, led by the Principal Investigator 
Stephen Agaba. He will work closely with the RU community outreach, clinical, laboratory, data 
management, and social science team leaders. All clinical and social science procedures, as well as 
diagnostic laboratory testing, will take place at RU according to RU SOPs and using RU source 
documents.  
 
A Trial Steering Committee was established, but a Data Safety Monitoring Board is not deemed 
necessary. The reason for this is that all products to be used in this trial are available on the market 
in Europe, the USA or Rwanda, and are considered low risk. The Trial Steering Committee will consist 
of the Chief Investigator, Principal Investigator, and external members listed in section 1: Protocol 
Team Roster. The Committee will be coordinated by the Chief Investigator (Prof. Janneke van de 
Wijgert) and will meet once prior to study initiation, once during data collection, and once after 
completion of data collection. The Committee will also be asked to provide input in study documents 
and procedures via email, and to review each SAE as they become available. In case of major safety 
concerns, the Trial Steering Committee Members may request the Chief Investigator to halt 
recruitment of the trial and/or to organize a formal data review of all available safety data. The 
responsibilities of and procedures related to the Trial Steering Committee are described in detail in 
its terms of reference. 
 
13.3 Protocol amendments 
 
This study will be conducted in full compliance with the most recent approved version of the study 
protocol. Protocol amendments will be reviewed and approved by the investigators and ethical 
review committees prior to implementing the amendment, except when necessary to protect the 
safety, rights and wellbeing of study participants. 

22 April 2015 
 
30 
13.4 GC(L)P monitoring 
 
This study will be implemented in accordance with the ICH-GCP and Good Clinical Laboratory 
Practice (GCLP) guidelines (1). We will implement the following monitoring procedures (these are 
described in detail in the study-specific monitoring plan): 
- 
The Chief Investigator will travel to Kigali to conduct the study training and study initiation visit. 
- 
During data collection, daily internal quality control procedures will be implemented in 
accordance with the RU SOP for Data Management and Quality Control (which is based on ICH-
GC(L)P). In addition, RU data staff will conduct monthly 100% source data verification. 
- 
A local independent monitor (Jeanine Nyinawabega) will conduct three monitoring visits during 
the study: approximately 6 weeks, 4.5 months and 8.5 months after the start of data collection.   
- 
The Chief Investigator will travel to Kigali again to conduct the study close-out visit. 
- 
The independent monitor may conduct additional monitoring visits if required. This will be 
decided by the Chief Investigator based on findings during previous monitoring visits. 
 
13.5 Study termination 
 
If the study is prematurely terminated or suspended, the Chief Investigator will promptly inform the 
investigators/institutions involved in the study, the ethics committees listed in section 10.1, the 
Rwanda Biomedical Centre National Research Committee, and the Rwanda Ministry of Health of the 
termination or suspension and the reason(s) for the termination or suspension. The RU Principal 
Investigator is responsible for informing active study participants of study termination or 
suspension. 
 
13.6 Dissemination of results 
 
Study results will be disseminated as follows: 
- To the study participants and community mobilisers via a report-back meeting at RU 
- To the research and public health community in Rwanda via presentations by RU or UoL staff at 
national meetings 
- To the international research and public health community via presentations by RU or UoL staff 
at UoL and international meetings, publications in scientific journals, and publications on the UoL 
and RU websites.  
 
Publications and authorships on publications will be governed by a separate data and specimen 
sharing agreement. The agreement will be written by the Chief Investigator with input from all other 
investigators and external advisors.  
 
13.7 Archiving 
 
Study records will be kept at RU according to the RU SOP for Archiving. RU will retain all study 
records for at least three years after completion of the last study visit. Documentation may not be 
destroyed without written approval from the Chief Investigator.  

22 April 2015 
 
31 
14. REFERENCES 
 
1. International Conference on Harmonisation Good Clinical Practice Guidelines. Accessed at 
http://www.ich.org/products/guidelines/efficacy/efficacy-single/article/good-clinical-
practice.html on 31 December 2014. 
2. Boskey E, Telsch K, Whaley K, et al. Acid production by vaginal flora in vitro is consistent with the 
rate and extent of vaginal acidification. Infect Immun 1999; 67:5170-5. 
3. Angus DC, van der Poll T. Severe sepsis and septic shock. New Engl J Med 2013; 369:840-51. 
4. Melin P. Neonatal group B streptococcal disease: from pathogenesis to preventive strategies. 
Clin Microbiol Infect 2011; 17:1294-303. 
5. Amsel R, Totten P, Spiegel C, et al. Nonspecific vaginitis - diagnostic-criteria and microbial and 
epidemiologic associations. Am J Med 1983; 74:14-22. 
6. Nugent R, Krohn M, Hillier S. Reliability of diagnosing bacterial vaginosis is improved by a 
standardized method of Gram stain interpretation. J Clin Microbiol 1991; 29:297-301. 
7. Leitich H, Kiss H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for 
adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol 2007; 21:375-90. 
8. van de Wijgert J, Morrison C, Cornelisse P, et al. BV and vaginal yeast, but not vaginal cleansing, 
increase HIV-1 acquisition in African women. J Acquir Immune Defic Syndr 2008; 8:203-10. 
9. Wolfe AJ, Toh E, Shibata N, et al. Evidence of uncultivated bacteria in the adult female bladder. J 
Clin Microbiol 2012: 50:1376. 
10. Fredricks D, Fiedler T, Marrazzo J. Molecular identification of bacteria associated with bacterial 
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11. Ravel J, Gajer P, Abdo Z, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad 
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15. APPENDICES 
 
15.1 
Visit flow chart 
 
 
 
 
*Successful BV or TV treatment is defined as oral metronidazole treatment for 7 days, after which 
there was no BV by Amsel criteria and no TV on wet mount. 
**Women may be enrolled when their NG and CT results are not yet known. However, a new 
participant will be randomised for each woman whose screening sample comes back positive for NG 
and/or CT after having been randomised. The NG/CT-positive women will be allowed to complete 
the study but their data may be removed from some analyses. 
 
 
 
Recruitment
Anonymous pre-eligibility checklist
Eligible: Screening visit (N at least 120)
Interview (incl BV knowledge); speculum exam; 
HIV/pregnancy/UTI/wet mount rapid tests and 
STI/Nugent
Treatment based on rapid tests
Eligible: Results visit
Treatment of remaining STIs and BV by Amsel 
criteria if not already treated. Determine 
eligibility; rescreening up to 3x
Max 3 days after successful oral 
metronidazole treatment for BV or TV*: 
Enrolment visit (N=60)
Face to face interview; speculum exam; 
wet mount, Nugent, TV culture, VMB 
assessments prior to randomisation
Randomise to 4 groups (N=4x15); 
initiate interventions
Day 7, Month 1, and Month 2 visits
Face-to-face interview; speculum exam; 
wet mount, Nugent, TV culture, VMB 
assessments
Terminate interventions at Month 2 
Month 6 visit
Face-to-face interview; speculum exam; 
wet mount, Nugent, TV culture, VMB 
assessments plus 
HIV/STI/UTI/pregnancy
Couse out study participation
Not eligible: Anyone 
who did not receive 
successful BV or TV 
treatment  with oral 
metronidazole*, was 
not free of other STIs at 
screening** or 
symptomatic vaginal 
candidiasis or UTI at 
enrolment, or did not 
pass other elig criteria.
Not eligible: HIV-positive or 
pregnant. Take vaginal swabs 
for VMB assessment without 
speculum.
BV knowledge 
interview (N=30) 
regardless of 
eligibility
Approximately 5 FGDs 
and 10 IDIs on 
adherence, 
acceptability, feasibility 

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15.2 
Schedule of assessments 
 
Procedures in clinic during visits 
Screening/
results 
Enrolment 
D7, M1, M2 
M6 
FGD/IDI 
Informed consent 
X 
X 
 
 
X 
Contact information 
X 
X 
X 
X 
X 
Assign PIN 
X 
 
 
 
 
Eligibility checklist 
X 
X 
 
 
 
Randomisation: initiate interventions 
 
X 
 
 
 
Cease interventions 
 
 
X (M2) 
 
 
Face-to-face interview, quantitative 
X 
X 
X 
X 
 
Discussion/interview, qualitative 
 
 
 
 
X 
Assess AEs and social harms 
 
X 
X 
X 
 
HIV pre- and post-test counselling 
X  
 
 
X 
 
HIV/STI/BV risk reduction counselling 
X  
X 
X 
X 
 
Blood collection (10 ml) 
X  
 
 
X  
 
Urine collection 
X  
X 
 
X  
 
Rapid HIV testing (whole blood)1 
X  
 
 
X  
 
Rapid pregnancy testing (hCG urine) 
X  
X  
[X] 
X  
 
Rapid UTI testing (urinalysis dipstick) 
X  
X  
[X] 
X  
 
Speculum exam: visual inspection 
X2 
X 
X 
X 
 
Speculum exam: vaginal pH + wet mount 
(TV, candida, BV by Amsel2) 
X2,3  
X3  
X3  
X3 
 
Speculum exam: vaginal swabs 
X  
X  
X  
X  
 
Speculum exam: endocervical swabs 
X2  
X  
[X] 
X 
 
Speculum exam: CVL 
X2 
X 
X 
X 
 
Provide results and treatment if appl1 
X 
X 
X  
X 
 
Laboratory procedures after visits 
 
 
 
 
 
Blood: syphilis and HSV-2 serology 
X  
 
 
X4 
 
Endocervical swabs: NG/CT PCR 
X  
[X]5 
[X]5 
X  
 
Vaginal swabs: TV InPouch culture 
X  
X 
X 
X  
 
Vaginal swabs: Nugent scoring 
X 
X 
X 
X 
 
Vaginal swabs: store for VMB MiSeq 
X 
X 
X 
X 
 
Laboratory procedures if funding permits: 
 
 
 
 
Endocervical swab: store for HPV 
X  
 
 
X  
 
Vaginal swabs: store for additional VMB 
assessments (e.g. qPCR, biofilm) 
X 
X 
X 
X 
 
CVL: store for additional VMB 
assessments 
X 
X 
X 
X 
 
Urine: store for urine microbiome 
 
X  
 
X  
 
AE=adverse event; BV=bacterial vaginosis; CT=Chlamydia trachomatis; CVL=cervicovaginal lavage; D7=Day 7 visit; FGD=focus group 
discussion; HPV=human papillomavirus; HSV-2=herpes simplex type 2; IDI=in depth interview; M1/2/6=Month 1/2/6 visit; NG=Neisseria 
gonorrhoeae; PIN=participant identification number; qPCR=quantitative PCR; STI=sexually transmitted infection; UTI=urinary tract 
infection; TV=Trichomonas vaginalis; VMB=vaginal microbiome. 
1. By the algorithm recommended in the most recent Rwandan HIV voluntary counselling and testing guidelines 
2. HIV-positive and pregnant women will not undergo a speculum exam unless clinically indicated but they will be asked to donate 
vaginal swabs and urine (left-over from the pregnancy and UTI testing) for VMB and urine microbiome assessments. 
3. BV by Amsel criteria will always be treated during the screening process but will only be treated when symptomatic during follow=up. 
Candida on wet mount will also only be treated when symptomatic. TV on wet mount will always be treated. 
4. For HSV-2 only when negative at baseline. 
5. Endocervical swabs will be tested for NG/CT by PCR at screening and M6, but may also be tested at enrolment and/or D7/M1/M2 if 
deemed necessary for clinical reasons or to accurately interpret the VMB findings. 
 
 

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15.3 Package inserts for metronidazole oral tablets, metronidazole vaginal gel, Ecologic 
Femi, and Gynophilus 
 
Metronidazole oral tablets 
 
  
 

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Metronidazole vaginal gel 
 
 
 
 
 

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Ecologic Femi 
 
 
 

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Gynophilus 
 

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