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Abstract
Introduction Group B streptococcus (GBS), or Streptococcus agalactiae, remains a leading cause of neonatal morbidity and mortality. Canadian guidelines advise universal maternal screening for GBS colonisation in pregnancy in conjunction with selective antibiotic therapy. This results in over 1000 pregnant individuals receiving antibiotic therapy to prevent one case of early-onset neonatal GBS disease, and over 20 000 pregnant individuals receiving antibiotic therapy to prevent one neonatal death. Given the growing concern regarding the risk of negative sequela from antibiotic exposure, it is vital that alternative approaches to reduce maternal GBS colonisation are explored.
Preliminary studies suggest some probiotic strains could confer protection in pregnancy against GBS colonisation.
Methods and analysis This double-blind parallel group randomised trial aims to recruit 450 pregnant participants in Vancouver, BC, Canada and will compare GBS colonisation rates in those who have received a daily oral dose of three strains of probiotics with those who have received a placebo. The primary outcome will be GBS colonisation status, measured using a vaginal/rectal swab obtained between 35 weeks’ gestation and delivery. Secondary outcomes will include maternal antibiotic exposure and urogenital infections. Analysis will be on an intention-to-treat basis.
Patient or public involvement There was no patient or public involvement in the design of the study protocol.
Ethics and dissemination This study protocol received ethics approval from the University of British Columbia’s Clinical Research Ethics Board, Dublin City University and Health Canada. Findings will be presented at research rounds, conferences and in peer-reviewed publications.
Trial registration number NCT03407157.
- Clinical Trial
- INFECTIOUS DISEASES
- Microbiology
- OBSTETRICS
- NEONATOLOGY
- BACTERIOLOGY
- Streptococcus agalactiae
- Pregnancy
- probiotics
- Infections disease
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- Clinical Trial
- INFECTIOUS DISEASES
- Microbiology
- OBSTETRICS
- NEONATOLOGY
- BACTERIOLOGY
- Streptococcus agalactiae
- Pregnancy
- probiotics
- Infections disease
STRENGTHS AND LIMITATIONS OF THIS STUDY
This study is a randomised, placebo-controlled trial, which is considered the ‘gold standard’ for testing treatment efficacy.
Both participants and researchers will be blinded to allocation arms to minimise bias and increase internal validity.
Pregnancy results in a significantly altered metabolic, immunological and hormonal state which may influence probiotic mechanisms of action. Our analysis will include strain-specific detection of each of the probiotics in the vaginal epithelium which will add to the body of literature on the influence of pregnancy on probiotic oral translocation and vaginal epithelium adhesion.
Sample is limited to one urban area which may limit generalisability.
Sample size may not be sufficient to allow for analysis for all subgroups.
Background and rationale
Group B streptococcus (GBS; Streptococcus agalactiae) is a beta-haemolytic gram-positive bacterium that often colonises the gastrointestinal and/or lower genital tract of humans of all ages. Although colonisation is most frequently benign, it can result in illness including invasive disease, particularly in the elderly and the young.1 Maternal GBS colonisation is present in 10%–35% of pregnancies and can result in maternal infection, stillbirth, and, most notably, is the leading cause of infectious neonatal sepsis and meningitis.2 Approximately 50% of infants born to those colonised with GBS will become colonised themselves. Most infants remain well; however, with no intervention, 1%–2% of colonised infants will become ill with GBS disease (GBSD), classified as early-onset (EOGBSD) when it manifests within the first week of life, or late-onset (LOGBSD) when it presents between day 7 and 3 months of age.1
Neonatal and infant GBSD is a worldwide concern and it is estimated that more than 300 000 cases occur annually resulting in more than 90 000 neonatal deaths.2 For survivors, long-term moderate to severe neurodevelopmental deficits affect more than 50% of those with a history of GBS meningitis.3 4 Under-resourced countries bear the greatest burden of neonatal and infant GBSD.3 However, even in resourced countries such as the USA and Canada, GBS remains a leading cause of neonatal and infant morbidity and mortality.5
The primary risk factor for EOGBSD has been well established to be maternal anogenital colonisation with transmission to the fetus/neonate occurring shortly before or during labour and delivery.6 EOGBSD is associated with significant neonatal morbidity including sepsis (71%), pneumonia (19%) and meningitis (11%). The mortality rate is also significant at 20%–30% for infants born prematurely (<37 weeks’ gestation) and 2%–3% in full-term infants (≥37 weeks’ gestation).1 Risk factors for LOGBSD are less clear but may include maternal anogenital colonisation, transmission via breastmilk,7 nosocomial transmission, particularly within the neonatal intensive care unit (NICU) environment8 and transmission from other GBS colonised individuals such as close family members.4
The standard of care in Canada at the time this protocol was finalised was to offer all pregnant individuals culture-based screening for GBS via an anogenital swab between 35 and 37 weeks’ gestation (universal screening). Intrapartum prophylactic antibiotics (IPA) is recommended in the intrapartum period for those who screen positive for GBS colonisation. If GBS status is unknown, IPA is recommended in the presence of preterm labour, preterm premature rupture of membranes (PPROM), prolonged rupture of membranes ≥18 hours or maternal fever in labour ≥38°C, all of which are increased risk factors for EOGBSD. Additionally, IPA is recommended regardless of colonisation status for those with a previous infant diagnosed with GBSD and those with GBS bacteriuria at any point in their current pregnancy.5
In settings that provide universal screening and IPA for GBS, the burden of EOGBSD has decreased5 9; however, the high numbers needed to treat to prevent GBS-related neonatal morbidity and mortality have created growing concern. Over 1000 pregnant individuals are needed to be treated with IPA in order to prevent one case of EOGBSD,10 11 and over 20 000 pregnant individuals are needed to be treated with IPA to prevent one death from EOGBSD.12 Maternal treatment with IPA simultaneously exposes and treats the fetus/neonate.13 As such, the above numbers needed to treat doubles to over 2000 to prevent one case of EOGBSD and over 40 000 to prevent one neonatal death from EOGBSD.
Antibiotic exposure carries in itself the potential for serious maternal14–19 and neonatal morbidity14 20–23 and is associated with a continued increase in antibiotic resistance.14 24
Emerging evidence shows that the neonatal microbiome is disrupted by perinatal exposure to antibiotics,25–27 and that this may be associated with poorer long-term health and neurodevelopmental outcomes,28 29 and increased risk of childhood diabetes, asthma, obesity and autoimmune disorders.30–34 Given the unintended maternal and infant consequences of antibiotic exposure, there is a growing interest in finding stratagies to reduce their use in pregnancy.
Probiotic supplements are preparations that contain viable, microbial agents that have been demonstrated to improve health.35 Typically, these products will contain freeze-dried (lyophilised) or live bacteria or yeasts. Probiotics confer health benefits through several mechanisms, including maintaining homeostasis of gut bacteria, acidifying mucosal surfaces and preventing pathogen adherence.36 Probiotics have been used to enhance and manipulate the human microbiome to reduce a wide range of communicable and non-communicable diseases.
At the time this protocol was finalised, probiotics had been studied extensively and considered safe and well tolerated in the pregnant population, with the exception of the severely immune compromised.37 Subsequent literature is providing increased detail on the efficacy and safety of probiotics in pregnancy. Examples include a systematic review and meta-analysis of 100 studies that found no mortality or serious adverse outcomes associated with the ingestion of probiotics in pregnancy.38 Conversely, a meta-analysis of seven studies on the effects of probiotics on gestational diabetes found an increased risk of preeclampsia in obese individuals (body mass index (BMI) >30).39 The strains used in our study are Health Canada approved, commercially available and do not list pregnancy as a contraindication for use.
Three probiotic strains were selected for this trial: Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC-14 and Streptococcus salivarius K12 based on research that suggests each has the potential to provide protective, inhibitory and/or antibacterial properties against GBS through a series of barriers, including adherence, self-aggregation, co-aggregation and interference via receptor binding and blocking mechanisms, and displacing pathogens. L. reuteri GR-1 and L. rhamnosus RC-14 are one of the most widely studied probiotic strains for urogenital health.40–42 There is clinical evidence of vaginal epithelial adhesion following oral administration resulting in a resurgence of commensal vaginal bacteria and a decrease in urinary tract infections (UTIs), bacterial vaginosis (BV) and candida.43 44 This is pertinent as GBS vaginal colonisation is found to be more prevalent in those with urogenital infections.45–47 Additionally, a small number of randomised trials have shown promise for the efficacy of L. reuteri GR-1 and L. rhamnosus RC-14 to reduce GBS colonisation in pregnancy and each reported no adverse outcomes. Statistical significance was reached in the study by Ho et al.48 In that study, 99 GBS positive participants, 42.9% of the probiotic arm converted to GBS negative status following an average of 3 weeks of third trimester intervention (two capsules daily) versus 18% in the placebo arm (p=0.007). Sharpe et al’s study with 113 participants with unknown status, although not statistically significant, showed a trend towards efficacy with a 15.8% GBS positive rate following 12 weeks of intervention from 23 to 25 weeks’ gestation (two capsules daily) in the probiotic arm versus 21.43% in the placebo arm (p=0.48).49 Two other trials failed to find a difference in GBS colonisation rates50 51; however, each only used one capsule a day. Despite no difference in GBS colonisation rates, Olsen et al did find a significant increase in vaginal commensals as a secondary outcome in those that had completed 14 days or more of supplementation. The authors questioned if a longer duration of probiotics and/or increased dosage would have had better efficacy in reducing GBS colonisation.50
S. salivarius has not yet been studied in pregnancy for GBS but it has been shown to secrete bacteriocin-like substances that have antibacterial effects against GBS52 and has also demonstrated the ability in a murine model to adhere to the vaginal epithelium.52 In addition, S. salivarius K12 has been shown to have antibacterial effects that eliminate pathological streptococcus in the oral cavity.53
Objectives
Our primary hypothesis is that those who take a daily oral supplementation of the study probiotics taken in pregnancy from 25 weeks’ gestation until delivery will have a lower incidence of maternal positive GBS colonisation status between 35 weeks’ gestation and delivery compared with those who take a placebo.
Our secondary research questions are to explore if participants taking a daily supplementation of the study probiotics will have a reduced incidence of maternal antibiotic exposure and urogenital infections (UTI, BV, candida) after 27 weeks’ gestation through 4–6 weeks postpartum compared with those who take a placebo.
Trial design
This is a double-blind, parallel group, placebo-controlled trial. Participants who consent to participate in the trial will be randomly allocated to the probiotic or the placebo group using a computer programme (Integrated System for Trial Allocation and Randomisation (iSTAR)) in random blocks of 2 and 4.
Methods: participants, interventions and outcomes
Study setting
Healthy pregnant individuals registered to deliver at either St. Paul’s Hospital or BC Women’s Hospital, Vancouver, British Columbia, Canada, and under the care of a regulated maternity care provider (midwife, obstetrician or family doctor) will be invited to participate in the study (see figure 1, study flow diagram). Those registered but who ultimately deliver outside of either hospital will still be included in the study. Study staff will meet with participants to explain the study and review participant procedures at their choice of designated study sites located at participating clinician offices and/or onsite at the BC Children’s Research Institute.
Study flow chart. For those assessed eligible, consent will be obtained between 23 and 25 weeks gestation. Participants will be blindly randomised via a computer-generated programme to either probiotic (n=225) or placebo (n=225). Participants will take their daily study supplements from 25 weeks gestation until infant delivery. Study swabs will be obtained between 23 and 25 weeks gestation (1 vaginal/rectal, 1 oral), 29–33 weeks gestation (1 vaginal/rectal) and 35 weeks gestation and delivery (1 vaginal/rectal, 1 oral). Participant questionnaires will be completed between 23 and 25 weeks gestation, 29–33 weeks gestation and 4–6 weeks postnatal. A telephone check-in with participants will take place at 27 weeks. Data will be collected from participant questionnaires and medical charts. Analysis will be by intention to treat. ITT, intention to treat; PP, per protocol.
Eligibility criteria
To be eligible for inclusion in the study, individuals must be over the age of 18, pregnant with a singleton fetus, at gestational between 23 and 25+0 weeks at consent, be registered for delivery at one of the participating centres and be under the care of a regulated maternity care provider. Individuals will be excluded from enrolling if they are unable to provide consent, have a fetus with known major anomalies, have significant immunosuppression, type I or type II diabetes (non-gestational), a previous infant with GBS (they will automatically be considered GBS positive for their pregnancy and advised to be treated with intravenous antibiotic therapy), GBS bacteriuria diagnosed in present pregnancy, plan to use oral or vaginal probiotic supplementation/therapy (capsules/tablets/lozenges/drinks) during their pregnancy (outside of dietary sources such as yoghurt, kimchi, kombucha), if they are enrolled in another study that involves the administration of a drug/product, or if they choose not to consume dairy products.
Interventions
Participants in the treatment group and the control group will undergo the same procedures in terms of initial assessment, obtaining consent, provision of products (probiotic or placebo), obtaining study swabs and data collection. Once assessed to be eligible and consent is obtained, participants will be admitted to the study between 23 and 25 weeks’ gestation and randomised to the treatment group or the control group. They will receive their unique study ID number and be given study supplements (placebos or probiotics). If required, additional supplements will be dispensed during participation.
Participants randomised to the treatment group will receive a combination of three probiotic strains as follows: capsules containing 2.5 billion colony forming units (CFUs) each at the time of manufacturing of L. rhamnosus GR-1 and L. reuteri RC-14 (Urex), and lozenges each containing 2.5 billion CFU at the time of manufacturing of S. salivarius K12 (Blis K12). Aside from the exclusion of the probiotics, the placebo ingredients will be the same except for the non-medical addition of Silicone-dioxide in the capsules. The placebos for each product will look, taste and be packaged identically to their counterparts.
Participants will be instructed to ingest two capsules per day and one lozenge per day. The lozenge is recommended to be consumed in the evening after oral hygiene care (eg, brushing teeth) and participants are asked to avoid drinking fluids or eating for as long as possible afterwards. If randomised to the treatment arm, this will allow for optimal seeding of the probiotic within the oral cavity. Participants can take the capsules at the same time or staggered in the day, with no restrictions on food or liquid.
The probiotics have a 2-year shelf life from the time of manufacturing. Over time, and if the product is stored in an unstable environment, viability may be reduced. Participants will be advised to keep study capsules and lozenges in a cool environment, optimally refrigerated. All study capsules and lozenges will be stored in a temperature-monitored refrigerator prior to being dispensed to participants.
The probiotics and placebos are being supplied by two companies, each with a strong history of product quality, manufacturing and distribution. The probiotics to be used in this study are approved by Health Canada and do not state pregnancy as a contra-indication for use. The lactobacilli are supplied by Chr. Hansen, a Denmark-based global bioscience company and the owners of one of the world’s largest commercial collections of bacteria. The supplier of the S. salivarius K12, Blis Technologies, solely manufactures and markets S. salivarius for which they were granted Generally Recognised as Safe status in 2011 by the US Food and Drug Administration.
Individual participants can withdraw at any point of the trial by personal choice, if they develop a serious adverse event (SAE) found to be directly related to taking the probiotic/placebo, if they are diagnosed with an immune condition requiring immune-suppressant drug therapy or if they are directed by a treating clinician to withdraw from the study for medical reasons. Data collected from any participant who is consented and randomised but prematurely withdraws from the study will be incorporated and analysed as per intention to treat (ITT).
The trial may be discontinued if there is concern that the intervention is creating harm, product(s) are compromised (without the ability to replace) or if the trial becomes unviable for any reason.
We do not anticipate that we will encounter serious, unexpected or unanticipated AEs in relation to the study supplements. The most common reported side effect of oral probiotic consumption is mild digestive symptoms such as increased flatulence.54 Participants will be informed that if this should occur, it will most likely resolve within a few days with the continued use of their supplement.
Some participants may find it inconvenient to follow the instruction to not eat or drink fluid for as long as possible after sucking their lozenge. If this is the case, they will be advised that they may eat or drink if they experience discomfort or distress from not doing so and that this would not exclude them from the study.
A supplement dose will be considered missed if not taken within a 12-hour time frame of the usual dosing time. Participants will be advised to leave any missed doses in the supplement packaging that will be returned to the study team as per the study procedures.
Participants will be encouraged to download and use the Medisafe app55 or an equivalent which will send regular reminders to take their medications/supplements. Alternatively, a paper calendar will be provided to track taking their daily doses of supplements.
Participants will be asked questions about their compliance at their 27-week phone call check-in and in questionnaires at the study midpoint and at study completion.
Participants will be asked to refrain from ingesting other probiotic supplements while participating in the study and if they are prescribed antibiotics, to take their supplements at least 2–3 hours before or after their antibiotic.
Participants will be asked in questionnaires about all prescribed and over the counter medications, supplements and herbal remedies taken during the trial and information on medications prescribed will be taken from participants’ records during the chart audit.
Outcomes
The primary outcome is GBS vaginal/rectal colonisation status ascertained through qPCR analysis of their study vaginal/rectal swab taken between 35 weeks and delivery.
Secondary outcomes include the incidence of maternal antepartum, intrapartum and postpartum antibiotic exposure and maternal urogenital infections (specifically UTIs, BV and candida). Previous research has shown efficacy with L. reuteri RC-14 and L. rhamnosus GR-1 in prevention and/or reduced recurrence of urogenital infections within the non-pregnant population.56 Urogenital infections such as candida may be an independent risk factor for GBS colonisation in pregnancy46; however, pregnancy results in an altered metabolic and immunologic state,57 58 which may impact probiotic mechanisms of action and the positive outcomes seen in the non-pregnant population. We will report the number of incidences of each that occur two or more weeks following the start of the intervention. These data will be collected through questionnaires completed by participants at intake, at 29–33 weeks and after delivery, and from a review of participants’ medical records.
Information will also be collected on maternal and neonatal conditions including gestational diabetes, preterm labour, preterm and pre-labour rupture of membranes, chorioamnionitis, maternal BMI, infant birth weight, prepartum and postpartum depression, APGAR (Appearance, Pulse, Grimace, Activity, Respiration) scores at 1, 5 and 10 min, (direct) infant exposure to antibiotics, admission to NICU and early-onset neonatal GBS infections. These data will be collected from a review of participants’ medical records and through questionnaires completed by participants at intake, at 29–33 weeks and 4–6 weeks postpartum.
Other outcomes in the analysis will include the presence of the three probiotic strains (L. rhamnosus GR-1, L. reuteri RC-14 and S. salivarius K12) in swabs taken over the course of the study to indicate the ability of ingested probiotics to survive the gastrointestinal tract and adhere to vaginal epithelial tissue within the altered pregnant state. Additionally, oral swabs obtained from participants pre-intervention (between 23 and 25 weeks’ gestation) and again between 35 weeks’ gestation and prior to delivery will be analysed using qPCR for the presence of GBS. Streptococci has been found to be the dominant micro-organism in the human oral cavity.59 The Human Microbiome Project found the transfer of oral bacteria to the gut is common60; however, little research has specifically investigated the colonisation rates of oral GBS and in particular, investigated if the oral presence of GBS reflects intestinal and genitourinary tract GBS colonisation. A study that included 90 pharyngeal cultures from healthy subjects found 11% were positive for GBS.61 Data collected from the oral swabs that are specific to GBS colonisation in the oral cavity will be compared with the vaginal/rectal GBS status of participants to help ascertain whether there may be a correlation between oral and vaginal GBS colonisation. The result of the oral swabs will not designate a participant as being GBS positive or negative in relation to their vaginal/rectal colonisation.
Participant timeline
The participant timeline is set out in table 1. Those interested in participating will be screened between 23 and 25 weeks for eligibility. If they are eligible and provide informed consent, they will be randomly allocated to the probiotic or placebo group. They will be provided with the study pack, supplements (probiotics or placebos) and swabs. Participant information will be set up on the online data collection platform, Research Electronic Data Capture (REDCap). Participants will complete the first questionnaire, provide oral and vaginal/rectal swabs between 23 and 25 weeks and begin to take their daily study supplements at 25 weeks. At or around 27 weeks’ gestation, participants will be contacted by a member of the research team by telephone to enquire how participation is going and compliance, and to answer any questions. They will meet with a member of the study team in person at 29–33 weeks, complete their second questionnaire and provide a vaginal/rectal swab. Between 35 weeks and delivery, participants will again provide an oral and a vaginal/rectal swab. The chart audit will then be completed using the information obtained from participant questionnaires and from hospital and clinician medical records.
Timeline for enrolment, intervention and assessments
Sample size
The prevalence of GBS positivity in 2015 among vaginal births in British Columbia was 21%.62 To detect an absolute decrease of 10% (from 21% to 11%), with 80% power and a type I error of 0.05, two-sided, our trial will require 225 participants in each trial arm, assuming a 10% dropout rate. Small trials of probiotics to date have been able to demonstrate a decrease in colonisation between 6% and 24%.48 49
Recruitment
The study will be publicised widely within the community using posters, brochures, local newsletters and a study website (https://www.opsipstudy.com/). Maternity care providers will be made aware of the study through presentations at individual department meetings and hospital rounds. Midwives, family doctors and obstetricians will be involved in the recruitment of participants and are represented on the research team.
Assignment of interventions: allocation
Sequence generation
Randomisation will be securely and centrally controlled using the web-based computerised randomisation platform iSTAR developed and run by the University of British Columbia’s PREempt team (PREgnancy Evidence, Monitoring, Partnerships and Treatment). iSTAR is a mobile-friendly and streamlined programme that integrates screening, randomisation, product dispensing, bottle management and reporting and validation tools. Eligible participants with written consent will be randomised via iSTAR using block sizes of 2 and 4 to either the probiotic or placebo arm.
Concealment mechanism
iSTAR will generate a unique study ID number for each participant with allocation arms only accessible by the iSTAR data manager.
Implementation
Study team members include the OPSiP clinical lead/coordinator, research assistants and study co-investigators. A study team member will review the study procedures and consent form with potential participants. If deemed eligible and with consent, a study team member will enter participant information into iSTAR. iSTAR will screen and confirm eligibility, randomise, generate the participant ID number and allocate the bottle numbers for dispensing. A study team member will print bottle labels as generated by iSTAR and dispense the study supplements to the participant.
Assignment of interventions: blinding
Who will be blinded?
Study participants, care providers, study team members and data analysts will be blinded to the allocation of participants. Allocation will be concealed until all participant swabs have been analysed and data entry is completed for all participants.
Procedure for unblinding if needed
Should there be an immediate need for a treating care provider to know a participant’s intervention allocation to ensure participant safety the facility to perform emergency unblinding will be available 24 hours per day. If possible, it is recommended that a co-investigator contact the principal investigator of the study to discuss the situation before breaking the participant’s randomisation code. Separate step-by-step guidelines on the unblinding process will be supplied to all centres and will be covered in initiation training.
Data collection and management
Plans for assessment and collection of outcomes
Data will be collected from participants at entry to the trial (23–25 weeks’ gestation, time 1), at 29–33 weeks’ gestation (study mid-point, time 2) and following delivery (time 3). Sources of data will include an initial assessment conducted by the study coordinator or research assistant at time 1, self-completed questionnaires at times 1, 2 and 3, oral swabs taken at times 1 and 3, and vaginal/rectal swabs at times 1, 2 and 3. Data will be collected from participants’ medical records following delivery (4–6 weeks postnatal, time 4) to corroborate, clarify or fill gaps in participants’ reports.
Data will be collected from all sources on the following:
Participant baseline characteristics, including parity.
Pregnancy history including incidence of UTIs, BV infections or vaginal yeast infections; antenatal antibiotic exposure and reason, maternal antibiotic exposure in labour, maternal antibiotic exposure postpartum and history of mental illness (depression).
Pregnancy outcomes including gestation at delivery, preterm rupture of membranes, place and type of delivery, care providers and perineal/vaginal/cervical tears.
Newborn outcomes including birth weight, infection, antibiotic exposure, APGARs and newborn intensive care unit admissions.
Participant-completed questionnaires will be used to collect data on demographics, relationship status, type of care provider (midwife, obstetrician, family doctor), history and impact of past GBS positive status, consumption of probiotic-rich foods/drinks, use of prescription, over the counter and/or natural remedies, occurrence of urinary or vaginal infections, perceived negative and positive side effects from probiotic supplements, compliance in taking the supplements, perception/impact of term GBS status, willingness to continue with the study, willingness to be contacted/enrolled in future studies and open comments/suggestions.
Plans to promote participant retention and complete follow-up
At the first visit with the research team, participants will be provided with a study pack. This will contain detailed information on the study, a timeline based on their expected due date that sets out what is required each week, instructions for each task required, information on their mid-point in-person meeting and contact details should they have any questions. Participants will be contacted by phone approximately 2 weeks into the study for a check-in. Participants will also be encouraged to contact the study team if they have any questions or concerns at any time. If participants withdraw from the study, data provided up to that time will be retained and included in the analysis.
Data management
The data for each participant will be handled in accordance with local regulatory legislation and Ethics Committee approval. REDCap will be used to record data relating to each participant on an electronic Case Record Form (eCRF). All data will be entered directly into the eCRF by the designated research staff at participating centres. Participants will be prompted to complete the three questionnaires at the appropriate time via an online interface with REDCap and data will be captured in their eCRF. Paper questionnaires will also be available for participants who do not have access to the internet and data will be entered from them by a member of the research team into their eCRF.
Data will be examined prior to data analysis, including range checks, and checks of consistency on data items obtained from more than one source.
Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use
Oral and vaginal/rectal swabs will be collected from participants. Participants will be provided with their study swabs and given detailed verbal and written instructions for taking the samples. For those not comfortable collecting the samples themselves, they may request a study clinician or their primary provider to do so. Once obtained, participants will be asked to return the samples to one of the collection study sites within 12 hours of taking the sample. The samples will be stored in a fridge until collected by a study team member and then stored securely at the OPSiP offices in a −80°C freezer to await analysis.
Analysis of the swabs will include qPCR and CFU counts of GBS and the three study strains and will be undertaken by a skilled laboratory technician. Primers and probes will be developed to match the specific strains of interest. Clear standard operating procedures will be developed for the analysis processes to ensure consistency and repeatability.
Statistical methods
Statistical methods for primary and secondary outcomes
Data will be exported from REDCap to SPSS V.27.0 (or an equivalent) for analysis. We will describe the characteristics of the sample using mean, SD and 95% CI for normally distributed variables, and median and IQR for non-normal distributions, and number and percentage for categorical variables. Hypothesis testing will use an ITT approach with a two-sided 0.05 significance level. For the primary outcome (GBS positive or negative at term) and other categorical variables, we will report relative rates and absolute risk differences and their 95% CIs, as estimated using log binomial regressions. For continuous outcomes, we will perform independent t-tests and treatment effect will be expressed as the mean difference with 95% CIs. Changes in scores on standardised scales will be assessed adjusting for baseline values using an analysis of covariance (ANCOVA) approach. To adjust for prognostic variables not balanced at baseline, we will undertake multivariable log-binomial regression for our binary outcomes and general linear regression modelling for continuous outcomes. We will perform a sensitivity analysis to account for missing outcome data using multiple imputations.
Interim analyses
There is no plan to conduct an interim analysis. However, an interim analysis may be considered if there is any concern that the intervention is creating harm, if there is any concern about whether the trial should continue or if the trial becomes unviable for any reason. An interim analysis will only be conducted following discussion and agreement from the principal/co-principal investigators.
Methods for additional analyses
We will undertake a subgroup analysis according to antibiotic use during pregnancy as antibiotic exposure may mitigate the effectiveness of the probiotics. We will examine subgroups by BMI status as pregnant individuals with obesity may have a higher likelihood of being GBS positive.63 Sufficient numbers permitting, we will also undertake a subgroup analysis according to the mode of delivery (caesarean vs vaginal birth) as caesarean birth does not prevent ascending colonisation occurring during pregnancy.64
Additional analyses will be used to examine the treatment effect by reported compliance with study supplements (per protocol analysis), treatment effect adjusted by intake GBS status (prior to beginning the intervention), to assess the potential impact of established versus new colonisation after initiation of supplements and treatment effect adjusted for use of other dietary probiotic sources.
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data
Where there are missing data, treatment effect will be imputed using the last observation carried forward.
Plans to give access to the full protocol, participant level-data and statistical code
Three years following the publication of results, the full deidentified dataset will be made available, pending approval from the research ethics committee. There are no contractual limitations to making such data available.
The study brochure and poster will be available for download on the study website. A watermarked consent form which details participant procedures and study details will also be available on the study website.
Oversight and monitoring
Composition of the coordinating centre and trial steering committee
The clinical lead will be responsible for the day-to-day coordination of the study, including publicising the study and meeting with clinicians and other interested parties to promote their support of the study. The clinical lead and study staff will be responsible for assessing participants for eligibility, discussing the details of the study with participants, obtaining consent, administering the study pack and study supplements (probiotics/placebos—blinded), overseeing data collection and arranging to pick up samples, conducting chart audits and responding to participant queries. The Trial Steering Group (comprising the principal and co-principal investigators, co-investigators and the trial coordinator) will meet regularly to oversee all aspects of the study, monitor progress, address any issues that may arise and obtain all necessary approvals.
Composition of the data monitoring committee, its role and reporting structure
A data safety monitoring board (DSMB) will be established to monitor the trial and review the study progress. The DSMB will receive bi-monthly reports pertaining to any AEs and meet once halfway through trial recruitment to review the safety data and monitor the progress of the trial, or sooner if warranted. The board members will be independent of the trial and free of conflicts with any of the investigative team.
AE reporting and harms
Data will be collected on any AEs that occur from the time of randomisation to 28 days of discontinuing the study supplements. Several means will be in place to capture any AEs and include healthcare provider notification, participant notification and semi-weekly chart audits. The letter provided to participants’ maternity care providers will include instructions on how to notify the study team of any AE. Participants will be advised to keep their study participation card with them and if seen by a healthcare provider outside of their primary maternity care provider to alert them to their participation in the study. Their study card will have contact information for that provider to report an AE. All participant admissions to their registered hospital of delivery will be captured by semi-weekly chart audits. All participants will be able to contact a member of the study team by phone and/or email to report an AE. Participants will also enter any AE into their mid-point and post-delivery questionnaires.
Data on AEs will be reviewed and recorded to ascertain if they may be related to the study supplements and reported accordingly. The clinical lead in conjunction with the qualified investigator will be responsible for determining whether an AE is expected or unexpected. An AE will be considered unexpected if the nature, severity or frequency of the event is not consistent with the risk information previously described for the intervention. Data will be collected and reviewed for the following AEs: preterm birth, PPROM, fetal anomalies (diagnosed after randomisation), fetal demise, stillbirth, chorioamnionitis, neonatal infection (including EOGBSD) and neonatal death. AEs will be categorised according to the likelihood that they are related to the study intervention.
The clinical lead will contact the primary investigator within 3 days of being made aware of any AE. Any AEs deemed to be severe and related to the study intervention will be notified to the DSMB and the Research Ethics Board (REB) within 24 hours. If any unexpected SAE occurs that is fatal or life threatening, Therapeutics Products Directorate will be notified no later than 7 days after the sponsor becomes aware of the information.
If any other unanticipated problem, as defined by the University of British Columbia (UBC) ethics board, is identified, it will be reported promptly to the REB, no later than 7 days after identification.
Frequency and plans for auditing trial conduct
The trial sponsor will be provided with annual updates on trial progress and annual updates and renewals will be submitted to the REB.
Plans for communicating important protocol amendments to relevant parties (eg, trial participants, ethical committees)
Changes were made to the original protocol following feedback from the Research Ethics Committee and Health Canada. In addition, several changes were made to the protocol in 2020, with the outbreak of the COVID-19 pandemic. New information and consent forms were prepared to reflect the changes and these and all changes were approved by the Research Ethics Committee.
No deviation from the protocol will be implemented without the prior review and approval of the health ethics board(s) except where it may be necessary to eliminate an immediate hazard to a research subject. In such case, the deviation will be reported to the health ethics board(s) as soon as possible.
Ethics and dissemination
Research ethics approval
This protocol was approved by the UBC Children & Women’s Research Ethics Board (ref: H17-02189) and further ratified by the Research Ethics Committee, Dublin City University (ref: DCUREC/2018/124). It was also approved by Health Canada (Natural and Non-prescription Health Products Directorate) and was registered on Clinicaltrials.gov.
Consent or assent
Individuals will be fully informed about the study and what participation entails in written materials and through a verbal discussion with a member of the research team before providing written consent to participate. They will be informed that they may withdraw from the study at any time and that their care will not be affected whether or not they choose to participate.
For those that meet eligibility criteria, a study team member will review the consent form with the participant to explain the study and participant procedures and allow the participant time for full consideration and consultation as required.
Consent for publication
Participants will be asked for their consent for findings to be published as deidentified and aggregated data.
Confidentiality
Participants will be identified by a unique participant ID number. Any personal identifiers will be kept within secure premises and secure systems. Personal identifiers will not be accessed remotely except through encrypted devices or if the device is held securely at the research site. Downloaded material will be deleted as soon as no longer required.
If eCRFs are transferred or stored offline using removable media (including laptops, portable hard drives and USB key drives), they will be encrypted with a password. The transferred material on the removable media will be deleted as soon as the data transfer is successfully completed. The investigators and iSTAR management will also have access to the database to monitor the data collection for all participants.
Any trial-related documents and Site Master Files including paper CRFs, SAE forms, copies of laboratory reports will be kept in a secure area or locked filing cabinets only accessible by study team members.
Identifying information will not be released without the written permission of the participant, except as necessary for monitoring, auditing or inspection by the relevant authorities. Published results will not contain any personal data that could allow identification of individual participants.
Access to data
The eCRFs entered by participants will be stored on the UBC/British Columbia’s Children’s Health Research servers and can only be accessed by OPSiP team members and iSTAR management. The database management system on the server is password protected. The servers will be backed up daily by Information Technology (IT) system administrators.
Provisions for post-trial care
Post-trial care will be provided by participants’ usual maternity care provider. Participants will be provided with a letter to give to their maternity care provider to inform them of their participation in the study and contact details for the study coordinator, should they have any concerns or queries.
Dissemination policy
The findings will be presented to those accessing local maternity services at the research sites through posters, social media, and to clinicians and other researchers through research rounds, at conferences and in peer-reviewed journals.
Discussion
This will be the first trial to study the impact of this combination of probiotic strains on maternal GBS colonisation. It will also be unique in that each probiotic strain will be examined for its ability to survive the gastrointestinal tract in pregnancy and adhere to vaginal epithelial tissue following oral consumption.
Trial status
This is version 2.3 of the research protocol. This study was launched in January 2020 and was paused on the outbreak of the COVID-19 pandemic to seek approval from the Research Ethics Committee to continue the study with a number of revisions to protect participants and researchers, as follows. All face-to-face contacts were replaced with video calls and non-contact arrangements were developed for the provision of supplements, study materials and the collection of samples. All revisions (to protocol and study documents) were approved by the Research Ethics Committee and the study was allowed to proceed in November 2020. Recruitment began again in January 2021. Recruitment has been slower than anticipated, largely due to the COVID-19 pandemic, and to date 168 participants have been recruited. This publication of this trial was delayed until now in order to preserve the novelty of the study (selection of strains, analysis approach) as the study is being conducted as a PhD study.
Ethics statements
Patient consent for publication
References
Footnotes
Contributors MB is the principal investigator. PJa is the co-principal investigator. KH conceived the study. KH, MB, PJo, CJ and BAP designed the protocol and made revisions following feedback from the REB and Health Canada, and in response to the COVID-19 pandemic. MB and KH adapted the protocol to meet the SPIRIT requirements. All authors reviewed the final draft and approved the final manuscript.
Funding The study is funded by the Alva Foundation and the Stollery Midwifery Research Initiative. The probiotics and placebos will be provided free of charge by the manufacturers (Chr. Hansen, and Blis). Neither the funders nor the supplement manufacturers had any role in the design of the study, nor will they have any role in the conduct (collection, analysis and interpretation of data) of the study or the production of any written reports or manuscripts.
Competing interests None declared.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.