Conditions: Infection Pregnancy
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 251
Healthy volunteers: yes

Brief summary:
The investigators wish to determine if oral probiotic supplementation during the second half of pregnancy decreases maternal GBS recto-vaginal colonization at 35-37 weeks' gestational age, thereby decreasing need for maternal antibiotic administration at time of labor. The importance of this study is that it may offer a safer alternative to antibiotic treatment of group B Streptococcus (GBS) colonized pregnant women.

Detailed description:
1. Screening: All pregnant women prior to 28 weeks gestational age. Patients who choose to enroll and who will not deliver at Lucile Packard Childrens Hospital at Stanford will sign release of medical information forms for study personnel to access pregnancy outcomes. Patients receiving obstetric care at any of the satellite research sites in Santa Cruz will also be offered enrollment in the study.
2. Women will continue regular and routine obstetric care and clinic visits.
3. Placebo vs probiotic daily regimen: We plan to begin administration of product and placebo at 20 weeks gestation, and no later than 28 weeks gestation until delivery. Once a women is enrolled in the study, she will be randomized to either the placebo or the probiotic group.
4. At the time of randomization, the patient will receive her month supply of 30 capsules; The allocation arm will be double-blinded.
5. The investigators will schedule the women for routine monthly obstetric visits (more often if clinically required) during which time they will also meet with one of the investigators. The investigator at each monthly visit will provide an additional monthly allotment of 30 capsules. The capsule bottle from the previous cycle will be collected and dated if there are capsules remaining in the bottle. Remaining capsules will be counted and refrigerated for future use.
6. The investigators will collect history data including safety data per the questionnaire and will document compliance with the study.
7. GBS recto-vaginal screening: The investigators will enroll the women in the study and we will perform the standard GBS colonization screening (using standard GBS recto-vaginal cultures) at 36 weeks.
8. Additionally, subjects may opt to have serial vaginal swabs collected to assess potential beneficial effects of probiotics on the vaginal microbiota and bacterial vaginosis (BV) status. Vaginal swabs will be collected (either by study personnel or self-collected by the study participant). Swabs will be inserted 1-2 inches into the vaginal introitus and spun for 20 seconds and then withdrawn. Swabs will be collected at the following time points: prior to probiotic/placebo initiation, every 1-4 weeks from time of enrollment to time of delivery, and postpartum serially up to 12 months. These swabs will be stored at -20 degrees Celsius or colder for additional microbiologic analyses.
9. Additionally, placental tissue may be collected at time of delivery for possible future microbiome and/or other analyses.
10. Women who suffer a premature rupture of the membranes, deliver before 36 weeks gestation, or go into labor before the GBS culture result is available, will receive the standard GBS antibiotic prophylaxis.
11. Labor: The patient will receive standard delivery and newborn care. Patients with a positive GBS culture will be treated with standard antibiotics in labor.
12. Postpartum and neonatal care: The patient will receive routine postpartum care per the obstetric team. Data regarding her postpartum course and neonatal outcomes will be collected.

Arms:
arm 1: Probiotic dietary supplement one capsule once per day until delivery. arm 2: Placebo capsule, one daily until delivery.

Interventions: intervention 1: Placebo intervention 2: Probiotic dietary supplement
Intervention descriptions: intervention 1: One placebo capsule daily. intervention 2: Dietary Supplement: Lactobacillus rhamnosus GR-1, Lactobacillus reuteri RC -14

Protocol document:
Prot_000.pdf: 
Oral Probiotic Supplementation and Group B 
Streptococcus Rectovaginal Colonization in 
Pregnant Women: a Randomized Double-blind 
Placebo-controlled Trial 
NCT01479478 
November 19, 2013  
 
Natali Aziz, MD, Principal Investigator 
Stanford University 
Stanford, California 94305 
 


Title 
 
Oral probiotic supplementation and Group B Streptococcus rectovaginal colonization in 
pregnant women: a randomized double-blind placebo-controlled trial.  
 
Introduction and Specific Aims  
 
Group B Streptococcus (GBS) is a leading cause of perinatal morbidity and mortality, 
including neonatal sepsis and maternal puerperium infections. In light of significant 
consequences of GBS colonization in pregnancy, GBS screening at 35-37 weeks’ 
gestational age and prophylaxis at time of labor is recommended in the United States. 
Although intrapartum antibiotic prophylaxis effectively prevents perinatal transmission in 
colonized pregnant women, numerous challenges in this management remain, including 
allergic reactions to antibiotic use, antimicrobial resistance development, potential 
unpredictability of transient colonization, as well as drug and administration costs. 
Identifying strategies that decrease the rate of GBS colonization would be beneficial in 
reducing such obstacles.  
 
Probiotics are live microorganisms that are thought to be healthy for the host organism.  
One of the mechanisms proposed for the potential benefit of probiotics includes balance 
of intestinal microbial environment and inhibition of pathogenic bacterial growth.  
Recently, lactobacilli-containing probiotics have been studied for use in intestinal and 
non-intestinal conditions, including urogenital infections.  Several studies have 
demonstrated that use of probiotics may reduce the incidence of bacterial vaginosis (BV).  
Given the potential benefits of probiotics in treatment of bacterial vaginosis, a similar 
beneficial effect of increased balance in normal vaginal microflora may reduce GBS 
colonization in pregnant women.  We hypothesize that supplementation with oral 
probiotics may reduce the rate of GBS colonization in term pregnant women. 
  
We propose the following specific aims to examine the aforementioned hypothesis:  
1. Compare GBS rectovaginal colonization rate in pregnant women at 35-37 weeks’ 
gestational age after oral supplementation with probiotics during the second and 
third trimesters with the rate in pregnant women who receive placebo.   
2. Compare perinatal outcomes related to infectious morbidity between the two 
groups.  
 
Background 
 
Group B streptococcus (GBS) is an encapsulated gram-positive diplococcus that 
frequently colonizes the human genital and gastrointestinal tracts, as well as the upper 
respiratory tract of young infants.  Approximately 10-30% of pregnant women in the 
United States are colonized with GBS in the vaginal or rectal area.(1) Colonization 
prevalence in pregnant women differs by ethnic groups, geographic locales, and age.(1)  
 
GBS is a leading cause of morbidity and mortality among newborn infants; is a common 
cause of maternal peripartum infections; and has been associated with adverse obstetric 


events. (2-8) GBS colonization of the mucous membrane is typically asymptomatic.  
Vertical transmission of GBS at the time of birth occurs in 50-70% of infants born to 
colonized mothers, and of these, 1-2% will develop early-onset invasive disease. (2)  
Transmission may also occur per an ascending route and cause intrauterine fetal 
infection.  Maternal colonization is the primary risk factor for GBS infection in neonates 
and young infants (younger than 90 days of age).(4,9) Other risk factors for neonatal 
infection include maternal GBS colonization density, with approximately 2.5 times 
greater risk of infant infection in pregnant women with heavy versus light 
colonization.(10)   
 
Neonatal infectious complications of GBS infection include bacteremia, sepsis, 
pneumonia, and meningitis, sometimes with permanent neurologic sequelae, as well as 
neonatal death.(2-4)  Maternal infections attributable to GBS in pregnancy include 
asymptomatic bacteriuria, urinary tract infections, intraamniotic infection or 
chorioamnionitis, and postpartum endometritis. Less commonly, pneumonia, puerperal 
sepsis, and bacteremia can occur.  GBS colonization has also been associated with 
obstetrical complications, including fetal demise, midgestation pregnancy loss, preterm 
labor, preterm premature rupture of membranes, and preterm delivery. (2-8)   
 
Consensus guidelines for prevention of perinatal GBS disease recommending either risk-
based or screening-based strategies have been issued since the early 1990’s by the 
American College of Obstetricians and Gynecologists (ACOG), American Academy of 
Pediatrics (AAP), and Centers for Disease Control and Prevention (CDC).  In 2002 
Schrag et al demonstrated that antenatal screening for GBS prevented approximately 
55% more cases of early-onset sepsis than the risk-based approach.(11) In August of 
2002 the CDC recommended universal prenatal screening for vaginal and rectal GBS 
colonization of all pregnant women at 35-37 weeks’ gestation for the prevention of 
perinatal GBS disease.(4) 
  
Universal prenatal screening at 35-37 weeks’ gestation with intrapartum antibiotic 
prophylaxis of colonized women during labor is thus far the most successful strategy for 
prevention of perinatal GBS disease.  However, there remain several challenges 
pertaining to screening and treatment considerations. Although the accuracy of late 
antenatal screening cultures performed within 5 weeks of delivery in predicting genital 
GBS colonization at time delivery have relatively high positive (87%) and negative 
(96%) predictive values, this identification is not perfect. (12)  In reality, greater than 
10% of women may receive unnecessary treatment, while a small minority may not 
receive appropriate intrapartum antibiotic prophylaxis.   
 
Overuse of antibiotics, which may lead to organism resistance and potential allergic 
reactions to antimicrobials, is another potential consequence of our current management 
strategy.   In women with rectovaginal GBS colonization at term, without use of 
intrapartum antibiotics, the incidence of invasive neonatal GBS disease ranges from 2 to 
3 cases per 1,000 live births. (3, 13) Thereby, large numbers of mothers and infants may 
be exposed to unnecessary antibiotics. Based on a 2002 Cochrane Database Review, it is 
estimated that 1,000 women must be treated with antibiotics to prevent 1.4 cases of 


disease. (14)  While administration of antibiotics does decrease GBS neonatal early-onset 
disease, in general the cost of large scale antibiotics use is out of proportion to the 
improvement in public health. Intravenous antibiotic therapy is costly and utilizes 
hospital personnel time.  Also, up to 10% of women administered antibiotics in labor will 
experience some form of allergic reaction; 1 in 100,000 will die from anaphylactic 
shock.(4, 15) Women who are allergic to penicillin and not candidates for cephalosporin 
therapy are treated with alternative antibiotic therapies such as erythromycin, 
clindamycin, and vancomycin. However, these are not well studied and may not achieve 
therapeutic levels in cord blood.  These alternative antibiotics have very limited human 
transplacental data and also may result in greater maternal adverse events.  (16, 17)  
Widespread exposure to antibiotics results in selection of resistant organisms as recently 
demonstrated by reports of increasing resistance to clindamycin (>20%) and 
erythromycin (>30%); this resistance may lead to reduction of antimicrobial 
effectiveness.(18-20)  Finally, risk of maternal candidiasis may increase with use of 
antibiotics, which may in turn lead to breastfeeding difficulties.(21)  Therefore, given 
these limitations to our current management of GBS during pregnancy and labor, 
strategies to decrease maternal colonization would be of tremendous benefit.    
 
According to the currently adopted definition by the Food and Agricultural Organization 
of the United Nations and World Health Organization, probiotics are defined as live 
microorganisms which when administered in adequate amounts confer a health benefit on 
the host.(22) Numerous recent studies have been conducted investigating the potential 
therapeutic benefits of probiotics.  It has been proposed that the potential beneficial effect 
of probiotics involves improvement of intestinal microbial balance and inhibition of the 
growth of pathogenic bacteria. Recently, lactobacilli-containing probiotics have been 
studied for use in a number of intestinal and nonintestinal conditions, including 
Clostridium difficile infection, inflammatory bowel diseases, and urogenital 
infections.http://www.femalepatient.com/html/arc/sig/urog/articles/035_08_042.asp - 2#2 
(23) Investigators suggest that imbalance in the normal vaginal bacterial flora may 
contribute to the pathogenesis of urogenital infections, especially BV and UTI. (24)  
Thereby, restoration of the normal vaginal flora with use of lactobacilli-containing 
probiotics has been proposed as a novel approach for the prevention and treatment of 
urogenital infections. (23)  
Genitourinary infections in women are often characterized by an alteration in the local 
flora, with a transformation from a predominance of lactobacilli to coliform pathogens. 
This can occur as a result of multiple factors, including hormone deficiency, sexual 
activity, and contraception.(25)  In order for probiotics to be effective in the prevention or 
treatment of urogenital infections, lactobacilli must exhibit antibacterial activity.(26) One 
antibacterial property of lactobacilli is the ability to maintain an acidic pH <4.5, which is 
conducive to lactobacilli replication and their subsequent production of antibacterial 
metabolites such as bacteriocin and hydrogen peroxide. It has been noted that different 
strains of lactobacilli produce varying amounts of these substances.(27)  Lactobacilli-
containing probiotics also offer protection by producing biosurfactants, which interfere 
with growth and adhesion of organisms such as Escherichia coli and Enterococcus 


faecalis to uroepithelial cells.(28) Finally, lactobacilli may also block adhesion or 
displace uropathogens that are adherent to vaginal epithelial cells.(29)  
Recent studies and reviews have demonstrated successful treatment of BV with 
probiotics in majority of trials.(30-39)   These trials generally included young, healthy, 
premenopausal women who were treated with lactobacilli-containing probiotics. Several 
studies included pregnant women, as well.(31,32)  Most protocols included probiotics   
administered intravaginally, however oral route has also proven successful for treatment 
of BV.(37) Studies differed by strains, concentrations, and preparations of lactobacilli 
used, including Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus 
reuteri, and Lactobacillus crispatus. The utility of probiotics for either prevention or 
adjunctive treatment of vulvovaginal candidiasis and urinary tract infections has yet to be 
proven, since most studies included in recent reviews showed no benefit and conflicting 
results.http://www.femalepatient.com/html/arc/sig/urog/articles/035_08_042.asp - 5#5 
(26,30) The conflicting data may be due to heterogeneity of the strains,   preparations of 
lactobacilli used, route of administration, and duration of treatment.   
Several recent studies have also reported probiotics to be safe for use during pregnancy 
without demonstrated adverse perinatal outcomes. (40, 41) However, a recent review of 
risks associated with probiotic use identified major and minor risk factors for associated 
infectious morbidities, including immunocompromised status, premature infants, central 
venous catheter, impaired intestinal epithelial barrier, administration of probiotic by 
jejunostomy, concomitant use of broad spectrum antibiotics, probiotics with high 
mucosal adhesion or known pathogenecity, and cardiac valvular disease. (42)  
Specifically, pregnancy was not included as a risk factor. While caution for adverse 
effects must be maintained with the growing use of probiotics, the majority of the 
available evidence to date suggests that probiotics are safe, except in limited 
circumstances. Larger studies have not yet reproduced these findings. Furthermore, no 
major side effects from the use of probiotics were reported in the studies on genitourinary 
infections. All preparations, regardless of strain and route of administration, were 
tolerable and caused minimal adverse effects in healthy, young, premenopausal women.  
 
Review of the literature does not demonstrate any specific strains of probiotic organisms 
that are known to be effective in displacing GBS either in the gut or in the reproductive 
tract and no study has investigated whether probiotics may effect rectovaginal 
colonization in pregnant women. However, the biological plausibility exists that there 
may be a beneficial effect of decreased GBS colonization given observed associations of 
reduction of other genital infections with probiotic therapy.   
 
Preliminary Studies 
 
In an empiric observational case series in two midwifery practices, 80 pregnant women 
were supplemented with oral probiotics.  Given the average colonization rates, we would 
expect 10-30% GBS colonization rate or 8-24 cases in this cohort. However, of the 80 
women who took oral probiotics throughout their pregnancies, only 1 woman had GBS 


colonization (1.25%) at time of screening between 35-37 weeks. (personal 
communication)    
 
 
Methods   
A. Design: prospective randomized double-blind placebo-controlled trial.  
 
B. Site: A collaborative trial including Stanford University School of Medicine/Lucile 
Packard Children’s Hospital (LPCH) Obstetric Clinic, Dominican Hospital/ Aptos 
Women’s Health (AWH) private Obstetrics-Gynecology practice in Santa Cruz, and 
private Midwifery Practice of Karen Ehrlich, a certified midwife and co-investigator of 
this study.  We plan to enroll the majority of subjects (approximately 2/3) at Stanford 
University School of Medicine/Lucile Packard Children’s Hospital Obstetric Clinic.    
 
C. Inclusion criteria:  
1. Pregnant women < 28 weeks’ gestation. 
2. 18 years of age or older.   
3. Singleton gestation.  
 
D. Exclusion criteria:  
1. Preexisting morbidity: Immunocompromised status (HIV +; 
malignancy; history of organ transplant; chronic steroid therapy; 
autoimmune disease requiring treatment during pregnancy, and other 
immunocompromised states); Type 1 diabetes and type 2 diabetes; 
congenital cardiac disease and cardiac valvular disease requiring antibiotic 
prophylaxis during procedure/labor; pulmonary disease (except mild 
asthma); renal disease; chronic hepatic disease (Hepatitis B, C); 
inflammatory bowel disease (Crohn’s disease or ulcerative colitis); 
stomach or duodenal ulcer; bowel resection, gastric bypass, and chronic 
indwelling venous, bladder, or gastric catheter.  
2. Multi-fetal gestation.  
3. Regular use of probiotics preparations in the 3 months prior to 
beginning the study treatment or use of any additional probiotics 
preparations (other than study treatment) at any time during the study 
period (including over the counter food supplements such as Activia, 
BioK, other oral or vaginal probiotics products (BUT not including other 
common forms of yogurt). 
4. Chronic (daily) use of broad spectrum antibiotics.  
5. History of infant with GBS sepsis.  
6. IUGR, Fetal Anomalies-major diagnosed at time of second trimester 
anatomy ultrasound  
7. Anticipated delivery <35 weeks for maternal/fetal indication 
8. Placenta previa or accreta (with anticipated delivery prior to 35 weeks) 
 
E. Outcomes:  
 
 
1. Primary: GBS colonization at 35-37 weeks   


2. Secondary: maternal ante- intra- and postpartum outcomes (urinary 
tract infections, chorioamnionitis, endometritis, cellulitis, bacteremia, 
sepsis, and other infectious morbidity) and neonatal outcomes (gestational 
age at delivery, APGAR scores, bilirubin levels, C-reactive protein, rule 
out sepsis evaluation, sepsis, pneumonia, meningitis, neonatal ICU 
admission, and length of hospital stay).  
 
F. Agents:  
1. Treatment arm: Study probiotic dietary supplement arm will consist of 
1 capsule (>1 billion CFU/capsule) daily including two strains, 
Lactobacillus rhamnosus GR-1   and Lactobacillus reuteri RC-14, which 
have been shown to reduce the incidence of bacterial vaginosis and 
candidiasis. (43) Those two strains are present in Jarrow’s Femdophilus, 
which is produced by Chr Hansen in Denmark under the brand name 
Urex. Shelf life is two years when stored at room temperature (max 25 
degrees Celsius).  
2. Control arm: Placebo arm will consist of one inert (heat inactivated) 
capsule daily.   
 
G. Study Drug and Placebo Supply, Transport, and Storage:  
1. Chr Hansen A/S will provide the active probiotic dietary supplement 
and placebo oral tablets for 372 patients (186 per arm of the trial.)  
However, the company providing the study probiotic dietary supplement 
and placebo will not have access to preliminary data during the conduct of 
the study and will not have the right of review of the results prior to 
publication.  In summary, 27,900 doses of product and 27,900 doses of 
placebo will be provided.  (Appendix 1) Capsules will be packed in 
aluminum tubes and these will then be packed in cardboard boxes and sent 
by courier from manufacturer to study site. The tubes containing the 
capsules will be packed such that no physical damage will occur to the 
individual capsules. Refrigeration is not necessary since bacteria count of 
minimum 1 billion is guaranteed for 2 years when stored at room 
temperature up to 25 degrees Celsius. Capsules should not be exposed to 
direct sunlight when stored. The manufacturer recommends 5 degrees 
Celsius storage to preserve bacterial potency if more than 6 months of 
storage is expected. Study participants will be instructed to store capsules 
outside of children’s reach and at room temperature and to avoid direct 
sun exposure to capsules. 
 
H. Patient Recruitment:  
1. Pregnant study subjects who meet inclusion criteria will be recruited 
prior to 28 weeks’ gestational age at one of the following collaborative 
sites by either study co-investigator or study staff:  LPCH Obstetrics 
Clinic and the collaborating sites of AWH Obstetrics and Gynecology 
Clinic, other private obstetric-gynecology offices in Santa Cruz, and 


homes of patients who have elected to receive prenatal care and delivery at 
home by Midwife Karen Ehrlich, one of the co-investigators of this study.      
 
I. Randomization Strategy:  
1. Manufacturer will send identical probiotic and placebo capsules.  The 
probiotic and placebo capsules will be originally packed in separate 
containers, labeled as designated probiotic versus placebo for each specific 
study enrollee (with study material information only available to the 
designated pharmacist at LPCH). The designated pharmacist at LPCH will 
be responsible for randomization assignment based on our pre-generated 
randomization table and distribution of study material to research 
coordinator for disbursement to enrollees at all sites.  
a. Investigators will receive individual DEIDENTIFIED study 
material (patient probiotic versus placebo tubes) coded by a unique 
random number.  
b. Each enrolled patient will receive supplies at study visit.. 
c. At conclusion of study, the participant randomization arm will 
be identified by the designated pharmacist.  
 
J. Compliance Assessment: 
1. Compliance with study drug (probiotic versus placebo) will be assessed 
by self-reported and provider mechanisms at monthly visits. 
2. Subjects will be asked by investigators how many capsules they have 
missed since the last monthly visit.  
3. Investigators will perform capsule count of the subject’s monthly 
distributed study drug.  
4. Investigators will provide subjects with a single aluminum tube of 30 
capsules for the next monthly period.  
 
 
I. Clinical Protocol:   
1. Investigators will recruit study participants up to 28 weeks’  
gestational age. 
2. Following informed patient consent, patients will be blindly randomized 
to either study probiotic dietary supplement or placebo per computer 
generated randomization table.   
3. A hard-copy questionnaire/data sheet will be generated for each patient 
and will be labeled with patient’s unique number, name and other data 
points that we intend to collect prospectively.  
4. Subjects will be initiated on probiotic or placebo consisting of one 
capsule daily between 20-28 weeks’ gestation and continued until 
delivery.  Once enrolled, patients will receive a study questionnaire to 
complete with study investigator(s) or study coordinator(s) and a monthly 
supply of probiotic dietary supplement vs. placebo either at enrollment or 
subsequent study visit.   Subjects will be instructed to store capsules away 


from sun and consume 1 capsule with food every day at the same time (as 
determined to be the most convenient time for that patient). 
5. Women diagnosed with GBS bacteriuria during the index pregnancy 
will be enrolled/maintained in the study, will have routine GBS cultures at 
35-37 weeks for study purposes, and will receive intrapartum intravenous 
antibiotics for GBS prophylaxis per routine standard of care. 
6. As is the current standard of care, subjects will undergo rectovaginal 
culture for GBS at 35-37 weeks’ gestation. Any subject who tests positive 
will be treated with intrapartum intravenous antibiotic prophylaxis per 
routine GBS management protocol.  
7. Women who deliver preterm will receive chemoprophylaxis unless their 
GBS status within the most proximate 4 weeks is known per routine GBS 
management protocol. We will obtain a culture prior to initiation of 
antibiotics in those women delivering preterm without known group B 
strep status. 
8. Study visits will be conducted during routine monthly prenatal 
appointments to assess adverse events, study treatment compliance, and to 
dispense subsequent 30-day supply of probiotic or placebo capsules. 
Patients will complete a study visit questionnaire with the investigator(s)  
or study coordinator(s). Patients will also receive an additional monthly 
allotment of 30 capsules. The capsule bottle from the previous cycle will 
be collected and dated if there are capsules remaining in the bottle. 
Remaining capsules will be counted and refrigerated for future use.  
9. Patient questionnaire/database assessing adverse events will be 
amended at each study visit, which will occur monthly.  The 
questionnaire/database will assess adverse events, including rash, 
significant nausea/vomiting, significant diarrhea, persistent fever, and 
compliance data.  The questionnaire will also remind patients that all 
adverse events must be reported to the Protocol Director immediately at 
all times. 
10. Additionally, subjects may opt to have serial vaginal swabs collected 
to assess potential beneficial effects of probiotics on the vaginal 
microbiota and BV status. Vaginal swabs will be collected (either by study 
personnel or  self-collected by the study participant). Swabs will be 
inserted 1-2 inches into the vaginal introitus and spun for 20 seconds and 
then withdrawn. Swabs will be collected at the following time points: prior 
to probiotic/placebo initiation, every 1-4 weeks from time of enrollment to 
time of delivery, and postpartum serially up to 12 months. These swabs 
will be stored at -20 degrees Celsius or colder for additional microbiologic 
analyses. 
11. Additionally, placental tissue may be collected at time of delivery 
for possible future microbiome and/or other analyses. 
12. Labor: The patient will receive standard delivery and newborn care. 
Patients with a positive GBS culture will be treated with standard 
antibiotics in labor. 


13. Postpartum and neonatal care: The patient will receive routine 
postpartum care per the obstetric team. Data regarding her postpartum 
course and neonatal outcomes will be collected. 
 
G. Sample size calculation:  
1. The baseline rectovaginal GBS colonization is approximately 25% in 
recent years in our practice, we have powered the study to identify a 40% 
reduction of GBS colonization to 15%.  Using a two-tailed alpha of 0.05, 
133 patients per arm (266 patients total) are needed to fulfill a power of 
80% to detect a difference of 40% from current baseline GBS colonization 
of 25%. 
2. Given anticipated 30% drop out/lost to follow-up and 10% preterm 
delivery prior to 35-37 weeks’ gestation, we propose increasing our 
sample size by 40% to account for this subject loss.  Therefore, we 
propose recruitment of 372 patients total (186 in each arm).  
 
H. Statistics:  
1. Analysis will be by intention to treat.  Demographic data, specific 
information regarding the course of labor, and outcome variables will be 
collected on printed and numbered data sheets and entered into a relational 
database (Access; Microsoft Corporation, Redmond, WA). All data 
management and analysis will be performed using STATA version 7 
software (StataCorp – College Station, TX).  Descriptive statistics will be 
performed.  All tests of significance will be 2-tailed and an alpha level of 
0.05 will be used. Categoric data will be analyzed by the uncorrected X2 
and Fisher exact test, as appropriate. Continuous data will be analyzed by 
the unpaired Student t test.  
 
I. Data safety and monitoring plans:  
1. The Protocol Director (PD) will be responsible for monitoring data 
collected. 
2.  Patient questionnaire/database assessing adverse events will be 
completed at each study visits, approximately every month.  The 
questionnaire will assess adverse events, including rash, significant 
nausea/vomiting, significant diarrhea, persistent fevers.  The 
questionnaire/database will also remind patients that such events must be 
reported to the Protocol Director immediately at all times.  
3. Serious adverse events will be reported to the PD within one week. An 
interim analysis will be conducted to when primary outcome data is 
available for 150 subjects.     
4. Data or events captured by the monitoring plan will be assessed 
monthly. 
5. The following will be monitored:  
a. Side effects related to consumption of the probiotic or placebo 
capsules 
b. Rates of compliance 


c. GBS colonization culture results 
d. Neonatal morbidity or mortality, including but not limited to 
infection and duration of hospitalization 
e. Maternal morbidity and mortality, including but not limited to 
postpartum infection, complications, and duration of 
hospitalization 
 
 
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Appendix A: Letter from Manufacturer confirming their agreement to supply of probiotic 
dietary supplement and placebo free of charge for this investigator-initiated study.  
 
Appendix B: Patient Questionnaire to be completed at monthly study visits.   
 
 
 
 
 
 
 
 
 
 
 
 

SAP_001.pdf: 
Oral Probiotic Supplementation and Group B 
Streptococcus Rectovaginal Colonization in 
Pregnant Women: a Randomized Double-blind 
Placebo-controlled Trial 
NCT01479478 
July 14, 2017 
 
Natali Aziz, Principal Investigator 
Stanford University 
Stanford, California 94305 
 
Statistical analysis plan 
Treatment group characteristics will be summarized and reported using numbers and percentages 
for categorical data, means and standard deviation for normally distributed data, and medians 
and interquartile ranges (IQR) for non-normally distributed data. Characteristics will be 
compared between the treatment groups using the standardized mean difference. 
Binary study outcomes will be compared between treatment groups using the Cochran-Mantel-
Haenszel test. Normally distributed outcomes will be compared using linear regression adjusting 
for site. Non-normally distributed outcomes will be compared using the van Elteren (vE) test; a 
stratified Wilcoxon-Mann-Whitney test. In all cases treatment effects, 95% confidence intervals 
and p-values will be reported. All analyses will be performed according to intention-to-treat. P-
values ≤.05 will be considered significant. All statistical analysis was performed using SAS 9.4.