Clinical trial NCT02748317
Conditions: Lower Urinary Tract Symptoms
Keywords: Spina Bifida Spinal Cord Injury Lower Urinary Tract Symptoms Probiotics Intermittent Catheterization
Sponsor: Medstar Health Research Institute
Start date: 2016-01-01; Completion date: 2018-10-31
Enrollment: 96 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
As a collaborative effort between MedStar National Rehabilitation Hospital (NRH)/MedStar Health Research Institute (MHRI), Children's National Medical Center (CNMC) Department of Urology, Children's Research Institute (CRI) Center for Genetic Medicine Research, and Georgetown University Medical Center, the overall objective of this study is to develop, validate, and assess a patient-initiated, probiotics-based, self management protocol that is initiated at the time of urinary symptoms. The self-management protocol will allow patients to manage urinary symptoms and avoid potentially unnecessary antibiotic use, and provide a readily-available means of maintaining health, function, and independence throughout the lifespan.

Detailed description:
Lower urinary symptoms are a common issue for individuals with neurogenic bladder, commonly occurring in the Spina bifida and Spinal Cord Injury population. In this study, probiotics will be introduced into the bladder to prevent UTIs. Introduction of probiotics will be determined by a validated symptom questionnaire (USQ-NB) and protocol (SMP-PRO).

This study will estimate the strength of the associations between successful implementation of the probiotic self-management program (USQ-NB and SMP-PRO) and urinary symptoms, bladder inflammation, and the urine microbiome. Investigators will conduct an 18-month study in which each participant will serve as his/her own control through 3 phases of study: 6-months usual care (baseline), 6-months probiotic intervention, and 6-months follow-up.

Participants will complete the Urinary Symptom Questionnaire weekly. After 6 months of baseline data collection, participants will receive the Lactobacillus (Culturelle GG, 20 billion live organisms for adults and 10 billion live organisms for children \<18 years of age), will be instructed on preparation and intravesicular instillation of the Lactobacillus, and will have a tutorial with a fellow consumer on use of the patient-initiated protocol. The protocol and Lactobacillus bladder instillation instructions (including a step-by-step video) will be available on the study website for 24/7 access and written instructions will be provided at the time of instruction.

For the intravesicular Lactobacillus instillation, participants will be instructed to mix the contents into sterile saline. After mixing, participants will draw up the liquid Lactobacillus mixture into a 60cc catheter tip syringe and instill via the intermittent catheter after fully emptying the bladder. Participants will be instructed not to catheterize for at least 4 hours after the bladder instillation. Participants will receive 10 Culturelle GG at the beginning of the treatment phase. At the end of each month, the coordinator or RA will ask how many remaining tablets the participant has, and if needed dispense the next supply of 10 tablets. Participants will be instructed to complete the USQ-NB weekly.

If/when urinary symptoms occur, subjects will be instructed to follow the protocol to determine whether to initiate intravesicular Lactobacillus instillation or be evaluated by a physician. The self-management protocol will also direct them to discontinue Lactobacillus instillation or be evaluated by a physician if symptoms remit, persist (after 2 instillations), or worsen. The maximum number of instillations is 2 over 28 hours. If participants are directed by the self-management protocol to seek medical attention or s/he feels the need for medical evaluation, s/he will be advised to obtain care as they typically would by their health care provider. Participants will be supplied with letters to be brought to their health care provider notifying them of the study and requesting sharing of urinalysis and urine culture results with the research team. A verified UTI will include those that resulted in antibiotic treatment by a health care professional. An additional urine sample for metagenomics will either be left with the health care provider for pick up by the research team, brought to the research site, or obtained by the RA at a mutually convenient site.

After completion of the 6-month patient-initiated, self-management protocol intervention period, participants will monitor symptoms weekly using the USQ-NB for the final 6-month phase of the 18-month study.

Arms: arm 1: Lactobacillus rhamnosus GG
Interventions: intervention 1: Lactobacillus rhamnosus GG
Intervention descriptions: intervention 1: For the intravesicular Lactobacillus instillation, participants will be instructed to mix the contents of 1 Lactobacillus capsule into 45 cc sterile 0.9% saline.5,6 After mixing, participants will draw up the 45 cc of the liquid Lactobacillus mixture into a 60cc catheter tip syringe and instill via the intermittent catheter after the last catheterization prior to going to bed. Participants will receive 10 Lactobacillus GG tabs at the beginning of treatment phase. At the end of each month, the coordinator or RA will ask how many remaining tablets the participant has, and if needed dispense the next supply of 10 tablets Participants will be instructed to complete the USQ-NB weekly.

Protocol documents:
Prot_SAP_000.pdf: 
 
The Impact of Self-Management with Probiotics on Urinary Symptoms and the Urine Microbiome in 
Individuals with Spinal Cord Injury and Spina Bifida Protocol 
5/18/2017 
Page 1 of 17 
 
The following includes all aspects of the protocol entitled, “The Impact of Self-Management with 
Probiotics on Urinary Symptoms and the Urine Microbiome in Individuals with Spinal Cord 
Injury and Spina Bifida” in which we propose use of intravesicular Lactobacillus to reduce 
urinary symptoms in people with neuropathic bladder.  
 
Specific Aim 2 Methods and Procedures: Develop and pilot a UTI Self-Management Protocol 
using Probiotics (SMPPRO) to reduce the severity, frequency and impact of urinary 
symptoms. The validated Urinary Symptom Questionnaire for people with Neuropathic Bladder 
(USQ-NB; developed in Aim 1 of this study, currently in progress IRB # 2012-187) will be the 
linchpin of the SMPPRO protocol. The principal utility of the SMPPRO will be in helping 
individuals identify the symptoms that signal recourse to increase fluid intake to reduce 
symptoms, initiate probiotic treatment, or seek medical attention.  
 
Development of the SMPPRO has been an iterative participatory design process. The 
multidisciplinary team began refining the decision tree underlying the protocol, concurrent with 
development of the USQ-NB, informed by patient focus groups, and later will be assessed 
through large scale national testing in conjunction with United Spinal Association. The prototype 
SMPPRO will be shared with a diverse sample of individuals with Spina Bifida (SB), Spinal Cord 
Injury (SCI), and Multiple Sclerosis to develop use cases and scenarios of use to further inform 
the protocol. These patients further informing the SMPPRO will include adults, youth, and 
children, both genders, persons who reuse catheters and those who don’t, among individuals with 
SCI, SB, and MS who use intermittent catheterization for their bladder management.  Participants 
will engage in semi-structured interviews in which they will be asked to recall the onset and 
progression of symptoms in their most recent episode of urinary symptoms. The interviewer will 
then ask participants to mentally step through the experience of urinary symptoms: 1) to confirm 
that the prototype SMPPRO can be triggered by the items composing the USQ-NB and 2) to 
elucidate the mechanics of implementing the protocol across the diversity of individuals with 
neuropathic bladder due to SB, SCI or MS. Interview data will be managed, coded, and 
thematically analyzed using NVivo 10 qualitative software. Use cases and scenarios of use will 
be developed from themes. The interdisciplinary team will refine and expand the SMPPRO to 
accommodate patient experience augmented by use cases and scenarios of use.  
 
 
THE FOLLOWING SECTIONS REPRESENT ASPECTS OF THE PROPOSED STUDY 
IN WHICH THE LACTOBACILLUS PROBIOTIC PROTOCOL WILL BE 
IMPLEMENTED BY PATIENTS. 
 
Once the SMPPRO has been revised and tailored to the patient experience, we will conduct a multi 
phase pilot study with 120 individuals with neuropathic bladder due to SB or SCI/ Multiple 
Scleorosis to study its safety, acceptability and perceived usefulness. Pilot data will include 
weekly USQ-NB and records of any urinary symptoms and UTIs that are experienced.  
 
Phase Ia: Safety study of a single bladder instillation of probiotic in adults. A subsample of 
healthy adults with neuropathic bladder who do not have urinary symptoms meeting the criteria 
for antibiotic treatment of urinary tract infection or initiation of the protocol will undergo a single 
intravesical instillation of Lactobacillus to determine the tolerability of the instillation, safety of a 
single instillation, and whether the instilled Lactobacillus persists in the urine. 
Study Design: We will recruit N=5 adult subjects with neuropathic bladder due to SCI or SB. 
Clinical Setting: MedStar National Rehabilitation Hospital  


 
The Impact of Self-Management with Probiotics on Urinary Symptoms and the Urine Microbiome in 
Individuals with Spinal Cord Injury and Spina Bifida Protocol 
5/18/2017 
Page 2 of 17 
 
Target Enrollment: 5 subjects at least 18 years of age with SCI or SB and at least 1-year post-
injury (if SCI). 
Informed Consent: Consent will by obtained by the PI, Study Coordinator, or Research Assistant.  
Inclusion Criteria: Inclusion criteria for safety study participants are: 1) age 18-50 years; 2) if 
SCI, at least 1-year duration; 3) neuropathic bladder, as determined by the attending physician; 4) 
utilizing intermittent catheterization for bladder management; and 5) community dwelling. 
Exclusion Criteria: Study candidates with the following will be excluded: 1) known genitourinary 
pathology beyond neuropathic bladder (i.e., vesicoureteral reflux, bladder or kidney stones, etc.); 
2) use of prophylactic antibiotics; 3) instillation of other intravesicular agents to reduce urinary 
symptoms or UTI (i.e., gentamycin); 4) psychologic or psychiatric conditions influencing the 
ability to follow instructions; 5) participation in another study in which results would be 
confounded; 6) pregnant or breastfeeding women; 7) individuals with a history of acquired or 
genetic immunodeficiencies; active, acute or chronic serious infections (e.g., viral hepatitides, 
HIV/AIDs); 8) individuals with cancer/autoimmune disorders; 9) serious allergy to any 
component or excipients in the live bacterial combination product; 10) no change in neurologic 
status in the previous 2 weeks; 11) taken antibiotic for any reason in the previous 2 weeks; 12) 
any patient with history of sensitivity or allergy to ampicillin or tetracycline; and 13) current 
urinary tract infection or urinary tract infection within the previous 2 weeks;  (as defined by 
Infectious Diseases Society of America CAUTI Guidelines, 2010. According to the Infectious 
Disease Society of America Guidelines for Diagnosis of Catheter-Associated UTI, the presence 
of the following urinary symptoms is required for UTI diagnosis in patients using intermittent 
catheterization: new onset or worsening fever, rigors, altered mental status, malaise, lethargy with 
no other identified cause, flank pain, costovertebral angle tenderness, acute hematuria, pelvic 
discomfort, increased spasticity, autonomic dysreflexia or sense of unease. The Guidelines 
specifically state that “the presence or absence of odorous or cloudy urine should NOT be used as 
an indication for urine culture or antimicrobial therapy” and “the presence, absence or degree of 
pyuria should NOT be interpreted as an indication for antimicrobial treatment.”  If a subject 
presents with any of these symptoms and there are ≥103 cfu/hpf bacterial growth on urine culture, 
the subject will be excluded and treated for a UTI according to clinical best practice. 
Baseline Data Collection: At baseline, subjects who do not report urinary symptoms (see below) 
other than cloudy or foul-smelling urine will complete the validated USQ-NB. Urine will be 
collected for urinalysis and urine culture (see below for methods of urine collection), however 
urinalysis results will be used for post hoc data collection only and not inclusion/exclusion in 
keeping with the recommendations of the IDSA.   
Intervention: Eligible patients will undergo 1 Lactobacillus (Culturelle GG, 20 Billion live 
organisms) instillation by study personnel at the study site. For the intravesicular Lactobacillus 
instillation, the contents of 1 Lactobacillus capsule will be mixed into 45 cc sterile 0.9% saline 
(adult bladder volume is 450cc, so this amount represents 10% of adult bladder volume). After 
mixing, study personnel will draw up the 45 cc of the liquid Lactobacillus mixture into a 60cc 
catheter tip syringe and instill via the intermittent catheter. Participants will be observed in the 
clinic for 30 minutes after instillation to assure that no adverse reactions occur.  
Post-Intervention Follow-Up: After completion of the one Lactobacillus instillation and 30 
minute observation period, participants will be asked to monitor for any symptoms using the 
USQ-NB daily for one week. They will be instructed to contact study personnel in the event of 
any urinary symptoms. They will then return to the clinic 5-10 days after instillation for repeat 
urinalysis and urine culture.  
 
Phase 1b: Safety study of single bladder instillation probiotic in children with SB or SCI. After 
Phase 1a is complete, accumulated safety data (inclusive of adverse events and laboratory results) 


 
The Impact of Self-Management with Probiotics on Urinary Symptoms and the Urine Microbiome in 
Individuals with Spinal Cord Injury and Spina Bifida Protocol 
5/18/2017 
Page 3 of 17 
 
will be presented to the DSMB. Phase 1b will be initiated only after review and approval of the 
Phase 1a safety data by the DSMB. Once approval demonstrates that a single dose of intravesiclur 
Lactobacillus has been determined to be safe in adults, the same safety study will be repeated in 
children. A subsample of healthy children with neuropathic bladder due to SB who do not have 
urinary symptoms meeting the criteria for antibiotic treatment or initiation of the protocol will 
undergo a single intravesical instillation of Lactobacillus to determine the tolerability of the 
instillation, safety of a single instillation, and whether the instilled Lactobacillus persists in the 
urine. 
Study Design: We will recruit N=5 children with neuropathic bladder due to SB or SCI. 
Clinical Setting: MedStar National Rehabilitation Hospital and Children’s National Medical 
Center. 
Target Enrollment: 5 subjects at least 6 years of age with SB or SCI, at least 1-year post-injury (if 
SCI). 
Informed Consent: Consent and assent will by obtained by the PI, Study Coordinator, or Research 
Assistant.  
Inclusion Criteria: Inclusion criteria for safety study participants are: 1) age >=6 years; 2) if SCI, 
at least 1-year duration; 3) neuropathic bladder, as determined by the attending physician; 4) 
utilizing intermittent catheterization for bladder management; and 5) community dwelling. 
Exclusion Criteria: Study candidates with the following will be excluded: 1) known genitourinary 
pathology beyond neuropathic bladder (i.e., vesicoureteral reflux, bladder or kidney stones, etc.); 
2) use of prophylactic antibiotics; 3) instillation of intravesicular agents to reduce UTI (i.e., 
gentamycin); 4) psychologic or psychiatric conditions influencing the ability to follow 
instructions; 5) participation in another study in which results would be confounded; 6) pregnant 
or breastfeeding; 7) individuals with a history of acquired or genetic immunodeficiencies; active, 
acute or chronic serious infections (i.g., viral hepatitides, HIV/AIDs); 8) individuals with 
cancer/autoimmune disorders; 9) serious allergy to any component or excipients in the live 
bacterial combination product; 10) no change in neurologic status in the previous 2 weeks; 11) 
taken antibiotic for any reason in the previous 2 weeks; and 12) current urinary tract infection or 
urinary tract infection within the previous 2 weeks (as defined by Infectious Diseases Society of 
America CAUTI Guidelines, 2010. According to the Infectious Disease Society of America 
Guidelines for Diagnosis of Catheter-Associated UTI, the presence of the following urinary 
symptoms is required for UTI diagnosis in patients using intermittent catheterization: new onset 
or worsening fever, rigors, altered mental status, malaise, lethargy with no other identified cause, 
flank pain, costovertebral angle tenderness, acute hematuria, pelvic discomfort, increased 
spasticity, autonomic dysreflexia or sense of unease. The Guidelines specifically state that “the 
presence or absence of odorous or cloudy urine should NOT be used as an indication for urine 
culture or antimicrobial therapy” and “the presence, absence or degree of pyuria should NOT be 
interpreted as an indication for antimicrobial treatment.”  If a subject presents with any of these 
symptoms and there are ≥103 cfu/hpf bacterial growth on urine culture, the subject will be 
excluded and treated for a UTI according to clinical best practice. 
Baseline Data Collection: Same as for Phase Ia Safety Study 
Intervention: Eligible patients will undergo 1 Lactobacillus pediatric dose (Culturelle GG 10 
Billion live organisms) instillation by study personnel at the study site. For the intravesicular 
Lactobacillus instillation, the contents of 1 Lactobacillus capsule will be mixed into sterile 0.9% 
saline using an age-based estimate of bladder capacity (see Table below). Similar to adults, this 
bladder instillation will represent 10% of the estimated maximum bladder volume based on age. 
 


 
The Impact of Self-Management with Probiotics on Urinary Symptoms and the Urine Microbiome in 
Individuals with Spinal Cord Injury and Spina Bifida Protocol 
5/18/2017 
Page 4 of 17 
 
Age (yrs) 
Estimated Bladder 
Capacity (ml) 
Volume Culturelle 
mixture infused (ml) 
# of particles 
delivered per dose 
(billions) 
6 
270 
27 
12.0 
7 
285 
28.5 
12.7 
8 
300 
30 
13.3 
9 
315 
31.5 
14.0 
10 
330 
33 
14.7 
11 
345 
34.5 
15.3 
12 
360 
36 
16.0 
13 
375 
37.5 
16.7 
14 
390 
39 
17.3 
15 
405 
40.5 
18.0 
16 
420 
42 
18.7 
17 
435 
43.5 
19.3 
18 
450 
45 
20.0 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Reference: Kaefer M1, Zurakowski D, Bauer SB, Retik AB, Peters CA, Atala A, Treves ST. 
Estimating normal bladder capacity in children. J Urol. 1997 Dec;158(6):2261-4. 
 
After mixing, study personnel will draw up the liquid Lactobacillus mixture into a 60cc catheter 
tip syringe and instill via the intermittent catheter. Participants will be observed in the clinic for 
30 minutes after instillation to assure that no adverse reactions occur.  
 
Post-intervention Follow-Up: Same as for Phase Ia Safety Study 
 
Phase II: Pilot study assessing the USQ-NB and SMPPRO 
We will recruit up to 120 persons from a national sample in who will use the SMPPRO in order to 
estimate the strength of the associations between successful implementation of the probiotic 
self-management program and urinary symptoms. We will conduct a 18-month study in 
which each participant will serve as his/her own control through 3 phases of study: 6-months 
usual care (baseline), 6-months probiotic intervention, and 6-months follow-up.  
Study Design: We will recruit N=up to 120 subjects with neuropathic bladder who will utilize the 
SMPPRO as directed, and we will follow patient satisfaction and urinary symptom outcomes, but 
not utilize urinalysis or urine culture (as this is the intended use of the SMPPRO in practice). 
Clinical Setting: Enrollment through a national database 
Target Enrollment:  Adult subjects at least 1-year post-injury and 18 years of age with 
SCI/Multiple Sclerosis (MS) or at least 18 years of age with SB or pediatric subjects at least 6 
years of age with SB or SCI. 
Recruitment Strategy: SCI/MS and SB subjects will be enrolled with assistance from United 
Spinal Association. Additionally, we will query the MedStar National Rehabilitation Hospital 
inpatient service, the 40+ MedStar National Rehabilitation Network outpatient sites, various 
MedStar Network databases, and Children’s National Medical Center (CNMC) SB database. 
Databases will be queried for participants who meet the eligibility criteria and have already 


 
The Impact of Self-Management with Probiotics on Urinary Symptoms and the Urine Microbiome in 
Individuals with Spinal Cord Injury and Spina Bifida Protocol 
5/18/2017 
Page 5 of 17 
 
provided HIPAA authorization and consent to be contacted for future research. Announcements 
and IRB-approved flyers will be posted on the United Spinal Association and study websites and 
at participating institutions. 
Informed Consent: Informed consent will be emailed or mailed (if not in person at MedStar NRH 
or CNMC) out to participants interested in the study. Paid return envelopes will be included for 
participants to return signed informed consent to Study Coordinator. At MedStar NRH and 
CNMC, consent will by obtained by the PI, Study Coordinator, or Research Assistant.  
Inclusion Criteria: Inclusion criteria for participants with SCI/ MS includes: 1) age≥18 years; 2) 
SCI/MS; 3) neuropathic bladder at least 1 year duration, as determined by the attending 
physician; 4) utilizing intermittent catheterization for bladder management; 5) a history of 2 or 
more UTIs in the past year; and 6) community dwelling. Inclusion criteria for participants with 
SB adults includes: 1) age≥18 years; 2) SB; 3) neuropathic bladder as determined by the 
attending physician; 4) utilizing intermittent catheterization for bladder management; 5) a history 
of 2 or more UTIs in the past year; and 6) community dwelling. Inclusion criteria for pediatric 
subjects includes: 1) age≥6 years; 2) SB or SCI; 3) neuropathic bladder at least 1 year duration 
(for SCI), as determined by the attending physician; 4) utilizing intermittent catheterization for 
bladder management; 5) a history of 2 or more UTIs in the past year; and 6) community dwelling. 
 
Exclusion Criteria: Study candidates with the following will be excluded: 1) known genitourinary 
pathology beyond neuropathic bladder (i.e., vesicoureteral reflux, bladder or kidney stones, etc.); 
2) use of prophylactic antibiotics; 3) instillation of intravesicular agents to reduce UTI (i.e., 
gentamycin); 4) psychologic or psychiatric conditions influencing the ability to follow 
instructions; 5) participation in another study in which results would be confounded; 6) pregnant 
or breastfeeding; 7) individuals with a history of acquired or genetic immunodeficiencies; active, 
acute or chronic serious infections (i.g., viral hepatitides, HIV/AIDs); 8) individuals with 
cancer/autoimmune disorders; 9) serious allergy to any component or excipients in the live 
bacterial combination product; 10) no change in neurologic status in the previous 2 weeks; 11) 
taken antibiotic for any reason in the previous 2 weeks; 12) any patient with history of sensitivity 
or allergy to ampicillin or tetracycline; and 13) current urinary tract infection or urinary tract 
infection within the previous 2 weeks (as defined by Infectious Diseases Society of America 
CAUTI Guidelines, 2010. According to the Infectious Disease Society of America Guidelines for 
Diagnosis of Catheter-Associated UTI, the presence of the following urinary symptoms is 
required for UTI diagnosis in patients using intermittent catheterization: new onset or worsening 
fever, rigors, altered mental status, malaise, lethargy with no other identified cause, flank pain, 
costovertebral angle tenderness, acute hematuria, pelvic discomfort, increased spasticity, 
autonomic dysreflexia or sense of unease. The Guidelines specifically state that “the presence or 
absence of odorous or cloudy urine should NOT be used as an indication for urine culture or 
antimicrobial therapy” and “the presence, absence or degree of pyuria should NOT be interpreted 
as an indication for antimicrobial treatment.”  If a subject presents with any of these symptoms 
and there are ≥103 cfu/hpf bacterial growth on urine culture, the subject will be excluded and 
treated for a UTI according to clinical best practice. 
Baseline Data Collection: After consent, participants will complete the Health History 
Questionnaire Thereafter, participants will complete the Urinary Symptom Questionnaire (USQ-
NB) weekly.  
Intervention: After 6 months of baseline data collection, participants will receive the 
Lactobacillus (Culturelle) kits. Participants will be instructed on preparation and intravesicular 
instillation of the Lactobacillus, and will have a tutorial with a fellow consumer on use of the 
patient-initiated SMPPRO. Participants will receive 10 Culturelle tablets the beginning of the 


 
The Impact of Self-Management with Probiotics on Urinary Symptoms and the Urine Microbiome in 
Individuals with Spinal Cord Injury and Spina Bifida Protocol 
5/18/2017 
Page 6 of 17 
 
treatment phase. At the end of each week, the coordinator or RA will ask how many remaining 
tablets the participant has and will instruct them to complete the USQ-NB weekly.  
If/when urinary symptoms occur, they will be instructed to follow the SMPPRO protocol to 
determine whether to initiate intravesicular Lactobacillus instillation or to seek medical attention. 
The self-management protocol will also direct them to discontinue Lactobacillus instillation or to 
seek medical attention if symptoms remit, persist or worsen. If participants are directed by the 
self-management protocol to seek medical attention or s/he feels the need for medical evaluation, 
s/he will be advised to obtain care as they typically would by their health care provider. 
Participants will be supplied with letters to be brought to their health care provider notifying them 
of the study and requesting sharing of urinalysis and urine culture results with the research team. 
A verified UTI will include those that resulted in antibiotic treatment prescribed by a health care 
professional. 
 
For the intravesicular Lactobacillus instillation, the contents of 1 Lactobacillus capsule will be 
mixed into 45 cc sterile 0.9% saline (adult bladder volume is 450cc, representing 10% of adult 
bladder volume).  After mixing, study participants will draw up the 45 cc of the liquid 
Lactobacillus mixture into a 60cc catheter tip syringe and instill via the intermittent catheter using 
sterile technique. For children volume is based on age (see table on page 4 of protocol).  
 
Study participant will recheck symptoms after 24 hrs (valid range 22-30 hrs) following the 
SMPPRO protocol and either perform another Lactobacillus instillation or seek medical attention, 
per the subjects choice. If symptoms still persist 4 hrs after second instillation, study participants 
are instructed to seek medical attention.  
 
Post-Intervention Follow-Up: After completion of the 6-month patient-initiated self-management 
protocol intervention period, participants will monitor symptoms weekly using the USQ-NB. 
 
Measurements:  
Health History Questionnaire: After subjects give consent, a brief medical history will be 
conducted using a structured questionnaire, to obtain demographic information and relevant 
family history (these items will be collected only at baseline), and genitourinary history 
Urinary Symptom Questionnaire (USQ-NB): A preliminary urinary symptom questionnaire 
has been developed with consumer input based on the most common typical and SCI/MS-specific 
urinary symptoms. The final version (developed in Specific Aim 1 IRB # 2014-187) will be used 
in this phase of the study. USQ-NB will be completed weekly.  All participants will receive a link 
to the questionnaire every Sunday morning. If they have not responded by Monday morning, they 
will receive another link. If they have not responded by Tuesday, the study participant will 
receive a phone call. If there is no response by noon Wednesday, it is considered a missed event. 
Patient satisfaction: We have developed a simple four item patient satisfaction “survey” that 
will be requested at the end of each timeframe. Participants will be asked to rate their satisfaction, 
thinking only about the preceding time period, with changes in the frequency of their symptoms; 
the amelioration of the severity of their symptoms; and changes in the impact of their symptoms. 
A 5-point Likert scale will be used for each of these items (not at all satisfied; not very satisfied; 
no perceived difference; satisfied; and very satisfied). A fourth item, “Can you estimate, using a 
scale from 0% <would absolutely never do this>, through 50% <might do this> to 100% <would 
absolutely always do this>, whether or not you would seek this intervention out and pay for it 
yourself if insurance did not pay for it”.   
 
Medical Information: 


 
The Impact of Self-Management with Probiotics on Urinary Symptoms and the Urine Microbiome in 
Individuals with Spinal Cord Injury and Spina Bifida Protocol 
5/18/2017 
Page 7 of 17 
 
 
If participants instill lactobacillus at any time during the intervention phase, and  after 
Lactobacillus instillation reports a UTI as an Adverse Event (AE) we will request their urinalysis 
and urine culture results to attempt to determine if Lactobacillus might have been the cause of the 
UTI. If determined necessary by  the PI, Co-PI and/or DSMB other AEs reported could require 
retrieval of medical records.  
 
Phase III: Pilot Study of Multiple (up to 2) Lactobacillus Bladder Instillations 
We will recruit 30 persons in whom we will collect urine up to 18 timepoints (up to monthly for 
the first year when not experiencing symptoms, up to two times when subject experience urinary 
symptoms, follow-up after either lactobacillus treatment or antibiotics, and three (3) months into 
the six (6) months follow-up) in order to estimate the strength of the associations between 
successful implementation of the probiotic self-management program and urinary 
symptoms, bladder inflammation, and the urine microbiome. We will conduct an 18-month 
study in which each participant will serve as his/her own control through 3 phases of study: 6-
months usual care (baseline), 6-months probiotic intervention, and 6-months follow-up  
Study Design: We will recruit N=30 subjects with neuropathic bladder who will contribute urine 
samples for analyses. 
Clinical Setting: MedStar National Rehabilitation Hospital and Children’s National Medical 
Center. 
Target Enrollment: 30 adult subjects at least 1-year post-injury and 18 years of age with SCI/MS 
or at least 18 years of age with SB  
Recruitment Strategy: SCI/MS and SB subjects will be enrolled with assistance from United 
Spinal Association. Additionally, we will query the MedStar National Rehabilitation Hospital 
inpatient service, the 40+ MedStar National Rehabilitation Network outpatient sites, various 
MedStar Network databases, and the CNMC Spina Bifida database. Databases will be queried for 
participants who meet the eligibility criteria and have already provided HIPAA authorization and 
consent to be contacted for future research. Announcements and IRB-approved flyers will be 
posted on the United Spinal Association and study websites and at participating institutions. 
Informed Consent: Consent will by obtained by the PI, Study Coordinator, or Research Assistant. 
All project staff are currently CITI certified and have passed all requirements for HIPAA 
certifications.  
Inclusion criteria for participants with SCI/SB/MS includes: 1) age≥18 years; 2) SCI/SB/MS; 3) 
neuropathic bladder at least 1-year duration, as determined by the attending physician; 4) utilizing 
intermittent catheterization for bladder management; 5) a history of 2 or more UTIs in the past 
year; 6) community dwelling, 7) Enrollment in Phase II 
Exclusion Criteria: Study candidates with the following will be excluded: 1) known genitourinary 
pathology beyond neuropathic bladder (i.e., vesicoureteral reflux, bladder or kidney stones, etc.); 
2) use of prophylactic antibiotics; 3) instillation of intravesicular agents to reduce UTI (i.e., 
gentamycin); 4) psychologic or psychiatric conditions influencing the ability to follow 
instructions; 5) participation in another study in which results would be confounded; 6) pregnant 
or breastfeeding; 7) individuals with a history of acquired or genetic immunodeficiencies; active, 
acute or chronic serious infections (i.g., viral hepatitides, HIV/AIDs); 8) individuals with 
cancer/autoimmune disorders; 9) serious allergy to any component or excipients in the live 
bacterial combination product; 10) no change in neurologic status in the previous 2 weeks; 11) 
taken antibiotic for any reason in the previous 2 weeks; 12) any patient with history of sensitivity 
or allergy to ampicillin or tetracycline; and 13) current urinary tract infection or urinary tract 
infection within the previous 2 weeks (as defined by Infectious Diseases Society of America 
CAUTI Guidelines, 2010 14) Not enrolled in Phase II. According to the Infectious Disease 
Society of America Guidelines for Diagnosis of Catheter-Associated UTI, the presence of the 


 
The Impact of Self-Management with Probiotics on Urinary Symptoms and the Urine Microbiome in 
Individuals with Spinal Cord Injury and Spina Bifida Protocol 
5/18/2017 
Page 8 of 17 
 
following urinary symptoms is required for UTI diagnosis in patients using intermittent 
catheterization: new onset or worsening fever, rigors, altered mental status, malaise, lethargy with 
no other identified cause, flank pain, costovertebral angle tenderness, acute hematuria, pelvic 
discomfort, increased spasticity, autonomic dysreflexia or sense of unease. The Guidelines 
specifically state that “the presence or absence of odorous or cloudy urine should NOT be used as 
an indication for urine culture or antimicrobial therapy” and “the presence, absence or degree of 
pyuria should NOT be interpreted as an indication for antimicrobial treatment.”  If a subject 
presents with any of these symptoms and there are ≥103 cfu/hpf bacterial growth on urine culture, 
the subject will be excluded and treated for a UTI according to clinical best practice. 
 
Participant Burden:   
Urine samples for urinalysis, urine culture and metagenomic sequencing will be obtained at up to 
monthly for the first year of study participation when not experiencing symptoms, at up to 2 
occurrences of urinary symptoms during SMPPRO intervention phase, and follow-up after either 
lactobacillus treatment or antibiotics and at follow-up, for a total of up to 18 samples per patient 
depending on the frequency with which urinary symptoms are experienced. When possible, our 
Research Assistant will meet with the patient at a mutually convenient location. 
 
 
 
 
Baseline Data Collection:  
Per IRB protocol # 2014-211 phase II, after consent, participants will complete the Health History 
Questionnaire, Urinary Symptom Questionnaire, In addition to questionnaires, all participants 
will provide a urine sample using sterile catheterization techniques. Thereafter, participants will 
complete the Urinary Symptom Questionnaire weekly.  
 
Intervention: 
 After 6 months of baseline data collection the intervention phase will begin. Prior to 
implementing the intervention protocol, participants will provide study staff with a urine sample 
either at MedStar NRH, CNMC, or when possible, the study RA will meet with the participant at 
a mutually convenient location. After urine sample is collected, study participants will follow the 
study protocol and complete USQ-NB weekly. If/when urinary symptoms occur, they will be 
instructed to follow the SMPPRO protocol to determine whether to initiate intravesicular 
Lactobacillus instillation or be evaluated by a physician. The self-management protocol 
(SMPPRO) will also direct them to discontinue Lactobacillus instillation or be evaluated by a 
physician if symptoms remit, persist or worsen. If participants are directed by the self-
management protocol to seek medical attention or s/he feels the need for medical evaluation, s/he 
will be advised to obtain care as they typically would by their health care provider. Participants 
will be supplied with letters to be brought to their health care provider notifying them of the study 
and requesting sharing of urinalysis and urine culture results with the research team. A verified 
UTI will include those that resulted in antibiotic treatment by a health care professional. An 
additional urine sample for metagenomics will either be left with the health care provider for pick 
up by the research team, brought to the research site, or obtained by the RA at a mutually 
convenient site. If participant is treating urinary symptoms at home with Lactobacillus, a urine 
sample will be dropped off either at MedStar NRH, CNMC or when possible, the study RA will 
meet with the participant at a mutually convenient location. 


 
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For the intravesicular Lactobacillus instillation, the contents of 1 Lactobacillus capsule will be 
mixed into 45 cc sterile 0.9% saline (adult bladder volume is 450cc, so this amount represents 
10% of adult bladder volume).  After mixing, study personnel will draw up the 45 cc of the liquid 
Lactobacillus mixture into a 60cc catheter tip syringe and instill via the intermittent catheter. For 
children volume is based on age (see table on page 4 of protocol). Study participant will recheck 
symptoms after 24 hrs (valid range 22-30 hrs) following the SMPPRO protocol and either complete 
another Lactobacillus instillation or seek medical attention. If symptoms still persist 4 hrs after 
second instillation, study participants should seek medical attention.  
 
Post-Intervention Follow-Up:  
After completion of the 6-month patient-initiated self-management protocol intervention period, 
participants will monitor symptoms weekly using the USQ-NB. At the end of follow-up, we will 
obtain another urine sample from all participants. 
 
Measurements:  
In addition to the measurements described in Phase II the following measurements will occur for 
phase III; 
Urine Acquisition: A 50-100 ml urine sample will be collected from each subject for (1) 
urinalysis and cultivation, and (2) urine microbiome by SMRT sequencing. Urine will be 
collected from a new, unused intermittent catheter.  
Urine Preparation: Collected urine will be immediately placed at 4°C. Within 6 hours, 
samples will be taken to CNMC for sequencing preparation. At CNMC, urine samples will be 
centrifuged at 4°C, 5,000 x g for 20 minutes. The supernatant will be aliquoted in 2 ml cryotubes 
and frozen at -20°C. 10ml PBS will be added to the pellet with the remaining supernatant and 
then centrifuged (5,000xg) at 4°C for 20 minutes. The pellets and aspirated PBS wash solution 
will be stored at -20°C. Pellets will later be transferred for DNA isolation and SMRT sequencing.  
Urinalysis: Urine samples will be assessed utilizing standard clinical microbiology 
semiquantitative chemical testing using commercial disposable test strips for glucose, bilirubin, 
ketone, specific gravity, blood pH, protein, urobilinogen, nitrite, and leukocyte esterase. After 
centrifuging for 5 minutes, microscopic examination for and quantification of WBCs, RBCs, 
epithelial cells, yeast, bacteria, Trichomonas vaginalis, sperm cells, mucous filaments and 
crystals will be performed using standard microbial techniques. 
Urine Culture: Standard urine culture microbiology will be performed. A measured amount is 
inoculated to each of the appropriate media. A calibrated loop designed to deliver a known 
volume (0.0 I ml per loopful) of urine is used. The sample will be mixed the thoroughly and the 
top of the container removed. A calibrated wire-inoculating loop is flamed and allowed to cool 
without coming in contact with any surface. The sterile loop is inserted into the urine sample 
vertically and urine is allowed to adhere to the loop. The loopful of urine is inoculated over 
MacConkey agar plates using standard methods. Similarly, a second loopful is collected and 
inoculated on a blood agar plate. The plates are incubated aerobically at 35-37oC for at least 24 
hours. The colonies and colony forming units (CFUs) are counted by multiplying with 100 (since 
0.01 ml loop was used). 
SMRT Sequencing Methods: Thawed urine samples will be clarified by low-speed 
centrifugation and bacterial genomic DNA will be isolated from pelleted bacteria using a DNeasy 
tissue extraction kit (Qiagen) according to manufacturer’s instruction. 16S rRNA genes will be 
amplified using universal primers 28F 5’- AGAGTTTGATCMTGGCTCAG-3’ and 1492R 5’-
ACCTTGTTACGACTT-3’.  These primers will amplify sequences that correspond to the 
sequence between positions 27 and 1492 in E. coli 16S rRNA and produce amplification products 
of approximately 1500 bp. These amplification products will be used for sequencing template 


 
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preparation using DNA Template Prep Kit 2.0 (Pacific Biosciences) followed by DNA 
Polymerase binding (DNA Polymerase Binding Kit P4, Pacific Biosciences). Briefly, ends of the 
16S rRNA will be repaired with the Klenow DNA polymerase, SMRT Bell adapters will be 
ligated with the PCR products, followed by annealing of sequencing primers and ternary complex 
formation with the P4 DNA polymerase. Ternary complexes will be loaded onto SMRT cells and 
sequenced on the Pacific Biosciences RSII instrument using DNA Sequencing Reagent 2.0 and 
one 90 min movie. Resulting circular consensus sequence (CCS) reads will be used for taxonomic 
classification and diversity measurements. The amount of ternary complex needed for the optimal 
loading of the zero molecular waveguides (ZMW) will be optimized by loading different amounts 
of ternary complex and measuring number of productive ZMWs. Sequenced rRNA genes will be 
analyzed and classified using Pathoscope.  We will use the following formula to determine the 
number of SMRT cells needed to detect most/all bacterial species in our samples: # of SMRT 
cells = (189 * 1 * 87/30,000).  Our preliminary data from 454 sequencing indicated the presence 
of approximately 189 bacterial species in samples from patients with neuropathic bladder. The 
ratio between most and least abundant bacterial species was 87. CCS protocol yields one accurate 
consensus read per sequenced rRNA gene, representing a member of the microbiome. Therefore, 
to ensure detection of most/all bacterial species in the sample and accurate sequence 
determination 1 SMRT cell per patient sample will be sufficient.  
     Patient satisfaction: We have developed a simple four item patient satisfaction “survey” that 
will be requested at the end of each timeframe. Participants will be asked to rate their satisfaction, 
thinking only about the preceding time period, with changes in the frequency of their symptoms; 
the amelioration of the severity of their symptoms; and changes in the impact of their symptoms. 
A 5-point Likert scale will be used for each of these items (not at all satisfied; not very satisfied; 
no perceived difference; satisfied; and very satisfied). A fourth item, “Can you estimate, using a 
scale from 0% <would absolutely never do this>, through 50% <might do this> to 100% <would 
absolutely always do this>, whether or not you would seek this intervention out and pay for it 
yourself if insurance did not pay for it”.    
 
Data and Safety Monitoring Plan 
A Data Safety Monitoring Board has been established. Data and Safety Monitoring Board 
(DSMB) reports will be prepared monthly during the Phase I “Baseline: Safety Evaluation of a 
Single Instillation in Adults” period, and then two times a year (or as requested by the DSMB) for 
Phase II.  Each report will consist of five sections, including: 1) recruitment, 2) adverse effects of 
the investigational product (i.e. the intravesicular Lactobacillus instillation), 3) completion of the 
Urinary Symptom Questionnaire (USQ-NB), 4) completion of, and adherence to, the Self-
Management Protocol (SMPPRO), and 5) data accrual and quality.  The recruitment section will 
include counts for screening (eligible v. ineligible), enrollment (consented v. declined), and 
retention (completion of phase milestones and transitions v. withdrawal or lost-to-follow-up).  For 
reporting purposes, subgroups will include age (adult v. child), neuropathic bladder etiology 
(Spinal Cord Injury v. Spina Bifida), and standard demographics (i.e. gender, race, and ethnicity).  
The adverse effects section will report all adverse outcomes that appear to be associated with the 
investigational product, and will include specific event details, relevant tests and laboratory 
results, relevant histories (including pre-existing and co-existing medical conditions), use of 
concomitant medications, and investigational product dose, frequency, route of instillation, timing 
of instillation, manufacturer’s lot number, and manufacturer’s expiration date (as appropriate for 
completion of the MedWatch FDA Form 3500A).  The USQ-NB completion section will report 
the frequency of on-line survey completion, with and without the need for study staff reminders.  
The SMPPRO completion/adherence section will report the frequency of on-line survey 
completion, with and without reminders, as well as the appropriateness/correctness of protocol 


 
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implementation, investigational product instillation, and medical care seeking.  The data accrual 
and quality section will report counts of completed surveys and case report forms at phase-
specific time points, as well as counts of specific responses to individual questions. 
 
H1. Compared with usual management, self-management of urinary symptoms with 
probiotics will reduce the frequency, severity and impact of urinary symptoms and UTIs 
(PRIMARY OUTCOMES).  The goal of aim 2 is to assess the effect of Lactobacillus 
supplementation on the frequency, severity and impact of urinary symptoms and UTIs.  This aim 
can be evaluated with all 100 participants in the SMP pilot study. We expect to find a significant 
difference in these primary outcomes between our three experimental timeframes (6 months prior 
to intervention, 6 months of SMPPRO intervention, and 6 month follow-up period).  Frequency of 
UTI will be defined as the number of verified UTIs occurring during the timeframe. Urinary 
symptoms will be assessed using the USQ-NB where frequency of symptoms, degree of severity, 
and impact on the individual, will be considered.   
We will estimate the average number of symptoms on the USQ-NB endorsed 
(irrespective of frequency, severity and impact) across the weeks during that time frame, for each 
participant. Thus, each participant will have a continuous outcome (average number of USQ-NB 
items endorsed). We will also compute the number of verified UTIs experienced by each 
participant. These two outcomes are the primary outcomes for our study and analysis; they 
depend solely on the face validity of the USQ-NB,and on clinically verified UTIs, and so will not 
be affected by a failure to find or establish formal measurement properties for the USQ-NB, 
should any occur. They will also be less subject to any variability in participant perceptions of 
frequency, severity or impact, which can complicate analytic assumptions and limit power and 
interpretability of change that might be perceived. We will describe these features of the sample 
over time but will not conduct inference tests on these ratings. 
We propose linear mixed-effects modeling to explore the effect of introduction to/use of 
the SMPPRO on the number of USQ-NB symptoms (averaged over the entire time frame) and the 
incidence of UTIs. Each model will include gender as a fixed effect, and time (baseline; SMPPRO 
use; post-SMPPRO observation) as the explanatory variable, and random effects for intercept 
(subject) only. This method allows for missing data and the inclusion of covariates where 
appropriate; however we plan post-hoc analyses of the modeling in order to pinpoint potential 
interaction effects (e.g., between age or sex and time).  The focus of the analyses is to determine 
whether (irrespective of individual starting values or specific slopes) mean USQ-NB item 
endorsement and UTI counts go down in the SMPPRO time frame relative to the baseline values 
(generally negative associations in each regression between the outcome and time) and whether 
these values stay flat (zero slope), continue to decrease (negative slope) or increase (positive 
slope) during the post-SMPPRO follow up.  
Power calculation: We plan to use mixed effects regression modeling for the most 
precision in our estimated effects of the SMPPRO on the USQ-NB symptom rate and UTI rate. 
However, it is difficult to predict the exact form of the final models since our understanding of 
the USQ-NB symptom makeup, and the univariate associations between gender or age (or any 
other of the demographic variables we will collect) and these two outcomes. Therefore, for the 
most conservative estimates, we use the most basic analytic method to estimate the worst-case 
scenario power calculation, a t-test. In this pair of primary outcomes, alpha is set at 0.025 for each 
power calculation (and for each inference test). We will have data from 100 participants; 
however, to protect against a possible 20% drop out rate over the course of the 18-month study, 
we will recruit 120 persons for this arm, hoping for a final N of 100. If we observe an average of 
25 items on the USQ-NB endorsed over the baseline period, then with alpha = 0.025 we would 
have 81% power to detect a reduction in this average number of items endorsed as low as five 


 
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(i.e., to detect a change from an average of 25 to an average of 20) during the SMPPRO period, 
assuming that the standard deviation of the average number of endorsed USQ-NB items is as high 
as 16. If we observe an average of one UTI per month in the initial 6-month period (for an 
average of six UTIs across this sample of 100 persons), then, with the alpha of 0.025 we would 
have 81% power to detect a reduction in this average number of UTIs as low as 1.25 (i.e., to 
detect a change from an average of 6 to an average of 4.75) during the SMPPRO period, assuming 
that the standard deviation of the number of UTIs is as high as 4. We will explore the consistency 
of the UTI variable with the assumption of a normal distribution, and if appropriate we may 
utilize Poisson modeling if the assumption of normality is violated. 
 
Additionally, because we have the potential to carefully describe the symptom profile for 
neuropathic bladder generally and for SCI/MS and SB specifically, we also plan secondary 
analyses based on the USQ-NB items. Frequency, severity, and impact of each of the USQ-NB 
symptoms will be estimated for each participant, within each of the study timeframes. Each of 
these symptom characteristics will be rated on an ordinal scale, so for each symptom on the USQ-
NB, the median frequency rating from their weekly USQ-NBs will be computed. These ordinal 
outcomes will be explored for similarities and differences across sex, age, injury (SB/SCI/MS), 
and other demographic variables that could be useful in diagnosis or management of USQ-NB 
symptoms by us and other investigators in future studies. Descriptive statistics for each primary 
outcome (frequency, duration, and severity of each UTI) and their change from baseline, will be 
produced for each time frame. We will generate preliminary (underpowered and not corrected for 
multiple comparisons) estimates of the strengths of associations between item frequency, 
severity, and impact with time, sex, age, satisfaction with the SMP-UTI, and etiology 
(SCI/MS/SB). If the reliability and validity evidence from Aim 1 and that national sample 
supports the estimation of latent factor scores on the USQ-NB factors, we will also compute 
preliminary estimates of association between these covariates and the factor scores, as well as the 
association between the SMP-UTI and changes in these factor scores. This estimation will only 
be appropriate if measurement invariance across groups is obtained in Aim 1, but since we would 
not yet have evidence of invariance over time, these estimates would be considered preliminary.  
 
H2. Compared with usual management, self-management of urinary symptoms with 
probiotics will reduce bladder inflammation (Secondary outcome). For those 20 participants 
in the subsample giving urine samples, bladder inflammation will be measured from each urine 
sample using a standard white blood count (WBC) from the urinalysis. WBC counts will be 
obtained only at the start of each timeframe, and so can be analyzed longitudinally as continuous 
variables representing the 6-month pre-treatment period and the 6 month-SMPPRO period. As such 
we plan a mixed effects linear regression model of WBC counts with time treated as the factor of 
interest.  This analysis will assess whether WBC is significantly different from the SMPPRO, and 
post-SMPPRO, relative to the baseline pre-treatment periods.   
 
H3. Compared with usual management, self-management of urinary symptoms with 
probiotics will reduce the need for antibiotics to treat UTI (Secondary outcome). The 
number of times antibiotics are used for each patient will be obtained from their medical records 
or self report. This value will be analyzed with the same three-point longitudinal mixed effects 
regression model with time as the factor of interest. Depending on the distribution of the outcome 
(i.e. given the count nature of this outcome, it may be Poisson distributed), we will utilize either 
linear or Poisson regression models.  This analysis will assess whether there is an association 
between UTI rate during use of the SMPPRO, and post-SMPPRO exposure, relative to UTI rate 
during the baseline pre-treatment period.   
 


 
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H4. Compared with usual management, self-management of urinary symptoms with 
probiotics will return the microbiome to baseline (Secondary outcome). For analysis of the 
microbiome, the fasta and fastq files of each sample’s CCS reads will be retrieved and processed 
using LUCY54 to filter out reads with low quality segments, as well as remove sequences that are 
shorter than the full length 16S rRNA (approximately 1500 bp). The sequences will then be 
aligned against the SILVA 16S rRNA database followed by subsequent implementation of 
Mothur’s Chimera Slayer  to filter out chimeric reads from sequences in each sample. The 
remaining sequences will undergo taxonomic classification. Bowtie2 will align each sequence 
read to a reference genome derived from the SILVA 16S database and output a SAM file. With 
the resulting SAM file, Pathoscope 51 is capable of characterizing the biological sample to the 
subspecies-level, generating an excel file with the proportions of each classified organism within 
the sample tabled. 
To determine whether bacterial diversity differs between study groups the Shannon Diversity 
Index will be used. The Shannon Index is a measure of sample diversity that takes into account 
total number of bacterial types in the sample and relative proportion of the bacterial types. From 
the subsample of 50 participants giving urine samples, subjects who did in fact utilize the SMP at 
least once during the 6-month SMP time frame will be stratified into two groups, those who 
responded to SMP and those who did not; we will define “response” as the observation of a 10% 
or more decrease in the average number of USQ items endorsed within any time frame, so that 
each of the 50 persons in this sub-sample will be characterized as either a responder (10% or 
more decrease) or a non-responder (0-9.99% decrease).  A scatter plot of response group versus 
Shannon Index will be produced, and a mixed effects linear regression model will test the 
individual (main) and interaction effects of response group and time on Shannon Index. A 
positive time coefficient is indicative of increased microbiome diversity and a negative time 
coefficient indicates reduces diversity. The magnitude of difference between the response groups 
will be determined using the Yue-Clayton theta similarity index (). The  index measures the 
difference in the number and relative abundance of bacterial species between samples4,5 when 
=1 two samples have identical community structure and when =0 indicates complete 
dissimilarity between samples. Pathoscope will be used to calculate abundance of different 
taxonomic groups at each time point. 
 
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