Citation
A Within-Subjects Comparison of Two Antegrade Flushing Regimens in Children

Material Information

Title:
A Within-Subjects Comparison of Two Antegrade Flushing Regimens in Children
Creator:
Jarczyk, Kimberly S
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (6 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Nursing Sciences
Nursing
Committee Chair:
PIEPER,PAMELA
Committee Co-Chair:
HORGAS,ANN L
Committee Members:
ELDER,JENNIFER HARRISON
SHUSTER,JONATHAN J

Subjects

Subjects / Keywords:
ace -- antegrade -- bowel -- continence -- enema -- fecal -- incontinence -- malone -- neurogenic -- pediatric
Nursing -- Dissertations, Academic -- UF
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Nursing Sciences thesis, Ph.D.

Notes

Abstract:
Fecal incontinence past the time of toilet training is devastating to affected children. Antegrade continence enema (ACE) therapy administered through a catheterizable stoma surgically placed in the cecum has helped children with intractable fecal incontinence attain continence for stool. Retrospective studies demonstrate favorable continence outcomes and prospective studies demonstrate improvement in quality of life on ACE therapy. There are no prospective trials comparing the effectiveness of different flushing regimens on continence. The purpose of this prospective study was to compare two distinct flushing regimens, saline and USP glycerin, in the immediate postoperative period in children requiring ACE therapy. The aims of this study were to a) identify the minimal administration frequency, titration time to reaching effective dose, and cost of two flushing solutions; b) compare which solution at an optimum dose was delivered in the least amount of time, with fewer side effects, while promoting the higher degree of fecal continence and quality of life; and c) process stool for down- stream identification of 16SrRNA microbiome gene.This prospective study utilized a repeated measures, single subjects alternating treatments withdrawal design in which all subjects were tested under all conditions and each subject acted as his or her own control. A within subjects cross-over design was embedded in the dosing and maintenance treatment comparison phases of the study. Subjects were randomly assigned to one of two treatment sequences. Five subjects were recruited between 2015 and 2017. All subjects had fecal incontinence secondary to low level spinal cord lesions. Only 1/5 subjects (20%) gained continence on saline while 4/5 subjects (80%) gained continence on USP glycerin. Moderate pain with flushing was consistently present in 1/5 subjects (20%) and 1/5 subjects (20%) was dropped from the study due to vegal symptoms. There was no electrolyte imbalance associated with either regimen. Saline was the more costly regimen. Findings did not reach statistical significance due in part to small sample size. Findings may have clinical relevance when applied to male children under the age of 8 with fecal incontinence due to low level spinal cord lesions. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2017.
Local:
Adviser: PIEPER,PAMELA.
Local:
Co-adviser: HORGAS,ANN L.
Statement of Responsibility:
by Kimberly S Jarczyk.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
LD1780 2017 ( lcc )

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A WITHIN SUBJECTS COMPARISON OF TWO ANTEGRADE FLUSHING REGIMENS IN CHILDREN By KIMBERLY SUSAN SMITH JARCZYK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIRE MENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017

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2017 Kimberly Susan Smith Jarczyk

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To my husband William Aden Cover, Jr and my son Adam Bennett Jarczyk

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4 ACKNOWLEDGMENTS I would like to thank my husband, William Aden Cover, Jr. for his unstinting support throughout my doctoral studies. His constant encouragement was foundational to the successful completion of my degree. I would like to thank my son, Adam Bennett Jarczyk for tak ing precious vacation time to fly home from Singapore to share his expertise in data visualization I would like to thank my Dissertation Committee Chair, Dr. Pam Pieper for patiently guiding me through the challenges of doctoral work. I would like to th ank Dr. Jenn i fer Elder for opening my horizons to the possibilities of single subjects research. I would like to thank Dr. Ann Horgas for assisting me in substruct ing Probabalistic Epigenesis thereby providing a theoretical framework f or my proposed body of research I would like to thank Dr. Jonathan Shuster for agreeing without hesitation to be my minor and external committee member and s haring his immense expertise in helping me refine my study design I would like to thank Mr. Matth ew Robinson for assisting in inferential data analysis I would like to thank Dr. Donald George and Dr. Mark Barraza for their clinical mentorship and for their encouragement and support in pushing me to reach beyond self imposed boundaries and achieve mo re than I thought I was capable of achieving I would like to thank Dr. Kathryn Blake for providing me with direction in completing my FDA and IRB applications. I would like to than k Dr. J ames Sylvester and Karl Mann for their instruction and oversight in learning the process of extracting DNA from the gut microbiome. In conclusion, I would like to thank my clinical colleagues and co workers

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5 Susan Carnall, Kelly Ez zell, Elizabeth Eminisor and Key e ra Boyce for their support and encouragement throughout my doctoral studies.

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6 TABLE OF CONTENTS page ACKNOWLE DGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATION S ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Epidemiology ................................ ................................ ................................ .......... 16 Background ................................ ................................ ................................ ............. 16 Theoretical Framework ................................ ................................ ........................... 19 Review of Pertinent Literature to Gu ide Theory Development ................................ 19 Physioanatomic Considerations ................................ ................................ ....... 20 Linkages between bowel, bladder, and pelvic floor function. ..................... 20 Nervous system associations ................................ ................................ ..... 23 Animal studies ................................ ................................ ............................ 23 Human studies ................................ ................................ ........................... 24 Developmental Considerations ................................ ................................ ......... 26 Pain and learning in children ................................ ................................ ...... 27 Toilet trai ning ................................ ................................ ............................. 28 Psychosocial Considerations ................................ ................................ ............ 29 Psychological associations ................................ ................................ ........ 2 9 Social associations ................................ ................................ .................... 30 Anthropometric and Nutritional Considerations ................................ ................ 31 Obesity ................................ ................................ ................................ ....... 31 Dietary considerations ................................ ................................ ............... 32 Genetic Considerations ................................ ................................ .................... 32 Summary of Pertinent Literature to Guide Theory Development ............................ 33 Identification of a Metaparadigm to Guide Theory Development ............................ 34 Probabilistic Epigenesis ................................ ................................ .......................... 35 Historical Overview ................................ ................................ ........................... 35 Constructs and Relational Statements ................................ ............................. 36 The Applicability of Probabalistic Epig enesis to Pediatric Incontinence Research 38 The Integrative Model of Pediatric Incontinence and Dysfunctional Elimination ..... 39 Prop osed Program of Research ................................ ................................ ............. 41 Problem ................................ ................................ ................................ .................. 41

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7 Purpose, Aims, and Hypothesis: ................................ ................................ ............. 43 Null Hypotheses for Aim 1 ................................ ................................ ...................... 43 Null Hypotheses for Aim 2 ................................ ................................ ...................... 44 Null Hypothesis for Aim 3 ................................ ................................ ........................ 44 2 LITERATURE REVIEW ................................ ................................ .......................... 47 Overview ................................ ................................ ................................ ................. 47 Studies Addressing the Effectiveness of ACE Therapy in Promoting Cont inence ................................ ................................ ................................ .... 47 Studies Addressing Side Effects Associated with ACE Therapy ...................... 53 Studies Addressing Gut Microbiota ................................ ................................ .. 55 Significance and Innovation ................................ ................................ .................... 58 3 METHODOLOGY ................................ ................................ ................................ ... 63 Design ................................ ................................ ................................ ..................... 63 Rationale for Utilization of Within Subjects Designs ................................ ............... 64 Inclusion and Exclusion Criteria ................................ ................................ .............. 68 Regu latory Considerations and Study Expenses ................................ .................... 69 Subjects ................................ ................................ ................................ .................. 70 Setting ................................ ................................ ................................ ..................... 70 Measures ................................ ................................ ................................ ................ 72 Materials ................................ ................................ ................................ ................. 75 High Volume Flush ................................ ................................ ........................... 76 Low Volume Flush ................................ ................................ ............................ 76 Procedures ................................ ................................ ................................ ............. 77 High Volume Regimen ................................ ................................ ..................... 77 Low Volume Regimen ................................ ................................ ...................... 78 Optimal Dose Regimen ................................ ................................ .................... 79 Regimen Comparative Phase ................................ ................................ ........... 79 Specimen Collection ................................ ................................ ......................... 80 Statistical Analysis ................................ ................................ ................................ .. 81 Social Validity ................................ ................................ ................................ ......... 85 Threats to Validity ................................ ................................ ................................ ... 87 4 FINDINGS ................................ ................................ ................................ ............... 93 Subject Characteristics ................................ ................................ ........................... 93 Aims ................................ ................................ ................................ ........................ 93 Null Hypotheses for Aim 1 ................................ ................................ ...................... 94 Frequency of Administration ................................ ................................ ............. 94 Titration Time ................................ ................................ ................................ ... 94 Cost Burden ................................ ................................ ................................ ..... 95 Null Hypotheses for Aim 2 ................................ ................................ ...................... 96 Continence ................................ ................................ ................................ ....... 96

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8 Infusion Time ................................ ................................ ................................ .... 96 Procedural Time ................................ ................................ ............................... 97 Side Effects ................................ ................................ ................................ ...... 97 Quality of Life ................................ ................................ ................................ ... 98 Electrolytes ................................ ................................ ................................ ....... 99 Stool Calprotectin ................................ ................................ ............................. 99 Flushing Preference ................................ ................................ ............ 100 5 DISCUSSION AND CONCLUSIONS ................................ ................................ .... 132 Aimes Specific Discussion of Outcomes ................................ ............................... 132 Frequency of Administration ................................ ................................ ........... 132 Flush Titration Time to Continence ................................ ................................ 133 Cost Burden ................................ ................................ ................................ ... 134 Fecal Continence ................................ ................................ ........................... 134 Infusion and Procedural Time ................................ ................................ ......... 137 Side Effects ................................ ................................ ................................ .... 138 Electrolytes ................................ ................................ ................................ ..... 138 Stool Calprotectin ................................ ................................ ........................... 139 Quali ty of Life ................................ ................................ ................................ 140 Study Limitations ................................ ................................ ................................ .. 140 Implications for Future Research and Practice ................................ ..................... 142 Probabalistic Epigenesis as a Framework for Pediatric Incontinence Research ................................ ................................ ................................ ..... 142 Implications for future research ................................ ................................ ...... 142 Implications for Clinical Practice ................................ ................................ ..... 144 Conclusions ................................ ................................ ................................ .......... 144 APPENDIX A THEORETICAL AND OPERATIONAL LINKAGES FOR RESEARCH ADDRESS ING INCONTINENCE ................................ ................................ .......... 146 B REQUIRED MATERIALS FOR FLUSHING PROCEDURE ................................ .. 147 C PROCEDURAL INSTRUCTIONS FOR COMPLETING SALINE FLUSH .............. 148 D PROCEDURAL INSTRUCTIONS FOR COMPLETING USP GLYCERIN FLUSH 149 E MICROBIOME DNA EXTRACTION AND PROCESSING FOR DOWNSTREAM ANALYSIS ................................ ................................ ................................ ............ 150 F THREATS TO STATISTICAL CONCLUSION VALIDITY ................................ ...... 151 G THREATS TO INTERNAL VALIDITY ................................ ................................ .... 152

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9 H SOCIAL THREATS TO INTERNAL VALIDITY ................................ ..................... 153 I THREATS POSED TO CONSTRUCT VALIDITY OF CAUSE AND EFFECT ....... 154 J NEMOU RS CLINICAL RESEARCH COMMITTEE APPLICATION AND REVIEW ................................ ................................ ................................ ................ 155 K FDA IND APPLICATION ................................ ................................ ....................... 158 L IAA AGREEMENT BETWEEN NEMOURS AND UNIVERSITY OF FLORIDA ..... 184 M MANAGEMENT OF RESEARCH PHARMACEUTICAL PRODUCTS .................. 186 N IRB SUBMISSION, APPROVAL AND APPLICATION FOR AND APPROVAL F OR AMMENDMENTS ................................ ................................ ......................... 192 O KJ001 WITHIN SUBJECTS ANALYSIS CALCULATIONS INCLUDING GRAPHS DETAILING STABILITY AND TREND ................................ .................. 242 P KJ002 WITHIN SUBJECTS ANALYSIS CALCULATIONS INCLUDING GRAPHS DETAILING STABILITY AND TREND ................................ .................. 252 Q KJ003 WITHIN SUBJECTS ANALYSIS CALCULATIONS INCLUDING GRAPHS DETAILING STABILITY AND TREND ................................ .................. 260 R KJ004 WITHIN SUBJECTS ANALYSIS CALCULATIONS INCLUDING GRAPHS DETAILING STABILITY AND TREND ................................ .................. 268 S KJ005 WITHIN SUBJECTS ANALYSIS CALCULATIONS INCLUDING GRAPHS DETAILING STABILITY AND TREND ................................ .................. 277 REFERENCES ................................ ................................ ................................ ............ 286 BIOGRAPHIC AL SKETCH ................................ ................................ .......................... 301

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10 LIST O F TABLES Table page 2 1 Summary of Published Flush Solution, Dosing, Administration Frequency and Procedural Time Outcomes ................................ ................................ ......... 59 3 1 Itemized Estimate for Maximum Flushing Costs For 6 Subjects ......................... 88 3 2 Level of Soiling ................................ ................................ ................................ ... 88 3 3 Dependent Variables ................................ ................................ .......................... 89 3 4 Dosing Strategy to Determine Minimum Volume and Frequency Necessary to Maintain Continence ................................ ................................ ........................... 90 3 5 Dosing Strategy to Determine Minim um Volume and Frequency Necessary to Minimize Side Effects ................................ ................................ ......................... 91 4 1 Individual Subject Data and Descriptive Statistics ................................ ............ 101 4 2 Cost Comparison Normal Saline vs USP Glycerin Flush ................................ .. 104

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11 LIST OF FIGURES Figure page 1 1 of the bidirectional coaction between levels of functioning over the course of developmen t. ................................ ................................ ................................ ...... 45 1 2 Reduced Model of Probabilistic Epigenesis adapted to test pediatric continence ................................ ................................ ................................ ......... 45 1 3 Model depicting specific interactions between organ systems contained within the neural constitutive element specific to incontinence and dysfunctional elimination ................................ ................................ ................... 46 3 1 Procedural timeline. ................................ ................................ ............................ 92 4 1 Frequency of flush administration ................................ ................................ ..... 103 4 2 Titration time to ac hieve continence ................................ ................................ 103 4 3 KJ001 Absolute frequency of incontinence graph ................................ ............. 105 4 4 KJ002 Absolute frequency of incontinence graph ................................ ............. 106 4 5 KJ003 Absolute frequency of incontinence graph ................................ ............. 107 4 6 KJ004 Absolute frequency of incontinence graph ................................ ............. 108 4 7 KJ005 Absolute frequency of incontinence graph ................................ ............. 109 4 8 KJ001 Frequency and severity of incontinence graph ................................ ...... 110 4 9 KJ002 Frequency and severity of incontinence graph ................................ ...... 111 4 10 KJ003 Frequency and severity of incontinence graph ................................ ...... 112 4 11 KJ004 Frequency and severity of incontinence graph ................................ ...... 113 4 12 KJ005 Frequency and severity of incontinence graph ................................ ...... 114 4 13 KJ001 Procedural time graph ................................ ................................ ........... 115 4 14 KJ002 Procedural time graph ................................ ................................ ........... 116 4 15 KJ003 Procedural time graph ................................ ................................ ........... 117 4 16 KJ004 Procedural time graph ................................ ................................ ........... 118

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12 4 17 KJ005 Procedural time graph ................................ ................................ ........... 119 4 18 KJ001 Abdominal pain graph ................................ ................................ ........... 120 4 19 KJ001 Cramping from flush graph ................................ ................................ .... 121 4 20 KJ002 Abdominal pain ................................ ................................ ...................... 122 4 21 KJ003 Abdominal pain ................................ ................................ ...................... 123 4 22 KJ003 Cramping with flush ................................ ................................ ............... 124 4 23 KJ004 Cramping with flush ................................ ................................ ............... 125 4 24 KJ005 Cramping with flush ................................ ................................ ............... 126 4 25 KJ001 Vagal symptoms graph ................................ ................................ .......... 127 4 26 Serum sodium levels ................................ ................................ ........................ 128 4 27 Serum potassium levels ................................ ................................ ................... 128 4 28 Serum chloride levels ................................ ................................ ....................... 129 4 29 Serum carbon dioxide levels ................................ ................................ ............. 129 4 30 Blood urea nitrogen levels ................................ ................................ ................ 130 4 31 Serum creatinine levels (low levels normal in children) ................................ .... 130 4 32 Serum calcium levels ................................ ................................ ........................ 131 4 33 Stool calprotectin levels ................................ ................................ .................... 131

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13 LIST OF ABBREVIATIONS A CE Antegrade continence enema BBD Bowel and bladder dysfunction FIC QOL Fecal Incontinence Quality of Life Measure in Children with Spinal Bifida GALT Gut associated lymphoid tissue IBD IOA Inflammatory bowel disease Interobserver Agreement Kg Kilogra m L Liter LUTD Lower urinary tract dysfunction mL Milliliter mmol Millimole OAB Overactive bladder Peds QL Pediatric quality of life qd Occurring or administered every day qod Occurring or administered every other day QOL Quality of life STC Slow transit constipation WBFPRS Wong Baker Faces Pain Rating Scale wk Occurring or administered weekly

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Doctor of Philosophy A WITHIN SUBJECTS COMPARISON OF TWO ANTEGRADE FLUSHING REGIMENS IN CHILDREN By Kimberly Susan Smith Jarczyk December 2017 Chair: Pamela Pieper Major: Nursing Sciences Fecal incontinence past the time of toilet training is devastating to affected children. Antegrade continence enema ( ACE ) therapy administered through a catheterizable stoma surgically placed in the cecum has helped children with intractable fecal incontinence attain continence for stool. Retrospective stud ies demonstrat e favorable continence outcomes and pr ospective studies demonstrate improvement in quality of life on ACE therapy T here are no prospective trials comparing the effectiveness of different flushing regimens on continence The purpose of this p rospective study was to compare two distinct flushing regimens, saline and USP glycerin, in the immediate postoperative period in children requiring ACE therapy. The aims of this study were to ( a) identify the minimal administration frequency titration ti me to reaching effective dose, and cost of two flushing solutions ; ( b) compare which solution at an optimum dose wa s delivered in the le ast amount of time, with fewer side effects, while promoting the highe r degree of fecal continence and quality of life; and ( c) process stool for down stream identification of 16SrRNA microbiome gene This prospective study utilize d a repeated measures, single subjects alternating treatments

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15 A B C B C B 1 withdrawal design in which all subjects were tested under all conditions and each subject act ed as his or her own control. A within subjects cross over design was embedded in the dosing A B C dosing and maintenance B C B 1 treatment comparison phase of the study S ub jects were randomly assigned to one of two treatment sequences Five subjects were recruited between 2015 and 2017. All subjects had fecal incontinence secondary to low level spinal cord lesions. Only one of five subjects (20%) gaine d continence on saline while four of five (80%) gained continence on USP glycerin. Moderate pain with flushing was consistently present in 1/5 subjects (20%) and 1/5 subject s (20%) was dropped from the study due to vagal symptoms. There was no electrolyte imbalance associated with either regimen Saline was the more costly regimen. Findings did not reach statistical significance due in part to small sample size. Findings may have clinical relevance when applied to male children under the age of 8 with fecal incontinence due to low level spinal cord lesions.

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16 CHAPTER 1 INTRODUCTION Epidemiology Incontinence and dysfunctional elimination impact a large percentage of children. Von Gontard, Heron, and Joinson (2011) reported a prevalence of 15.5% nocturnal enu resis and 7.8% diurnal enuresis in 7 year olds. Reports detailing the prevalence of pediatric constipation world wide range from 0.7% to 29.6% with a median of 12% (Mugie, Benninga, & Di Lorenzo, 2011). Constipation accounts for 25 % of referrals to pedi atric gastroenterologists. Estimates of the prevalence of childhood fecal incontinence range from 1% to 3% (Culbert & Banez, 2007). The majority of children with incontinence, constipation, and lower urinary tract symptoms do not have an organic or structu ral etiology for their symptoms. The prevalence of incontinence due to organic or structural etiologies is difficult to determine. Bischoff, Levitt, and Pena (2009) estimate there are 200,000 infants with m yelo meni ng ocele and 8,500 infants with anorectal m alformation born each year worldwide. M yelomeningocele is a part of a larger grouping of neurogenic etiologies including sacral agenesis, trauma, and tumor that often carry a poor functional prognosis for both urinary and fecal incontinence. Anorectal malf ormations are associated with a poor functional prognosis for fecal incontinence. Background and fecal continence. Incontinence in children past the expected time of toilet t raining is devastating and has been associated with substantial decrease in quality of life, increased abuse, bullying, anxiety, depression, greater social problems, and poorer

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17 school performance (Kaugars et al., 2010; Youssef, Langseder, Verga, Mones, & R osh, 2005). Incontinence is a subset within the broader category of dysfunctional elimination that includes chronic constipation, urinary frequency and urgency, and recurring urinary tract infection. Bower (2008) identified multiple domains including self esteem, mental health, independence, family functioning social interaction, and body image that were adversely affected by bladder and bowel dysfunction with or without incontinence. Results from a multicenter prospective trial compared the impact of f unctional constipation to the impact of functional constipation plus fecal incontinence in four different age domains on quality of life using five instruments including Pediatric Quality of Life Inventory, Pediatric Quality of Life Family Impact Module, Functional Disability Inventory Parent Version, Pediatric Inventory for Parents, and Pediatric Symptom Checklist Parent Report. The sample included 410 subjects ranging from 2 to 18 years of age. The four age domains were 2 to 4 years, 5 to 7 years, 8 to 12 years, and 13 to 18 years. Findings indicated children with combined constipation and fecal incontinence had worse family functioning that deteriorated as the child aged, significantly higher parental stress, and significantly lower emotional and b ehavioral well being when compared to the children who suffered with functional constipation alone. Adolescents with constipation and fecal incontinence in particular were adversely impacted and had a substantially worse quality of life as measured by lowe r scores (0 minimum, 100 maximum). When the Pediatric Quality of Life scores in this study sample (57), were compared to reported norms in healthy adolescents (83), Inflammatory Bowel Disease (79), chronically ill children (77), and children with acute ex acerbation of a chronic illness (79), the degree of devastation on the quality of life of

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18 affected adolescents caused by fecal incontinence becomes explicit. When the Pediatric Quality of Life scores of children with constipation and fecal incontinence gre ater than 8 years of age were compared to published Pediatric Quality of Life scores in other studies, scores of affected children were worse than children with Type 1 Diabetes, cancer in remission, heart disease, and transplant recipients (Kovacic et al., 2015). Bowel, bladder, and pelvic floor dynamics are closely interrelated. They share the same embryonic origins, are innervated from the same level of the spinal cord, and are all modulated by the central nervous system (Shafik, 1 984). Because dynamics o f the bowel, bladder, and pelvic floor are closely interrelated, organic or functional disruption in one of these systems can create a dysfunctional pattern of elimination affecting all of them. Incontinence and disordered elimination, whether it is primar ily structural, organic, or functional in etiology, is further complicated by developmental, psychological, and social problems. Therefore, it is important to address the physical, developmental, psychological, and social issues comprehensively to achieve better outcomes. In the North American pediatric system of health care delivery, bowel, bladder, and psychiatric issues are managed within the subspecialties of gastroenterology, urology, and psychiatry/psychology respectively. Providers in these subspeci alties do not share any forum in which they normally interact. They do not generally interface clinically and do not have journals, subspecialty societies, or national conferences in common. The care l subspecialties with resulting fragmentation of services Improved outcomes for this population of children will require a comprehensive and integrated understanding of the condition and a holistic approach to care.

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19 Theoretical Framework In an effort to find a unifying theory or framework to guide practice, an extensive review of the literature was conducted including searches in MEDLINE, Ovid MEDLINE, Cochrane Central Register of Controlled Trials, ERIC, EMBASE, CINAHL, and PsycINFO with the assistance o f two librarians from different organizations using over 25 search terms. Review of the references at the back of journal articles was completed. A textbook on nursing theory (Glanz, Rimer, & Viswanath, 2008) and several nursing theory websites were review ed. The search did not reveal a theoretical framework or model addressing incontinence and/or dysfunctional elimination in children. Review of Pertinent Literature to Guide Theory Development Pediatric dysfunctional elimination is a multifactorial, multi dimensional, complex interplay of factors including physioanatomic, neurological, developmental, psychosocial, environmental, and genetic determinants. Theory is needed to integrate these factors into a coherent whole that would enable clinicians to provid e comprehensive and effective care. The interrelationships between these factors have been demonstrated to varying degrees in the literature. The mechanisms involved in these interactions are poorly understood. A review of the literature was undertaken to gain an overview of pertinent concepts and their interrelationships to help frame the beginning of a theory for pediatric dysfunctional elimination and continence.

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20 Physioanatomic Considerations Linkages between bowel, bladder and pelvic floor function. T here is a well established association between bowel and bladder dysfunction. The symptom complex was initially termed dysfunctional elimination syndrome by Koff and colleagues in 1998 and was later renamed bowel and bladder dysfunction (BBD) by the Intern Koff, Wagner, & Jayanthi 1998 ). This disorder constitutes a symptom spectrum of lower urinary tract dysfunction (LUTD) that occurs concomitantly with constipation and/or fecal incontinence (Queir oz Machado, Monteiro, Pecanha, & Garcez da Fonseca, 2015; Santos, Lopes, & Koyle, 2017). Constipation is the presenting symptom in as many as 5% of visits to the pediatrician and 25% of visits to pediatric gastroenterologists (Loening Baucke, 1996). Estim ates of voiding dysfunction prevalence in the pediatric population range from 2 25%, with voiding dysfunction as the presenting symptom in as many as 40% of referrals to pediatric urologists (Feldman & Bauer, 2006; Vaz et al., 2006). It is estimated that 3 0% of children with constipation have lower urinary tract symptoms (Loening Baucke, 1997). C onstipation and occult megarectum have been linked to reversible urinary tract abnormalities including incomplete bladder emptying, urinary frequency, recurring uri nary tract infection, daytime and nighttime urinary incontinence and upper urinary tract dilation (Belman, 1998; Dohil, Roberts, Verrier Jones, & Jenkins, 1994; Hodges & Anthony, 2012; Neumann, DeDomenico, & Nogrady, chick, 1986). Findings from a population based study in Brazil demonstrated constipated children were 6.8 times more likely to have symptoms of lower urinary tract dysfunction, including incontinence, withholding,

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21 infrequent voiding, urgency, and dysuria w hen compared to their non constipated peers. Infrequent voiding and withholding behavior were independent predictors of constipation. In addition, an increase in constipation severity was associated with an increase in lower urinary tract symptom severity (Sampaio et al., 2016). Veiga et al. (2013) found that in addition to lower urinary tract dysfunction, 54.9% of children with overactive bladder (OAB) were constipated and approximately three times more likely to develop constipation than children without OAB. Of interest, although there is a well documented correlation between LUTD and retentive constipation, two retrospective studies have demonstrated an association between OAB and non retentive fecal soiling (Burgers et al., 2013; Combs, Van Batavia, Cha n, & Glassberg, 2013). Dysfunctional elimination has been associated with both delayed resolution and post operative recurrence of vesicoureteral reflux in children (Chen, Mao, Homayoon, & Steinhardt, er, & Yazbeck, 1986; Koff, 1992; Koff, Wagner, & Jayanthi, 1998; Upadhyay et al., 2003). Rectal distention has been shown to heighten the sensation of bladder fullness at lower volumes (De Wachter & Wyndaele, 2003). Treatment of constipation has been show n to be beneficial in promoting the resolution of urinary tract symptoms including recurring urinary tract infection, urinary frequency and urgency, dysuria, nocturnal enuresis and daytime urinary incontinence (Chrzan, Klijn, Vijverberg, Sikkel, & De Jon g, 2008; Loening Baucke, 2005) Although counterintuitive, one study demonstrated treatment of OAB with anticholinergics resulted in amelioration of fecal incontinence with or without constipation in 75% of treated children (Coombs et al., 2013).

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22 Althoug h exact mechanisms have not been elucidated, shared embryological origins, close anatomic proximity similar storage and emptying functions, cross and common innervation have been implicated in comorbid bowel and bladder dysfunction. The gastrointestinal system and urinary tract emerge from a shared cloaca dividing in the seventh week of gestation. An extensive nerve and vascular supply support the evolving tissue. Small changes in this differen tiation process could lead to functional defects in both systems (Kaplan et al., 2013; Malykhina, Brodie, & Wilcox, 2016). Shafik (1984) discussed the similarities in muscle composition, evacuation function, and continence mechanisms between the bladder n eck and rectal sphincter with the pelvic floor acti oordination of these individual intrahiatal outlets with the pelvic floor is necessary to maintain patterns of functional elimination and continence. Using this model, dysfunc tional changes in elimination dynamics are thought to be caused by physical obstruction at the level of the pelvic floor. Support of the pelvic floor as a key contributor to elimination dynamics is supported by an extensive body of research demonstrating t he effectiveness of pelvic floor biofeedback Animated pelvic floor biofeedback has been shown to be particularly effective in treating dysfunctional elimination syndrome in children (Desantis, Leonard, Preston, Barrowman, & Guerra, 2011 ; Herndon, Decambre & Mckenna, 2001; Kaye & Palmer, 2008; Mulders, Cobussen Boekhorst, de Gier, Feitz & Kortmann, 2011 ). Neuromuscular involvement seen in cerebral palsy and degenerative neuromuscular disorders has the potential to negatively impact voiding and defecation d ynamics

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23 leading to dysfunctional elimination due to the inability to coordinate the muscles of elimination. N ervous system a ssociations Although poorly understood, outcomes from human and animal studies suggest the presence of bladder distal gut cross sen cross talk discreet afferent signals from the bowel and bladder are transmitted through convergent dorsal root ganglia and both superficial and deeper lumbosacral spine neurons for modulation of routine pelvic organ function. T his cross talk, or neural circuitry, also provides a pathway for the abnormal function of one pelvic organ to cause dysfunctional changes in another pelvic organ. Shared peripheral innervation and neuronal mechanisms in the presence of neural plasticity in early childhood allows physical insult or psychological perturbation to effect the restructuring and connectivity of neural circuitry affecting bowel and bladder function. Animal studies Penzzone, Liang, and Fraser (2005) measured bowel and bladder smoo th and striated muscle reflexes during voiding and rectal distention in rats using external urethral and anal sphincter electromyography (EMG). They measured external anal EMG phasic firing during voiding synchronous with external urethra sphincter activit y with phasic firing and tonic bursts independent of urethral sphincter activity. Colorectal distention in the absence of bladder irritation produced no change in abdominal wall EMG activity. Colorectal distention in the presence of acute bladder irritatio n resulted in abdominal wall EMG activity at much lower distention pressures, suggesting colonic afferent sensitization. Acute colonic irritation resulted in a 66% increase in bladder

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24 contraction frequency, suggesting lower urinary tract afferent sensitiza tion. Findings from this study provided evidence of a bidirectional cross sensitization of the lower bowel and lower urinary tract. Researchers induced colonic inflammation in rats via enema administration of trinitrobenzenesulfonic acid and found signifi cant attenuation in the amplitude of bladder contractions that returned to normal with resolution of the inflammatory stimulus (Noronha, Akbarali, Malykhina, Foreman, & Greenwood Van Meervald, 2007). Ustinova, Fraser, and Pezzone (2010) tested the effects of colonic irritation on the mechano chemo sensitive properties of bladder afferents in rats and found colonic irritation increased firing rates over resting rates by two fold. In addition, a higher percentage of bladder afferents responded at lower dist ention pressures in the presence of colonic irritation. Wyndaele and colleagues (2013) evaluated the functional response of rat bladders to differing levels of colorectal distention and demonstrated reversible and pressure dependent effects of colorectal d istention on the bladder. Noxious levels of colorectal distention resulted in an initiation of an inhibitory rectovesical reflex demonstrated by an increased micturition volume threshold and a decreased bladder contraction, providing a possible explanation for increased bladder capacity and acontractility in the constipated child. Human studies A within subjects study evaluated urodynamic parameters completed in adult women with rectal balloon insufflation to 150 mL repeated in the same subjects without b alloon insufflation (Panayi et al., 2011). They demonstrated statistically significant findings in the subjects during balloon insufflation, including a 46% decrease in volume

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25 infused at first sensation, 33% decrease in volume infused at first desire, and 26% reduction in maximum bladder capacity. Of note, 13% of subjects who had been diagnosed with OAB were found to have detrusor over activity in the presence of balloon insufflation that was absent when the rectum was empty. Chang, Hsieh, and Yang (2012) compared post void residuum urine volumes in children with constipation versus children without constipation and found constipation resulted in a statistically significant increase in absolute urine volume, a higher rate of residual >20 mLs, and a lower vo iding efficiency. I nvestigators combine d colonic motility and urodynamic studies in humans to explore the relationship of bowel and bladder dynamics between children with severe constipation and those with bladder dysfunction. Lucanto, Bauer, Hymen, Flore s, & Di Lorenzo (2000) evaluated children with severe constipation and bladder dysfunction and found abnormal colonic motility suggestive of neuropathy in all of the children and abnormal urodynamics in 90.0% of the children studied. Burgers et al. (2010) investigated the effect of rectal distention on urodynamic parameters and found it significantly impacted bladder capacity, sensation, and instability in an unpredictable manner regardless of the presence of a history of constipation. A study evaluating colonic transit time in children with refractory constipation and LUTD noted abnormal urodynamic features in 86.7% of affected subjects with 66.7% categorized as detrusor over activity and voiding dysfunction, 16.7% categorized as isolated detrusor activit y, and 8.3% categorized as isolated voiding dysfunction. Colonic transit time was abnormal in 80% of constipated subjects, with 60% categorized as slow transit and 20% as outlet dysfunction A high prevalence of slow transit constipation ( STC ) was found in children with refractory constipation. There was a significant

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26 association between STC and OAB indicating the presence of a common neuropathy that affects both the colon and lower urinary tract (Queiroz Machadoet al., 2015). The bowel, bladder, and pelvic floor are innervated by the spinal cord at level s S1 through S4. I njury or disruption in spinal cord function at any of these levels has the potential to significantly impact bowel and bladder function. T he concept of neuroplasticity may have significant relevance in dysfunctional elimination in the neurologically intact child. Afferent impulses generated from the bowel, bladder, or pelvic floor in respons e to aversive stimuli like pain may cause a significant change in efferent stimuli resulting in end or gan change. There is evidence to sugge st the brain, not the bladder, i s the primary site of model to explain the association between encopresis, urinary incontinence, and psy chiatric comorbidities postulating a central defect in the anterior cingulate gyrus or prefrontal cortex. Vortex based mapping analysis revealed a decrease in frontal grey matter in adults with a history of enuresis. Von Gontard & Hollman (2004) found chi ldren with comorbid functional urinary incontinence and encopresis had a significantly higher rate of abnormal electroencephalography. Developmental Considerations Central nervous system maturation is an essential consideration in issues related to pediat ric continence and functional elimination. To successfully achieve independence in toileting, a child must have attaine d a certain level of cognitive and fine and gross motor development. Any significant cognitive or muscular disorder or delay

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27 has the pote ntial to prevent, substantially delay, or necessitate extraordinary means for achieving continence in the affected child. Pain and learning in children Conventional thought regarding the origins of dysfunctional elimination in children identifies painful elimination as the primary cause. Pain is an aversive stimulus. Young children learn by cause and effect and make the association between pain and elimin ation. Children will avoid things that cause pain so they begin to actively withhold out of fear of p ain. The function of the lower bowel is fluid reabsorption and storage of waste. When a child withholds stool, the stool becomes hard causing further discomfort during defecation, thereby reinforcing the withholding behavior. The r ectum dilates to accommod ate retained stool. The stool increases in caliber exacerbating the cycle. As the rectum accommodates to the increased stool burden, the urge to defecate is blunted and may vanish so the child can go for longer periods of time without passing a bowel motio n. Fear and pain result in dyssynergic pelvic floor dynamics. Instead of increasing intra abdominal pressure and relaxing the muscles of the pelvic floor, the child tightens the pelvic floor resulting in incomplete emptying of the bowel and bladder. Over t ime, loose stool from higher up in the colon leaks around the mass of hard stool in the rectal vault causing encopresis. The mass of stool in the rectal vault further compromises bladder dynamics compressing the bladder, decreasing its functional capacity, and causing an earlier sensation of bladder distention (Culbert & Banez, 2007; Halachmi & Farhat, 2008). The same basic dynamic is seen with children who have pain with urination. Dysuria leads to withholding behavior and dyssynergic voiding dynamics that is often the root cause for urinary incontinence and recurring urinary tract infection. It is

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28 interesting to note that despite the accepted conventional wisdom proposing pain as a major precipitating factor in the development of dysfunctional elimination, there are no publications focused on aggressive treatment of constipation and dysuria as a preventive strategy. Toilet training Although achieving continence is a universal milestone for the normally developing child, there is limited and conflicting evid ence regarding the ideal age for ini tiation of toilet training, required skills, and sequencing of those skills necessary for successful toilet training in the normal child (Joinson et al., 2009; Klassen et al., 2006; Schrum et al., 2002). Conventional wis dom has been to allow the child to determine when they are ready to initiate toilet tra ining. The combination of improved diapers that prevent the child from experiencing a significant degree of discomfort when incontinent has resulted in children toilet t raining at a later age. Recent studies suggest earlier toilet training may be beneficial in preventing dysfunctional elimination. Joinson et al. (2009) found initiation of toilet training past 24 months of age was associated with increased odds of daytime urinary incontinence in school age children. Children with neuromuscular disorders, urologic and anorectal malformations, spinal cord anomalies, spinal cord trauma or tumor, nonneurogenic neu rogenic bladder, and megarectum are at high risk for urinary a nd/or fecal incontinence that is very difficult to manage using conservative measures (Tobias, Mason, Lutkenhoff, Stoops, & Ferguson, 2008). In a large portion of children with neurogenic bowel and bladder, extraordinary measures need to be employed to gai n continence including clean intermittent catheterization and either retrograde enemas using continence apparatus or

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29 antegrade continence enemas (ACE) administered through a catheterizable stoma using the appendix, a tubularized portion of the bowel, or a low profile button device from the abdominal wall into the cecum. Effectiveness of ACE therapy has been demonstrated in many retrospective studies with overall continence rates in children ranging from 55.7 % to 98% with a mean time to first relapse lasti ng as long as 121.9 +/ 29.7 months (Bani Hani, Cain, King, & Rink, 2008; Basson et al. 2014; Curry, Osborne, & Malone, 1999; Dey et al. 2003; Driver et al. 1998; King, Sutcliffe, Southwell, Chait, & Hutson, 2005; Driver et al. 2001; Mousa et al., 2006; Ok & Kurzrock, 2011; Peeraully et al ., 2014; Randall, Coyne, & Jaffray, 2014; Siddiqui, Fishman, Bauer, & Nurko, 2011; Sinha, Grewal, & Ward, 2008; Thomas et al., 2006; VanderBrink et al. 2012 ; Yardley et al., 2009). Prospective studies have demonstrated a significant improvement in somatic functioning, psychosocial functioning, and quality of life (QOL) following an ACE procedure (Aksnes et al., 2002; Mousa et al., 2006; Tiryaki, Ergun, Celik, Ulman, & Avanoglu, 2010). There are a number of standard inte rventions such as dietary manipulation, timed emptying, suppository use or retrograde enema therapy that are initially attempted prior to instituting more extraordinary measures. However, there is no research detailing the appropriate and most effective s equencing or combination of conservative therapies in children Psychosocial Considerations Psychological associations Recent studies have demonstrated a high rate of psychiatric comorbidities including attention deficit disorder in children suffering from urinary and fecal incontinence. The rate of psychiatric comorbidities in children with nocturnal enuresis is

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30 as high as 30%, diurnal enuresis 40%, and fecal incontinence 50% (Von Gontard, Baeyens, Van Hoecke, Warzak, & Bachmann, 2011). Von Gontard and Ho llmann (2004) found daytime urinary incontinence was associated with executive function, attention, separation anxiety, opposition, and conduct disorders while fecal incontinence was associated with attention, activity, obsession, compulsion, and oppositi on disorders Children with fecal incontinence were more likely to bully or be bullied and engage in antisocial behaviors than children who were continent (Joinson, Heron, Butler, & Von Gontard, 2006; Zavadenko, Kolobova, & Suvorinova, 2011). There is evi dence to support comprehensive evaluation and treatment of underlying attention and psychiatric issues improve treatment outcomes in children with dysfunctional elimination and incontinence (Williamson, Gower, & Ulzen, 2011; Von Gontard, Baeyens, Van Hoeck e, Warzak, & Bachmann, 2011) Social associations Maternal depression and anxiety were associated with daytime wetting and soiling in children (Joinson et al., 2008). Having a family member with a neuropsychiatric disorder placed the child without a neuro psychiatric disorder at higher risk for urinary tract symptoms (Franco, 2011). Sexual abuse is a potential causative factor in fecal incontinence. Children with fecal soiling, although not predictive of sexual abuse, had a higher rate of sexual abuse than children who had not been abused (Mellon, Whiteside, & Friedrich, 2006; Morrow, Yeager, & Lewis, 1996). It is unclear if fecal incontinence in sexually abused children results from pain with defecation following anal penetration that leads to withholding b ehavior with subsequent

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31 encopresis or is used intentionally by the child as a protective mechanism to discourage access by the sexual predator. Anthropometric and Nutritional Considerations Obesity There is a significantly higher rate of obesity in child ren with constipation and encopresis (Pashankar & Loening Baucke, 2005; Teitelbaum, Sinha, Micale, Yeung, & Jaeger, 2009 ; Wagner, Equit, Niemczyk, & von Gontard, 2015 ) Fishman, Lenders, Fortunato, Noonan, and Nurko (2004) reported a higher incidence of fe cal soiling in obese children. Of note, 50% of the soiling in their sample subjects was non retentive. A study conducted by van der Baan Slootweg, Liem, Bekkali, van Aalderen, Pels Rijcken, Di Lorenzo, and Benninga (2011) confirmed a higher incidence of co nstipation in children with obesity. A small percentage of those children had delayed colonic transit. There is a well known correlation between obesity and sleep apnea (Barone et al., 2009; Verhulst et al., 2007). C hildren with disordered breathing have an increased rate of nocturnal enuresis (Alexopoulos et al., 2006; Bascom et al., 2011; Brooks & Topol, 2003; Umlauf & Chasens, 2003). Children with voiding dysfunction have close to twice the rate of obesity found in the general population (Erdem, Lin, Kogan, & Feustel, 2006). Elevated BMI (including both overweight and obese categories) has been associated with a higher incidence of lower urinary tract symptoms, bladder bowel dysfunction, and a poorer treatment response in children (Guven, Giramonti, & Kogan, 2007). Chang, Chiang, Lin, Hsieh, and Yang (2015) identified a higher risk of overactive bladder in children with an elevated BMI. Fraga and colleagues (2017) confirmed a significant association between obesity and a positive Dysfunctional

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32 Voiding Symptoms Scale score. Findings from their study further defined the association of lower urinary tract function in obese children to symptoms of bladder emptying only. Arlen, Cooper, and Leong (2017) found obese children with a BMI greater than the 85 th percentile were greater than 3 times more likely to fail treatment for symptoms of lower urinary tract dysfunction. Obese children with febrile urinary tract infection were more likely to have a higher grade of vesicoureteral reflux and more frequent occu rrence of renal cortical defects than non obese children (Byun et al., 2016). In addition, the body habitus in obese girls make them more prone to vaginal pooling of urine resulting in small volume daytime urinary incontinence. Dietary considerations Alth ough ubiquitous in practice, there is no evidence to support the use of fiber in the active treatment of constipation in children (Tabbers et al., 2014) Outcomes in clinical practice, as yet not reported in the literature, suggests fiber, which acts as a bulking agent, can exacerbate constipation in the child who actively withhold s stool or has anismus. There is preliminary evidence to support the use of probiotics including Lactobacillus casei rhamnosus Lcr35 and Bifidobacterium breve in the treatment of constipation in children (Bu, Chang, Ni, Chen, & Cheng, 2007; Tabbers, De Milliano, Roseboom, & Benniga, 2011). Genetic Considerations N umerous diseases and birth defec ts have a formal genetic linkage associated with abnormal bowel and bladder function. Formal genetic linkages have not been

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33 of defecation and should be considered in the eval uation of the constipated child (Peeters, Benninga, & Hennekam, 2011). Formal genetic risks have been identified for nocturnal and diurnal enuresis (Von Gontard, Heron, & Joinson, 2011). Summary of Pertinent Literature to Guide Theory Development The li terature identifies multiple physioanatomic, psychological, social, environmental, and genetic factors that contribute to incontinence and dysfunctional elimination in children and supports the ex istence of multiple, diverse, poorly understood associations between those factors. The anatomic model identifies obstruction at the level of the pelvic floor as the driver in bowel and bladder dysfunction. The bladder distal gut cross sensitization model identifies discreet afferent signals from the bowel and blad der transmitted through convergent dorsal root ganglia, and both superficial and deeper lumbosacral spine neurons as the driver for modulation of pelvic organ function. The n eurocentric model identifies the brain as the primary driver of dysfunctional elim ination by making the association between bowel, bladder and psychiatric co morbidities. The aversive stimulus model identifies the interplay of pain and development as the drivers in dysfunctional elimination in children. There is very little evidence el ucidating the contribution of genetics to dysfunctional elimination. D irect causes of neurogenic incontinence are better understood but the inter play of associated factors, most effective therapies, and the rapy sequ encing has not been established. A numbe r of studies clearly elucidate the impact of dysfunctional elimination on quality of life. Very few studies address the contributions of environment and social considerations to incontinence and dysfunctional elimination. Prevention of

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34 incontinence and dys functional eliminati on is not addressed. No unifying model promotes understanding of this issue Pediatric incontinence and dysfunctional elimination is a complex entity that negatively impacts the lives of a large number of children. Current practice and research addressing this issue is highly fragmented resulting in less than optimal care for this population. Preliminary review of the literature has demonstrated some possible linkages between the factors that comprise dysfunctional elimination, but reve al substantial holes. There is a need to integrate practice related research into a comprehensive theory that would allow for a holistic and coherent approach to children with incontinence and dysfunctional elimination. A theory addressing pediatric incont inence and dysfunctional elimination is needed to serve as a guide for practice and future research. Identification of a Metaparadigm to Guide Theory Development Use of a metaparadigm to provide a global conceptual framework would be helpful in organizing a theory for children with incontinence and dysfunctional patterns of elimination. The metaparadigm would need to encompass the complexity of the different levels of interactions within and between organismic systems and organismic environmental interacti ons within the context of a developing child. v on Bertalanffy (1969) defined a living organism as a hierarchical open system that maintains itself in a dynamic equilibrium and evolves toward higher complexity by continuous exchange of matter with the envir onment that is attained and maintained through equifinal circular feedback processes. Gilbert Gottlieb, a developmental psychologist, derived the developmental psychobiological metatheory of Probabilistic Epig enesis from Ludwig

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35 von Bertalanff eory in which he conceived the developing organism a s an open system consisting of four levels of analysis with reciprocity of influences within and be tween each level (Gottlieb, 1991 ). The first three of the four levels of analysis or constructs are orga nismic and include genetic (DNA, RNA), neural (cytoplasm, cell, tissue, organs, organ systems, organism), and behavioral (prenatal and postnatal) levels. The fourth level of analysis is environmental which is further subdivided into physical, social, and cultural groupings (Gottlieb, 1991; Gottlieb, 1998; Gottlieb, 2002; Gottlieb, 2007 ). Probab i listic Epigenesis provides constructs and relational statements that allow for the explanation and exploration of the multidimensional structural and functional pro perties of pediatric incontinence and dysfunction elimination within a developmental framework. Probabilistic Epigenesis Historical O verview functions during the course of indiv Epigenesis was initially conceptualized as the predetermined, nonreciprocal development of the structure function dyad whereby genetic activity determined structural maturation which in turn determined functio n, activity, or experience in a feed forward manner. In this view, the genome contains the blueprint that determines the master plan for the developing organism. Simplistically RNA pathway in development is thought to be unidirectional, encapsulated, and immune to the influence of supragenetic feedback (Gottlieb, 199 1; Gottlieb, 1998; Gottlieb, 2001). Gottlieb (1991 ) proposed

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36 epigenesis as probabilistic determination with active int eraction and feedback among constitutive elements creating a bidirectional, hierarchical, constructive, reciprocal ; structural maturation ; and function, activity, or experience. In this view, genes are iner t and require supragenetic input to function during no rmal development. Simplistically, the genetic activity (DNA processes and neural and behavioral functions. DNA is inert and requires intracellular signals or feedback from constitutive elements originating from outside the cy toplasm to turn DNA on or off in protein production thereby regulating genetic expression. It is coaction above the level of DNA RNA that drives cellular differentiation a nd function. The genome is a part of the development physiological system responsive to intracellular, extracellular, and extra organismic influences. Morphogenesis i s not caused or controlled solely by genes, but rather genes enable morphogenesis contributing to production of traits and features in the developing organism. Organisms wit h the same genome can demonstrate significant phenotypic variation under differing ontogenetic conditions (Gottlieb, 1998 ; Gottlieb, 2002; Gottlieb, 2007 ). Const ructs and Relational Statements Gottlieb (2002) defined Probab i listic Epigenesis as : individu al development characterized by an increase of complexity of organization i.e., the emergence of new structural and functional properties and competencies at all levels of analysis (molecular, subcellular, cellular, organismic) as a consequence of hori zontal and vertical coactions among its parts, including organism environment coactions (p. 159 16 0).

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37 The constructs are levels of analysis and include genetic, neural, behavioral, and environmental. Horizontal coaction is defined as interactions that occu r at the same level within an organism. Vertical coaction is defined as interactions that occur at different levels within the organism and between the organism and environment. Horizontal and vertical coactions are reciprocal allowing constitutive elemen ts to hierarchical and holistic manner. Behavioral and organic outcomes of development are a consequence of at least two specific components of coaction. Development is caused by interaction between at least two constitutive elements, not the elements themselves, and is temporal in nature as shown in Figure 1 1 Gottlieb (2001 ) further defined the coaction between the constitutive elements which includes function or activity at all levels of analysis. Experience serves three purposes to (a ) maintain function (maintenance function), (b ) temporarily regulate or facilitate emerging features (facilitative function), and (c ) induce or create fe atures within a developing system that would not occur in the absence of that particular coaction (inductive function). Within the theoretical framework of Probab i listic Epigenesis, developmental causality is nonlinear and emergent and is therefore not obv ious. Plasticity or malleability occurs at a cellular level as well as at neural, psychological, and behavioral level s in the developing organism. Plasticity of the determinants of behavioral adaptability. This plasticity allows coaction to drive of

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38 exposed to all possible environments. The phenotypic change precedes and drives morphologic physiologic change that eventually may drive genetic change (Gottlieb, equifinality, developing organisms can start at different points but reach the same endpoint ; alternatively, organisms can share the same starting point and reach the same end point by different pathways. Multipotentiality maintains that each cell in the body has equal genetic potential because each cell contains the total genetic complement (Gottlieb, 2001). There is a wealth of biological experimentation in insects, animals, and humans that support Probabalistic Epigenesis and are clearly elucidated in various manuscripts ( Gottlieb, 1991; Gottlieb, 1992; Gottlieb, 1998; Gottlieb, 2001 ; Gottlieb, 2002; Gottlieb, 2007). The Applicability of Probabalistic Epigenesis to Pediatric Incontinence Research The overarching principles contained within the construct s and relational statements of Probabalistic Epigenesis are broad, but are highly applicable to the derivation of a global theory of pediatric incontinence and dysfunctional habits of elimination and can be subjected to experimental confirmation. Review of the literature addressing pediatric incontinence and dysfunctional elimination demonstrates the complex interplay of factors including genetic, physioanatomic, behavioral, and environmental determinants within the context of emerging development that can be logically reformulated to fit the hierarchical constructs of Probabalistic Epigenesis. The relational statements proposing interaction and feedback among constitutive elements creating reciprocal, lateral and horizontal coaction among genetic activity, structural maturation, and function promulgated within Probabalistic Epigenesis provid e a logical

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39 explanation for existing interac tions and a framework for exploration of the multidimensional structural and functional properties of pediatric incontinence and dysfunction elimination. The Integrative Model of Pediatric Incontinence and Dysfunctional Elimination As a starting point in the development of a theoretical framework for pediatric continence, a restructured model of the Theory of Epigenes is will be (1991 ) original metatheoretical model is depicted in Figure 1 1 W hile all encompassing, this theory was too abstract to be empirically tested. His theory was therefore substructed to allow formal evaluation of epigenetic trends testing ho rizontal and vertical interactions in the four constructs including genetic, neural, behavior, and environment as shown in Figure 1 2 The model looks at the vertical interaction of genetics, neural activity and environment on behavior at a single point i n time and the vertical and horizontal interaction of genetics, neural activity, environment and behavior longitudinally. Behavior is determined by horizontal or vertical interaction of at least two constitutive elements within or between constructs. Each constitutive element may serve in the role of dependent variable, independent variable, or control. The specific interaction between organ systems contained within the neural constitutive element specific to incontinence and dysfunctional elimination is further defined in Figure 1 3. The epigenetic principals of plasticity, equifi nality, multipotentiality, norm of reaction range, and behavioral neophenotypes as defined by Gottlieb are essential principals in the new substructed theory. The substructed t heory is comprehensive enough in scope to subsume the anatomic, bladder distal gut cross sensitization, neurocentric, and aversive stimulus models thus far elucidated in the literature and

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40 provide context for as yet to be identified genetic and phenotypic contributions. Using Gibbs (1972) system of theory construction as modified by Dulock and Holzemer (1991), it is possible to clearly demonstrate theoretical and operational linkages between Probabalistic Epigenesis and proposed research addressing pediatr ic incontinence and dysfunctional elimination as shown in Appendix A. This substructed, new theory is parsimonious, allows for incorporation of multiple paradigms spanning genetic and epigenetic regulation of gene expression to environmental influences on treatment outcomes, facilitates temporal aspects of cross sectional and longitudinal design, is broad enough conceptually to encourage transdisciplinary and translational science, provides an integrated framework that views dysfunctional voiding and defeca tion dynamics as two expressions of the same entity, and functions to inform clinicians in the configuration of services and treatment of children with incontinence and dysfunctional elimination. Pediatric incontinence and dysfunctional elimination is a complex entity that negatively impacts the lives of a large number of children with devastating effect. Current practice and research addressing this issue is highly fragmented resulting in less than opt imal care for this population. Current research suppo rts the existence of multiple, interrelated associations between dysfunctional voiding and defecation dynamics suggesting both are different manifestations of the same syndrome that when integrated and embedded within the context of psychological, social, environmental, and genetic factors provide the beginnings of a comprehensive theoretical framework addressing incontinence and/or dysfunctional elimination in children. There is a need for a comprehensive theory that allow s for a translational, transdisc iplinary, and

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41 coherent approach to children with incontinence and dysfunctional elimination. Such a theory could serve as a guide for practice and future translational research. New theories need to provide an expansive and dynamic guide that facilitates t emporal exploration of symptom clusters, symptom trajectories and interventional outcomes (Brant, Beck, and Miaskowski, 2010). Probab i listic Epigenesis provides a metaparadigm that allows for the derivation of a theory of pediatr ic incontinence and dysfun ctional elimination within a developmental framework that provides a coherent, coordinated, and holistic approach to research a nd improves the outcomes and lives of effected children. Proposed Program of Research As a starting point in the pediatric conti nence program of research, the Integrative Model of Pediatric Incontinence and Dysfunctional Elimination will be used to evaluate epigenetic trends testing horizontal and vertical coaction in three of the four constructs including neural activity, behavior and environment as shown in Figure 1 2. Genetic activity will be considered a constant backdrop. The research will explore the vertical coaction of environment and neural activity on behavior at a single point in time and the vertical and horizontal coa ction of environment and neural activity on behavior l ongitudinally. Problem ACE therapy administered through a catheterizable stoma surgically placed in the cecum has helped children with intractable fecal incontinence attain continence for stool. There a re a number of retrospective studies demonstrating the effectiveness of ACE therapy and several prospective studies that demonstrate improvement in quality

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42 of life following an ACE procedure. There are case reports detailing morbidity and mortality associa ted with different flushing regimens. However, there are no prospective trials comparing the effectiveness of different flushing regimens on continence. The catheterizable stoma used for the antegrade administration of enema solution is frequently made by bringing the appendix out through the abdominal wall or by placing a skin level device (button) into the cecum. The appendix and cecum have significant amounts of gut associated lymphoid tissue, have high concentrations of microbiota, and serve an essenti al immune function. ACE therapy administration through the appendix or into the cecum has the potential to disrupt the gut microbial ecosystem causing dysbiosis and immune dysfunction. The effects of such ACE administration on colonic microbiome and mucosa l immunity have not been investigated. Three aims with corresponding hypotheses have been identified as a starting point for a program of research and theory development in pediatric incontinence framed by the Integrative Model of Pediatric Incontinence and Dysfunctional Elimination. ACE therapy including identification of an optimal dosing regimen is the independent variable representing the Environment construct. The bowel, gut microbiome, electrolyte balance, and immune function represent the Neural Ac tivity construct. Continence and quality of life represent the Behavioral Construct. This study will explore vertical coactions between Environment (ACE therapy) and Neural Activity (bowel) on Behavior (continence and quality of life). Exploration of horiz ontal coactions between Environment and Neural Activity include measurement of side effects and changes in electrolyte balance, the gut microbiome, and markers of mucosal immune function.

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43 Purpose, Aims, and Hypothesis The purpose of this prospective pilot study was to compare two distinct flushing regimens, one high volume saline flush and one low volume USP glycerin flush, in the immediate postoperative period in children requiring ACE therapy. Findings from this study provided a comparative analysis of t hese two regimens serving as a starting point to guide practice and serving as a foundation for prospective, randomized, controlled trials. The aims of this study were to (a) identify the minimal administration frequency titration time to reaching effecti ve dose, and cost of two flushing solutions ; (b) compare which solution at an optimum dose is delivered in the le ast amount of time, with fewer side effects, while promoting the highe r degree of fecal continence and quality of life; and (c) to collect and process stool for future analysis to determine if administration of antegrade enema solution through an appendicostomy/cecostomy affects gut microbiota and immune function. Null Hypotheses for Aim 1 1. There will be no differences in frequency of administrati on necessary to gain and maintain continence between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (primary aim). 2. There will be no differences in titration tim e to reaching effective dose between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). 3. There will be no differences in cost between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade cont inence therapy (secondary aim).

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44 Null Hypotheses for Aim 2 1. There will be no difference in continence between two different ACE flushing regimen s using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (primary aim). 2. There will be no difference in infusion time between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). 3. There will be no difference in procedural time between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring ante grade continence therapy ( secondary aim). 4. There will be no difference in side effects between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy ( secondary aim). 5. T here will be no difference in parent/patient satisfaction as measured by the Fecal Incontinence and Constipation Quality of Life Measure in Children with Spina Bifida (FIC QOL) between two different ACE flushing regimens using normal saline and normal sali ne with USP glycerin in children requiring antegrade continence therapy ( secondary aim). Null Hypothesis for Aim 3 1. There will be no difference in the effect of two different ACE flushing regimens using normal saline and normal saline with USP glycerin on c olonic microbiota in children requiring antegrade continence therapy. 2. There will be no difference in the effect of two different ACE flushing regimens using normal saline and normal sali ne with USP glycerin on mucosal immune function in children requiring antegrade continence therapy In Chapter 2, research results from studies evaluating the effects of ACE flushing regimens on continence, side effects, and gut microbiota are reviewed. Chapter 3 reviews the design, measures, and analysis utilized in this stu dy. Chapter 4 reports the findings from this study. Chapter 5 contains a discussion of findings.

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45 Figure 1 1. Gilbert Model of Probabi listic Epigenesis D e piction of the bidirectional coaction between levels of functioning over the course of de 2002, Oxford University Press, New York, N.Y., p186. Copyright date by Taylor Francis Group LLC Books. Reprinted with permission. Figure 1 2. R educed Mod el of Probabi listic Epigenesis adapted to test pediatric c ontinence D epiction of the bidirectional coaction between levels of functioning over the course of development modified to represent vertical interaction of genetics, neural activity, and environment on behavior at a single poi nt in time and the vertical and horizontal interaction of genetics, neural activity, environment and behavior longitudinally. Adapted from University Press, New York, N.Y., p186. Copyright date by Taylor Francis Group LLC Books. Modified and r eprinted with permission.

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46 Figure 1 3. Model depicting specific interactions between organ systems contained within the neural constitutive element specific to incontinence and dysfunctional e liminati on d epiction of the bidirectional coaction between levels of functioning over the course of development modified to specify the interactions between organ systems contained within the neural constitutive element specific to incontinence and dysfunctional e limination adapted from University Press, New York, N.Y., p186. Copyright date by Taylor Francis Group LLC Books. Modified and reprinted with permission

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47 CHAPTER 2 LITERATURE REVIEW Over view Fecal incontinence in children past the expected time of toilet training has been associated with increased anxiety and depression, more social problems, worse school performance, an increased incidence of abuse and bullying and a significantly poore r quality of life (Kaugars et al., 2010; Kovacicet al., 2015; Youssef, Langseder, Verga, Mones, & Rosh, 2005). It is particularly difficult to manage fecal incontinence using conservative measures in children with neuromuscular disorders, anorectal malform ations, spinal cord injuries, spinal cord trauma or tumor, megarectum, and slow transit constipation. The Malone or antegrade continence enema (ACE) procedure was popularized over 20 years ago as a means of helping children with intractable fecal incontine nce attain stool continence. A catheterizabl e stoma from the abdominal wall into the cecum is cons tructed using the appendix, a tu bularized portion of the bowel, or a low profile button device. The stoma allows for antegrade administration of enema solutio n into the colon. Studies Addressing the Effectiveness of ACE Therapy in Promoting Continence A number of retrospective studies evaluate overall continence rates in children fol lowing ACE therapy. Findings from these studies are highly variable. Short term reported success rates range from 55.7 to 98% with long term outcomes ranging from 14 to 41% abandonment rate at 5 years to a mean time to first relapse being as long as 121.9 +/ 29.7 months (Aspirot, Fernandez, Di Lorenzo, Skaggs, & Mousa, 2009; Bani Ha ni, Cain, King, & Rink, 2008; Basson et al., 2014; Becmeur et al., 2008; Chu, Balsara, Routh, Ross, & Wiener, 2013; Church, Simah, Wild, Teitelbaum, & Ehrlich, 2017; Curry,

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48 Osborne, & Malone, 1999; Chong et al. 2016; Dey et al., 2003; Dolejs, Smith, Sheplo ck, Croffie, & Rescorla, 2017; Driver et al., 1998; Freeman et al., 2014; Hoekstra et al. 2011; King, Sutcliffe, Southwell, Chait, & Hutson, 2005; Large et al. 2017; Levitt, Soffer, & Pena, 1997; Marshall, Hutson, Anticich, & Stanton, 2001; Matsuno et al. 2010; Mousa et al., 2006; Ok & Kurzrock, 2011; Peeraully et al., 2014; Randall et al., 2014; Siddiqui, Fishman, Bauer, & Nurko, 2011; Sinha et al., 2008; Thomas et al., 2006; VanderBrink et et al., 2013; Yardley et al., 2009). The high variability in ACE effec tiveness outcomes in these retrospective studies could have been due to a number of confounds. Study designs were uniformly retrospective and sample size was highly variable and often small. There was wide variability in the underlying organic and structur al disorders in the study population s of children who received ACE therapy including, but not limited to, neurogenic etiologies including myelomeningocele, lipoma, tethering, caudal regression syndrome, spinal cord tumor or trauma, and transverse myelitis ; ; anorectal malformation including imperforate anus, perineal fistula, vestibular fistula, vaginal rectal atresia without fistula, cloaca, and C urarino triad ; 22q11syndrome ; cerebral palsy ; prune belly syndrome ; intractable fecal inc ontinence ; slow transit constipation ; intractable functional constipation ; and colonic pseudo obstruction. The extensive variability in organic, structural, and functional etiologies could affect outcomes. Additional potential confounds include subject age at the time of surgery ; type and placement of the stoma ; colonic transit time : presence of outlet dysfunction ; type and timing of post operative complications ; caregiver and child motivation compliance and education ; co morbid conditions such as autism ; mitochondrial disease ; metabolic disorders ; chromosomal abnormalities ; VACTERL ;

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49 psychiatric disorders, etc. ; and flush solution type, volume, and frequency of administration. Kuizenfa Wessel, Mousa, Benniga, and Di Lorenzo (2016) published an overview of existing literature excluding articles without a clear definition of addressing ACE outcomes, sample size less than 20, and patients over 25 years of age. They identified 21 articles that met inclusion criteria. In addition, they surveyed 23 pediatric gas troenterologists and surgeons worldwide with acknowledged expertise in ACE therapy. The authors concluded there are differing opinion s regarding indications for the procedure, preoperative evaluation, preferred flushing regimens, and definitions of success Two retrospective studies evaluated ambulatory status as a potential confounding variable for achieving continence in children with spina bifida receiving ACE therapy. Vande Velde and colleagues (2007) evaluated 80 children ranging from 5 to 18 years of age and did not find an association between level of lesion or ambulatory status and continence rates. Large et al. (2017) evaluated 115 children greater than 8 years of age who had undergone an ACE procedure and found 65.1% of ambulatory children achieve d full fecal continence compared to 44.2% of their non ambulatory counterparts. Two retrospective studies were identified that addressed age at the time of ACE stoma construction and initiation of flushing. Stenstrom, Graneli, Salo, Hagelsteen, and Arnbjo rnsson (2013) presented findings from a retrospective study of 21 of 164 children with anorectal malformation who underwent appendicostomy for bowel management. Median age at the time of stoma construction was 4 with a range of 1 to 6 years. Observation ti me ranged from 0.5 to 14 years. The goal was for every child with

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50 anorectal malformation to achieve fecal continence by the start of school. The major indication for ACE stoma construction surgery was persistent fecal incontinence and failure to adhere to a retrograde enema regimen. Postoperatively, 15/17 children (88%) were continent for stool. Post operative infection rate was reported at 29%, higher than infection rates reported in older populations. However, stomal stenosis (12%), surgical revision (6%) and retrograde stomal leakage (0%) were considerably lower than respective rates reported in older populations. Sixteen families were satisfied with the appendicostomy. The remaining family felt it was too early to evaluate. Freeman, Simha, Jarboe, Ehrli ch, and Teitelbaum (2014) presented findings from a study of 35 children to determine if ACE stoma construction and initiation of flushing prior to starting school (< 6 years of age) would improve functional stooling and quality of life. Children were divi ded into two groups based on age at which ACE therapy was initiated. Families of children who had initiation of ACE therapy prior to 6 years of age were compared to families of children who had initiation of ACE therapy after 6 years of age. All were prosp ectively surveyed using a 17 item questionnaire addressing stool habits and a 7 item Pediatric QOL survey, both of which had been adapted from previous tools. Psychometric properties of the original and adapted tools were not addressed. Total stooling, def ined continence, and stool pattern scores were calculated from the total survey. Of the 35 patients identified, 20 families could not be reached and one did not wish to participate, leaving 14 patients who were successfully surveyed. Of the 14 children sur veyed, six were under 6 (3.68 +/ 1.02) years of age and eight were older than 6 (10.30 +/ 1.02) years of age at the time of surgery. Absolute continence rates could not be determined based on reported results. However, stooling and continence

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51 scores were significantly better in the <6 year old group. QOL scores showed significant improvement post operatively in both groups, with the < 6 age group showing a Many different ACE flushing regimens are p rescribed in practice, including licorice root, mineral oil, treacle/milk mix, tap water, normal saline, twice normal saline, polyethylene glycol solution, phosphate soda solution, bisacodyl, and USP liquid glycerin (Bani Hani et al., 2008; Becmeur et al., 2008; Chu et al., 2013; Dolejs et al., 2017; Hyde, Coulthard, Jaffray, Vallely, & Harding, 2008; Marshall et al., 2001; Large et al., 2017; Matsunoet al., 2010; Siddiquiet al., 2011; Stenstrom et al., 2013 ; Yerkeset al., 2001; Youssef, Barksdale, Griffiths Flores, & Di Lorenzo, 2002 ; Vande Velde, 2007) A number of studies were identified that to some extent report flushing solution use dosing, frequency of administration and procedural time. A summary of th is data is contained in Table 2 1. Kuizenga Wessel et al. (2016) reported survey data of 23 pediatric gastroenterologists and surgeons with expertise in ACE therapy. They found that 16 (70%) started the flushing regimen with saline; 5 (22%) tap water, polyethylene glycol, or sodium phosphate; and on ly 2 (8%) a stimulant using either USP glycerin or bisacodyl. Nineteen respondents added a stimulant only when the initial antegrade variable. Forty eight percent started at 10 20 mg/kg, 22% used 0 249 mL, 22% used 250 500 mL, and 8 % tailored the volume to the child. All but one respondent began the regimen with daily flushing. An infusion duration ranging from 0 to 15 minutes was preferred by 62% of respondents, with 38% prefe rring 16 to 30 minutes. A total

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52 procedural time (inclusive of flush infusion ranging from 30 to 60 minutes following flush infusion was preferred by 65% of respondents, with 35% limiting procedural time to under 30 minutes. A single prospective study was i dentified that addressed comparative effectiveness of ACE flushing regimens This study utilized colonic manometry to compare motor response of three stimuli (meal, antegrade saline infusion, and antegrade bisacodyl administration) on the number of high am plitude contractions and motility index in 13 pediatric patients. Findings in this study demonstrated that there was no significant difference in parameters after ingestion of a meal or saline infusion at 10 to 20 mg/kg with a maximum volume of 700 mL. Bi sacodyl at a dose of 0 .2 mg/kg with a maximum dose of 10 mg significantly increased the motility index and high amplitude propagated contractions when compared to meal or saline (Gomez, Mousa, Liem, Hayes, & Di Lorenzo, 2010). Bani Hani, Cain, King, and Ri nk (2008) identified timing of accidents as the most important factor in troubleshooting flushing regimen failure. Siddiqui et al. (2011) postulated an inverse relationship between increased commode time and long term ACE adherence A number of prospective trials demonstrate significant improvement in somatic functions, psychosocial functioning, and quality of life (QOL) after an ACE procedure (Aksnes et al., 2002; Churchet al., 2017; Hoekstraet al., 2011; Mousa et al., 2006; Tiryaki, Ergun, Celik, Ulman, & Avanoglu, 2010). Identifying a successful flushing regimen is determined by individual clinician preference and often requires multiple attempts before success is achieved. There are no prospective studies comparing the effectiveness of type, dose/volu me, or frequency of different flushing regimens in preventing incontinence to inform practice.

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53 Studies Addressing Side Effects Associated with ACE Therapy Several case reports detail morbidity and mortality associated with a particular flushing solution, i ncluding hypocalcemia and hyperphosphatemia, following retrograde administration of phosphate enemas in children (Helikson, Parham, & Tobias, 1997; Ismail, Al Mutairi, & Al Anzy, 2000), hypernatremia following retrograde administration of hypertonic saline (Schreiber & Stone, 1999), and water intoxication following retrograde enema therapy using tap water (Chertow & Brady, 1994). Solution composition, volume, retention time, and underlying electrolyte imbalances are all factors that increase morbidity and m ortality (Yerkes et al., 2001). Several case series describe a variety of side effects of flushing regimens, including pain with stomal intubation, nausea, vomiting, abdominal cramping, sweating, dizziness, and pallor (Bani Hani et al., 2008 ; Dey et al., 2 003; King et al., 2005). A single retrospective study evaluated the safety of a specific flushing solution. Yerkes et al. (2001) evaluat ed the safety of tap water ante grade flush in 71 patients using serum electrolytes obtained pre and post operatively a s the dependent variable. The timing and place of the laboratory evaluation varied widely due to the retrospective nature of their study. The ACE flush was administered at home every day or every other day at volumes ranging from 300 to 1,000 mL. Clinicall y insignificant electrolyte abnormalities attributed to the flush included minor deviations in serum sodium or serum chloride in 18 patients. More significant hyperchloremia (107 to 113 mmol/L with upper limits set at 105 mmol/L) was noted in 12 patients. Significant hypernatremia and hyperchloremia was noted in a single subject who used softened water to flush, which corrected when the flush was changed to untreated water.

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54 A single prospective study was identified detailing and comparing adverse effects associated with different antegrade flushing regimens. ACE flush using twice normal saline has been associated with unpleasant symptoms including thirst and generally feeling unwell during the flush. Hyde et al., (2008) conducted a within subjects double blind crossover study on four children comparing antegrade administration of three commercially prepared flush solutions: water, normal saline, and twice normal saline. Each solution was administered on separate days. Each flush was spaced two days apart t o prevent the potential confound of carry over effects. Each child was randomized to one of six treatment sequences to prevent the potential confound of order effects. The study protocol was complex. Blood pressure, pulse, peripheral toe temperature, and capillary refill were measured at baseline prior to initiation of flush and at, 20, 40 and 60 minutes following flush administration. Blood was drawn at baseline, 20, and 60 minutes for hemoglobin, serum creatinine, electrolytes, albumin, total protein, os molality, renin activity, and arginine vasopressin concentration. Weight was measured before and after lavage with urine collected for electrolytes, creatinine, and osmolality, facilitating calculation of fractional excretion of water and sodium. Flush ef fluent was measured and analyzed for sodium and osmolality. Symptoms were recorded in real time. A paired t test was used to analyze within subjects data. Between subjects data was analyzed using an unpaired t test. Findings in this study demonstrated no c hange in any of the measured parameters with saline flush. Using water to flush resulted in a transient decrease in plasma osmolality of 7.3 mmol/kg at 20 minutes (lower level than NS, p=0.02) and a decrease in urine sodium and osmolality not observed with administration of the other flushing solutions. Administration of twice

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55 normal saline resulted in an increase in plasma sodium of 2.5 mmol/l at 20 minutes (higher than NS, p=0.03), a rise in plasma proteins of 2.3 g/l, and a decrease in the flush effluen t sodium concentration by one third, suggesting a shift of approximating 10 mL/kg of plasma water into the colonic lumen. The plasma sodium level increase of 2.5 mmol/l was insufficient to attribute the increase to sodium absorption alone. The drop in effl uent sodium concentration was attributed to two processes including colonic absorption of sodium from the twice normal saline and water shifting from plasma to the colonic lumen. Two children reported thirst suggesting the potential start of dehydration. T hirst occurred only during the twice normal saline flush. The same investigators also evaluated the composition of home made saline and twice normal saline flush solution from five families. Each family collected five independent samples of both home mad e flush solutions for sodium assay. Investigators found sodium concentrations in both the home made normal saline and twice normal saline solutions were 45% higher than the target value with a wide with in family variation. Investigators in this study fou nd 30 mL/kg of normal saline was as effective as 20 ml/kg of twice normal saline in achieving continence without the potential hazards of using a large volume of hypertonic solution (Hyde et al., 2007). Studies Addressing Gut Microbiota The catheterizabl e stoma used for antegrade administration of enema solution is frequently made by bringing the appendix out through the abdominal wall or by placing a button into the cecum. The appendix and cecum have significant amounts of gut associated lymphoid tissue (GALT), have high concentrations of microbiota, and serve an essential immune function (Penders, Stobberingh, Van den Brandt, & Thijs, 2007).

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56 The appendix has higher concentrations of microbial biofilms compared to other areas of the colon, serves as a saf e house for symbiotic gut flora, and functions to preserve gut microbiota through re inoculation with normal flora following gastrointestinal infections (Bazar, Lee, & Yun, 2004; Bollinger, Barbas, Bush, Lin, & Parker, 2007; Gebbers & Laissue, 2004; Kawani shi, 1987; Smith et al., 2009). The human gut contains 10 14 bacteria (Jia, Li, Zhao & Nicholson, 2008) Bacterial composition varies along the bowel axis, with further differentiation of luminal or adherent microcolonies that lead to development of biofil ms. Factors that influence microbial composition include pH, transit time, bile acids, pancreatic enzymes, mucus composition, nutrient consumption, medication, environment, bacterial adhesion capacity, and metabolic capacity. The most important function of the gut microbiome is colonization resistance, which is accomplished through competition for nutrients and secretion of bacteriocins (Penders et al., 2007). Microbiota function to degrade dietary substances and enhance digestive efficiency while providing nutrients to the microbes themselves. These microbes are essential for host physiology but in turn pose a threat of opportunistic invasion by resident bacteria with resulting pathologies. The immune system maintains a delicate homeostatic and symbiotic b alance protecting host microbial ecosystem dualism through the mechanism of stratification and compartmentalization. Stratification minimizes direct contact between gut microbiota and the intestinal epithelial surface. Compartmentalization utilizes anatomi c adaptation to confine bacteria that breach the mucosal surface to limit systemic immune system exposure. Immune system response to microbiota plays an important role in host vulnerability to disease. Host microbial symbiosis and dysbiosis are extraordina rily

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57 complex phenomena. The immune system controls the composition, diversity, and location of gut microbiota while the microbiota has a profound effect on lymphoid tissue formation and immune system development (Forchielli & Walker, 2005). Microbiota have been shown to have protective properties against autoimmune disease and, conversely, can cause inflammation and metabolic dysregulation in an immune compromised host (Hooper, Littman, & Macpherson, 2012; Nicholson et al., 2012). Disruptions in gut microbi ota due to diet, including infant feeding regimens, microbial inoculations, and antibiotics, can alter mucosal immunity and mechanisms involved in regulating immune tolerance outside the gastrointestinal tract (Noverr & Huffnagle, 2004). Microbiome composi tion imbalance has been associated with diverse disorders including cancer, inflammatory bowel disease ( IBD ) atopy, asthma, obesity, and autism (Barker, 2012). The appendix is a secondary lymphoid organ that is an important constituent of the mucosa assoc iated lymphoid tissue system; it has a pronounced function in children. Neonatal appendectomy in rabbits impaired mucosal immunity (Dasso & Howell, 1997). Long term effects of appendectomy include moderate immune function changes in part due to a decrease in immunoglobulin increased risk of acute myocardial infarction (Anderson, Olaison, Tysk, & Ekbom, 2003; Andreu Ballester et al., 2007; Janszky, Mukamal, Dalman, Hammar, & Ah nve, 2011). ACE therapy administered through the appendix or into the cecum has the potential to disrupt the gut microbial ecosystem causing dysbiosis and immune dysfunction. Effects of appendiceal or cecal administration of ACE on colonic microbiome and mucosal immunity has not been investigated.

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58 Significance and Innovation A large body of literature demonstrates ACE therapy can be effective in helping children with intractable fecal incontinence attain continence for stool with resulting significant impr ovement in quality of life. However, findings regarding effectiveness are highly variable. This variability may be due to what is used to flush. No prospective trials compare the efficacy and adverse effects of different flushing regimens. No studies evalu ate the effects of appendicos tom y/cecostomy flush on gut microbiota. Findings from this study provide d a comparative analysis of two different regimens and will serve as a starting point to guide practice and provide a foundation for additional prospective randomized, controlled trials.

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59 Table 2 1. Summary of Published Flush Solution, Dosing, Administration Frequency and Procedural Time Outcomes Authors # of Subjects Methods Solution Volume mL/kg Total Volume (mL) Infusion Time in Minutes Procedure Time i n Minutes Frequency of Administration Bani Hani, A. H. et al., 2008 n = 236 Retrospective Tap water 100 1,000 1 x day chart review additives 10.5% 642 mineral oil MiraLAX USP glycerin Becmeu r, F. et al., 2008 n = 22 Survey 250 1000mL 5 60 10 90 4 X week patient recall 700 mL 25 80 (qd q1wk) Chu, D.I. et al., 2013 n = 23 Retrospective Glycerin 30 mL of M 60 chart review USP glycerin mixed in 50 mL of tap water followed by 30 mL of tap water Dolejs, S.C. et al., 2017 n = 93 Retrospective Normal saline + IQR 16 30 chart review Bisacodyl if slow 22 motility, i ncrease to USP glycerin, IQR 18 35 Polyethylene 24 Glycol

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60 Table 2 1. Continued. Authors # of Subjects Methods Solution Volume mL/kg Total Volume (mL) Infusion Time in Minutes Procedure Time in Minutes Frequency of Administration Large, T. et al., 2017 n = 115 Data mined from clinical records of patients in a separate QOL study 7% required 40 60 additives 45 including Polyethylene Glycol, Castile Soap, glyce rin, castor oil and senekot Levitt, M.A. et al., 1997 n = 20 Retrospective chart review Normal saline 30 45 Matsuno, D. et al., 2010. n=25 Retrospective chart review T ap water 400 1500 36.7 +/ 23.1 4.2 +/ 4.0 x wk Range 1 7 x wk Peeraully, M.R., 2014 n=40 Retrospective Combination of saline & phosphate 808 +/ 612 20 60 1 x d in 77.5% of patients enema solution in 85% of sample 1 x qod in 15% of patients Variety of flush solutions in 15% of sample

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61 Table 2 1. Continued. Authors # of Subjects Methods Solution Volume mL/kg Total Volume (mL) Infusion Time in Minutes Procedure Time in Minutes Frequency of Admini stration Randall, J., 2014 n=203 Maintained a prospective data base 1998 2013 Normal Saline (48%) 4 x wk Range 1 7 x wk Klean Prep (29%) Bisacodyl (22%) Not Specified (1%) Siddiqui, A. A. et al., 2011 n=105 Retrospective Started on 23 +/ 0.7 847 +/ 55 12.1 +/ 1.2 51.7 +/ 3.5 5 +/ 0.3 d/wk chart review Saline (31%) Changed to Golytely if needed, if inadequate response used additives 34% including Bisacodyle 28% or other 7% (glycerin, phosphosoda, magnesium citrate) Sinha, C.K., 2008 n=48 Retrospective Bisacodyl Compared outcomes of 48 patie nts to the pooled outcomes of 676 patients from 24 studies published from 2002 2007 Normal Saline 180 3,000 5 60 Golytely M 516 M 42 Soap Water Glycerol and other flush combinations

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62 Table 2 1. Conti nued. Authors # of Subjects Methods Solution Volume mL/kg Total Volume (mL) Infusion Time in Minutes Procedure Time in Minutes Frequency of Administration Stenstrom, P. et al., 2013 n = 21 Questionnaire Saline Klyx 35 85 15 60 1 x day patient recall Movicol Range: 11 80 Range: 200 3000 M 45 (3xd 1 x wk) Vande Velde, S .,e t al., 2007 n = 16 Retrospective Tap water 60 240 Chart review 150 weekly Yerkes, E. B ., et al., 2001 n = 71 Retrospective Tap water 300 1,000 1 x day to 550 1 x qod

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63 CHAPTER 3 METHODOLOGY Design This prospective study utilize d a repeated measures, single subjects alternating treatments A B C B C B 1 withdrawal design in which all subjects we re tested under all conditions and each subject act ed as his or her own control (Gast, 2010; Janosky, Leininger, Hoerger, & Libkuman, 2009; Kazdin, 2011; Portney & Watkins, 2009). A within subjects cross over desi gn wa s embedded in the dosing B C and main tenance B C B 1 treatment comparison phase of the study (Chow & Brady, 1994; Janosky, Leininger, Hoerger, & Libkuman, 2009; Jones & Kenward, 2003) Subjects were randomly assigned to one of two treatment sequences to control for the possibility of order effects The treatment was replicated across 5 subjects randomized to 2 treatment groups The patient and investigator could not be blinded to flushing regimens contents due to the difference in dosing volume The high volume flushing regimen was comprised of nor mal saline alone. The low volume regimen was comprised of USP glycerin with a small volume of normal saline used as diluent. Volume and frequency of administration was structured to find the lowest dosing of each regimen sufficient to maintain continence. Baseline data A serve d as the control and was obtained pre operatively. Ethics prohibit ed return to a no treatment baseline phase as this would have result ed in multiple daily episodes of fecal incontinence. The first B C phase of the study evaluate d dos e response relationship and w as used to identify the optimal dose and frequency of ACE administration for normal saline and USP glycerin with a very low volume of saline as diluent When the optimal dose was identified, the child continue d on that dose for two weeks to insure treatment stability and effectiveness If

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64 continence was not achieved within the dosing guidelines, the child was trialed on the alternate flushing regimen to determine if continence with minimal side effects could be achieved and if s o, at what dose, but did not continue in the maintenance phase of the study. To prevent statistical bias from subject loss due to treatment failure, each child who completed the dosing phase was randomized to a second t reatment sequence once they had achie ved continence with minimal side effects in the dosing phase of the study The last phase of the study ( B C B 1 ) compare d continence, presence and severity of side effects, quality of life, and flush effects on stool microbiome of both regimens administe red for consecutive weeks each ( B C ) at optimal dose and administration frequency. The final two weeks of the study ( B 1 ) consisted of reintroduction of the flush administered in the B phase. Rationale f or Utilization of Within Subjects Design s Any rese arch design is a tool used to answer a question. Strategies, design choice, and use of design elements should be based on how best to answer the question at hand (Kazdin, 2011). The purpose of experimental design is to control the effects of random error a nd bias (Piantadosi, 2005). However, no design completely eliminates either. Whatever the chosen research method, good design and procedures are necessary to prevent confounding and improve causal inference (Shadish et al., 2002). Skillful statistical anal ysis cannot salvage significant design faults or increase the validity of a poorly designed study (Janosky et al., 2009). Skillful utilization of design elements can increase internal validity and causal inference (Cook & Campbell, 1979). Both single subje ct and between group research make and test predictions about treatment effects, the first by evaluating treatment effects on an individual, the second

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65 by addressing group mean and variance (Kazdin, 2011). Single subjects and group designs, in there most rigorous form, decrease the plausibility that rival hypo theses resulted in the experimental outcome, improv ing the quality of inference. (Cook & Campbell, 1979; Kazdin, 2011; Shadish, Cook, & Campbell, 2002). A randomized controlled trial (RCT) is conside red the gold standard for intervention research (Piantadosi, 2005). However, a RCT is not the only standard for causal inference (Kazdin, 2011). Reliance on large numbers makes application of a RCT with small groups or rare disease problematic (Janosky et al., 2009). The focus of this research involve d instillation of a solution through an appediceal stoma. A procedure used for over a century and widely popularized over 20 years ago. Case reports and retrospective studies detail widely divergent effectiven ess rates (Bani Hani, Cain, King, & Rink, 2008; Dey et al., 2003; Mousa et al., 2006; Siddiqui, Fishman, Bauer, & Nurko, 2011: Yardley et al., 2009). The literature and involved clinicians have identified the need for prospective trials comparing ACE flush ing regimens. Despite the identified need fo r prospective trials, only two have been identified to date. The first addressed comparative effectiveness of ACE flushing regimens utiliz ing colonic manometry to compare the motor response of three stimuli (meal antegrade saline infusion, and antegrade bisacodyl administration) on the number of high amplitude contractions and motility index in 13 pediatric patients (Gomez et al., 2010) The second compared safety parameters of a single flush of saline, twice norm al saline, and tap water ( Hyde et al., 2008). Th e lack of prospective trials is in large part because the small size and heterogeneity of this population d oes not lend itself to a large N study. Many

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66 clinical questions go unanswered due to over reliance o n RCT large N methodology (Kazdin, 2011). This population is an exemplar of that problem. Th is study compar ed two flushing regimens utiliz ing a cross over design embedded in a single subject A B C B 1 design. Both methods are experimental and lay a foundation for causal inference (Chow & Liu, 2014; Elder, 1997; Portney & Watkins, 2009). In both designs the subject acted as his or her own control minimizing within subject variability and ensuring the highest possible degree of equivalence across treatment conditions, thereby, allowing greater precision and efficient estimates of treatment effects increasing internal validity and causal inf erence (Janosky et al., 2009; Piantadosi, 2005; Portney & Watkins, 2009). In both methods subjects were randomized to treatment sequence. Randomization decrease d the threat of order effects in both methods, increase d group equivalency in the cross over des ign, minimize d variability in measurement due to subject or period differences, and increas ed internal validity and causal inference (Chow & Liu, 2014; Jones & Kenward, 2003). Single subjects design is an inductive, experimental methodology with controlle d introduction and manipulation of an independent variable Single subjects design promotes exploration of inter subject variability without the introduction of error inherent in group methodology in the absence of subject homogeneity. This method allows f or experimentation in situations in which a limited number of subjects are available, a circumstance that significantly limits the usefulness of large N studies. It allows for isolation of individual response to interventions and identification of valuable information from outliers that would be obscured or lost in group methodology. Single subjects design also allows for time series observations of response providing continuous and

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67 often a more accurate representation of the dependent variable of interest that may be compromised when the data are collected as an isolated snapshot in group methods (Elder, 1997). This study wa s ideally suited to single subject repeated measures design, because children requiring an ACE procedure comprise a very small popula tion with widely disparate anatomic and physiologic causative factors making sample homogeneity difficult. I nclusion of heterogeneous subjects allow ed differentiation of subject characteristics that could have impact ed response to treatment. The design all ow ed for frequency, volume, and dose adjustment of each flushing regimen when indicated. The ability to adjust the treatment regimen facilitate d dose response comparison and aid ed in identifying which flushing regimen require d the minimal dose and administ ration frequency, wa s accomplished in the least amount of time, and ha d the fewest side effects while achieving continence. Because this design allow ed for repe ated measurement over time, it wa s particularly helpful when studying comparisons between sever al treatments and wa s more sensitive to variations in treatment response that might otherwise have be en missed using group methodology (Gast, 2010; Janosky et al., 2009; Kazdin, 2011). Subjects w ere limited to children who were scheduled for a cecostomy o r appendicostomy ensuring their gut wa s nave to the effects of a flushing regimen and allow ed for a true no treatment baseline. Flush effects were reversible, which made this intervention amenable to a withdrawal design. There were no known carry over ef fects associated with either flushing regimen that would have impacted treatment effect on ed to a physiologic

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68 baseline between treatments, negating the need for a wash out period. S pecimen c ollection facilitate d comparison of treatment effects on gut microbiota, electrolytes, and stool calprotectin that occur red after an active wash out period at the completion of each flushing regimen negating any carry over effects. Given the pragmatic iss ues involved in answering the research question at hand, the chosen methods and design elements strengthen ed the demonstration of the counterfactual and ma de implausible potential threats to validity, lending credence to the assumption that intervention ef fects were due to the treatment and not random error or bias. Inclusion and Exclusion Criteria The goal in this study was to recruit six children, ages 3 to 1 2 years, recruited (NCC) and the Pediatric Spinal Defects Clinic in Jacksonville, Florida. Five children, ages 3 to 7 years, were recruited. Children were selected by purposive sampling and include d those who were scheduled to have ACE stoma construction and require d regular antegrade enema adminis tration to maintain continence. C hildren with preexisting electrolyte imbalance, chronic high rectal tone, quadriplegia, renal or cardiac disease, or those who require d prophylactic antibiotics, c ould n ot communicate, or ha d significant cognitive delay tha t would interfere with their ability to fully participate in the study were excluded from the study In addition, p arents of participants were required to have English language competency and a willing ness and ab ility to participate in administration or ov ersight of the flushing regimen and data collection for a minimum of twelve consec utive weeks.

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69 Regulatory Considerations and Study Expenses This study confer red no greater than minimal risk as categorized by the National Institutes of Health (National Ins titutes of Health [NIH], 1998). However, it involve d vulnerable subjects and require d full Institutional Review Board (IRB) ap prov al with legal guardian informed consent by at least one parent and child assent for children age 7 and above (Knox & Burkhart, 2007; Pieper, 2008). In addition, although normal saline and USP glycerin have been used for over two decade s in the clinical setting as ACE flushing solutions, administration of either solution through a ACE stoma is considered off label use and therefor e the study required FDA approval. It is a policy of N CC for any investigator conducting research involving patients seen in one of their facilities, to submit their proposal to the for review and approval of s tudy design and analysis prior to submission to the IRB. The application for the Nemours Clinical Research Review Committee was successfully completed and approval was granted to proceed with the study following FDA and IRB approval. (Appendix J) T he propo sal was submitted to the FDA and an IND l etter of exemption was obtained ( A ppendix K ) The study was subsequently entered in the Clinicaltrails.gov website An IRB application was completed for both UF and N CC The UF IRB deferred to N CC because the study was to be completed at N CC An IRB authorization agreement was completed, and the contract was signed by all relevant parties (Appendix L ) As a part of the N CC IRB application process, a submission for research involving investigational drugs and biologi cs and management of research pharmaceutical products was complete d and approved. (Appendix M ) A submission was completed and successfully met the requirements for the In stitutional Biosafety

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7 0 Committee. Nemours IRB approval wa s obtained and renewed annual ly (APPENDIX N ) The requirement for formal educational presentations to the clinical staff that would be involved with the children recruited in this study were successfully completed The cost for five subjects and c ompensation to participants was approx imately $6029.34. An itemized account of costs is detailed in Table 3 1. Subjects Subject characteristics data w ere collected and include d : (a) age in years and months, (b) diagnosis, including specific description of level of spinal cord lesion or anal rectal malformation when appropriate, (c) reason for ACE procedure, (d) surgical date for stoma formation, (e) type of stoma, (f) type of an e nter o stomal device, (g) comorbid conditions, and (h) results of previous pertinent test results, including upper gastrointestinal ( GI ) study with small bowel follow through, unprepped barium enema, sitz marker study, and an o rectal manometry. Parent characteristics include d : (a) age, (b) ethnicity, (c) marital status, (d) family composition (including number of childr en or other individuals living in the household), (e) occupation, and (f) highest level of education. Setting Once parental consent and child assent were obtained, baseline data w ere collected daily for a minimum of 2 weeks prior to surgery Baseline data includ ed frequency and volume of episodes of fecal soiling, and frequency and severity of abdominal pain. Blood samples for electrolyte and stool for calprotectin and microbiota were obtained in the preoperative period. The initial stool specimen s for a na lysis w ere collected prior to initiation of any pre operative bowel prep. Postoperatively, the child

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71 was randomly assigned to either the saline or USP glycerin protocol. A clinician from the surgical division at N CC met with the parent and child in the imm ediate postoperative period after surgical clearance ha d been obtained for initiation of the first flush. During th e initial flush the surgical clinician reviewed the flush protocol in detail including materials and procedures. The child receive d the fir st antegrade infusion during that in patient visit. A home visit w as scheduled by the investigator for the initial home flush The reliability or accuracy and consistency of measurements w ere verified using interobserver agreement (IOA) during the first ho me visit Gross method w as used to calculate IOA comparing investigator and parent/child concurrent observations including flush time, procedural time, and number/ level of soiling. IOA was calculated by dividing the smaller number by the larger number and multiplying by 100. At no point was there a significant discrepancy in observational accuracy as demonstrated by a calculated IOA below 80 % that would have required additional observer training to achieve a calculated IOA of 80% or higher (Gast, 2010). Th e initial plan was to ascertain IOA at the initial home visit and at each clinic visit. Procedural reliability w as ascertained for each procedural variable to assure the intervention was being implemented as described in the methods section of the proposal Procedural fidelity w as calculated by dividing the number of observed behaviors by the number of planned behaviors and dividing by 100 (Gast, 2010). IOA and procedural fidelity for antegrade infusion and data collection techniques conducted by the parent w as checked by the investigator and documented at the initial home visit. Procedural fidelity was completed prior to the start of each regimen change during the patients clinic visit for sample collection Error in

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72 study design did not facilitate IOA dur ing the subsequent clinic visits scheduled with each phase change. Measures Dependent variables at baseline include d : (a) the numb er and severity of episodes of fecal soiling per day with fecal soiling scored based on volume of accidents with 0 = no accid ent, 1 = a small volume accident classified as a smear, 2 = a moderate volume accide nt not visible through clothing and 3 = any accident visible through clothing, (b) frequency and severity of abdominal pain were recorded daily and measured using the Wong Baker Faces Pain Rating Scale as the age appropriate visual analog sc ale (c) serum electrolytes, (d) stool for calprotectin (e ) qual ity of life measured by the FI C Q O L and (f ) collection and p rocessing of stool for future analysis utiliz ing molecular t echniques for identification of 16SrRNA gene sequence in stool samples obtained pre operatively to identify and quantify phylogenetic groups (Penders et al., 2007). Dependent variables obtained post operatively following initiation of cecos to my/appendicos tomy flush in the dosing phase includ e : (a) flush administration time in minutes per flush, (b) total procedural time from start of flush to completion of colonic emptying in minutes per flush, ( c ) the numb er and severity of episodes of fecal soiling per day with fecal soiling scored based on volume of accidents with 0 = no accidents, 1 = a small volume accident classified as a smear, 2 = a moderate volume accide nt not sufficient in volume to be visible through clothing and 3 = any accident of sufficient volume to be visible through the clothing, (d ) timing of the accident when possible (e) frequency and severity of abdominal pain was recorded daily and

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73 measured using the Wong Baker Faces Pain Rating Scale (WBFPRS) as the age appropriate visual analog sca le (f ) serum electrolytes collected at the end of each dosing phase, ( g ) stool for calprotectin collected at the end of each dosing phase, ( h ) collection of stool samp l e at the end of each dosing phase for down stream analysis utiliz ing molecular techniq ues for identification of 16SrRNA gene sequence to identify and quantify phylogenetic groups (Penders et al., 2007) and ( i ) q ual ity of life measured by the FIQo L was to be completed at the completion of each maintenance phase. Only 1 of 5 subjects complete d the maintenance phase so FIQoL data w ere not completed on 4 of 5 subjects Fecal soiling score is detailed in Table 3 2 Dependent variables including type of sample or instrument, sample characteristics, and measurement and data level are explicated in Table 3 3 A dministration time in minutes per flush was defined as the time at which the tubing connected to the bag or syri nge holding the flush solution was unclamped and the cecostomy fluid start ed to infuse into the pa tient to the time the infusion wa s completed (no more fluid left in the bag/syringe or tubi ng). The total procedural time wa s defined as the time the flush start ed to infuse into the subject and end ed following passage of stool when the child ha d sat on the commode for 5 minutes with no a dditional stool pass age. Both administration and total procedural time s w ere measured using duration per occurrence direct observational with the recording completed by the parent. Total v olume w as recorded wi th each flush in mL. Accidents w ere defined as non toilet elimination, which was tracked and tallied as the number of underwear soiled with stool with documentation of the severity of the accident and the estimated tim ing of

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74 each accident using event recording. Dependent variables were measured and rec orded by the parent using a data collection sheet specifically designed for this study. Side effects w ere measured using the Wong Baker FACES P ain Rating Scale The WBFPRS has undergone extensive testing is preferred by children, and has well establishe d psychometrics in the pediatric population ( Tomlins on, von Baeyer, Stinson, & Sung, 2010; Wong & Baker, 1988). The scale ranges from 0 ( = very happy without pain ) to 10 ( = the worst pain imaginable ) Each pain level is associated with a facial expression. The child is asked to choose the face that best describes his/her level of discomfort. The WBFPRS was used to evaluate the presence and severity of flush side effects including abdominal pain, abdominal cramping, and nausea. The parent was instructed to c all if the child wa s having accidents or discomfort greater than a 4 on the WBFPRS associated with the flushing regimen. Documentation of severity of side effects was completed by the parent on a data collection form The investigator kep t results of all electronic communication from each subject that served as a research log that document ed and detail ed any event that cause d a change in level, stability, or trend of the dependent variable not related to the intervention, for example treatment with antib iotics or an intercurrent illness. The number of episode s of fecal soiling per week exclude d accidents caused by viral, bacterial, or drug induced enteritis; these were recorded and documented as confounds (Portney & Watkins, 2009). The Fecal Incontinence and Constipation Quality of Life Measure in Ch ildren with Spinal Bifida (FIC QO L) was used to assess child and parental perception of social validity (Nanigian e t al., 2008) The tool was administered preoperatively during the

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75 baseline period and at the end of each flushing regimen in the comparati ve phase of the study. The FIC QO L is a 51 item questionnaire with established validity and reliabilit y in families of children with spina b ifida who are incontinent for stool. Th is instrument measures those aspect s of daily living that are significantly impacted by fecal incontinence. Of the 51 items, four address subject and family demographics. The remaining 47 items are divided into seven groupings that include bowel program, diet, symptoms, travel and socializa tion, family relationships, caregiver support and emotional impact, and financial impact (Nanigian et al., 2008; Ok & Kurzrock, 2011) Only one subject w as continent on both flushing regimens and completed the study so the FIC QO L data w ere extremely limi ted. In addition to the FIC QO L, a simple qualitative question w as directed to the children at the end of the study to ascertain which flushing regimen they prefer and why. Materials To ease access, e ach child had an appropriate low profile button in plac e with the device specific access tube. Children with an appendicostomy had a Chait button in place with the device s pecific access tube. Materials for both flushing regimens are detailed in Appendix B A monitored temperature storage area was identified for storage of the flushing solution. The cabinet was locked with restricted key access. The temperature in the storage cabinet was maintained between 15 and 30 degrees Centigrade and monitored on a continuous basis using a MCC USB 501 LCD thermometer whic h allowed for storage and downloading of temperature data. No excursions occurred in ambient temperature. A temperature mon itoring log was kept A dispensing log was kept detailing the solution, volume dispensed, and lot numbers each

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76 time flush solution wa s dispensed to a subject. Funding was obtained to purchase the necessary equipment for isolating the microbiome DNA from samples to flash freeze for downstream identification of the 16SrRNA gene in stool samples. High Volume Flush For the high volume flus h, the normal saline was infused using a 1,000 mL enteral feeding bag with drip chamber and roller clamp. During the infusion, the bag w as hung from an IV pole or a hook on the bathroom wall located 5 feet above the toilet seat on which the child sat durin g the flush. Tub ing from the enteral feeding bag was hooked to the low profile device access tubing and primed with the high volume flush to remove all air in the tube prior to hooking the access tubing to the low profile device and infusing the solution. The s tep by step procedural directions for the p arents were reviewed verbally and provided through written instructions. The written procedure is located in Appendix C The written instructions were included in a parent notebook to reinforce parent teachin g and serve d as a check off list to document procedural integrity which w as checked during the initial home visit and with each subsequent clinic visit at the time of every change in phase. Low Volume Flush For the low volume flushing regimen, a calibrate d six ounce p lastic container with a screw top was used to mix USP glycerin with normal saline. The mixed solution w as then poured into a 60 mL catheter tipped syringe attached to the low profile device access tubing. The tubing w as primed prior to hooking it to the low profile device. The parent h el shoulder level while the flush wa s infusing. Al l components for each flush w ere at room

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77 temperature prior to mixing and infus ing the solution. A stop watch was used to measure the time taken to complete the procedure. The stop watch was started at the beginning of the infusion and was stopped when the child defecated and ha d not passed any additional stool for at least 5 minutes. T he time in min utes and seconds from start to completion of the infusion w as documented on a log designed specifically for this study. Following the flush, the tubing and bag or syringe w as washed in warm soapy water, rinsed, and allowed to air dry. Step by step parental procedural directions are listed in Appendix D The written instructions were included in a parent notebook to reinforce parent teaching and serve d as a check off list to document procedural integrity which w as checked during the initial home visit and wi th each subsequent clinic visit at the time of every change in phase. Procedures ere obtained preoperati vely. Data at baseline (A) include d continence data, serum electrolytes and stool calprotectin. The initial stool sample f or microbiota analysis was obtained pre operatively prior to initiation of any bowel prep. The child was randomized to one of the two treatment sequences pre operatively. The process was restricted random assignment to force equal sample size and w as accom plished using the SAS random number generator ensuring subject assignment results in equal group size. High Volume Regimen The high volume regimen (B) consist ed of a normal saline flush at a starting dose of 10mL /kg infused every other day and adjusted as detailed in Table 3 4 until stability of target outcomes wa s achieved. At any point the investigator received notification that

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78 the subject ha d had an episode of fecal soiling t he dosing strategy was increased by 5 mL /kg increments with a subsequent inc rease in frequency, if needed so as not to exceed a maximum dose of 500 mL administered daily for a child under five years of age and 1000 mL administered daily for a child over 5 years of age. If the child d id not attain continence on the maximum dose, t hey were trialed on the alternate regime but did not proceed to the maintenance phase of the study. I f the child ha d side effects greater than 4 on the W BFPR S at the starting dose of 10mL /kg, flush volume was incrementally decreased as needed by 2.5 mL/kg to the lowest dose of 5 mL/kg daily as detailed in T able 3 5 The goal was to find the lowest effective dose and flushing freque ncy with minimal side effects. If the dose necessary to minimize side effects result ed in episodes of fecal soiling or the chil d continue d to have side effects greater than 4 on the W BFP R S at the lowest dose of administration, the child w as trialed on the alternative flushing regimen, but did not proceed to the maintenance phase of the study Low Volume Regimen The low volume reg imen (C) consist ed of USP glycerin diluted in normal saline prior to antegrade instillation through the low profile device. The child start ed on an every other day dose of 20 mL of USP glycerin and >20 mL of saline (used as diluent at a dose sufficient to allow the solution to easily infuse through the ACE access tubing) and adjusted as detailed in Table 3 4 unt il stability of target outcome wa s achieved. At any point the investigator was notified the child wa s having episodes of fecal soiling greater than one smear per week the volume of USP glycerin was increased in 5 to 10 mL increments with subsequent increase in frequency of administration if needed so

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79 as not to exceed 4 0 mL of USP glycerin administered daily. If the child d id not attain continence on the maximum dose of USP glycerin he w as trialed on the alternative flushing solution, but did not proceed to the maintenance phase of the study. If the subject was having side effects greater than 4 on the WBFPRS at the starting dose of 20mL of USP glyce rin the volume of the USP glycerin was decreased as needed by 5 mL increments until were less than 3 on the WBFPRS or the lowest dose of 5 mL daily wa s reached as detailed in Table 3 5 The goal wa s to find the lowest effective dose a nd flushing freque ncy with minimal side effects. If the dose necessary to minimize effects result ed in episodes of fecal soiling greater than one smear per week or the child continue d to have side effects greater than 4 on the WBFPRS at the lowest dose of administration, the child w as trialed on the alternative flushing solution, but did not proceed to the maintenance phase of the study Optimal Dose Regimen Once the optimal dose was established, the child w as maintained on that dose and frequency for at least 2 weeks or until stability in continence wa s achieved. The child w as then scheduled to come into the clinic for a visit once the above criteria ha d been met at which time the child was flushed, labs w ere drawn a stool sample collected and the proc edure for the next regimen was reviewed and procedural fidelity was obtained Regimen Comparative Phase Following completion of the dose response phase, the comparative portion of the study beg an by administering either the established eff ective dose of n ) or the established ef ). Patients w ere randomized for a

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80 second time to either a B 1 or a C 1 sequence The last flush in the dose response sequence was stopped an d the initial flush in the compar ative treatment phase w as introduced the following day at the previously established minimum effective dose and administration frequency. Children remain ed on treatment for 4 weeks at which point the treatment w as withdrawn. Children w ere then placed on th e next treatment in the sequence at the pre established effective dose and frequency for 4 weeks. The second flush w as then withdrawn and the init i al flush in the sequence w as reintroduced for an additional two weeks ( B 1 or C 1 ) At the conclusion of the study, the child w as placed on the flushing regimen of his/her choice that maintained continence Specimen Collection Stool sample s w ere obtained during the baseline phase prior to any pre operative bowel prep and addit ional stool samples were obtained at the completion of each phase. Stool calprotectin was completed on the preoperative sample and the postoperative samples collected at the completion of each dosing phase for a total of 3 samples. Stool collection for dow nstream microbiota analysis was obtained preoperatively and following the completion of each dosing and maintenance phase for a total of 5 samples. Blood samples for electrolytes w ere drawn prior to surgery and following surgery at the end of each dosing p hase at the time stool samples were obtained for a total of three samples. Funding was secured and accounts with outside laboratories were set up. Blood samples and stool for calprotectin were sent for analysis The investigator received bench training in the lab to process the stool samples for isolation of microbiome DNA. S tool samples for microbiota analysis w ere collected, labeled with patient identifier, date, time, and flush composition. Each stool

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81 sample for microbiota analysis w as processed by the i nvestigator to purify and extract microbial DNA for sequencing, flash frozen to 80 o C, and archived for future downstream batch analysis using 16SrRNA as a taxonomic marker (Barker, 2012) as detailed in Appendix E The procedural timeline is detailed in F igure 3 1 Statistical Analysis The strength of an interrupted time series design is that effects of the intervention under investigation can be repeatedly and reliably measured over time. Time series parameters include mean intercept level, slope, and ad ditional non linear changes in shape. If the treatment is effective, it will result in a change in parameters that reverse with treatment withdrawal. Independent variable impact on time series parameters is based on the degree of change elicited in level, slope, or cycle of the measures process. The ability to determine independent variable effects is a function of a stable baseline and the number of baseline, intervention, and post intervention data points (Biglan, Ary, & Wagenaar, 2000; Janosky, Leininger Hoerger, & Libkuman, 2009; Shadish, Cook, & Campbell, 2002). In this study, each regimen w as administered at optimum dosing for a minimum of 2 weeks at the end of the titration phase Each flushing regimen was administered for 4 weeks during the comparat ive phase for a total of 6 weeks per flushing regimen for those subjects who progressed potentially yielding a total of at least 12 weeks or more of observations for those subject s who completed the study Data w ere used to facilitate analysis and dose r esponse adjustment during the B C or C B dosing phase. D ata w ere graphed on an equal interval line graph with the proportion of ordinate and abscissa scaled at a 2:3 ratio to ensure consistency of data

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82 presentation and prevent data distortion during visual analysis. D ependent measures w ere placed on the ordinate scale with time by day on the abscissa scale. Separation of the baseline, both dose response and the three comp arative phases of the study were designated by a bold vertical line D os ing regimen ch anges were designated by a thin dotted vertical line. Each phase change w as labeled with solution name Each dosing change was labeled with mL per dose and the total days of administration for the dose Independent variable effects on target behaviors were analyzed using visual analysis. Analysis within each condition include d : (a) condition length defined as the number of d ays contained within each phase, (b) level stability with a stability envelope calculated using the median and a stability criterion of 80% of the data points falling within 20 % of the calculated median for the phase, (c) relative change in level, (d) absolute change in level, (e) estimation of trend direction using split middle method, and (f ) identification of multiple paths within tren ds if present (Gast, 2010; Hartmann et al., 1980; Ma, 2006; Kazdin, 2011; McCleary, Meidinger, & Hay, 1980; Portney & Watkins, 2009). Testing in multiple subjects allow ed for analysis of replication of treatment effects. In addition to visual analysis of time series parameters, analysis was to include inferential procedures used to compare interventional effects and increase the reliability of visual methods analysis. The independent variable wa s nominal and dichotomous. Dependent variables were comprised of either interval or ratio level measurements. The dosing and comparative phase of the study wa s a 2 treatment crossover design with each child receiving both treatments. Each child who completed the dosing phase of the study was to be randomly assigned to either a C B C 1 or B C B 1 treatment sequence with half of

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83 the subjects allocated to each sequence (Portney & Watkins, 2009). The plan was to use i nferential statistics for two tailed hypothesis testing, which employs use of a sampling distributio n combined with the laws of probability to make statistical inference regarding rejection or failure to reject the null hypothesis (Polit, 2010). Statistical testing is based on the hypothetico deductive method where the null hypothesis states there is an absence of a relationship between the two populations at a given level of confidence. The object is to reject the null hypothesis at a fixed probability. The probability of committing an error i of significance, the probability level established as the risk of making a type 1 error, sets the threshold against which the p value is measured, and is generally set at .05. Failure to reject the null hypothesis occurs if the probability value p is > .05 and the confidence interval contains zero. Confidence interval is used to estimate precision of the effect. Effect size is used to determine the magnitude of the treatment difference (Polit, 2010; Rempher & Silkman, 2007). Initial consideration was given to testing t outcomes of interest that included number of soiling episodes, level of soiling, abdominal pain, procedural side effects, infusion time, pr ocedural time, electrolyte balance, and fecal calprotectin, using a two tailed, two sample pooled variance t test with a significance level set at 0.05. Because a cross over design is comparing two treatment sequences, the groups are independent (Chow & L iu, 2014). Confounding by carry over and direct by period interaction is a potential with cross over designs, which if present, can bias treatment effects (Jones & Kenward, 2003; Senn, 2002; Shuster, 2007). If inferential procedure

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84 was to be used in data a naly sis a decision had to be made regarding which test would best eliminate conditional bias and minimize variance. Jones et al. (2003) and Sen (2002) have suggest ed use of a one sample t test for analysis of cross over designs ( y= treatment two t reatme nt one ). Shuster (20 1 7) has advocate d a two sample t test in the analysis of a randomized 2 treatment cross over design irrespective of treatment order y = ( period two period one ) /2 Analysis of a one sample t test in a cross over design ignores treatme nt ordering. Two sample t test analysis compares ordering and yields over, results from a one sample or two sample t test will yield unbiased estimates of and vari should be similar using either the one sample or two sample method. However, the one sample method does not account for carry over effects increasing variance. If sample sizes but not the two sample t test will be biased. The decision was made to use the 2 sample method to lend precision in the presence of carry over effects, and precision and ac (Shuster, 2009). Due to small sample size, the treatment effects did not reach statistical significance so d escriptive statistics were used including median, mean, range and standard deviation to d escribe effects of intervention on the outcomes of interest. Neither visual analysis nor inferential statistics alone will infer causality. Strictly speaking, correlation does not infer causation (Polit, 2010). Causal inference is primarily influenced by t he research design. Visual analysis and statistical procedures are helpful in measuring the effects of potential causes (Kazdin, 2011). Appropriate use of

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85 statistics aid in the inference of causality by quantifying the effect chance plays on conclusions ( Hill, 1965) This study was prospective allowing for the determination of temporal precedence. Statistical and visual analysis was used to assess contiguity with respect to presumed cause and effect. Strategic use of design elements were used to limit alt ernative explanations for findings. The first phase of the study evaluate d dose response which assess ed biological gradient. The design assesse d treatment response across multiple subjects, which provide d support for consistency across contexts. Social Validity Attaining continence is a highly socially significant issue for any child but particularly difficult to attain using conservative measures in children with neuromuscular disorders, anorectal malformations, spinal cord injuries, spinal cord trauma or tumor, megarectum, and slow transit constipation. ACE therapy has been shown to be effective in helping children with intractable fecal incontinence attain continence for stool with resulting significant improvement in independence and quality of life. However, research findings regarding short and long term effectiveness are variable (Aspirot, Fernandez, Di Lorenzo, Skaggs, & Mousa, 2009; Bani Hani, Cain, King, & Rink, 2008; Basson et al., 2014; Becmeur et al., 2008; Chu, Balsara, Routh, Ross, & Wiener, 2013; Church, Simah, Wild, Teitelbaum, & Ehrlich, 2017; Curry, Osborne, & Malone, 1999; Chong, Featherstone, Sharif, Cherian, Cuckow, Mushtaq, De Coppi, Cross, & Curry, 2016; Dey et al., 2003; Dolejs, Smith, Sheplock, Croffie, & Rescorla, 2017; Driver et al ., 1998; Freeman et al., 2014; Hoekstra,Kuijper, Bakx, Heij, Aronson, & Benninga, 2011; King, Sutcliffe, Southwell, Chait, & Hutson, 2005; Large, Szymanski, Whittman, Misseri, Chan, Kaefer, Rink and Cain, 2017; Levitt, Soffer, &

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86 Pena, 1997; Marshall, Hutson Anticich, & Stanton, 2001; Matsuno, Yamazaki, Shiroyanagi, Ueda, Suzuki, Nishi, Hagiwara, & Ichiroku, 2010; Mousa et al., 2006; Ok & Kurzrock, 2011; Peeraully et al., 2014; Randall et al., 2014; Siddiqui, Fishman, Bauer, & Nurko, 2011; Sinha et al., 2008; T homas et al., 2006; VanderBrink et al., 2013; Yardley et al., 2009). This variability may be due to what is used to flush. In addition, there are no comparative studies documenting the time necessary to complete the flushing procedure. Sitting on the commod e for one hour versus 20 minutes represents a major difference in age appropriate expectations for a 6 year old child. In a child, procedural time may be inversely related to adherence and negatively impact effectiveness of the procedure. Currently the fl ushing regimen for each child is based on clinician preference and can be a lengthy process of trial and error. Identifying effective dose and frequency of commonly used flushing solutions or medication regimens and comparing effectiveness of the differen t flushing regimens will benefit any child requiring ACE therapy promoting continence at an earlier stage in their therapy and cost savings for the family by decreasing cost s associated with ineffective trials and additional time spent in protective garmen ts. This study allow ed implementation of ACE flush by the parent and eventually the child in th e home after research support was withdrawn. Social importance and acceptability of the treatment mediates the relationship between the prescribed therapy and implementation. Treatment acceptability and parental willingness to carry out the procedure is directly related to the likelihood of procedural fidelity and effectiveness. (Jones, Eyberg, Adams, & Boggs, 1998; Kazdin,

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87 2011). U pon completion of the study, a simple question w as directed to the children to ascertain which flushing regimen they preferred Threats to Validity Causal inference is predicated on covariation in the independent and dependent variables. The source of that covariation is central to i nferring cause (Cook & Campbell, 1979). Confounding or threats to internal validity occur when the covariation is not due to the independent variable, but to another variable not accounted for in the causal pathway (Susser, 2001). Multiple threats to inter nal validity can operate simultaneously (Cook & Campbell, 1979). A central task of any researcher is to identify and trenchantly examine all the potential confounds, assess the plausibility of the identified threats, and determine how best to deal with the m (Shadish et al., 2002). A sound study is designed to limit plausible alternative explanations achieved through utilization of proper control that strengthens the inference that the outcome is due to treatment (Janosky et al., 2009). Reliability is esse ntial to validity. In single subject research, emphasis is placed on the reliability of measurement and consistency in implementation of procedures. Reliability of the individual collecting data w as verified using interobserver agreement (IOA) at the initi al home visit Procedural fidelity w as ascertained to assure interventions were reliably implemented with each phase change (Kazdin, 2011; Gast, 2010). Typology used for assessing potential sources of confounding include threats to: (a) statistical conclu sion validity, (b) internal validity, and (c) construct validity (Portney & Watkins, 2009). Assessment of potential threats to statistical conclusion validity are detailed in Appendix F Assessment of potential threats to internal validity are detailed in Appendix G Assessment of potential social threats to internal validity are detailed in

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88 Appendix H Assessment of potential threats to construct validity are detailed in Appendix I Table 3 1. Itemized Estimate for Maximum Flushing Costs For 6 Subjects N ormal USP BMP + Stool Saline Glycerin Diluent Draw Fee Calpro tectin Lido caine Tega derm Stipend Cost per $3.65/L $126.55 / $3.65/ L $11.98 + $123.45 $32.86/ $18.76/ $25.00 Unit 3.78 L $3.00 T ube B ox Estimate 1 L/d 60 mL /d 60 mL/d 3 3 4 2 6 Units x 70 d x 70 d x 70 d Total Cost/ $255.55 $140.61 $15.33 $44.94 $370.35 $150.0 0 Subject Total Cost/ $1,533.00 $843.66 $91.98 $269.64 $2,222.10 $131.44 $37.52 $900.0 0 Six Subject s BMP Basic Metabolic Panel Table 3 2. Level of Soiling Code Description 0 No soiling 1* Smear of Stool 2* Moderate volume accident no t visible through clothing 3* Any accident large enough to be visible through clothing *Change d ose with any soiling

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89 Table 3 3 Dependent Variables Sample/Instrument Variable Measurement Data Level Blood BMP Electrolyte Balance Ratio Stool Calprotectin Mucosal Inflammation Ratio Stool Colonic microbiome Metagenomic Profiling 16SrRNA Ratio F IC QOL Parent/child quality of life Symptoms Rating Scale Ordinal WBFPRS Abdominal Pain Symptom Rating Scale Ordinal WBFPRS Procedural side effects Symptom Rating Scale Ordinal Stop Watch Infusion Time Minutes Ratio Stop Watch Procedural Time Minutes R atio

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90 Table 3 4 Dosing Strategy to Determine Minimum Volume and Frequency Necessary to Maintain Continence *Change dose with any soiling > Level 0 Incrementally increase volume of saline or glycerin unti l maximum dose in reached Stopping rule for saline is a maximum of 500 mL flush volume for children 5 years of age and under and 1,000 mL flush volume in children over 5 years of age who do not achieve continence at maximum dose Stopping rule for USP glyce rin is incontinence at a maximum dose of 40 mL qd every day qod every other day q3d every third day Normal Saline (High volume regimen) USP Glycerin + Normal Saline as Diluent (Low volume regimen) B1 = 10mL/kg maximum dose 1000 mL qod C1 = 20 mL + > 20 mL qod B2 = 10mL/kg maximum dose 1000 mL q3d C2 = 20 mL + > 20 mL q3d B3 = 10mL/kg maximum dose 1000 mL qd C3 = 20 mL + > 20 mL qd B4 = 15 mL/kg maximum dose 1000 mL qod C4 = 25 mL + > 40 mL qod B5 = 15 mL/kg maximum dose 1000 mL q3d C5 = 25 mL + > 40 mL q3d B6 = 15 mL/kg maximum dose 1000 mL qd C6 = 25 mL + > 40 mL qd C7 = 30 mL + > 50 mL qod C8 = 3 0 mL + > 50 mL q3d C9 = 3 0 mL + > 50 mL qd

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91 Table 3 5. Dosing Strategy to Determine Minimum Volume and Frequency Necessary to Minimize Side Effects Normal Saline* (High volume regimen) USP Glycerin + NS as Diluent* (Low volume regimen) B1 = 10 mL/kg maximum dose 1000 mL qod C1 = 20 mL + > 20 mL qod B7 = 7.5 mL/kg maximum dose 1000 mL qod C10 = 15 mL + > 15 mL qod B8 = 7.5 mL/kg maximum dose 1000 mL q3d C11 = 15 mL + > 15 mL q3d B9 = 7.5 mL/kg maximum dose 1000 mL qd C12 = 15 mL + > 15 mL qd B10 = 5.0 mL/kg maximum dose 1000 mL qod C13 = 10 mL + > 10 mL qod B11 = 5.0 mL/kg maximum dose 1000 mL q3d C14 = 10 mL + > 10 mL q3d B12 = 5.0 mL/kg maximum dose 1000 mL qd C15 = 10 mL + > 10 mL q d C16 = C17 = C18 = 5 mL + > 5 mL qod. 5 mL + > 5 mL q3d 5 mL + > 5 mL qd Change dose with any side effects > 3 on the WBFPRS or with any soiling > Level 0 Incrementally decrease volume of saline or glycerin until minimum dose in reached Stopping rule for saline is a continued pain or incontinence at a minimum dose of 5.0 mL/kg Stopping rule for USP glycerin is continued pain or incontinence at a minimum dose of 5.0 mL/ qd every day qod every other day q3d every third day

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92 A Baseline NT No treatment B Saline dose response phase C USP Glycerin dose response phase Initial trial of saline effectiveness phase Initial trial of USP glycerin effectiveness phase B 1 Second trial of saline effectiveness phase C 1 Second Trial of U SP glycerin effectiveness phase BMP Basic Metabolic Profile SIM S tool immune marker Visits a Procedural fidelity and inter rater reliability will be measured at each visit FIC QOL Fecal Incontinence and Constipation Quality of Life Measure in Childre n with Spina Bifida QQ Qualitative question asked of the child at the end of the study Abd Pain Abdominal Pain Admin T Administration time Proc T Procedural time ISE Infusion side effects Figure 3 1. Procedural t imeline

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93 CHAPTER 4 FINDINGS Sub ject Characteristics Five subjects between the ages of 3 and 7 years were recruited to participate in the study. All subjects were male. Two subject s were Caucasian, one was Hispanic, one was African American and one was Asian and had recently been adopted from China. Four of the five subjects had lumbosacral myelomeningocele and were ambulatory. The remaining child had cord transection below T 10 from a motor vehicle accident and was wheelchair bound. All five subjects had a patulous anus and had failed re trograde suppository administration or retrograde enema therapy using an assistive device. Aim s The p rimary aim s of this study w ere to determine (a) if there was a diffe rence in minimal administration frequency (b) titration time to reaching effective d ose, and (c) whi ch solution at an optimum dose was delivered in the le ast amount of time, with fewer side effects, while promoting the highe r degree of fecal continence Secondary aims were to (a) compare quality of life metrics, (b) determine the cost dif ference between two flushing solutions and (c) collect and process stool for future analysis to determine if administration of antegrade enema solution through an appendicostomy/cecostomy affects gut microbiota and immune function. Visual analysis was use d to analyze within subjects data. Sample size was too small to produce statistically significant findings using inferential statistics. Between subjects data w ere analyzed using descriptive statistics including median, mean, standard deviation and range. A power analysis was conducted to estimate the necessary sample size needed in a future study to minimize

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94 the probability of committing a Type II error (1 ) and power = .80 To correct for data dependence among means, a correlation b etween two means was calculated and applied to make a direct comparison of effect sizes Null Hypotheses for Aim 1 Frequency of Administr ation Null Hypothesis 1.1 There will be no differences in frequency of administration necessary to gain and maintain continence between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring anteg rade continence therapy (primary aim). One hundred percent of subjects ( five of five ) required daily flushing with saline. Sixty percent ( three of five ) required daily flushing with USP Glycerin. Twenty percent ( one of five ) maintained continence on every other day flushing with USP Glycerin. Twenty percent ( one of five ) maintained continence on every third day flushing w ith USP Glycerin. Of those subjects who achieved continence on saline during the dosing phase ( two of five ) 100% of subjects (two of two) required daily flush administration with saline to maintain continence, only 50% of subjects ( two of four ) required daily flushing with USP gl ycerin to maintain continence A visual comparison of the administration frequency of normal saline versus USP gl ycerin is graphically represented in Figure 4 1. Inferential statistics did not yield statistical significance. Titration Time Null Hypothesis 1.2. There will be no difference in titration time to reach effective dose between two different ACE flushing re gimens using normal saline and

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95 normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). Titration time between subjects and between flush solutions was highly variable Analysis exclud ing titration times for the s ubjects who did not achieve continence revealed t itration time s ranged from 52 to 53 days for the two subjects who initially achieved continence on saline during the dosing phase of the study Individual subject data and descriptive statistics are detaile d in Table 4 1 A visual comparison of titration time for normal saline versus USP glycerin including all subjects is found in Figure 4 2 Cost Burden Null Hypothesis 1.3 There will be no difference in cost between two different ACE flushing regimens usin g normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). Normal saline flush was more expensive than USP Glycerin flush both on a per dose and per week basis. The average cost per saline flush was $2.59 as compared to $1.19 per flush of USP glycerin. Using the USP glycerin resulted in a n average reduction of 54% in cost per dose. A verage weekly cost for the saline flushing regimen was $18.16 as compared to $4.89 for USP glycerin. Using USP glyce rin resulted in an average reduction of 73 % in cost per week. Cost per subject is detailed in Table 4 2 The difference in cost did not reach statistical significance.

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96 Null Hypotheses for Aim 2 Continence Null Hypothesis 2. 1. There will be no difference in continence between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (primary aim). T here was a difference in continence rates between the two flushing regimens. The difference did not reach statistical significance Descriptive statistics were calculated on the last data point in the final phase of each flush Only 20% of subjects ( one of five ) gained and maintained continence on saline. Eighty percent of subject s ( four of five ) gained continence on USP Glycerin Severity of daily soiling was greater on saline when compared to glycerin Power analysis conducted using data from this = 0 .5, power of .80 correlation between two means of .598, and effect size of 1.554 estimated a sample size of 11 would be needed to minimize the risk of a Type II error to (20%) Individual subject dat a and de scriptive statistics are detailed in Table 4 1. The graphed continence data used in visual analysis to evaluate absolute frequency of incontinence is detailed in Figures 4 3 t hrough 4 7 with graphed severity data detailed in Figures 4 8 t hrough 4 12 With in subjects c alculations including stability and trend graphs are located in subject specific Appendi ces O through S Infusion Time Null Hypothesis 1.2 There will be no difference in infusion time between two different ACE flushing regimens using normal s aline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim).

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97 The infusion time of USP glycerin was less in 80% of subjects ( four of five ) when compared to normal saline. Infusion time data were included in procedural time graphs and calculations. Procedural Time Null Hypothesis 1. 3 There will be no difference in procedural time between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrad e continence therapy (secondary aim). Descriptive statistics were calculate d on the last data point in the final phase of each flush. Procedural time with normal saline was less in 60% of subjects ( three of five ) when compared to USP glycerin Procedural time was equivalent for normal saline and USP glycerin in 20% of subjects ( one of five ). Procedure time on USP glycerin was less in 20% of subjects ( one of five ) when compared to normal saline Individual subject data and descriptive statistics and are de tailed in Table 4 1. The difference in procedural time did not reach statistical significance The graphed data used in visual analysis to evaluate procedural time is detailed in Figures 4 1 3 through 4 17. Within subjects c a lculations for procedural data in cluding stability and trend graphs are located in subject specific Appendices O through S. Side Effects Null Hypothesis 2. 4 There will be no difference in side effects between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). Descriptive statistics were calculated on the last dat a point in the final phase of each flush. The difference in pain between the two flushing regimens did not reach

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98 statisti cal significance Side effects were minimal with only 20% of subjects ( one of five ) experiencing consistent cramping with USP glycerin. The cramping was 4 or less on the WBFP RS was consistently relieved with defecation, and did not necessitate dosing adjus tment Of interest, this is the only subject that documented consistent pain at baseline. No subjects had consistent cramping with normal saline flush One subject had a significant episodes of abdominal pain secondary to constipation that improved with fl ushing. One subject had a single significant degree of cramping on a higher than prescribed dose of glycerin that resolved with defecation and did not recur Only 20% of subjects ( one of five ) had a v a gal response to USP glycerin The response was severe e nough the child was dropped from the study The child had started taking iron supplementation, had missed several flushes and was quite constipated. The child was cleaned out with Golytely and placed back on USP glycerin without further side effects. It i s interesting to note that constipation appeared to be the major cause of side effects. Individual subject dat a and descriptive statistics are detailed in Table 4 1. The graphed data used in visual analysis to evaluate side effects is detailed in Figures 4 3 through 4 17. Within subjects calculations for side effects data including stability and trend graphs are located in subject specific Appendices O through S Quality of Life Null Hypothesis 2. 5. There will be no difference in parent/patient satisfactio n as measured by the Fecal Incontinence and Constipation Quality of Life Measure in Children with Spina Bifida (FIC QOL) between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade con tinence therapy (secondary aim).

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99 Only one of five subjects achieved and maintained continence on saline. The remaining four subjects either did not progress to or complete the maintenance phase of the study. By study design, the FIC QOL tool was administe red in the comparative phase of the study and therefore was not completed by 80% of subjects. Thus any difference in parent/patient satisfaction between flushing regimens could not be assessed. Electrolytes There were no abnormalities in electrolytes asso ciated with either normal saline or USP glycerin flush A visual comparison of the effects of saline and USP glycerin flushing solutions on serum electrolytes including sodium, potassium, chloride, carbon dioxide calcium, blood urea and nitrogen, and crea tinine are graphically represented in Figure s 4 26 through 4 31. Stool Calprotectin Null Hypothesis 3.2 There will be no difference in the effect of two different ACE flushing regimens using normal saline and normal sali ne with USP glycerin on mucosal immune function in children requiring antegrade continence therapy Stool samples for calprotectin were obtained directly following saline flush One subject had a significant rectal prolapse and did not pass stool following the saline flush completed in cl inic prior t o transition to glycerin so levels are available in only four of five subjects for saline Of the four remaining samples, c alprotectin levels were elevated in 75% of subjects ( three of four ) and decreased in 25% of subjects ( one of four ) in re sponse to saline Changes in calprotectin levels on saline rang ed from a decrease of 31 % to an increase of 2,550 % when compared to baseline In one subject levels

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100 exceeded the upper limits of normal on s aline, but did not reach a level of clinical concer n that would necessitate stopping ACE therapy. Stool samples for calprotectin were obtained in 100% ( five of five ) of subjects following USP glycerin flush. C alprotectin levels were elevated in 80% of subjects ( four of five ) and decreased in 20% o f subj ects ( one of five ) on glyceri n Changes in calprotectin levels on glycerin rang ed from a decrease of 58.6 % to an increase of 379 % compared to baseline levels Individual subject data and descriptive statistics and are detailed in Table 4 1. Stool c alprot e c tin levels are gra phically presented in Figure 4 32 Flushing Pr eference Three of five ( 60% ) subjects preferred normal saline ; two ( 40% ) had no preference. None of the subjects who preferred saline could articulat e their reasons for doing so

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101 Ta ble 4 1: Individual Subject Data and Descriptive Statistics Titratio n Titration Continence Continence Dosing Dosing Procedure Procedure Time Time Rates Rates Frequency Frequency Time Time Days Days Per Day Per Day Minutes Minutes Saline Glycer in Saline Glycerin Saline Glycerin Saline Glycerin KJ001 ** 53 46 0 0 Daily Daily 22 57 KJ002 *** 54 37 1 0 Daily Q3d 20 90 KJ003 *** 52 33 1 0 Daily QOD 42 102 KJ004 ** 12 79 4 1 Daily Daily 35 35 KJ005 ** 39 53 4 0 Daily Daily 50 30 Mean 42 49.6 2 0.2 33.8 62.8 Median 52 46 0 0 35 57 Range 12 to 54 33 to 79 0 to 4 0 to 1 20 to 50 30 to 102 SD 17.85 18.19 1.87 0.45 12.85 32.24 Data calculated on last data point in final phase of each dosing re gimen **Randomized to start on Normal Saline ***Randomized to start on USP Glycerin

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102 Table 4 1: Continued Pain Pain Stool Stool Weekly Weekly Severity Severity Calprotectin Calprotectin Cost Cost WBFPRS WBFPRS Dollars Dollars Saline Glycerin* Saline Glycerin Saline Glycerin KJ001 ** 0 0 413.4 217.4 21.70 10.92 KJ002 *** 0 0 148.9 34.7 25.55 1.9 KJ003 *** 0 2 54.1 211.2 17.92 1.95 KJ004 ** 0 0 119.1 57.9 12.81 10.92 KJ005 ** 0 0 Missing 277.4 12.81 7.77 Mean 0 0. 4 183.88 159.72 18.16 6.69 Median 0 0 134 211.2 17.92 7.77 Range 0 to 0 0 to 4 119.1 to 413.4 34.7 to 277.4 17.92 to 25.55 1.90 to 10.92 SD 0 0.89 158.05 107.03 5.58 4.54 Data calculated on last data point in final phase of each dosing regimen **Rando mized to start on Normal Saline ***Randomized to start on USP Glycerin

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103 Figure 4 1: Frequency of flush a dministration Figure 4 2 : Titration t ime to a chieve c ontinence 1 1 1 1 1 2 1 3 1 1 0 1 2 3 SALINE USP GLYCERIN FREQUENCY OF DOSING IN DAYS COMPARITIVE FREQUENCY OF FLUSH ADMINISTRATION KJ001 KJ002 KJ003 KJ004 KJ005 1=daily 2=qod 3=q3d X X 52 X 53 53 X 33 37 46 0 10 20 30 40 50 60 70 80 90 5 4 3 2 1 DAYS SUBJECTS TITRATION TIME TO CONTINENCE USP Glycerine Saline x = Failure to gain continence on flush

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104 Table 4 2 Cost Comparison Normal Saline vs USP Glycerin Flush Subject # NaCl 0.9 % USP Glycerin + Cost/ Dose Cost/Dose Weekly Cost Weekly Cost Dose (mL/day) Dose & Diluent (mL/day) NaCl 0.9% USP Glycerin NaCl 0.9% USP Glycerin KJ001 850 40 mL + 60 mL/d $3.10 $1.56 $21.70 $10.92 KJ002 1000 25 mL + 30 mL/q3d $3.65 $0.95 $25.55 $1.90 KJ003 700 20 mL + 30 mL/qod $2.56 $0.78 $17.92 $1.95 KJ004 500 40 mL + 60 mL/d $1.83 $1.56 $12.81 $10.92 KJ005 500 30 mL + 30 mL/d $1.83 $1.11 $12.81 $7.77 *Cost calculation based on pricing of normal saline at $3.65/L and USP glycerin at $33.48/L qod every other day q3d every third day

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105 Figure 4 3: KJ001 Absolute frequency of incontinence g raph Intercurrent Illness

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106 Figure 4 4: KJ002 Absolute frequency of incontinence g raph

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107 Figure 4 5 : KJ003 Absolute f requency of incontinence g raph

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108 Figure 4 6: KJ004 Absolute frequency of incontinence g raph

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109 Figure 4 7: KJ005 Absolute f requency of i ncontinence g raph D iscontinued Osmotic Agent

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110 F igure 4 8: KJ001 Frequency and s everity of incontinence g raph

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111 Figure 4 9: KJ002 Frequency and severity of incontinence g raph

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112 Figure 4 10: KJ003 Frequency and severity of incontinence g raph

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113 Fi gure 4 11: KJ004 Frequency and severity of incontinence g raph

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114 Fi gure 4 12: KJ005 Frequency and severity of incontinence g raph

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115 Fi gure 4 13: KJ001 Procedural time g raph

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116 Figure 4 14: KJ002 Procedural time g raph

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117 Figure 4 15: KJ003 Procedural time g raph

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118 Figure 4 16 : KJ004 Procedural t ime g raph

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119 Figure 4 17: KJ005 Procedural time g raph

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120 Note: Severe episode of abdominal pai n due to constipation relieved with defecation following flush Figure 4 18: KJ001 Abdominal p ain g raph Abdominal pain associated with constipation

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121 Figure 4 19 : KJ001 Cramping from flush g raph

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122 Figure 4 20: KJ002 Abdominal p ain

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123 Note: Significant abdominal pain in baseline may indicate exist ing predisposition to abdominal discomfort Figure 4 21: KJ003 Abdominal p ain

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124 Figure 4 22: KJ003 Cramping with f lush

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125 Figure 4 23: KJ 00 4 Cramping with f lush

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126 Fi gure 4 24: KJ005 Cramping with f lush

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127 Figure 4 2 5 : KJ001 Vagal symptoms g raph

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128 Figure 4 2 6 : Serum sodium l evels Figure 4 2 7 : Serum potassium l evels 130 132 134 136 138 140 142 144 146 Baseline Saline USP Glycerine SERUM SODIUM LEVELS KJ001 KJ002 KJ003 KJ004 KJ005 Lower Limit Upper Limit 0 1 2 3 4 5 6 Baseline Saline USP Glycerine SERUM POTASSIUM LEVELS KJ001 KJ002 KJ003 KJ004 KJ005 Lower Limit Upper Limit

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129 Figure 4 2 8 : Serum chloride l evels F igure 4 29 : Serum carbon d ioxide l evels 92 94 96 98 100 102 104 106 108 110 112 Baseline Saline USP Glycerine SERUM CHLORIDE LEVELS KJ001 KJ002 KJ003 KJ004 KJ005 Lower Limit Upper Limit 0 5 10 15 20 25 30 35 Baseline Saline USP Glycerine CARBON DIOXIDE LEVELS KJ001 KJ002 KJ003 KJ004 KJ005 Lower Limit Upper Limit

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130 Figure 4 3 0 : Blood urea nitrogen l evels Figure 4 3 1 : Serum creatinine l evels ( l ow levels normal in children ) 0 5 10 15 20 25 Baseline Saline USP Glycerine BLOOD UREA NITROGEN LEVELS KJ001 KJ002 KJ003 KJ004 KJ005 Lower Limit Upper Limit 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Baseline Saline USP Glycerine CREATININE KJ001 KJ002 KJ003 KJ004 KJ005 Lower Limit Upper Limit

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131 Figure 4 3 2 : Serum calcium l evels Figure 4 3 3 : Stool c alprotectin l evels 7.5 8 8.5 9 9.5 10 10.5 Baseline Saline USP Glycerine CALCIUM LEVELS KJ001 KJ002 KJ003 KJ004 KJ005 Lower limit Upper Limit KJ001 KJ002 KJ003 KJ004 KJ006 Baseline 15.6 84 78.5 22.6 57.9 Saline 413.4 148.9 54.1 119.1 USP Glycerine 217.4 34.7 211.2 57.9 277.4 Limit 162 162 162 162 162 0 50 100 150 200 250 300 350 400 450 mcg/g STOOL CALPROTECTIN LEVELS

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132 CHAPTER 5 DISCUSSION AND CONCLUSIONS Aimes Specific Discussion of Outcomes Frequency of A dministration The ability to minimize frequency of flush administration is a clinically significant issue One goal of ACE therapy is to maximize effectiveness while minimizing procedural intrusiveness. Minimizing flush frequency is one means to decrease the time commitment required to maintain continence. I n the single study reporting outcom es of antegrade administration of USP glycerin 84% of subjects required an every other day or less flush frequency to maintain continence (Chu et al., 2013) In this study, a ll five subjects required daily dosing when using normal saline with only one of five achieving and maintaining continence on saline. Of the four subjects who gained and maintained continence on USP glycerin, 50% required a n every other day or less flush frequency. Failure of subjects to gain continence on saline negated the utility o f the results. M inimizing the time required to achieve p redictable continence on any flush regimen has implications for procedural acceptability on the part of the child and quality of life for both the child and family. Compared to the Chu et al (2013) data f indings from this study may under estimate the flush frequency required to gain and maintain continence using USP glycerin. Findings in this study did not reach statistical significance due to sample size but may have clinical utility. Building on p reviously reported outcomes, use of USP glycerin may result in predictable continence at a d ecrease d frequency of administration in male children under 8 years of age wit h low lying spinal cord lesions t hereby decreasing the overall time burden and cost a ssociated with a successful ACE flushing regimen. Further studies are needed to

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133 confirm administration frequency providing clinically useful information that has potential to guide clinical care and improve procedural acceptability Flush Titration Time t o Continence Sawin and Thompson (2009) reported themes from a qualitative study documenting seven stress and struggle in finding an effective bowel management program for their children with spina bifida. The parents described a long journey compl icate d by the uncertainty of soiling and lack of responsiveness on the part of health care providers. Parents described the devastating impact of soiling on the affected child ren including embarrassment, continu al battering of self esteem, and school issu es. They described the constant struggle and stress caused by the unpredictable soiling. Finally they described either immense frustration with failure to find an effective bowel management program or life changing joy when they found a program that resul ted in predictable continence for their child The importance of expeditious achievement of predictable continence for affected children and their families cannot be overstated. Unfortunately, f indings in th is study were confounded by numerous factors including medication s illness, dietary indiscretion, and failure in the timely reporting of episodes of fecal incontinence to the investigator resulting in prolongation of the titration period and/or delays in dosing adjustment. Time to continence was fu rther complicated by only two of five subjects achieving continence on saline during the dosing trial. These confounds render flush titration time to continence data irrelevant.

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134 Cost Burden S aline is the costlier antegrade treatment regimen when compared to USP glycerin B ecause only 1/ 5 children achieved continence on sa line, the results are confounded. However, it is conceivable that t hose children who failed on saline may have achieved continence on higher doses increasing the cost differential. The ar gument could be made for mixing saline at home considerably decreasing the cost of the saline flushing solution Of concern Schreiber et al (1999) reported a death due to fatal hypernatremia following inaccurate home compounding and flush administration of a twice normal saline solution concentration Hyde et al., (2008) prospectively evaluated the composition of homemade saline and twice normal saline flush solution from five families and found sodium concentrations in both the homemade normal saline and twice normal saline solutions were 45% higher than the target. Multiple conversation with peers working at different pediatric institutions suggest few continue to allow home compounding of saline due to electrolyte imbalance secondary to improper solutio n composition. While home compounding saline may reduce flush solution cost, it has been associated with increased morbidity and mortality which can be prevented with the use of premixed standardized flush solution s Findings in this study do not reach sta tistical significance due to sample size, but in all five subjects, administration of saline was more expensive than USP glycerin suggesting use of USP glycerin instead of saline may reduce the cost burden associated with ACE flushing regimens Fecal Con tinence Findings from this study demonstrated a difference in the effectiveness of normal saline and USP glycerin flush es in achieving continence. Only two of five subjects

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135 gained continence on normal saline during the dosing phase and only one of five (20 %) maintained continence on normal saline in the maintenance phase. Four of five subjects gained and maintained continence on USP glycerin (80%). The continence rate achieved on USP glycerin in this study is less than the 95% continence rate reported by C hu and colleagues in 2013. The dosing range was similar in both studies (continence dose = 28.75mL and Mdn = 27.5mL in this study versus a =29mL, and Mdn = 30mL in the Chu study ). The protocol in this study utilized normal saline as a diluent versus ta p water in the Chu study. The difference in reported success rates in the two studies may be due in part to the difference in sample size (5 versus 23), age ( 3 to 7 years versus a mean age of 8.8 years ) and the tim ing of data collection In this study failure was defined as a single accident at maximum flush volume, whereas subjects in the Chu study had to have been on therapy for greater than 6 months and could have failed multiple times prior to reaching the 6 month mark. As an exemplar of that point, t he single child that failed on glycerin during this study is now continent on glycerin raising the overall success rate to 100% on glycerin flush measured at greater than 6 months follow up The failure of subjects in this study to gain and main tain continence on saline differs from reported outcomes in previous studies. Published ACE effectiveness outcomes on saline were highly variable but uniformly higher than those reported in this study. It is difficult to place the findings from this study within the context of reported outcomes from previous retrospective studies. This in part stem s from differing definitions of what constitutes continence, procedural and dosing consideration s sample size and composition, wide variability in diagnoses lea ding to the need for ACE

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136 therapy, and publication content and clarity. Two retrospective studies were identified that reported continence rates us ing normal saline without additives. Siddiqui et A l (2011) rep o rted a success rate of 33% on saline at flush volumes ranging from 845 +/ 68 mL in 66 patients with age range of 11.2 +/ 5 years at time of antegrade stoma construction. Becmeur (2008) reported a success rate of 64% on flush volumes ranging from 250mL to 1000 mL ( mean 700 mL ) in 29 patients 18 mal e s and 11 females, ranging in age from 3 to 21 years in patients with a wide variety of diagnoses leading to the need for ACE therapy Kuizenga Wessel et al. (2016) reported survey data of 23 pediatric gastroenterologist s and surgeons with expertise in AC E therapy. preference for enema flush volume was variable. Forty eight percent started at 10 20 mg/kg, 22% used 0 249 mL, 22% used 250 500 mL, and 8% tailored the volume to the child. The higher rate of failure of subjects to gain and maintain continence on saline in this study may have been due to limiting the volume of the saline flush to 500mL in children 5 years of age or younger and 1000 mL to children over 5 years of age although saline flush volume reflected current standard of care In addition, o ther factors that may have resulted in a discrepancy include a homogenous sample with regard to gender and diagnosis as compared to a wide v ariety of diagnos e s represented in other studies a younger sample age range and a more restrictive defi nition of continence with immediate determination of flush failure at the time of any soiling on maximum dosing. Kuizenga Wessel et al. (2016) found that 16 (70%) started the flushing regimen with saline; 5 (22%) tap water, polyethylene glycol, or sodium phosphate; and only 2

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137 (8%) a stimulant using either USP glycerin or bisacodyl. Nineteen respondents added a stimulant only when the initial antegrade solution was not effective. The importance of a chieving continence in the immediate post operative period following an ACE procedure should not be underestimated. Findings in this study did not reach statistical significance but are clinically relevant. It may be reasonable for clinicians caring for a male child under eight years of age with a neurogenic bowel and patulent sphincter secondary to a spinal cord lesion lower than T 10 to consider starting the child on USP glycerin as opposed to saline. Infusion and Procedural Time In a survey of expert clinicians conducted by Kuizenga Wessel et al. (2016) a ll bu t one respondent began the regimen with daily flushing An infusion duration ranging from 0 to 15 minutes was preferred by 62% of respondents with 38% preferring 16 to 30 minutes. A total procedural time ranging from 30 to 60 minutes following flush infus ion was preferred by 65% of respondents with 35% limiting procedural time to under 30 minutes. Although findings in this study suggest saline has the lower procedural time in minutes ( =33.8, Mdn=35 ) when compared to USP glycerin ( = 62.8, Mdn=57), b ecause only one of five subjects gained continence on saline, the difference in procedural time is clinically irrelevant. Because the timing of accidents did not occur directly following the flush but occurred the following day prior to the start of the next da y s scheduled flush, the decreased procedural time does not seem to be related to the poorer flushing outcomes on saline

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138 Side E ffects Several case series describe a variety of side effects from different flushing regimens, including pain with stomal intub ation, nausea, vomiting, abdominal cramping, sweating, dizziness, and pallor (Dey et al., 2003; King et al., 2005; Bani Hani et al., 2008). One of five children experienced persistent cramping with USP glycerin flush. The cramping was short in duration and immediately relieved with passage of stool. It is interesting to note that this child experienced abdominal discomfort in the baseline phase of the study which may indicate a predisposition to abdominal pain. Of interest, t he limited episodes of pain and cramping during saline administration either occurred in the immediate post operative period ( one subject) or were correlated with episodes of constipation ( two subjects) One child failed USP glycerin due to vagal symptoms. The child had missed several scheduled flushes and started on elemental iron supplementation In addition, h is osmotic agent was stopped while he was on normal saline in an effort to achieve continence and had not been restarted. USP glycerin was stopped and he was cleaned out with Go lytely. He had no vagal symptoms on USP glycerin prior to the missed doses and successfully returned to a glycerin flushing regimen with the addition of an osmotic agent following the clean out without recurrence of vagal symptoms suggesting a significant colonic stool burden secondary to missed doses and medication may have been responsible for the vagal symptoms. Electrolytes The bowel is a substantive absorptive surface. Instillation of solution into the cecum has the potential to caus e electrolyte imbal ance. Deaths have been reported due

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139 to electrolyte imbalance from retrograde and antegrade enema solution s (Schreiber & Stone, 1999) Several case reports detail morbidity and mortality associated with flushing solution s including hypocalcemia and hyperph osphatemia following retrograde administration of phosphate enemas in children (Helikson, Parham, & Tobias, 1997; Ismail, Al Mutairi, & Al Anzy, 2000) and water intoxication following retrograde enema therapy using tap water (Chertow & Brady, 1994). Soluti on composition, volume, retention time, and underlying electrolyte imbalances are all factors that increase morbidity and mortality (Yerkes et al., 2001). Hyde et al., (2008) prospectively demonstrated clinically significant changes in serum electrolytes a ssociated with antegrade enema administration of tap water and twice normal saline. N o clinically significant electrolyte imbalance was associated with antegrade administration of saline. Outcomes in this study confirm previous findings that antegrade admi nistration of normal saline does not result in electrolyte imbalance. A finding from this study that has not been previously reported is the absence of electrolyte abnormalities with antegrade administration of USP glycerin Neither normal saline nor USP glycerin flush at the doses and freque ncy of administration used in this s t udy resulted in any abnormality in serum electrolytes increasing clinician confidence that both solutions may be safe ly administered as ACE flush solution in the well hydrated chil d with normal transit time, Stool Calprotecti n Previous research has not explored the effects of ACE therapy on gut health. All subjects had an elevation of stool calprotectin levels in response to at least one flushing regimen. The highest increase in st ool calprotectin was seen following saline flush.

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140 E levations above baseline suggest the presence of a low lying inflammatory process. I n no instance did elevations reach a level of clinical concern requiring cessation of the study Further stu dy is needed to better define the long term course and clinical relevance. Quality of Life A number of prospective studies have demonstrated the positive impact of ACE therapy on the quality of life for children and their families (Aksnes et al., 2002; Bower, 2008; K augers et al., 2010; Ok & Kurzrock, 2011; Youssef et al., 2005) Subject attrition due to treatment failure resulted in incomplete survey completion. Study Limitations Both t he limited number of enrolled subjects and failure of the majority of those enroll ed to progress to the study maint enance phase thereby limit ing the number of collected data points, pos ed a substantial threat to statistical validity result ing in insufficient power. Reliability of measures, reliability of treatment, and heterogeneity o f subjects did not pose a threat to statistical validity. Although there was no voluntary loss of subjects, a consequence of treatment failure was substantial attrition resulting in an A B C design instead of an A B C B B 1 design as intended. Failure of subjects to complete the study was inductively relevant but deductively posed a threat to internal validity. Only one subject completed the study and was continent on both solutions so replication of treatment effects did not occur in the majority of subjects Failure of replication weaken ed the design increasing the plausibility that other factors may have influenced the outcome. A legitimate argument could be made that confidence in the outcomes would have been significantly strengthen ed by allow ing the three subjects

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141 who initially failed on saline to progress to the maintena nce phase of the study to determin e if the ir failure on saline could be replicated. However, ethics prohibited the reintroduction of a treatment they had previously failed tha t had a high probability of causing recurrence of soiling. I ntercurrent illness, medications, dietary indiscretion and to a limited degree, inconsistency in treatment adherence, posed historical threats to internal validity. Interobserver agreement ( IOA ) was completed at protocol initiation but could not be completed at every change in phase due to a study design flaw P rocedural fidelity was completed at each phase change, to some degree offsetting instrumentation threat to internal validity. The short ened length of data collection and follow up could have posed a threat to construct validity of cause and effect. However, the length of data collection on a single treatment was sufficient to reduce the p otential for carry over effects. O rder effects were minimized by randomization. There were no identified social threats to internal validity. Inability to blind to treatment increased the risk of experimental bias. S ingle subjects methodology is often used to evaluate operant behavior. Fecal incontinence in the children in this study was secondary to neurogenic bowel. Maintaining continence was not under their volitional control and could not be achieved without extraordinary intervention. This in s ome ways complicates use of trad itional within subjects vi sual analysis where timing for the institution of treatment and treatment changes are based on behavioral stability, trend, and trend direction. Issues such as a stable baseline or whether the rate of incontinence is accelerating or decelerating in the ba seline phase is not applicable to this population. All subjects were

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142 incontinent at baseline. The purpose of the study was to assess if subjects could gain continence on one or both flushing regimens. Any single episode of fecal incontinence or pain grea ter than a 4 on the pain scale were unacceptable and resulted in an immediate dosing change making the traditional methods of visual analysis such as d ata stability a nd trend relevant only in those instances in which the subjects gained and maintained con tinence in the presence of minimal side effects Implications for Future Research and Practice Probabalistic Epigenesis as a Framework for Pediatric Incontinence Research Probablistic Epigenisis provided a theoretical framework for the study of ACE thera py in children. The overarching principles contained within the constructs and relational statements of Probabalistic Epigenesis appear to be highly applicable to the study of pediatric incontinence and dysfunctional habits of elimination The restructure d model facilitated experimental confirmation. Findings from this study suggest there is both a vertical and horizontal coaction of the constructs of Environment (ACE flush) on Neural Activity (electrolyte balance, and gut health as measured by calprotecti n), and Behavior (continence). Further studies are needed test the applicability and fit of Probabalistic Epigenesis in pediatric incontinence research. Implications for Future R esearch Findings from this study are promising but the sample size was insuffi cient to reach statistical significance. Further study is needed. The children who require ACE therapy to maintain continence constitute a small population. The case can be made to use inductive methodology utiliz ing single subjects design with replicatio n between subjects The limited population makes the study of ACE therapy well suited to within

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143 subjects methodology. The case can be made to compare flushing regimens deductively using a cross over design. The study of ACE therapy is well suited to a cros s over design, which facilitates obtaining the same number of observations with fewer patients and the same precision of estimation with fewer observations resulting in a significant savings in resources when compared to a parallel group design. One of the major disadvantages of a cross over design is carry over which if not accounted for, may lead to errors in interpretation. The impact of carry over can be minimized by incorporation of a wash out period and use of appropriate statistical analysis. Using a two sample method versus a one sample method produces a smaller variance and eliminates conditional bias when sample sizes differ by order Calculating treatment effects by ignoring the washout and post washout values and using the last data point (post test) differences only will substantially decrease variance. Increased precision and efficiency in analysis will allow estimation of treatment effects in the presence of a smaller sample size thereby saving resources ( Jones et al., 2003; Senn, 2002; Shust er, 2017). Future research comparing flushing ACE flushing regimens would be well served by utilization of a cross over design embedded in a single subjects design and analyzing the cross over using a two sample method and post test differences. Conducti ng a multicenter study would ac celerate enrollment, thereby producing information on treatment effects in a shorter time frame. The des ign would benefit from simplification of the titration scheme which could conceivably shorten the study duration and mini mize the burden of data collection on families.

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144 Implications for Clinical Practice The results of this study did not reach clinical significance but the effect size suggests clinical utility. This study is the first study to prospectively compare two ant egrade flushing regimens. Findings of interest in this study included (a) comparative treatment outcomes with substantial treatment failure on normal saline when compared to USP glycerin, (b) decreased frequency of administration in two subjects using USP glycerin versus saline, (c) discomfort with USP glycerin flush in a single subject that did not occur with any subject on normal saline flush, (d) saline was the costlier flushing regimen, (e) no evidence of electrolyte imbalance with either flushing regim en, and (f) elevations in stool calprotectin compared to baseline that did not reach a level of clinical concern requiring cessation of the study, but is of interest and will require further study to better define course and determine relevance. Conclusion s This study compares two ACE flushing regimens. The ACE procedure has been used for well over two decades. Case reports and retrospective studies detail widely divergent effectiveness rates. The literature and involved clinicians have identified the need for prospective trials comparing ACE flushing regimens but n one have been undertaken to date. This is in large part because the small size and heterogeneity of this population does not lend itself to a large N study. Many clinical questions go unanswered due to over reliance on randomized controlled trial large N methodolog y. This population is an exemplar of that problem Use of a cross over design embedded in single subjects methodology has started to provide clinically useful information that could ult imately improve care for children with neurogenic bowel on ACE therapy

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145 Both single subject and between group research make and test predictions about treatment effects, the first by evaluating treatment effects on an individual, the second by addressing g roup mean and variance (Kazdin, 2011). Single subjects and group designs, in the i r most rigorous form, rule out or make implausible rival hypothes e s for the experimental outcome improving quality of inference (Cook & Campbell, 1979; Kazdin, 2011; Shadish Cook, & Campbell, 2002). In this study, t he sample size was too small to deductively determine inferential significance Due to early treatment failure within the bounds of ethical considerations t he inductive case for which flushing regimen is more eff ective could not be made by replicating the intervention within subjects However, the argument could be made that r eplication of findings between subjects has started to inductively suggest that i n male children under 8 years of age with neurogenic b owel due to low lying spinal cord lesions, USP glycerin can be safely administered and may be more effective than normal saline in achieving continence.

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146 APPENDIX A THEORETICAL AND OPERATIONAL LINKAGES FOR RESEARCH ADDRESSING INCONTINENCE

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147 A PPENDIX B RE QUIR ED MATERIALS FOR FLUSHING PROCEDURE Required Flushing Materials Flushing Regimen Materials Normal Saline One liter containers of Normal Saline (NS) premixed to 0.09% One 1,000 mL enteral feeding bag with drip chamber and roller clamp One Christmas tree a dapter connected to the end of the enteral feeding bag tubing A hook hung on the wall beside or behind the commode at a height of 5' from the floor One appropriate access tube for Chait or Mickey low profile device Warm tap water and mild liquid dish deter gent to wash equipment after each use One stop watch One clip board with data collection sheets and pen attached with string to the clipboard USP Liquid Glycerin mixed with Normal Saline One appropriate access tube for Chait or Mickey low profile device One 60 mL catheter tipped syringe One 8 ounce bottle with screw top to mix liquid glycerin and Normal Saline One liquid bisacodyl enema solution in pre packaged 10 mg aliquots One 10 mL syringe for measuring liquid glycerin solution One liter containers of NS premixed to 0.09% One stop watch One clip board with data collection sheets and pen attached with string to the clipboard

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148 APPENDIX C PROCEDURAL INSTRUCTIONS FOR COMPLETING SALINE FLUSH Steps Parental Instructions for Saline Flushing Regimen 1 As semble equipment: One liter container of Normal Saline(NS) premixed to 0.09% Each container will be marked by the investigator with ACE flush contents and a lot number One 1,000 mL enteral feeding bag with drip chamber and roller clamp One Christmas tree adapter connected to the end of the enteral feeding bag tubing A hook hung on the wall behind the commode at a height of 5' from the floor One appropriate access tube for Chait or Mickey low profile device S top Watch C lip Board with data collection sheets and pen attached with string to the clipboard 2 Warm tap water and mild liquid dish detergent to wash equipment after each use 3 Make sure flushing solution is at room temperature 4 Insert the Christmas tree adapter into the end of the end of the feeding b ag tubing making sure the fit is secure 5 Make sure the roller clamp is closed on the feeding bag tubing 6 Unscrew cap from the top of the feeding bag by rotating in a counter clockwise direction 7 Pour room temperature liquid from the 2 liter mixing bottl e into the feeding bag 8 Screw the cap to the top of the feeding bag by turning in a clockwise direction until secure 9 Hold the feeding bag with liquid in one hand at shoulder height and put the end of the tubing in the sink 10 Using your other hand slowly loosen the roller clamp on the feeding bag tubing until the liquid fills the tube and then reclamp by tightening the roller clamp to stop the flow of liquid 11 Hang the feeding bag on the wall hook above the commode 12 Position child comfortably on commod e using a toilet seat insert and a foot stool if needed 13 Give child toys/books for distraction 14 Hook the end of the Christmas tree adapter into the access tubing and secure the access tubing to the button 15 Unclamp the roller clamp to start the flow o f the flushing solution and immediately start the stop watch 16 If your child complains of cramping or discomfort, slowly tighten the roller clamp to slow the flow of the flush 17 When the flush solution has infused, write the time from the stopwatch onto the record sheet under but do not stop the stopwatch 18 Once the flush has infused, disconnect the tubing and rinse with a mixture of mild dish soap and tap water followed by tap water alone and hang back on the hook to air dry 19 Once your chi ld has passed a bowel motion and 5 minutes has gone by without any additional stool output, stop the watch and record the time on the record sheet under column "Completion Time"

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149 APPENDIX D PROCEDURAL INS TRUCTIONS FOR COMPLETING USP GLYCERIN FLUSH

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150 A PP ENDIX E MICROBIOME DNA EXTRACTION AND PROCESSING FOR DOWNSTREAM ANALYSIS 1 2 3 4 5 6 Ensure two heat blocks are available and set at 70 c and 95 c Obtain a cup of ice to store the weighed sample until InhibitEX buffer is added Ensure Ethanol has been add ed to b uffers AW1 & AW2 Mix all buffers before use If a precipitate has formed in the InhibitEX buffer or AL b uffer, dissolve by incubating at 37 c All steps to be performed at room temperature, except otherwise noted 7 8 9 10 11 Ensure the sample is kep t on ice until the addition of InhibitEX buffer Weigh the stool sample in a 15 mL conical tube or homogenizer and place on ice Add 10 volumes of InhibitEX buffer (e.g., add 10 mL buffer to 1 g stool) Vortex vigorously for 1 minute or until stool sample is thoroughly homogenized. If the stool sample is too viscous, use a homogenizer until no solid sample is visible 12 13 14 15 16 Transfer 2 mL of lysate into a 2 mL microcentrifuge tube Incubate suspension at 95 C for 5 minutes, then vortex 15 seconds Ce ntrifuge sample at full speed for 1 minute to pellet stool particles Pipette 15 L proteinase K into a new 1.5 mL microcentrifuge tube Transfer 200L of the supernatant from step 13 into the 1.5Ml microcentrifuge tube containing proteinase K 17 18 19 20 A dd 200 L Buffer AL and vortex 15 seconds Incubate 70C for 10 minutes. Then tap spine to bring condensate down Add 200 L 100% ethanol to lysate and mix by vortexing Transfer the lysate from step 18 to a QIAamp spin column. Centrifuge full speed 1 min 21 22 23 24 25 Place the spin column in a new 2 mLcollection tube. Discard THE tube containing the filtrate Add 500 L Buffer AW1. Centrifuge full speed for 1 minute Place the spin column in a new 2 Ml collection tube. Discard the tube containing the filtra te Add 500 L Buffer AW2. Centrifuge full speed for 3 minutes Place the spin column in a new 2 mL tube. Discard the tube containing the filtrate. 26 Centrifuge full speed for 3 minutes to remove any residual Buffer AW2 27 28 Place the spin column into a n ew 1.5 mL microcentrifuge tube Pipette 200 L Buffer ATE directly onto the membrane. Incubate for 5 minutes. Centrifuge full speed for 1 minute to elute DNA. 28 Freeze at 80 for downstream analysis

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151 APPENDIX F THREATS TO STATISTICAL CONCLUSION VAL IDITY Threat Assessment of Potential Threat & Proposed Control Insufficient Power (Increases probability of a Type I error) Within subject analysis the strength of the ability to determine treatment effects requires a stable baseline, and is a functio n of the number of baseline and intervention data points (Cook & Campbell, 1979). This study design features a prolonged pre operative baseline assessment and a minimum of 6 weeks of daily measurement for each treatment variable meeting the criteria for su fficient power analysis Cross over analysis: The study will use 6 subjects randomized to 2 treatment orders. Although the design confers greater precision in estimating treatment difference and generally requires fewer subjects, the limited number of subj ects will limit the power of the analysis increasing the probability of a Type II error (Piantadosi, 2005). Reliability of Measures (Impacts the relationship between variables) All biological samples will be run in a single accredited laboratory. All f ecal samples will be collected and stored for downstream analysis by the procedure detailed in the protocol (Shadish et al., 2002). Reliability of Treatment Implementation (May result in underestimation in treatment effect. Procedural fidelity will be asc ertained each procedural variable at specified times throughout the study. Written instruction forms detailing interventions will be given to the family (Gast, 2010). Random Heterogeneity of Respondents (Increases variability in outcome, increasing error variance) Within subject analysis should not pose a problem if baseline is stable as the baseline reflects background variable effects (Shadish et al., 2002) Cross over analysis: Period confounds should be accounted for using the two sample cross over t test (Shuster, 2007) Both designs use subjects as their own control substantially decreasing variance due to subject heterogeneity (Cook & Campbell, 1979). Violated assumptions Within Subject analysis violates statistical assumptions that error re siduals are independent, normally distributed, and homoscedastic. Serial dependency can by modeled and removed. Cross over analysis treatment sequences considered independent. Carry over effects should not be a factor due to design considerations but ar e accounted for using the two sample t test (Shuster, 2007) two n over or underestimation of the magnitude and statistical significance of treatment effects

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152 APPENDIX G THREATS TO INTERNAL VALIDITY Threat Assessment of Potential Threat & Proposed Control Ambiguous Temporal Precedence (cause precedes effect) This is a prospective study. Ascertainment of temporal precedence and ambiguity regarding direction of causal inference is not a risk Attrition (subject mortality) Attrition will have a significant effect on data analysis. Careful follow up and provision of su pplies may discourage subject fatigue and drop outs. A second randomization to treatment has been instituted after the dose response phase just before the treatment phase to insure randomization scheme if attrition occurs due to ineffective treatment or si de effects (Portney & Watkins, 2009) Maturation (confounding due to passage of time) This study is several months in duration which should preclude maturational confounding. In addition, randomization to treatment sequence should minimize maturation as a potential bias (Shadish et al., 2002) History (Confounding events other than the independent variable) Effects of significant diet change or infection causing diarrhea will confound treatment results. A diary will be kept documenting any obvious histori cal confounds. In those instances, treatment length will be extended if necessary (Gast, 2010). Use of a match control that did not receive treatment would strengthen the design (Cook & Campbell, 1979) but would be unethical. In addition, disease instabil ity may confound treatment effects. This population generally has a chronic but stable disease and should provide a stable sample to compare treatment effects (Piantadosi, 2005). Order Effects (Position in sequence) & Carry Over Effects (Effects from prev ious phase impacting current phase) Order effects which will be controlled by randomization to one of two treatment sequences (Kazdin, 2011). Carry over effects should not confound continence. There is a prolonged active washout period between serum and s tool sampling Regression (Tendency of extreme scores to regress toward the mean) Testing Should not pose a threat in this study Should not pose a threat in this study Instrumentation (reliability of measurement) IOA will be checked at key points throu ghout the study and calculated using gross method. If IOA is <80%, additional observer training will be implemented. The family will have report forms to help standardize and quantify responses. (Gast, 2010; Kazdin, 2011) Internal validity questions if th e evidence support s a causal relationship between treatment and outcome variable s (Portney & Watkins, 2009).

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153 APPENDIX H SOCIAL THREATS TO INTERNAL VALIDITY Threat Assessment of Potential Threat & Proposed Control Diffusion and Imitation of Treatment M any of the families who will be enrolled in this study interact socially a ttend a support group together, and attend a multidisciplinary clinic in which they share a waiting room. Subjects are not blinded to treatment. Parents and children enrolled in the study may be influenced by parent or child preference of individuals already on ACE therapy. Blinding is not possible due to obvious differences in flush volume (Portney & Watkins, 2009) Compensatory Equalization of Treatments Subject or parental prefer ence for one treatment over another based on intangibles not measured in the study may influence treatment implementation and/or evaluation of dependent measures. Blinding is not possible due to obvious differences in flush volume (Portney & Watkins, 2009) Compensatory Rivalry All subjects will receive all treatments so no subject should receive what they view as a less than desirable treatment negating the threat of compensatory rivalry (Portney & Watkins, 2009) Resentful Demoralization All subjects will receive all treatments so no subject should receive what they view as a less than desirable treatment negating the threat of resentful demoralization (Portney & Watkins, 2009) Social Validity questions if there is evidence to support a causal relati onship between treatment and outcome variable s that are influenced by social interactions or the treatment specifics (Portney & Watkins, 2009).

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154 APPENDIX I THREATS POSED TO CONSTRUCT VALIDITY OF CAUSE AND EFFECT Threat Ass essment of Potential Threat & Proposed Control Operational definitions Efforts were made to ensure the construct of fecal incontinence was clearly defined. All measurement of continence, side effects, and physiologic outcomes are measured at an interval s cale or higher minimizing the risk of miss inference (Portney & Watkins, 2009) Levels of Construct Dosing regimen adjustment is built into the design allowing do s e response and level of effect determination mitigating threat of levels of construct (Cook & Campbell, 1979) Delayed Causation & Interaction Slow diffusion of treatment causing a delay in treatment effect should not be problematic (Shadish et al., 2002). Carry over and order effects have been adequately controlled for in both design and analys is (Jones & Kenward, 2003; Shuster, 2007) Length of Follow Up Repeated measures over time negates concerns regarding potential bias due to misleading short term data (Portney & Watkins, 2009) Experimenter Bias Treatment or problems solving will be stand ardized across subjects. Inability to blind to investigator to treatment may increase risk of this threat (Portney & Watkins, 2009) Psychometric Soundness of Instruments measures the construct it i s intended to measure (DeVon et al., 2007, p. 162). The Fecal Incontinence and Constipation on Quality of Life instrument has established validity and reliability for children with constipation and fecal incontinence due to neurogenic etiology and their c aregivers (Ok & Kurzrock, 2011). The Wong Baker FACES pain Rating Scale has well established validity and reliability in children (Tomlinson, Von Baeyer, Stinson, & Sung, 2010; Wong & Baker, 1988))

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155 APPENDIX J NEMOURS CLINICAL RESEARCH COMMITTEE APPLICAT ION AND REVIEW

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158 APPENDIX K FDA IND APPLICATION

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159 Table of Contents Introductory Statement and General Investigational Plan ................................ ................................ ........... 1 Chemistry, Manufacturing and Control Information ................................ ................................ ................... 2 Pharmacology Toxicology Information ................................ ................................ ................................ .......... 3 ................................ ................................ ................................ ................................ .... 9 Clini cal Protocol ................................ ................................ ................................ ................................ ................ 9 Summary of Previous Human Experience with the Investigational Drug ................................ ................. 19 References ................................ ................................ ................................ ................................ ........................ 21 Additional Information Appendix A Dr. Pam Pieper Curriculum Vitae Appendix B Dr. Donald George Curriculum Vitae Appendix C Kimberly S. Jarczyk, MSN CPNP Curriculum Vitae Appendix D Dr. Mark Barraza Curriculum Vitae Appendix E Dr. Jonathan Shuster Curriculum Vitae Appendix F Letters of Acceptance from Data and Safety Monitoring Board Members

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160 Introductory Statement and General Investigati onal Plan Broad Objectives and Planned Duration of the Proposed Clinical Investigation Fecal incontinence in children past the expected time of toilet training has been associated with increased anxiety and depression, more social problems, worse school p erformance, and an increased incidence of abuse and bullying (Kaugars et al., 2010; Youssef, Langseder, Verga, Mones, & Rosh, 2005) It is particularly difficult to manage fecal incontinence using conservative measures in children with neuromuscular disor ders, anorectal malformations, spinal cord injuries, spinal cord trauma or tumor, megarectum, or slow transit constipation. The Malone or antegrade continence enema (ACE) procedure was popularized over 20 years ago as a means of helping children with int ractable fecal incontinence attain stool continence. A catheterizable stoma from the abdominal wall into the cecum is constructed using the appendix, a tubularized portion of the bowel, or a low profile button device. The stoma allows for antegrade adminis tration of enema solution into the colon. Many different prescribed ACE flushing regimens are used in practice, including licorice root, mineral oil, treacle/milk mix, tap water, normal saline, polyethylene glycol solutions with and without added electroly tes, phosphate soda solution, bisacodyl, and USP liquid glycerin (Bani Hani et al., 2008; Marshall et al., 2001; Youssef, Barksdale, Griffiths, Flores, & Di Lorenzo, 2002). A large body of literature demonstrates ACE therapy can be effective in helping ch ildren with intractable fecal incontinence attain continence for stool with resulting significant improvement in quality of life (Aksnes et al., 2002; Kaugars, Silverman, Kinservik, Heinze, Sander & Sood, 2010; Mousa et al., 2006; Tiryaki, Ergun, Celik, U lman, & Avanoglu, 2010). However, findings regarding effectiveness are highly variable. Short term reported success rates range from 79 to 98% with long term outcomes ranging from 41% abandonment rate at 5 years to a mean time to first relapse lasting as long as 121.9 +/ 29.7 months (Aspirot, Fernandez, Di Lorenzo, Skaggs, & Mousa, 2009; Bani Hani, Cain, King, & Rink, 2008; Becmeur et al., 2008; Curry, Osborne, & Malone, 1999; Dey et al., 2003; King, Sutcliffe, Southwell, Chait, & Hutson, 2005; Marshall, Hutson, Anticich, & Stanton, 2001; Mousa et al., 2006; Ok & Kurzrock, 2011; Siddiqui, Fishman, Bauer, & Nurko, 2011; Thomas et al. 2006; Yardley et al., 2009). T his variability may be due to what is used to flush. Identifying a successful flushing regimen is determined by individual clinician preference and often requires multiple attempts before success is achieved. There are no prospective studies comparing the side effects or effectiveness of type, dose/volume, or frequency of different flushing regimen s in preventing incontinence to inform practice. The catheterizable stoma used for antegrade administration of enema solution is frequently made by bringing the appendix out through the abdominal wall or by placing a button into the cecum. The appendix a nd cecum have significant amounts of gut associated lymphoid tissue (GALT), have high concentrations of microbiota, and serve an essential immune function (Anderson, Olaison, Tysk, & Ekborn, 2003; Andreu Ballester et al. 2007; Barker, 2012; Dasso & Howell 1997; Forchielli & Walker, 2005; Hooper, Littman & Macpherson, 2012; Janszky, Mukamal, Dalman, Jammar & Ahnve, 2011; Nicholson et al. 2012; Noverr & Huffnagle, 2004; Penders, Stobberingh, Van den Brandt, & Thijs, 2007). The appendix has higher concent rations of microbial biofilms compared to other areas of the colon, serves as a safe house for symbiotic gut flora, and functions to preserve gut microbiota through re inoculation with normal flora following gastrointestinal infections (Bazar, Lee, & Yun, 2004; Bollinger, Barbas, Bush, Lin, & Parker, 2007; Gebbers & Laissue, 2004; Kawanishi, 1987; Smith et al. 2009). The human gut contains 10 14 bacteria (Jia, Li, Zhao & Nicholson, 2008). Bacterial composition varies along the bowel axis, with further diffe rentiation of luminal or adherent microcolonies that lead to development of biofilms. Factors that influence microbial composition include pH, transit time, bile acids, pancreatic enzymes, mucus composition, nutrient consumption, medication, environment, b acterial adhesion capacity, and metabolic capacity. The most important function of the gut microbiome is colonization resistance, which is accomplished through competition for nutrients and secretion of bacteriocins (Penders et al., 2007). Microbiota funct ion to degrade dietary substances and enhance digestive efficiency

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161 while providing nutrients to the microbes themselves. These microbes are essential for host physiology but in turn pose a threat of opportunistic invasion by resident bacteria with resulti ng pathologies. ACE therapy administration through the appedix or into the cecum has the potential to disrupt the gut microbial ecosystem causing dysbiosis and immune dysfunction. No studies evaluate the effects of appendicosomty/cecostomy flush on gut mucoal immunity and microbiota. The proposed research is significant in that it is the first prospective study to compare the effectiveness and tolerability of two commonly used ACE flushing regimens, and the first study to explore ACE flushing impact on t he gut microbiome. Findings from this study have the potential to provide both clinical and biological insights into ACE administration safety and start to build a foundation of scientific evidence that could increase ACE effectiveness rates from 78% towar d 98% or better, and provide a foundation for additional prospective, randomized, controlled trials. We seek approval from the Food and Drug Administration to investigate and compare two distinct flushing regimens, one high volume saline flush and one low volume USP glycerin flush, in the immediate postoperative period in children requiring ACE therapy. We have chosen normal saline and USP glycerin because both have been widely used, have a proven safety record over a long time frame, and are available ove r the counter for retrograde enema administration in infants and children. Both have been widely used clinically for antegrade enema administration with no case reports detailing any adverse outcome. We could not find any evidence that either USP glycerin or normal saline have been withdrawn from investigation or marketing in any country for any reason related to safety and effectiveness. The proposed pilot study is prosp ec tive and utilize s a cross over design embedded in a single subjects design employing six subjects randomized to two treatment sequences. The primary aims of this study are to 1) identify the minimal administration frequency and titration time to reaching effective dose; 2) compare which solution at an optimum dose is delivered in the leas t amount of time, with fewer side effects, while promoting the higher degree of fecal continence and quality of life; and 3) determine if administration of antegrade enema solution through an appendicostomy/cecostomy affects gut microbiota and immune funct ion. This study will involve children ages 3 to 12 years recruited from subspecialty clinics at Nemours entities in Florida and the Pediatric Community Spinal Defects Clinic in Jacksonville, Florida. Children will be selected by purposive sampling and wil l include those who are scheduled to have an ACE stoma and will require regular antegrade enema administration to maintain continence. Excluded will be children with preexisting electrolyte imbalance, chronic high rectal tone, quadraplegia, renal or cardia c disease, and those who cannot communicate or have significant cognitive delay that would interfere with their ability to fully particiate in the study. Parents must have English language competency and be willing and able to participate in administration or oversight of the flushing regimen and data collection for a minimum of 20 consecutive weeks. Single subjects within and between subjects data will be analyzed using visual analysis. The cross over data will be analyzed using inferential statistics emp lying two tailed hypothesis testing. Findings from this study will serve to provide a comparative analysis of two different regimens that will serve as a starting poing to guide practice and provide a foundation for additional prospective, randomized, cont rolled trials. Chemistry, Ma nu facturing, and Control Information Drug/Active Ingredients, Pharmacertical Class, Structural formula, Formulation, Route of Administration Chemical name: Glycerol or 1,2,3 propa n etriol Common name: Glycerin Empirical formul a: C 3 H 8 O 3 Glycerol is the backbone common to all triglycerides. Glycerol is a water miscible trihydroxy sugar alcohol used in pharmaceuticals, foods, and personal care products that has over 1500 known uses and is vitually non toxic to human health and the environment. It is available over the counter and widely used for retrograde

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162 enema administration in infants and children. Orally, after hydrolysis of glycerol esters in the intestine, it is readily absorbed in the intestine. It enters the glycolic pathwa y after conversion in the liver to to glycerol 3 phophate eventually yielding pyruvic acid WHO, 2002). As a suppository it is poorly absorbed acts to hydrate stool in the rectum by osmotic absorption of water from the mucosa, acts as a lubricant to softe n stool, and gen e rally causes mechanical stimulation of the anocolonic reflex promoting passage of retained stool. It is unclear the extent to which the hygroscopic or local irr i tant action is responsible for the laxative effect (Pharmacists, 2011; Rosso ff, 1974, & WHO, 2002) Structural formula of Synthetic USP Glycerin: Manufacturing Information HM Glycerin USP 6OZ. The NDC# is 62011 0115 01 and the brand is Healthmart. Normal Saline The NDC# is 00338 0049 04 and the brand is Baxter. Environ ment environment, the volume and frequency of admi nistration in this proposal does not substantially increase the active moiety and does not substantially alter or increase the concentration or distribution of the substance, metabolites, or products of degradation in the environment. To the best of the in extraordinary circumstances exist with the proposed used of glycerin and saline that would adversely affect the quality of the human environment. Pharmacology and Toxicology Information Animal Studies Using Glycerin There are no animal studies evaluating instillation of glycerin into the cecum Sample Dose Route Outcome Reference 6 rabbits 18,700 mg/kg bw Occlusive dermal application for 8 hours No deaths United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html 12 female rats 27,260 mg/kg bw Gavage Muscle spasm, convulsions, lung congestion & death (3) United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 :

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163 Survivors normal within 2.5 hrs of dosing http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html Mice & Guinea pigs Not reported Gavage Tremor and convulsions with hyperemia of pylorius, small intestine and cerebtral meninges United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : http://www.chem.un ep.ch/irptc/sids/OECD SIDS/sidspub.html Rabbits 4 mL over 30% BSA 8 hr/day for 90 days Topical No irrita tion United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html 6 r abbits 0.1 mL Occular instillation Very low potential to irritate eyes United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html 24 male Guinea pigs 0.1 mL of 0.1% in NS qod x 20 days Injection No indication of sensitization after 2 week exposure free period Environment Programme: Screening Informat ion Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html Rats 1.75 mL of 50%/100 g bw SQ injection Severe hemolysis followed by necrosis of tubular portions of n ephrons reversible within 6 12 wks European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015 : http://esis.jrc.ec.europa.eu/ Rats 1 mL 100%/100 g bw Intra peritoneal instillation Severe convulsions, hemoglobinuria, renal damage died within 2 hrs of injection European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of: February 18, 2015 http://esis.jrc.ec.europa.eu/

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164 Rats 1 mL 100% or 50%/100 g bw SQ injection Hemoglobinuris, renal tubular necrosis, some convulsions with 100% European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015 : http://esis.jrc.ec.europa.eu/ Rats 100% & 5 0% at 1 mL/100 g bw IV Severe Convulsions and death in all animals European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015 : http://esis.jrc.ec.europa.eu/ Rabbit Aq 100% Anterior chamber of eye Inflammation and edema of cornea & damage of endothelial cells Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 Rabbits Aq 50% Anterior chamber of eye Significantly less reaction but visibly deh ydrates lens can capsule wrinkling Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 Rabbit Aq 30% for 20 min, 50% for 10 min or 92% for 4 min Anterior chamber of ete Normal deturgescence after 30% and 50% expposure but endothelium destruction with 92% > 30 min Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 Rabbit with corneal traume Aq 43% dilution for 30 min x 20 d Opthalmic Edema of conjunctiv a las ting for several hrs Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 Rats 14 d 5d/wk 6hr/d Mean conc 1000, 1930 3910 mg/cu m Respirable aerosol Minimal to mild squamous metaplasia of epiglottis greatest at higest test dose No systemic effects United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html

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16 5 Rats 13 wk 6hr/d 5d/wk 0, 33, 165, & 662 mg/cu m Respirable aerosol Minimal to mild squamous metaplasia of epiglottis considered local irritant effect United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html Rats 10% for 9 d IV Degeneration in renal tubular epithelium return to normal after 19 d NOWAK H ET AL; PATOL POL 30 (1): 61 (1979) Grow ing pigs 8 4.51% added to feed for 138 d Feeding trial Pigs can be fed up to 10% crude glycerin with no effects on performance, carcass composition, or meat quality Lammers PJ et al; J Anim Sci 86 (11): 2962 2970 (2008) Rats 0 up to 60,000 mg/kg bw /d, for 20 wks oral At 5000 mg/kg bw marked hydropic and fatty degenration of liver parenchymal cells WHO/FAO: Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances ( 56 81 5) (2002). Available from, as of February 18, 2015 : http://www.inchem.org/pages/jecfa.html Mice 6 to 8 wks Given carcinogen followed by 0, 0.5 or 1% glycerol or water until 1 yr of age oral Lower incidence of liver and lung tumor after glycerin no adverse treatment effects WHO/FAO: Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances (56 81 5) (2002). Available from, as of February 18, 2015 : http://www.inchem.org/pages/jecfa.html Rats 0 to 20,000 mg/kg bw /d oral No significant treatment related effects WHO/FAO: Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, t riols, and related substances (56 81 5) (2002). Available from, as of February 18, 2015 : http://www.inchem.org/pages/jecfa.html

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166 Rats 4,000 to 10000 mg/kg bw for 2 years Oral No adverse effects up to 10,000 mg/kg bw United Nations Environment Programme: Sc reening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html Rats 5%, 10%, & 20 % for 12 to 24 months Oral Glycerol does no t initiate tumor development in rats United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html Mice 5% in drinking water for 1 20 wee ks after sq injection of 4 NQO Oral Enhance s lung tumor development mainly adenomas. Tumor development independent from pulmonary cell kinetics United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) A vailable from, as of February 18, 2015 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html Rats 7 generation s 15000 mg/kg bw d Oral Pups of treated dams mean weight 20% less than controls Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances (56 81 5) (2002). Available from, as of February 18, 2015 : http://www.inchem.org/pages/jecfa.html Rats Mice Rabbits Levels up to 1310, 1280, 1180 mg/kg bw d aily during part of gestational period oral No maternal or tetragenic effects seen at highest dose level tested United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 20 15 : http://www.chem.unep.ch/irptc/sids/OECD SIDS/sidspub.html Summary of Previous Human Studies Clinical Trials Involving USP Glycerin Dose Route Effects Reference

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167 Not resported Not reported Very slight diuresis in healthy individuals receiving a single dose May produce tissue dehydration and decreases in CSF pressure McEvoy, G.K. (ed.). American Hospital Formulary Service Drug Information 93. Bethesda, MD: American Society of Hospital Pharmacists, Inc., 1993 (Plus Supplements, 1993)., p. 1773 Not rep orted. E mployees engaged in glycerol manu facturing Environ mental exposure No significant difference in sperm count or sperm quality parameters when compared eith controls European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition) Available from, as of February 18, 2015 : www.inchem.org/documents/sids/sids/ 56 815 0.05 mL of 10% solution for 21 days Dermal patch test Slight irritation at 48 hrs and maximum rating of 4 on a 9 point scale at day 14 of a 21 day application European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015 : www.inchem.org/documents/sids/sids/ 56 815 Acute and chronic (42 d) ingestion Oral Increase in plasma glycerides in males only following a cute ingestion and in both males and females with chronic ingestion (significantly greater increase in males) European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015 : www.inchem.org/docu ments/sids/sids/ 56 815 Orange juice mixed with 30 mL of 95% glycerol after each of 3 daily meals Oral No overt signs of toxicity or chage in food consumption European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available fr om, as of February 18, 2015 : www.inchem.org/documents/sids/sids/ 56 815 Repeated application of 100% solution Occular Extensive changes to appearance of endothelium that disappear ed within 90 minutes of application Grant, W.M. Toxicology of the Eye. 3rd ed Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 24,000 mg/kg b w d for 50 days Oral Slight t endency toward increase in weight WHO/FAO: Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Alip hatic acyclic diols, triols, and related substances (56 81 5) (2002).

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168 Available from, as of February 18,2015, : http://www.inchem.org/pages/jecfa.html Not reported. W orkers in foam rubber facory Dermal patch testing No sensiti z ing effects United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015 : echa .europa.eu/ Case Reports Subject Dose Route & Purpose Effects Reference 46 y.o. male 500 mL of 10% solution Alter ed sensorium, generalized seizures, focal neurologic signs Managed conservatively and recovered within 48 hrs case represents rare presentation of overdose with an otherwise safe drug used in neurology Singh R et al; Neurol India 49 (3): 320 1 (2001) M ale Adjunctive glycerin test used in the diagnosis of 20 40dB hearing loss in univolved ear during standard testing that resolved within 3 days Mattox DE, Goode RL; Arch Otolaryngol 104 (6): 359 61 (1978) 73 y.o. male Oral solution use d to treat elevated IOP Developed severe pulmonary edema 45 minutes after administration Almog Y et al; Ann Ophthalmol 18 (1): 38 9 (1986) 72 y.o. male Dose should not exceed 1.5 g/kg bw in Klockhoff test for diagnosis of suspected P atient received 3.88 3.95 g/kg bw Progressive neurological signs and pathologically elevated se rum concentration of triglycerides (3,465 mg/dl) Andresen H et al; Clin Toxicol (Phila) 47 (4): 312 6 (2009) 3 y.o. male 0.5 1.0 g/kg Unique intolerance inclu ding mental changes, N&V, hypoglycemia and loss of consciousness following IV European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of

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169 administration with spontaneous recovery after 30 min February 18, 2015 : echa .europa.eu/ Not reported Rectal administration prior to coronary artery bypass Acute colonic ischemia European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015 : echa .europa.eu/ 82 y .o. hypertensive and senile female 200 mL 50% solution for primary angle closure glaucoma Headache, shaking of arm, quivering of eyes and nausea European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of Feb ruary 18, 2015 : echa .europa.eu/ 68 y.o. female d i a betic 280 mL of 50% solution Severe daibetic acidosis within 3 days of ingestion European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 201 5 : echa .europa.eu/ / The route of administration influences toxicity of glycerol in humans. Toxic effects from oral administration include nausea and vomiting. Glycerol has a CNS dehydration effect. Intraocular pressure begins to fall at plasma concentrat ions of 10 mmoles per liter. Concentration and dilutant used also influence toxicity. Use of saline as dilutant diminishes the toxic effects of glycerol. Toxic effects following intraperitoneal and subcutaneous administration are albuminuria, hemoglobinuri a, anemia and renal damage. European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of July 18, 2011: http://esis.jrc.ec.europa.eu/ No information is included in this section under 31 2.55 Clinical Protocol/ General Investigational Plan Specific Aims The purpose of this prospective pilot study is to compare two distinct flushing regimens, one high volume saline flush and one low volume USP glycerin flush, in the immediate postoperative period in children requiring ACE therapy. Findings from this study will provide a comparative analysis of these two regimens that will serve as a starting point to guide practice and serve as a foundation for additional prospective, randomized, controlled trials. The aims of this study are to 1) identify the minimal administration frequency and titration time to reaching effective dose; 2) compare which solution at an optimum dose is delivered in the least amount of time, with fewer side effects, while prom oting the higher degree of fecal continence and

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170 quality of life; and 3) determine if administration of antegrade enema solution through an appendicostomy/cecostomy affects gut microbiota and immune function. Null Hypotheses for Aim 1: 1. There will be n o differences in frequency of administration necessary to gain and maintain continence between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy ( primary aim ). 2 There will be no differences in titration time to reaching effective dose between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). Null Hypot heses for Aim 2: 1. There will be no difference in continence between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy ( primary aim ). 2. There will be no dif ference in procedural time between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). 3. There will be no difference in side effects between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). 4. There will be no difference in parent/patient satisfaction as measured by the Fecal Incontinen ce and Constipation Quality of Life Measure in Children with Spina Bifida (FIC QOL) between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). Nul l Hypothes e s for Aim 3: 1. There will be no difference in the effect of two different ACE flushing regimens using normal saline and normal saline with USP glycerin on colonic microbiota in children requiring antegrade continence therapy. 2. There will be no difference in the effect of two different ACE flushing regimens using normal saline and normal saline with USP glycerin on gut mucosal health in children requiring antegrade continence therapy Design This prospective study will utilize a repeated measures, single subjects alternating treatments A B C withdrawal design in which all subjects are tested under all conditions and each subject acts as his or her own control. A within subjects cross ment comparison phase of the study Subjects will be randomly assigned to one of two treatment sequences to control for the possibility of order effects. The treatment will be replicated across 6 subjects randomized to 3 subjects per group. The patient an d investigator cannot be blinded to flushing regimens contents due to dosing considerations. The high volume flushing regimen will be comprised of normal saline alone. The low volume regimen will be comprised of USP glycerin with a small volume of normal s aline used as diluent. Volume and frequency of administration will be structured to find the lowest dosing of each regimen sufficient to maintain continence. Baseline data A will serve as the control and will be obtained pre operatively. Ethics prohibit re turn to a no treatment baseline phase as this would result in multiple daily episodes of fecal incontinence. The first B C phase of the study will evaluate dose response relationship and will be used to identify the optimal dose and frequency of ACE admi nistration for normal saline and normal saline with USP glycerin. When the optimal dose has been identified, the child will continue on that dose for two weeks to insure treatment stability and

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171 effectiveness. If continence cannot be achieved within the dos ing guidelines, the child will be trialed on the alternative therapy but will not progress to the maintenance phase of the study. To prevent statistical bias from subject loss due to treatment failure, each child will be randomized to a second treatment se quence once they study will compare the effectiveness of the two regimens at optimal dose and administration frequency. Rationale for Utilization of W ithin Subjects Designs Any research design is a tool used to answer a question. Strategies, design choice, and use of design elements should be based on how best to answer the question at hand (Kazdin, 2011). The purpose of experimental design is to contro l the effects of random error and bias (Piantadosi, 2005). Both single subject and between group research make and test predictions about treatment effects, the first by evaluating treatment effects on an individual, the second by addressing group mean and variance (Kazdin, 2011). Single subjects and group designs, in the i r most rigorous form, rule out or make implausible rival hypothes e s for the experimental outcome improving quality of inference. (Cook & Campbell, 1979; Kazdin, 2011; Shadish, Cook, & Ca mpbell, 2002). A randomized controlled trial (RCT) is considered the gold standard for intervention research (Piantadosi, 2005). However, a RCT is not the only standard for causal inference (Kazdin, 2011). Reliance on large numbers makes application of a R CT with small groups or rare disease problematic (Janosky et al., 2009). The focus of this research involves instillation of a solution through an appediceal stoma a procedure used for over a century and widely popularized over 20 years ago. Case report s and retrospective studies detail widely divergent effectiveness rates (Bani Hani, Cain, King, & Rink, 2008; Dey et al., 2003; Mousa et al., 2006; Siddiqui, Fishman, Bauer, & Nurko, 2011 ; Yardley et al., 2009). The literature and involved clinicians have identified the need for prospective trials comparing ACE flushing regimens. None have been undertaken to date. This is in large part because the small size and heterogeneity of this population does not lend itself to a large N study. Many clinical question s go unanswered due to over reliance on RCT large N methodology (Kazdin, 2011). This population is an exemplar of that problem. The proposed study comparing two flushing regimens utilizes a cross over design embedded in a single subject A B C Liu, 2014; Elder, 1997; Portney & Watkins, 2009). In both designs the subject acts as his or her own control minimizing within subject variability and ensuring the highest possible degree of equivalence across treatment conditions, thereby allowing greater precision and efficient estimates of trea tment effects increasing internal validity and causal inference (Janosky et al., 2009; Piantadosi, 2005; Portney & Watkins, 2009). In both methods subjects are randomized to treatment sequence. Randomization will decrease the threat of order effects in bo th methods, increase group equivalency in the cross over design, and minimize variability in measurement due to subject or period differences, increasing internal validity and causal inference (Chow & Liu, 2014; Jones & Kenward, 2003). Single subjects de sign is an inductive, experimental methodology with controlled introduction and manipulation of an independent variable. Single subjects design promotes exploration of inter subject variability without the introduction of error inherent in group methodolog y in the absence of subject homogeneity. It allows for isolation of individual response to interventions and identification of valuable information from outliers that would be obscured or lost in group methodology. Single subjects design also allows for ti me series observations of response, providing continuous and often a more accurate representation of the dependent variable of interest that may be compromised when the data is collected as an isolated snapshot in group methods (Elder, 1997). This study is ideally suited to single subject repeated measures design, because children requiring an ACE procedure comprise a very small population with widely disparate anatomic and physiologic causative factors making sample homogeneity difficult. Inclusion of he terogeneous subjects will allow differentiation of subject characteristics that impact response to treatment. The design allows for frequency, volume, and dose adjustment of each flushing regimen when indicated. The ability to adjust the treatment regimen facilitates dose response comparison and will aid in identifying which flushing regimen requires the minimal dose and administration frequency, is accomplished in the least amount

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172 of time, and has the fewest side effects while achieving continence. Because this design allows for repeated measurement over time, it is particularly helpful when studying comparisons between several treatments and is more sensitive to variations in treatment response that might otherwise be missed using group methodology (Gast, 2010; Janosky et al., 2009; Kazdin, 2011). Subjects will be limited to children who are scheduled for a cecostomy or appendicostomy, ensuring their gut is nave to the effects of a flushing regimen and allowing a true no treatment baseline. Flush effects are reversible, making this intervention amenable to a withdrawal design. There are no known carry over effects associated with either flushing regimen that would impact treatment effect on continence. Specimen collection facilitating comparison of treatm ent effects on gut microbiota, electrolytes, and stool calprotectin occurs after an active wash out period at the completion of each flushing regimen, negating any carry over effects. Given the pragmatic issues involved in answering the research question a t hand, the chosen methods and design elements strengthen demonstration of the counterfactual and make implausible potential threats to validity, lending credence to the assumption that intervention effects are due to the treatment and not random error or bias. Subjects This study will involve six children, ages 3 to 12 years, recruited from subspecialty clinics at Nemours entities in Florida and the Pediatric Spinal Defects Clinic in Jacksonville, Florida. Children will be selected by purposive sampling a nd will include those who are scheduled to have an ACE stoma and will require regular antegrade enema administration to maintain continence. Subjects will be limited to children who are scheduled for a cecostomy or appendicostomy, ensuring their gut is na ve to the effects of a flushing regimen and allowing a true no treatment baseline. Excluded will be children with preexisting electrolyte imbalance, chronic high rectal tone, quadriplegia, renal or cardiac disease, or those who require prophylactic antibio tics, cannot communicate, or have significant cognitive delay that would interfere with their ability to fully participate in the study. Parents must have English language competency and be willing and able to participate in administration or oversight of the flushing regimen and data collection for a minimum of twenty + consecutive weeks. Setting Once parental consent and child assent have been obtained, baseline data will be collected daily for a minimum of 2 weeks prior to surgery, including frequency a nd volume of episodes of fecal soiling, and frequency and severity of abdominal pain. Blood samples for electrolyte and stool for calprotectin and microbiota will be obtained in the immediate preoperative period. The initial stool specimens for analysis wi ll be collected prior to initiation of any pre operative bowel prep. Postoperatively, the child will be randomly assigned to either the saline or USP glycerin protoco l. The process will be restricted random assignment to force equal sample size and will be accomplished using the SAS random number generator ensuring subject assignment results in equal group size. A member of the research team will meet with the parent and child in the immediate postoperative period after surgical clearance has been obtaine d for initiation of the first flush. During that time, the flush protocol, including materials and procedures will be reviewed in detail The child will receive the first antegrade infusion during that in patient visit. A member of the research team will be available for each subsequent flush during the hospitalization. This will allow the family to gain competency in a controlled environment and familiarize themselves with the prescribed protocol and procedures prior to transition to the home setting. The reliability or accuracy and consistency of measurements will be verified using interobserver agreement (IOA) calculated by gross method If there is a significant discrepancy in observational accuracy, as demonstrated by a calculated IOA below 80%, additi onal observer training will be provided until the calculated IOA is 80% or higher (Gast, 2010). Procedural reliability will be ascertained for each procedural variable to assure the intervention is being implemented as described in the methods section of t he proposal. Measures

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173 Dependent variables at baseline will include: (a) number of episodes of fecal soiling. In addition, fecal soiling will be scored based on fre quency and volume of accidents ( 0 = no soiling, 1 = smear, 2 = moderate volume accident not visible through clothing per week, 3 = any accident visible through clothing ) (b) frequency and severity of abdominal pain recorded daily and measured using the Wong Baker Faces Pain Rating Scale as the age appropriate visual analog scale, (c) serum elect rolytes, (d) stool for calprotectin, (e) quality of life measured by the FIQoL and (f) utilization of molecular techniques for identification of 16SrRNA gene sequence in stool samples obtained intra operatively to identify and quantify phylogenetic groups (Penders et al., 2007). Dependent variables obtained post operatively following initiation of cecosotmy/appendicostomy flush will include: (a) administration time in minutes per flush, (b) total procedural time from start of flush to completion of colon ic emptying in minutes per flush, (c) volume of solution in mL (d) number of episodes of fecal soiling In addition, fecal soiling will be scored based on freq uency and volume of accidents (0 = no soiling, 1 = smear, 2 = one moderate volume accident not vi sible through, 3 = any accident visible through clothing), (e) frequency and severity of abdominal pain recorded daily and measured using the Wong Baker Faces Pain Rating Scale as the age appropriate visual analog scale (f) number and frequency of side eff ects per week with severity of side effects measured using the Wong Baker Faces Pain Rating Scale as the age appropriate visual analog scale, (g) serum electrolytes, (h) stool for calprotectin, (i) quality of life measured by the FIQoL, and (j ) utilizatio n of molecular techniques for identification of 16SrRNA gene sequence in stool samples to identify and quantify phylogenetic groups (Penders et al., 2007). Fecal soiling score is detailed in Table 1.1. Dependent variables including type of sample or instr ument, sample characteristics, and measurement and data level are explicated in Table 1.2 Study timetable is explicated in Table 1.3. Table 1.1 Level of Soiling 0 No soiling 1* S mear 2* One moderate volume accident not visible through clothing 3* A ny accident visible through clothing *Change dose with any soiling > Level 0 T able 1.2 Dependent Variables Sample/Instrument Variable Measurement Data Level Blood BMP Electrolyte Balance Ratio Stool Calprotectin Mucosal Inflammation Ratio Stool Colonic microbiome Metagenomic Profiling 16SrRNA Ratio FIC QOL Parent/child quality of life Symptoms Rating Scale Ordinal WBFPRS Abdominal Pain Symptom Rating Scale Ordinal WBFPRS Procedural side effects Symptom Rating Scale Ordinal Stop Watch Infusi on Time Minutes Ratio Stop Watch Procedural Time Minutes Ratio Administration time in minutes per flush will be defined as the time at which the tubing connected to the bag or syringe holding the flush solution is unclamped and the cecostomy fluid start s to infuse into the patient to the time the infusion is completed (no more fluid left in the bag/syringe or tubing). The total procedural time is defined as the time the flush starts to infuse into the subject and ends following passage of stool when the child has sat on the commode for 5 minutes with no additional stool passage. Both administration and total procedural times will be measured using duration per occurrence direct observational recording completed by the parent or child. Volume and dose will be calculated with each flush. Accidents will be defined as non toilet elimination, which will be tracked and tallied as the number of pairs of underwear soiled with stool with documentation of accident severity and the estimated time of each accident usi ng event recording. Dependent variables will be measured and recorded by the parent or child using a data collection sheet specifically

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174 designed for this study and reviewed weekly with the study coordinator to encourage completion of all relevant data. The investigator will keep a research log documenting and detailing any event that may cause a change in level, stability, or trend of the dependent variable not related to the intervention, for example treatment with antibiotics or an intercurrent illnes s. The number of episodes of fecal soiling per week will exclude accidents caused by illness, medications, or dietary indescretion. Side effects will be measured using the Wong Baker FACES Pain Rating Scale (WBFPRS). The WBFPRS has undergone extensive test ing, is preferred by children, and has well established psychometrics in the pediatric population (Tomlinson, von Baeyer, Stinson, & Sung, 2010; Wong & Baker, 1988).The scale ranges from 0 (very happy without pain) to 10 (the worse pain imaginable). Each p ain level is associated with a facial expression. The child is asked to choose the face that best describes his/her level of discomfort. The WBFPRS will be used to evaluate the presence and severity of flush side effects including abdominal cramping, naus ea, and vomiting. The parent will call if the child is having accidents or discomfort greater than a 4 on the WBFPRS associated with the flushing regimen. Documentation of severity of side effects will be completed by the parent and child on a data collect ion form by viral, bacterial, or drug induced gastroenteritis; these will be recorded and analyzed as confounds (Portney & Watkins, 2009). The Fecal Incontinence and Constipation Quality of Life Measure in Children with Spinal Bifida (FIC QO L) will be use d to assess child and parental perception of social validity (Nanigian et al., 2008). The tool will be administered preoperatively during the baseline period and at the end of each flushing regimen in the comparative phase of the study. The FIC QO L is a 51 item questionnaire with established validity and reliability in families of children with spina bifida who are incontinent for stool. This instrument measures those aspects of daily living that are significantly impacted by fecal incontinence. Of the 51 i tems, four address subject and family demographics. The remaining 47 items are divided into seven groupings that include bowel program, diet, symptoms, travel and socialization, family relationships, caregiver support and emotional impact, and financial im pact (Nanigian et al., 2008; Ok & Kurzrock, 2011). In addition to the FIC QO L, a simple qualitative question will be directed to the children at the end of the study to ascertain which flushing regimen they prefer and why.

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175 Table 1.3 Timeline for Completio n Overview of Timeline Baseline Dose Response Phase Flush Effectiveness Phase Weeks: 1 2 3 4 5 6 7 8 9 10+ 11 12 13 14 15 16 17 18 19 20 Randomize X X Order: A NT NT B C B B B B C C C C C B C C C C B B B B B B 1 B B B B C C C C B 1 B 1 C C 1 C C C C B B B B C 1 C 1 Visits: Hospital X Home X Clinic X X X X X Biomarker Stool X X X X X BMP X X X SIM X X X Instrument FIC QOL X X X Measures: Soiling X X X X X X X X X X X X X X X X X X X X Abd Pain X X X X X X X X X X X X X X X X X X X X Admin T X X X X X X X X X X X X X X X X X X Proc T X X X X X X X X X X X X X X X X X X ISE X X X X X X X X X X X X X X X X X X Cost X X X X X X X X X X X X X X X X X X X X A Baseline NT No treatment B Saline do se response phase C USP Glycerin dose response phase Initial trial of saline effectiveness phase Initial trial of USP glycerin effectiveness phase B 1 Second trial of saline effectiveness phase C 1 Second Trial of USP glycerin effectiveness p hase Stool Metagenomic Profiling 16SrRNA (collected and batched for downstream analysis) BMP Basic Metabolic Profile SIM Stool Inflammatory Marker (Calprotectin) Visits Procedural fidelity and inter rater reliability will be measured at each visit FIC QOL Fecal Incontinence and Constipation Quality of Life Measure in Children with Spina Bifida Abd Pain Abdominal Pain Admin T Administration time Proc T Procedural time ISE Infusion side effects

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176 Dosing High Volume Regimen : The high volume regimen (B) consists of a normal saline flush at a starting dose of 10mL/kg infused every other day and adjusted until stability of target outcomes is achieved. At any point the subject is h aving episodes of fecal soiling the dosing strategy will be incr eased by 5 to 10 mL/kg increments with a subsequent increase in frequency, if needed, so as not to exceed 20 mL/kg and a maximum dose of 500 mL in children 5 years of age and under and 1000 mL administered daily in children older than 5 If the child does not attain continence on the maximum dose, he/she will be trialed on the alternate flushing solution but will not progress to the maintenance phase of the study. If the child is having side effects greater than 4 on the WBFPR S at the starting dose of 10mL /kg, flush volume will be incrementally decreased as needed by 2.5 mL/kg to the lowest dose of 5 mL/kg daily. The goal is to find the lowest effective dose and flushing frequency with minimal side effects. If the dose necessary to minimize effects results in episodes of fecal soiling or the child continues to have side effects greater than 4 on the WBFPRS at the lowest dose of administration, the child will be dropped from the study. The decision tree for dose adjustment of Normal Saline is detailed in Tabl es 1. 4 and 1. 5 Low Volume Regimen : The low volume regimen (C) will consist of USP glycerin diluted in normal saline prior to antegrade instillation through the low profile device. The child will start on an every other day dose of 20 mL of USP glycerin a nd >20 mL of saline (used as diluent at a dose sufficient to allow the solution to easily infuse through the ACE access tubing) and adjusted until stability of target outcome is achieved. At any point the child is having episodes of fecal soiling, the volu me of USP glycerin will be increased in 10 mL increments with subsequent increase in frequency, if needed, so as not to exceed 4 0 mL of USP glycerin administered daily. If the child does not attain continence on the maximum dose of USP glycerin, he/she wil l be placed on the alternate flushing regimen but will not progress to the maintenance phase of the study. If the subject is having side effects greater than 4 on the WBFPRS at the starting dose of 20mL of USP glycerin, the volume of the USP glycerin will 4 or less on the WBFPRS or the lowest dose of 5 mL daily is reached. The goal is to find the lowest effective dose and flushing frequency with minimal side effects. If the dose neces sary to minimize effects results in episodes of fecal soiling greater than one smear per week or the child continues to have side effects greater than 4 on the WBFPRS at the lowest dose of administration, the child will placed on the alternative flushing r egimen but will not advance to the maintenance phase of the study. The decision tree for dose adjustment of USP glycerin is detailed in Tables 1. 4 and 1. 5

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177 Table 1. 4 Dosing Strategy to Determine Minimum Volume and Frequency Necessary to Maintain Conti nence in the Absence of Side Effects *C hange dose with any soiling > Level 0 as defined in Table 1.5 Table 1. 5 Dosing Strategy to Determine Minimum Volume and Frequency Necessary to Minimize Side Effects and Maintain Continence Normal Saline* (High volume regimen with maximum dose for less t han 5 and over 5 yrs. ) USP Glycerin + NS as Diluent* (Low volume regimen) B1 = 10 mL/kg maximum dose 500 or 1000 mL qod C1 = 20 mL + > 20 mL qod B7 = 7.5 mL/kg maximum dose 500 or 1000 mL qod C10 = 15 mL + > 15 mL qod B8 = 7.5 mL/kg maximum dose 500 o r 1000 mL q3d C11 = 15 mL + > 15 mL q3d B9 = 7.5 mL/kg maximum dose 500 or 1000 mL qd C12 = 15 mL + > 15 mL qd B10 = 5.0 mL/kg maximum dose 500 or 1000 mL qod C13 = 10 mL + > 10 mL qod B11 = 5.0 mL/kg maximum dose 500 or 1000 mL q3d C14 = 10 mL + > 10 m L q3d B12 = 5.0 mL/kg maximum dose 500 or 1000 mL qd C15 = 10 mL + > 10 mL qd C16 = C17 = C18 = 5 mL + > 5 mL qod. 5 mL + > 5 mL q3d 5 mL + > 5 mL qd *Cha nge dose with any side effects > 4 on the WBFPRS or soiling Level 1 as defined in Table 1.5 Optimal Dose Regimen : Once the optimal dose has been established, the child will be maintained on that dose and frequency for at least 2 weeks or until stability in dependent measures without significant variability or tre nd is achieved. The child will be scheduled to come into the clinic for a visit once the above criteria have been met at which time labs will be drawn and a stool sample collected. Regimen Comparative Phase : Following completion of the dose response phas e, the comparative portion of the study will begin by The last flush in the dose response sequence will be withdrawn and the initial flush in the comparative treatment phase will be introduced the following day at the previously established minimum effective dose and frequency. Children w ill remain on treatment for 4 weeks at which point the treatment will be withdrawn. Children will then be placed on the next treatment in the sequence at the pre established effective dose and frequency for 4 weeks. The second flush will then be withdrawn and the initial flush in the sequence will be on the flushing regimen of his/her choice. Normal Saline (High volume regimen ) USP Glycerin + Normal Saline as Diluent (Low volume regimen Max dose for children 5 and under is 30 mL + > 30 mL qd ) B1 = 10mL/kg maximum dose 1000 mL qod C1 = 20 mL + > 20 mL q od B2 = 10mL/kg maximum dose 1000 mL q3d C2 = 20 mL + > 20 mL q3d B3 = 10mL/kg maximum dose 1000 mL qd B4 = 15 mL/kg maximum dose 1000 mL qod B5 = 15 mL/kg maximum dose 500 or 1000 mL q3d B6 = 15 mL/kg maximum dose 500 or 1000 mL qd C3 = 20 mL + > 20 mL qd C4 = 25 mL + > 30 mL qd C5 = 25 mL + > 30 mL qod C6 = 25 mL + > 30 mL q3d C7 = 30 mL + > 40 mL qod C8 = 30 mL + > 40 mL q3d C9 = 30 mL + > 40 mL qd C10 = 4 0 mL + > 50 mL qod C11 = 4 0 mL + > 50 mL q3d C12 = 4 0 mL + > 50 mL qd

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178 Data Evaluation Data will be obtained and dose respo nse adjustment will be made during the B C or C B phase when indicated. Once data has been colleceted, it will be graphed on an equal interval line graph with the proportion of ordinate and abscissa scaled at a 2:3 ratio to ensure consistency of data prese ntation and prevent data distortion during visual analysis. Dependent measures will be placed on the ordinate scale with time by day on the abscissa scale. Separation of dose response and comparative phases of the study will be designated by a bold vertica l line and high and low dose regimen changes by a thin vertical line. Each phase change will be labeled with solution name, dose, and frequency. Independent variable effect on target behaviors will be analyzed using visual analysis. Analysis within each co ndition will include: (a) condition length defined as the number of data points contained within each phase, (b) level stability with a stability envelope calculated using the median and a stability criterion of 80% of the data points falling within 15% of the calculated median for the phase, (c) relative change in level, (d) absolute change in level, (e) estimation of trend direction using split middle method, (f) trend stability with a stability envelope using the same criteria as level stability, and (g) identification of multiple paths within trends if present. Analysis between conditions will include: (a) the number of variables that have changed between adjacent conditions, (b) change in trend direction between conditions, (c) assessment if trend chan ge is in keeping with intervention goals, (d) assessment for change in trend stability, (e) assessment of immediacy of effect in change in level and trend, (f) calculation of absolute and median level change, (g) calculation of percentage of non overlappin g and overlapping data points and ( h) percentage of data points exceeding the median (Gast, 2010; Hartmann et al., 1980; Ma, 2006; Kazdin, 2011; McDowall, McCleary, Meidinger, & Hay, 1980; Portney & Watkins, 2009). Testing in multiple subjects will allow f or analysis of replication of treatment effects. Analysis will include between series strategies comparing data points including frequency, mean occurrence, and immediacy and magnitude of effect within and between treatment conditions. In addition to visu al analysis of time series parameters, inferential procedures will be used to compare interventional effects and increase the reliability of visual methods analysis. The independent variable is nominal and dichotomous. Dependent variables are comprised o f either interval or ratio level measurements. The comparative phase of the study is a 2 treatment crossover design with each child sequence with h alf of the subjects allocated to each sequence (Portney & Watkins, 2009). The flushing including number of soiling episodes, level of soiling, abdominal pain, procedural side effects, infusion time, procedural time electrolyte balance, fecal calprotectin, and quality of life will be analyzed using a two tailed, two sample pooled variance t test with a significance level set at 0.05. Confidence intervals will be calculated to provide precision of mean differences e stimates (Polit, 2010). The two sample t test will be used to test treatment difference s in the cross over design. Because the design is comparing two treatment sequences, the groups are independent (Chow & Liu, 2014). Confounding by carry over and direct by period interaction is a potential with cross over designs which if present, can bias treatment effects (Jones & Kenward, 2003; Senn, 2002; Shuster, 2007). Jones et. al. (2003) and Sen (2002) suggest use of a one sample t test for analysis of cross over designs (treatment one is subtracted from treatment two). Shuster (2007) advocates a two sample t test in the analysis of a randomized 2 treatment cross over design (period two is subtracted from period one irrespective of treatment order). Analysis of a one sample t test in a cross over design ignores treatment ordering. Two sample t test analysis compares ordering and yields one sample or two s ample t test will yield unbiased estimates of and variance when the sample size is equal method. However, the one sample method does not account for carry over effects increasing variance. If but not the two sample t test will be biased. Using the 2 sample method will lend precision in the presence of carr y over characteristics will be described, when appropriate, using frequency distribution and graphed using either histograms or pie charts. Chang es in gut microbiota will be analyzed using descriptive statistics (Polit, 2010). Summary of Previous Hu man Experience with the Investigational Drug

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179 Retrospective Studies and Case Reports Addressing Antegrade Colonic Flush Administration Pediatric Sub jects Solution & Dose Side Effects from Flush Reference 40 Dose not reported 1) GoLYTLEY 2) Liquirice root 3) Oil/water mix 4) Treacle/milk mix 5) Water only 6) Oil only None Marshall, J., Hutson, J. M., Anticich, N., & Stanton, M. P. (2001). Antegr ade continence enemas in the treatment of slow transit constipation. Journal of Pediatric Surgery 36 1227 1230. doi:10.1053/jpsu.2001.25768 62 1) Polyethylene Glycol with electrolytes (50 1,000 mL) 2) Phosphate enema 3) NS alone (50 1000 mL) None D ey, R., Ferguson, C., Kenny, S. E., Shankar, R. R. (2003). After the honeymoon Medium term outcome of antegrade continence enema procedure. Journal of Pediatric Surgery 38 65 68. doi:10.1053/jpsu.2003.5001 2 26 Flush s olution not reported. Dosage ( 250 1000 mL ) None Becmeur, F., Demarche, M., Lacreuse, I., Molinaro, F., Kauffmann, I., Moog, R., Rebeuh, J. (2008). Cecostomy button for antegrade enemas: Survey of 29 patients. Journal of Pediatric Surgery 43 1853 1857. doi:10.1016/jpedsurg.2008.03.028 71 Tap water (300 1000 mL) Minor deviations in serum sodium and serum chloride present in 18/71 patients Significant hypernatremi and hyper chloremia i n Yerkes, E. B., Rink, R. C ., King, S., Cain, M. P., Kaefer, M., & Casale, A. J. (2001). Tap water and the Malone antegrade continence enema: A safe combination? Journal of Urology 166 1476 1478.

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180 1/71 us ed softened tap water 236 1)Tap water alone volume (100 to 1000 ) 2) 60 mL of USP glycerin & 60 mL of NS 3) GoLYTELY 1 liter 4) MiraLAX 17 grams mixed in 250 mL tap water 5)Mineral oil 30 mL None Bani Hani, A. H., Cain, M. P., King, S., & Rink, R. C. (2008). Tap water irrigation and additives to optimize success with the Malone antegrade continence enema: T he Indiana University algorithm. Journal of Urology 180 1757 1760.doi:10.1016/j.juro.2008.04.074 105 1) Normal saline or GoLYTELY(23 +/ 0.7 mL/kg) 2) USP glycerin dose not reported 3), bisacodyl dose not reported 4) magnesium cirate dose not repo rted 5) phosphosoda dose not reported None Siddiqui, A. A., Fishman, S. J., Bauer, S. B., & Nurko, S. (2011). Long term follow up of patients after antegrade continence enema procedure. Journal of Pediatric Gastroenterology and Nutrition 52 574 580 23 USP glycerin 10 60 mL & 15 775 mL tap water with a total volume of irrigation solution ranging from 30 800 mL None Chu, D., Balsara, Z.R., Routh, J.C., Ross, S.S., & Wiener, J.S. (2012). Experience with glycerin for antegrade continence enema in patie nts with neurogenic bowel. The Journal of Urology, 189,690 693. 1 C ase report Hypertonic saline Death Schreiber, C. K., & Stone, A. R. (1999). Fatal hypernatremia associated with the antegrade continence enema procedure. Journal of Urology 162 1433 1 434.

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181 References Aksnes, G., Diseth, T. H., Helseth, A., Edwin, B., Strange, M., Aafos, G., & Emblem, R. (2002). Appendicostomy for antegrade enema: Effects on somatic and psychosocial functioning in children with myelomeningocele. Pediatrics 109 48 4 489. doi:10.1542/peds.109.3.484 Anderson, R. E., Olaison, G., Tysk, C., & Ekbom, A. (2003). Appendectomy is followed by increased risk of Gastroenterology 124 40 46. doi:10.1053/gast.2003.50021 Andreu Ballester, J. C., Perez Griera, J. Ballester, F., Colomer Rubio, E., Ortiz Tarin, I., & Penarroja, O. C. (2007). Secretory immunoglobulin A (sig A) deficiency in serum of patients with GALTectomy (appendectomy and tonsillectomy). Clinical Immunology 123 289 297. doi:10.1016/j.clin.2007. 02.004. Aspirot, A., Fernandez, S., Di Lorenzo, C., Skaggs, B., & Mousa, H. (2009). Antegrade enemas for defecation disorders: Do they improve the colonic motility? Journal of Pediatric Surgery 44 1575 1580. doi:10.1016/j.jpedsurg.2008.11.061 Bani Hani, A. H., Cain, M. P., King, S., & Rink, R. C. (2008). Tap water irrigation and additives to optimize success with the Malone antegrade continence enema: The Indiana University algorithm. Journal of Urology 180 1757 1760.doi:10.1016/j.juro.2008.04.074 Barke r, M. (2012). Taking stock of the human microbiome and disease. Science 336 1246 1247. the immune intelligence network. Medical Hypothesis 63 752 758. doi:10.1016/j.mehy.2004.04.008 Becmeur, F., Demarche, M., Lacreuse, I., Molinaro, F., Kauffmann, I., Moog, R.,...Rebeuh, J. (2008). Cecostomy button for antegrade enemas: Survey of 29 patients. Journal of Pediatric Surgery 43 1853 1857. Bollin ger, R. R., Barbas, A. S., Bush, E. L., Lin, S. S., & Parker, W. (2007). Biofilms in the large bowel suggest an apparent function of the human veriform appendix. Journal of Theoretical Biology 249 826 831. doi:10.1016/j.jtbi.2007.08.032 Chow, S., & Liu, J. (2014). Design and analysis of clinical trials: Concepts and methodologies (3rd ed.). Hoboken, NJ: Wiley. Cook, T. D., & Campbell, D. T. (1979). Quasi experimentation: Design and analysis issues for field settings Boston, MA: Houghton Mifflin. Curry, J I., Osborne, A., & Malone, P. S. (1999). The MACE procedure: Experience in the United Kingdom. Journal of Pediatric Surgery 34 338 340. Dasso, J. F., & Howell, M. D. (1997). Neonatal appendectomy impairs mucosal immunity in rabbits. Cellular Immunology 182 29 37. After the honeymoon Medium term outcome of antegrade continence enema procedure. Journal of Pediatric Surgery 38 65 68. doi:10.105 3/jpsu.2003.50012 Elder, J. H. (1997). Single subject experimentation for psychiatric nursing. Archives of Psychiatric Nursing XI (3), 133 138. Forchielli, M. L., & Walker, W. A. (2005). The role of gut associated lymphoid tissues and mucosal defense. Bri tish Journal of Nutrition 93 S41 S48. doi:10.1079/BJN20041356 Gast, D. L. (2010). Single subjects research methodology in behavioral sciences New York, NY: Routledge, Taylor & Francis Group. Gebbers, J., & Laissue, J. (2004). Bacterial translocation in the normal human appendix parallels the development of the local immune system. Annals New York Academy of Sciences 1029 337 343. doi:10.1196/annals.1309.015 Gomez, R., Mousa, H., Liem, O., Hayes, J., & Di Lorenzo, C. (2010). How do antegrade enemas work ? Colonic motility in response to administration of normal saline solution into the proximal colon. Journal of Pediatric Gastroenterology and Nutrition 51, 741 746. doi:10.1097/MPG.0b013e318e75d18 Hartmann, D. P., Gottman, J. M., Jones, R. R., Gardner, W. Kazdin, A. E., & Vaught, R. S. (1980). Interrupted time series analysis and its application to behavioral data. Journal of Applied Behavior Analysis 13 543 559.

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182 Hooper, L. V., Littman, D. R., & Macpherson, A. J. (2012). Interactions between the microbi ota and the immune system. Science 336 1268 1273. doi:10.1126/science.1223490 Janosky, J. E., Leininger, S. L., Hoerger, M. P., & Libkuman, T. M. (2009). Single subject designs in biomedicine New York, NY: Springer Science+Business Media. Janszky, I., Mukamal, K. J., Dalman, C., Hammar, N., & Ahnve, S. (2011). Childhood appendectomy tonsillectomy, and risk for premature acute myocardial infarction A nationwide population based cohort study. European Heart Journal 32 2290 2296. doi:10.1093/eurheartj /ehr137 Jones, B., & Kenward, M. G. (2003). Design and analysis of cross over trials (2nd ed.). Boca Raton, FL: Chapman & Hall/CRC. Kaugars, A. S., Silverman, A., Kinservik, M., Heinze, S., Reinemann, L., Sander, M., Sood, M. (2010). on the effect of constipation and fecal incontinence on quality of life. Journal of Pediatric Gastroenterology and Nutrition 51 747 752 doi: 10.1097/MPG.0b013e3181de0651 Kawanishi, H. (1987). Immunocompetence of normal human appendiceal lymphoid cells: I n vitro studies. Immunology 60 19 28. Kazdin, A. E. (2011). Single case research designs: Methods for clinical and applied settings (2nd ed.). New York, NY: Oxford University Press. Kelley, M. L., Heffer, R. W., Gresham, F. M., & Elliot, S. N. (1989). De velopment of a modified treatment evaluation inventory. Journal of Psychopathology & Behavioral Assessment 11 235 247 King, S. K., Sutcliffe, J. R., Southwell, B. R., Chait, P. G., & Hutson, J. M. (2005). The antegrade continence enema successfully treat s idiopathic slow transit constipation. Journal of Pediatric Surgery 40 1935 1940. doi:10.1016/j.jpedsurg.2005.08.011 Knox, C. A., & Burkhart, P. V. (2007). Issues related to children participating in clinical research. Journal of Pediatric Nursing 22 310 318. doi:10.1016/jpedn.2007.02.004 Ma, H. (2006). An alternative method of quantitative synthesis of single subject researches: Percentage of data points exceeding the median. Behavior Modification 30 598 617. doi:10.1177/0145445504272974 Marshall, J ., Hutson, J. M., Anticich, N., & Stanton, M. P. (2001). Antegrade continence enemas in the treatment of slow transit constipation. Journal of Pediatric Surgery 36 1227 1230. doi:10.1053/jpsu.2001.25768 McDowall, D., McCleary, R., Meidinger, E. E., & Hay R. A. (1980). Interrupted time series analysis Thousand Oaks, CA: Sage. Mousa, H. M., Van Den Berg, M. M., Caniano, D. A., Hogan, M., Di Lorenzo, C., & Hayes, J. (2006). Cecostomy in children with defecation disorders. Digestive Diseases and Sciences 5 1 154 160. doi:10.1007/s10620 006 3101 7 Nanigian, D. K., Nguyen, T., Tanaka, S. T., Cambio, A., DiGrande, A., & Kurzrock, E. A. (2008). Development and validation of the Fecal Incontinence and Constipation Quality of Life measure in children with spina b ifida. Journal of Urology 180 1770 1773. doi:10.1016/j.juro.2008.03.103 National Institutes of Health (NIH). (1998). NIH policy and guidelines on the inclusion of children as participants in research involving human subjects Retrieved from: http://grant s.nih.gov/grants/guide/notice files/not98 024.html Nicholson, J. K., Holmes, E., Kinross, J., Burcelin, R., Gibson, G., Jia, W., & Pettersson, S. (2012). Host gut microbiota metabolic interactions. Science 336 1262 1267. Noverr, M. C., & Huffnagle, G. B. (2004). Does the microbiota regulate immune responses outside the gut? TRENDS in Microbiology 12 562 568. doi:10.1016/j.tim.2004.10.008 Ok, J., & Kurzrock, E. A. (2011). Objective measurement of quality of life changes after ACE Malone using FICQOL surv ey. Journal of Pediatric Urology 7 389 393. doi:10.1016/j.jpurol.2011.02.012 Penders, J., Stobberingh, E. E., Van den Brandt, P. A., & Thijs, C. (2007). The role of the intestinal microbiota in the development of atopic disorders. Allergy 62 1223 1236. doi:10.1111/j.1398 9995.2007.01462.x Pharmacists, American Society of Health System. (2011). Drug Information 2011 Bethesda, MD. Piantadosi, S. (2005). Clinical trials: A methodologic perspective (2nd ed.). Hoboken, NJ: Wiley. Pieper, P. (2008). Ethical utilitarianism. Pediatric Nursing 3 319 323. Polit, D. F. (2010). Statistics and data analysis for nursing research (2nd ed.). New York, N.Y.: Pearson.

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183 Portney, L. G., & Watkins, M. P. (2009). Foundations of clinical research: Applications to practice (3rd ed.). Upper Saddle River, NJ: Pearson Prentice Hall. Rossoff, I. S. (1974). Handbook of v eterinary d rugs New York NY : Springer. Schreiber, C. K., & Stone, A. R. (1999). Fatal hypernatremia associated with the antegrade continence enema procedure. Journal of Urology 162 1433 1434. Senn, S. (2002). Cross over trials in clinical research (2nd ed.). West Sussex, England: Wiley. Shadish, W. R., Cook, T. D., & Campbell, D. T. (2002 ). Experimental and quasi experimental designs for generalized causal inference Boston, MA: Houghton Mifflin. Shuster, J. J. (2007). Design and analysis of experiments. In W. T. Ambrosius (Ed.), Topics in biostatistics (pp. 235 259). Totowa, NJ: Humana Pr ess. Shuster, J. J. (2009). Student t test for potentially abnormal data. Statistics in Medicine 28 2170 2184. doi :10.1002/sim.3581 Siddiqui, A. A., Fishman, S. J., Bauer, S. B., & Nurko, S. (2011). Long term follow up of patients after antegrade contine nce enema procedure. Journal of Pediatric Gastroenterology and Nutrition 52 574 580. Smith, H. F., Fisher, R. E., Everett, M. L., Thomas, A. D., Bollinger, R. R., & Parker, W. (2009). Comparative anatomy and phylogenetic distribution of the mammalian cec al appendix. Journal of Evolutionary Biology 22 1984 1999. doi:10.1111/j.1420 9101.2009.01809.x Thomas, J. C., Dietrich, M. S., Trusler, L., DeMarco, R. T., Pope, J. C., Brock, J. W., & Adams, M. C. (2006). Continent catheterizable channels and the timin g of their complications. Journal of Urology 176 1816 1820. doi:10.1016/S0022 5347(06)00610 0. Tomlinson, d., von Baeyer, C., Stinson, J., & Sung, L. (2010). A systematic review of faces scales for the self report of pain intensity in children. Pediatric s, 126 e1168e1198. doi:10.1542/peds.2010 1609. European Journal of Pediatric Surgery 20 405 407. W orld Health Organization (WHO) Expert Committee of Food. (2002). Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances. Retrieved from: http://www.inchem.org/pages/jecfa.html Wong, D., & Baker, C. (1988). Pain in children: Comparison of assessment scales. Pediatric Nursing, 14 (1), 9 17. Yardley, I. E., Pauniaho, S., Baillie, C. T., Turnock, R. R., Coldicutt, P., Lamont, G. L., & Kenny, S. E. (2009). After the honeymoon comes divorce: Lon g term use of the antegrade continence enema procedure. Journal of Pediatric Surgery 44 1274 1277. doi:10.1016/j.jpedsurg.2009.02.030 Yerkes, E. B., Rink, R. C., King, S., Cain, M. P., Kaefer, M., & Casale, A. J. (2001). Tap water and the Malone antegrad e continence enema: A safe combination? Journal of Urology 166 1476 1478. Youssef, N. N., Barksdale, E., Griffiths, J. M., Flores, A. F., & Di Lorenzo, C. (2002). Management of intractable constipation with antegrade enemas in neurologically intact child ren. Journal of Pediatric Gastroenterology and Nutrition 34 402 405.

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184 APPENDIX L IAA AGREEMENT BETWEEN NEMOURS AND UNIVERSITY OF FLORIDA

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186 APPENDIX M MANAGEMENT OF RESEARCH PHARMACEUTICAL PRODUCTS

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192 APPENDIX N IRB SUBMISSION APPROVAL AND APPLICATION FOR AND APPROVAL FOR AMMENDMENTS Title: A Within Subjects Comparison of Two Antegrade Flushing Regimens in Children Principal Investigator : Kimberly S. Jarczyk, MSN,CPNP, Pam Pieper, PhD, ARNP, PPCNP BC Co Investigator(s): Donald George, MD, James Sylvester, PhD, Mark Barraza, MD, Tara Spruill, RN, BSN, Karl Mann BS, Kaitlyn Smith, RN, BSN Abstract: F ECAL INCONTINENCE PA ST THE TIME OF TOILE T TRAINING IS DEVAST ATING TO AFFECTED CHILDREN A NTEGRADE CONTINENCE ENEMA (ACE) THERAPY ADMINISTER ED THROUGH A CATHETERIZABLE STOMA SURGICALLY PLACED I N THE CECUM HAS HELP ED CHILDREN WITH INTRACTABLE FECAL IN CONTINENCE ATTAIN CO NTINENCE FOR STOOL T HERE ARE A NUMBER OF RETROSPECTIVE STUDIE S DEMONSTRATING THE EFFECTIVENESS OF ACE THERAPY AND SEVERAL PRO SPECTIVE STUDIES THA T DEMONSTRATE IMPROV EMENT IN QUALITY OF LIFE FOLLOWING AN ACE PROCEDURE T HERE ARE NO PROSPECT IVE TRIALS EVALUATIN G THE EFFECTIVENESS OF DIFFERENT FLUSHING REGIMENS T HE CATHETERIZABLE ST OMA USED FOR THE ANT EGRADE ADMINISTRATIO N OF ENEM A SOLUTION IS FREQUE NTLY MADE BY BRINGIN G THE APPENDIX OUT T HROUGH THE ABDOMINAL WALL OR BY PLACING A SKIN LEVEL DEVICE ( BUTTON ) INTO THE CECUM ACE THERAPY ADMINISTRATION THROU GH THE APPENDIX OR I NTO THE CECUM HAS TH E POTENTIAL TO DISRU PT THE GUT MICROBIA L ECOSYSTEM CAUSING DYSBIOSIS A ND IMMUNE DYSFUNCTIO N T HE EFFECTS OF ACE ADMINISTRATION ON CO LONIC MICROBIOME AND MUCOSAL IMMUNITY HA VE NOT BEEN INVESTIG ATED T HIS STUDY WILL COMPA RE A HIGH VOLUME NOR MAL SALINE FLUSH AND A LOW VOLUME USP GLYCERIN FLUSH T HE PROPOSED PILOT ST UDY IS PROSPECTIVE A ND UTILIZES A CROSS OVER DESIGN EMBEDDED IN A SINGLE SUBJECTS DESIGN EMP LOYING T WELVE SUBJECTS RANDO MIZED TO TWO TREATMENT SEQUENCES T HE PRIMARY AIMS OF T HE STUDY ARE TO COMP ARE WHICH SOLUTION GIVEN AT AN OPTIMA L DOSE AND FREQUENCY IS DELIVERED IN THE LEAST AMOUNT OF TIM E WITH FEWER SIDE EFFECTS WHILE PROMOTING THE HIGHER DEGREE OF FE CAL CONTINENCE AND Q UALITY OF LIFE AND TO DETERMINE IF ADMINIS TRATION OF ANTEGRADE ENEMA SOLUTION THRO UGH AN APPENDICOSTOMY / CEC OSTOMY AFFECTS GUT M ICROBIOTA AND GUT IM MUNE FUNCTION T HIS STUDY WILL INVOLVE CHILDRE N AGES 3 TO 12 YEARS RECRUITED FRO M SUBSPECIALTY CLINI CS AT N EMOURS C HILDREN S S PECIALTY CARE AND TH E P EDIATRIC S PINAL D EFECTS C LINIC IN J ACKSONVILLE F LORIDA C HILDREN WILL BE SELECTED BY PURPOSIVE SAMPLING A ND WILL INCLUDE THOS E WHO ARE SCHEDULED TO HAVE AN ACE STOMA AND WILL REQU IRE REGULAR ANTEGRAD E ENEMA ADMINISTRATI ON TO MAINTAIN CONTINENCE E XCLUDED WILL BE CHIL DREN WITH PREEXISTIN G ELECTROLYTE IMBALA NCE CHRONIC H IGH RECTAL TONE QUADRIPLEGIA RENAL OR CARDIAC DI SEASE AND THOSE WHO CANNO T COMMUNICATE OR HAVE SIGNIFICANT COGNITIV E DELAY THAT WOULD I NTERFERE WITH THEIR ABILITY TO FULLY PARTICIPATE IN THE STUDY P ARENTS MUST HAVE E NGLISH LANGUAGE COMP ETENCY AND BE WI LLING AND ABLE TO PA RTICIPATE IN ADMINIS TRATION OR OVERSIGHT OF THE FLUSHING REG IMEN AND DATA COLLECTION FOR A MINIMUM OF 20 CONSECUTIVE WEEKS S INGLE SUBJECTS WITHI N AND BETWEEN SUBJECTS DAT A WILL BE ANALYZED U SING VISUAL ANALYSIS T HE CROSS OVER DATA WI LL BE ANALYZED USING INFER ENTIAL STATISTICS EM PLOYING TWO TAILED HYPOTHESIS TE STING F INDINGS FROM THIS STUDY WILL SERVE TO PROVIDE A COMPARATIVE ANALYSIS OF TWO DIFFERENT RE GIMENS THAT WILL SERVE AS A STARTING POINT TO G UIDE PRACTICE AND PR OVIDE A FOUNDAT ION FOR ADDITIONAL PROSPECTI VE RANDOMIZED CONTROLLED TRIALS

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193 Background State the Problem Fecal incontinence in children past the expected time of toilet training has been associated with increased anxiety and depression, more social problems, worse sc hool performance, and an increased incidence of abuse and bullying (Kaugars et al., 2010; Youssef, Langseder, Verga, Mones, & Rosh, 2005). It is particularly difficult to manage fecal incontinence using conservative measures in children with neuromuscular disorders, anorectal malformations, spinal cord injuries, spinal cord trauma or tumors, megarectum, or slow transit constipation. The Malone, or antegrade continence enema (ACE), procedure was popularized over 20 years ago as a means of helping children wi th intractable fecal incontinence attain stool continence. A catheterizable stoma from the abdominal wall into the cecum is constructed using the appendix, a tubularized portion of the bowel, or a low profile button device. The stoma allows for antegrade a dministration of enema solution into the colon. A large body of literature demonstrates ACE therapy can be effective in helping children with intractable fecal incontinence attain continence for stool with resulting significant improvement in quality of li fe. However, findings regarding effectiveness are highly variable. This variability may be due to what is used to flush. No prospective trials compare the effectiveness and adverse effects of different flushing regimens. No studies evaluate the effects of appendicosomty or cecostomy flush on gut microbiota. Justification for Conducting the Study The proposed research is significant in that it is the first prospective study to compare the effectiveness and tolerability of two commonly used ACE flushing regi mens and the first study to explore ACE flushing impact on the gut microbiome. Findings from this study have the potential to provide both clinical and biological insights into ACE administration safety and start to build a foundation of scientific evidenc e that could increase ACE effectiveness rates from 78% toward 98% or better and provide a foundation for additional prospective, randomized, controlled trials. Synopsis of the Literature Studies Addressing the Effectiveness of ACE Therapy in Promoting Cont inence A number of retrospective studies evaluate overall continence rates in children following an ACE. Findings from these studies are highly variable. Short term reported success rates range from 79 to 98% with long term outcomes ranging from 41% abando nment rate at 5 years to a mean time to first relapse lasting as long as 121.9 +/ 29.7 months (Aspirot, Fernandez, Di Lorenzo, Skaggs, & Mousa, 2009; Bani Hani, Cain, King, & Rink, 2008; Becmeur et al., 2008; Curry, Osborne, & Malone, 1999; Dey et al., 20 03; King, Sutcliffe, Southwell, Chait, & Hutson, 2005; Marshall, Hutson, Anticich, & Stanton, 2001; Mousa et al., 2006; Ok & Kurzrock, 2011; Siddiqui, Fishman, Bauer, & Nurko, 2011; Thomas et al., 2006; Yardley et al., 2009). A number of prospective trial s demonstrate significant improvement in somatic functions, psychosocial functioning, and quality of life (QOL) after an ACE procedure (Aksnes et al., 2002; Mousa et al., 2006; Tiryaki, Ergun, Celik, Ulman, & Avanoglu, 2010). Many different prescribed AC E flushing regimens are used in practice, including licorice root, mineral oil, treacle/milk mix, tap water, normal saline, polyethylene glycol solution, phosphate soda solution, bisacodyl, and USP liquid glycerin (Bani Hani et al., 2008; Marshall et al., 2001; Youssef, Barksdale, Griffiths, Flores, & Di Lorenzo, 2002). A single prospective efficacy study utilized colonic manometry to compare the motor response of three stimuli (meal, antegrade saline

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194 infusion, and antegrade bisacodyl administration) on the number of high amplitude contractions and motility index in 13 pediatric patients. Findings in this study demonstrated that there was no significant difference in parameters after ingestion of a meal or saline infusion at 10 to 20 mg/kg with a maximum vol ume of 700 mL. Bisacodyl at a dose of 0.2 mg/kg with a maximal dose of 10 mg significantly increased the motility index and high amplitude propagated contractions when compared to meal or saline (Gomez, Mousa, Liem, Hayes, & Di Lorenzo, 2010). Bani Hani, Cain, King, and Rink (2008) identified timing of accidents as the most important factor in troubleshooting flushing regimen failure. Siddiqui et al. (2011) postulated an inverse relationship between increased commode time and long term ACE adherence. Iden tifying a successful flushing regimen is determined by individual clinician preference and often requires multiple attempts before success is achieved. There are no prospective studies comparing the effectiveness of type, dose/volume, or frequency of diffe rent flushing regimens in preventing incontinence to inform practice. Studies Addressing Side Effects Associated with ACE Therapy Several case reports detail morbidity and mortality associated with a particular flushing solution, including hypocalcemia and hyperphosphatemia, following retrograde administration of phosphate enemas in children (Helikson, Parham, & Tobias, 1997; Ismail, Al Mutairi, & Al Anzy, 2000), hypernatremia following retrograde administration of hypertonic saline (Schreiber & Stone, 1999 ), and water intoxication following retrograde enema therapy using tap water (Chertow & Brady, 1994). Solution composition, volume, retention time, and underlying electrolyte imbalances are all factors that increase morbidity and mortality (Yerkes et al., 2001). Several case series describe a variety of side effects of flushing regimens, including pain with stomal intubation, nausea, vomiting, abdominal cramping, sweating, dizziness, and pallor (Dey et al., 2003; King et al., 2005; Bani Hani et al., 2008). A single retrospective study evaluated the safety of a specific flushing solution. Yerkes et al. (2001) evaluated the safety of tap water antegrade flush in 71 patients using serum electrolytes obtained pre and post operatively as the dependent variable. The timing and place of the laboratory evaluation varied widely due to the retrospective nature of their study. The ACE flush was administered at home every day or every other day at volumes ranging from 300 to 1,000 mL. Clinically insignificant electroly te abnormalities attributed to the flush included minor deviations in serum sodium or serum chloride in 18 patients. More significant hyperchloremia (107 to 113 mmol/L, with upper limits set at 105 mmol/L) was noted in 12 patients. Significant hypernatrem ia and hyperchloremia was noted in a single subject who used softened water to flush, which corrected when the flush was changed to untreated water. There have been no prospective trials detailing and comparing adverse effects associated with different ant egrade flushing regimens. Studies Addressing Gut Microbiota The catheterizable stoma used for antegrade administration of enema solution is frequently made by bringing the appendix out through the abdominal wall or by placing a button into the cecum. The appendix and cecum have significant amounts of gut associated lymphoid tissue (GALT), have high concentrations of microbiota, and serve an essential immune function (Penders, Stobberingh, Van den Brandt, & Thijs, 2007). The appendix has higher concentratio ns of microbial biofilms compared to other areas of the colon, serves as a safe house for symbiotic gut flora, and functions to preserve gut microbiota through re inoculation with normal flora

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195 following gastrointestinal infections (Bazar, Lee, & Yun, 2004; Bollinger, Barbas, Bush, Lin, & Parker, 2007; Gebbers & Laissue, 2004; Kawanishi, 1987; Smith et al., 2009). The human gut contains 10 14 bacteria (Jia, Li, Zhao & Nicholson, 2008). Bacterial composition varies along the bowel axis, with further different iation of luminal or adherent microcolonies that lead to development of biofilms. Factors that influence microbial composition include pH, transit time, bile acids, pancreatic enzymes, mucus composition, nutrient consumption, medication, environment, bacte rial adhesion capacity, and metabolic capacity. The most important function of the gut microbiome is colonization resistance, which is accomplished through competition for nutrients and secretion of bacteriocins (Penders et al., 2007). Microbiota function to degrade dietary substances and enhance digestive efficiency while providing nutrients to the microbes themselves. These microbes are essential for host physiology but, in turn, pose a threat of opportunistic invasion by resident bacteria with resulting pathologies. The immune system maintains a delicate homeostatic and symbiotic balance protecting host microbial ecosystem dualism through the mechanism of stratification and compartmentalization. Stratification minimizes direct contact between the gut mic robiota and the intestinal epithelial surface. Compartmentalization utilizes anatomic adaptation to confine bacteria that breach the mucosal surface to limit systemic immune system exposure. Immune system response to microbiota plays an important role in h ost vulnerability to disease. Host microbial symbiosis and dysbiosis are extraordinarily complex phenomena. The immune system controls the composition, diversity, and location of gut microbiota while the microbiota has a profound effect on lymphoid tissue formation and immune system development (Forchielli & Walker, 2005). Microbiota have been shown to have protective properties against autoimmune disease and, conversely, can cause inflammation and metabolic dysregulation in an immune compromised host (Hoop er, Littman, & Macpherson, 2012; Nicholson et al., 2012). Disruptions in the gut microbiota due to diet, including infant feeding regimens, microbial inoculations, and antibiotics, can alter mucosal immunity and mechanisms involved in regulating immune tol erance outside the GI tract (Noverr & Huffnagle, 2004). Microbiome composition imbalance has been associated with diverse disorders including cancer, inflammatory bowel disease (IBD), atopy, asthma, obesity, and autism (Barker, 2012). The appendix is a sec ondary lymphoid organ that is an important constituent of the mucosa associated lymphoid tissue system; it has a pronounced function in children. Neonatal appendectomy in rabbits impaired mucosal immunity (Dasso & Howell, 1997). Long term effects of append ectomy include moderate immune function changes in part due to a decrease in immunoglobulin production, myocardial infarction (Anderson, Olaison, Tysk, & Ekbom, 2 003; Andreu Ballester et al., 2007; Janszky, Mukamal, Dalman, Hammar, & Ahnve, 2011). ACE therapy administered through the appendix or into the cecum has the potential to disrupt the gut microbial ecosystem causing dysbiosis and immune dysfunction. Effects of appendiceal or cecal administration of ACE on colonic microbiome and mucosal immunity has not been investigated. Synopsis of previous animal and human studies involving USP Glycerin There are no animal studies evaluating instillation of glycerin into the cecum. A composite of animal studies involving alternative routes of glycerin administation are included in Appendix A. There are no prosepective human trials evaluating instillation of glycerin into the cecum. A

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196 composite of human studies involving a lternative routes of glycerin admnistration is included in Appendix B. The Proposed Research as a logical Progression Toward Solving the Problem Attaining continence is a highly socially significant issue for any child but particularly difficult to attai n using conservative measures in children with neuromuscular disorders, anorectal malformations, spinal cord injuries, spinal cord trauma or tumors, megarectum, or slow transit constipation. ACE therapy has been shown to be effective in helping children wi th intractable fecal incontinence attain continence for stool with resulting significant improvement in independence and quality of life. However, research findings regarding short and long term effectiveness are variable (Aspirot, Fernandez, Di Lorenzo, S kaggs, & Mousa, 2009; Bani Hani, Cain, King, & Rink, 2008; Becmeur et al., 2008; Curry, Osborne, & Malone, 1999; Dey et al., 2003; King, Sutcliffe, Southwell, Chait, & Hutson, 2005; Marshall, Hutson, Anticich, & Stanton, 2001; Mousa et al., 2006; Ok & Kurz rock, 2011; Siddiqui, Fishman, Bauer, & Nurko, 2011; Thomas et al., 2006; Yardley et al., 2009). This variability may be due to what is used to flush. There are no prospective comparative studies documenting effectiveness rate, frequency and severity of si de effects, or the time necessary to complete the flushing procedure. Sitting on the commode for one hour versus 20 minutes represents a major difference in age appropriate expectations for a 6 year old child. In a child, procedural time and side effects may be inversely related to adherence and negatively impact effectiveness of the procedure. In addition, there is no information on the effects of ACE flush on bowel health and the microbiome. Currently, the flushing regimen for each child is based on clin ician preference and achieving continence can be a lengthy process of trial and error. This study is the next step in helping children using ACE flushing regimens attain continence while minimizing side effects and procedural time. It has the potential to identify effective dose and frequency and compare side effects of commonly used flushing solutions. Promoting continence at an earlier stage in therapy will decrease costs associated with ineffective trials and additional time spent in protective garments. In addition, this study makes beginning attempts to evaluate the effects of ACE therapy on gut microbiota and colonic health Specific Aims Aims The purpose of this prospective pilot study is to compare two distinct flushing regimens, one high volume sal ine flush and one low volume USP glycerin flush, in the immediate postoperative period in children requiring ACE therapy. Findings from this study will provide a comparative analysis of these two regimens that will serve as a starting point to guide practi ce and serve as a foundation for additional prospective, randomized, controlled trials. The aims of this study are to (1) identify the minimal administration frequency and titration time to reaching effective dose; (2) compare which solution at an optimum dose is delivered in the least amount of time, with fewer side effects, while promoting the higher degree of fecal continence and quality of life; and (3) determine if administration of antegrade enema solution through an appendicostomy/cecostomy affects g ut microbiota and mucosal health.

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197 Hypotheses Null Hypotheses for Aim 1 1. There will be no differences in frequency of administration necessary to gain and maintain continence between two different ACE flushing regimens using normal saline and normal s aline with USP glycerin in children requiring antegrade continence therapy ( primary aim ). 2. There will be no differences in titration time to reach an effective dose between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). Null Hypotheses for Aim 2 1. There will be no difference in degree of continence between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy ( primary aim ). 2. There will be no difference in procedural time between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegra de continence therapy (secondary aim). 3. There will be no difference in side effects between two different ACE flushing regimens using normal saline and normal saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). 4. There will be no difference in parent/patient satisfaction as measured by the Fecal Incontinence and Constipation Quality of Life Measure in Children with Spina Bifida (FIC QOL) between two different ACE flushing regimens using normal saline and norm al saline with USP glycerin in children requiring antegrade continence therapy (secondary aim). Null Hypothesis for Aim 3 1. There will be no difference in the effect of two different ACE flushing regimens using normal saline and normal saline with USP g lycerin on colonic microbiota in children requiring antegrade continence therapy. 2. There will be no difference in the effect of two different ACE flushing regimens using normal saline and normal saline with USP glycerin on gut mucosal health in children requiring antegrade continence therapy Research Plan This prospective study will utilize a repeated measures, single subjects alternating treatments A B C subject acts as his or her own control (Gast, 2010; Janosky, Le ininger, Hoerger, & Libkuman, 2009; Kazdin, 2011; Portney & Watkins, 2009). A within subjects cross over design is Janosky, Leininger, Hoerger, & Libkuman, 2009;Jones & Kenward, 2003) The treatment will be replicated across twelve subjects randomized to six subjects per group. Randomization will be open label. The patient and investigator cannot be blinded to flushing regimens contents due to dosing considerations. The high volume flushing regimen will be comprised of normal saline alone. The low volume regimen will be comprised of USP glycerin with a small volume of normal saline used as diluent. Volume and frequency of administration will be structured to find the low est dosing of each regimen sufficient to maintain continence. Baseline data A will serve as the control and will be obtained pre operatively. Ethics prohibit return to a no treatment

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198 baseline phase as this would result in multiple daily episodes of fecal i ncontinence. The first B C phase of the study will evaluate dose response relationship and will be used to identify the minimum dosing volume and frequency delivered in the least amount of time with fewest side effects while promoting fecal continence of ACE administration for normal saline and normal saline with USP glycerin. When the optimal dose has been identified, the child will continue on that dose for 2 weeks to insure treatment stability and effectiveness. If continence cannot be achieved within the dosing guidelines, the child will be placed on alterntive therapy but will not progress to the maintenance phase of the study. To prevent statistical bias from subject loss due to treatment failure, each child will be randomized to a second treatment s equence once they administration frequency (see Study Schedule, p. 18). In clusion and Exclusion Criteria This study will involve twelve children ages 3 to 12 years recruited from subspecialty clinics at Florida. Children will be selected b y purposive sampling and will include those who are scheduled to have an ACE stoma and will require regular antegrade enema administration to maintain continence. Excluded will be children with preexisting electrolyte imbalance, chronic high rectal tone, q uadriplegia, renal or cardiac disease, or those who require prophylactic antibiotics, cannot communicate, or have significant cognitive delay that would interfere with their ability to fully participate in the study. Parents must have English language comp etency and be willing and able to participate in administration or oversight of the flushing regimen and data collection for a minimum of 20 consecutive weeks. Step by Step Procedure Once parental consent and child assent have been obtained, the child wil l be randomly assigned to either the saline or USP glycerin protocol. The family will be provided with a notebook containing the procedural flushing guidelines, data collection sheets, a stop watch, and flushing solution. Baseline data A will be collected daily for a minimum of two weeks prior to surgery, including frequency and volume of episodes of fecal soiling, and frequency and severity of abdominal pain. Blood samples for electrolyte and stool for calprotectin and microbiota will be obtained in the i mmediate preoperative period. Initial stool specimens for gut microbiota analysis will be collected prior to initiation of any pre operative bowel prep. Per standard of care, a nurse from Nemours Pediatric Surgery will meet with the parent and child in th e immediate postoperative period after surgical clearance has been obtained for flush initiation. During that time, the nurse will review the flush protocol in detail, including materials and procedures. The child will receive the first antegrade infusion during that in patient stay. The nurse will be available if needed for each subsequent flush during the hospitalization. This will allow the family to gain competency in a controlled environment and familiarize themselves with the prescribed protocol and p rocedures prior to transition to the home setting. Data will not be collected during the hospital admission since learning the flushing procedure and post operative abdominal discomfort will pose confounds to measures of flush time and side effetcs. The in vestigator will make a home visit for assistance in administration of the first flush following discharge. During that visit, flushing and data collection procedures will be reviewed. To reiterate, t he first B C phase of the study will evaluate dose resp onse relationship and will be used to identify the optimal dose and frequency of ACE administration for normal saline and normal saline with USP glycerin. When the optimal dose has been identified for each flush

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199 solution, the child will continue on that do se for 2 weeks to insure treatment stability and effectiveness. If continence cannot be achieved within the dosing guidelines, the child will be placed on the alternative regimen but will not advance to the maintenance phse of the study. After completeion of the B C phase, once they have achieved continence on optimal dosing with minimal side effects, the child will be randomized to a second treatment sequence. The second imal dose and administration frequency. After the completion of each phase, the child will come in for a clinic visit. During that visit, the laboratory evalaution will be completed and the new flushing regimen will be reviewed. Documentation will be comp leted by the parent and child on a data collection form and reviewed weekly with the study coordinator to encourage completion of all relevant data. The investigator will keep a research log documenting and detailing any event that may cause a change in l evel, stability, or trend of the dependent variable not related to the intervention, for example treatment with antibiotics or an intercurrent illness. The number of episodes of fecal soiling per week will exclude accidents caused by viral, bacterial, or d rug induced gastroenteritis; these will be recorded and analyzed as confounds (Portney & Watkins, 2009). Interobserver Agreement and Procedural Fidelity The reliability or accuracy and consistency of measurements will be verified using interobserver agreem ent (IOA). Gross method will be used to calculate IOA comparing investigator and parent/child concurrent observations including flush time, procedural time, and number/level of soiling. IOA will be calculated by dividing the smaller number by the larger nu mber and multiplying by 100. If there is a significant discrepancy in observational accuracy, as demonstrated by a calculated IOA below 80%, additional observer training will be provided until the calculated IOA is 80% or higher (Gast, 2010). Procedural re liability will be ascertained for each procedural variable to assure the intervention is being implemented as described in the methods section of the proposal. Procedural fidelity will be calculated by dividing the number of observed behaviors by the numbe r of planned behaviors and dividing by 100 (Gast, 2010). IOA and procedural fidelity for antegrade infusion and data collection techniques conducted by the parent will be checked by the investigator and documented at each in patient visit. A home visit wil l be scheduled by the investigator for initial home infusion. IOA and procedural fidelity will be checked during the first home visit and subsequent clinic visits scheduled with each phase change. Measures operatively. Data at baseline (A) will include continence data, serum electrolytes, and immune markers. The initial stool sample for microbiota analysis will be obtained pre operatively prior to initiation of any bowel prep. Dependent variables at baseline will include: (a) number of episodes of fecal soiling. In addition, fecal soiling will be scored based on frequency and volume of accidents (0 = no soiling, 1 = a smear, 2 = one moderate volume accident that would be insufficient in volume to be visible t hrough clothing if the child wears regular underwear, 3 = any large volume accident that would be visible through clothing if the child wears regular underwear; fecal soiling score is detailed in Table 1, (b) frequency, and severity of abdominal pain recor ded daily and measured using the Wong Baker Faces Pain Rating Scale (WBFPRS) as the age appropriate visual analog scale, (c) serum electrolytes, (d) stool for calprotectin, (e) quality of life measured by the Fecal Incontinence and Constipation Quality of Life Measure in Children with Spinal Bifida (FIC QOL), and (f) stool samples obtained and stored for later microbial DNA analysis using

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200 16SrRNA molecular techniques to identify 16SrRNA gene sequence to identify and quantify phylogenetic groups (Penders et al., 2007). Dependent variables obtained post operatively following initiation of cecosotmy/ appendicostomy flush will include: (a) administration time in minutes per flush, (b) total procedural time from start of flush to completion of colonic emptying in minutes per flush, (c) volume of solution in mL/dose, (d) number of episodes of fecal soiling. In addition, fecal soiling will be scored based on frequency and volume of accidents (0 = no soiling, 1 = smear, 2 = moderate volume accident that would be in sufficient in volume to be visible through clothing if the child wears regular underwear, 3 = any large volume accident that would be visible through clothing if the child wears regular underwear (e) frequency and severity of abdominal pain recorded daily and measured using the WBFPRS as the age appropriate visual analog scale, (f) number and frequency of side effects per week with severity of side effects measured using the WBFPRS as the age appropriate visual analog scale, (g) serum electrolytes, (h) sto ol for calprotectin, (i) quality of life measured by the FIC QOL, and (j) stool samples obtained and stored for later microbial DNA analysis using 16SrRNA molecular techniques to identify 16SrRNA gene sequence to identify and quantify phylogenetic groups ( Penders et al., 2007). Dependent variables, including type of sample or instrument, sample characteristics, and measurement and data level, are explicated in Table 2 Table 1 Level of Soiling 0 No soiling 1* smear 2* moderate volume accident per week that would be insufficient in volume to be visible through clothing if the child wears regular underwear 3* any large volume accident that would be visible through clothing if the child wears regular underwear el 1 Table 2 Dependent Variables Sample/Instrument Variable Measurement Data Level Blood BMP Electrolyte balance Ratio Stool Calprotectin Mucosal inflammation Ratio Stool Colonic microbiome Metagenomic profiling 16SrRNA Ratio FIC QOL Parent/child q uality of life Symptoms rating scale Ordinal WBFPRS Abdominal pain Symptom rating scale Ordinal WBFPRS Procedural side effects Symptom rating scale Ordinal Stop Watch Infusion time Minutes Ratio Stop Watch Procedural time Minutes Ratio Administration time in minutes per flush will be defined as the time at which the tubing connected to the bag or syringe holding the flush solution is unclamped and the cecostomy fluid starts to infuse into the patient to the time the infusion is completed (no more flui d left in the

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201 bag/syringe or tubing). The total procedural time is defined as the time the flush starts to infuse into the subject and ends following passage of stool when the child has sat on the commode for 5 minutes with no additional stool passage. Bot h administration and total procedural times will be measured using duration per occurrence direct observational recording completed by the parent or child. Volume and dose will be recorded with each flush in mL/dose. Accidents will be defined as non toilet elimination, which will be tracked and tallied as the number of pairs of underwear soiled with stool with documentation the severity of the accident and the estimated time of each accident using event recording. Dependent variables will be measured and re corded by the parent using a data collection sheet specifically designed for this study. Side effects will be measured using the Wong Baker FACES Pain Rating Scale (WBFPRS); see Appendix C). The WBFPRS has undergone extensive testing, is preferred by chil dren, and has well established psychometrics in the pediatric population (Tomlinson, von Baeyer, Stinson, & Sung, 2010; Wong & Baker, 1988). The scale ranges from 0 (very happy without pain) to 10 (the worse pain imaginable). Each pain level is associated with a facial expression. The child is asked to choose the face that best describes his/her level of discomfort. The WBFPRS will be used to evaluate the presence and severity of flush side effects, including abdominal cramping, nausea, vomiting, sweating, dizziness, and pallor. The parent will call if the child is having flushing regimen associated accidents or discomfort greater than a 4 on the WBFPRS. Documentation of side effect severity will be completed by the parent and child on a data collection form and reviewed weekly with the study coordinator to encourage completion of all relevant data. The investigator will keep a research log documenting and detailing any event that may cause a change in level, stability, or trend of dependent variables not r elated to the intervention, for example treatment with antibiotics or an intercurrent illness. Those episodes of fecal soiling will exclude accidents caused by viral, bacterial, or drug induced gastroenteritis; these will be recorded and analyzed as confou nds (Portney & Watkins, 2009). The Fecal Incontinence and Constipation Quality of Life Measure in Children with Spinal Bifida (FIC QOL; see Appendix D) will be used to assess child and parental perception of quality of life impact and as an indirect measur e of social validity (Nanigian et al., 2008). The tool will be administered preoperatively during the baseline period and at the end of each flushing regimen in the comparative phase of the study. The FIC QOL is a 51 item questionnaire with established val idity and reliability in families of children with spina bifida who are incontinent for stool. This instrument measures aspects of daily living significantly impacted by fecal incontinence. Of the 51 items, four address subject and family demographics. The remaining 47 items are divided into seven groupings that include bowel program, diet, symptoms, travel and socialization, family relationships, caregiver support and emotional impact, and financial impact (Nanigian et al., 2008; Ok & Kurzrock, 2011). In a ddition to the FIC QOL, a simple qualitative question will be directed to the children at the end of the study to ascertain which flushing regimen they prefer and why. For the high volume flush, the normal saline will be infused using a 1,000 mL enteral fe eding bag with drip chamber and roller clamp. During the infusion, the bag will be hung from a hook located 6 feet above the floor on a wall to the side of or behind the commode. Tubing from the enteral feeding bag will be hooked to the low profile device access tubing and primed with the high volume flush to remove all air in the tube prior to hooking access tubing to the low profile device and infusing the solution. Step by step procedural directions for the parents are located in Appendix E. The instruc tions will be used to reinforce parent teaching and will serve as a check off list to document procedural integrity which will be evaluated during the initial training

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202 session, each additional hospital visit, the initial home visit, and with each subsequen t home visit made with every change in phase. For the low volume flushing regimen, an 8 ounce plastic bottle with a screw top will be used to mix USP glycerin with normal saline. The mixed solution will then be poured into a 60 mL catheter tipped syringe a ttached to the low profile device access tubing. The tubing will be primed prior to hooking it to the low profile device. The child or parent will initially hold the ush infuses. All components of the flush will be at room temperature prior to mixing and infusing the solution. Step by step procedural directions for the parents are listed in Appendix F. For both the high and low voume flush, a stop watch will be used t o measure the time it takes to complete the procedure. The stop watch will be started at the beginning of the infusion, the time in minutes and seconds from start to completion of the infusion will be documented. The stop watch will be stopped when the chi ld feels the flush has been effective and he/she has not passed any additional stool for at least 5 minutes. Total time will be documented on the log. Following the flush, the tubing and bag or syringe will be washed in warm soapy water, rinsed, and allowe d to air dry. Recruitment and Consent Subjects will be recruited from the population of children who have failed to achieve continence for stool using conservative means; are being followed clinically in Nemours Gastroenterology, Urology, Continence, or Su rgery clinics or the community based Pediatric Spinal Defects clinic; and are scheduled for appedicosotmy/cecosotomy. This study confers no greater than minimal risk as categorized by the National Institutes of Health (National Institutes of Health [NIH], 1998). However, it involves vulnerable subjects and will require full Institutional Review Board (IRB) approval with legal guardian informed consent by at least one parent and child assent for children age 7 and above (Knox & Burkhart, 2007; Pieper, 2008) The study has been approved by the Nemours CRRC, but will require approval of the Nemours IRB. Targeted Study Population and Research Setting This study will involve twelve children ages 3 to 12 years recruited from subspecialty clinics at Nemours Chil Florida. Children will be selected by purposive sampling and will include those who are scheduled to have an ACE stoma and will require regular antegrade enema administration to maintain continence. Indentification and Recruitment of Subjects Subjects will be indentified during routine clinic visits in pedatric gastroenterology, general surgery, continence, or spinal defects clinic. When the determination is made that a child is going to have an appedicosotomy/cecosotomy and meets study inclusion criteria, the clinician seeing the family in clinic will apprise them of the study. If they are interested in learning more, Kim Jarczyk will be notified and will meet with the family or contact the family by phone to explain the study in detail. Protection of PHI Consents and patient completed data sheets will be stored in a locked file box housed within a locked cabinet in an office that remains locked when it is not in use. The da ta will be entered in REDCap and identifiable data will be accessible only to Kimberly Jarczyk. Dr. Shuster, Dr. Pieper, and Safety and Monitoring Board members will have access de identified data. A quires dosing adjustiment will be

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203 progress on therapy will be included as a part of the notes associated with each clinic visit following each phase completio n. Any data used in reporting results will be de identified. Consent Process If the family would like to particpate, Kimberly Jarczyk or another of the investigators will attend the family while they are in clinic to review the study protocol and consent. The family will be provided with a copy of the consent to review. All questions will be answered. If the family is still interested in participation, consent and assent for children seven years and older will be obtained prior to enrollment in the study. Subject Number In a single subjects repeated measures design, the strength of the ability to determine treatment effects requires a stable baseline and is a function of the number of baseline and intervention data points (Cook & Campbell, 1979). This stu dy design features a prolonged pre operative baseline assessment and a minimum of 6 weeks of daily measurement for each treatment variable at optimal dosing, meeting criteria for sufficient power analysis. For the cross over design, t here is insufficient e xisting data to conduct a power analysis for sample size estimation. A purpose of this pilot study is to collect data that will allow for power analysis and optimal sample size determination. Description of Drugs Treatment, Intervention and Observation Hig h Volume Regimen The high volume regimen (B) consists of a normal saline flush at a starting dose of 10mL/kg infused every other day and be adjusted until stability of target outcomes is achieved. The overall goal is to maximize dose at minimum frequency with minimal side effects. If the child is continent on an every other day dose, the administration frequency will be dropped to every third day. If they have breakthrough incontinence on an every third day dosing schedule, the frequency will be increased back to every other day and if needed to every day. At any point the subject is having episodes of fecal, the dosing strategy will be increased by 5 10 mL/kg increments starting at an administration frequency of every other day with a subsequent increase in frequency, if needed, so as not to exceed 20 mL/kg and a maximum dose of 500mL a day for children under 5 years of age and 1000 mL for children over 5 years of age administered daily. If the child does not attain continence on the maximum dose, he/she will be placed on the alternative flush but will not progress to the maintenance phase of the study. If the child is having side effects greater than WBFPRS level 4 at the starting dose of 10mL/kg, flush volume will be incrementally decreased as needed by 2.5 mL/kg to the lowest dose of 5 mL/kg daily. The goal is to find the lowest effective dose and flushing frequency with minimal side effects. If the dose necessary to minimize effects results in episodes of fecal soiling greater than one smear per week or the child continues to have side effects greater than WBFPRS level 4 at the lowest dose of administration, the child will be placed on the alternate flushing regimen but will not progress to the maintenance phase of the study. The decision tree for dose a djustment of Normal Saline is detailed in Tables 3 and 4 Treatment, Intervention and Observation Low Volume Regimen The low volume regimen (C) will consist of USP glycerin diluted in normal saline prior to antegrade instillation through the low profile de vice. The child will start on an every other day dose of 20 mL of USP glycerin and >20 mL of saline (used as diluent at a dose sufficient to allow the solution to easily infuse through the ACE access tubing) and be adjusted until stability of target outcom e is achieved. The families will record the amount of saline used as diluent. The

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204 overall goal is to maximize dose at minimum frequency with minimal side effects. If the child is continent on an every other day dose, the administration frequency will be dr opped to every third day. If they have breakthrough incontinence on an every third day dosing schedule, the frequency will be increased back to every other day and if needed to every day. At any point the child is having episodes of fecal soiling, the vol ume of USP glycerin will be increased in 5 mL increments starting at an administration frequency of every other day with subsequent increase in frequency, if needed, so as not to exceed 4 0 mL of USP glycerin administered daily. If the child does not attai n continence on the maximum dose of USP glycerin, he/she will be placed on the aslternative flushing regimen but will not advance to the maintenance phase of the study. If the subject is having side effects greater than WBFPRS level 4 at the starting dose of 20mL of USP glycerin, the volume of the USP glycerin will be decreased as needed by 5 mL increments daily is reached. The goal is to find the lowest effectiv e dose and flushing frequency with minimal side effects. If the dose necessary to minimize effects results in episodes of fecal soiling greater than one smear per week or the child continues to have side effects greater than WBFPRS level 4 at the lowest do se of administration, the child will be placed on the alternaitve flushing regimen abut will not advance ot the maintenance phase of the study. The decision tree for dose adjustment of USP glycerin is detailed in Tables 3 and Table 4. Table 3 Dosing St rategy to Determine Minimum Volume and Frequency Necessary to Maintain Continence in the Absence of Side Effects Normal Saline (High volume regimen with maximum dose for less than 5 and over 5 yrs.) USP Glycerin + Normal Saline as Diluent (Low volume regi men Max dose for children 5 and under is 30 mL + > 30 mL qd) B1 = 10mL/kg maximum dose 500 or 1000 mL qod C1 = 20 mL + > 20 mL qod B2 = 10mL/kg maximum dose 500 or 1000 mL q3d C2 = 20 mL + > 20 mL q3d B3 = 10mL/kg maximum dose 500 or 1000 mL qd B4 = 15 mL/kg maximum dose 500 or 1000 mL qod B5 = 15 mL/kg maximum dose 500 or 1000 mL q3d B6 = 15 mL/kg maximum dose 500 or 1000 mL qd C3 = 20 mL + > 20 mL qd C4 = 25 mL + > 30 mL qod C5 = 25 mL + > 30 mL q3d C6 = 25 mL + > 30 mL qd C7 = 30 mL + > 40 mL qod C8 = 30 mL + > 40 mL q3d C9 = 30 mL + > 40 mL qd C10 = 35 mL + > 50 mL qod C11 = 35 mL + > 50 mL q3d C12 = 35 mL + > 50 mL qd

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205 Table 4 Dosing Strategy to Determine Minimum Volume and Frequency Necessary to Minimize Side Effects and Maintain Continence Normal Saline* (High volume regimen with maximum dose for less than 5 and over 5 yrs.) USP Glycerin + NS as Diluent* (Low volume regimen) B1 = 10 mL/kg maximum dose 500 or 1000 mL qod C1 = 20 mL + > 20 mL qod B7 = 7.5 mL/kg maximum dose 500 or 1000 mL qod C10 = 15 mL + > 15 mL qod B8 = 7.5 mL/kg maximum dose 500 or 1 000 mL q3d C11 = 15 mL + > 15 mL q3d B9 = 7.5 mL/kg maximum dose 500 or 1000 mL qd C12 = 15 mL + > 15 mL qd B10 = 5.0 mL/kg maximum dose 500 or 1000 mL qod C13 = 10 mL + > 10 mL qod B11 = 5.0 mL/kg maximum dose 500 or 1000 mL q3d C14 = 10 mL + > 10 mL q 3d B12 = 5.0 mL/kg maximum dose 500 or 1000 mL qd C15 = 10 mL + > 10 mL qd C16 = C17 = C18 = 5 mL + > 5 mL qod. 5 mL + > 5 mL q3d 5 mL + > 5 mL qd Optimal Dose Regimen Once the optimal dose has been established, the child will be maintained on that dose and frequency for at least 2 weeks or until stability in dependent measures without significant variability or trend is achieved. The child will be schedule d to come into the clinic for a visit once the above criteria have been met, at which time labs will be drawn and a stool sample collected. The volume of solution in mL/ dose will be recorded for each treatment phase. Comparative Phase Following completion of the dose response phase, the comparative portion of the study will to withdrawn and the initial flush in the comparative treatment phase will be introduced the following day at the previously established minimum effective dose and frequency. Children will remain on treatment for 4 weeks, at which point the treatment will be withdrawn. Children will then be placed on the next treatment in the sequence at the pre established effective dose and frequency for 4 weeks. The second fl ush will then be withdrawn and the initial flush in the sequence will be reintroduced for an additional 2 weeks (B study, the child will be placed on the flushing regimen of his/her choice. Procurement, Storage, and Management of Research Pharmaceutical Products Both normal saline (NDC# 00338 0049 04, 0.9% Sodium Chloride Irrigatio n, USP 500mL, Baxter brand) and USP glycerin (HM Glycerin USP, 16 ounces, NDC# is 62011 0115 01, HealthMark brand) are non controlled and are available over the counter. Both solutions will be purchased from the Pavilion Pharmacy in the Pavilion Building at Baptist Medical Center on 836 Prudential Drive, STE 110 in Jacksonville, Florida. Participants will not be charged for either solution. The normal saline and USP glycerin will be dispensed in their original containers. Because the route of administrat ion constitutes an off label use of both medications, the containers will be labeled using regulation compliant medication labels. Only a PI or Co Investigator will handle study drug. The PI will train those Co Investigators designated in the IRB proposal in the proper storage, labeling, and dispensing of study drug. The study drug will be stored at ambient temperature (15 to 30 degrees Centigrade or 59 to 86 degrees Fahrenheit

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206 with protection from moisture and light). The drug will be stored in a locked ca binet in the medication room in the Continence Clinic on the 3 rd a separate cabinet from other drugs that are stocked i n the clinic for patient care use. The cabinet is locked with restricted key access. The temperature in the cabinet in which the drug is stored will be monitored on a continuous basis using a MCC USB 501 LCD thermometer which allows for continuous monitori ng, storage, and downloading of temperature data. Staff will monitor the device daily. Following weekends or holidays, the stored temperature data will be reviewed. Any temperature excursions will be discussed with a pharmacist prior to dispensing the stor ed flush solution. A daily hard copy temperature log will be kept. If a significant variance from the acceptable temperature range is found, the principal investigator will be notified, who will contact the pharmacist for instructions before any of the s tored drug is dispensed. Shipment receipt and product inventory including lot # and expiration dates will be stored in the regulatory binder with a separate log maintained for participant product accountability. Both normal saline and USP glycerin are non hazardous. The volume and frequency of administration in this proposal does not substantially alter or increase the concentration or distribution of the substance, metabolites, or products of degradation in the environment. To the best of the s knowledge, no extraordinary circumstances exist with the proposed use of saline and glycerin that would adversely affect the quality of the human environment. Excess solution will be disposed of by the family through regular household plumbing. Nature of Experimental Control Any research design is a tool used to answer a question. Strategies, design choice, and use of design elements should be based on how best to answer the question at hand (Kazdin, 2011). The purpose of experimental design is to contro l the effects of random error and bias (Piantadosi, 2005). However, no design completely eliminates either. Whatever the chosen research method, good design and procedures are necessary to prevent confounding and improve causal inference (Shadish et al., 2 002). Skillful statistical analysis cannot salvage significant design faults or increase the validity of a poorly designed study (Janosky et al., 2009). Skillful utilization of design elements can increase internal validity and causal inference (Cook & Cam pbell, 1979). Both single subject and between group research make and test predictions about treatment effects, the first by evaluating treatment effects on an individual, the second by addressing group mean and variance (Kazdin, 2011). Single subjects an d group designs, in their most rigorous form, rule out or make implausible rival hypotheses for the experimental outcome improving quality of inference. (Cook & Campbell, 1979; Kazdin, 2011; Shadish et al., 2002, Cook, & Campbell, 2002). A randomized cont rolled trial (RCT) is considered the gold standard for intervention research (Piantadosi, 2005). However, a RCT is not the only standard for causal inference (Kazdin, 2011). Reliance on large numbers makes application of a RCT with small groups or rare dis eases problematic (Janosky et al., 2009). The focus of my research involves instillation of a solution through an appediceal stoma, aprocedure used for over a century and widely popularized over 20 years ago. Case reports and retrospective studies detail widely divergent effectiveness rates (Bani Hani, Cain, King, & Rink, 2008; Dey et al., 2003; Mousa et al., 2006; Siddiqui, Fishman, Bauer, & Nurko, 2011: Yardley et al., 2009). The literature and involved clinicians have identified the need for prospective trials comparing ACE flushing regimens. None have been undertaken to date. This is in large part because the small size and heterogeneity of this population does not lend itself to a large N study.

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207 Many clinical questions go unanswered due to over relianc e on RCT large N methodology (Kazdin, 2011). This population is an exemplar of that problem. The proposed study comparing two flushing regimens utilizes a cross over design embedded in a single subject A B C and lay a foundation for causal inference (Chow & Liu, 2014; Elder, 1997; Portney & Watkins, 2009). In both designs the subject acts as his or her own control, minimizing within subject variability and ensuring the highest possible degree of equivalence a cross treatment conditions, thereby,allowing greater precision and efficient estimates of treatment effects, increasing internal validity and causal inference (Janosky et al., 2009; Piantadosi, 2005; Portney & Watkins, 2009). In both methods, subjects are randomized to treatment sequence. Randomization will decrease the threat of order effects in both methods, increase group equivalency in the cross over design, and minimize variability in measurement due to subject or period differences, increasing interna l validity and causal inference (Chow & Liu, 2014; Jones & Kenward, 2003). Single subjects design is an inductive, experimental methodology with controlled introduction and manipulation of an independent variable. Single subjects design promotes explorati on of inter subject variability without the introduction of error inherent in group methodology in the absence of subject homogeneity. It allows for isolation of individual response to interventions and identification of valuable information from outliers that would be obscured or lost in group methodology. Single subjects design also allows for time series observations of response, providing continuous and often a more accurate representation of the dependent variable of interest that may be compromised wh en the data is collected as an isolated snapshot in group methods (Elder, 1997). This study is ideally suited to single subject repeated measures design because children requiring an ACE procedure comprise a very small population with widely disparate anat omic and physiologic causative factors, making sample homogeneity difficult. Inclusion of heterogeneous subjects will allow differentiation of subject characteristics that impact response to treatment. The design allows for frequency, volume, and dose adju stment of each flushing regimen when indicated. The ability to adjust the treatment regimen facilitates dose response comparison and will aid in identifying which flushing regimen requires the minimal dose and administration frequency, is accomplished in t he least amount of time, and has the fewest side effects while achieving continence. Because this design allows for repeated measurement over time, it is particularly helpful when studying comparisons between several treatments and is more sensitive to var iations in treatment response that might otherwise be missed using group methodology (Gast, 2010; Janosky et al., 2009; Kazdin, 2011). Subjects will be limited to children who are scheduled for a cecostomy or appendicostomy, ensuring their gut is nave to the effects of a flushing regimen and allowing a true no treatment baseline. Flush effects are reversible, making this intervention amenable to a withdrawal design. There are no known carry over effects associated with either flushing regimen that would i mpact physiologic baseline between treatments. Specimen collection facilitating comparison of treatment effects on gut microbiota, electrolytes, and stool cal protectin occurs at the completion of each flushing regimen, negating any carry over effects. Given the pragmatic issues involved in answering the research question at hand, the chosen methods and design elements strengthen demonstration of the counterfact ual and make implausible potential threats to validity, lending credence to the assumption that intervention effects are due to the treatment and not random error or bias.

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208 Potential threats to statistical validity are detailed in Appendix G. Potential thre ats to internal vailidity are detailed in Appendix H. Potential social threats to internal validity are detailed in Appendix I. Randomization Procedures Subjects will be randomly assigned to one of two treatment sequences to control for the possibility o f order effects. To prevent statistical bias from subject loss due to treatment failure, each child will be randomized to a second treatment sequence once they have achieved continence on optimal dosing with minimal side effects. The process will be restri cted random assignment to force equal sample size and will be accomplished using the SAS random number generator. Physical Examinations, Laboratory Evaluation, Radiographic Evaluation, Special Test Procedures, Surveys, Questionaires A history and physical examination will be completed at each clinic visit. Stool samples will be obtained during the baseline period prior to any pre operative bowel prep; additional stool samples will be obtained at the completion of each phase. Blood samples for electrolytes and immune markers will be drawn prior to surgery and, subsequently, following surgery after completion of B and C phases. Blood samples and stool for calprotectin will be sent for analysis; stool samples for microbiota analysis will be labeled with the p atient identifier, date, time, and flush composition. Each stool sample for microbiota analysis will be processed by the investigator with oversight by Karl Mann and Dr. Sylvester to purify and extract microbial DNA for sequencing, flash frozen to 80 o C, and archived for downstream batch analysis using 16SrRNA as a taxonomic marker (Barker, 2012). Responsible Party and Setting for Obtaining Information The families will be responsible for keeping a log both pre and post operatively. Kim Jarczyk will be re sponsible for collecting and entering the data in to REDCap for future analysis. Data will collected in the clinic and home settings. How Will Activities Answer the Study Questions T he B C/C B phase of the study will evaluate the dose response relationshi p and will be used to identify the optimal dose and frequency of ACE administration for normal saline and normal saline with USP glycerin. at optimal dose and administration frequency. The Basic Metabolic Panel will evalute flush effects on electolyte balance. Stool calprotectin will be used as a gut inflammatory marker. Molecular techniques for identification of 16SrRNA gene sequence in stool samples will be used to identify and quantify microbiome phylogenetic groups. Severity of side effects will be measured using the WBFPRS as the age appropri ate visual analog scale. The FIC QOL will be used to assess child and parental perception of quality of life impact and as an indirect measure of social validity. A simple qualitative question will be addressed to the child upon study completion to determi n

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209 Study Schedule Overview of Timeline Baseline Dose Response Phase Flush Effectiveness Phase Weeks: 1 2 3 4 5 6 7 8 9 10+ 11 12 13 14 15 16 17 18 19 20 Randomize X X Order: A NT NT B C B B B B C C C C C B C C C C B B B B B B 1 B B B B C C C C B 1 B 1 C C 1 C C C C B B B B C 1 C 1 Visits: Hospital X Home X Clinic X X X X X Biomarker Stool X X X X X BMP X X X SIM X X X Instrument FIC QOL X X X QQ X Measures: Soiling X X X X X X X X X X X X X X X X X X X X Abd Pain X X X X X X X X X X X X X X X X X X Admin T X X X X X X X X X X X X X X X X X X Proc T X X X X X X X X X X X X X X X X X X ISE X X X X X X X X X X X X X X X X X X Cost X X X X X X X X X X X X X X X X X X X X A Baseline NT No treatment B Saline dose response phase C USP Glycerin dose response phase Initial trial of saline effectiveness phase Initial trial of USP glycerin effectiveness phase B 1 Second trial of saline effectiveness phase C 1 Second Trial of USP g lycerin effectiveness phase Stool Metagenomic Profiling 16SrRNA (collected and batched for downstream analysis) BMP Basic Metabolic Profile SIM Stool Inflammatory Marker (Calprotectin) Visits Procedural fidelity and inter rater reliability will be measured at each visit FIC QOL Fecal Incontinence and Constipation Quality of Life Measure in Children with Spina Bifida QQ Qualitative question asked of the child upon study completion Abd Pain Abdominal Pain Admin T Administration time Proc T P rocedural time ISE Infusion side effects

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210 Procedures to Protect Privacy Consents and patient completed data sheets will be stored in a locked file box housed within a locked cabinet in an office that remains locked when it is not in use. The data will be entered in REDCap and accessible only to Kimberly Jarczyk. Pam Pieper and Jonathan Shuster will have access to deidentified data in REDCap. Safety and Monitoring Board members will have access only to de identified data. Any data used in reporting res ults will be de identified. protected electronic medical record. A summary will be included in the notes associ ated with each clinic visit following each phase How research interventions differ from standard therapies and alternatives if they exist This research study in cludes clinical treatments and procedures that would be required during normal clinical care for children who require an appendicostomy or cecostomy for treatment of fecal incontinence. The protocol will compare two common ACE flushing solutions that have been used clinically for many years, but have not been studied. A child undergoing appendicostomy/cecostomy would be placed on an ACE flush, most likely using one of the two solutions used in this study. Dose and frequency would be adjusted in much the sa me manner it would be in this study. Children who were not enrolled in the study would not have blood drawn for a Basic Metabolic Panel or stool samples collected for calprotectin and storage for later microbial DNA analysis using 16SrRNA. Families would n ot complete a quality of life survey and children would not be asked to answer an open ended question regarding flushing preference. Statistical Analysis Describe Statistical Analysis The strength of an interrupted time series design is that effects of t he intervention under investigation can be repeatedly and reliably measured over time. Time series parameters include mean intercept level, slope, and additional non linear changes in shape. If the treatment is effective, it will result in a change in para meters that reverse with treatment withdrawal. Independent variable impact on time series parameters is based on the degree of change elicited in level, slope, or cycle of the measures process. The ability to determine independent variable effects is a fun ction of a stable baseline and the number of baseline, intervention, and post intervention data points (Biglan, Ary, & Wagenaar, 2000; Janosky, Leininger, Hoerger, & Libkuman, 2009; Shadish, Cook, & Campbell, 2002). In this study, each subject will have a minimum of 2 weeks of data collection pre operatively, allowing for establishment of a stable baseline. Postoperativey, after titration, each regimen will be administered at optimum dosing for a minimum of 2 weeks at the end of the titration phase and 4 we eks during the comparative phase, for a total of 6 weeks per flush regimen at optimal dosing, yielding a total of at least 12 weeks of observations at the therapuetic dose and frequency threshold per subject. Data will be obtained and graphed at the time of each flush to facilitate analysis and dose response adjustment during the B C or C B phase, when indicated. Data will be graphed on an equal interval line graph with the proportion of ordinate and abscissa scaled at a 2:3 ratio to ensure consistency of data presentation and prevent data distortion during visual analysis. Dependent measures will be placed on the ordinate scale with time by day on the abscissa scale.

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211 Separation of dose response and comparative phases of the study will be designated by a b old vertical line and high and low dose regimen changes by a thin vertical line. Each phase change will be labeled with solution name, dose, and frequency. Independent variable effect on target behaviors will be analyzed using visual analysis. The indepe ndent variable is nominal and dichotomous. Dependent variables are comprised of either interval or ratio level measurements. The comparative phase of the study is a two treatment crossover design with each child receiving both treatments. Each child will b e outcomes of interest, including number of soiling episodes, level of soiling, abdominal pain, procedural side effects, infusion time, procedural time, electrolyte balance, fecal calprotectin, and quality of life will be analyzed using inferential statistics. Data Analyses Analysis within each condition will include: (a) con dition length, defined as the number of data points contained within each phase; (b) level stability with a stability envelope calculated using the median and a stability criterion of 80% of the data points falling within 15% of the calculated median for t he phase; (c) relative change in level; (d) absolute change in level; (e) estimation of trend direction using split middle method; (f) trend stability with a stability envelope using the same criteria as level stability; and (g) identification of multiple paths within trends, if present. Analysis between conditions will include: (a) number of variables that have changed between adjacent conditions, (b) change in trend direction between conditions, (c) assessment if trend change is in keeping with interventi on goals, (d) assessment for change in trend stability, (e) assessment of immediacy of effect in change in level and trend, (f) calculation of absolute and median level change, (g) calculation of percentage of non overlapping and overlapping data points, a nd (h) percentage of data points exceeding the median (Gast, 2010; Hartmann et al., 1980; Ma, 2006; Kazdin, 2011; McDowall, McCleary, Meidinger, & Hay, 1980; Portney & Watkins, 2009). Testing in multiple subjects will allow for analysis of replication of t reatment effects. Analysis will include between series strategies comparing data points, including frequency, mean occurrence, and immediacy and magnitude of effect within and between treatment conditions. In addition to visual analysis of time series para meters, inferential procedures will be used to compare interventional effects and increase the reliability of visual methods analysis. Inferentail analysis will be accomplished using a two tailed, two sample pooled variance t test with a significance leve l set at 0.05. Confidence intervals will be calculated to provide precision of mean differences estimates (Polit, 2010). The two sample t test will be used to test treatment difference in the cross over design. Because the design is comparing two treatment sequences, groups are independent (Chow & Liu, 2014). Confounding by carry over and direct by period interaction is a potential with cross over designs, which if present, can bias treatment effects (Jones & Kenward, 2003; Senn, 2002; Shuster, 2007). Jones et. al. (2003) and Sen (2002) suggest use of a one sample t test for analysis of cross over designs (treatment one is subtracted from treatment two). Shuster (2007) advocates a two sample t test in the analysis of a randomized two treatment cross over des ign (period two is subtracted from period one irrespective of treatment order). Analysis of a one sample t test in a cross over design ignores treatment ordering. Two sample t test analysis compares ordering and yields potentially useful data on carry over sample or two sample t test will yield unbiased estimates of and variance when the sample size

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212 ther the one or two sample method. However, the one sample method does not account for carry over effects, estimates in the one, but not the two, sample t te st will be biased. Using the two sample method will lend precision in the presence of carry over effects, and precision and accuracy in the described, when appropr iate, using frequency distribution and graphed using either histograms or pie charts. Changes in gut microbiota will be analyzed using descriptive statistics (Polit, 2010). Neither visual analysis nor inferential statistics alone will infer causality. Stri ctly speaking, correlation does not infer causation (Polit, 2010). Causal inference is primarily influenced by the research design. Visual analysis and statistical procedures are helpful in measuring the effects of potential causes (Kazdin, 2011). Appropri ate use of statistics aid in the inference of causality by quantifying the effect chance plays on conclusions (Hill, 1965). The proposed study is prospective allowing for the determination of temporal precedence. Statistical and visual analysis will be use d to assess contiguity with respect to presumed cause and effect. Strategic use of design elements limits alternative explanations for findings. The first phase of the proposed study evaluates dose response, which will assess biological gradient. The desig n assesses treatment response across multiple subjects, which may provide support for consistency across contexts. Stopping Rules The parent will call if the child is having accidents or discomfort greater than WBFPRS level 4 associated with the flushin g regimen and the regimen will be changed. The child will be put on the alternate flushing regimen if the child has discomfort greated than WBFPRS level 4, cannot achieve and maintain continence, or has a clinically significant electrolyte imbalance or cal protectin elevation. Data and Safety Monitoring Plan This study has a Data and Safety Monitoring Board (DSMB). Although this is a low risk study and, based on previous studies, we do not anticipate any serious adverse effects for USP glycerin or normal sal ine at the doses we plan to use, as suggested by the IRB, we have established an expert and independent three member DSMB. Kathryn Blake, Pharm D, Principal Research Scientist, Center for Pharmacogenomics and Translational Research at Nemours Children's Sp ecialty Care in Jacksonville, Florida; Laurie Duckworth, PhD, ARNP, Clinical Associate Professor, Director of Clinical Research, University of Florida College of Nursing; and Salik Taufiq, MD, Division of Pediatric Gastroenterology and Nutrition at Nemours Specialty Care in Jacksonville, Florida have agreed to be members of this expert panel. All are Nemours or University of Florida employees, are exceptionally qualified to serve on this expert panel, and are not otherwise involved in this study. Letters from these members agreeing to be part of the DSMB expert panel are located in Appendices K, L, and M, respectively. The DSMB will assess the safety and efficacy of study procedures, and monitor the overall conduct of the study. De identified gene rated data will be made available to the panel. We will report our progress to the DSMB every 6 months. Any adverse events, abnormal laboratory results, or problems that arise during the conduct of this study will be reported at the time of the occurrence. The study may be modified or stopped based on its assessment of the participant safety needs. The members of the DSMB will review the study adverse event experience, and provide written reports to us following each scheduled DSMB review. Periodic DSMB rep orts will be forwarded to the IRB as the need arises.

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213 Possible Discomforts and Risks Risks This study does not entail any additional physical, physiological, or social risks over standard of care therapy. Discomforts ACE flush can be associated with elec trolyte imbalance, abdominal cramping or nausea. A dditional discomforts above those often experienced during standard care include the discomfort associated with venipunctures to obtain BMP levels. These will be collected three times over the course of the study. Economic Risks This study does not entail additional economic risks to the participant above what would be encountered during standard of care treatment. The participant or his/her insurer will pay for regularly scheduled clinic visits and supplie s associated with the ACE flush. These are considered standard therapy. Procedures to Minimize Discomforts and Risks Electrolytes will be drawn to monitor for electrolyte imbalance. If a participant experiences any side effects from an ACE flushing soluti on, the dose will be adjusted. If side effects do not resolve on a lower dose, the participant will not continue in the study and will be placed on an alternative ACE flush solution. Children will be supplied with topical Lidocaine+Prilocaine to decrease t he discomfort associated with venipuncture. C ompensation to participants will include (a) costs of supplies for stool collection and calprotectin analysis, (b) laboratory and basic metabolic panel (BMP) analysis fees, (c) costs of topical Lidocaine+Priloc aine and tegaderm used to minimize discomfort during venipuncture, (d) and $25 stipend for each scheduled study visit (maximum total of $125). Adolescent participants will receive a $5 stipend for each scheduled study visit (maximum total of $25). Risk Ben efit Analysis Fecal incontinence in children past the expected time of toilet training has been associated with poor outcomes including increased anxiety and depression, more social problems, worse school performance, and an increased incidence of abuse an d bullying (Kaugars et al., 2010; Youssef, Langseder, Verga, Mones, & Rosh, 2005). Helping children achieve continence can be life altering. Fecal incontinence is particularly difficult to manage using conservative measures in children with neuromuscular d isorders, anorectal malformations, spinal cord injuries, spinal cord trauma or tumors, megarectum, or slow transit constipation. The ACE procedure was popularized over 20 years ago as a means of helping children with intractable fecal incontinence attain s tool continence. A large body of literature demonstrates ACE therapy can be effective in helping children with intractable fecal incontinence attain continence for stool with resulting significant improvement in quality of life. However, findings regarding effectiveness are highly variable. This variability may be due to the flushing regimen utilized. No prospective trials compare the effectiveness and adverse effects of different flushing regimens. No studies evaluate the effects of appendicosomty or cecos tomy flush on gut microbiota. Currently, identifying a successful flushing regimen is determined by individual clinician preference and often requires multiple attempts before success is achieved. There are no prospective studies comparing the effectivenes s of type, dose/volume, or frequency of different flushing regimens in preventing incontinence to inform practice. This study involves minimal risk above standard therapy and has

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214 the potential to benefit participants as well as contribute to our understand ing of the effectiveness of both flushing regimens, benefiting future children requiring ACE therapy. Justification for Conducting the Study The proposed research is significant in that it is the first prospective study to compare the effectiveness and tol erability of two commonly used ACE flushing regimens and the first study to explore ACE flushing impact on gut microbiome. Risks associated with this study are low and do not constitute a substantial threat above risk exposure in standard of care therapies Findings from this study have the potential to provide both clinical and biological insights into ACE administration safety. They will provide a foundation of scientific evidence to serve as a starting point to guide practice and for additional prospecti ve, randomized, controlled trails. Benefits to Subjects Participants may or may not personally benefit from taking part in this study. However, each child will be closely monitored throughout the study and his/her treatment will be adjusted to find the bes t possible volume of flush that works in the least amount of time with the fewest side effects. Benefits to Future Populations Information from this study will help us start to understand how the two ACE flushing regimens compare with regard to side effec ts and effectiveness. A better understanding will eventually lead to better ACE treatment outcomes for other children who require ACE flushing to gain and maintain continence for stool. Conflict of Interest There are no real or potential conflicts of int erest for any investigator in this study. None of the investigators hold a patent or license pending for any of the study medications, materials, or processes utilized in this study nor is there an intent to file a patent application at a later date. None of the investigators hold stock, give presentations, or serve as a consultant for any company that produces the drugs or materials used in this study.

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219 References Aksnes, G., Diseth, T. H., Helseth, A., Edwin, B., Strange, M., Aafos, G. & Emblem, R. (2002). Appendicostomy for antegrade enema: Effects on somatic and psychosocial functioning in children with myelomeningocele. Pediatrics 109 484 489. doi:10.1542/peds.109.3.484 Anderson, R. E., Olaison, G., Tysk, C., & Ekbom, A. (2003). A ppendectomy is followed by Gastroenterology 124 40 46. doi:10.1053/gast.2003.50021 Andreu Ballester, J. C., Perez Griera, J., Ballester, F., Colomer Rubio, E., Ortiz Tarin, I., & Penarroja, O. C. (2007). Secretory immun oglobulin A (sig A) deficiency in serum of patients with GALTectomy (appendectomy and tonsillectomy). Clinical Immunology 123 289 297. doi:10.1016/j.clin.2007.02.004. Aspirot, A., Fernandez, S., Di Lorenzo, C., Skaggs, B., & Mousa, H. (2009). Antegrade e nemas for defecation disorders: Do they improve the colonic motility? Journal of Pediatric Surgery 44 1575 1580. doi:10.1016/j.jpedsurg.2008.11.061 Bani Hani, A. H., Cain, M. P., King, S., & Rink, R. C. (2008). Tap water irrigation and additives to optim ize success with the Malone antegrade continence enema: The Indiana University algorithm. Journal of Urology 180 1757 1760.doi:10.1016/j.juro.2008.04.074 Barker, M. (2012). Taking stock of the human microbiome and disease. Science 336 1246 1247. Bazar, sensory organ of the immune intelligence network. Medical Hypothesis 63 752 758. doi:10.1016/j.mehy.2004.04.008 Becmeur, F., Demarche, M., Lacreuse, I., Molinaro, F. Kauffmann, I., Moog, R.,...Rebeuh, J. (2008). Cecostomy button for antegrade enemas: Survey of 29 patients. Journal of Pediatric Surgery 43 1853 1857. Biglan, A., Ary, D., & Wagenaar, A. C. (2000). The value of interrupted time series experiments for c ommunity research. Prevention Science 1, 31 49. Bollinger, R. R., Barbas, A. S., Bush, E. L., Lin, S. S., & Parker, W. (2007). Biofilms in the large bowel suggest an apparent function of the human veriform appendix. Journal of Theoretical Biology 249 82 6 831. doi:10.1016/j.jtbi.2007.08.032 Chertow, G. M., & Brady, H. R. (1994). Hyponatraemia from tap water enema. Lancet 344 748. Chow, S., & Liu, J. (2014). Design and analysis of clinical trials: Concepts and methodologies (3rd ed.). Hoboken, NJ: Wiley. Cook, T. D., & Campbell, D. T. (1979). Quasi experimentation: Design and analysis issues for field settings Boston, MA: Houghton Mifflin. Curry, J. I., Osborne, A., & Malone, P. S. (1999). The MACE procedure: Experience in the United Kingdom. Journal of Pediatric Surgery 34 338 340. Dasso, J. F., & Howell, M. D. (1997). Neonatal appendectomy impairs mucosal immunity in rabbits. Cellular Immunology 182 29 37. R. R. (2003). After the honeymoon Medium term outcome of antegrade continence enema procedure. Journal of Pediatric Surgery 38 65 68. doi:10.1053/jpsu.2003.50012

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220 Elder, J. H. (1997). Single subject experimentation for psychiatric nursing. Archives of P sychiatric Nursing 11 133 138. Forchielli, M. L., & Walker, W. A. (2005). The role of gut associated lymphoid tissues and mucosal defense. British Journal of Nutrition 93 S41 S48. doi:10.1079/BJN20041356 Gast, D. L. (2010). Single subjects research met hodology in behavioral sciences New York, NY: Routledge, Taylor & Francis Group. Gebbers, J., & Laissue, J. (2004). Bacterial translocation in the normal human appendix parallels the development of the local immune system. Annals New York Academy of Scien ces 1029 337 343. doi:10.1196/annals.1309.015 Gomez, R., Mousa, H., Liem, O., Hayes, J., & Di Lorenzo, C. (2010). How do antegrade enemas work? Colonic motility in response to administration of normal saline solution into the proximal colon. Journal of P ediatric Gastroenterology and Nutrition 51, 741 746. doi:10.1097/MPG.0b013e318e75d18 Hartmann, D. P., Gottman, J. M., Jones, R. R., Gardner, W., Kazdin, A. E., & Vaught, R. S. (1980). Interrupted time series analysis and its application to behavioral data Journal of Applied Behavior Analysis 13 543 559. Helikson, M. A., Parham, W. A., & Tobias, J. D. (1997). Hypocalcemia and hyperphosphatemia after phosphate enema use in a child. Journal of Pediatric Surgery 32 1244 1246. Hooper, L. V., Littman, D. R. & Macpherson, A. J. (2012). Interactions between the microbiota and the immune system. Science 336 1268 1273. doi:10.1126/science.1223490 Ismail, E. A., Al Mutairi, G., & Al Anzy, H. (2000). A fatal small dose of phosphate enema in a young child with n o renal or gastrointestinal abnormality. Journal of Pediatric Gastroenterology and Nutrition 30 220 221. Janosky, J. E., Leininger, S. L., Hoerger, M. P., & Libkuman, T. M. (2009). Single subject designs in biomedicine New York, NY: Springer Science+Bus iness Media. Janszky, I., Mukamal, K. J., Dalman, C., Hammar, N., & Ahnve, S. (2011). Childhood appendectomy, tonsillectomy, and risk for premature acute myocardial infarction A nationwide population based cohort study. European Heart Journal 32 2290 2296. doi:10.1093/eurheartj/ehr137 Jai, W., Li, H., Zhao, L., & Nicholson, J. (2008). Gut microbiota: A potential new territory for drug targeting. Nature Reviews 7, 123 129. Jones, B., & Kenward, M. G. (2003). Design and analysis of cross over trials (2n d ed.). Boca Raton, FL: Chapman & Hall/CRC. Kaugars, A. S., Silverman, A., Kinservik, M., Heinze, S., Reinemann, L., Sander, M., Sood, M. (2010). quality of life. Journal of Pedi atric Gastroenterology and Nutrition, 51 747 752 doi:10.1097/MPG.0b013e3181de0651 Kawanishi, H. (1987). Immunocompetence of normal human appendiceal lymphoid cells: In vitro studies. Immunology 60 19 28. Kazdin, A. E. (2011). Single case research desig ns: Methods for clinical and applied settings (2nd ed.). New York, NY: Oxford University Press. Kelley, M. L., Heffer, R. W., Gresham, F. M., & Elliot, S. N. (1989). Development of a modified treatment evaluation inventory. Journal of Psychopathology & Beh avioral Assessment 11 235 247

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221 King, S. K., Sutcliffe, J. R., Southwell, B. R., Chait, P. G., & Hutson, J. M. (2005). The antegrade continence enema successfully treats idiopathic slow transit constipation. Journal of Pediatric Surgery 40 1935 1940. doi :10.1016/j.jpedsurg.2005.08.011 Knox, C. A., & Burkhart, P. V. (2007). Issues related to children participating in clinical research. Journal of Pediatric Nursing 22 310 318. doi:10.1016/jpedn.2007.02.004 Ma, H. (2006). An alternative method of quantitat ive synthesis of single subject researches: Percentage of data points exceeding the median. Behavior Modification 30 598 617. doi:10.1177/0145445504272974 Marshall, J., Hutson, J. M., Anticich, N., & Stanton, M. P. (2001). Antegrade continence enemas in the treatment of slow transit constipation. Journal of Pediatric Surgery 36 1227 1230. doi:10.1053/jpsu.2001.25768 McDowall, D., McCleary, R., Meidinger, E. E., & Hay, R. A. (1980). Interrupted time series analysis Thousand Oaks, CA: Sage. Mousa, H. M., Van Den Berg, M. M., Caniano, D. A., Hogan, M., Di Lorenzo, C., & Hayes, J. (2006). Cecostomy in children with defecation disorders. Digestive Diseases and Sciences 51 154 160. doi:10.1007/s10620 006 3101 7 Nanigian, D. K., Nguyen, T., Tanaka, S. T., Ca mbio, A., DiGrande, A., & Kurzrock, E. A. (2008). Development and validation of the Fecal Incontinence and Constipation Quality of Life measure in children with spina bifida. Journal of Urology 180 1770 1773. doi:10.1016/j.juro.2008.03.103 National Insti tutes of Health (NIH). (1998). NIH policy and guidelines on the inclusion of children as participants in research involving human subjects Retrieved from http://grants.nih.gov/grants/guide/notice files/not98 024.html Nicholson, J. K., Holmes, E., Kinross, J., Burcelin, R., Gibson, G., Jia, W., & Pettersson, S. (2012). Host gut microbiota metabolic interactions. Science 336 1262 1267. Noverr, M. C., & Huffnagle, G. B. (2004). Does the microbiota regulate immune responses outside the gut? TRENDS in Microbi ology 12 562 568. doi:10.1016/j.tim.2004.10.008 Ok, J., & Kurzrock, E. A. (2011). Objective measurement of quality of life changes after ACE Malone using FICQOL survey. Journal of Pediatric Urology 7 389 393. doi:10.1016/j.jpurol.2011.02.012 Penders, J ., Stobberingh, E. E., Van den Brandt, P. A., & Thijs, C. (2007). The role of the intestinal microbiota in the development of atopic disorders. Allergy 62 1223 1236. doi:10.1111/j.1398 9995.2007.01462.x Pharmacists, American Society of Health System. (20 11). Drug Information 2011 Bethesda, MD. Piantadosi, S. (2005). Clinical trials: A methodologic perspective (2nd ed.). Hoboken, NJ: Wiley. and utilitarianis m. Pediatric Nursing 3 319 323. Polit, D. F. (2010). Statistics and data analysis for nursing research (2nd ed.). New York, N.Y.: Pearson. Portney, L. G., & Watkins, M. P. (2009). Foundations of clinical research: Applications to practice (3rd ed.). Uppe r Saddle River, NJ: Pearson Prentice Hall. Rossoff, I. S. (1974). Handbook of veterinary drugs New York, NY: Springer. Schreiber, C. K., & Stone, A. R. (1999). Fatal hypernatremia associated with the antegrade continence enema procedure. Journal of Urolog y 162 1433 1434.

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222 Senn, S. (2002). Cross over trials in clinical research (2nd ed.). West Sussex, England: Wiley. Shadish, W. R., Cook, T. D., & Campbell, D. T. (2002). Experimental and quasi experimental designs for generalized causal inference Boston, MA: Houghton Mifflin. Shuster, J. J. (2007). Design and analysis of experiments. In W. T. Ambrosius (Ed.), Topics in biostatistics (pp. 235 259). Totowa, NJ: Humana Press. Shuster, J. J. (2009). Student t test for potentially abnormal data. Statistics in M edicine 28 2170 2184. doi:10.1002/sim.3581 Siddiqui, A. A., Fishman, S. J., Bauer, S. B., & Nurko, S. (2011). Long term follow up of patients after antegrade continence enema procedure. Journal of Pediatric Gastroenterology and Nutrition 52 574 580. Sm ith, H. F., Fisher, R. E., Everett, M. L., Thomas, A. D., Bollinger, R. R., & Parker, W. (2009). Comparative anatomy and phylogenetic distribution of the mammalian cecal appendix. Journal of Evolutionary Biology 22 1984 1999. doi:10.1111/j.1420 9101.2009 .01809.x Thomas, J. C., Dietrich, M. S., Trusler, L., DeMarco, R. T., Pope, J. C., Brock, J. W., & Adams, M. C. (2006). Continent catheterizable channels and the timing of their complications. Journal of Urology 176 1816 1820. doi:10.1016/S0022 5347(06)0 0610 0. Tomlinson, d., von Baeyer, C., Stinson, J., & Sung, L. (2010). A systematic review of faces scales for the self report of pain intensity in children. Pediatrics, 126 e1168e1198. doi:10.1542/peds.2010 1609. Tiryaki, S., Ergun, O., Celik, A., Ulman, European Journal of Pediatric Surgery 20 405 407. World Health Organization (WHO) Expert Committee of Food. (2002). Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances. Retrieved from http://www.inchem.org/pages/jecfa.html Wong, D., & Baker, C. (1988). Pain in children: Comparison of assessment scales. Pediatric Nursing, 14 (1), 9 17. Yardley, I. E., Pauniaho, S., Baillie, C. T., Turnock, R. R., Coldicutt, P., Lamont, G. L., & Kenny, S. E. (2009). After the honeymoon comes divorce: Long term use of the antegrade continence enema procedure. Journal of Pediatric Surgery 44 1274 1277. doi:10.1016/j.jpedsurg.2009.02.030 Yerkes, E. B., Rink, R. C., King, S., Cain, M. P., Kaefer, M., & Casale, A. J. (2001). Tap water and the Malone antegrade continence enema: A safe combination? Journal of Urology 166 1476 1478. Youssef, N. N. Barksdale, E., Griffiths, J. M., Flores, A. F., & Di Lorenzo, C. (2002). Management of intractable constipation with antegrade enemas in neurologically intact children. Journal of Pediatric Gastroenterology and Nutrition 34 402 405.

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223 Appendices Append ix A Compilation of Animal Studies Using Glycerin Appendix B Compilation of Human Studies Using Glycerin Appendix C Wong Baker FACES Pain Rating Scale Parental Instructions for Saline Flushing Regimen Appendix D Fecal Incontinence & Quality of Life Measure in Children with Spina Bifida Appendix E Parental Instructions on Normal Saline Flush Administration Appendix F Parental Instructions on USP Glycerin Flush Administration Appendix G Threats to Statistical Conclusion Validity Appendix H Threats to Intern al Validity Appendix I Social Threats to Internal Validity

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224 Appendix A Animal Studies Using Glycerin (There are no animal studies evaluating instillation of glycerin into the cecum) Sample Dose Route Outcome Reference 6 rabbits 18,700 mg/kg bw Occlusive dermal application for 8 hours No deaths United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html 12 femal e rats 27,260 mg/kg bw Gavage Muscle spasm, convulsions, lung congestion & death (3) Survivors normal within 2.5 hrs of dosing United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html Mice & Guinea pigs Not reported Gavage Tremor and convulsions with hyperemia of pylorius, small intestine and cerebtral meninges United Nations Environment Programme: Screening In formation Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html Rabbits 4 mL over 30% BSA 8 hr/day for 90 days Topical No irritation United Nations Environment Prog ramme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html 6 rabbits 0.1 mL Occular instillation Very low potential to irritate eyes Unite d Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html 24 male Guinea pigs 0.1 mL of 0.1% in NS qod x 20 days Injection No indication of sensitization after 2 week exposure free period Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sids pub.html

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225 Rats 1.75 mL of 50%/100 g bw SQ injection Severe hemolysis followed by necrosis of tubular portions of nephrons reversible within 6 12 wks European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: http://esis.jrc.ec.europa.eu/ Rats 1 mL 100%/100 g bw Intra peritoneal instillation Severe convulsions, hemoglobinuria, renal damage died within 2 hrs of injection European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (200 0 CD ROM edition). Available from, as of: February 18, 2015 http://esis.jrc.ec.europa.eu/ Rats 1 mL 100% or 50%/100 g bw SQ injection Hemoglobinuris, renal tubular necrosis, some convulsions with 100% European Chemicals Bureau; IUCLID Dataset, Glycerol (5 6 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: http://esis.jrc.ec.europa.eu/ Rats 100% & 50% at 1 mL/100 g bw IV Severe Convulsions and death in all animals European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD RO M edition). Available from, as of February 18, 2015: http://esis.jrc.ec.europa.eu/ Rabbit Aq 100% Anterior chamber of eye Inflammation and edema of cornea & damage of endothelial cells Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 Rabbits Aq 50% Anterior chamber of eye Significantly less reaction but visibly dehydrates lens can capsule wrinkling Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 R abbit Aq 30% for 20 min, 50% for 10 min or 92% for 4 min Anterior chamber of ete Normal deturgescence after 30% and 50% expposure but endothelium destruction with 92% > 30 min Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Pu blisher, 1986., p. 463 Rabbit with corneal trauma Aq 43% dilution for 30 min x 20 d Opthalmic Edema of conjunctiva lasting for several hrs Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 Rats 14 d 5d/wk 6hr/d Mean conc 1000, 1930 3910 mg/cu m Respirable aerosol Minimal to mild squamous metaplasia of epiglottis greatest at higest test dose No systemic effects United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html

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226 Rats 13 wk 6hr/d 5d/wk 0, 33, 165, & 662 mg/cu m Respirable aerosol Minimal to mild squamous metaplasia of epiglottis considered local i rritant effect United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html Rats 10% for 9 d IV Degeneration in renal tubular epithelium return to normal after 19 d NOWAK H ET AL; PATOL POL 30 (1): 61 (1979) Grow ing pigs 84.51% added to feed for 138 d Feeding trial Pigs can be fed up to 10% crude glycerin with no effects on performance, carcass composition, o r meat quality Lammers PJ et al; J Anim Sci 86 (11): 2962 2970 (2008) Rats 0 up to 60,000 mg/kg body weight/d for 20 wks oral At 5000 mg/kg bw marked hydropic and fatty degenration of liver parenchymal cells WHO/FAO: Expert Committee on Food Additives. S ummary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances (56 81 5) (2002). Available from, as of February 18, 2015: http://www.inchem.org/pages/jecfa.html Mice 6 to 8 wks Given carcinogen followed by 0, 0.5 or 1% glycerol or water until 1 yr of age oral Lower incidence of liver and lung tumor after glycerin no adverse treatment effects WHO/FAO: Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Ser ies 48: Aliphatic acyclic diols, triols, and related substances (56 81 5) (2002). Available from, as of February 18, 2015: http://www.inchem.org/pages/jecfa.html Rats 0 to 20,000 mg/kg body weight/d oral No significant treatment related effects WHO/FAO : Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances (56 81 5) (2002). Available from, as of February 18, 2015: http://www.inchem.org/pages/jecfa.ht ml Rats 4,000 to 10000 mg/kg bw for 2 years Oral No adverse effects up to 10,000 mg/kg bw United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.u nep.ch/irptc/sids/OEC DSIDS/sidspub.html Rats 5%, 10%, & 20 % for Oral Glycerol does not initiate tumor development in rats United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18,

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227 12 to 24 months 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html Mice 5% in drinking water for 1 20 weeks after sq injection of 4 NQO Oral Enhances lung tumor development mainly adenomas. Tumor development independent from p ulmonary cell kinetics United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html Rats 7 generations 15000 m g/kg bw/d Oral Pups of treated dams mean weight 20% less than controls Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances (56 81 5) (2002). Availabl e from, as of February 18, 2015: http://www.inchem.org/pages/jecfa.html Rats Mice Rabbits Levels up to 1310, 1280, 1180 mg/kg bw daily during part of gestational period oral No maternal or tetragenic effects seen at highest dose level tested United Nation s Environment Programme: Screening Information Data Sheets on Glycerol (56 81 5) (March 2002) Available from, as of February 18, 2015: http://www.chem.unep.ch/irptc/sids/OEC DSIDS/sidspub.html

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228 Appendix B Summary of Previous Human Studies Involving USP Gl ycerin Dose Route Effects Reference Not resported Not reported Very slight diuresis in healthy individuals receiving a single dose May produce tissue dehydration and decreases in CSF pressure McEvoy, G.K. (ed.). American Hospital Formulary Service Drug Information 93. Bethesda, MD: American Society of Hospital Pharmacists, Inc., 1993 (Plus Supplements, 1993)., p. 1773 Not reported. Employees engaged in glycerol manu facturing Environ mental exposure No significant difference in sperm count or sperm qua lity parameters when compared eith controls European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: www.inchem.org/documents/sids/sids/ 5 6815 0.05 mL of 10% solution for 21 days Dermal patch test Slight irritation at 48 hrs and maximum rating of 4 on a 9 point scale at day 14 of a 21 day application European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: www.inchem.o rg/documents/sids/sids/ 5 6815 Acute and chronic (42 d) ingestion Oral Increase in plasma glycerides in males only following acute ingestion and in both males and females with chronic ingestion (significantly greater increase in males) European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: www.inchem.org/documents/sids/sids/ 5 6815 Orange juice mixed with 30 mL of 95% glycerol after each of 3 daily meals Oral No overt signs of toxicity or chage in food consumption European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: www.inchem.org/documents/sids/sids/ 5 6815 Repeated application of 100% solution Occular Extensive c hanges to appearance of endothelium that disappeared within 90 minutes of application Grant, W.M. Toxicology of the Eye. 3rd ed. Springfield, IL: Charles C. Thomas Publisher, 1986., p. 463 24,000 mg/kg bw d for 50 days Oral Slight tendency toward increase in weight WHO/FAO: Expert Committee on Food Additives. Summary of Toxicological Data of Certain Food Additives Series 48: Aliphatic acyclic diols, triols, and related substances (56 81 5) (2002). Available from, as of February 18,2015,: http://www.inchem.org/pages/jecfa.htm l

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229 Not reported. Workers in foam rubber facory Dermal patch testing No sensitizing effects United Nations Environment Programme: Screening Information Data Sheets on Glycerol (56 8 1 5) (March 2002) Available from, as of February 18, 2015: echa .europa.eu/

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230 Case Reports Subject Dose Route & Purpose Effects Reference 46 y.o. male 500 mL of 10% solution Altered sensorium, generalized seizures, focal neurologic signs Managed conse rvatively and recovered within 48 hrs case represents rare presentation of overdose with an otherwise safe drug used in neurology Singh R et al; Neurol India 49 (3): 320 1 (2001) Male Adjunctive glycerin test used in the diagnosis of 20 40dB hearing loss in univolved ear during standard testing that resolved within 3 days Mattox DE, Goode RL; Arch Otolaryngol 104 (6): 359 61 (1978) 73 y.o. male Oral solution used to treat elevated IOP Developed severe pulmonary edema 45 minutes afte r administration Almog Y et al; Ann Ophthalmol 18 (1): 38 9 (1986) 72 y.o. male Dose should not exceed 1.5 g/kg bw in Klockhoff test for diagnosis of disease Patient received 3.88 3.95 g/kg bw Progressive neurological signs and pat hologically elevated serum concentration of triglycerides (3,465 mg/dl) Andresen H et al; Clin Toxicol (Phila) 47 (4): 312 6 (2009) 3 y.o. male 0.5 1.0 g/kg Unique intolerance including mental changes, N&V, hypoglycemia and loss of consciousness followi ng IV administration with spontaneous recovery after 30 min European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: echa .europa.eu/ Not reported Rectal administration prior to coronary artery bypass Acute colonic ischemia European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: echa .europa.eu/ 82 y.o. hypertensive and senile female 200 mL 50% solution for primary angle closure glaucoma Headache, shaking of arm, quivering of eyes, and nausea European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of February 18, 2015: echa .europa.eu/ 68 y.o. female diabetic 280 mL of 50% s olution Severe daibetic acidosis within 3 days of ingestion European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available

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231 from, as of February 18, 2015: echa .europa.eu// Retrospective Studies and Case Reports Addressing Antegrade Colonic Flush Administration Pediatric Subjects Solution & Dose Side Effects from Flush Reference 40 Dose not reported 1) GoLYTLEY 2) Liquirice root 3) Oil/water mix 4) Treacle/milk mix 5) Water only 6) Oil only None Marshall, J., Hutson J. M., Anticich, N., & Stanton, M. P. (2001). Antegrade continence enemas in the treatment of slow transit constipation. Journal of Pediatric Surgery 36 1227 1230. doi:10.1053/jpsu.2001.25768 62 1) Polyethylene Glycol with electrolytes (50 1,000 mL) 2) Phosphate enema 3) NS alone (50 1000 mL) None Dey, R., Ferguson, C., Kenny, S. E., Shankar, K. R., Coldicutt, P., Baillie, C. T., honeymoon Medium term outcome of antegrade continence enema procedure. Journal of Ped iatric Surgery 38 65 68. doi:10.1053/jpsu.2003.50012 26 Flush solution not reported. Dosage (250 1000 mL) None Becmeur, F., Demarche, M., Lacreuse, I., Molinaro, F., Kauffmann, I., Moog, R., Rebeuh, J. (2008). Cecostomy button for antegrade enemas: Su rvey of 29 patients. Journal of Pediatric Surgery 43 1853 1857. doi:10.1016/jpedsurg.2008.03.028 71 Tap water (300 1000 mL) Minor deviations in serum sodium and serum chloride present in 18/71 patients Significant hyper natremia and hyper chloremia i n 1/71; used softened tap water Yerkes, E. B., Rink, R. C., King, S., Cain, M. P., Kaefer, M., & Casale, A. J. (2001). Tap water and the Malone antegrade continence enema: A safe combination? Journal of Urology 166 1476 1478. 236 1)Tap water alone volu me (100 to 1000) 2) 60 mL of USP glycerin & 60 mL of NS 3) GoLYTELY 1 liter None Bani Hani, A. H., Cain, M. P., King, S., & Rink, R. C. (2008). Tap water irrigation and additives to optimi ze success with the Malone antegrade continence enema: The Indiana University algorithm. Journal of

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232 4) MiraLAX 17 grams mixed in 250 mL tap water 5)Mineral oil 30 mL Urology 180 1757 1760.doi:10.1016/j.juro.2008.04.074 105 1) Normal saline or GoLYTELY(23 +/ 0.7 mL/kg) 2) USP glycerin dose not reported 3), bisacodyl dose not reported 4) magnesium cirate dose not reported 5) phosphosoda dose not reported None Siddiqui, A. A., Fishman, S. J., Bauer, S. B., & Nurko, S. (2011). Long term follow up of patients after antegrade continence enema procedure. Journal of P ediatric Gastroenterology and Nutrition 52 574 580 23 USP glycerin 10 60 mL & 15 775 mL tap water with a total volume of irrigation solution ranging from 30 800 mL None Chu, D., Balsara, Z.R., Routh, J.C., Ross, S.S., & Wiener, J.S. (2012). Experien ce with glycerin for antegrade continence enema in patients with neurogenic bowel. Journal of Urology, 189,690 693. 1 Case report Hypertonic saline Death Schreiber, C. K., & Stone, A. R. (1999). Fatal hypernatremia associated with the antegrade contine nce enema procedure. Journal of Urology 162 1433 1434. The route of administration influences toxicity of glycerol in humans. Toxic effects from oral administration include nausea and vomiting. Glycerol has a CNS dehydration effect. Intraocular pressur e begins to fall at plasma concentrations of 10 mmoles per liter. Concentration and dilutant used also influence toxicity. Use of saline as dilutant diminishes toxic effects of glycerol. Toxic effects following intraperitoneal and subcutaneous administrati on are albuminuria, hemoglobinuria, anemia, and renal damage. European Chemicals Bureau; IUCLID Dataset, Glycerol (56 81 5) (2000 CD ROM edition). Available from, as of July 18, 2011: http://esis.jrc.ec.europa.eu/

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234 Appendix D Fecal Incontine nce & Quality of Life Measure in Children with Spina Bifida

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237 Appendix E Steps Parental Instructions for Saline Flushing Regimen 1 Assemble equipment: One liter container of Normal Saline (NS) premixed to 0.09% Each container will be marked by th e investigator with ACE flush contents and a lot number One 1,000 mL enteral feeding bag with drip chamber and roller clamp One Christmas tree adapter connected to the end of the enteral feeding bag tubing The bag will be hung from a hook located 6 feet ab ove the floor on a wall to the side of or behind the commode One appropriate access tube for Chait or Mickey low profile device Stop watch Clip board with data collection sheets and pen attached with string to the clipboard 2 Warm tap water and mild liqu id dish detergent to wash equipment after each use 3 Make sure flushing solution is at room temperature 4 Insert the Christmas tree adapter into the end of the feeding bag tubing, making sure the fit is secure 5 Make sure the roller clamp is closed on the feeding bag tubing 6 Unscrew cap from the top of the feeding bag by rotating in a counter clockwise direction 7 Pour room temperature liquid from the 2 liter mixing bottle into the feeding bag 8 Screw the cap onto the top of the feeding bag by turning in a clockwise direction until secure 9 Hold the feeding bag with liquid in onehand at shoulder height and put the end of the tubing in the sink 10 Using your other hand, slowly loosen the roller clamp on the feeding bag tubing until the liquid fills the tube and then reclamp by tightening the roller clamp to stop the flow of liquid 11 Hang the feeding bag on the wall hook above the commode 12 Position child comfortably on commode using a toilet seat insert and a foot stool, if needed 13 Give child toys/books f or distraction 14 Hook the end of the Christmas tree adapter into the access tubing and secure the access tubing to the button 15 Unclamp the roller clamp to start the flow of the flushing solution and immediately start the stop watch 16 If your child comp lains of cramping or discomfort, slowly tighten the roller clamp to slow the flow of the flush 17 When the flush solution has infused, write the time from the stopwatch onto the record 18 Disconnect th e tubing and rinse with a mixture of mild dish soap and tap water followed by tap water alone and hang back on the hook to air dry 19 Once your child has passed a bowel motion and 5 minutes has gone by without any additional stool output, stop the watch an d record the time on the record sheet under column "Completion Time"

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238 Appendix F Parental Instructions for USP Glycerin and Normal Saline* 1 Assemble equipment: One appropriate access tube for Chait or Mickey low profile device 60 mL catheter tipped syri nge One 8 ounce bottle with screw top to mix glycerin and NS USP Glycerin dispensed in a resealing container appropriate for liquids Glycerin container will be marked by the investigator with ACE Flush and a lot number One 30 mL syringe for measuring liqui d glycerin One liter container of NS premixed to 0.09% Stop watch Clip board with data collection sheets and pen attached with string to the clipboard 2 Warm tap water and mild liquid dish detergent to wash equipment after each use 3 Using the 8 ounce bot tle with screw top pour ______mL of USP glycerin and ________ mL of NS into the bottle making sure the NS is at room temperature 4 Recap the bottle making sure the cap is secure; shake the bottle until the liquids are well combined 5 Attach the catheter ti p of the 60 mL syringe to the access tubing 6 Hold the 60 mL syringe with liquid in one hand and put the end of the tubing in the sink 7 Pour room temperature liquid from the 8 ounce mixing bottle into the 60 mL syringe 8 When the flush solution reaches th e end of the tubing, pinch off the tubing to stop the flow of liquid 9 Position child comfortably on commode using a toilet seat insert and foot stool if needed 10 Give child toys/books for distraction 11 Attach and secure the access tubing to the low prof ile device 12 Unpinch the tubing to start the flow of the flushing solution and immediately start the stop watch 13 Hold the syringe at the child's shoulder height 14 If your child complains of cramping or discomfort, lower the height of the syringe to slo w the flow of the flush 15 Raise or lower the syringe to adjust the flow of liquid. The higher the syringe the faster the liquid will go in 16 When the flush solution has infused, write the time from the stopwatch onto the record t do not stop the stopwatch 17 Once the flush has infused, disconnect the tubing and rinse the syringe and tubing with a mixture of mild dish soap and tap water followed by tap water alone 18 Once your child has passed a bowel motion and 5 minutes has gone by without any additional stool output stop the watch and record the time on the record sheet under column "Completion Time"

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239 Appendix G Threats to Statistical Conclusion Validity Threat Assessment of Potential Threat & Proposed Control Insufficient Po wer (Increases probability of a Type I error) Reliability of Measures (Impacts the relationship between variables) Reliability of Treatment Implementation (May result in underestimation in treatment effect. Random Irrelevancies in the Experimen tal Setting (May inflate error) Random Heterogeneity of Respondents ( Increases variability in outcome, increasing error variance) Violated assumptions Within subject analysis the strength of the ability to determine treatment effects requires a stable baseline, and is a function of the number of baseline and intervention data points (Cook & Campbell, 1979). This study design features a prolonged pre operative baseline assessment and a minimum of 6 weeks of daily measurement for each treatment va riable meeting the criteria for sufficient power analysis Cross over analysis: The study will use 6 subjects randomized to 2 treatment orders. Although the design confers greater precision in estimating treatment difference and generally requires fewer su bjects, the limited number of subjects will limit the power of the analysis increasing the probability of a Type II error (Piantadosi, 2005). All biological samples will be run in a single accredited laboratory. All fecal samples will be collected and s tored for downstream analysis by the procedure detailed in the protocol (Shadish et al., 2002). Procedural fidelity will be ascertained each procedural variable at specified times throughout the study. Written instruction forms detailing interventions wi ll be given to the family (Gast, 2010). Within subject analysis should not pose a problem if baseline is stable as the baseline reflects background variable effects (Shadish et al., 2002) Cross over analysis: Period confounds should be accounted for using the two sample cross over t test (Shuster, 2007) Both designs use subjects as their own control substantially decreasing variance due to subject heterogeneity (Cook & Campbell, 1979). Within Subject analysis violates statistical assumptions th at error residuals are independent, normally distributed, and homoscedastic. Serial dependency can by modeled and removed. Cross over analysis treatment sequences considered independent. Carry over effects should not be a factor due to design considerat ions but are accounted for using the two sample t test (Shuster, 2007) which result in over or underestimation of the magnitude and statistical significance of treatment effects.

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240 Appendix H Threats to Internal Validity Threat Assessment of Potential Threat & Proposed Control Ambiguous Temporal Precedence (cause precedes e ffect) Attrition (subject mortality) Maturation (confounding due to passage of time) History (Confounding events other than the independent variable) Order Effects (Position in sequence) & Carry Over Effects (Effects from previous phase impacting current phase) Regression (Tendency of extreme scores to regress toward the mean) Instrumentation (reliability of measurement) Testing This is a prospective study. Ascertainment of temporal precedence and ambiguity regarding direction of cau sal inference is not a risk Attrition will have a significant effect on data analysis. Careful follow up and provision of supplies may discourage subject fatigue and drop outs. A second randomization to treatment has been instituted after the dose respo nse phase just before the treatment phase to insure randomization scheme if attrition occurs due to ineffective treatment or side effects (Portney & Watkins, 2009) This study is several months in duration which should preclude maturational confounding. In addition, randomization to treatment sequence should minimize maturation as a potential bias (Shadish et al., 2002) Effects of significant diet change or infection causing diarrhea will confound treatment results. A diary will be kept documenting any obv ious historical confounds. In those instances, treatment length will be extended if necessary (Gast, 2010). Use of a match control that did not receive treatment would strengthen the design (Cook & Campbell, 1979) but would be unethical. In addition, dise ase instability may confound treatment effects. This population generally has a chronic but stable disease and should provide a stable sample to compare treatment effects (Piantadosi, 2005). Order effects which will be controlled by randomization to one of two treatment sequences (Kazdin, 2011). Carry over effects should not confound continence. There is a prolonged active washout period between serum and stool sampling Should not pose a threat in this study IOA will be checked at key points througho ut the study and calculated using gross method. If IOA is <80%, additional observer training will be implemented. The family will have report forms to help standardize and quantify responses. (Gast, 2010; Kazdin, 2011) Should not pose a threat in this stu dy Internal validity questions if there evidence to support a causal relationship between the treatment and outcome variable (Portney & Watkins, 2009).

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241 Appendix I Social Threats to Internal Validity Threat Assessment of Potential Threat & Proposed Contr ol Diffusion and Imitation of Treatment Compensatory Equalization of Treatments Compensatory Rivalry Resentful Demoralization Many of the families who will be enrolled in this study interact socially. Attend a support group together, and atte nd a clinic multidisciplinary clinic in which they share a waiting room. Subjects are not blinded to treatment. Parents and children enrolled in the study may be influenced by parent or child preference of individuals who are already on ACE therapy. Blindi ng is not possible due to obvious differences in flush volume (Portney & Watkins, 2009) Subject or parental preference for one treatment over another based on intangibles not measured in the study may influence treatment implementation and/or evaluation o f dependent measures. Blinding is not possible due to obvious differences in flush volume (Portney & Watkins, 2009) All subjects will receive all treatments so no subject should receive what they view as a less than desirable treatment negating the threa t of compensatory rivalry (Portney & Watkins, 2009) All subjects will receive all treatments so no subject should receive what they view as a less than desirable treatment negating the threat of resentful demoralization (Portney & Watkins, 2009) Social V alidity questions if there is evidence to support a causal relationship between the regarding treatment specifics (Portney & Watkins, 2009).

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242 APPENDIX O KJ001 WITHIN SUBJECTS ANALYSIS CALCULATIONS INCLUDING GRAPHS D ETAILING STABILITY AND TREND

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301 BIOGRAPHIC AL SKETCH Kim graduating with a Master of Science deg ree in the spring of 1994 and a Doctor of Philosophy in the fall of 2017. Her clinical specialization and research focus are dysfunctional elimination in children. Kim is a practicing pediatric nurse practitioner and the Clinical Director of the Dysfunctio nal Elimination Program at Nemours Pediatric Specialty Care in Jacksonville, Florida This program is the first reported independent nurse practitioner run referral center in a free standing pediatric subspecialty ambulatory setting in the country and the first pediatric program to combine subspecialty services including urology, gastroenterology, and psychology in a single point of care for this population.