Clinical trial NCT02903030
Conditions: Autism Spectrum Disorders Anxiety
Keywords: Gastrointestinal Microbiome
Sponsor: Ohio State University
Start date: 2016-08-01; Completion date: 2017-12-01
Enrollment: 13 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
A randomized pilot trial of a probiotic for quality of life in autism spectrum disorder (ASD), targeting gastrointestinal (GI) symptoms.

Detailed description:
The physical and mental/emotional health of people with autism spectrum disorder (ASD) are closely connected. The emerging data on immune abnormalities and gut microbiome differences, and interactions of the genome with these suggest a possible etiological link between physical and mental dysfunction, especially the gastrointestinal (GI) dysfunction and severe anxiety that many individuals with ASD manifest. The investigators have preliminary clinical evidence that children with ASD \& GI symptoms differ in microbiome composition and function from neurotypical children with GI symptoms. The investigators hypothesize that altered host-microbial signals, which include altered fecal neurotransmitter gamma-aminobutyric acid (GABA) levels contribute towards anxiety and sensory over-responsivity in ASD. Our preliminary findings also show that probiotic Visbiome Extra Strength, improves GI and pain symptoms, correlating with altered gut microbiome composition and related metabolites (the macrobiome). The proposed crossover trial will explore the possibilities of this new appreciation of the microbiome-mental/physical function connection for ASD, GI dysfunction, and anxiety. If altering the gut microbiome results in better GI and emotional function, it could improve the quality of life for children with ASD and their parents. A pilot trial with 12 children with ASD will test feasibility for a proposed three-site crossover randomized clinical trial (RCT) of probiotics (beneficial bacteria including Lactobacilli \& Bifidobacteria) in 60 children 3-12 years old with ASD, GI dysfunction, \& anxiety. In a balanced crossover children will be randomized 1;1 to Visbiome or placebo first, 8 weeks per condition with 3 weeks washout between. The investigators have access to significant fecal microbiome and metabolome data from NIH-funded Human Microbiome Projects (HMP) on similar-age healthy and irritable-bowel children, with and without ASD. These will help leverage our understanding of macrobiome changes that correlate with functional improvement of GI and abdominal pain symptoms. Pilot study efficiency will also benefit from those HMPs having already collected and analyzed baseline stools for some children with ASD, thus saving significant costs for baseline stool analyses for the pilot.

Arms: arm 1: The probiotic mix (VISBIOME) will be mainly Bifidobacteria and Lactobacilli, in view of the previously reported encouraging clinical studies and safety data. arm 2: Placebo matched to probiotic.
Interventions: intervention 1: Maltose (placebo) intervention 2: Visbiome Extra Strength
Intervention descriptions: intervention 1: Maltose with a trace amount of silicon dioxide intervention 2: It is a mix of 8 strains of beneficial bacteria that should improve gut flora, improving GI function and hopefully anxiety

Protocol documents:
Prot_SAP_000.pdf: 
Probiotic for QOL in ASD 
 
 
Probiotics for Gastrointestinal Symptoms and Quality of Life in 
Autism: a placebo-controlled pilot trial 
NCT02903030 
 
Dr. L. Eugene Arnold, MD, MEd 
5/19/2016 
 
 
 
 
 
 


Probiotic for QOL in ASD 
 
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Protocol for Pilot of Probiotic for Quality of Life in Autism 
 
Introduction & Overview.  The physical and mental/emotional health of people with autism 
spectrum disorder (ASD) are closely connected. The emerging data on immune abnormalities 
and gut microbiome differences, and interactions of the genome with these suggest a possible 
etiological link between physical and mental dysfunction, especially the gastrointestinal (GI) 
dysfunction and severe anxiety that many individuals with ASD manifest. We have preliminary 
clinical evidence that children with ASD & GI symptoms differ in microbiome composition and 
function from neurotypical children with GI symptoms.  We hypothesize that altered host-
microbial signals, which include altered fecal neurotransmitter gamma-aminobutyric acid 
(GABA) levels contribute towards anxiety and sensory over-responsivity in ASD.  Our 
preliminary findings also show that probiotic Visbiome, formerly known as VSL#3, improves GI 
and pain symptoms, correlating with altered gut microbiome composition and related 
metabolites (the macrobiome). The proposed crossover trial will explore the possibilities of this 
new appreciation of the microbiome-mental/physical function connection for ASD, GI 
dysfunction, and anxiety. If altering the gut microbiome results in better GI and emotional 
function, it could improve the quality of life for children with ASD and their parents. A pilot trial 
with 12 children with ASD will test feasibility for a proposed three-site crossover randomized 
clinical trial (RCT) of probiotics (beneficial bacteria including Lactobacilli & Bifidobacteria) in 60 
children 3-12 years old with ASD, GI dysfunction, & anxiety. In a balanced crossover children 
will be randomized 1;1 to Visbiome or placebo first, 8 weeks per condition with 3 weeks washout 
between. We have access to significant fecal microbiome and metabolome data from NIH-
funded Human Microbiome Projects (HMP) on similar-age healthy and irritable-bowel children, 
with and without ASD. These will help leverage our understanding of macrobiome changes that 
correlate with functional improvement of GI and abdominal pain symptoms. Pilot study efficiency 
will also benefit from those HMPs having already collected and analyzed baseline stools for 
some children with ASD, thus saving significant costs for baseline stool analyses for the pilot. 
 
Specific Aims of the Pilot: 
Aim 1:  To test feasibility of the planned RCT. 
Hypothesis 1A:  12 children aged 3-12 years with ASD, GI dysfunction, and anxiety can be 
recruited from underserved and minority populations in a 15-month pilot. 
Hypothesis 1B:  Retention during the full crossover will be 75% or more. 
Hypothesis 1C:  Adherence to taking the probiotic and placebo will be 80% or more. 
Hypothesis 1D:  Participant satisfaction will be good (mean score matching the anchor 
“satisfied” on the satisfaction scale). 
Aim 2:  Confirm safety of Visbiome in ASD ( supported by numerous published reports 
showing no adverse effects in patients with GI disorders). 
 
Hypothesis 2:  There will be no serious adverse events attributable to study treatment. 
Aim 3.  To explore effects of probiotics on quality of life (QOL) and gastrointestinal (GI) function 
in children age 3-12 with ASD, GI dysfunction, and anxiety. 
Hypothesis 3:  Children with ASD randomly assigned to receive VISBIOME will have 
nominally better GI QOL on the GI module of the PedsQOL scale (primary outcome) than 
those assigned to placebo, with average 25% or greater improvement or improvement to the 
normal range.. 
Aim 4:   
To explore effects of probiotics on anxiety in 3-12 year-olds with ASD. 
Hypothesis 4:  Compared to baseline, children with ASD will have nominally better emotional 
stability (less anxiety) when taking Visbiome than when taking placebo, with average 25% or 
more improvement.or improvement to the normal range on the anxiety scale.. 
Aim 5: To confirm that the 3 week washout period is adequate for GI symptoms to return to pre-
treatment baseline.  
Hypothesis 5: By week 11, the score of the GI module of PedsQOL will be substantially closer to 
the baseline score than to the week 8 score for the group that received Visbiome first.  


Probiotic for QOL in ASD 
 
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Exploratory Aim 6:  To explore the extent to which the candidate probiotic (VISBIOME) can 
change the composition of gut microbiome and stool neutotransmitters in 3-12 year-olds 
with ASD.  Responders in GI function or anxiety will be compared to nonresponders in 
microbiome change/retention. 
If 3 of the 4 feasibility hypotheses plus Hypothesis 2 plus one of the 2 clinical outcome 
hypotheses are upheld, the RCT will be justified. If washout is not complete in 3 weeks, it 
will be adjusted to a longer period for the RCT, based on the pilot data collected monthly. 
 
Background & Significance 
The healthy human gut contains trillions of bacteria from diverse bacterial species, most being 
harmless or beneficial (Lozupone et al., 2012).  The beneficial bacteria protect from pathogenic 
microbes, assist in metabolism and immune balance, and enable healthy GI functioning.  
Probiotics are defined by the Food and Agricultural Organization/World Health Organization, as: 
"Live microorganisms which when administered in adequate amounts confer a health benefit” 
[WHO, 2001].  “To be classified as a probiotic, the organism must have scientifically proven 
beneficial physiologic effects, must be safe for human consumption, must be stable in bile and 
acid, and must be able to adhere to the intestinal mucosa” [AACE Nutrition Guidelines Task 
Force, 2003].  The most common types are lactic acid bacteria and bifidobacteria.  
 
In a meta-analysis of 5 RCTs in irritable bowel syndrome, Tiequn et al (2015) found a 
significant relative “risk” of improvement with Lactobacillus over placebo of 7.69 (p=0.0008), 
17.62 in adults (p=0.00001) and 3.71 in children (p=0.04) without side effects.  Two RCTs in 
neurotypical children with constipation, one in infants, found benefits (Sadeghzadeh et al, 2014; 
Coccorullo et al, 2010). Three other trials using a single organism compared to laxative did not.  
Probiotics produce neurotransmitters, such as GABA and serotonin, which act on the 
brain-gut axis via the vagus, and have been dubbed “Psychobiotics” in this role (Dinan et al, 
2013). The benefits reported for irritable bowel syndrome, depression, and chronic fatigue 
syndrome may also be related to anti-inflammatory actions (Dinan, 2013). Both GABA and 
serotonin are involved in actions of FDA-approved anxiolytic drugs. Rodent studies suggest that 
some psychobiotics (e.g., Lactobacillus helveticus and Bifidobacterium longum) can be 
anxiolytic (Bercik, Premysl, et al., 2011; Bercik, Park, et al., 2011; Messaoudi et al. 2011. In a 4-
week trial in healthy women (Tillisch et al, 2013), fermented milk product and a probiotic 
(Bifidobacterium animalis subsp lactis, Streptococcus thermophiles, Lactobacillus bulgaricus, & 
Lactococcus lactic) affected activity of brain centers controlling emotion and sensation, 
documented by fMRI with an emotional faces task (p=0.004).  
In a neurotypical human study, Diop (2008) found that following a stressful event, 
Lactobacillus helveticus Rosell-52 and Bifidobacterium longum Rosell-175 resulted in more 
improvement in abdominal pain and nausea/vomiting than placebo, but with no difference on 
psychiatric symptoms. In another study, healthy volunteers randomly assigned to probiotic 
experienced significant improvement on global distress, somatization, depression, anger-
hostility, and problem solving, and had decreased urinary free cortisol levels compared to 
controls [Messaoudi, et al. 2011]. In a pilot study, patients with chronic fatigue syndrome 
randomly assigned to a Probiotic (Lactobacillus casei strain Shirota, LcS) reported significant 
decreases in anxiety symptoms compared to controls (Rao et al., 2009). In a study in children, 
Christian et al (2015) found an association between child temperament and gut microbiome. In 
a RCT of the 8-strain formulation (VSL#3/Visbiome) to be used in this study, Kim et al (2005) 
found significant reduction of bloating in adults with irritable bowel syndrome.  Using the same 
formulation in a placebo-controlled crossover trial (6 weeks each condition, 2-week washout) in 
59 neurotypical children with irritable bowel syndrome, Guandalini et al (2010) found significant 
benefit for abdominal pain/discomfort, bloating/gassiness, and life disruption. 
 
Studies in ASD 
In an ATN registry study, Mazurek et al (2012) found that 24% of children with ASD had 
chronic GI problems, most commonly constipation and abdominal pain; and those with GI 
problems had higher rates of anxiety and sensory over-responsivity (p<.0001). Further, those 


Probiotic for QOL in ASD 
 
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with multiple GI problems had significantly more anxiety and over-responsivity than those with 
only one (p<.0001). De Angelis et al (2013) compared the fecal microbiota and metabolome of 
children with Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), autistic 
disorder (AD), and healthy children (HC). The main bacterial phyla (Firmicutes, Bacteroidetes, 
Fusobacteria, and Verrucomicrobia) were significantly (P<0.05) different among the three 
groups 
Our own preliminary data (Fig. 1) are supportive of these findings, with a decreased 
Faecalibacteria representation in ASD subjects. Our preliminary studies also show that 
Visbiome, formerly branded VSL#3, the probiotic to be used in this study, restores 
Faecalibacteria abundance in patients with GI disease and pain who respond favorably to this 
probiotic.  Changes in unclassified Ruminococcaceae are also evident in ASD children, 
especially in mucosa-associated communities (Fig. 1).  We have demonstrated that these types 
of bacteria are potent GABA producers using a novel decarboxylation conversion of dietary 
glutamate, modulating anxiety and sensory over-responsivity in preclinical models.  In further 
support of microbial neurotransmitter production being associated with GI symptoms and pain in 
ASD, we performed a 14 day case-controlled trial (including a healthy sibling) and demonstrated 
that stool GABA levels strongly correlated with GI symptoms and pain episodes in an ASD child 
(Fig. 2).  These findings build on earlier reports that fecal free amino acids and volatile organic 
compounds are markedly affected in PDD-NOS and especially AD. Tomova et al (2015) found a 
significant decrease of the Bacteroidetes/Firmicutes ratio and elevation of the amount of 
Lactobacillus in autistic disorder, potentially associated with altered content of GABA-producing 
bacteria, but this was not investigated.  West et al (2013) reported a 20% improvement in 
autistic symptoms in an open trial of a 4-bacteria probiotic formulation. 
Thus, there are 4 encouraging literature themes: 1) Probiotics appear to improve GI 
function and emotional symptoms such as anxiety and depression in rodent models and in 
neurotypical humans; 2) Children with ASD have a high rate of GI dysfunction and a gut 
microbiome/macrobiome that differs from healthy controls; 3) GI problems were significantly 
associated with anxiety and sensory over-responsivity in ASD; 4) An open trial of probiotic in 
ASD suggested mild improvement in autistic symptoms.  Taken together, these suggest the 
need for a randomized, placebo-controlled trial in ASD focusing on quality of life as 
affected by GI function & the emotional instability/anxiety that most children with ASD have 
trouble with, but also exploring possible benefit for socialization, communication, and interests. 
  
Preliminary Work 
Collaborators in this proposal have studied stool and mucosal microbiome communities in ASD 
children and have also investigated the effect of a specific probiotic (VSL#3, now called 
VISBIOME) in greating irritable bowel syndrome (IBS). VISBIOME (ExeGi Pharma LLC, 
Gaithersburg, MD) is made up of 4 strains of lactobacilli (L. casei, L. plantarum, L. acidophilus, 
and L. delbrueckii subsp. bulgaricus), 3 strains of bifidobacteria (B. longum, B. infantis, and B. 
breve), 1 strain of Streptococcus thermophiles, and starch.  Although previously described as 
separate species, B. infantis and B. longum are now considered to be biotypes or subspecies of 
the same organism (B. longum). Lactobacillus bulgaricus has been renamed L. delbrueckii.  
Subjects took 2 packets daily (900 billion bacteria) for either 4 or 8 weeks, mixed in a cold food 
or noncarbonated beverage. See figures 1-3.  Safety data included all 21 subjects. No deaths, 
infections with component bacteria, unscheduled IBS-related health care visits, change in 
frequency or severity of events, or reported causality occurred in subjects while taking VSL#3.  
There were 88 AEs reported; all were rated as not serious according to the FDA rating scale. 
The AEs were deemed not related to VSL#3 as the symptoms reported as AEs are common 
symptoms in IBS. Bloating was the most common AE reported, followed by having ≥ 4 stools in 
a 24-hour period. 
 
 
 
 


Probiotic for QOL in ASD 
 
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 Figure 3. Changes in 
the composition of the most abundant members of the gut microbiome and the VSL#3 
component species as a function of treatment duration. Bacterial community profiles were 
generated from shotgun metagenomic sequence data and Metaphlan2 profiling. Subjects are 
organized by treatment and pre- and post-probiotic samples are adjacent to one another (i.e., 
C1 vs C2). Other taxa (not specifically shown) make up the balance of each specimen’s 
community (i.e., contribute the remaining balance, wherein the sum of each sample is 100%). In 
the majority of subjects (10 of 12), VSL#3 species were detected in the post-probiotic sample, 
Figure 1: The colonic mucosa-associated 
microbiome shows distinct community-
associated shifts in ASD.  In a 16S rDNA 
deep sequencing study of mucosal biopsies 
from 14 children with ASD (red triangles), 
12 with functional abdominal pain (FAP, 
green squares) and 9 healthy (blue circles) 
children, principal coordinate component 
plots demonstrated significantly different 
community composition in ASD children 
(left) including a depletion of 
Faecalibacteria and enrichment of 
Ruminococcaceae (right).      


Probiotic for QOL in ASD 
 
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and in all subjects, the relative abundances of the most abundant community members were 
altered following VSL#3 consumption. 
Innovation: This will be the first study to our knowledge of probiotic effect on quality of life and 
anxiety as well as GI function in ASD. It will include cutting-edge comprehensive stool analyses. 
 
Research design & Method for Pilot 
In a balanced crossover randomized feasibility pilot trial, 12 children aged 3-12 years with ASD, 
GI dysfunction, and anxiety will be randomly assigned 1:1 to probiotics first  or  placebo 
matched to probiotic first for 8 weeks then a 3-week washout followed by the opposite 
condition..  
 
Inclusion Criteria: Participants (both sexes) must: 1. have DSM-5 ASD on clinical evaluation 
by a doctoral-level diagnostician, confirmed by Autism Diagnostic Interview-Revised or Autism 
Diagnostic Observation Schedule; 2. be between 3 and 12 years old; 3. have >2 mo. abdominal 
pain, constipation, diarrhea, and/or vomiting, with an item-mean score >2 on at least one scale 
of the GI module of the PedsQL scale;  4. have clinical anxiety symptoms with an item mean of 
>1.0 (0-3 scale) on the new Autism Anxiety Scale.  They will be recruited from minority, poor, 
inner city, or rural populations. 
Exclusion Criteria: 1. Antibiotics in 2 months prior to enrolling; 2. Prior bowel surgery; 3.. 
Chronic serious medical condition (e.g., diabetes); 4. Weight or height < 3rd %ile for age; 5. 
Chronic anti-inflammatory use within 2 months prior to enrolling;  6. History of inflammatory 
bowel disease, Celiac disease, or eosinophilic disorders (e.g., eosinophilic esophagitis); 7. 
Already taking probiotics  within the previous 6 months. 
 
Recruitment:  For efficiency in the pilot we will take advantage of screening and initial GI & 
ASD assessment of an ongoing Autism Speaks study of ASD microbiome (Williams & Savidge). 
The baseline stool samples for a considerable number in that study have already been collected 
and analyzed, saving that expense for the pilot. We will aim recruitment efforts to underserved 
minority, inner-city, and rural populations. The Catchment area for the Nisonger Center UCEDD 
and NCH includes the Appalachian SE quadrant of OH and adjacent parts of WV and KY. 
Columbus has 45,000 Somali-Americans with a high rate of autism. We will check the 
race/ethnicity, zip codes, and income of the patients in the genome studies with already 
documented GI problems and a stool sample done and, with IRB approval, contact those who 
are eligible as underserved, to offer them participation in the study. 
Family Engagement: Thomas Hess, leader of the NCH Family Advisory Committee, 
provided input to the protocol. Families are partners in the research. Parents are recognized as 
the experts on their own child’s behavior and feelings; their frank appraisals of effects are 
necessary for successful results. Parent stress changes will be measured as an outcome. Their 
perceptions of the experience on a satisfaction survey at the end will inform implementation of 
the RCT. They will be given the results and will be asked to help disseminate the findings. 
 
Diagnostic Measures: One or the other will be done by a research-reliable clinician (if not 
already done by a reliable administrator) at screen in addition to clinical diagnosis:  
Autism Diagnostic Observation Schedule (ADOS) (Rutter et al., 2004; Lord, Rutter et al, 2012) 
is an investigator-based assessment that places the child in naturalistic social situations 
demanding specific responses. Behaviors are coded for social communication, social 
relatedness, play, imagination, and repetitive behaviors.  
Autism Diagnostic Interview-Revised (ADI-R).(Rutter, et al, 2003): The ADI-R “short version” 
(40-item algorithm) is a highly-structured method of eliciting information from a parent to confirm 
a clinical impression of autism in children and adults.  
 
Outcome Measures (see also Table 1, Schedule of Measures):   


Probiotic for QOL in ASD 
 
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The primary outcome measure is the Gastrointestinal (GI) Module of the Pediatric 
Quality of Life Inventory (PedsQL) (Varni et al., 2001;  (Varni, Burwinkle, & Seid, 2006; Varni et 
al, 2014).  It is a 74-item survey with 14 scales (# of items): stomach pain & hurt (6 items), 
discomfort when eating (5), food & drink limits (6), trouble swallowing (3), heartburn/reflux (4), 
nausea/vomiting (4), gas & bloating (7), constipation (14), blood in poop (2), diarrhea (7), worry 
about going poop (5), worry about stomachaches (2), medicines (4), and communication (5). 
Report forms for specific age ranges assess the parent’s perception of the child’s GI function 
and/or symptoms during the last month on a 5-point scale from 0 (never a problem) to 4 (almost 
always a problem).  Items are reverse-scored and transformed to a 0-100 scale so lower scores 
reflect worse GI dysfunction Response choices are in Likert-scale format ranging from 0 to 4 
(0=Never, 1=Almost Never, 2=Sometimes, 3=Often, 4=Almost Always). There are 4 versions, 
for ages 2-4 years, 5-7 years, 8-12 years, and 13-18 years.  We will use the age-normed scale 
appropriate for each child; once selected, the same scale will be used throughout that child’s 
participation. Clear written instructions will be reviewed with the parent for all scales. 
An important secondary measure is Target Symptom Rating (Arnold et al, 2003), for 
which parents are asked to name the 2 problems of most concern to them at baseline; a 
clinician helps the parent quantify and describe the problem (frequency, duration, severity, 
interference with daily life) at baseline. At subsequent visits the clinician reminds the parent of 
the previous description and helps them again quantify/describe the current state. A panel of 
blind clinicians reviews the descriptions and rates each on a 9-point scale relative to baseline, 
from remission to disastrously worse, with 5=no change. These ratings are averaged, capturing 
the issues of most concern to parents across families. For purposes of this study, one of the 2 
problems will be required to pertain to GI function, and will be analyzed separately as well as 
being averaged into the overall symptom rating. 
The main measure of emotional stability/anxiety is the new Parent Anxiety Checklist--
ASD (Scahill, Lecavalier, Bears, & Aman, 2015) developed by the NIMH-funded consortium, 
“Toward outcome measurement of anxiety in youth with autism spectrum disorders”: Using the 
Child and Adolescent Symptom Inventory’s anxiety items as its nucleus, investigators have 
expanded the scale’s content following a review of the literature and after conducting 6 focus 
groups with parents of children with ASD. This scale will likely become the standard for use in 
randomized clinical trials. We are fortunate to have the most recent (41-item) version available.  
The Aberrant Behavior Checklist (ABC) (Aman et al., 1985a, 1985b) is a 58-item parent 
rating on a 0-3 scale with five subscales: 1) Irritability (includes agitation, aggression, and self-
injury, 15 items); 2) Social Withdrawal (16 items); 3) Stereotypies (7 items); 4) Hyperactivity (16 
items); and 5) Inappropriate Speech (4 items), (Aman et al., 1985a; Aman et al. 1985b; Aman et 
al., 1987, Brown, Aman, & Havercamp, 2002). The ABC is commonly used in ASD RCTs. 
Social Responsiveness Scale (SRS) (Costantino et al., 2003). This 65-item rating scale 
measures the severity of autism spectrum symptoms as they occur in natural social settings.  
Completed by a parent or teacher in 15 to 20 minutes, the SRS provides a clear picture of a 
child's social impairments, assessing social awareness, social information processing, capacity 
for reciprocal social communication, social anxiety/avoidance, and autistic preoccupations and 
traits. It is appropriate for use with children from 4 to 18 years of age and will detect changes in 
core ASD symptoms.  
The abbreviated Children’s Sleep Habits Questionnaire (CSHQ) (Owens, Spirito, & 
Mcguinn, 2000) will be a secondary outcome measure.  It includes 33 items rated 
retrospectively over the previous week by parents. Eight subscales include: (1) bedtime 
resistance (2) sleep onset latency, (3) sleep duration, (4) anxiety around sleep, (5) night 
awakenings, (6) sleep disordered breathing, (7) parasomnias and (8) morning waking/daytime 
sleepiness. In a recent study of the Autism Treatment Network, 75% of the participants 
analyzed had a CSHQ score >41, the clinical threshold for sleep problems (Hollway, Aman, and 
Butter, 2013). Sleep greatly affects quality of life for both children and parents, and it is 
important to detect any changes in this important vegetative function. 
 
The Parenting Stress Index Short Form (PSI) (Abidin,1995) will be given at baseline & 8 
wk.  The PSI is used to evaluate the degree of stress in the parent-child relationship. The Short 


Probiotic for QOL in ASD 
 
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Form has 36 items from the full length PSI, rated on a 5-point scale from 1 = strongly disagree, 
to 5 = strongly agree. It is completed in 10-15 minutes. The PSI may be used for parents of 
children up to 12 years. It yields a Total Score and three domain scores. This will detect effect 
on parental stress and QOL. 
Vital signs, concomitant treatments, & adverse events will be collected each visit. Fecal 
bacterial DNA and metabolites will be measured at baseline and end of each condition. 
Microbiome will be characterized by sequencing the 16S rRNA gene and extensive 
bioinformatics analysis of bacterial composition, diversity, and community structure.  PCR will 
assist 16S sequencing efforts to quantify probiotic loads in stool specimens.  LC- and GC-mass 
spectrometry will identify metabolites of interest in anxiety, as well as a panel of microbial-
produced neurotransmitters (GABA, serotonin, indoles). Complete multi-omic analysis of a 
subset of children in each order of condition, integrating microbiome, metabolome, and clinical 
metadata, will allow the identification of correlative profiles with anxiety in ASD. 
 
Treatment:  The probiotic mix (VISBIOME) will be mainly Bifidobacteria and Lactobacilli (8 
species listed under “Preliminary work”), in view of the previously reported encouraging clinical 
studies and safety data. Probiotic and matched placebo will be supplied by the company in 
powder packets containing 900 billion organisms, a half packert to be taken twice daily mixed 
with food.  
Adherence will be measured by packet counts of returned probiotic and placebo 
containers. An additional check on adherence for a subset of children will be change in specific 
VISBIOME bacterial content in gut microbiome composition. 
 
Statistical Considerations:  During the pilot phase, the focus will be on assessment of 
feasibility (meeting at least 3 of the specific items for Aim 1), safety (Aim 2; defined as no 
serious AEs attributed to the study treatment) and some suggestion of improvement in the 
PedsQOL GI scale, the primary outcome of the study, or anxiety, the chief secondary outcome.  
Sample size is limited by the pilot budget.  If we confirm that the 3 week washout period is 
reasonable (Aim X) then the intent is to pool these results with the larger randomized study for 
the final analysis.   
 
See RCT section for protection of human subjects.  Briefly, the study uses a commercially 
available product with safety evidence; locks & passwords insure confidentiality.  
 


Probiotic for QOL in ASD 
 
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Specific Aims for Stage 2, Randomized ClinicalTrial: 
Aim 1 (Primary Aim):  To test effects of probiotics (beneficial bacteria contained in VISBIOME:  
Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus 
acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus bulgaricus, & 
Streptococcus thermophilus) on gastrointestinal quality of life (GI-QOL) in 3-12 year-olds with 
ASD, GI dysfunction, and anxiety.  
Hypothesis 1:  Children with ASD will improve significantly more on the GI module of the 
PedsQOL when receiving probiotics than when receiving placebo.  
Aim 2 (Secondary);  
To test effects of probiotics on emotional stability in 3-12 year-olds with ASD & anxiety. 
Hypothesis 2:  Children with ASD will improve significantly more in anxiety when taking 
probiotics than when taking placebo. 
Aim 3 (Exploratory):  To examine moderator & mediator effects of gut macrobiome and related 
GI function. 
Exploratory Hypothesis 3A:  Children with more severe baseline GI complaints will have 
more improvement in GI function and greater placebo-controlled anxiety improvement with 
probiotics than those with fewer GI complaints. 
Exploratory Hypothesis 3B:  Children with ASD whose GI function improves more will 
have greater improvement of anxiety than those with less GI improvement.   
Exploratory Hypothesis 3C:  Children with ASD whose fecal analysis shows a greater 
enrichment in probiotic, especially Faecalibacteria, with reduced stool GABA levels will have 
greater improvement of GI function, and anxiety than those with less probiotic bacteria. 
Exploratory Aim 4:  To explore effects of probiotics and prebiotics on social 
communication, social responsiveness, and scope of interests). 
As part of several independent NIH-funded Human Microbiome Projects (HMP) on healthy and 
IBS children aged 7-12 years, we already have access to significant fecal microbiome and 
metabolome data to leverage our understanding of macrobiome changes that correlate with 
functional improvement of GI and abdominal pain symptoms associated with constipation (the 
major GI complaint in children with ASD). 
 
Research design & Methods 
In a crossover design, 60 children age 3-12 years with ASD and impairing emotional instability 
will be randomly assigned 1:1 to probiotics or probiotic-matched placebo first for 8 weeks, with a 
3-week washout followed by the opposite condition. Randomization will be stratified by site, and 
blocked over time with blocks varying in size.   
 
Inclusion/exclusion criteria, treatment, and measures will be the same as in the pilot.  See 
the schedule of measures below. 
 
Treatment will be identical with the pilot except that if the pilot data indicate that 3 weeks 
washout is not sufficient, the washout period will be lengthened as indicated by pilot data. In the 
unexpected event that the washout takes more than 7 weeks, the design will be changed to 
parallel groups and the sample expanded. We have a commitment from the ExeGI company to 
provide active 8-strain probiotic and matched placebo  We have initiated conversation about 
an IND with Paul David at the FDA so that this can be wrapped up by the project start 
date. 
.   
Recruitment:  All 3 clinical sites have established recruitment sources and strategies, being the 
main referral centers for respective metropolitan areas. Local ATN family advisory committees 
will assist with recruitment. All sites are tertiary referral centers for a large low-income inner city 
population, large numbers of minorities, and some rural patients. Although restricted to 
underserved minorities, lower SES, and rural populations, we anticipate a broader pool of 
potential subjects than usual because this trial does not require withdrawing from medication. 
The OSU site will recruit through the NCH gastroenterology clinic, where co-PI Kent Williams 


Probiotic for QOL in ASD 
 
9 
 
sees 4-6 new children with ASD and GI complaints each week. NCH (and OSU’s Nisonger 
Center UCEDD) draw from the southeastern third of Ohio, north and west West Virginia, and 
Eastern Kentudky, a large Appalachian rural area, as well as inner city Columbus.  
The University of Pittsburgh site will recruit study participants primarily from the Merck Child 
Outpatient Clinic. In accordance with ATN recommendations, children served at the Merck 
Program are regularly screened for both GI and Sleep problems. This allows program MDs and 
clinicians to readily identify children who may require intervention for GI symptoms. In addition, 
we will work with the Department of Gastroenterology at Children’s Hospital of Pittsburgh to also 
identify potential study participants. The aim will be to recruit underserved minority, inner-city 
and rural populations. The catchment area for the program includes western PA, northern WVA, 
and eastern Ohio. This regions includes metropolitan, suburban and rural areas. 
The Children’s Hospital of Philadelphia (CHOP) site will recruit primarily from established 
patients at the institution’s Regional Autism Center (RAC) or from a registry housed at the 
Center for Autism Research (AutismMatch).  Children evaluated and treated at RAC are 
routinely screened for GI problems and behavioral challenges, allowing the clinical team to 
easily identify and refer potential study participants. Families enrolled in AutismMatch have 
expressed interest in participating in research studies related to ASD and can be prescreened 
for comorbid GI problems.  Finally, the AS-ATN Philadelphia site has two actively engaged 
pediatric gastroenterologists who will aid in identifying and referring potential 
participants.  CHOP’s catchment area spans Southeastern PA, New Jersey, and Delaware, with 
patients representing urban, suburban, and rural communities.  
Family Engagement: Thomas Hess, leader of the NCH ATN family advisory committee 
(FAC), has already provided input, and other sites’ FAC input will be incorporated before the 
RCT. Families are partners in the research and will be given a plain-language summary of the 
study. Parents are experts on their own child’s behavior and feelings; their frank appraisals of 
effects are essential for successful results. Parent stress changes will be measured.  Their 
perceptions of overall usefulness on a satisfaction survey will inform future research. 
 
Schedule of Measures 
Measure 
Screen 
Baseline 
4 Wk 
8 Wk 
11 
Wk 
15 Wk 
19 Wk 
Minutes 
ADOS or ADI-R 
     X 
 
 
 
 
 
 
45-150 
Medical Hx, PE 
     X 
 
 
 
 
 
 
20 
Demographics 
 
     X 
 
 
 
 
 
10 
Vital Signs 
     X 
     X 
   X 
     X 
   X 
      X 
     X 
5 
Concomitant Meds 
     X 
     X 
   X 
     X 
   X 
      X 
     X 
2-5 
Adverse Events 
 
     X 
   X 
     X 
   X 
      X 
     X 
2-10 
 
 
 
 
 
 
 
 
 
Parent Anxiety Checklist-
ASD 
     X 
     X 
   X 
     X 
   X 
      X 
     X 
10 
GI module of PedsQL 
     X 
     X 
   X 
     X 
   X 
      X 
     X 
10-15 
Target Symptoms 
 
     X 
   X 
     X 
   X 
      X 
     X 
10 
Aberrant Behavior 
Checklist 
 
     X 
   X 
     X 
   X 
      X 
     X 
10 
 
 
 
 
 
 
 
      
 
Social Responsiveness 
Scale 
  
     X 
    
     X 
   X   
       
     X 
15-20 
Sleep Questionnaire 
(CSHQ) 
       
     X 
        
     X 
   X             
     X 
15 
Stool Sample 
 
     X 
 
     X 
   X 
 
     X 
 
Parent Stress Index 
 
     X 
 
     X 
   X 
 
     X 
10-15 
Adherence packet 
counts 
 
 
  X 
     X 
 
      X 
     X 
 
Satisfaction 
Questionnaire 
 
 
 
      
 
 
     X 
5-10 


Probiotic for QOL in ASD 
 
10 
 
  
Statistical Considerations:   
Analysis.  With our cross-over design we will have (in children completing the study), a total of 6 
measurements, three each for period 1 and period 2, plus a screening value.   We intend to 
model the results using a generalized mixed models analysis of variance, with the average of 
the screen (if available) and baseline of period 1 included as a predictor of the outcome, and 
subject as a random factor.  There are several specific effects which will be tested:  (a): is there 
a significant change for period 2 baseline from period 1, which determines whether we need to 
incorporate a term for treatment order in the analysis; and (b) is treatment and time x treatment 
(together) significant predictors of outcome?  We hypothesize that the two terms together 
(treatment and time x treatment) together will provide a significant improvement, and speculate 
that the time x treatment interaction will not be significant, which would imply that the treatment 
benefit has (largely) occurred with 4 weeks of treatment, based on pilot data presented above in 
IBD.  As suggested by the reviewer, time will be treated as a categorical factor in the primary 
analysis, and a continuous factor in a sensitivity analysis.  Site will be included as a random 
effect, and either a site x treatment or a site x treatment x time interaction will be considered 
depending on the results of the time and time x treatment interactions in the analysis of the 
primary and major secondary endpoints. We intend to combine data from both the pilot phase 
and the larger randomized study in this analysis if the washout period is confirmed in the pilot 
phase.   The primary outcome will be the level of the  PedsQOL GI module  Important 
secondary outcomes will be the change for anxiety and Target symptom ratings, . Other 
secondary outcomes include the sleep scale SRS, and ABC.   
A major secondary analysis will assess the impact of the underlying GI complaint.  The precise 
complaints will be determined based on the data availability, but based on prior data we expect 
to be using two variables: constipation (yes/no) and pain (yes/no). These analyses will focus on 
an interaction of the GI complaint flags with the primary predictor of outcome (treatment or time 
x treatment, as determined in the initial analysis). 
Additional analyses will study the relationship of baseline GI complaints, change in GI 
complaints, or in the subset of patients with microbiome data, change in GI flora with changes in 
GI function, anxiety, and other variableds.  Treatment will not be included as a predictor in these 
analyses.  Instead we will focus solely on the underlying biological variables as predictors.   As 
an illustration of the approach, we would use the change in GI complaints at 4 weeks as a 
predictor of overall pediatric QOL at 4 weeks and change in GI complaints at 8 weeks as 
predictor of pediatric QOL at 8 weeks in the generalized linear model framework, taking into 
account that these are repeated measures.   
Sample Size Considerations 
We will recruit a total of 60 participants to the second stage study.  We plan to pool the 12 
participants from the first stage with these 60 participants, assuming that the designs are 
compatible, and provide power calculations both with (and without) such pooling.   Assuming a 
sample size of 72 (60) and approximately 85% completion of the study, we would have a total of 
61 (51) participants with complete data.   As a very simplified approach to estimating the power 
of the study, a paired t-test would have 90% power to detect a difference of 0.43 (0.47) 
population standard deviations (alpha=0.05, two-tailed).  We expect that the power for the 
proposed analysis proposed would be somewhat higher (i.e. smaller differences could be 
detected with the same power) because of the additional measurements available for 


Probiotic for QOL in ASD 
 
11 
 
participants completing the study and because we would expect to have partial data even from 
participants dropping-out of the study. Importantly, we would be able to detect relatively 
moderate effect even in subgroups of size 30 (25) of 0.53 (0.61) such as when the effects are 
considered only in participants with pain or constipation as the major complaint. 
. 
 
Microbial Composition:  
Processing and Analysis of 16S Sequence Data and targeted metabolite analysis. 
Bacterial DNA will be analyzed through the Illumina pyrosequencing pipeline. Amplification will 
target the 16S rDNA gene of the bacterial genome, specifically a ~500 bp amplicon spanning 
the V1-V3 region of the gene. Each sample will be amplified using a barcoded primer, which 
yields a unique sequence identifier tagged onto each individual sample library. Processing for 
next generation sequencing will be performed, including library generation, amplicon 
purification, and emulsion PCR. Sequencing by synthesis utilizing the Illumina MiSeq sequencer 
will yield roughly >50,000 reads per sample. All generated sequences will be quality filtered to 
remove mismatched sequences (to primer or molecular identifier), short reads, and low quality 
sequences (including ambiguous base calls). The remaining quality-checked sequence set will 
be analyzed via QIIME using a reference database. Multiple analyses will be performed on the 
16S data, including determination of bacterial diversity, evenness, richness, and relative 
abundance of the bacteria identified in each sample. QIIME and supervised machine learning 
will be employed to characterize changes in relative abundance of specific organisms and 
overall bacterial diversity based on disease classification. 
For targeted metabolite analysis of stool GABA and serotonin, selective reaction 
monitoring (SRM) by Liquid Chromatography/Mass Spectrometry (LC/MS/MS) will be used as 
established in the Savidge laboratory.  
 
Analysis and integration of microbiome and clinical metadata: 
Metadata collected on all patients is listed above Our project goal is to contrast 16S rDNA 
metagenome and targeted metabolites with clinical metadata, including responsiveness to 
probiotics in the crossover trial.  The TCMC has been instrumental in developing 16S rDNA 
gene and whole genome shotgun sequencing as part of the Human Microbiome Project (HMP) 
since its inception.  Correlation of clinical metadata with multi-omic data will be assessed by 
univariate analysis and multivariate regression analyses using STATA or SAS software. 
Univariate statistics will include the Mann-Whitney U test for nonparametric continuous data and 
χ2 for comparisons of proportions among categorical data.  Normality of data will be assessed 
using the Shapiro-Wilk test. A multivariate logistic regression model will be constructed using a 
forward stepwise process with all variables associated with a p-value < 0.20 on univariate 
analysis included in the final model. The goodness of fit of the final model will be assessed 
using the Hosmer-Lemeshow test. All tests will use a p-value <0.05 to be statistically significant. 
 
Human Subjects Considerations 
Subjects will be boys and girls age 3-12 with ASD, chronic or recurrent GI dysfunction 
(constipation, abdominal pain, diarrhea, or bloating) and anxiety symptoms.  Per RFA 
requirements, they will be recruited exclusively from underserved populations (minority, poor, 
inner-city, or rural) at each site. Children are involved because that is where we expect the most 
benefit of the treatment. 
The treatment, assessments, and procedures are minimal risk, using a commercially available 
dietary supplement that we have found safe in preliminary studies, including in children with 
ASD. In 21 subjects there were no serious adverse events, including no infections with 
component bacteria, unscheduled IBS-related health care visits, change in frequency or severity 
of events, or reported causality while taking VSL#3  (Visbiome). There were 88 total AEs 
reported; they were deemed not related to VSL#3 because the symptoms reported as AEs are 


Probiotic for QOL in ASD 
 
12 
 
common symptoms in IBS. Bloating was the most common, followed by having ≥ 4 stools in a 
24-hour period. 
There could be some initial bloating or other GI discomfort, but one reason the children 
are trying this treatment is that they already have GI dysfunction. Two RCTs of irritable bowel 
syndrome, one in adults and a crossover study in children, both showed improvement of 
bloating.The risk of discomfort will be minimized by giving it with food. The only physical 
specimens collected are stools. 
The risk to confidentiality is minimized by the usual data and source document security 
of locked paper files and password-protected electronic files. The parental nuisance and time 
cost of multiple assessments at 7 visits to the center are ameliorated by reimbursement for 
transportation and time. Possible psychological distress from a question is ameliorated by 
informing that if a question is distressing, it can be skipped. Weekly teleconferences with a 
clinical panel will review possible adverse events. The value of breakthrough knowledge to be 
gained and possible individual participant benefit far outweigh the risk. 
 
Resources and Environment forTwo-Phase Study of Probiotics for Quality of Life through 
Gastrointestinal and Emotional Stability in Youth with Autism Spectrum Disorders, GI 
Problems, & Anxiety: Single-Site Pilot Trial Followed by 3-Site Randomized Clinical Trial 
The lead co-PIs are  L. Eugene Arnold, M.D.,  Professor Emeritus of Psychiatry at the 
Nisonger Center UCEDD, Ohio State University, and Kent Williams, M.D.,  Assistant Professor 
of Pediatrics and Director of Endoscopy, in the Department of Pediatric Gastroenterology, 
Hepatology, and Nutrition at Nationwide Children's Hospital (NCH), both in Columbus. For the 
follow-on RCT, the PIs and sites will be  Benjamin Handen, Ph.D., Center for Autism and 
Developmental Disorders, University of Pittsburgh, and Amanda Bennett M.D., M.P.H., Division 
of Developmental and Behavioral Pediatrics, Children’s Hospital of Philadelphia. An important 
collaborator for both the pilot and RCT will be Tor Savidge, Ph.D., Texas Children’s Microbiome 
Center, Baylor College of Medicine, Houston.  Thus the disciplines represented include 
psychiatry, gastroenterology, psychology, developmental/behavioral pediatrics, & microbiomics  
 
The OSU/NCH site in Columbus has a University Center of Excellence in Developmental 
Disorders (UCEDD, Nisonger Center) as well as an ATN site and is a tertiary referral center for 
about a third of the state of OH.  NCH is the only children’s hospital in central OH. Nisonger 
Center has a well-established clinical trials program with successful execution of both federal 
and industry trials, both pharmacologic and complementary/alternative treatments. It is one of 
the Research Units in Pediatric Psychopharmacology (RUPP) Autism Network sites and has all 
the requisite space, exam rooms, equipment, computers, etc. needed. Nisonger Center has 
special expertise in assessment in ASD, having developed the ABC, NCBRF, C-SHARP, etc. 
 
Tor Savidge at the Baylor Texas Children’s Microbiome Center  has already worked 
productively with  co-PI Kent Williams and has all equipment, software, and other resources 
needed for analysis and interpretation of stool samples for microbiome and metabolome.  
 
The other two clinical sites are well established centers for study of autism. Pittsburgh 
and Ben Handen have done numerous clinical trials, both pharmacological and behavioral. 
University of Pittsburgh is an ATN site and is well-known for psychiatric research. It is the 
tertiary referral center for western PA and northern WV, and has all the necessary clinical, 
equipment, and personnel resources. CHOP has the largest pediatric health care network in the 
U.S., is tertiary referral center for much of Eastern PA, and has all necessary resources for 
carrying out the RCT. The PI there is a leader in investigating anxiety in autism. 
All sites have an ATN family advisory committee to help with recruitment. 
 


Probiotic for QOL in ASD 
 
13 
 
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