Symptom Clusters in Children With Exacerbation-prone Asthma
Phase: PHASE2
Conditions: Asthma in Children
Sponsor: Emory University

Study Protocol 
 
 
 Symptom Clusters in Children with Exacerbation-prone Asthma 
 
 
Protocol Date: August 3, 2023 
 
 
NCT04002362 

1
Symptom clusters in children with exacerbation-prone asthma
STUDY PROTOCOL
Sponsor: 
National Institutes of Health, National Institute of Nursing Research
R01 NR018666
ClinicalTrials.gov Identifier: 
NCT04002362
Principal Investigator:
Anne M. Fitzpatrick, Ph.D.
Emory University Department of Pediatrics
Children’s Healthcare of Atlanta
Atlanta, Georgia
Version 5
August 3, 2023

2
Summary of Protocol Changes
Version 4.1, 1/17/22
•
Changed the types of blood tubes for blood sample collection (to make laboratory 
processing easier). Blood draw volumes remain the same.
Version 4, 5/18/21
•
Changed age of eligibility from 8-17 years to 6 up to 21 years of age
•
Expanded definition of “exacerbation” for eligibility purposes 
•
Added option for a single study visit with telephone/email follow-ups and review of 
medical records
•
For participants who consent to the 48 week follow-up, added option for visits 3, 4 and 5 
to be completed by telephone
Version 5, 8/3/23
•
Added two additional specific aims and their analysis plans to the study protocol that will 
be funded through an Administrative Supplement to the parent grant.
A. Background
Asthma symptom control is suboptimal in the majority of children in the United States,1-3 
despite widespread availability of asthma controller medications and standardized treatment 
guidelines.4, 5 While deaths from asthma have declined,6 53.7% of children with asthma continue 
to experience an exacerbation each year7 and the associated public health burden is 
substantial. Indeed, national surveillance data in children highlight disturbing trends of 
decreased primary care visits for asthma,8 increased emergency department visits for asthma 
exacerbations,9 and rising asthma costs estimated at $82 billion annually.10-13 School 
absenteeism14, 15 and decreased productivity in working parents12 are also consequences of 
childhood asthma that negatively impact affected children and their families.16, 17
While the factors responsible for poor 
asthma symptom control are complex and include 
limited access to care,18 poor adherence to 
preventative asthma medications3, 19 and 
exposures to environmental allergens and irritants 
such as tobacco smoke,20-22 it is also recognized 
that children with exacerbation-prone asthma are 
a heterogeneous group23, 24 with differing clinical 
outcomes and longitudinal disease trajectories.25, 
26 Symptoms (defined as subjective 
sensations) can also be quite varied within and 
among affected children. Whereas some 
children have persistent, troublesome 
respiratory symptoms, others have respiratory 
Figure 1. Symptom domains (large circles) and 
potential symptom clustering (overlap areas) in 
children with exacerbation-prone asthma. 
Peer
relationships
Family
relationships
Positive affect/
well-being
Physical
activity
Fatigue
f ct/t/
Depression/
sadness
Anxiety/
fear
ess
ess
ess
ess
Anger
Pain
Dyspnea
Wheeze
y
alalalalalalalalalalalalalal
Physical
function
/
A
/
A
/
A
/
A
/
A
/
A
/
A
/
A
/
A
/
A
Stress
Mental Health
Ph
i
Cough
Physical Health
Social Health
D
Sleep
SYMPTOM
CLUSTERS

3
symptoms only with upper respiratory infections.26, 27. Mental health symptoms and social health 
symptoms have been inadequately characterized in this population, but some children with 
asthma also report depression and anxiety28, 29 and impaired family functioning and 
relationships30, 31 that may further worsen asthma outcomes. However, prior studies are limited 
by a narrow focus on individual symptoms in isolation. To date, there has been no attempt to 
identify symptom clusters (defined as two or more concurrent symptoms independent of 
other clusters) (Figure 1) in children with exacerbation-prone asthma. 
Poor understanding of symptom clusters is a major shortcoming in asthma symptom 
science. In other chronic disorders such as cancer, compared with a single symptom, symptom 
clusters of pain, fatigue, sleep disturbance and mood disturbance significantly worsen patient-
reported outcomes of functional status and quality of life.32 There is also emerging evidence that 
interventions for one symptom within a cluster (i.e., cognitive-behavioral therapy for pain) 
reduce the severity of other symptoms within that cluster (i.e., fatigue and sleep disturbance).33 
Because children with exacerbation-prone asthma rarely report a single symptom, greater 
knowledge of the assessment (and ultimately management) of symptom clusters in these 
children has the potential to significantly improve individualized treatment and clinical outcomes. 
B. Study hypothesis/aims
We propose a 48-week cohort study (N=173) to test the overarching hypothesis that 
symptom clusters and their associated inflammatory and metabolic pathways predict 
corticosteroid treatment responsiveness (primary objective outcome) and quality of life (patient-
reported secondary outcome) in children 6 up to 21 years with exacerbation-prone asthma.
Aim 1. Identify and phenotype symptom clusters and assess their temporal trajectories in 
children with exacerbation-prone asthma. 
Hypotheses: A cluster of children with the poorest physical, mental, and social health 
symptoms will be identified by latent class analysis; this cluster will have stable symptom 
presentations over time.  
Aim 2. Determine associations between symptom clusters and clinical outcomes in 
children with exacerbation-prone asthma. 
Hypotheses: Children with the poorest physical, mental, and social health symptoms will 
have the poorest corticosteroid treatment responsiveness and quality of life.
Aim 3. Identify inflammatory and metabolic pathways underlying symptom clusters in 
children with exacerbation-prone asthma. 
Hypotheses: Symptom clusters will be distinguished by systemic and airway 
inflammatory cytokine profiles and metabolomic biomarkers.
Dr. Fitzpatrick is also receiving supplemental funding to pursue two additional aims related to 
climate change. Climate change poses a significant threat to respiratory health in patients with 
asthma. The effects are direct, through extreme heat or humidity, and indirect, through 

4
synergistic disturbances in air quality and airborne allergens. For example, rising temperatures 
accelerate production of pollutants such as ozone, fine particulate matter (PM2.5), polycyclic 
aromatic hydrocarbons, and volatile compounds, which alter airway function and host defense 
through epigenetic modifications in immune cells, airway eosinophil infiltration and epithelial 
inflammation. Heavy metals, nitrates and sulfur pollutants can also agglomerate on the surface 
of pollen grains and may increase allergenic potential and absorption of these toxicants once 
inhaled. 
The effects of climate change and air pollution are not equal. Children are more susceptible. 
Children have a higher respiratory rate and minute ventilation, which increases pollutant 
inhalation, and more narrow airways, which amplify particulate trapping, absorption, and 
epithelial inflammation and oxidative stress. The negative effects of climate are increased 
further in socially vulnerable populations. Black and Hispanic children are more likely to live 
in lower opportunity neighborhoods due to systemic structural racism and historic red lining 
practices, are exposed to more pollutants, and have more severe asthma exacerbations 
attributable to air pollution. They are also projected to have a 1.3-fold risk of living in a 
neighborhood with the highest projected increases in asthma due to increased PM 
concentrations. In these neighborhoods, fears about crime and safety and limited recreational 
green space also force children indoors. However, poorer housing standards allow for increased 
infiltration of outdoor pollutants into the home. Impaired ventilation or filtration also results in 2-5 
higher pollutant concentrations when compared to what is experienced outdoors. 
Through supplemental funding, the study team will also test the central hypothesis that climate 
change-induced disruptions in air quality worsen asthma inflammation, symptoms, and 
longitudinal outcomes in school-age children with asthma and that these effects are mediated 
by social vulnerability. Two additional aims to be pursued are below:
Aim 4. Determine associations between climate, air quality, symptoms, and outcomes in 
children with exacerbation-prone asthma during two climate seasons. 
Hypothesis: Children with greatest social vulnerability will have more air pollution exposure, 
increased symptoms, poorer lung function, more exacerbations and decreased QOL during 
each season.
Aim 5. Determine the biological impact of climate and pollutants in children with 
exacerbation prone asthma. Children with greatest social vulnerability and the highest air 
pollution exposure will have differentially expressed cytokines and metabolomic features. 
This project involves a multidisciplinary team with a history of collaboration and is aligned with 
the NINR Strategic Plan to “Advance Symptom Science Research” in chronic conditions through 
biobehavioral research.32, 34 The study population, children with exacerbation-prone asthma, has 
also been understudied in this regard. This project is expected to: 1) refine knowledge regarding 
the identification and clinical utility of symptom clusters in these children, and 2) identify 
mechanisms underlying symptom clusters that can be targeted in future interventional studies, 
to reduce the high morbidity and improve quality of life.

5
C. Significance
Importance and barriers. Asthma currently affects more than 6 million children less 
than 18 years in the United States (8.3% prevalence) and the majority of these children have 
poor symptom control.7 In 2016, more than 3.2 million children with asthma experienced an 
exacerbation necessitating unscheduled care.7 Exacerbations consist of narrowing of the airway 
lumen, inflammation, and mucus plugging that can be deadly. Even in non-fatal cases, 
exacerbations may lead to progressive loss of lung function.35-37 To date, the specific factors 
(and associated symptom profiles) that contribute to asthma exacerbations in children are 
unclear.38 Major barriers in the pediatric asthma field include inadequacies of animal models in 
recapitulating human asthma, a paucity of basic/translational studies, developmental differences 
that prohibit extrapolation of data from adults to children, and few studies of long-term outcomes 
in affected children.39 
Knowledge gap. In June 2015, the NINR/Office of Rare Disease Research/NCATS held 
a workshop on “Advancing Symptom Science Through Symptom Cluster Research.” While the 
majority of evidence presented at this workshop originated from oncology patients, it was 
recognized that symptom clusters also occur in other chronic conditions such as asthma and 
result in decrements in functional status and quality of life.32 Studies of symptom clusters (and 
ultimately development of personized strategies for symptom management) could advance 
care. However, this area of research is only emerging; existing studies tend to be cross-
sectional in nature, ignoring the temporal component of disease progression.32 To date, there 
has also been no attempt to identify and phenotype symptom clusters in asthma. Limited 
symptom science in asthma is a major shortcoming given the chronicity and burden of the 
disorder that this study is designed to address. 
D. Innovation
Novel concepts. Whereas other studies have focused on single symptom manifestations 
(i.e., wheeze or anxiety) in children with exacerbation-prone asthma, this would be the first 
study to:
Identify and phenotype symptom clusters in children with exacerbation-prone 
asthma using validated, age-appropriate PROMIS measures and a novel statistical 
analytical technique in symptom science research, latent transition analysis, to 
address temporal symptom trajectories,
Examine whether identified symptom clusters predict objective and patient-reported 
clinical outcomes (corticosteroid treatment responsiveness and quality of life) that are 
important to both physicians and affected patients, and
Link symptom clusters with inflammatory/metabolic pathways that could be targeted 
with future interventions.

6
The results of this study could have a significant impact on the assessment and 
management of children with asthma through identification of high-risk children who might 
benefit from personalized medicine approaches. While national asthma treatment guidelines do 
recognize the importance of respiratory symptoms, these guidelines provide no guidance 
regarding the assessment and management of non-respiratory symptoms in affected children.4, 5 
To address these critical 
knowledge gaps, we will 
perform extensive clinical 
characterization and 
state-of-the-art laboratory 
analyses of children with 
exacerbation-prone 
asthma. We will also use 
an adaptation of the 
conceptual framework 
proposed by Brant et al.40 
that incorporates temporality (Figure 2). Our framework proposes that: 1) children with 
exacerbation-prone asthma and a unique mixture of covariates possess an equally unique set of 
inflammatory/metabolic pathways that are each associated with symptoms, 2) those symptoms 
synergize positively or negatively in a symptom cluster that may or may not evolve over time, 
and 3) symptom clusters impact asthma outcomes.
Application of novel technology. The technology and the statistical techniques that we 
will utilize for this study are cutting-edge. We can multiplex up to 100 biomolecules with 
magnetic bead technology in 35 microliters of plasma.41 With only 2.5 microliters of plasma 
(from a starting volume of only 40 microliters), we can perform chemical partitioning with 
hydrophilic interaction liquid chromatography (using a novel Merck SeQuant ZIC-HILIC column 
that is well-known for high-performance selectivity, reproducibility and robustness) for hundreds 
of small molecules including amino acids and metabolites. This may lead to identification of new 
biomarkers and opportunities for technology transfer. Our core laboratory facilities are also 
state-of-the-art (see letters of support). Ultimately, the analyses performed will capitalize upon a 
rich sample of highly characterized children with exacerbation prone-asthma. Thus we will have 
the rare opportunity to link symptom clusters with inflammatory and metabolic pathways and key 
outcomes of clinical relevance to both physicians and patients. 
E. Study population 
We will enroll a cohort of 173 children age 6 up to 21 years with exacerbation-prone 
asthma. Participants will be given the option to either: 1) complete a single study visit, with 
telephone/email contacts and a review of their electronic medical record for up to 48 weeks after 
enrollment, or 2) complete the 48-week cohort study with scheduled study visits. Participants 
who select option 2 with follow-up over 48 weeks will also have the option to complete visits 3, 4 
and 5 by telephone.
Covariates:
Age
Sex
Race
Ethnicity
SES
Medications
Exposures
Sensitization
Family
relationships
Positive
affect
Fatigue
Depression
Anxiety
F
Anger
Pain
i
Dyspnea
ctctctctctctctctctctct
F
Wheeze
Physical
function/
activity
Stress
Dys
Dys
D
Cough
Sleep
Exacerbation-
prone asthma
Inflammatory/
metabolic
pathways
ough
Sle
ough
ough
ough
ough
ough
ough
o gh
y
h
Peer
relationships
Symptoms
Symptom
clusters
Treatment response
Quality of life
Symptom
trajectories
m
trtrtrtrtrtrtrt
S
Figure 2. Conceptual framework. 

7
Eligibility criteria include the following:

Age 6 to less than 18 years at the enrollment visit

Physician diagnosis of asthma, and  

History of an asthma exacerbation in the previous 12 months, defined as either:
o
Treatment with systemic corticosteroids,* or
o
Increase in rescue medication use (i.e., abuterol or inhaled corticosteroid) for 24 
hours or more, or
o
One or more missed school days due to asthma symptoms, or
o
An unscheduled visit for asthma at either a physician’s office, urgent care, 
hospital emergency room, or
o
Hospitalization for asthma.
*In the event of a recent exacerbation treated with systemic corticosteroids or 
requiring hospitalization, the first study visit will be postponed until two weeks 
after the last dose of systemic corticosteroids (i.e., prednisone or prednisolone).
These criteria should ensure a study population with sufficient disease burden, since 
several prior studies in children have identified these risk factors as significant predictors of poor 
asthma control and poor future outcomes.42 Indeed, several prior studies in children have 
identified a recent exacerbation as the strongest risk factor for subsequent exacerbations 
regardless of disease severity or use of controller medications.26, 43-46 In the third iteration of the 
NHLBI Severe Asthma Research Program (SARP, Fitzpatrick co-investigator), exacerbations 
occurred in 39.7% of enrolled children by one year (unpublished data), similar to reports in other 
cohorts.47, 48 In SARP, an exacerbation in the previous year was also associated with an 8.94-
fold increase in the odds of future exacerbation by one year (95% CI: 4.37, 18.69). Poor asthma 
symptom control at baseline was similarly associated with increased odds of future exacerbation 
(OR 2.65, 95% CI: 1.44, 4.82), but there was no additive effect with prior exacerbation. 
Therefore, we have elected to enroll participants irrespective of asthma symptom control. This 
will not only facilitate enrollment of participants, but will also promote symptom heterogeneity 
(which is essential for symptom clustering) and the likelihood of biomarker detection (since 
inflammation will also be heterogeneous). 
Exclusion criteria for Option 1, a single study visit with telephone/email and 
electronic medical record follow-up for up to 48 weeks, include: 

Pregnancy,

Current smoking, 

Congenital disorders or deformities of the chest wall, lungs or airways, 

History of premature birth <35 weeks gestation, or

No medical care provider for asthma. 
Exclusion criteria for Option 2, a 48-week cohort study with scheduled study 
visits, include: 

Previous allergic reaction to systemic corticosteroids,

Hepatic, biliary, or renal disease that can interfere with drug metabolism/excretion,

8

Chronic medical disorders that may increase the risk of drug-related injury, including 
osteogenesis imperfecta (increased risk of fracture with corticosteroids), or Crohn’s 
disease, ulcerative colitis, juvenile rheumatoid arthritis, clotting disorders, or Factor 
deficiency (increased risk of bleeding with corticosteroid therapy), 

Pregnancy,

Current smoking, 

Congenital disorders or deformities of the chest wall, lungs or airways, 

History of premature birth <35 weeks gestation, 

Unwillingness to receive triamcinolone, 

No medical care provider for asthma, or 

Planning to relocate before study visit 2.
F. Rationale for heterogeneous study population
In children, asthma symptoms are complex and result from a variety of triggers including 
allergens, irritants, pollutants, microbes, viruses, psychological stressors and emotions.43, 44 
These triggers induce airway changes such as mucus production, hyperresponsiveness, 
smooth muscle hypertrophy and constriction that result in respiratory symptoms (and 
consequent physical, mental and social health symptoms). However, triggers act through 
different pathways (Figure 3) and likely yield different symptom clusters. For example, allergen 
sensitization is associated with Type-2 (i.e., Th2) eosinophilic inflammation and is thought to be 
a pivotal risk factor for persistent and severe wheezing 49, 50 and airflow obstruction.51 By 
contrast, children with non-Type-2 neutrophilic inflammation tend to wheeze only with 
respiratory viruses and often have cessation of asthma symptoms during later childhood.52 
Children with non-Type-2 inflammation also have increased IL-8 expression during the course of 
the exacerbation,53-55 which may reflect innate immune impairment in response to viral56 or 
bacterial57 exposures. 

9
Nonetheless, 
the paradigm of “Type-
2” versus “non-Type-2” 
inflammation is not 
quite as simplistic as 
depicted and there can 
be synergy of triggers 
and pathways. For 
example, in addition to 
being a pivotal risk 
factor for asthma 
persistence, Type-2 
inflammation has also 
been associated with 
increased severity of 
upper and lower 
respiratory tract 
infections and a 
greater risk of hospitalization for human 
rhinovirus infection58, 59 Psychological 
stress can also skew T cells toward a Th2 phenotype, since norepinephrine suppresses Th1 
cytokines (i.e., IFN-γ) via binding to adrenergic receptors.60 Additionally, stress promotes DNA 
methylation and alters expression of numerous genes that regulate behavioral, autonomic, 
neuroendocrine, and immunological responses.61 Chronic stress further exerts direct effects on 
airway smooth muscle through downregulation of the β2-adrenergic receptor,62 possibly through 
dysfunction of the pituitary adenylate cyclase-activating polypeptide 1 pathway which has been 
previously implicated in children with anxiety63 and asthma.64 We therefore anticipate that the 
synergy of physical respiratory symptoms with mental and social health symptoms 
augments inflammation and results in poorer asthma outcomes compared to children with 
respiratory symptoms alone. Identification of symptom clusters and their biological features may 
ultimately advance knowledge of inflammation (and treatment) in children with exacerbation-
prone asthma. 
G. Rationale for inflammatory measures
In clinical practice, many children with asthma have ongoing airway inflammation and 
poor symptom control despite best therapy attempts with controller medications such as inhaled 
corticosteroids. Inflammation is driven by white blood cell release of cytokines and chemokines, 
which in turn, promote generation of reactive oxygen species (ROS) and free radicals that 
regulate inflammation through complex interactions with cysteine residues on the surface of 
proteins. These protein modifications may or may not be reversible65 and could result in 
sustained inflammation and tissue damage. For example, nitration and chlorination of catalase 
results in impairment of the enzymatic catalyzation and breakdown of hydrogen peroxide,66 
whereas nitration of manganese superoxide dismutase and glutathione-S-transferase inhibits 
Type-2 inflammation
Non Type-2
inflammation
Allergens
Irritants, pollutants,
microbes, viruses
Macrophages
Antigen
presenting cell
Th0
Th1
Th2
Th17
CrTh2, IL-4,
IL-5, IL-6,
IL-10, IL-13,
IL-25
IL-2, IL-4,
IL-10
CCR4, CCR6, IL-17A,
IL-17F, IL-21, IL-22,
IL-24, IL-26
TGF-β, IL-1β,
IL-6, IL-23, IL-33
IFN-γ, IL-12
CCR5, CXCR3,
TNF-α, IL-2,
IFN-γ, IL-12
Hyperresponsiveness, remodeling,
mucus production and smooth
muscle constriction and hypertrophy
Th2
Stress, Emotions
10, IL 13,
25
Eosinophils
Neutrophils
Mast cell
IgE
SYMPTOMS
Figure 3. Asthma triggers (yellow) and pathways 
contributing to symptoms/symptom clustering. 

10
detoxification of superoxide and other electrophilic substances.67, 68 Another example involves 
glutathionylation of the p50 subunit of the transcription factor, nuclear factor kappa B, which is 
associated with repressed DNA binding activity.69 
There is also a wide body of literature demonstrating the inhibitory effects of ROS on 
corticosteroid responsiveness, through direct actions on the glucocorticoid receptor (GR), 
resulting in a sustained pro-inflammatory state.70 Ligand binding of the GR in the cytoplasm 
results in translocation of the activated GR complex into the nucleus, where it binds to the DNA 
glucocorticoid response element and regulates gene transcription through recruitment of histone 
deacetylase (HDAC) complexes.71, 72 Whereas increased histone acetyltransferase activity 
promotes acetylation of histones and inflammatory gene transcription, removal of acetyl groups 
by HDACs results in gene silencing.73, 74 Like any protein, the GR contains several cysteine 
residues that maintain its structure and function; these are also highly sensitive to ROS and can 
form disulfide bonds that inhibit ligand binding to the receptor75 or cause the GR to assume a 
folded conformation,76 inhibiting nuclear translocation and DNA binding.77, 78 These actions 
might account for prior observations of GR dysfunction (with unattenuated proinflammatory 
cytokine release and increased levels of glucocorticoid hormones) in patients with severe 
asthma79-82 and also in patients with chronic stress and anxiety,83 depression84, and fatigue.85 
We anticipate that inflammation will be greatest in the cluster of children with the poorest 
physical, mental, and social health symptoms; we also anticipate that this inflammation 
(from symptom synergy) will be less responsive to systemic corticosteroid treatment, 
resulting in persistently poor quality of life. 
H. Rationale for triamcinolone acetonide injection
At the completion of the baseline visit, a subset participants who consented to the 48 week 
cohort study will receive intramuscular triamcinolone acetonide (Kenalog, 1 mg/kg, up to 40 
mg maximum) administered deep in the gluteal muscle by a trained registered nurse. 
Corticosteroids are the cornerstone of treatment for asthma;4, 5 intramuscular triamcinolone was 
selected as the treatment of choice over oral prednisolone or inhaled corticosteroids to allow for 
characterization of corticosteroid insensitivity without the confounding effects of poor adherence 
or inadequate drug delivery or absorption. We further considered the pharmacology of 
corticosteroids. Because triamcinolone has 20% more glucocorticoid activity than 
prednisolone,86 triamcinolone 1 mg/kg will ensure that participating children have a sufficient 
dose of corticosteroid to achieve the desired potential anti-inflammatory effects. In previous 
studies of adults, a single dose of 80 mg of triamcinolone significantly improved lung function 
and asthma symptoms in the majority of patients.87-89 Given several previous studies in 
children90-95 and our own published studies of triamcinolone responsiveness in children with 
asthma,79, 96, 97 a dose of 1 mg/kg of triamcinolone should elicit a clinical response. Because 
inclusion is limited to children with a recent exacerbation (many of whom will have poor 
symptom control and all of whom are at risk for future exacerbation), all participants have the 
potential to benefit from this procedure. The safety of our dosing strategy has also been well 
established. In our previous study (Fitzpatrick R01NR012021),96 the triamcinolone injection was 
well tolerated and no unexpected adverse events were reported. Expected adverse events 

11
within 24 to 48 hours of triamcinolone receipt included tenderness (25%), warmth (3%), and 
mild itching (2%) at the injection site. No activity limitation from injection pain was reported and 
there were no reports of subcutaneous atrophy following the injection. Other unrelated adverse 
events included dizziness (n = 1), atopic dermatitis (n = 1), facial rash (n = 1), headache (n = 1), 
nausea and vomiting (n = 1), pharyngitis (n = 1), and cough (n = 1). The distribution of adverse 
events did not differ according to asthma severity or treatment response group, and each event 
resolved within 14 days.96 In the proposed study, the one-time administration of 1 mg/kg of 
triamcinolone (40 mg maximum) has documented safety and will result in a significantly smaller 
cumulative corticosteroid dose than that used by other pediatric investigators.90-92 

12
I. Overview of study design
We will enroll a cohort of 173 children age 6 up to 21 years with exacerbation-prone 
asthma. Participants will be given the option to either: 1) complete a single study visit, with 
telephone/email contacts and a review of their electronic medical record for up to 48 weeks after 
enrollment, or 2) complete the 48-week cohort study with scheduled study visits (Appendix 1). 
Participants who select option 2 with follow-up over 48 weeks will also have the option to 
complete visits 3, 4 and 5 by telephone.
  
Option 1: A single study visit with telephone/email and electronic medical record follow-
up for up to 48 weeks
Visit 1 (enrollment) will involve informed consent and participant characterization for the 
purpose of symptom cluster identification. After this visit is completed, the participant’s medical 
record will be reviewed for up to 48 weeks to assess for asthma exacerbations requiring medical 
treatment. Telephone/email contacts will also be attempted at 24 weeks and 48 weeks after this 
visit to ensure that asthma exacerbations treated outside of a healthcare facility or other asthma 
exacerbations that are managed at home are accounted for.
Option 2: A a 48-week cohort study with scheduled study visits
Visit 1 (enrollment) will involve informed consent and participant characterization for the 
purpose of symptom cluster identification and receipt of systemic triamcinolone acetonide. Visit 
2 will involve assessment of triamcinolone responsiveness. Visits 3, 4 and 5 are follow-up visits 
to assess the temporal stability of the symptom clusters. Visits 3, 4 and 5 can be completed in 
person or over the telephone.
This research is considered a clinical trial, because it involves an intervention 
(triamcinolone injection), and the study is designed to evaluate the efficacy of the intervention 
on symptoms and quality of life. The Emory Office of Clinical Research will facilitate the
registration, periodic updates, and results reporting within ClinicalTrials.gov for the duration of 
the study.
J. Outcome measures
Asthma exacerbations are an outcome of interest for all participants. 
For participants enrolled in the 48-week cohort study, the primary outcome is 
corticosteroid treatment responsiveness, assessed 14 days after systemic triamcinolone 
administration (at visit 2). Responsiveness will be determined from the mean difference in the 
Asthma Control Questionnaire (ACQ) score between visit 1 (baseline) and visit 2 (14 days), with 
a reduction of 0.5 considered a minimally important difference.98, 99 

13
The secondary outcome for participants enrolled in the 48-week cohort study is quality of 
life, assessed by the PROMIS Pediatric Asthma Impact Scale (PAIS) at visits 3, 4 and 5. As 
with all PROMIS measures, the PAIS is scored on the T-score metric, with higher scores 
reflecting more of the concept being measured. On the T-score metric, 50 is the mean of the 
reference population and 10 is the standard deviation; thus scores of 40 and 60 are one 
standard deviation lower and higher than the mean of the reference population, respectively.
K. Visit-specific procedures
Detailed procedures are listed below and also appear in the Appendix (Appendix I).
Option 1: A single study visit with telephone/email and electronic medical record follow-
up for up to 48 weeks
Baseline visit (Visit 1), study week 0 
1.
Informed consent obtained
2.
Eligibility determined based upon inclusion and exclusion criteria
3.
Brief physical examination (by RN) including height and weight performed
4.
Medical history obtained
5.
Urine pregnancy testing 
6.
Administer Demographic questionnaires 
7.
Asthma control evaluation (Asthma Control Questionnaire and Asthma Control Test)
8.
Administer PROMIS symptom questionnaires 
9.
Measure exhaled nitric oxide
10. Perform spirometry with oscillometry (with bronchodilator reversibility)
11. Perform venipuncture for:
a.
Total IgE (Children’s Healthcare of Atlanta)
b.
Inhalant allergen specific IgE (Children’s Healthcare of Atlanta)
c.
Complete blood count with differential (Children’s Healthcare of Atlanta)
d.
Plasma and white blood cells for aim 3 (Fitzpatrick laboratory)
12. Perform sputum induction with samples stored for aim 3 (Fitzpatrick laboratory)
Telephone/email contact at 24 weeks and 48 weeks
1.
Medical history obtained
2.
Asthma control evaluation (Asthma Control Questionnaire, Asthma Control Test, 
Pediatric Asthma Impact Questionnaire)
Option 2: A 48-week cohort study with scheduled study visits
Baseline visit (Visit 1), study week 0 
1.
Informed consent obtained
2.
Eligibility determined based upon inclusion and exclusion criteria
3.
Brief physical examination (by RN) including height and weight performed
4.
Medical history obtained

14
5.
Urine pregnancy testing 
6.
Administer Demographic questionnaires 
7.
Asthma control evaluation (Asthma Control Questionnaire and Asthma Control Test)
8.
Administer PROMIS symptom questionnaires 
9.
Measure exhaled nitric oxide
10. Perform spirometry with oscillometry (with bronchodilator reversibility)
11. Perform venipuncture for:
a.
Total IgE (Children’s Healthcare of Atlanta)
b.
Inhalant allergen specific IgE (Children’s Healthcare of Atlanta)
c.
Complete blood count with differential (Children’s Healthcare of Atlanta)
d.
Plasma and white blood cells for aim 3 (Fitzpatrick laboratory)
12. Perform sputum induction with samples stored for aim 3 (Fitzpatrick laboratory)
13. Triamcinolone acteonide injection
Steroid Responsiveness visit (Visit 2), Study Week 2 (window +7 days)
1.
Brief physical examination (by RN) including height and weight performed
2.
Medical history obtained
3.
Urine pregnancy testing
4.
Asthma control evaluation (Asthma Control Questionnaire and Asthma Control Test)
5.
Administer PROMIS symptom questionnaires 
6.
Measure exhaled nitric oxide
7.
Perform spirometry with oscillometry (with bronchodilator reversibility)
8.
Perform venipuncture for:
a.
Complete blood count with differential (Children’s Healthcare of Atlanta)
b.
Plasma and white blood cells for aim 3 (Fitzpatrick laboratory)
9.
Perform sputum induction with samples stored for aim 3 (Fitzpatrick laboratory)
Follow-up visits (Visits 3 and 4), Study Week 16, 32 (window +4 weeks)
1.
Brief physical examination (by RN) including height and weight performed*
2.
Medical history obtained
3.
Urine pregnancy testing*
4.
Asthma control evaluation (Asthma Control Questionnaire and Asthma Control Test)
5.
Administer PROMIS symptom questionnaires 
6.
Measure exhaled nitric oxide*
7.
Perform spirometry with oscillometry* 
*procedure will not be performed if follow-up occurs by telephone
Final follow-up visit (Visit 5), Study Week 48 (window +4 weeks)
1.
Brief physical examination (by RN) including height and weight performed*
2.
Medical history obtained
3.
Urine pregnancy testing*
4.
Asthma control evaluation (Asthma Control Questionnaire and Asthma Control Test)
5.
Administer PROMIS symptom questionnaires 

15
6.
Measure exhaled nitric oxide*
7.
Perform spirometry with oscillometry (with bronchodilator reversibility)*
8.
Perform venipuncture:*
a.
Complete blood count with differential (Children’s Healthcare of Atlanta)
b.
Plasma and white blood cells for aim 3 (Fitzpatrick laboratory)
9.
Perform sputum induction with samples stored for aim 3 (Fitzpatrick laboratory)*
*procedure will not be performed in follow-up occurs by telephone
L. Procedural details
Asthma control evaluation. Asthma control and associated respiratory symptoms will 
also be assessed with the Asthma Control Questionnaire (ACQ) and the Asthma Control Test 
(ACT). The ACQ can be completed by children down to 6 years with strong measurement 
properties when administered by a trained interviewer.98 The ACQ is sensitive to short-term 
changes in asthma control and has well-documented reliability and cross-sectional and 
longitudinal validity in diverse asthma populations across a wide range of severities.100 This 7-
item questionnaire includes questions related to daytime and nocturnal symptoms, short-acting 
bronchodilator use, and lung function during the clinic visit on that day. A higher score indicates 
poorer asthma control.101 The ACT can be completed directly by children 12 years and older; 
children 6-11 years will complete the Childhood ACT (C-ACT). Both tools have been extensively 
validated and have excellent measurement properties.4, 102-107 For both tests, individual items 
about respiratory symptoms (5 and 7 in total for ACT and C-ACT, respectively) are summed; 
scores of 19 or less reflect uncontrolled asthma.
PROMIS symptom questionnaires. Symptoms will be assessed with validated 
PROMIS measures for children. PROMIS measures will be completed directly by children age 
8-21 years, with caregiver assistance for children 6-7 years. The following PROMIS short-form 
measures will be utilized:

Pediatric Life Satisfaction (4a)

Pediatric Meaning and Purpose (4a)

Pediatric Physical Stress Experience (4a)

Pediatric Physical Activity (4a)

Pediatric Positive Affect (4a)

Pediatric Psychological Stress Experiences (4a)

Pediatric Family Relationships (4a)

Pediatric Asthma Impact (8a)

Pediatric Anger (5a)

Pediatric Anxiety (8a)

Pediatric Depressive Symptoms (8a)

Pediatric Fatigue (10a)

Pediatric Peer Relationships (8a)

16
Spirometry with Oscillometry. Spirometry for measurement of lung function will be 
performed with a portable spirometer (MGC Diagnostics® CPFS/D USB™) according to 
technical standards.108 The best of three forced vital capacity maneuvers will be interpreted 
according to population reference standards.109 Oscillometry will be performed in cooperative 
participants (typically age 3 and older) with the TremoFlo C-100 device (Thorasys). Participants 
will wear a noseclip and take tidal breaths through a mouthpiece for 20 seconds. Airway 
resistance and reactance will be recorded.
Bronchodilator reversibility testing. Participants will receive 4 inhalations of albuterol 
from a metered dose inhaler (MDI) and valved holding chamber and spirometry will be 
repeated 15 minutes later. 
Exhaled nitric oxide. Exhaled nitric oxide will be measured with a commercial device 
(NIOX VERO®, Circassia) using recommended standards.110 The procedure is non-invasive 
and considered to be an indirect measurement of airway inflammation. Participants will be 
instructed to take in a deep breath and blow air out at a constant pressure. One 
measurement will be taken and recorded.
Venipuncture.  Blood will be collected by venipuncture as outlined in the table below. 
Participants will have the option to receive anesthetic cream prior to venipuncture.
Test
Where 
performed
Test 
code
Visits
Collection tube
Blood 
collection 
volume
Total IgE and 
Specific IgE
Children’s
TIGE, 
INHALA 
Panel
1
Gold top SST 
1.5 mL
Complete blood 
count with differential
Children’s
CBCD
1,2,5
Lavender EDTA
1 mL
Cytokines, 
metabolomics
Fitzpatrick lab
1,2,5
Lavender EDTA
Blue tempus
~6 mL
~3 mL (+6 
mL solution)
Table 2. Blood collection procedures
Urine collection for pregnancy testing. Urine will be collected in females of child-
bearing potential by clean-catch methods for the purpose of pregnancy testing. Unused urine 
will be discarded.
Sputum induction. Sputum induction will be performed in participants 12 years and 
older after pre-treatment with 4 inhalations of albuterol sulfate delivered through a MDI and 
holding chamber. 3% saline (substitutions permitted) will be inhaled through an ultrasonic 
nebulizer for a maximum of 12 minutes, with breaks every 2 minutes. To ensure safety, the 
procedure will only be performed on children with a post-bronchodilator FEV1 of >70% 
predicted; spontaneous sputum samples may be collected on children with an FEV1 below 

17
these limits. A physician will be available during the challenge; study staff will calculate and 
record the FEV1 value that equals both a 10% and 20% fall in lung function based upon 
the recorded post-bronchodilator FEV1 values. Participants will not discharged until their FEV1 
is within 10% of their post bronchodilator FEV1 and will be given albuterol sulfate (2 inhalations 
as needed) for this purpose. Sputum samples will be processed by the Fitzpatrick laboratory.
Triamcinolone acetonide injection. Each participant who meets inclusion/exclusion 
criteria will receive triamcinolone acetonide intramuscularly at the end of visit 1. Children 6-
21 years will receive 1 mg/kg triamcinolone acetonide (up to 40 mg maximum). 
Triamcinolone acetonide will be administered as a single intramuscular dose deep in the 
gluteal region. 
M. Non-study drugs
Because this study is not a treatment trial, any medication considered necessary for the 
medical management and welfare of the participant may be given at any time. Participation in 
this study will also not prohibit access to new or novel therapies. Medications will not be 
withheld.
N. Drug supplies
The study intervention, triamcinolone acetonide (Kenalog for injection), is FDA-
approved for the treatment of asthma in children. There is no IND associated with this study. 
The drug will be administered consistent with the instructions listed in the accompanying 
package insert.
The following drug supplies will be provided by the research pharmacy:
Triamcinolone acetonide (Kenalog® for injection), 1 mg/kg, 40 mg max
Albuterol sulfate (90 mcg/inhalation) for bronchodilator reversibility testing and sputum 
induction
3% saline for sputum induction (substitutions and compounding permitted)
O. Study failure
Given the observational nature of this study, there are no specified criteria for study 
failure.  Communication with each participant’s primary caregiver will be ongoing. 
P. Availability of study personnel
The study physician (Dr. Claudia Morris) will be available for discussion with families 24 
hours/day should uncertainty or questions arise regarding the study. Communication regarding 
the study, receipt of triamcinolone, and laboratory results will be sent to the child’s primary care 
provider. At all study visits, coordinators will emphasize the importance of maintaining contact 
with the child’s primary care provider.

18
Q. Recruitment and Retention 
Prospective enrollees will be enrolled at Children’s Healthcare of Atlanta, which serves 
an urban population with diverse demographic features including race, ethnicity and 
socioeconomic status. Eligible participants will be identified from within the Children’s 
Healthcare of Atlanta system by ICD-10 codes for wheezing and asthma. We will obtain a 
HIPAA waiver from the Emory and Children’s Healthcare of Atlanta IRBs for this purpose (i.e., 
identification of eligible participants). Once the list of eligible participants is generated, IRB-
approved mailings will be sent to these families informing them of the study and the phone 
number/email address of the study coordinator to use if they would like more information. We 
will also run printed advertisements in Atlanta Parent Magazine and contact previous study 
patients/caregivers (who have given us permission to do so) to see if they are interested.  
We anticipate no problems with our recruitment approach since the burden of asthma and 
wheezing disorders is significantly higher in Georgia versus other states. Indeed, the current 
asthma prevalence in children in Georgia is 12.1% compared to a pooled estimate of 9.0% in 38 
other states.111 Our planned recruitment strategies were also previously utilized in our NHLBI 
AsthmaNet studies and yielded a large number of eligible participants who were interested in 
research. Emory University, under the leadership of Dr. Anne Fitzpatrick, was one of the lead 
recruitment sites for multiple NHLBI longitudinal asthma studies.
Retention of research participants is essential for this longitudinal study. In our 
experience, the best retention method for participants in long-term clinical studies is education 
and continual “reminders.” For all participants enrolled in our clinical research studies, including 
the study proposed here, newsletters are distributed every 3 months (by mail or email) that 
include educational material about respiratory health, bulletins of important issues that affect 
patients (flu vaccines), and study updates. We also have several additional methods to 
encourage continued involvement in our studies, including distribution of materials with our 
logos and contact numbers and other continual “reminders” of study involvement (refrigerator 
magnets, pens, mugs, t-shirts or blankets). Other retention efforts will focus on ease of the 
majority of visits and informational rewards (such as asthma education). Visits will be at times 
convenient to the parents, many of whom are employed. Appointments after school will also be 
available. Study team members will be available to answer questions about asthma. The study 
physician will also be available by phone during off-hours to aid in the management of adverse 
events including asthma exacerbations. 
Participants will also be paid for their time and participation in the study. While we will 
enroll 173 children, we expect some dropout to occur. Study visits will be pro-rated accordingly, 
and families will be paid at the end of each visit. Parking passes are also provided so that 
families do not have to pay for parking. In the event that transportation is a challenge, we also 
pay for taxis to pick up participants at their homes and drive them to the study site.

19
R. Risks and benefits
Asthma exacerbation. The inclusion criteria require that all participants have a history 
of asthma exacerbation, and therefore we can expect a similar pattern of illness (i.e., a high 
probability of subsequent exacerbation) in the year of study enrollment. While we do not 
anticipate that children enrolled in this study will have significant worsening of asthma 
symptoms as a result of the study procedures, children enrolled in this trial may develop an 
asthma exacerbation of sufficient severity to warrant emergency medical care. Hospitalization 
will be considered a Serious Adverse Event, and be reported to local IRBs in the usual manner. 
Parent-completed questionnaires (medical history, demographics) and child-
completed questionnaires (asthma control, PROMIS symptoms). Participants will complete 
a variety of questionnaires at each study visit. There are minimal risks to answering these 
questionnaires, although they may be considered long with repeating questions. For certain 
questions (such as those that ascertain family education or income level), participants will be 
allowed to decline from responding. Some of the questionnaires pertaining to depression and 
anxiety may also highlight significant yet undiagnosed mental health disorders. We will 
encourage all children to discuss their concerns with their parents, teachers or physicians. In 
more severe cases, we will also identify available support services through Children’s 
Healthcare of Atlanta and share these with participating children and their families. In severe 
cases where suicidal ideation is stated or presumed, emergency referrals will be made in 
accordance with Children’s Healthcare of Atlanta policies and procedures.
 There is a rare risk that information from a study can be disseminated in ways that can 
risk the privacy of a person with attendant social and occupational harm. However, this risk is 
not as great for conditions like asthma, which are common. Regardless, care will be taken to 
ensure confidentiality. Research records will be held in locked cabinets or secure storage 
rooms.  All transmission of data to the study co-investigators for analysis is by study ID code 
only. Publication of results will involve aggregate data only so that individual participants cannot 
be identified. On occasion it may be necessary, for legal reasons or for good clinical practice, for 
third parties such as the FDA, IRB, or NIH to review medical records that are identified by name. 
However, this is not a common occurrence, and every effort will be made by the investigators to 
provide confidentiality for such audits.
Urine pregnancy testing. Women of child-bearing potential will submit a urine sample 
at each study visit for pregnancy testing. Pregnancy is an exclusion since intramuscular 
triamcinolone acetonide is classified as category C for pregnant women and may be associated 
with virilizing effects on the human fetus. In the event that a female participant becomes 
pregnant during this study, she will be withdrawn immediately and referred to her primary 
physician for further treatment.  The perinatal outcomes of any pregnancies will be reported. 
This will be considered an adverse event and will be reported to the local IRB. The 
ascertainment of sexual activity, use of birth control, and administration of pregnancy tests to 
girls age 11 and older is a sensitive issue. In the NHLBI’s Childhood Asthma Management 
Program (CAMP),112 parents were informed in the initial consent process that the study 

20
coordinators or physicians may ask the participants confidential questions about their health or 
administer pregnancy tests. Our medical team and research staff are versed in the sensitive 
acquisition of this information from adolescents and exchange experiences and effective 
communication techniques. Whether a pregnancy needs to be reported to the participant’s 
parent (legal guardian) is a matter of local hospital policy and state law that will not be 
addressed as a separate study policy, but we strongly advocate the practice of informing 
participant’s parents with the consent of the participant and referral for appropriate prenatal 
care. 
Spirometry with Oscillometry. Participants will undergo spirometry with oscillometry 
(lung function tests) at each study visit. These tests can cause children to become light-headed 
or dizzy. The risk of syncope is minimized by having participants sit during the procedure. These 
tests may also result in shortness of breath or cough. Treatment (albuterol sulfate) will be 
available if these symptoms occur. However, in the Childhood Asthma Management Program,113 
in approximately 12,000 spirometry tests in children age 6-15 years, there were no serious 
adverse events.  
Bronchodilator reversibility testing. Children will undergo spirometry with 
bronchodilator reversibility testing at visits 1, 2 and 5. This involves administration of 4 
inhalations of albuterol sulfate through a metered dose inhaler with a mouthpiece. Side effects 
of albuterol administration may include nervousness, hyperactivity, increased heart rate, 
nausea, or headache.  These side effects are temporary and should resolve within 15 to 30 
minutes.
Exhaled nitric oxide determination.  Exhaled nitric oxide testing will be performed at 
each study visit. Exhaled nitric oxide testing can cause similar risks as spirometry but these 
risks are less intense and are even more rare. Exhaled nitric oxide tests involve breathing into a 
device at a flow rate that is held constant by an in-line resistance valve. As a result, participants 
occasionally perceive an inability to freely exhale, or similar to spirometry, may complain of light-
headedness. 
Venipuncture.  Blood will be collected by venipuncture for the following: 1) total and 
allergen-specific IgE, 2) complete blood count with differential, 3) cytokine analyses, and 4) 
metabolomic analyses. The blood draw will involve a maximum of 12 mL (about a tablespoon) 
and this volume is well below our local IRB guidelines of no more than 1 mL/kg. Therefore, the 
risk of anemia is extremely small. This blood will be collected with a butterfly needle and 
aliquoted into the necessary tubes to avoid participant burden related to multiple needle 
insertions. A maximum of three attempts will be made to obtain the blood sample. The volume 
of blood is not expected to be an issue. To minimize risk to the participants, fasting will not be 
required. Risks of venipuncture can include brief pain during the needle insertion, local bruising 
at the collection site, and small hematomas. Rarely, children can get light-headed or even faint. 
Venipuncture can also cause anxiety in children. Therefore all pediatric venipunctures will only 
be performed by pediatric phlebotomists who are trained in pediatric blood collection methods, 
which differ from those in adults by the use of topical anesthetic creams and small gauge 

21
needles. In our experience from several local and multi-center pediatric asthma studies which 
involve blood draws, the most effective strategy for dealing with children is honest explanation 
of the procedure and associated feelings/sensations (such as “pressure, prick, pinch”), how long 
those sensations last, strategies to overcome those sensations (singing, thinking about “happy” 
thoughts), and other coping strategies. 
Sputum induction. Sputum induction will be performed in participants 12-21 years of 
age at visits 1, 2 and 5 for cytokine and metabolomic assays. Induced sputum can lead to 
increased wheezing, coughing and dyspnea and thus, sputum induction will be halted if 
participants become increasingly symptomatic or do not tolerate the procedure. Our manual of 
procedures includes frequent measurements of peak expiratory flow to prevent excessive 
bronchospasm during sputum induction and the safety of this procedure in moderate-severe 
obstructive lung disease (asthma) has been published.114 Additionally, the following safety 
procedures will be followed: 1) a physician will be available during the procedure, 2) all 
participants will be pre-treated with 4 inhalations of albuterol before the procedure, and 3) only 
participants with a post-bronchodilator forced expiratory volume in one second (FEV1) of >70% 
predicted will undergo the procedure. Spontaneous sputum samples may be collected on 
children with an FEV1 below these limits. Study staff will calculate and record the peak flow 
and FEV1 value that equal both a 10% and 20% fall in lung function based upon the 
recorded post-bronchodilator peak flow and FEV1 values. Children will not be discharged until 
FEV1 is within 10% of their post bronchodilator FEV1.
Triamcinolone injection. Triamcinolone acetonide injectable suspension (Kenalog®, 40 
mg/mL) provides a synthetic corticosteroid with anti-inflammatory action. Triamcinolone 
acetonide is FDA-approved and indicated for children 6 years of age and older with asthma. The 
suggested initial dose for children 6-12 years of age is 40 mg, although dosage depends more 
on the severity of symptoms than on age or weight.  For this protocol, children will receive 1 
mg/kg in one intramuscular dose (up to 40 mg maximum).  Although local side effects of 
intramuscular administration have been reported, including pigmentation changes and 
subcutaneous and cutaneous atrophy, these side effects are minimized by deep injection into 
the gluteal muscle. All triamcinolone acetonide injections will be administered by an experienced 
pediatric registered nurse. Systemic side effects with a single triamcinolone acetonide injection 
(40 mg max) are rare and  triamcinolone acetonide injection is thought to be generally safe in 
children.  Recent studies of much higher doses of intramuscular triamcinolone acetonide in 
children with nephrotic syndrome (~2 mg/kg)115 and difficult-to-treat asthma (60 mg)90, 91 have 
revealed little side effects and good tolerance of the medication, with no reported incidence of 
cataracts, diabetes, added weight gain, or arterial hypertension. Although repeated use of 
triamcinolone was associated with slightly decreased growth velocity during the active treatment 
phase (2 mg/kg injection each month for up to 36 months), growth velocity returned to normal 
after discontinuation of triamcinolone therapy.115 Nonetheless, participating children will be 
screened thoroughly by the study physician for the presence of any co-morbid conditions that 
may increase the risk of triamcinolone acetonide administration. Children receiving 
triamcinolone acetonide will be educated about signs of potential complications and will have 

22
24-hour access to an on-call pediatric physician. Participants will also undergo a comprehensive 
evaluation 2 weeks after the triamcinolone acetonide is administered.
Triamcinolone acetonide is a pregnancy category C medication and will not be 
administered to pregnant adolescents due to potential virilizing effects on the fetus.
Children receiving triamcinolone acetonide injection can experience localized pain at the 
injection site. This risk will be minimized through the application of topical ethyl chloride 
anesthetic prior to injection.  
Potential benefits. While some study participants may receive no direct benefit from 
participating in this study, others may benefit from the close monitoring of their respiratory 
health, specialized asthma education, and general evaluation of their condition, including 
aeroallergen sensitization profiles. Some families also achieve psychological benefit from 
participating in an important research study and from interaction with the study staff.  There is a 
possibility that the knowledge obtained from participation in this study (i.e., allergen sensitization 
profiles, lung function results, symptom profiles and corticosteroid treatment responsiveness) 
will directly benefit the majority of the participating children and their families. 
All research procedures will be completed free of charge to the research participants and 
their families. All research data will also be shared with the caregivers (or the legal guardians). 
Data that may influence the clinical care of the participant, such as allergen sensitization reports 
and complete blood counts, will also be shared with the participant’s primary care physician. 
Every child enrolled in this research protocol will also undergo a medical respiratory evaluation 
at several time points and the information obtained from this study may be useful in the 
development of specialized respiratory care. It is also possible that the knowledge obtained from 
this study, such as identification of biomarkers, may assist in the creation of novel asthma 
therapies in the future. 
S. Data and Safety Monitoring Plan
This protocol has a Data and Safety Monitoring Plan that was reviewed and approved by the 
NIH/NINR and the IRB. This Plan is available as an attachment in the IRB application and will 
adhere to all aspects of the protocol. The DSMP attachment provides an overall framework for 
monitoring including :1) Monitoring Entity, 2) Procedures for Monitoring Safety and Minimizing 
Risk, 3) Data Quality, Management, Confidentiality and Security, 4) Adverse Events and 
Unanticipated Problems, and 5) Assessment of External Factors or Relevant Information.
 
T. Sample size 
The sample size is 173 participants age 6 up to 21 years, which accounts for a 
conservative attrition rate of 15% (final N=147). The following assumptions were used for 
sample size calculations:

Identification of 4 symptom clusters resulting in 3 degrees of freedom,

23

>80% desired power for a chi-square test of multiple proportions,

Two-tailed significance level of 0.05 if the effect size (W) is small-to-medium by 
Cohen’s convention116 and equivalent to W=0.275 (small effect: W=0.10, 
corresponding odds ratio=1.49; medium effect: W=0.30, corresponding odds 
ratio=3.45), and 

Sample size allocation ratio of 1-2 per cluster group and sample sizes of at least 20 
per group.
The sample size calculations with the 
above parameters should achieve at least 80% 
power as shown in Table 3.
For the secondary outcome, The sample 
size of 173 children necessary for the primary 
outcome should achieve >85% power to detect a 
difference of 0.2 between group means 
(considered a small effect size d)116 with 
standard deviation of 0.20. Power >80% is 
achieved if the mean difference between groups 
and associated standard deviation is slightly 
larger but still small (0.25-0.30). In our prior study of triamcinolone administration to children 
with asthma,96 mean ( standard deviation) quality of life scores (on a different instrument, the 
Asthma Quality of Life Questionnaire) were 6.17 ( 0.75) and 5.07 ( 0.99), respectively. If 
similar differences are noted between two symptom cluster groups, we should have >90% 
power for the primary outcome and >95% for the secondary outcome if the groups have at least 
20 participants. With smaller group sizes of only 13 participants, power would be >81% and 
>89% for the primary and secondary outcomes, respectively.
U. Statistical analysis plan
Plan for parent R01 study (aims 1-3)
Latent transition analysis (LTA) is a cluster-based model that allows for integration of 
repeated data.117 Discrete latent classes of individuals are identified based on their
shared characteristics across a set of observed categorical variables and models are allowed to 
change over time between classes. Parameters estimated in LTA include class membership 
probabilities and item response probabilities, which are used to characterize the group 
structures. LTA also produces a transition probability matrix, estimating probabilities of 
membership in the same group at each time point (entries along the diagonal) and probabilities 
of transitioning to a different group over time (entries off the diagonal).118, 119 Parameters are 
combined mathematically into a likelihood function, and the set that maximizes the likelihood 
function determines the membership of each individual in one of the latent classes. 
Drop-out
df
Power if effect size is:
0.250
0.275
0.300
0.325
0.350
10% (N=156)
3
0.749
0.833
0.897
0.941
0.969
4
0.703
0.795
0.867
0.920
0.956
5
0.664
0.760
0.840
0.900
0.942
12.5% (N=151)
3
0.734
0.820
0.886
0.933
0.963
4
0.687
0.780
0.855
0.911
0.949
5
0.648
0.744
0.826
0.889
0.934
15% (N=147)
3
0.721
0.808
0.877
0.926
0.959
4
0.674
0.767
0.844
0.903
0.943
5
0.634
0.731
0.814
0.878
0.927
Table 3. Estimated power.

24
Because LTA is aimed at characterizing transition over time, it requires variability in all 
variables. Therefore, we will perform LTA with patient-reported symptoms (global, mental, social 
and physical health categories) and measures of asthma disease activity (ACQ, ACT, lung 
function, exhaled nitric oxide, blood eosinophils) measured at baseline and visits 3, 4 and 5 (see 
Appendix 1). Age, sex, race, ethnicity, socioeconomic status, medications, exposures and 
allergic sensitization measured at the baseline visit will be considered stable and treated as 
covariates in the LTA model.120 All participants will be included in the analysis since missing 
data for the variables are handled in the procedure, with data assumed to be missing completely 
at random.121 The test of the null hypothesis that data are missing completely at random 
(Missing Completely At Random test in the SAS LTA procedure) will be evaluated; if non-
significant, missingness does not affect the interpretation of the results. The number of classes 
will be statistically determined using the Bayesian information criterion (BIC) and class 
prevalence if BICs are of a similar magnitude, to avoid a solution leading to low prevalent 
groups (<5%). To assess the stability of the group structure, goodness of fit of the “constrained” 
LTA model in which identical item–response probabilities across time are estimated (i.e., the 
same cluster structure is forced into multiple time points) will be compared with the 
“unconstrained” LTA model in which item response probabilities are estimated at each time 
point. To further address sex as a biological variable, models will also be fitted by sex, with 
statistical comparison of results observed in boys and girls. All analyses will be performed with 
SAS software version 9.4 (SAS Institute) using the PROC LTA procedure.122, 123 
The primary outcome will be assessed after assignment of individuals to the latent 
classes that they had the highest probability of belonging to (based on the posterior probability 
provided by the model at baseline). A straightforward analysis of the primary outcome will follow 
and involves comparison of the proportion of children achieving a minimally importance 
difference in ACQ of at least 0.5 in each of the latent classes via a Chi-Square test.
The secondary outcome, quality of life, will be compared with generalized estimating 
equations at visits 3, 4 and 5 using latent class assignment as the predictor. Analyses will be 
evaluated in light of pertinent covariates such as age, sex, race, ethnicity, socioeconomic status, 
medications, exposures and allergic sensitization. A 0.05 significance threshold will be utilized 
for secondary analyses without adjustment for multiple comparisons.
Metabolomic data, cytokine data, and amino acid data will be quantitative, linear mixed 
effects models will be used to determine the association between these symptom cluster group 
assignment.  
Plan for supplemental R01 funding (aims 4-5)
Aim 4 will determine associations between neighborhood air quality indices (predictor variables) 
and patient-reported asthma control and symptoms, lung function, exacerbation rates and QOL 
(outcome variables) during two separate seasons (spring/summer versus fall/winter) in children 
stratified by high- versus low-to-moderate social vulnerability. Prespecified outcomes are 
continuous and will be evaluated with linear regression. Analyses will be stratified by social 

25
vulnerability (high versus low-to-intermediate) and season (spring/summer versus fall/winter). 
Models will be generated for air quality variables on the visit date and for each of the five lag 
days prior to the visit. Given the relatively small number of participants and high number of air 
quality predictor variables, we will use ElasticNet to select the most important variables in 
Python. Variables with a Pearson correlation coefficient >0.8 will be removed to minimize 
redundancy. Models will be evaluated considering pertinent covariates such as age, asthma 
medications and tobacco smoke exposure. To further address sex as a biological variable, 
models will also be fitted by sex, with statistical comparison of results observed in boys and 
girls. Analyses will assume a type 1 error rate of 0.05 without adjustments for multiple testing 
(exploratory study). For exploratory dichotomized outcomes that are not pre-specified, such as 
exacerbation occurrence or QOL scores below a threshold, partial least squares regression and 
discriminant analysis could be performed to determine the air quality features that provide the 
largest separation between outcomes. Temporal stability of the results would be assessed by 
repeating analyses during the second season. 
Aim 5 will determine whether children with greatest social vulnerability and the highest air 
pollution exposure will have differentially expressed cytokines and metabolomic features. 
Cytokine and metabolomics data will be integrated using the R package Multiple Co-inertia 
(MCIA) to identify novel biomarkers of climate-related outcomes. These co-inertial analyses 
(CIA) are similar to the sparse Partial Least Squares (sPLS) approach used to integrate multi-
omics data in the analyses pipeline from the popular package mixOmics by maximizing the 
covariance between eigenvectors and efficiently determining key features driving variance in 
paired multi-omics samples. Unlike sPLS, CIA allows us to project multi-omic data in the same 
space, reducing the need to generate several paired multi-omic integration modules.
V. Timeline
Pre-
award
Year 1 
first 3 
months
Year 1
last 9 
months
Year 
2
Year 
3
Year 
4
Year 
5
Start-up activities
Institutional Review Board approval
X
Georgia CTSA study approval (for 
utilization of the Pediatric Research 
Center)
X
Develop study case report forms 
X
Develop study database 
X
Develop participant characterization  
Manual of Procedures 
X
Develop laboratory sample processing 
Manual of procedures
X
Study team training
X
Participant-related activities
Creation of advertisements, mailings
X
Screening and recruitment
X
X
X
X
X
Participant enrollment
X
X
X
X
Participant follow-up (48 week study)
X
X
X
X
X
Laboratory procedures

26
Cytokine analyses
X
X
X
X
X
Metabolomic analyses
X
X
X
X
X
Statistical analyses
Preliminary analyses for progress 
reporting
X
Primary/secondary outcome analyses
X
Dissemination
   Research conference presentation
X
X
Manuscript development
X
X
X
Grant submission
Future related studies
X
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34
Appendix I. Schedule of study procedures for the 48 week cohort study (option 2). 
Procedures that will not be performed if telephone follow-up vistis are requested are 
indicated by an (X).
 
6-21 years with diagnosed
asthma and an asthma
exacerbation in the past
12 months
Procedure
Consent
X
Vital signs, physical exam
X
X
(X)
(X)
(X)
Medical history review
X
X
X
X
X
Urine pregnancy testing
X
X
(X)
(X)
(X)
Demographic questionnaires
X
Asthma evaluation (ACQ/ ACT)
X
X
X
X
X
PROMIS questionnaires
X
X
X
X
X
Spirometry
X
X
(X)
(X)
(X)
• Bronchodilator reversibility
X
X
(X)
Exhaled nitric oxide
X
X
(X)
(X)
(X)
Venipuncture
X
X
(X)
• Total IgE, specific IgE
X
• Complete blood count/diff
X
X
(X)
• Blood samples stored (aim 3)
X
X
(X)
Sputum induction
X
X
(X)
• Sputum sample stored (aim 3)
X
X
(X)
Triamcinolone injection
X
Visit
(week)
(window)
Visit 1*
Baseline
Steroid
response
Follow-
up
* Can be split into two visits (V1A and V1B, with triamcinolone at V1B)
Visit 2
(2)
(+7 days)
Follow-
up
Follow-
up
Visit 3
(16)
(+4 weeks)
Visit 4
(32)
(+4 weeks)
Visit 5
(48)
(+4 weeks)
Eligibility
Characterization
Mechanisms