Very Low Birthweight (VLBW) Preterm Infant Skin Health With Bundled Care in the Neonatal Intensive Care Unit (NICU): A Randomized Controlled Trial
Phase: NA
Conditions: Preterm Infants
Sponsor: Duke University

 
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Research Summary  
NCT03370757 
 
1. Protocol Title – Very Low Birth Weight (VLBW) Preterm Infant Health Outcomes with 
Bundled Care in the Neonatal Intensive Care Unit: A RCT 
 
2. Purpose of the Study – Explore the impact of bundling nursing care activities on the overall 
health of VLBW preterm infants who receive bundled care in a Level IV Neonatal Intensive 
Care Unit (NICU). Specific study aims include: 
 
Aim 1. Evaluate differences in infant health when diaper changes occur at 3- versus 6-hours 
during 3-hour bundled care.  
RQ1. What caregiving activities other than diaper changes are included in bundled care 
events and do they differ when diaper changes are bundled every 3 versus 6 hours.  
RQ2. Does infant physiological stability (∆ heart rate, ∆ respiratory rate, ∆ oxygen 
saturation, # apnea # bradycardias, # desaturations, feeding tolerance) differ between 
3- and 6-hour bundled care diapering?  
RQ3. Does infant weight gain over time differ between 3- and 6-hour bundled care 
diapering? 
RQ4. Within the 6-hour bundled diaper care group, does infant physiological stability 
(∆ heart rate, ∆ respiratory rate, ∆ oxygen saturation) differ between bundled care with 
and without a diaper change (3 versus 6-hours)?  
 
Aim 2. Evaluate differences in infant skin health between 3- and 6-hour bundled care 
diapering at two sites (buttocks and chest).  
RQ1. Does skin transepidermal water loss (TEWL) differ over time and at the end of 6 
weeks between 3- and 6-hour bundled care diapering on the chest and buttocks? 
RQ2. Do neonatal skin condition scores differ over time and at the end of 6 weeks 
between 3- and 6-hour bundled care diapering? 
RQ3. Does skin pH differ over time and at the end of 6 weeks between 3- and 6-hour 
bundled care diapering? 
RQ4. Does the skin microbiota differ over time and at the end of 6 weeks between 3- 
and 6-hour bundled care diapering? 
 
Aim 3. Evaluate the acceptability and feasibility of conducting a bundled care intervention 
where the frequency of one care activity, diaper care, is standardized.  
 
RQ1. What are the perceptions of parents and providers about the criteria necessary for 
conducting a diaper change in the NICU? 
RQ2. What are the perceived barriers of parents and providers to longer timeframes 
between diaper changes? 
 
Exploratory health outcomes will examine the incidence of late onset sepsis and urinary 
tract infections in both bundled care groups. 
 
3. Background & Significance –  
Progress in perinatal medicine has resulted in an increased survival rate of very preterm 
infants (typically ≤ 1500 grams and < 34 weeks). However, preterm infants’ growth and 
developmental outcomes vary with illness severity and degree of neurological insult (Boyle et 
al., 2012; Ge et al., 2013) and unpredictable variations in outcomes exist even among healthy 
preterm infants (Schneider et al., 2014; Vohr, 2014).  Preterm infants also have poorer 

 
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orientation, self-regulation, and reflexes and more excitability, hypotonia, and hypertonia at 40 
weeks postmenstrual age (PMA) when compared to term infants (Pineda et al., 2013). The 
variations in preterm infants’ outcomes have led to the supposition that the NICU environment 
may negatively impact the health and development of these infants and significant research has 
been devoted to examination of light, noise, and caregiving interventions (Boo et al., 2002; 
Brandon et al., 2017; Brandon et al., 2002; Morag & Ohlsson; 2013).  
In addition, individualized developmental care has become the norm in most NICUs since 
developmental care was first promoted by Dr. Heidelise Als in the 1980s (1982). A body of 
literature has emerged to support the observation of infant behavioral organization (infant cues) 
in response to the stress of the NICU environment to guide caregiving to minimize infant stress 
and optimize infant outcomes. The overall goal of individualized developmental care is to 
promote the short- and long-term health and development of high risk infants including 
interventions that promote the well-being of an infant’s family (Macho, 2017). Consequently, 
many NICUs have implemented neuroprotective strategies to reduce over simulation, promote 
sleep and facilitate brain development in all high-risk infants (Gressens et al., 2002; Lucas-
Thompson et al., 2008).   
One neuroprotective strategy includes the grouping of care activities around a single 
caregiving event described as “clustering” or “bundling care”. Individualized developmental care 
dictates that a bundled care event is halted if an infant displays significant behavioral stress 
cues. Yet, the number and type of caregiving activities that are included in bundled care and the 
timeframe between bundled care events has not been systematically studied. While painful and 
intrusive interventions like heel sticks are often described as a source of infant stress (Lucas-
Thompson et al., 2008) routine interventions like diaper changes can also be a high stress event 
(Mörelius et al., 2006). Current bundled care typically includes a diaper change, but the 
timeframes between bundled care events differ based upon infant illness severity, 
postmenstrual age, and individual NICU practice standards.  Currently no operational definition 
exits around the expected length of time that categorizes a bundled care event from a non-
bundled care event. In our pilot work of five extremely low birth weight (ELBW) infants we 
identified a caregiving event as including at least two nursing care activities and lasting at least 
five minutes. Over the first 14 days of life in the NICU, 161 bundled care events were identified, 
averaging 4.7 events per day per infant.  The average duration for bundled care events greater 
than 5 minutes was 10.8 minutes. The time between bundled care events was 2.9 hours.  
The inclusion and exclusion of certain care activities in any individual care event is often 
dictated by the infant’s treatment plan or needs, but some activities like diapering may be 
optional. Understanding the impact of when to include optional, yet stress provoking 
interventions, will allow us to minimize overall environmental stress in hospitalized VLBW 
infants.  It is unclear how the different times used in bundled care affect overall infant health 
including skin health in the diapered area.  Therefore, given the lack of data around bundled 
care, we aim to explore the impact of bundling diaper care activities on overall health of VLBW 
preterm infants. We will include a focus on skin health because it is important to understand the 
benefits of decreased infant stress and any potential skin health trade-offs associated with 
longer versus shorter time between diapering care. Findings from this study will allow us to 
better understand the relationship between neonatal skin health while providing developmentally 
appropriate bundled care.  
 
4. Design & Procedures  
 
a. Design and Setting. A randomized controlled designed will be used to evaluate the 
effect of bundled diaper care on overall infant and skin health. Infants will be recruited from the 
Level IV Intensive Care Nursery (ICN) at Duke University Hospital.  The ICN is a Level IV, 67-
bed nursery and one of the area’s leading referral centers for infants born prematurely. The 

 
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Duke ICN has an average daily census of 90% and annually admits 150 VLBW infants. The ICN 
practices individualized developmental care with standard bundled care intervals that vary 
based upon an infant’s birth weight and feeding status (every 3, 4, or 6 hours).  Infants who are 
admitted or transferred to one of the ICN three stepdown units will also be considered if eligible 
and will continue to receive the intervention when transferred including the Special Care 
Nursery at Durham Regional Hospital.  The principal investigator has been a clinical nurse 
specialist affiliated with the ICN since 1993 and has the support of the nursery medical and 
nursing team to conduct this research including the Division of Neonatology, the ICN Intensive 
Care Developmental Care Team and the Medical Director.   
 
b. Participants. A maximum of 80 VLBW infants will be recruited to ensure at least 25 
infants in each study group with complete data.  Ten parents of infant participants (5 in each 
study group) and 10 health care providers will participate in interviews about the acceptability 
and feasibility of the diapered bundled care intervention. The goal of this study is to enrich the 
literature with estimation of values and variances of the outcomes measured as well as how 
these values change over time.  With a total sample size of 50 VLWB infants, we are confident 
that we will be able establish effect to inform a larger subsequent study. Because most of the 
infants are expected to receive care up to 6-weeks, we expect little missing data or participant 
attrition. 
 
Infant Inclusion criteria are: 
1. Must be participating in the ICN standard 3-hour bundled care 
2. Must wear a breathable diaper 
3. Must be ≤ 32 weeks gestation at birth  
4. Infants are expected to remain hospitalized for at least 4 weeks 
Infant Exclusion criteria are: 
1. Neonatal Abstinence Syndrome  
2. Humidified incubator 
3. Diaper rash 
4. Pre-existing or genetic skin conditions  
5. Severe illness requiring 1:1 nursing care: e.g. minimal stimulation protocol, high 
frequency ventilation, vasopressor drug support, or body cooling. 
 
Infants will be recruited and enrolled as soon as they meet study criteria (see Figure 1. 
Study Schemata). Data collection will begin following study enrollment and will continue until 6 
weeks following randomization (see Table 1). 
 
Figure 1. Study Schemata  
 
c. Intervention: Infants will be 
randomly assigned and stratified by birth 
weight (≤ 800 grams, >800 grams to 1150 
grams, > 1150 grams) to either 3- or 6- hour 
bundled diaper care. All infants will receive 
bundled care per unit standards until they 
are eligible for 3-hour bundled care. Infants 
typically transition to 3-hour bundled care 
once they are receiving interval feedings 
every three hours. This transition usually 
occurs before 3 weeks chronological age in 
infants >800 grams at birth and by 6 weeks 
Screen for Eligibility Following Delivery 
3-hour standard bundled 
care infants 
Randomized to: 
6-hour Bundled 
Diaper Care group 
3-hour Bundled 
Diaper Care group 

 
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for infants ≤ 800 grams. Other care activities included in the standard 3-hour bundle vary based 
upon infant treatment needs, but usually include a diaper change. The only difference in a 3- 
hour and 6-hour bundled care event will be the timing of the diaper change.  Infants will receive 
a diaper change at either 3 or 6 hours.  While 3-hour bundled care is standard of care, this is a 
clinical environment in which caregiving cannot be expected to occur exactly every three hours 
to the minute, therefore both expected and actual times for the bundled care events will be 
collected to include in data analysis as needed. Infants who are receiving the 6-hour bundled 
diaper care will continue to receive 3-hour care interventions for all other interventions or as 
dictated by infant care needs. At the 3-hour caregiving interval in which the 6-hour bundled care 
infants are not due for a diaper change, the nurse will peek into the diaper to ensure there are 
no stool present.  If stool is present, a diaper change should occur and the deviation from the 
protocol noted in the bedside documentation. 
 
d. Procedures: A neonatal provider will give approval for participation in the study.  
Parents will be approached for consent after eligibility screening is completed (≤ 32 weeks 
gestation).  The principal investigator is a clinical nurse specialist and nurse scientist who has 
been affiliated in the Intensive Care Nursery since 1993. Study personnel will explain the study, 
allow time for questions, have a parent sign the consent form, and a copy will be given to the 
parent.  Parents will provide consent for their infant’s participation in the study and will give 
permission for themselves to participate in an interview about the study, if selected. Five parents 
from each group will purposively selected to participate in an interview.  Parents will be selected 
to include parents with infants < 1000 grams at birth and parents with infant ≥ 1000 grams at 
birth. 
Following consent, the infant will be monitored until all inclusion and exclusion criteria are 
met.  At that time randomized to an intervention group and data collection will be initiated.  The 
intervention will continue for six weeks at which time collection of data will stop and bundled 
diapered care will resume according to unit protocol.  If the infant no longer meets inclusion 
criteria the intervention will be discontinued and routine data collection will be discontinued.  The 
exclusion criteria will be noted, e.g. use of diaper cream, and data collection regarding any new 
illness characteristics or treatments will continue.  If an infant requires a diaper change due to a 
stool or other needed procedure this will be recorded on a bedside card. 
Ten nurses will be recruited for participation in interviews around intervention acceptability at 
the end of the study. 
 
e. Data Collection Measures 
Study data will include maternal and infant demographics and medical history extracted from 
the medical record to describe the sample and for needed covariate data, physiologic data (e.g. 
heart rate, respiratory rate, oxygen saturation), and biomarkers of skin health (pH, 
transepidermal water loss, NSCS, microbiome).  See Figure 2 below for the timeframe for the 
collection of physiologic data and contents and length of bundled care.  Table 1 for the 
constructs, measures, and timing and frequency of data collection. 
 
Figure 2. Timeline 

 
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i. 
Medical Record Data: Participant participation is documented in the electronic medical 
record and data extraction from the electronic medical record can be accessed through 
the Duke Health System EPIC or through a Virtual Pin Network.  Flow sheets, lab results 
and provider notes will be reviewed for the required data. 
 
 
Table 1. Data Collection 
Construct 
Measure/ 
Instrument 
Variable 
Time frame 
 
Descriptive data to describe the sample 
Maternal 
history 
EMR1 
Age, medical hx. of skin disease, 
Enrollment 
Infant 
characteristics 
EMR 
GA2, birth weight, diagnoses, antibiotics, delivery 
mode, lab values as required to describe skin health 
(e.g. albumin, protein, bilirubin) 
All bundled care event time frames from study entry 
to study discharge (expected vs. actual bundled 
times) 
During bundled care—respiratory support, changes in 
support, FiO2 changes, average Daily FiO2, caffeine 
 
Enrollment, 3x/week, or as 
available 
 
Aim 1. Infant Health 
Bundled care 
Observation, 
videotape 
& EMR 
Specific interventions (e.g. suctioning, positioning, 
offering pacifier, diapering) 
Length of bundled care events 
Time between bundled care events 
 
Observer 3x/week for the 
3-hour diaper group and 
4s/week for the 6-hour 
group to allow for one 
within group comparison; 
Videotaped 1x/week 
Daily; retrieved 3x/week 
Physiologic 
stability 
GE monitor 
1-minute values of heart rate, respiratory rate and 
oxygen saturation 15 minutes before and after each 
observed bundled care event 
3x/week for the 3-hour 
diaper group and 4s/week 
for the 6-hour group to 
allow for one within group 
comparison 
 
EMR 
# bradycardia events daily 
Daily; retrieved 3x/week 
 
EMR 
# apneic events daily 
Daily retrieved 3x/week 
 
EMR 
# desaturation events daily 
Daily retrieved 3x/week 
 
EMR 
Feeding tolerance (emesis, gastric residual volume-
ml, feeding interruptions) 
Daily retrieved 3x/week 
Weight gain 
EMR 
Daily weight in grams 
Daily retrieved 3x/week 
 
Aim 1. Covariates 
Development 
EMR 
Postmenstrual age (PMA) 
 
Severity of 
illness 
Technology 
Burden 
Total Score 0-100 
Retrieved 3x/week to 
coincide with observation 
 
EMR 
Time in minutes since the end of the last bundled 
event 
With each observation 

 
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Aim 2. Skin Health (buttocks and chest) 
Acid mantle 
development 
pH 
0.00 to 14.00. 
3x/week with observation 
Water loss 
TEWL 
Dermalab 
TEWL3  
3x/week with observation 
Skin status 
NSCS4 
NSCS-range 3-9 
Twice daily retrieved 
3x/week 
Microbiome 
 
Microbiota pattern 
3x/week with observation 
 
Aim 2. Covariates 
 
EMR 
PMA 
 
 
EMR 
Delivery mode 
 
 
EMR 
Time since last bath 
3x/week with observation 
 
EMR 
Daily urine output 
3x/week with observation 
 
EMR 
Time in minutes since the last bundled event 
 
 
EMR 
Stool pattern (categorical) 
3x/week with observation 
 
EMR 
Feeding type (formula vs. breast and route; enteral 
vs. nipple) 
3x/week with observation 
 
Bedside 
checklist 
Diaper size and brand 
3x/week with observation 
 
Stool 
Scavenged  
1x/week as available 
 
Aim 3. Feasibility 
Intervention 
acceptability 
and feasibility 
Interviews 
 
Parent perceptions 
 
Provider perceptions 
End of infant data 
collection; 
At study end 
 
Exploratory Health Outcomes 
Late onset 
sepsis 
EMR 
Presence or absence 
Two weeks after end of the 
study 
Urinary tract 
infection 
EMR 
Presence or absence 
Two weeks after end of the 
study 
1EMR=electronic medical record; 2GA=gestational age at birth; 3TEWL=transepidermal water loss; 
4NSCS=neonatal skin condition score. 
 
ii. Physiologic measures: Heart rate, respiratory rate, and pulse oximetry will be 
measured continuously with a GE Cardiopulmonary monitor (standard monitor at DUH) 
with acquisition from the server, sent to the PI weekly for all enrolled subjects in a data 
secured zip file. 1-minute values for each 24-hour observation in which a bundled care 
data collection occurred will be extracted and the required time frames will be extracted 
for analysis.  
 
iii. Bundled Care: The content of bundled care will be determined through the 
observational coding of all caregiving activities during weekly observations (3 
times/week for 3-hour diapered and 4 times/week for the 4-hour diapered intervention 
group; see intervention section above) and 12-hour videotaping (1 time per week for 
each group) of bundled care events. The length of time for each bundled care event will 
be operationally defined as beginning when the nurse’s hands enter the incubator and 
will end when the nurse’s hands leave the incubator following caregiving.  
 
iv. Microbiome: One microbiome sample will be taken from the buttocks immediately after 
the diaper is removed and before the skin is cleaned, if there is NO stool present.  A 
second swab will be collected from the chest at the same time. The following procedure 
will be used for all microbiome samples.  Needed supplies will include a tube of sterile 
solution of PBS + 0.1%Tween 20 and a Nova Biostorage FLOQ swab with cuvette.  
a. Remove the swab from the package 
b. Insert the tip of the swab into the sterile PBS + 0.1% Tween 20 solution until 
moistened. 

 
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c. Before the buttocks is cleaned, swab the left and right buttocks with two back and 
forth strokes on each buttocks for a total of four strokes.  
d. Place the swab tip into the cuvette and bend at scored break point until the tip 
remains untouched in the cuvette 
e. Seal the cuvette, and label it with the subject number, site, visit number, date, 
time, initials of the individual taking the swab. 
f. 
Place swabs into a -20°C (±5°C) freezer and store until end of study. 
 
v. pH: The Extech PH100 meter will be used to measure skin pH in the range of 0.00 to 
14.00. The small flat surface electrode provides a non-intrusive accurate measure of pH. 
The instrument’s probe will be placed at the center of the test site (either chest or diaper) 
and measurements will be taken. Each measurement will be performed by holding the 
probe flush over the skin at the test site. The measurement is taken instantly and will be 
recorded into RedCap via iPad. 
 
vi. Trans Epidermal Water Loss: TEWL values will be collected using the Dermalab.  We 
will use non-invasive skin probes to perform each assessment of transepidermal water 
loss (TEWL). TEWL will be measured using the DermaLab® TEWL probe (Cortex 
Technology, Hadsund, Dermark), which consists of an open probe with paired sensors 
placed at different distances from the skin. Humidity and temperature are measured in 
each sensor to calculate vapor pressure gradients. The difference between two vapor 
pressure gradient measures is representative of TEWL at that point on the skin (Grove, 
Grove, Zerwick, & Pierce, 1999).  Environmental humidity levels will also be measured 
using the DermaLab® before obtaining the TEWL measures. The average humidity 
readings from the two sensors will be recorded as environmental humidity. Individual 
probe covers will be used for each infant to prevent any cross contamination between 
patients. Each measurement will be performed by holding the probe flush over the skin 
at the test site for at least sixty (60) seconds and the average value (in g/m2*hr) will be 
recorded. Probe covers are used to prevent cross contamination between patients, in 
addition, the probe will be thoroughly cleaned with 70% ethanol prior to and between 
participants to minimize cross-contamination or microbial transmission. The ethanol will 
be allowed to air dry prior to use of the probe.  
 
vii. Neonatal Skin Condition Score (see Figure 2): The NSCS will be collected twice daily 
(day and night shift by staff nurses) while infants are participating in the study. The 
NSCS is a reliable and valid measure of skin condition (Lund & Osborne, 2004) 
 
Figure 2. NEONATAL SKIN CONDITION SCORE (NSCS)  
Dryness  
1 = Normal, no sign of dry skin  
2 = Dry skin, visible scaling  
3 = Very dry skin, cracking/fissures  
Erythema  
1 = No evidence of erythema  
2 = Visible erythema, <50% body surface  
3 = Visible erythema, ≥50% body surface  
Breakdown  
1 = None evident  
2 = Small, localized areas  
3 = Extensive  
Note: perfect score = 3, worst score = 9.  

 
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viii. Parent and Provider Interviews: Parents will be asked to participate in an interview 
about the acceptability of the intervention after their infant completes the study.  Ten 
parents (5 in each study group) will be interviewed.  The interview is not expected to 
last more than 30 minutes, but will largely be under the direction of the parent (see 
Appendix A for the interview guide).  Ten providers to include at least two 
neonatologists, two neonatal nurse practitioners and six bedside nurses, will be asked 
to participate in an individual interview at the time of study completion about the 
acceptability and feasibility of the intervention (see Appendix B for the interview guide).   
 
5. Selection of Infant Subjects 
 
Infant Inclusion criteria are: 
a. Must be participating in the ICN standard 3-hour bundled care 
b. Must wear a breathable diaper 
c. Must be ≤ 32 weeks gestation  
d. Infants are expected to remain in ICN at least 2 weeks 
Infant Exclusion criteria are: 
1. Neonatal Abstinence Syndrome  
2. Humidified incubator 
3. Diaper rash 
4. Pre-existing or genetic skin conditions  
5. Severe illness requiring 1:1 nursing care: e.g. minimal stimulation protocol, high 
frequency ventilation, vasopressor drug support, or body cooling. 
 
6. Subject Recruitment and Compensation 
Study personnel will check the daily admission log for eligible patients. Clinical staff 
known to the parent/guardian of the potential subject will introduce the research study to the 
infant’s family. If the parent/guardian expresses interest in the study, the research staff will 
approach him/her to explain the study and request consent.  There will be no compensation for 
study participation, however infants will be given a developmental toy at the end of the study.  
 
7. Consent Process   
 
For any score of 2 under dryness, erythema or breakdown, 
that includes the diapered area, the study will be 
discontinued. 

 
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Subject’s Capacity to Give Legally Effective Consent 
For infants, consent will be obtained from the parent or guardian following approval of the 
infant’s provider using see Section 14 of the e-IRB submission form and complete the questions 
in that section.  The PI, research coordinator or other study personnel (who is either an RN or 
has a PhD (or both) and is trained in study and consent procedures) will conduct the consent 
process in the study as outlined by GCP guidelines.  
Parents will be approached for pre-consent if their infant is ≤ 32 weeks gestation. The 
infants’ medical provider, or a member of the Neonatal Perinatal Research Unit will seek 
permission from the mother or father for the research staff to discuss the study with them. Then, 
research staff (i.e. PI, study coordinator or research assistant) will contact the parent in person 
while visiting the ICN. If contact cannot be made with parents during visiting hours, parents will 
be contacted via phone. Parents will be approached at the bedside of their infant. Parents will 
be offered the option to discuss the study at their infant's bedside or in an available conference 
or meeting room on the unit.  If others are present, the parent is asked whether she would prefer 
for the research member to discuss the study in front of her visitors or at another location (e.g. 
unit conference room behind a closed door). 
We estimate that the consent process will take about 30-60 minutes. Parents will be allowed 
as much time as needed to decide whether or not to enroll the infant in the study.  Parents will 
be encouraged to ask questions at any time before their infant’s participation in the trial. The 
study team will present the consent form in its entirety, with special emphasis on the voluntary 
nature of research, and the ability of subjects to withdraw from the study at any time. No one 
who is obtaining consent is directly involved in the supervision of the providers who are being 
asked to participate in the study or involved in the direct care of the infants whose parents are 
being asked to participate in the study. The research staff will closely follow the IRB and GCF 
guidelines..  
Spanish speaking parents/guardians of eligible infants will be consented using two translators 
that are not involved in the study, one to present the consent form in Spanish and one to assure 
that what is presented is correct. The translator will translate the entire English consent form 
verbally to the potential study subject. The translator will relay questions to the study coordinator 
from the subject and the subject will have questions answered prior to signing the consent 
document. For Spanish speaking parents/guardians, the parent/guardian and witness will be 
asked to sign the Spanish short form. The witness and the person obtaining consent must sign 
the English consent form. 
 
Providers will be consented using the same IRB and GCF guidelines described above. 
Provider consent should take 15-30 minutes. 
 
8. Risk/Benefit Assessment  
Risks associated with the intervention and data collection will be minimal. All of the study 
interventions including a less frequent diaper change are considered acceptable standard of 
care.  The skin condition will be monitored twice daily to identify any potential change in the skin 
of the diapered area and if the NSCS is ≥ 5 due to dryness, erythema or skin breakdown in the 
diapered are, the intervention will be discontinued, the infant’s provider will be consulted 
regarding the need for treatment and the infant will be monitored until resolution of the problem. 
The infant stress associated with stimulation due to data collection should be minimal.  Data 
collection processes for the biomarkers should not cause discomfort and handling will be kept to 
a minimum.  However, all research staff will have neonatal experience and be trained in 
developmentally appropriated infant handling and positioning. 
The small risk of loss of confidentiality will be reduced by assigning code numbers to the 
samples.  The key linking the identity of the subject to the code number will be separately 
maintained in the Research Office, under lock and key. 

 
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There may be a direct benefit to the subjects participating in the study including less 
physiological stress during caregiving or improved skin health.  However, actual benefits will not 
be clear until future larger studies are conducted. By improving our understanding of the 
relationship between bundled diaper care and infant health outcomes more targeted 
interventions may be developed for general health, development and skin care.  
 
9. Costs to the Subject  
There will be no cost to study participants.  
 
10. Data Analysis and Statistical Considerations  
Initially, we will conduct descriptive analyses, including examination of means and proportions 
and the measures of variability for each variable overall and by bundled care groups. The 
descriptive analyses will be followed by modeling approaches to respond to the research 
questions. Tests of statistical significance will be two-sided and the threshold for statistical 
significance will be P < 0.05. 
 
Aim 1. Evaluate differences in infant health when diaper changes occur at 3- versus 6-
hours? 
 
RQ1.  What caregiving activities other than diaper changes are included in bundled care events 
and do they differ when diaper changes are bundled every 3 versus 6 hours  
Analysis: Each day bundled care is observed, a list of activities will be marked as occurred (1) 
or did not occur (0).  Each infant will have 18 days of data denoting whether an activity occurred 
or did not occur.  Hierarchical clustering of repeated data with an appropriate algorithm that 
producing stable and accurate clusters will be used will be to determine the groupings of 
activities that occur in the sample.  The frequency of the clusters between the intervention 
groups will be compared. 
 
RQ2.  Does infant physiological stability (∆ heart rate, ∆ respiratory rate, ∆ oxygen saturation, # 
apnea # bradycardias, # desaturations, feeding tolerance) differ between 3- and 6-hour bundled 
care diapering? 
Analysis: Heart rate, respiratory rate and oxygen saturation values captured a minute apart for 
each observed bundled care with a diaper change will be graphically displayed in various 
constructions to be able to compare the general trends of change in these outcomes between 
intervention groups, among periods of the bundled care (before, during and after) and over 
weeks.  Further, we will create a daily average of each outcome by period (before, during, and 
after).  Using a repeated measures analysis, we will test for difference among the periods and 
intervention group and all pairwise period comparisons between groups.  PMA and infant illness 
severity (Technology Burden Score) will be used as a covariate. We will control for multiple 
comparisons. 
 
A Poisson Generalized linear model for repeated measure will be use to estimate the daily 
number of apnea, bradycardias, and desaturations events over the 18 days of bundled care 
observations with a diaper change.  The need for a random intercept or slope will be assessed.   
 
Log(# of Apnea Eventsij) = b0 + b1[I(6HrBCi)] + b2(Dayij) + b3[(I(6HrBCi))×(Dayij) + b4(PMAij) + 
b5(TechBurij), 
where Log((# of Apnea Eventsij) is the logarithm of the number of apnea events for individual i at 
day j.  b0 is the mean log of apnea events for participants who receive 3-hour diaper changes 

 
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and are mean postmenstrual age and with mean technology scores at the start of the study; b1 
is the change in log number of apnea events for those receiving 6-hour diaper changes; 
I(6HrBCi) is an indicator for participants receiving bundled care and 6-hour diaper changes; b2 is 
the change in log of the number of apnea events for each day of bundled care; Dayij is the 
indicator of the day the number of apnea events was recorded; b3 is the additional change in log 
of the number of apnea events over days for those receiving bundled care and 6-hour diaper 
changes; PMAij is the difference from the baseline mean postmenstrual age of the ith participant 
at the jth day; TechBurij is the difference from the baseline mean technology burden score for 
the ith participant at the jth day; b4 and b5 are the expected changes in log number of events for 
postmenstrual age and technology burden scores, respectively.  The estimate of interest is b3.  
The feeding tolerance outcome will be estimated in a similar manner using the appropriate 
distribution of the outcome and link function for the model. 
 
RQ3.  Does infant weight gain over time differ between 3- and 6-hour bundled care diapering? 
Analysis: The trajectories of weight measured across the 18 days of bundle care will be fitted 
using a linear mixed model with a random intercept.  We will first determine the best trajectory fit 
for weight over the 18 days.  Once we select an appropriate trend for the data a model similar to 
the one described below will be estimated. 
 
Weightij = b0 + bi0 + b1[I(6HrBCi)] + b2(Dayij) + b3[(I(6HrBCi))×(Dayij) + b4(PMAij) + b5(TechBurij) + 
Ɛij, 
where Weightij is the weight for individual i at day j.  b0 is the mean weight of participants who 
receive 3-hour diaper changes and are mean postmenstrual age and with mean technology 
scores at the start of the study; bi0 is the individual baseline weights;  b1 is the change in weight 
for those receiving 6-hour diaper changes; I(6HrBCi) is an indicator for participants receiving 
bundled care and 6-hour diaper changes; b2 is the change in weight for each day of bundled 
care; Dayij is the indicator of the day the weight was recorded; b3 is the additional change in 
weight over days for those receiving bundled care and 6-hour diaper changes; PMAij is the 
difference from the baseline mean postmenstrual age of the ith participant at the jth day; 
TechBurij is the difference from the baseline mean technology burden score for the ith 
participant at the jth day; b4 and b5 are the expected changes in weight for postmenstrual age 
and technology burden scores, respectively; and Ɛij represents variance components to be 
estimated. The estimate of interest is b3. 
 
RQ4.  Within the 6-hour bundled diaper care group, does infant physiological stability (∆ heart 
rate, ∆ respiratory rate, ∆ oxygen saturation) differ between with and without a diaper change (3 
versus 6-hours)? 
Analysis: One day of the week for those individuals assigned to the 6-hour diaper changes will 
be randomly selected to have heart, respiratory, and oxygen rates at 3-hour without a diaper 
change to compare to the same day’s outcomes at the 6-hour diaper change.  The values 
captured a minute apart for each observed bundled care will be graphically displayed in various 
constructions to be able to compare the general trends of change in these outcomes within the 
6-hour diaper change group, among periods of the bundled care (before, during and after) and 
over weeks.  Further, we will create a daily average of each outcome by period (before, during, 
and after).  Using these computed values, we will create a weekly average of the 3 outcomes by 
period (before, during, and after).  Using an analysis for repeated measures from a matched 
sample, we will test for difference among the periods and intervention group and all pairwise 
period comparisons between treatment groups.  We will control for multiple comparisons. 
 

 
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Aim 2. Evaluate differences in infant skin health between 3- and 6-hour bundled care 
diapering at two sites (buttocks and chest)? 
 
RQ1. Does skin transepidermal water loss (TEWL) differ over time and at the end of 6 weeks 
between 3- and 6-hour bundled care diapering on the buttocks and chest? 
Analysis: The trajectories of TEWL measured across the 18 days of bundle care will be fitted 
using a linear mixed model with a random intercept.  We will first determine the best trajectory fit 
for TEWL over the 18 days.  Once we select an appropriate trend for the data a model similar to 
the one described for weight will be estimated.  We will remove technology burden scores and 
add time varying covariates for time since last bath, urine output, stool pattern, and feeding type 
indicator. 
 
RQ2. Do neonatal skin condition scores differ over time and at the end of 6 weeks between 3- 
and 6-hour bundled care diapering? 
Analysis: We will assess the distribution of the neonatal skin condition score.  Based on the 
distribution we will select the most appropriate link function to fit a generalized linear model with 
repeated measures.  The models to be estimated will be similar to the example model we 
presented for number of apnea events, including the appropriate link function and distribution.  
Further, we will remove technology burden scores and add time varying covariates for time 
since last bath, urine output, stool pattern, and feeding type indicator. 
 
RQ3. Does skin pH differ over time and at the end of 6 weeks between 3- and 6-hour bundled 
care diapering on the buttocks and chest? 
Analysis: We will assess the distribution of the pH of chest and buttocks.  Based on the 
distribution, we will select the most appropriate link function to fit a generalized linear model with 
repeated measures.  The models to be estimated will be similar to the example model we 
presented for number of apnea events, including the appropriate link function and distribution.  
Further, we will remove technology burden scores and add time varying covariates for time 
since last bath, urine output, stool pattern, and feeding type indicator.  Separate models will be 
estimated for pH of the chest and pH of the buttocks. 
 
RQ4. Skin Microbiota 
Analysis: Mr. DNA in Shallowater, TX will provide the microbiome sample analysis services to 
determine composition of skin microbiome by 16S rRNA pyrosequencing. Funding for the 
microbiome analyses will come directly from Kimberly Clark’s budget. Pyrosequencing data will 
be analyzed with a workflow based on QIIME software1.  Data will be compiled and relative 
percentages of microbes will be determined for each individual sample.  Data will also be 
compiled at each individual taxonomic level according to the NCBI taxonomy criteria.  
Significance differences in relative abundance of specific taxa between the intervention groups 
will be calculated using the Kruskal Wallis test.  
 
To assess the extent to which taxonomic diversity at the family and genus level can be used to 
distinguish microbial communities associated with skin swab samples; Principal Component 
Analysis (PCoA) will be used.  Rarefaction, Chaol, Ace and Shannon indexes will be performed 
on the data to evaluate if diversity and richness of populations are different among subjects 
within the different intervention and over time. Demographic and other clinical data will be 
evaluated for basic correlation with any observed microbiome changes.   
 
Exploratory Health outcomes 
The time to the occurrence of urinary tract infections and late onset sepsis will be measured 
from the start of the study to two weeks after study completion.  Because of the small 

 
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sample size, we estimate the risk of urinary tract infections and late onset sepsis by 
intervention using the log rank test.  
 
Aim 3. Analysis of interview transcripts will entail the review of transcripts to develop codes and 
the subsequent application of codes to all transcripts. Coded data will be reviewed to identify 
themes that characterize parents’ and providers’ of the acceptability and feasibility of the 
intervention. Data analysis activities will be supported by the use of Atlas.ti, a software program 
that supports the systematic coding and review of data. Rigor throughout the coding process will 
be maintained through a memo writing to recording thoughts, decisions, progression of code list, 
and identification of categories, and verifying coding results and interpretation with members of 
the research team. 
 
Data and Safety Monitoring  
There will be minimal risk associated with the study. The only study intervention is the timing 
of the diaper change. A record of study data for each subject will be maintained in the Research 
Office. Study records will be labeled with a code number. The key linking the identity of the 
subject to the code number will be separately maintained in the Research Office, under lock and 
key. No data that includes subjects’ names, addresses, medical record number or other 
identifiers will leave Duke.  Based upon the executed DTA with Kimberly Clark, microbiome 
samples will be sent via secure transfer for data analysis.  The DTA will be uploaded under the 
full protocol section of the IRB.  
 
Privacy, Data Storage, and Confidentiality   
Study records (hard copy and electronic) and biologic samples will be labeled with a code 
number to reduce the small risk of loss of confidentiality.  The key linking the identity of the 
subject to the code number will be separately maintained in the School of Nursing Research 
Office, under lock and key. No data that includes subjects’ names, addresses, medical record 
number or other identifiers will leave Duke.  All data will be collected and de-identified for data 
analysis. Data will be downloaded into a secure file and de-identified and stored on a secure 
server for analyses at Duke School of Nursing. All completed study documents will be stored in 
the study office within Duke School of Nursing in a locked cabinet. 
 
Microbiome samples will be processed for total genomic DNA using standardized 
procedures by Mr. DNA in Shallowater, TX. All samples send to Mr. DNA will be de-identified. 
Processed samples will be destroyed by Mr. DNA within two years of completed analysis. 
Unused and unprocessed samples will be stored long-term at -80°C in a locked room at in Julia 
Walker’s lab.  
 
 
 
 
 

 
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