Conditions: Staph Aureus Colonization Staph Aureus Infection
Keywords: Staphylococcus aureus Infection Colonization Transmission Neonate
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 307
Healthy volunteers: yes

Brief summary:
This trial will test the hypothesis that treating parents of neonates requiring NICU care with intranasal mupirocin and topical chlorhexidine bathing will reduce the spread of S. aureus from parents to neonates.

Detailed description:
The TREAT PARENTS Trial, or Treating Parents to Reduce NICU Transmission of S. aureus, is a placebo-controlled, double-masked, randomized clinical trial to test the hypothesis that treatment of S. aureus colonized parents with intranasal mupirocin and topical chlorhexidine gluconate antisepsis will decrease neonatal S. aureus acquisition. All neonates admitted to the Johns Hopkins Hospital and Johns Hopkins Bayview Medical Center NICUs will be pre-screened and parents will be approached for enrollment in the study. After consent and baseline screening, 400 neonate-parent pairs will be randomized; only neonates who have a parent colonized with S. aureus will be randomized. Parents will receive a 5 day treatment with intranasal mupirocin plus topical chlorhexidine gluconate antisepsis or placebo.

After recruitment and informed consent, parents will undergo pre-randomization screening. If both parents screen negative for S. aureus colonization, the neonate will be ineligible for the randomization and parents will be informed that the parents are not colonized at that time with S. aureus. If either parent screens positive for S. aureus, then both parents as a pair will be eligible for randomization to one of the two possible masked treatment arms. The neonate-parent "pair" will be the unit of randomization and each parent will be allocated to the same group if both consent.

After the baseline neonate testing for S. aureus colonization, repeat testing will be performed every 7 days for the neonates until the neonate acquires S. aureus colonization or is discharged from the NICU.

Arms:
arm 1: Participants will apply 2% intranasal mupirocin twice a day for five days and cleanse with 2% chlorhexidine cloths once a day for five days. arm 2: Participants will apply 2% petrolatum intranasal placebo ointment twice a day for five days and cleanse with 2% non-medicated soap placebo cloths once a day for five days.

Interventions: intervention 1: Mupirocin and Chlorhexidine intervention 2: Placebo ointment and placebo cloths
Intervention descriptions: intervention 1: None intervention 2: None

Protocol document:
Prot_SAP_000.pdf: 
 
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Treating Parents to Reduce NICU 
Transmission Of Staphylococcus aureus 
(TREAT PARENTS Trial) 
 
 
 
 
 
 
 
 
 
 
NCT02223520 
 
 
 
 
 
 
 
November 30, 2018 
 
 
 
 
 
 
 
 
 
 
 
 


 
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Key Study Personnel 
Aaron M. Milstone, MD MHS 
Associate Professor of Pediatric Infectious 
Diseases and Epidemiology 
Johns Hopkins University 
Associate Hospital Epidemiologist 
Johns Hopkins Hospital   
 
Karen Carroll, MD 
Professor Of Pathology 
Johns Hopkins University 
Director Clinical Microbiology Laboratory  
Johns Hopkins Hospital   
 
Susan Aucott, MD 
Associate Professor of Pediatrics 
Johns Hopkins University 
Medical Director, Neonatal ICU 
Johns Hopkins Hospital   
 
Elizabeth Colantuoni, PhD 
Johns Hopkins University 
615 N. Wolfe St., Room E3539 
 
Annie Voskertchian, MPH 
Senior Research Program Coordinator II 
Johns Hopkins University, School of Medicine 
Pediatrics, Infectious Disease 
 
Danielle Koontz, MAA 
Research Program Coordinator 
Johns Hopkins University, School of Medicine 
Pediatrics, Infectious Disease 
 
Maureen M. Gilmore, MD 
Assistant Professor of Pediatrics, JHU 
Director of Neonatology, JHBMC 
Johns Hopkins Bayview Medical Center 
 
Dina Khamash, MD 
Research Fellow 
Johns Hopkins University, School of Medicine 
Pediatrics, Infectious Disease 
 
 
 
 


 
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TABLE OF CONTENTS 
 
 
 
 
 
 
Section/Title Page #  
1. Synopsis  
 
 
 
 
 
 
 
 
 
 4 
2. Background and Rationale 
 
 
 
 
 
 
 
 5  
A. Background 
 
 
 
 
 
 
 
 
  
 5  
B. Rationale 
 
 
 
 
 
 
 
 
 
 
 5 
C. Significance of the Proposed Research  
 
 
 
 
 
 6  
3. Study Purpose, Objectives and Hypotheses 
 
 
 
 
 
 6 
A. Purpose 
 
 
 
 
 
 
 
 
 
  
 6  
B. Objectives  
 
 
 
 
 
 
 
 
  
 6 
C. Primary Hypothesis 
 
 
 
 
 
 
 
 
 6 
D. Secondary Hypotheses 
 
 
 
 
 
 
 
  
 6 
4. Study Procedures  
 
 
 
 
 
 
 
 
 6 
  
 
A. Summary  
 
 
 
 
 
 
 
 
 6 
 
B. Intervention  
 
 
 
 
 
 
 
 
 7 
 
C. Enrollment  
 
 
 
 
 
 
 
 
 7 
 
 
1. Population and Setting 
 
 
 
 
 
 
 7 
 
 
2. Eligibility (withdrawal) 
 
 
 
 
 
 
 8 
 
 
3. Recruitment 
 
 
 
 
 
 
 
 8 
 
 
4. Consent 
 
 
 
 
 
 
 
 
 8 
 
5. Screening   
 
 
 
 
 
 
 
 9 
6. Allocation 
 
 
 
 
 
 
 
 
10  
7. Study Initiation and baseline data collection 
 
 
 
11 
8. Discontinuation of Treatment and Subject Withdrawal  
 
 
11 
9. Unblinding  
 
 
 
 
 
 
 
12 
 
D. Follow-up  
 
 
 
 
 
 
 
 
15 
5. Primary and Secondary Endpoints and Outcomes 
 
 
 
 
15 
A. Primary Endpoint and Outcome – Neonatal acquisition of S. aureus  
 
15 
B. Secondary Endpoint and Outcomes  
 
 
 
 
 
16 
6. Risks and Benefits 
 
 
 
 
 
 
 
 
18 
A. Potential risks 
 
 
 
 
 
 
 
 
 
18 
B. Protection against risks 
 
 
 
 
 
 
 
 
19 
C. Data Safety Monitoring Board 
 
 
 
 
 
 
 
19 
D. Disclosing results   
 
 
 
 
 
 
 
 
20 
E. Potential Benefits  
 
 
 
 
 
 
 
 
21  
7. Specimen Collection and Laboratory Methods 
 
 
 
 
21 
A. Specimen Collection 
 
 
 
 
 
 
 
22 
B. Laboratory Methods  
 
 
 
 
 
 
 
22 
8. Data Collection and Data Monitoring 
 
 
 
 
 
 
24 
9. Statistical Analysis Plan  
 
 
 
 
 
 
 
26 
A. Sample size and power estimates  
 
 
 
 
 
26 
B. Interim Analysis 
 
 
 
 
 
 
 
 
27 
C. Statistical analysis  
 
 
 
 
 
 
 
28 
10. Payment and Remuneration 
 
 
 
 
 
 
 
31 
11. Costs 
 
 
 
 
 
 
 
 
 
 
31 
 
 


 
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1. Synopsis 
More than 33,000 healthcare associated infections (HAI) occur in neonatal intensive 
care units (NICU) each year in the United States. HAIs are estimated to result in $28-45 
billion in healthcare costs annually. In addition to the short-term costs of HAIs, neonatal 
infections contribute to devastating neurologic disabilities and poor growth outcomes. 
Staphylococcus aureus (S. aureus) is the second most common pathogen causing HAIs 
in neonates. Out of 57,000 very low birth weight (VLBW) infants, an estimated 3.7% 
develop bloodstream or central nervous system S. aureus infections annually with an 
attributable mortality of 25%. Despite aggressive measures to prevent S. aureus 
infections in neonates, the burden of S. aureus disease remains high in this population. 
Parents, rather than healthcare workers, may be a key reservoir from which neonates 
acquire S. aureus colonization in the NICU. This paradigm is consistent with a changing 
NICU environment where skin-to-skin contact between parents and neonates is 
encouraged and may promote S. aureus transmission, while at the same time, common 
hospital infection prevention measures have reduced healthcare worker transmission of 
S. aureus. The long term objective of the proposed study is to prevent HAIs in neonates, 
especially those caused by S. aureus. This protocol describes the TREAT PARENTS 
Trial (Treating Parents to Reduce Neonatal Transmission of S. aureus), a randomized, 
masked, placebo-controlled trial that will measure the effect of treating parents with short 
course intranasal mupirocin and topical chlorhexidine antisepsis on acquisition of S. 
aureus colonization and infection in neonates. The findings of the proposed study could 
provide a new tool for HAI prevention in the NICU.  
 
 


 
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2. Background and Rationale  
 
A. Background  
 
The Centers for Disease Control and Prevention (CDC) estimate that approximately 1.7 
million healthcare associated infections (HAIs) occur in U.S. hospitals, including more 
than 33,000 in the neonatal intensive care unit (NICU). Staphylococcus aureus is a 
leading cause of HAIs in NICUs. Despite appropriate therapy, neonatal S. aureus 
infections can have long-term sequelae including poor neurodevelopmental and growth 
outcomes. Preterm infants with infections are 30% more likely to have a lower mental 
developmental index, an important predictor of intelligence quotient. Immunization 
against S. aureus, either active or passive, is years or decades away from results that 
can impact clinical practice.  
 
S. aureus is the second leading cause of catheter-associated bloodstream infections 
(CABSIs) in NICUs. Unlike other ICU populations such as adult intensive care units in 
which the S. aureus CABSIs decreased by 50% from 1997-2007, the incidence of S. 
aureus CABSIs in U.S. NICUs increased by 50% from 1998-2008. In a recent review of 
8,444 very low birth weight infants in U.S. NICUs, 3.7% had S. aureus bacteremia or 
meningitis. Therefore, of the approximately 57,000 VLBW infants in the U.S. in 2011, an 
estimated 2,100 developed S. aureus bacteremia or meningitis, and more than 500 died 
from the infection. There are no national estimates of outcomes associated with other 
neonatal S. aureus infections, including pneumonia, so these projections likely 
underestimate the burden of actual disease in this population.  
 
B. Rationale  
 
Up to 40% of neonates acquire S. aureus in the first two months of life.  Vertical 
transmission of S. aureus is rare, but postnatal transmission from mother to healthy 
infants is common in the first few months of life.  Although healthcare workers have been 
implicated as a source of spreading S. aureus in NICUs, they are often not the source 
for transmission of S. aureus in NICUs. In addition to HCWs, parents have been 
identified as sources of S. aureus transmission. 
 
Our preliminary data suggest that the majority of MRSA strains acquired by neonates in 
our NICU were not acquired from healthcare workers, so parents, rather than healthcare 
workers, may be the key reservoirs from which neonates acquire S. aureus colonization 
in the NICU. This finding is consistent with a changing NICU environment where skin-to-
skin contact between parents and neonates is encouraged and may promote S. aureus 
transmission, while common hospital infection prevention measures have reduced 
healthcare worker transmission of S. aureus. We will test whether detection and 
treatment of S. aureus colonized parents with intranasal mupirocin and topical 
chlorhexidine bathing will decrease the risk of their infant acquiring S. aureus in the 
NICU. Reducing S. aureus burden has been shown to decrease infections.  Rather than 
a patient-directed approach (screening and treating S. aureus colonized neonates) 
which has major limitations in the neonatal population, we propose a parent-directed 
approach that eliminates or delays a neonate’s exposure. Similar to vaccinating 
pregnant mothers with influenza vaccine to prevent disease in newborn infants, we 
propose to engage parents in preventing HAIs in their children.   


 
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C. Significance of the Proposed Research 
 
Among 30,000 patients who acquire HAIs in NICUs across the U.S. each year, more 
than 500 VLBW infants alone die from S. aureus infections. Novel strategies are needed 
to prevent HAIs and alleviate the billions of dollars in short-term healthcare costs and the 
long-term neurologic disabilities in children that survive neonatal infections. This 
proposal builds on our previous work, will test a strategy aimed at reducing HAIs, and 
uses a novel approach for this unique and vulnerable neonatal population. The findings 
of this proposal could change HAI prevention in the NICU from one that focuses on 
healthcare workers and the environment to one that recognizes and highlights parents 
and visitors as important sources of exposure to pathogens that contribute to HAIs. 
 
3. Study Purpose, Objective and Hypotheses 
 
A. Purpose: The goal of this project is to determine the effectiveness of decolonizing 
parents of neonates in reducing neonatal acquisition of S. aureus and neonatal S. 
aureus infections in the NICU.   
 
B. Objectives: To compare the effect of treating parents with short course intranasal 
mupirocin and topical chlorhexidine bathing or placebo on acquisition of S. aureus 
colonization in neonates. To compare the relatedness of S. aureus strains colonizing 
parents and S. aureus strains acquired by their neonates in the NICU. 
 
 
C. Primary Hypothesis 
 
Treating parents of neonates requiring NICU care with intranasal mupirocin and 
topical chlorhexidine bathing will reduce the spread of S. aureus from parents to 
their neonates.  
 
D. Secondary Hypotheses 
 
Many neonates acquire S. aureus in the NICU from their parents 
 
Treating S. aureus colonized parents of neonates requiring NICU care with 
intranasal mupirocin and topical chlorhexidine bathing will reduce neonatal S. 
aureus infections.  
  
Treating S. aureus colonized parents of neonates requiring NICU care with 
intranasal mupirocin and topical chlorhexidine bathing will reduce parental S. 
aureus colonization. 
 
4. Study Procedures  
 
A. Summary  
The TREAT PARENTS Trial, or Treating Parents to Reduce Neonatal Transmission 
of S. aureus, is a placebo-controlled, double-masked, randomized clinical trial to test 
the hypothesis that treatment of S. aureus colonized parents with intranasal 


 
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mupirocin and topical chlorhexidine gluconate antisepsis will decrease neonatal S. 
aureus acquisition. All neonates admitted to the Johns Hopkins Hospital NICU and 
the Johns Hopkins Bayview Medical Center NICU will be pre-screened and parents 
will be approached for enrollment in the study.  After consent and baseline 
screening, approximately 400 neonate-parent pairs will be randomized. Parents will 
receive a 5 day treatment with intranasal mupirocin plus topical chlorhexidine 
gluconate antisepsis or placebo.  
 
B. Intervention  
 
 
Subjects (parents of neonates) will receive an ointment and cloths with standardized 
instructions for use. The instructions will be the same for all subjects. Approximately 
1 cm ribbon of ointment will be self-administered by inserting a new cotton-tipped 
applicator into each anterior naris. A new cotton-tipped applicator will be used for 
each naris followed by gentle massaging of the naris to ensure distribution of the 
ointment. This procedure will be performed twice daily for five days. Verbal and 
printed instructions will be provided to parents. Study staff will demonstrate 
application of mupirocin ointment to the anterior nares. Parents will self-administer all 
doses of the intervention. Subjects will also be given pre-packaged cloths for daily 
skin cleaning. Each package contains 6 cloths. Each cloth will be used to wipe a 
designated body area (arms, legs, chest and neck, back and perineum) once a day 
for 5 days. Study staff will demonstrate how to apply CHG to the skin and instruct 
parents to not rinse after using the cloths. Participants will be instructed to perform 
the baths on the same 5 days they apply the intranasal ointment.  
 
Compliance Monitoring 
Verbal and printed instructions will be provided to parents. A member of the research 
team will contact each participant daily during treatment to review compliance. 
Contact will be made at the bedside or by text, email or phone call at the preference 
of the participant. Participants will be asked to return the medication tube (mupirocin 
or placebo) at the end of the treatment period and any unused cloths. 
 
 
C. Enrollment 
 
1. Patient Population and Setting 
The Johns Hopkins Hospital (JHH) NICU is a 45 bed NICU in a quaternary care 
center that admits approximately 700 neonates per year. The Johns Hopkins 
Bayview Medical Center (Bayview) NICU is a 25-bed, level III unit with 
approximately 375 admissions per year. Neonates admitted to the JHH and 
Bayview NICUs and their parents or legal guardians will be screened for 
eligibility. We will define parents as the biologic mother and the father. In the 
event that one of the parents is not available or does not visit the child in 
the NICU, we will ask the available parent to identify a primary visitor of the 
child in the NICU. Only 2 parents or one parent and one primary visitor will 
be enrolled for each neonate. In the protocol below, parents will refer to 
either a parent or a designated primary visitor. 
 


 
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2. Eligibility  
 
Inclusion criteria for parent-neonate pairs 
i) 
Neonate has never had a prior clinical or surveillance culture grow 
S. aureus  
ii) 
Neonate was transferred from another hospital or admitted from 
home and had admission screening cultures for S. aureus 
colonization that were negative (if admission cultures were not 
performed, they will be performed as part of the pre-randomization 
screening process) 
iii) 
Parent(s) is(are) able to visit the child at the bedside 
iv) 
Parent(s) test positive for S. aureus at screening 
v) 
Neonate has anticipated stay longer than 5 days in the NICU (if 
estimated stay is unclear, parents can be screened for S. aureus 
colonization and decision to randomize can be delayed until 
hospital day 3 or 4 after reassessment of anticipated stay).   
vi) 
Parents is(are) willing to be randomized 
vii) 
No documented or reported allergies to any agent used in either 
treatment regimen   
viii) 
Able to perform written informed consent 
 
Exclusion criteria for parent-neonate pairs 
i) 
Allergies to any agent used in either treatment regimen   
ii) 
Neonate has had a prior clinical or surveillance culture grow S. 
aureus 
iii) 
Neonate admitted to NICU from home and is greater than 7 days 
of age 
iv) 
Neonate admitted to NICU from another hospital and is greater 
than 7 days of age 
v) 
Neonate is a ward of the State 
vi) 
Not able to provide written informed consent 
 
3. Recruitment 
 
We will pre-screen patients for eligibility (including anticipated length of stay, 
prior culture growing S. aureus). A member of the study team will approach 
the parent at the bedside and request participation as outlined below. If 
parents are not available at the bedside, a study team member will call the 
parents to arrange a time to meet and discuss participation in the study.  
 
4. Consent 
 
The consent process will be completed at the bedside in the neonate’s 
private room or in a private area of the NICU or in the mother’s private room 
in cases where the mother is still admitted to the labor and delivery ward. The 
consent form will be read aloud to any participant that has difficulty with 
reading. The consent has been developed on an 8th grade reading level. 
Each parent will be consented for participation. Only neonates who have a 


 
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parent colonized with S. aureus will be randomized, and a copy of the 
consent form will be placed in the child’s chart; therefore, participants will be 
informed that people outside the study may become aware that one of the 
parents is colonized with S. aureus, but nowhere in the child’s chart will it be 
documented which parent is colonized. A copy of the signed consent form will 
be made available to each participant, a copy will be placed in the neonate’s 
bedside chart, and a copy will be kept in a regulatory binder. Parents who are 
not colonized will not be randomized; however, they will be asked to continue 
to participate in the study by completing a follow-up questionnaire 
approximately four weeks after their screening date. If parent does not allow 
long-term storage of biospecimen for future research they will be asked for 
consent to store bacteria collected from biospecimens.  
 
Assessment of Protocol Comprehension Prior to Signing Consent 
 
After the consent form is read, a member of the research team will evaluate 
comprehension with three protocol specific questions: 1) “What is the name 
of the bacteria or germ that we are trying to get rid of?”; 2) “How does this 
study plan to get rid of this bacteria or germ?”; 3) “How will you be assigned 
to receive either antibacterial cloths and ointment versus a plain cloth and 
ointment with no antibiotic?”  We will deem the subject to have an acceptable 
level of comprehension based on their ability to answer those questions. We 
will review the corresponding section of the consent form with the participant 
if any of the responses are unsatisfactory and ask the questions again to 
ascertain comprehension. 
 
 
5. Screening 
 
The PI and/or a member of the research team will provide information 
regarding the study, answer any questions and obtain informed consent. The 
PI will be available in person or via telephone to answer any questions when 
a member of the research team is present for the consent. As part of the 
informed consent document, the subject will receive information on how to 
contact a study team member to discuss culture results or any noted 
treatments that may be related to S. aureus. Once consented, a member of 
the research team will assign a screening number (SN) to the subject. If the 
cultures are positive, the family unit will be enrolled and assigned a study 
identification number (SIN). This will be the primary mode of identification 
throughout the study. The SIN will identify the parent and the neonate (or 
neonates in the case of multiple gestations). The SIN will also identify both 
parents (if both consent to participate as both will be assigned to the 
treatment group). The SIN and/or SN will appear on the consent form, the 
data collection form, as well as all S. aureus cultures. If the cultures test 
negative, the family unit will not be randomized to treatment. However, parent 
participation in the study will not end. They will be asked to continue their 
participation in the study by completing a follow-up questionnaire 
approximately four weeks after their initial screening visit. 
 


 
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During the screening visit, a member of the research team will perform the 
necessary swabs to be sent for culture. The parent(s) who consent to 
undergo testing will be screened by obtaining a swab from the following 
anatomical sites: bilateral anterior nares, the throat, the groin/perineum, and 
the peri-anal area. A standardized check-list for obtaining and processing 
swabs will be used. For neonates transferred from an outside hospital or from 
home that did not have surveillance cultures obtained on admission, 
screening cultures will be performed by obtaining a swab from the following 
anatomical sites: bilateral anterior nares, umbilicus, groin, and peri-anal area. 
 
All swabs will receive a study number pre-printed on a label. The SN will not 
identify the patient’s name, medical record number or other identifying 
information. The SN will be recorded on the informed consent documents and 
the surveillance swabs. Before sending the swabs, a member of the research 
team will take a brief “time-out” to reassess that all paperwork is completed, 
that swabs are appropriately labeled with SN, and that all consent forms and 
swabs contain the same SN. 
 
Laboratory methods for determining whether a patient has S. aureus 
colonization are described below in Section 7.B. S. aureus colonization will 
be defined as a culture from any anatomic site that grows S. aureus. 
 
  
6. Allocation 
 
Randomization and masking: This study has taken into consideration the 
guidelines for reporting of clinical trials as recommended by the CONSORT 
report to inform the study design. After recruitment and informed consent, 
parents will undergo pre-randomization screening. If both parents screen 
negative for S. aureus colonization, the neonate will be ineligible for the 
randomization and parents will be informed that they are not colonized at that 
time with S. aureus. If either parent screens positive for S. aureus, then both 
parents as a pair will be eligible for randomization to one of the two possible 
masked treatment arms. The neonate-parent “pair” will be the unit of 
randomization and each parent will be allocated to the same group if both 
consent. Because couples can re-expose each other after treatment 
(especially in households) we have chosen to treat both parents even if only 
one parent is colonized with S. aureus. To prevent potential feelings of guilt 
or one parent blaming another if only one parent is colonized, the parents will 
be told that one or both is colonized with S. aureus and they are eligible for 
randomization. Discordant parents will not be informed which is colonized 
and which is not colonized as we will treat both regardless. 
 
Arm 1 will include assignment to masked intranasal mupirocin plus 
chlorhexidine gluconate cloths for topical antisepsis. Arm 2 will include 
assignment to masked placebo ointment and placebo cloths for skin 
antisepsis. Stratified permuted-block randomization will be performed using R 
statistical software to achieve balanced allocation of subjects within study site 
(Johns Hopkins Hospital vs. Bayview) and within strata of birth weight 


 
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(greater than or equal to or less than 1500g). Use of varying block sizes (4, 6 
and 8) will decrease the risk of imbalance. Neonates of multiple gestations 
and their parents will be randomized as a single family unit. Investigators and 
participants will be masked to treatment assignment. The treating clinicians of 
the neonates will be masked to treatment assignment. The investigational 
drug pharmacy will use the randomization sequences to prepare supply kits. 
As subjects are enrolled, they will receive the next kit in sequence based on 
the study site and birth weight of the neonate. Each kit will contain either 
active drug or placebo. Active chlorhexidine-impregnated cloths, active 
mupirocin ointment and their respective placebos will be identified only by lot 
numbers, and the respective lot numbers will only be known to the 
investigational drug pharmacist.  
 
 
7. Study initiation and baseline data collection 
 
After results of the pre-randomization screening are completed, a member of 
the research team will contact the subject(s) to review the results and 
randomize the positive family units into either treatment or placebo group. 
After reviewing consent and the study with the parent(s), treatment will 
commence immediately upon assignment to treatment arm. At that time, the 
research team will perform the necessary baseline swabs to be sent for 
culture from the neonate. Neonates’ screening cultures will be performed by 
obtaining a swab from the following anatomical sites: bilateral anterior nares, 
umbilicus, groin, and peri-anal area. Study team members will also gather 
residual samples from the clinical laboratory collected during routine clinical 
care. Parents will be asked about some demographic and medical history 
information for the researcher to complete on the case report form. Additional 
information will be obtained from the neonate’s medical record (e.g. birth 
history, maternal prenatal labs).  
 
Those neonates that test positive for S. aureus colonization at time of 
randomization will not be eligible for the primary outcome analysis and they 
will not contribute to the sample size required to power the primary outcome. 
Participants will be informed that their child is colonized with S. aureus, but 
participants will be asked to continue the assigned treatment, so secondary 
outcome data can be collected.  
 
8. Discontinuation of Treatment and Subject Withdrawal 
 
Discontinuation of Intranasal Treatment 
Study treatment may be discontinued for any of the following reasons:  
 
 
If participant(s) exhibits severe or life threatening side effects (Grade 3 or 
higher) that require subject to seek medical care. Side effects include 
pain, blistering, urticarial rash or hypersensitivity, trouble breathing, loss 
of consciousness, swelling of the tongue and airways, or other life 
threatening reactions judged by the treating provider. If the treating 


 
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provider and/or PI determines that the reaction is not related to study 
treatment, the subject will restart the treatment.  
 
Subject(s) request discontinuation of treatment. 
 
Discontinuation of Topical Bathing Treatment 
Study treatment may be discontinued for any of the following reasons: 
 
 
If participant(s) exhibits severe, or life threatening side effects (Grade 3 or 
higher) that require subjects to seek medical care. Side effects include 
pain, blistering, urticarial rash or hypersensitivity, trouble breathing, loss 
of consciousness, swelling of the tongue and airways, or other life 
threatening reactions judged by the treating provider. Mild skin irritation 
such as itching, dryness, and transient redness will not be reasons to stop 
treatment. If the treating provider and/or PI determines that the reaction is 
not related to study treatment, the subject will restart the treatment 
 
Subject(s) request discontinuation of treatment 
 
If reaction occurs to topical bathing, but not the ointment, then the participant 
may discontinue the bathing and continue the ointment application after 
discussion with the study team.  
 
In the case that a subject discontinues study nasal and/or topical bathing 
treatment(s), the parent(s) and their neonate(s) will be followed for swab 
collection, bacterial cultures, and medical history data collection as outlined in 
the protocol until they meet the criteria for study completion, unless the 
parent withdraws from the study and/or withdraws consent from disclosure of 
future information. In this case, the investigator may retain and continue to 
use any data collected before the subject withdrew their consent.  
 
Subject Withdrawal 
 
Participants may withdraw from study for any of the following reasons: 
 
Subject requests withdrawal of consent for further participation in study 
 
Subject meets criteria for withdrawal based upon investigator discretion 
(e.g. safety, behavioral, or administrative reasons) 
 
If patients discontinue treatment or withdraw from the study the primary reason 
for discontinuation/withdrawal must be provided and documented in the CRF. 
Discontinuation of treatment and/or withdrawal due to adverse event will be 
distinguished from withdrawal due to other causes and recorded on the 
appropriate adverse event CRF page.   
 
9. Unblinding 
 
In the event of a medical emergency where knowledge of the participant’s 
blinded treatment is critical to their medical management, the blind may be 
broken by the investigator. Before breaking the blind, the investigator must 
determine whether or not knowledge of the blinded information will alter the 
immediate care of the participant. In some instances, it may be possible to 


 
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treat the participant by assuming they are receiving active product. The 
investigator will discuss the decision to unblind with the DSMB.   
Blinding will only be broken for appropriate medical reasons such as 
managing treatment of a patient in an emergency or if required for reporting 
to regulatory authorities. Any event for which the study blind is broken will be 
reported as an SAE. The accompanying SAE report should include a 
comment about breaking the blinding. 
 


 
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Screen parents for S. aureus 
Randomization 2 
Baseline screening of neonate for S. 
aureus colonization  
Parents of Neonates admitted to The Johns Hopkins NICU 
Consents and Fulfills Eligibility 
Yes 
Not colonized 
Standardized regimen – 
Active product 
N=200 
Standardized regimen – 
Placebo 
N=200 
Weekly screening of neonate3 and 
biweekly/monthly screening of 
parents 4 
No 
Primary Outcome 
Acquisition of concordant S. aureus 
colonization prior to NICU discharge 
Secondary Outcomes 
Microbiology and Molecular 
Epidemiology 
Colonized1 
1Either parent colonized with S. aureus  
2 Unit of randomization is Parent-Neonate(s) Grouping 
3 Final screening at time of NICU discharge 
4 Final Screening at time neonate meets primary outcome 
 
 
Enrollment 
Allocation 
Follow-up 
Analysis 
Figure 1. Overview of Study Design 
Discontinued 
from study 
Neonate positive for S. aureus at 
baseline 
 
=Screen Fail 
Secondary Outcomes 


 
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D. Follow-up 
 
Parent Evaluation Time Points: After randomization, the study team will 
provide the study drugs, study drug application instruction sheets and 
informational material. All participants will begin the 5-day treatment course at 
that time. During the 5-day treatment period, a study team member will contact 
the study participant daily at the bedside, on the phone, by email or by text 
message to assess compliance with treatment and to answer any questions. 
When available, parent(s) will be re-tested for colonization at 2-week intervals 
from randomization for the first 8 weeks and then every four weeks until 
discharge. Follow up swabs may be taken within a week of scheduled time 
points, based on participant availability.  Anatomic sites to be tested and 
frequency of testing are detailed in Table 1. Peri-anal swabs will only be collected 
at enrollment and final screening. The final visit and testing will be performed at 
the time of a neonate’s discharge from the NICU or at the time the child is 
identified to have acquired S. aureus.  
 
Neonate Evaluation Time Points: After informed consent and randomization of 
parents, the neonate will undergo baseline testing to determine baseline S. 
aureus colonization status. This testing will occur on study day 1, the same day 
that parents begin treatment. Screening cultures will be performed by obtaining a 
swab from the following anatomical sites: anterior nares, the umbilicus, groin, 
and peri-anal area. If the nares site is not available to swab, the throat will be 
swabbed instead. Those neonates who test positive for S. aureus colonization at 
time of randomization will not be included in the primary analysis. After baseline 
testing, repeat testing will be performed every 7 days. Study samples will be 
saved and processed in the lab. The final visit and testing will be performed at 
the time of a neonate’s discharge from the NICU.   
 
 
Table 1 
Frequency of Parent Swab Collection 
Site 
Screening 
Weeks 2, 4, 6, 8, 12, 
16, 20, 24, 28,…. 
Time Neonate 
Acquires S. 
aureus 
Nares 
X 
X 
X 
Throat 
X 
X 
X 
Groin 
X 
X 
X 
Peri-anal 
X 
 
X 
 
 
Table 2 
Frequency of Neonate Swab Collection 
Site 
Parent 
Randomization 
Weeks 1, 2, 3, 4,…. 
NICU Discharge 
Nares 
X 
X 
X 
Umbilicus 
X* 
X* 
X* 
Groin 
X 
X 
X 
Peri-anal 
X 
X 
X 


 
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          *Umbilicus will only be tested if the neonate has attached umbilical cord.  
 
5. Primary and Secondary Endpoints and Outcomes  
  
 
 
A. Primary: Primary outcome is neonatal acquisition of S. aureus strain that is 
concordant to parental S. aureus strain as determined by periodic surveillance 
cultures or a culture collected during routine clinical care that grows S. aureus. 
 
Acquisition will be defined as meeting two criteria:  
 
1) A neonate who had baseline surveillance cultures that were negative 
for S. aureus  
2) A neonate who has a subsequent surveillance culture or culture 
collected during routine clinical care that grows S. aureus.   
 
Concordant strains must meet the following criteria: 
 
1) Strains that are related using pulsed-field gel electrophoresis (PFGE) 
analysis, as described below. Isolates will be considered related if 
their patterns have ≤3 band differences.  Isolates with >3 band 
differences will be considered epidemiologically different strain types. 
A neonate who has a subsequent surveillance culture or culture 
collected during routine clinical care that grows S. aureus.   
2) Alternative typing methods will be used to further discriminate highly 
prevalent strains or those that are not typable by PFGE.  
3) The same strain from the initial parent screening is identified from the 
neonate. 
 
Primary outcome ascertainment – We will measure each neonate’s baseline S. 
aureus colonization status at the time of study enrollment. As described above, neonates 
will then have swabs collected weekly after enrollment and at time of discharge from 
multiple anatomic sites by a study team member. We also will use data from cultures 
collecting during routine clinical care in the NICU. These cultures include cultures of 
nasal swabs collected every Tuesday as part of an active S. aureus surveillance 
program and other cultures collected during routine clinical care (e.g. blood cultures, 
respiratory cultures, wound cultures, etc.). Cultures collected by the study team and 
cultures collected during routine clinical care with both be used to identify S. aureus 
acquisition in neonates. Of note, nasal surveillance swabs collected as part of the 
hospital surveillance program are plated on selective media plates to detect S. aureus 
colonization, as previously reported [1]. 
 
Reducing the risk of bias in outcome ascertainment: Clinicians will continue to send 
cultures to evaluate neonates for possible infection as part of routine clinical 
management. The primary care providers in the NICU will not participate in the study 
and will be masked to the parent-neonate pair assignment. Maintaining masking of the 
clinicians will reduce the chance that practices for obtaining clinical cultures will differ 
between the two groups.  
 
The observation period, or survival time, is the time that a neonate is at-risk of 
concordant S. aureus acquisition. We will calculate survival time as 1) the interval 


 
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between randomization and the date of final culture (either at time of final surveillance 
culture or at time of discharge culture) for neonates who did not acquire concordant S. 
aureus and 2) the interval between randomization and date of first positive culture for 
neonates that acquire concordant S. aureus.  
 
B. Secondary outcomes of interest include: 
 
1. Neonatal acquisition of S. aureus as determined by periodic surveillance 
cultures or a culture collected during routine clinical care that grows S. 
aureus. 
 
Acquisition will be defined as meeting two criteria:  
 
1) A neonate who had baseline surveillance cultures that were negative 
for S. aureus  
2) A neonate who has a subsequent surveillance culture or culture 
collected during routine clinical care that grows S. aureus.   
 
Outcome ascertainment – same as for primary outcome 
 
Observation period or survival time – same as for primary outcome but does 
not require the S. aureus strain to match that of the parent.  
 
2. Neonatal S. aureus infection as determined by cultures collected during 
routine clinical care  
 
A S. aureus infection will be defined using criteria established by the 
Centers for Diseases Control and Prevention National Healthcare Safety 
Network and will be determined using results of cultures sent during routine 
clinical care (other than surveillance cultures).   
 
Secondary outcome ascertainment - Two study team members that are 
blinded to treatment assignment will review all clinical cultures for S. aureus 
during the treatment and control period and determine if the culture 
represents an infection due to S. aureus. A third study team member will 
review the case in the event of discordant results. 
 
The observation period, or survival time, is the time a neonate is at-risk of 
S. aureus infection. We will calculate survival time either by 1) the interval 
between randomization and the date the neonate is discharged from the 
NICU or death for neonates who did not develop a S. aureus infection or 2) 
the interval between randomization and date of positive culture for neonates 
that develop S. aureus infection. Neonates will not be included if they have an 
infection after meeting the primary outcome of concordant S aureus 
colonization or the secondary outcome of neonatal acquisition of S aureus. 
 
 
3. Eradication of S. aureus colonization in parents following treatment 
 


 
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Outcome ascertainment – Parents will be monitored as described above in 
section 4.D to monitor S. aureus colonization over time. Parents will be tested 
at baseline, and then every two weeks for the first two months, if available, 
while neonate remains in the NICU, monthly starting on third month their 
neonate remains in the NICU, at the time the infant acquires S. aureus 
colonization in the NICU, and at time of discharge from the NICU for a 
neonate that did not acquire S. aureus colonization in the NICU.  
 
4. Natural history of S. aureus colonization in parents receiving placebo 
 
Outcome ascertainment – same as above for parents receiving placebo 
  
5. Adverse reactions to treatment 
 
Outcome ascertainment – Participants will be instructed to report any rash, 
blister, itching, redness, swelling or other skin irritation, swelling of the face or 
limbs, difficulty breathing or swallowing, and any significant discomfort or 
illness that occurs during treatment. Study staff will specifically ask for these 
symptoms during the 5-day treatment period and after the treatment period at 
Day 6, and will report any adverse event that is thought to be related to the 
study on the Adverse Event Log as well as any other perceived adverse 
events.  
 
6. Feasibility and Feedback 
 
Outcome ascertainment - Using compliance data and parent reporting, we 
will also determine if the intervention is feasible for this population. 
Compliance with treatment is already being measured. In addition, after 
completing the 5 day intervention at Day 6, parents will be asked several 
questions about the ease of the cloth and ointment use as well as their 
comfort level and other feelings about the treatment. During the follow-up 
testing of parents, we will ask for any comments, suggestions or concerns 
from the parents about their general experience as a participant in the study. 
This data will be both quantitative and qualitative and can help determine if 
the intervention is feasible for future use. The study team may on occasion 
audio record responses to these questions. 
 
7. Attitudes and Behavior 
 
Outcome ascertainment – In order to examine the impact of S. aureus 
education and knowledge of S. aureus colonization, all consented parents will 
be asked to complete a follow-up questionnaire. The questionnaire will 
assess differences in behavior between ineligible parents (negative for S. 
aureus) and eligible parents (at least one parent positive for S. aureus and 
enrolled in study). In the questionnaire, parents will be asked about their 
interactions and behaviors with their newborns in the NICU, whether their 
hand hygiene and/or bathing routines have changed, and about modifications 
made to medication use over the course of the four weeks since their initial 
screening visit.  


 
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6. Risks and Benefits 
 
A. Potential Risks  
The risks for this study are minimal. There is a very small risk of bleeding associated 
with swabbing mucosal membranes; however, our institution has a long standing history 
of obtaining nares surveillance cultures for S. aureus and peri-anal surveillance cultures 
to detect vancomycin resistant enterococci without any reported adverse events. Throat 
swabs can make people gag but are routinely performed during clinical evaluations of 
pharyngitis without adverse events. Standard precautions will be applied for protection of 
the research team member during specimen collection. All parent swabs will be collected 
in a private room. Groin and peri-anal swabs will be self-collected by parents following 
instructions from the study team. If the parents prefer, the groin and peri-anal swabs can 
be collected by a study team member. A gown will be provided for the parent to protect 
modesty and privacy during collection if applicable.    
 
This decolonization procedure is widely used in individuals with S. aureus colonization in 
certain settings such as pre-operative, recurrent infections, and those in the intensive 
care unit. This treatment has been well tolerated in all populations, with rare adverse 
events. Allergic reactions to mupirocin and/or chlorhexidine are uncommon. Patients 
with known allergy to any product ingredient will be excluded from the study. All agents 
are FDA approved for the target population and we have received an FDA exemption 
from IND. A number of recent studies have applied decolonization to households rather 
than just individuals, as close contacts can serve as reservoirs for recurrent colonization 
after treatment. Based on reports that treating households versus just individuals may 
have a greater impact on S. aureus, we have decided to treat both parents even if one is 
not colonized. To protect parent’s privacy and prevent potential feelings of guilt or one 
parent blaming the other, we will not disclose discordant results to parents. Therefore, 
care providers will not know if a particular parent is colonized simply based on 
enrollment in the study, as it might be “the other parent” that is colonized. 
 
Mupirocin Ointment Possible Adverse Effects:  Local adverse effects may be 
associated with mupirocin application. Mupirocin ointment may cause itching, pain, 
stinging, and burning. Local effects from the nasal formulation have included epistaxis, 
rhinitis, taste perversion, pharyngitis, burning, and cough.  Gastrointestinal side effects 
have included nausea (1.1%), abdominal pain (<1%), and diarrhea (<1%). Dry mouth 
has been reported with mupirocin nasal (<1%). Nervous system side effects associated 
with intranasal mupirocin have included headache (9%). Dizziness has been reported 
with mupirocin ointment (<1%). Ocular side effects associated with mupirocin nasal 
ointment have included blepharitis (<1%). 
 
Chlorhexidine Gluconate-impregnated Cloths Possible Adverse Effects: The soap 
may cause burning and dryness of the skin.   It may cause burning of the eyes, but it’s 
not recommended to be used above the neck. Skin erythema and roughness, dryness, 


 
20 
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sensitization, allergic reactions are possible, but rare. 
  
B. Protection against Risks  
 
Protection against loss of information: We will institute strict procedures to maintain 
confidentiality. All patients will be assigned a study identification number.  We will 
maintain a master list of patients with unique study identifiers. The master list will be 
maintained on a password protected computer or institutional network drive. Data will be 
entered into REDCap (http://www.project-redcap.org) which is a secure, web-based 
application designed exclusively to support data capture for research. The Johns 
Hopkins University is a member of the REDCap consortium and this application is freely 
available to consortium members. REDCap provides: 1) an intuitive interface for data 
entry (with data validation); 2) audit trails for tracking data manipulation and export 
procedures; 3) automated export procedures for seamless data downloads to common 
statistical packages (SPSS, SAS, Stata, R); 4) procedures for importing data from 
external sources; and 5) advanced features, such as branching logic and calculated 
fields. To maximize quality control, staff will be trained by the PI in all data collection and 
entry procedures. The PI will have access to all data upon entry into REDCap. The 
research coordinator will monitor data collection by checking data for completeness. All 
research staff will be instructed regarding the security of data and will maintain the 
highest ethical standards in protocol adherence and data collection. After all data are 
collected, analyzed, and published, linkage between patients and their unique identifier 
will be destroyed in accordance with local regulations with the exception of data that 
have been collected for hospital surveillance activities. During the course of the study, 
information collected will not be disclosed to anyone other than the study personnel. No 
sensitive information will be collected. Following completion of each qualitative interview, 
all audiotapes (digital recordings) will be transcribed and marked with the session title 
and each subject’s SID number. No identifying information will appear on the label of the 
media. All transcribed media will be stored on a secure Johns Hopkins server. Audio 
recordings with identifiers will be destroyed after being transcribed. 
 
Protection against adverse events from treatment: As discussed above, treatment of 
S. aureus colonized individuals with intranasal mupirocin and chlorhexidine gluconate-
impregnated cloths is routinely performed in many clinical settings. Very rare cases of 
anaphylaxis have been reported. Parents will be given written and verbal instructions for 
proper use. The research team will contact parents daily to review compliance with 
treatment, to answer questions, and to monitor for adverse events. Safety data including 
expected and unexpected events will be provided to the DSMB for review. Any 
unexpected events will be submitted immediately to the IRB.  
 
The study team recognizes that subjects may seek advice about issues beyond the 
scope of this study and will refer all non-study related health issues to the patient’s 
treating clinician.   
 
C. Data Safety Monitoring Board 
 
A Data Safety Monitoring Board (DSMB) will be assembled to oversee this study.  
 


 
21 
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The DSMB will consist of the following members: 
1. Anthony Harris M.D., M.P.H.: Dr. Harris is a Professor of Medicine and Epidemiology 
at the University of Maryland and will be a member of the DSMB for his expertise in 
S. aureus, epidemiologic methods, adult infectious diseases, and clinical trials.  
2. Michael A. Rosenblum, Ph,D: Dr. Rosenblum is an Assistant Professor of 
Biostatistics in the Johns Hopkins Bloomberg School of Public Health. He is 
experienced in the design and analysis of clinical trials.  
3. Mary Leppert, M.D.:  Dr. Leppert is a member of the medical staff in the department 
of Neurodevelopmental Medicine at the Kennedy Krieger Institute in Baltimore, 
Maryland and an Assistant Professor of Pediatrics at Johns Hopkins University 
School of Medicine. Dr. Leppert is studying the parent experience in the NICU using 
the Parent Stress Index. She will serve as a member of the DSMB for her expertise 
in developmental pediatrics. 
4. Neal Halsey, M.D: Dr. Halsey is a Professor of Pediatrics and International Health at 
the Johns Hopkins Bloomberg School of Public Health. Dr. Halsey will be a member 
of the DSMB for his extensive expertise in pediatric clinical trials and infectious 
diseases. 
 
The DSMB will review safety data related to adverse events associated with treatment of 
parents and interim analysis of the primary outcome. The duties will include: 
1. Meeting before study initiation to review the research protocol and plans for data 
safety monitoring. 
2. Assessment of data quality and timeliness. 
3. Monitor fidelity to study protocol 
4. Review participant recruitment and retention 
5. Protection of the confidentiality of the trial data and results of the monitoring. 
6. Sharing recommendations and assessment with the investigative team. 
7. Assessing any reported unexpected adverse events that may impact the safety 
of the trial.   
8. Make recommendations to the IRB and investigative team concerning trial 
continuation or modification based on interim results as outlined below.  
 
 
D. Disclosing results 
 
Plan for Neonates Testing Positive for S. aureus and disclosure of results: 
The Johns Hopkins Hospital has an active program to identify neonates that are 
colonized with S. aureus. A protocol exists such that colonized neonates are 
decolonized to reduce the spread of S. aureus in the NICU and the risk of infection in 
individual patients. If a culture collected during the research study grows S. aureus, this 
information will be provided to the clinical team.  
 
Plan for Parents Testing Positive for S. aureus and disclosure of follow-up results:  
Upon enrollment, all subjects will be provided a brief structured orientation and 
education session regarding S. aureus by a member of the research team.  All patient 
questions will be answered and educational handouts will be provided with references 
for additional information. Part of the informed consent will include an understanding that 
parents will be made aware of their S. aureus colonization status. If either or both 
parents are colonized, parents will be informed that one or both parents are colonized, 


 
22 
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but we will not share which parent to protect confidentiality. By participating in the trial, 
others may also become aware that one of both of the parents are colonized simply by 
their participation in the trial. We will explain that approximately 30% of healthy adults 
are colonized with S. aureus. We do not take special precautions or treat parents 
differently that are colonized with S. aureus. Additionally, we will enroll both parents 
even if only one parent is colonized, which will aid in protecting the privacy of 
participants as care providers will not know if a particular parent is colonized simply 
based on enrollment in the study, as it might be “the other parent” that is colonized. 
 
Once parents are randomized to an assigned treatment, they will continue to be 
screened for S. aureus colonization throughout the study. The results of subsequent 
testing will not be made available to the parent. Disclosing subsequent testing results 
may lead to unmasking of the study. Parents will be informed that these results will not 
be available until the study is complete and the randomization code is un-blinded for 
data analysis. Parents will be provided with a means to contact the study team to learn 
their testing results and/or randomization group.  
 
Parents with discordant culture results should not become aware of each other’s 
colonization status. To decrease or eliminate guilt issues, the study team will not 
disclose which parent is colonized and will educate parents on the ubiquity of S. aureus 
and emphasize that parents are only one possible source of transmission. 
 
 
E. Potential Benefits  
 
There may be no direct benefit to the subjects in this study; however, information 
obtained in this research may lead to a better understanding of how neonates acquire S. 
aureus in the NICU and whether eradicating colonization in parents reduces neonatal S. 
aureus acquisition and infection.  
 
 
7. Specimen Collection and Laboratory Methods 
 
A. Specimen Collection 
 
    Swabs will be collected as outlined below:  
 
Neonates 
Using standardized methodology, four flocked swabs will be obtained from  
1. Bilateral nares: by inserting a swab 0.5-1cm into each naris and rotating the 
swab 3 times (if nares is not available, study team will collect a throat swab 
instead by inserting a swab in the pharynx and swabbing the region of the 
tonsils with a quick rotating motion three times on each side).  
2. Groin: The groin will be swabbed in a back and forth motion 3 times on each 
side. 
3. Umbilicus: For children who still have an umbilical stump, the base of the 
umbilical cord will be swabbed in a circular motion 3 times (if there is no 
umbilical cord, there will be no umbilicus swab). 


 
23 
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4. Peri-anal area: The peri-anal area will be swabbed in a circular motion 3 
times 
 
Parents 
Using standardized methodology, four flocked swabs will be obtained from  
1. Bilateral nares: Study staff will collect these by inserting a swab 0.5-1cm into 
each naris and rotating the swab three times 
2. Throat: Study staff will collect this by inserting a swab in the pharynx and 
swabbing the region of the tonsils with a quick rotating motion three times on 
each side. 
3. Groin: Parents will be instructed by study staff on how to collect these 
samples. Parents will be able to choose whether they prefer to self-collect the 
swab or have the study team member collect the swab. 
4. Peri-anal: Parents will be instructed by study staff on how to collect these 
samples. Parents will be able to choose whether they prefer to self-collect the 
swab or have the study team member collect the swab. 
 
B. Laboratory Methods 
 
Processing of cultures collected by the study team 
 
       
 
Parents’ Screening Cultures:  
1. An aliquot (10µL) from each culture specimen will be plated on MSSA Select 
(Bio-Rad Laboratories, Redmond, WA) selective and differential medium to 
detect S. aureus. Presence of mauve colored colonies after 24 hours of 
incubation will be confirmed as S. aureus by Gram’s stain and coagulase testing. 
In parallel, 10µL of each specimen will also be plated on a sheep blood agar 
plate. Residual specimen (Amies media) will be transferred to cryovials and 
stored at -700C. All cultures that grow S. aureus will be archived in Tryptic Soy 
broth with 20% glycerol at -700C 
2. To enhance detection of S. aureus, an aliquot (100µL) of medium will be 
inoculated into Tryptic Soy broth with 6.5% NaCl and incubated overnight for 18-
24 hours at 350C. 10µL of broth will be inoculated onto sheep blood agar and 
MSSA Select (Bio-Rad Laboratories, Redmond, WA) and processed as 
described above to detect S. aureus. Isolates recovered by this method will be 
archived in Tryptic Soy broth with 20% glycerol at -700C. 
3. To distinguish methicillin-resistant S. aureus (MRSA) and methicillin-susceptible 
S. aureus (MSSA), isolates will be tested on the BD Phoenix Microbial 
identification system and with other phenotypic testing methods. Screening 
cultures obtained from parents will not be tested in real-time to distinguish MRSA 
and MSSA. Parents who are colonized with MRSA will not be identified. There is 
no standard of care in terms of how to manage parents or visitors that are 
colonized with MRSA. We do not screen visitors and there is no consensus that 
colonized visitors should be treated any differently other than following standard 
precautions (hand washing, covering wounds, etc.). 
 
 
Neonates’ Baseline Colonization Cultures: 
 


 
24 
Version 13-11/30/2018 
 
Specimens from neonates will be processed as described in 7.B.2-7.B.3 above (no 
direct plating, i.e. all specimens will be inoculated into broth before plating).  
 
Follow-up Cultures for both Neonates and Parents: 
 
Follow-up cultures for both neonates and parents will be processed as described in 
7.B.2-7.B.3 above (no direct plating). 
 
                  Processing of cultures obtained during routine clinical care 
 
1. As usual, all cultures sent to the Johns Hopkins Microbiology laboratory will be 
processed according to Clinical and Laboratory Standards Institute guidelines. All 
clinical cultures that grow S. aureus will be archived in Tryptic Soy broth with 
20% glycerol at -700C.  
 
S. aureus strain typing:  
 
1. Strains of S. aureus will be compared using pulsed-field gel electrophoresis 
(PFGE) analysis. DNA will be extracted by standard methods and the restriction 
enzyme digested using Sma1.  A Staphylococcus aureus subspecies NCTC 
8325 will be used as a control strain with a molecular weight ladder at the 
beginning and end of each gel.  Control isolates, including all USA PFGE strain 
types, will be used for comparison.  Using 1% pulsed-field certified agarose gel 
(BioRad Laboratories, Hercules, CA) electrophoresis will be performed on the 
CHEF-DR III (BioRad Laboratories, Hercules, CA) with 0.5X TBE buffer at 14oC.  
The gels will be stained with ethidium bromide and scanned using a molecular 
analysis fingerprinting software (Fingerprinting II Version 3.0; BioRad 
Laboratories, Hercules, CA).  If PFGE using standard enzymes was unable to 
yield an interpretable fingerprint, PFGE was then performed using an alternative 
method utilizing Achromopeptidase (Wako Bioproducts, Richmond, VA) as the 
lysis enzyme in order to obtain an interpretable fingerprint. Gel images will be 
stored and within and between run comparisons will be performed for each group 
of tested isolates. Isolates will be considered related if their patterns have ≤3 
band differences.  Isolates with >3 band differences will be considered 
epidemiologically different strain types.   
 
2. Alternative S. aureus strain typing method: Because some S. aureus strains are 
genetically similar by PFGE, such as strain USA300, validation of highly 
prevalent and concordant strains will be carried out using a separate assay such 
as a PCR assay coupled with electrospray ionization-mass spectrometry 
(PCR/ESI-MS), whole genome or next generation sequencing, or multi-locus 
sequence typing. Isolates with no band differences will be considered identical, 
but those with 1-3 band differences will be considered for additional testing 
based on strain prevalence.  
3. If strains could not be distinguished by PFGE analysis, multi-locus sequence 
typing (MLST) will be performed using whole genome sequencing. After DNA 
extraction, a sequence library will be generated using the Nextera DNA Library 
Preparation Kit and sequenced by Miseq (Illumina Inc, San Diego, CA). 
Sequenced fragments will be assembled using SPAdes and aligned to reference 


 
25 
Version 13-11/30/2018 
 
S. aureus genome with Pavian (Johns Hopkins University, Baltimore, MD) for 
quality assessment. Strain type will be assigned by The Bacterial Analysis 
Pipeline (Illumina Inc, San Diego, CA) based on the alleles of seven 
housekeeping genes, including arcc, aroe, glpf, gmk, pta, tpi, and yqil. Two 
isolates will be considered concordant if both had identical alleles for all seven 
genes. 
 
8. Data Collection and Data Monitoring 
 
Prior to any work being performed on this research proposal it will be submitted for 
review and approval by the Institutional Review Board at JHU. We will maintain a master 
list of patients with unique study identifiers. The master list will be maintained on a 
password protected computer or institutional network drive. Data will be entered into a 
database that will be stored on a secure server at the Johns Hopkins University. All 
research staff will be instructed as to how to maintain the highest ethical standards in 
protocol adherence and data collection. Standard backup procedures are in place at the 
institution to prevent catastrophic loss of data. Validity and accuracy checks will be put in 
place as data are entered into the database using preset field choices where applicable. 
Edits of the data will be performed routinely to look for missing data, outliers, unusual or 
inconsistent values. We will collect information on all neonates and parents enrolled in 
the study.  
 
For parents, using standard questions that will be incorporated into the case report 
form, we will collect their date of birth, race, ethnicity, gender, educational level, 
underlying medical conditions (especially skin diseases that predispose to S. aureus 
colonization), current medications, exposure to recent antibiotics, recent healthcare 
exposures, other household members, past use of mupirocin, and dates of, and 
treatments for any prior S. aureus infections. 
 
For neonates, we will collect basic demographic information on all patients, including 
chronologic age (date of birth), birth weight, mode of delivery, gestational age, race, 
ethnicity, gender, antimicrobials (antibiotics, antifungals, antivirals) administered, other 
interventional or clinical medicines that may impact S. aureus colonization, presence of 
central venous catheter, respiratory support, feeding method (breastfeeding, orogastric 
tube, nasagastric tube), dates of NICU admission and discharge, primary reason for 
admission (primary condition), where neonate was admitted from (home, other hospital, 
inborn), procedures, number of trips to the operating room or radiology, on NP or 
resident service, number of consulting teams, APGAR scores, practice and timing of 
kangaroo care, days in isolette vs. open crib or bed, dates and results of all S. aureus 
surveillance cultures and other cultures sent during routine clinical care. 
 
Compliance with Hand Hygiene 
The JHH and JHBMC Departments of Hospital Epidemiology and Infection Control 
monitor compliance with hand hygiene on a monthly basis. We will have access to these 
data.  
 
 
 
 


 
26 
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            Adverse events collection and reporting  
As described above, participants on treatment will be instructed to report any rash, 
blister, itching, redness, swelling or other skin irritation, swelling of the face or limbs, 
difficulty breathing or swallowing, and any significant discomfort or illness that occurs 
during treatment.  Study staff will specifically ask for these symptoms every day while 
monitoring compliance with treatment and will report any adverse event that is thought to 
be related to the study on the Adverse Event Log as well as any other perceived adverse 
events. Neonates will be monitored for Serious Adverse Events. 
 
Adverse Event Definitions -  
 
Adverse Event (AE): An adverse event will be defined as an unusual and 
undesirable symptom or sign that occurs in parent participants during the clinical 
study. Adverse events include those clinically significant laboratory values and 
test results, concomitant illness, accident, medical occurrence or worsening of 
existing medical condition that emerge during study participation. 
 
Serious Adverse Event (SAE): A Serious AE is any untoward medical 
occurrence that at any dose produces any of the following outcomes in neonate 
and parent participants: 
 
Results in death; 
 
Is life threatening (defined as an event in which the subject was at risk of 
death at the time of the event; it does not refer to an event which 
hypothetically might have caused death if it were more severe); 
 
Requires inpatient hospitalization or causes prolongation of existing 
hospitalization (see NOTE below for exceptions); 
 
Results in persistent or significant disability/incapacity; 
 
Is a congenital anomaly/birth defect (note: reports of congenital 
anomalies/birth defects must also be reported on the Pregnancy 
Supplemental Form); 
 
Is an important medical event (defined as a medical event(s) that may not be 
immediately life threatening or result in death or hospitalization but, based 
upon appropriate medical and scientific judgment, may jeopardize the subject 
or may require intervention [e.g., medical, surgical] to prevent one of the 
other serious outcomes listed in the definition above). Examples of such 
events include, but are not limited to, intensive treatment in an emergency 
room or at home for allergic bronchospasm; blood dyscrasias or convulsions 
that do not result in hospitalization.) 
 
NOTE: For the purposes of this trial, the following hospitalizations will 
not be considered SAEs:  
 
Admissions as per protocol for a planned medical/surgical procedure; 
 
Routine health assessment requiring admission for baseline/trending of 
health status (e.g. post-partum complications, such as wound infection or 
bleeding); 
 
Medical/surgical admission for purposes other than remedying ill health state 
and was planned prior to entry into the study. Appropriate documentation is 
required in these cases; 


 
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 
Admission encountered for another life circumstance that carries no bearing 
on health status and requires no medical/surgical intervention (e.g., lack of 
housing, economic inadequacy, care-giver respite, family circumstances, 
administrative). 
 
Non-Serious Adverse Event: A non-serious adverse event is any adverse event 
not classified as serious.  All non-serious AEs will be reported to the IRB on an 
annual basis with the continuing review. 
 
Adverse Event Grading: The Division of AIDS Table of Grading Severity of Adult 
and Pediatric Adverse Experiences (DAIDS) Version 2 will be used to assess 
severity and provide the grade for each AE that is reported. The investigator will 
evaluate the severity of any adverse event not identified within the below criteria 
using the following definitions: 
 
 
Mild  (Grade 1)- Mild symptoms causing no or minimal interference with usual 
social & functional activities with intervention not indicated 
 
Moderate (Grade 2)- Moderate symptoms causing greater than minimal 
interference with usual social & functional activities with intervention indicated 
 
Severe (Grade 3) - Severe symptoms causing inability to perform usual social & 
functional activities with intervention or hospitalization indicated 
 
Potentially life threatening (Grade 4) - symptoms causing inability to perform 
basic self-care functions with intervention indicated to prevent permanent 
impairment, persistent disability. 
 
Death (Grade 5): Indicates death. 
 
AEs graded as Grade 3 or higher which are possibly, probably, or definitely attributable 
to the use of the investigational drug will be recorded and monitored until the event 
has resolved to meet the definition of ‘mild’.  
 
All adverse events will be further evaluated for attribution as per the following: 
 
 
Unrelated: Adverse event is clearly not related to the investigational agent  
 
Unlikely:  Adverse event is doubtfully related to the investigational agent  
 
Possibly:  Adverse event is possibly related to the investigational agent  
 
Probably: Adverse event is probably related to the investigational agent  
 
Definitely: Adverse event is definitely related to the investigational agent  
 
Participants who withdraw from the study, but who have AEs grade > “moderate” which 
are possibly, probably, or definitely attributable to the investigational drug will have 
AEs recorded and monitored until the event has resolved. 
 
9. Statistical Analysis Plan 
 
A. Sample size and power estimates: 
 
The primary outcome will be time to concordant S. aureus-colonization. Time to 
concordant S. aureus colonization will be expressed using an indicator variable for 
concordant colonization and time to concordant colonization. The a priori assumed 


 
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control group concordant colonization rate is 10% and power calculations are based on 
the unadjusted Cox proportional hazards model where the primary covariate is the 
treatment group indicator.  In time to event analyses, power is driven by the number of 
events; i.e. number of concordant colonizations.  Given our assumed control group 
concordant colonization rate of 10%, 38 events are required to detect a 60 percent 
reduction in the hazard of concordant colonization (Hazard Ratio: HR 0.4) in the 
intervention group with 80% power and Type I error rate of 5%.  The 38 events includes 
a roughly 2% inflation for pre-planned interim analyses (see Section 9.B).   Given that 
we expect 5% of the parent-neonate pairs will be non-singleton births, we increased the 
number of required events to 40 to account for the possible clustering of concordant 
colonization among non-singleton neonates. Some neonates may test positive at 
baseline for S. aureus after randomization and will not be eligible for the analysis of the 
primary outcome. Similarly, if one neonate from a multiple gestation birth is positive for 
S. aureus at baseline and another neonate is not, then the negative infant will be eligible. 
Eligible parent-neonate pairs will be assigned to either treatment or control as a unit until 
40 events are confirmed. Prior to the start of the study, given our a priori assumption of a 
10% concordant colonization rate in the control group and a HR of 0.40, we anticipated 
recruiting 400 parent-neonate pairs to achieve the required 40 concordant colonizations. 
With 400 parent-neonate pairs, we have roughly 80% power to detect an absolute risk 
difference of at least 7% for the secondary analysis of the indicator of concordant 
colonization within 90 days after randomization.   
 
 
 
 
B. Interim Analyses  
 
Several interim analyses will be performed.  We will perform interim analyses for efficacy 
based on the number of accumulated primary outcomes.  Specifically, we will perform an 
interim analysis after 20 and 30 neonates have been identified to have the primary 
outcome of concordant colonization.  After accruing 20 neonates with the primary 
outcome, the standard Cox proportional hazards model with main effect of treatment will 
be fit to estimate the treatment effect, i.e. the hazard ratio comparing the hazard of 
concordant colonization comparing the intervention and control groups, and the test 
statistic for the treatment effect will be computed using the methods described in Section 
Table 3. Power estimates based on anticipated outcomes  
 
 
Assumption about 
outcomes among twins 
Time to concordant colonization 
Power to detect a 
difference if concordant 
colonization rate in 
treated group is 2% 
 
HR detectable 
with 90% power 
 
HR detectable 
with 80% power 
Independent 
(38 outcomes) 
0.35 
0.40 
0.89 
Dependent, 5% twins 
(40 outcomes) 
0.35 
0.40 
0.87 
Dependent, 10% twins 
(42 outcomes) 
0.34 
0.39 
0.85 
 


 
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9.C.     The DSMB will review the results of the Cox proportional hazards model to 
determine if the trial will stop for efficacy.  The minimum criteria for stopping for efficacy 
will be if the  test statistic for the hazard ratio is less than Zr = -2.74.      If the trial 
continues, after accruing 30 neonates with the primary outcome, the Cox proportional 
hazards model will be fitted.  The DSMB will review the results of these statistical 
analyses to determine if the trial will stop for efficacy.  The minimum criteria for stopping 
for efficacy will be if the test statistic for the hazard ratio is less than Zr = -2.36.  If the trial 
continues, after accruing the 40 neonates with the primary outcome, the treatment effect 
will be deemed statistically significant if the test statistic for the primary analysis as 
described below falls within the rejection region defined by Zr = -2.03.    
 
To define the above rejection regions, we applied an approximation to the error spending 
function of O’Brien and Fleming (f(t) = min{αt3, α}) such that we spend the overall Type I 
error rate of 0.05 as 0.0062, 0.0148 and 0.029 after accruing 20, 30 and 40 patients, 
respectively.  The derived rejection regions at each interim analysis (i.e. Zr) are based on 
the hazard ratio (equivalent to the log-rank test comparing the survival experience in the 
treatment groups) using an approximation to the information available at each interim 
analysis (i.e. number of accumulated events at the interim analysis divided by 4).    
 
In addition to the interim analyses conducted to determine whether the trial should stop 
early for efficacy, the DSMB may request additional analyses to be performed to 
determine whether the trial should stop for futility after accruing 20 and 30 concordant 
colonizations.   
 
C. Statistical analysis plan 
 
Final analysis methods and changes in the analyses from those described in the 
protocol will be documented in the Statistical Analysis Plan prior to locking the database 
and unmasking the assignment.  A CONSORT flow diagram will be created.  In addition, 
descriptive statistics will be calculated to describe the feasibility of the study including 
number of parent-neonate units approached by a study team member, proportion of 
parents that consent, proportion of parents colonized among consented, proportion of 
parents colonized who subsequently do not complete the treatment, and the distribution 
of adherence, as defined below, to the randomized treatment.   
 
 
Analyses will be conducted according to the modified intention to treat (mITT) principal.  
The same day parents are randomized, the neonate will undergo baseline testing to 
determine baseline S. aureus colonization status. Neonates who test positive for S. 
aureus colonization at the time of randomization do not satisfy the inclusion criteria and 
will not be included in the analysis; hence defining the modified intention to treat 
analysis. Exploratory analyses of baseline characteristics for the treatment groups will be 
evaluated by Student t test for continuous data and Pearson’s 2 test or Fisher Exact test 
for categorical data.  No missing data should occur for baseline parent and neonate 
variables.  No missing primary or secondary outcomes are expected.  However, it is 
possible that neonates have no subsequent cultures between the baseline culture and 
NICU discharge, e.g. a neonate enters the study on Friday and is discharged on Sunday 
prior to their scheduled surveillance culture (Friday in this example) or discharge culture.  
Based on our definition of observation time (see Sections 5.A and 5.B), such neonates 


 
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will contribute no observation time for the analysis of the primary and secondary 
outcomes.  A sensitivity analyses will be conducted where the observation time for these 
neonates will be defined as the time from randomization to NICU discharge.  All tests will 
be 2-sided with a type-1 error rate set at 0.05. Data will be managed and analyzed using 
Stata, version 13.0 (Stata Corp., College Station, TX) and R: A language and 
environment for statistical computing (R Foundation for Statistical Computing, Vienna, 
Austria, 2014). 
               
The primary analysis will evaluate our hypothesis that detection and treatment of S. 
aureus colonized parents with intranasal mupirocin and topical chlorhexidine gluconate 
bathing (Treatment Group) will decrease the risk of a child acquiring S. aureus with a 
concordant strain in the NICU within 90 days of randomization relative to the control 
parents (Control Group). The parent-neonate pair will be the unit of measure.  For 
neonates that do not acquire concordant colonization within 90 days of randomization, 
their survival time will computed as the time from randomization to the date of final 
culture (surveillance or discharge) within 90 days of randomization. We will define the 
treatment effect as the relative hazard of concordant colonization comparing the 
Treatment to Control group and will estimate this hazard ratio using the standard Cox 
proportional hazards model that includes only a main term for treatment.  To account for 
the possibility of clustering within multiple gestation parent/neonate dyads, standard 
error estimates and bias-corrected and accelerated (BCa) confidence intervals for the 
log-hazard ratio will be derived using a bootstrap procedure. To preserve the correlation 
structure within the dyads, the bootstrap procedure will generate 10,000 bootstrap 
samples by sampling with replacement the parent/neonate dyads, as opposed to 
resampling the neonates.  The analysis described above will be conducted at each pre-
planned interim analysis with the test statistic derived from the estimated log hazard ratio 
from the Cox model and corresponding bootstrap standard error estimate, and the 
confidence level for the BCa bootstrap confidence intervals will be determined by the 
rejection region definitions specified in the Interim Analyses section (see Section 9.B).  
 
Planned analysis of secondary outcomes will include the following:  a) Repeat the 
primary analysis where time to concordant colonization will not be censored at 90 days 
after randomization; time will be defined as the time from randomization to the first of 
concordant colonization or last available culture (clinical, surveillance or NICU 
discharge), b) Define the treatment effect as the difference in the proportion of neonates 
acquiring concordant S. aureus by 4 and 8 weeks after randomization comparing the 
Treatment to Control group; this treatment effect will be estimated by taking the 
difference in the observed proportions comparing the Treatment and Control groups with 
standard error estimates and confidence intervals derived from the bootstrap procedure 
described above, c) Using the methods described above, estimate the treatment effect 
on the acquisition of S. aureus (regardless of concordant status), treated as both time to 
acquisition and the binary indicator for any acquisition by 4 and 8 weeks, and d) Using 
the methods described above, estimate the treatment effect on neonatal S. aureus 
infection, treated as both time to infection and the binary indicator for infection by 4 and 
8 weeks,  For the analysis of the S. aureus infection, we will first consider only S. aureus 
infections that occur prior to a neonate being identified as acquiring S. aureus 
colonization. For example, if a neonate acquires S. aureus colonization and then has a 
subsequent infection 7 days later, this infection will not be included. Only clinical cultures 
that occur before or on the same day as the first positive surveillance culture will be 


 
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considered. The NICU has a policy to decolonize all S. aureus colonized neonates which 
will impact the risk of infection in those that are first identified as colonized. Despite the 
possible independent impact of neonate decolonization on S. aureus infection risk, we 
recognize the importance of reporting all S. aureus infections in study patients; therefore, 
we will repeat the analysis, including S. aureus infections that occur at any time during a 
neonate’s admission in the NICU.  Lastly, a descriptive analysis of neonate mortality and 
bloodstream infections will be conducted, estimating the rate of occurrence separately 
for each treatment group. 
 
Adverse events, as defined in Section 8, will be reported overall and separately by 
treatment group.  Comparisons across treatment groups will be made using Fisher’s 
Exact tests.    
 
In addition, a series of planned secondary analyses will be conducted including 
subgroup analyses for the primary outcome, baseline-covariate adjusted analyses for 
the primary and secondary outcomes, per-protocol (PP) analyses for the primary and 
secondary outcomes and evaluation of patterns of loss and reacquisition of S. aureus 
among the parents (both treated and control).  
 
Planned subgroup analyses include repeating the analysis for the primary outcome 
within strata defined by i) neonates with MRSA vs. those with MSSA, ii) collection site of 
colonization for the parent/caregivers, categorized as nares alone (i.e. all cultured 
parent/caregivers colonized in the nares) vs. nares + other (i.e. at least one 
parent/caregiver colonized in the nares) vs. non-nares; iii) LGA (large for gestational 
age) vs. SGA (small for gestational age); and iv) birthweight (< 1500g vs. ≥ 1500g).  The 
subgroup analyses will be based on Cox proportional hazards models that include a 
main term for treatment, a main term for subgroup (binary indicator) and the interaction 
between treatment and subgroup.  Confidence intervals and the hypothesis test for no 
interaction will be derived based on the bootstrap procedure defined above.   
 
We will explore the prognostic power of three pre-specified baseline covariates collected 
at the time of NICU admission which, if found to be prognostic, could be used in 
subsequent trials to improve the precision of the estimated treatment effect (Rubin et al 
2008).  Specifically, we will derive baseline-covariate adjusted estimates of the treatment 
effects defined for the primary and secondary outcomes. The baseline covariates include 
birth weight (treated as a continuous variable),  an indicator for whether the neonate was 
born at the Johns Hopkins Hospital or the Johns Hopkins Bayview Medical Center 
(inborn) or admitted to the NICU from home or an outside hospital (outborn).  For the 
survival outcomes, we will utilize the method developed by Lu and Tsiatis (2008) 
implemented in the R package “speff2trial”.  The method produces an estimate of the 
relative hazard of concordant colonization comparing the Treatment to Control group 
analogous to the hazard ratio parameter obtained from fitting a Cox proportional hazards 
model with a treatment indicator as the only covariate.  In addition, the test for this 
relative hazard equates to applying the log-rank test in the case of proportional hazards. 
The method relies on constructing models for the treatment indicator and censoring 
indicator that account for chance imbalance in the selected baseline covariates.  We will 
include all of the selected baseline covariates in the analysis as main effects. The large 
sample property of this estimator guarantees that if any of the selected baseline 
covariates are correlated with the outcome, the method yields an estimate of the relative 


 
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hazard that has statistical variance that is smaller or equal to the results of the Cox 
model. The estimate of the treatment effect will be constructed using the neonate as the 
unit of analysis.  Similar to the methods proposed for the primary and secondary 
analyses, standard errors and confidence intervals for the covariate-adjusted estimate of 
the treatment effect will be based on a bootstrap approach to preserve the correlation 
structure within multiple gestation dyads.  For the binary outcomes, e.g. acquiring 
concordant S. aureus by 4 weeks after randomization, we will utilize novel methods 
proposed by Rotnitzky et al in 2012 and described in further detail in Colantuoni and 
Rosenblum (2014) that account for the specified baseline covariates.  Similar to the 
methods proposed by Lu and Tsiatis (2008), the goal is to leverage information in the 
baseline covariates to produce an estimate of the treatment effect (i.e. absolute risk 
difference) that is as precise as or more precise than the unadjusted estimator of the 
treatment effect.  We will follow exactly the algorithm detailed in Colantuoni and 
Rosenblum (2014) where the model for the treatment indicator we will be a logistic 
model with main effects of the specified baseline covariates. 
 
We will conduct a per protocol (PP) analysis.  Compliance to treatment will be defined 
first for the individual parent and then for the family or parent/neonate dyad (Table 4).  
Individual parents will be classified as: confirmed compliant where the parent returns a 
treatment kit with ointment tubes with a broken seal; reported compliant where the 
parent gave responses to questions about feasibility and potential adverse effects (see 
Section 4.B compliance monitoring); and non-compliant where a) kits were returned 
unused, b) the parent reported non-use, or c) parent was lost to follow up without 
reporting use.  We will define compliance to treatment for the individual parent as 
confirmed compliant, with a sensitivity analysis including reported compliant in the 
definition of compliance to treatment.  Family compliance will be defined as both parents 
satisfying the compliance to treatment definition, with a sensitivity analysis where we 
include partially compliant families, i.e. if the parent identified to be colonized with S. 
aureus is compliant based on the individual parent definition and the non-colonized 
parent is non-compliant.  Baseline characteristics of the parents, including age, race, 
education level, and recent antibiotic use, will be compared across the individual parent 
compliance definitions, overall and separately by treatment group. Baseline 
characteristics of the ITT and PP neonates will be compared, overall and separately for 
each treatment group. The analysis of the primary and secondary outcomes described 
above will be repeated among the subset of parent/neonate dyads who are observed to 
be compliant.  Note, that we will assume that all placebo group patients are compliant.  
We will conduct the PP subset analysis using this assumption and then separately 
incorporating the compliance definitions above to the placebo group as well.  
Conditioning on compliance, a post-randomization variable, may introduce bias into any 
treatment comparisons.  Therefore, we will also implement a causal inference approach 
using available baseline parent and neonate variables within an inverse probability 
weighted approach to estimate the treatment effect for the primary and secondary 
outcomes, where the treatment effect compares what would have happened if all 
patients receiving the treatment were compliant vs. if all patients receiving the placebo 
were compliant.    
 
 
  
 


 
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Table 4: Definition of individual parent and family compliance to treatment 
 
Individual parent compliance assessment 
Category 
Sub-category  
Definition 
Compliant 
Confirmed compliant 
seal broken on nasal ointment 
 
Reported compliant 
participants reported use 
Non-compliant 
Confirmed non-compliant 
kits were returned unused 
 
Reported non-compliant 
parent reported non-use 
 
Presumed non-compliant 
parent was lost to follow up 
without reporting use 
 
Family- level compliance assessment 
Category 
Individual 1 compliance   
Individual 2 compliance   
Compliant 
Compliant 
Compliant 
Partially compliant+ 
Compliant (SA colonized) 
Non-compliant (not SA colonized) 
Non-compliant 
 
Compliant (not SA colonized) 
Non-compliant (SA colonized) 
Non-compliant 
 
Non-compliant 
Non-compliant 
+partially compliant if S. aureus colonized parent is compliant, even if the non-colonized 
parent was non-compliant 
Lastly, within each treatment group, we will use standard survival analysis methods (e.g. 
Kaplan-Meier survival function curve) to explore the loss of and potential reacquisition of 
S. aureus among the parents, separately for each treatment group.  Cox proportional 
hazards models will be used to describe the association between loss of and 
reacquisition of S. aureus with baseline parent characteristics and treatment. 
10.  Payment and Remuneration  
 
Each study participant will receive gift cards worth $10 dollars at screening, when each 
kit is returned, if the participant completed treatment, and at Week 4, or at the final 
surveillance screening, whichever occurs first, up to a maximum of $30 total. 
 
11. Costs 
There are no direct costs to the patient in this study.  All study related supplies will be 
provided. 
 
 
References: 
1. 
Akinboyo, I.C., et al., Epidemiology and risk factors for recurrent Staphylococcus aureus 
colonization following active surveillance and decolonization in the NICU. Infect Control Hosp 
Epidemiol, 2018. 39(11): p. 1334-1339.