A Phase IIb Randomized, Placebo-controlled, Double-blind Trial of Azithromycin to Eradicate Ureaplasma Respiratory Tract Infection in Preterm Infants
Phase: PHASE2
Conditions: Ureaplasma Infections
Sponsor: University of Maryland, Baltimore

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Azithromycin to Prevent Bronchopulmonary Dsyplasia in Ureaplasma-
infected Preterms 
A Phase IIb randomized, placebo-controlled, double-blind trial of azithromycin to eradicate 
Ureaplasma respiratory tract infection in preterm infants 
 
Principal Investigator: Rose Marie Viscardi, M.D.,  
Department of Pediatrics, University of Maryland School of Medicine Baltimore, MD  
 
Supported by:  
The National Institute of Child Health and Human Development  
R01 HD067126 
 
NCT: 01778634 
 
Sponsor of IND 78990: Rose M. Viscardi 
 
 

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Statement of Compliance 
This study will be carried out in accordance with the US Code of Federal Regulations (CFR), 
local regulations, and Good Clinical Practice (GCP) as required by the following: 
 U.S. Code of Federal Regulations applicable to clinical studies (45 CFR 46; and 21 CFR 
including part 50 and 56 concerning informed consent and IRB regulations, and 21 CFR 11 
concerning electronic records. 
 International Conference on Harmonisation (ICH E6); 62 Federal Register 25691 (1997)  
 NICHD Clinical Terms of Award  
All individuals responsible for the design and conduct of this study have completed Human 
Subjects Protection Training and are qualified to be conducting this research prior to the 
enrollment of any subjects. CVs for all investigators and sub-investigators participating in this 
trial are on file in a central facility (21 CFR 312.23 [a] [6] [iii] [b] edition).  
 

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 Signature Page 1 
The signature below constitutes approval of this protocol and the attachments, and provides the 
required assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements, applicable US federal regulations and (ICH E6) guidelines.  
 
Principal Investigator: __Rose Marie Viscardi, M.D.______________________________  
 
 
Print/Type 
 
Signed: 
 
 
Date: 
 
Name/Title 
 
 
The Lead Principal Investigator (Protocol Chair) should sign Signature Page 1.  A copy of this 
Signature Page 1 should be filed with the holder of Regulatory documents and a copy should be 
maintained at the site. 

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Signature Page 2 
The signature below constitutes approval of this protocol and the attachments, and provides the 
required assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements, applicable US federal regulations and (ICH E6) guidelines.  
 
Investigator(s) of Record: _______________________________________________________  
 
 
Print/Type 
 
Signed: 
 
 
Date: 
 
Name/Title 
 
 
Additional Investigators: ______________________________________________________  
 
 
Print/Type 
 
Signed: 
 
 
Date: 
 
Name/Title 
 
 
 
The Principal Investigator or Investigator(s) of Record from each participating clinical site 
should sign the Signature Page 2 as appropriate. This Signature Page 2 should be maintained at 
each clinical site. 
 
 
 

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Tool Revision History 
Version Number 1.2 
Version Date 1/10/13 
Summary of Revisions Made: 
 
Version Number 1.3 
Version Date 3/8/13 
Summary of Revisions Made (Changes in italics):  
1. Added List of Case Report Forms to be used as source documents (section 10.2) 
2. Updated Consent Form to include time of consent to investigator signature line 
3. Provided updated Case Report Forms in Appendix F 
Version Number 1.4 
Version Date 3/21/2013 
Summary of Revisions Made:  
1. Changed the timing of long-term follow-up from 18-22 months to 22-26 months adjusted 
age 
2. In response to FDA Drug Safety Communication dated 3/12/2013 concerning 
Azithromycin and the risk of potentially fatal heart rhythms, have added conditions that 
may increase the risk for prolonged QT in stopping rules for individual subjects 
(summarized in Background p. 14-15 and Section 5.1.4). 
3. Consent form procedures section revised to include the conditions that may increase the 
risk for prolonged QT that would cause delay in drug receipt or discontinuation of the 
drug. 
4. Corrected the timing of the hearing screen and physiologic BPD assessment to 36 
weeks or pre-discharge in study schedule (Table 3). 
5. Revised consent process, to include parental consent for banking (storage) of 
Ureaplasma samples isolated from positive subjects and contact for future studies 
(sections 4.3 and 6.2.2). 
Version Number 2.0 
Version Date 4/14/2014 
Summary of Revisions Made:  
1. Change in eligibility criteria: Eliminated appropriate for gestational age (AGA) from 
inclusion criteria. Ten percent of potential subjects <29 weeks gestation are small for 
gestational age (SGA). Based on 2 prior prospective studies of Ureaplasma respiratory 
colonization, the risk of Ureaplasma respiratory colonization and BPD are similar for 
AGA and SGA infants<29 weeks when delivery for maternal indications are excluded. 
This should not impact risks for subjects and should not significantly change the 
anticipated respiratory colonization rate of enrolled subjects. 
2. Added additional pulmonary follow-up at 12 months adjusted age to section 6.3.5, study 
schedule and consent form procedures section. 
3. Clarify calculation of dosing volumes for study drug (Section 5.2.4) 
4. Revised serious adverse event reporting procedures (Section 7.4) for all SAEs 
regardless of relationship to study drug to be reported to the sponsor within 5 business 
days. The sponsor will forward all SAE reports to DSMB chair who will review and 

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determine whether to request treatment assignment. A monthly cumulative summary of 
SAEs will be submitted to the DSMB chair as previously described. 
5. Specific requirements for safety reports for NEC cases are included in section 7.4. 
6. Clarify that subjects will be followed for safety (morbidities of prematurity, adverse 
events, serious adverse events) until 44 weeks post-menstrual age (PMA), discharge, or 
transfer whichever comes first. 
7. Added Appendix E. Severity Assessment of Clinical Laboratory Values 
8. Oregon Health and Science University has been dropped as a participating site. 
Version Number 3.0 
Version Date 5/28/2015 
Summary of Revisions Made:  
1. Added information to risk section (p. 18 and 38) concerning new published information 
concerning an association of oral azithromycin and increased risk for idiopathic 
hypertrophic pyloric stenosis in infants. The consent form was revised to include this 
new information (see page 65). 
2. Since additional planned azithromycin drug measurements have been completed, 
residual blood collection procedure will be discontinued. 
3. Added Christiana Care Health System, Newark, DE as a study site. 
 
Version Number 4.0 
Version Date 1/13/2016 
Summary of Revisions Made: 
1. Gestational age eligibility criteria change: This revision changes the gestational age (GA) 
eligibility from 240-286 wk to 240-266 wk. This change is requested with approval from the 
DSMB to limit enrollment to the gestational age stratum with the highest Ureaplasma 
respiratory colonization rate and to reduce the exposure of non-colonized infants to the 
potential risks associated with azithromycin. To date, 94 infants have been enrolled in 
the trial; 60 subjects were in the lower gestational age stratum 240-266 wk and 34 
subjects were in the higher gestational age stratum 270-286 wk. The Ureaplasma 
respiratory colonization rate at study entry was 54% in the lower GA stratum in contrast 
to 16% colonization rate in more mature infants in the higher GA stratum. Using a 2-
sided α level of 0.05 and assuming 20% of subjects are twins and 5% drop-out rate, the 
study will have power of 0.8 to detect a 40% difference in the primary outcome of 
survival with Ureaplasma clearance with enrollment of 30 Ureaplasma-positive infants in 
each group (See Table 6, Section 9.2, p. 46). The change in eligibility criteria to limit 
enrollment to the lower GA stratum will reduce the number of total enrollment from the 
originally planned 180 to 140 to obtain 60 Ureaplasma-positive subjects. All infants 
enrolled and randomized in the trial including those already enrolled with gestational age 
270-286 will be followed until 22-26 month adjusted age. Revisions have been underlined 
in the text. 
 
Version Number 5.0 
Version Date 1/302017 
Summary of Revisions Made: 
1. Added Ages and Stage Questionnaire-3 (ASQ-3) as Neurodevelopmental Assessment 
Tool: Since some subjects are unable to return for in-person neurodevelopmental 
assessments (neuromotor exam and BSID-III), the ASQ-3 parental questionnaire has 

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been added as an option since it can be administered by phone, is easily scored, and 
has high sensitivity and negative predictive value to detect neurodevelopmental 
impairment in preterm infants <32 weeks gestation at 22-26 months adjusted age 1 
(page 39). 
2. Adjusted windows for follow-up assessment: Due to the challenges in reaching some 
parents within the windows for pulmonary assessments at 6, 12, and 22-26 months 
adjusted age, and neurodevelopmental assessments at 22-26 months adjusted age, the 
window for the questionnaires now extend from 5 months adjusted age for the 6 month 
pulmonary assessment through 30 months adjusted age (page 38).  
3. Cranial Ultrasound (CUS) and Retinopathy of Prematurity (ROP) results confirmation: In 
order to confirm accuracy and completeness of CUS and ROP results, all CUS and ROP 
reports for all subjects will be reviewed blinded to treatment assignment during site 
visits. In addition, independent review of CUS images may be requested for confirmation 
if there are differences in rates of severe IVH (>Grade 2) by site or treatment assignment 
(page 41).  
 
 
 

 
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TABLE OF CONTENTS 
Page 
Azithromycin to Prevent Bronchopulmonary Dsyplasia in Ureaplasma-infected Preterms ...... 1 
TABLE OF CONTENTS .............................................................................................................. 8 
PARTICIPATING STUDY SITES .......................................................................................................... 11 
PRÉCIS ........................................................................................................................................... 13 
1. STUDY OBJECTIVES ........................................................................................................... 15 
1.1 
Primary Objective ................................................................................................................. 15 
1.2 
Secondary Objectives ........................................................................................................... 15 
2. BACKGROUND AND RATIONALE ........................................................................................ 16 
2.1 BPD: Major Morbidity of Prematurity ....................................................................................... 16 
2.2 
Study Rationale .................................................................................................................... 22 
3. STUDY DESIGN .................................................................................................................. 23 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS ............................................................. 23 
4.1 
Inclusion Criteria .................................................................................................................. 23 
4.2 
Exclusion Criteria .................................................................................................................. 23 
4.3 
Study Enrollment Procedures ............................................................................................... 24 
5. STUDY INTERVENTIONS .................................................................................................... 25 
5.1 
Interventions, Administration, and Duration ........................................................................ 25 
5.2 
Handling of Study Interventions ........................................................................................... 28 
5.3 
Concomitant Interventions ................................................................................................... 29 
5.3.1 
Allowed Interventions .......................................................................................................... 29 
5.3.2 
Required Interventions None ............................................................................................... 29 
5.3.3 
Prohibited Interventions: Any other macrolide antibiotic (e.g. erythromycin, clarithromycin, 
clinically prescribed azithromycin) .................................................................................................... 29 
5.4 
Adherence Assessment ......................................................................................................... 29 
6.2.1 
Screening Evaluation ............................................................................................................ 31 
6.3 
Enrollment, Baseline, and/or Randomization .......................................................................... 33 
6.3.4 
Blinding ................................................................................................................................. 34 
6.3.5 
Follow-up Visits ..................................................................................................................... 34 
6.3.6 
Completion/Final Evaluation ................................................................................................. 39 
7. SAFETY ASSESSMENTS ...................................................................................................... 40 
7.1 
Specification of Safety Parameters ....................................................................................... 40 
7.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters ................. 40 
7.3 
Adverse Events and Serious Adverse Events ......................................................................... 41 
7.4 
Reporting Procedures ........................................................................................................... 44 
7.5 
Follow-up for Adverse Events ............................................................................................... 46 
7.6 
Safety Monitoring ................................................................................................................ 46 
8. INTERVENTION DISCONTINUATION .................................................................................. 46 
9. STATISTICAL CONSIDERATIONS ......................................................................................... 47 
9.1 
General Design Issues ........................................................................................................... 47 
9.3  
Definition of Populations ..................................................................................................... 49 
9.4 
Interim Analyses and Stopping Rules .................................................................................... 49 
9.5 
Outcomes ............................................................................................................................. 49 

 
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9.5.1 
Primary Outcome ................................................................................................................. 49 
9.5.2 
Secondary Outcomes ............................................................................................................ 49 
9.6 
Data Analyses ....................................................................................................................... 49 
10. DATA COLLECTION AND QUALITY ASSURANCE ............................................... 52 
10.1 
Data Collection Forms ................................................................................................... 52 
10.2 
Data Management ............................................................................................................. 53 
10.3 
Quality Assurance .............................................................................................................. 55 
10.3.1 
Training ............................................................................................................................... 55 
10.3.2 
Quality Control Committee ................................................................................................ 55 
10.3.3 
Metrics ............................................................................................................................... 55 
10.3.4 
Protocol Deviations ............................................................................................................ 55 
10.3.5 
Monitoring .......................................................................................................................... 55 
11. PARTICIPANT RIGHTS AND CONFIDENTIALITY ................................................................. 56 
11.1 
Institutional Review Board (IRB) Review ............................................................................ 56 
11.2 
Informed Consent Forms .................................................................................................... 56 
11.3 
Participant Confidentiality .................................................................................................. 56 
11.4 
Study Discontinuation ........................................................................................................ 56 
12. COMMITTEES .................................................................................................................. 56 
13. PUBLICATION OF RESEARCH FINDINGS ............................................................................ 57 
14. REFERENCES .................................................................................................................... 57 
15. SUPPLEMENTS/APPENDICES ........................................................................................... 63 
16. CONSENT FORM .............................................................................................................. 63 
WHAT ARE MY RESPONSIBILITIES IF I TAKE PART IN THIS RESEARCH? ............. Error! 
Bookmark not defined. 
POTENTIAL RISKS/DISCOMFORTS: ................................... Error! Bookmark not defined. 
POTENTIAL BENEFITS .......................................................... Error! Bookmark not defined. 
COSTS TO PARTICIPANTS .................................................... Error! Bookmark not defined. 
PAYMENT TO PARTICIPANTS ............................................. Error! Bookmark not defined. 
CONFIDENTIALITY AND ACCESS TO RECORDS .............. Error! Bookmark not defined. 
RIGHT TO WITHDRAW ......................................................... Error! Bookmark not defined. 
CAN I BE REMOVED FROM THE RESEARCH? ........................... Error! Bookmark not defined. 
UNIVERSITY STATEMENT CONCERNING RESEARCH RISKS ..... Error! Bookmark not 
defined. 
17. 
OTHER APPENDICES .............................................................................................................. 70 

 
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STUDY TEAM ROSTER 
Rose M. Viscardi, M.D., Principal Investigator 
110 S. Paca Street, 8th floor 
Baltimore, MD 21201 
Office phone: 410-706-1913 or 410-328-6003 
Fax: 410-706-0404 or 410-328-1076 
Cell Phone: 410-916-5392 
E-mail: rviscard@peds.umaryland.edu 
 
Michael L. Terrin, M.D. Co-PI 
660 W. Redwood Street, HH 140 
Baltimore, MD 21201 
Phone: (410) 706-6139  
Fax: (410) 706-4400 
E-mail: mterrin@epi.umaryland.edu 
 
Natalie L. Davis, M.D., M.M.Sc. 
110 S. Paca Street, 8th floor 
Baltimore, MD 21201 
Office phone: 410-706-1913 or 410-328-6003 
Fax: 410-706-0404 
E-mail: ndavis@peds.umaryland.edu 
 
Laurence S. Magder, Ph.D., Study Statistician 
University of Maryland School of Medicine 
Howard Hall, Room 114B 
Phone 410-706-2153 
Fax: 410-706-3808 
E-mail: lmagder@epi.umaryland.edu 
 
Alexander Agthe, M.D., Medical Monitor 
110 S. Paca Street, 8th floor 
Baltimore, MD 21201 
Office Phone: 410-328-6003 
Fax: 410-328-1076 
E-mail: aagthe@peds.umaryland.edu 
 
Susan Jean Dulkerian, M.D., Mercy Medical Center Site Investigator 
University of Maryland School of Medicine 
110 S. Paca Street, 8th floor 
Baltimore, MD 21201 
Office Phone: 410-328-6003 
Fax: 410-328-1076 
E-mail: sdulkerian@mdmercy.com 
 
University of Maryland and Mercy Medical Center Study Coordinators: 
Lisa Janofsky, RN ejanofsky@peds.umaryland.edu 
Ashley Bathgate, MS, abathgate@peds.umaryland.edu 
110 S. Paca Street, 8th floor 

 
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Baltimore, MD 21201 
Office phone: 410-328-6003 
Fax: 410-328-1076 
 
University of Maryland Coordinating Laboratory (address for shipping samples): 
Howard Hall Rm 342 
660 W. Redwood St. 
Baltimore, MD 21201 
Phone: 410-706-1915 
Fax: 410-706-0404 
E-mail: rviscard@peds.umaryland.edu 
 
 
Clinical and Translational Research Informatics Center 
J. Kathleen Tracy, PhD., Director  
ktracy@epi.umaryland.edu 
 
William Seymour, Project Manager 
410-706-6842 
wseymour@epi.umaryland.edu 
 
Help Desk: Research.support@epi.umaryland.edu 
 
University of Alabama Diagnostic Mycoplasma Laboratory 
Director: Ken B. Waites, M.D. 
Technical Staff: 
Donna Crabb dmcrabb@uab.edu 
Amy Ratliff aratliff@uab.edu 
Lynn Duffy lynnduff@uab.edu 
Phone: 205-934-9142 
Fax: 205-975-9256 
Website: mycoplasma.uab.edu 
PARTICIPATING STUDY SITES 
Namasivayam Ambalavanan, M.D. 
University of Alabama at Birmingham 
176F Suite 9380, Women and Infants Center 
619 South 19th Street 
Birmingham, AL 35249-7335 
Phone: Office (205) 934 4680 Lab (205) 934 0751 or 996 5419 
Fax: Office (205) 934-3100 Lab (205) 996 2333 
Email: ambal@uab.edu 
 
Pamela Donohue, ScD 
Johns Hopkins School of Medicine 
Charlotte R. Bloomberg Children's Center 
1800 Orleans Street, Room 8527 
Baltimore, MD 21287 
Phone: 410 955-5232 

 
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Fax: 410 955-0298 
E-mail: pdonohue@jhmi.edu 
 
Susan Aucott, M.D. 
Johns Hopkins School of Medicine 
Charlotte Bloomberg Children's Center 
1800 Orleans Street/Room 8517 
Baltimore, MD 21287 
Phone: 410-955-5259 
Fax: 410-955-0298 
E-mail: saucott@jhmi.edu 
 
David A. Kaufman, M.D. 
University of Virginia School of Medicine 
PO Box 800386, Charlottesville, VA, 22908-0386 
Phone: 434-924-5428 
Fax: 
E-mail: DAK4R@hscmail.mcc.virginia.edu 
 
Karen D. Fairchild, M.D. 
University of Virginia School of Medicine 
PO Box 800386, Charlottesville, VA, 22908-0386 
Phone: 434-924-5428 
Fax: 
E-mail: KDF2N@hscmail.mcc.virginia.edu 
 
Deborah Tuttle, M.D. 
Stephen Eppes, M.D. 
Christiana Care Health Services 
4765 Olgetown Stanton Rd. 
Newark, E 19713 
Phone: (302) 733-2166 
Fax: 
E-mail: DTuttle@Christianacare.org 
 
Jörn-Hendrik Weitkamp, MD 
Associate Professor of Pediatrics 
Director for Patient-Oriented Research 
Division of Neonatology 
Vanderbilt University Medical Center 
Monroe Carell Jr. Children¹s Hospital at Vanderbilt 
2215 B Garland Ave, 1125 MRB IV / Light Hall (Mail), 1135K MRB IV / Light 
Hall (Office) 
Nashville, TN  37232-0656 
Tel (615) 322-3476, Lab (615) 343-9297, Fax (615) 343-6182 
Email: hendrik.weitkamp@vanderbilt.edu 

 
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PRÉCIS  
Study Title: A Phase IIb randomized, placebo-controlled, double-blind trial of 
azithromycin to eradicate Ureaplasma respiratory tract infection in preterm infants 
Objectives  
Ureaplasma spp. respiratory tract colonization is a risk factor for the development of the 
chronic lung disorder bronchopulmonary dysplasia (BPD) in preterm infants 2,3. 
Preliminary data suggests that Ureaplasma-colonized infants may be at continued 
increased risk for adverse pulmonary outcomes in the first year of life. Trials of antibiotic 
therapy with erythromycin in the first few weeks of life often failed to eradicate 
respiratory colonization and the development of BPD in Ureaplasma-infected infants 4-6. 
There are no current effective therapies for the treatment of Ureaplasma in preterm 
infants. Our long-term objective is to develop therapies to eradicate Ureaplasma from 
the lungs of preterm infants and prevent or ameliorate Ureaplasma-mediated lung 
inflammation, injury, altered developmental signaling, and fibrosis. 
The azalide antibiotic azithromcyin (AZI) has immunomodulatory properties that make it 
an ideal candidate for therapy to prevent Ureaplasma-mediated lung injury in preterm 
infants. Azithromycin 1) inhibits neutrophil influx and chemoattractant/cytokine release 
in murine lung injury models 7-9; 2) exhibits higher potency than erythromycin against 
clinical Ureaplasma isolates in vitro 10; and 3) is preferentially concentrated in alveolar 
epithelial lining fluid and macrophages 11,12. In a neonatal murine Ureaplasma infection 
model, AZI, but not erythromycin treatment, improved survival and reduced lung 
inflammation 13.  
We hypothesize that intravenous (IV) AZI therapy will prevent BPD in Ureaplasma-
colonized preterm infants by accelerating pathogen clearance and/or down-
regulating the pulmonary inflammatory response. We conducted a Phase I open-
label, pharmacokinetic (PK) study characterizing the single dose PK, safety, tolerability, 
and biologic effects of 10 and 20 mg/kg IV AZI in mechanically ventilated 24-28 wk 
gestation preterm neonates who are at high-risk for Ureaplasma respiratory tract 
colonization and BPD. Pharmacokinetic data from 25 neonates (12 dosed with 10 mg/kg 
iv and 13 dosed with 20 mg/kg iv) were analyzed using a population modeling 
approach. A two-compartment structural model with the clearance and volume of 
peripheral compartment (V2) allometrically scaled on body weight best described the 
PK of AZI in preterm neonates. Although both single dose regimens appeared to be 
safe, the single 10 mg/kg dose was insufficient to eradicate Ureaplasma or to suppress 
pulmonary inflammatory responses. Pharmacokinetic simulations indicate that even 
multiple dose administration of 10 mg/kg AZI would be inadequate to maintain AZI 
plasma concentrations above the Ureaplasma MIC50. Despite apparent clearance of 
ureaplasmas in all infected subjects treated with 20 mg/kg single dose compared to 
43% failure rate in the 10 mg/kg group 14, simulations suggest that multiple azithromycin 
dose regimens may be needed to maintain a plasma concentration of azithromycin 
above MIC50 to clear the organisms.  
We then enrolled 15 subjects from 6 sites in a Phase IIa open-label, pharmacokinetic 
study characterizing the PK, safety, and biologic effects of 20 mg/kg x 3 days AZI 
regimen. Although there was no difference in physiologic BPD rates among the three 
azithromycin dose groups, the duration of supplemental oxygen was significantly shorter 
and there was a trend towards shorter duration of mechanical ventilation for the 20 

 
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mg/kg multi-dose group. Compared to the single dose groups, the 20 mg/kg mulit-dose 
effectively eradicated the Ureaplasma in all subjects who were colonized pre-dose. The 
20 mg/kg x 3 day regimen appeared safe, with no deaths or serious adverse events 
attributed to the drug. The aims of the current protocol will address the next steps 
preparatory to Phase III safety and efficacy trials of AZI in the preterm population. The 
study population includes infants 240-266 weeks gestation due to the high rate of 
Ureaplasma spp. respiratory tract colonization and BPD in this subpopulation. 
Aim 1: To determine the safety and microbiological efficacy of a multiple dose course of 
IV AZI to eradicate respiratory tract Ureaplasma infection that might lead to physiologic 
BPD in high risk preterm neonates in a Phase IIb multi-center, randomized, double-
blind, placebo-controlled trial.  
 Primary outcome: To compare the Ureaplasma clearance rate in AZI-treated vs 
placebo-treated infants  
 Secondary Outcomes: To analyze the potential of multiple dose AZI to prevent 
physiologic BPD sufficiently to justify a Phase III clinical trial 
o To evaluate the relationship of microbiologic eradication of Ureaplasma 
respiratory tract colonization in AZI-treated infants and clinical outcome of physiologic 
BPD at 36 wks post-menstrual age 
o To analyze the effect of IV AZI on other secondary endpoints including overall 
mortality, number of days of mechanical ventilation and oxygen supplementation, 
exposure to non-study antibiotics, and incidence of co-morbidities of prematurity 
 
Aim 2: To refine the population PK model of the multiple doses of azithromycin in 
preterm infants 24-28 weeks gestation. 
Aim 3: To analyze the potential of multiple dose AZI to improve the pulmonary 
outcomes at 6, 12, and 22-26 months adjusted age assessed as number and duration 
of hospitalizations, number of physician and emergency visits for respiratory illness, 
frequency of pulmonary medication use, and Infant Lung Health Questionnaire parental 
report of recurrent wheezing or chronic cough. 
Aim 4: To assess the impact of multiple dose AZI on risk for neurodevelopmental 
impairment at 22-26 months adjusted age. 
Design and Outcomes  
The study design will be a double-blind, placebo-controlled clinical trial to test the 
efficacy and safety of azithromycin 20 mg/kg x 3 days to eradicate Ureaplasma spp 
from the respiratory tract of preterm infants 240-266 weeks gestation exposed to 
positive pressure ventilation. The primary outcome will be survival with microbiological 
eradication of Ureaplasma defined as survival to discharge or transfer with 3 negative 
cultures obtained post-therapy. Secondary outcomes will include physiologic BPD at 36 
weeks post-menstrual age (PMA), overall mortality, incidence of co-morbidities of 
prematurity such as intraventricular hemorrhage, periventricular leukomalacia, 
necrotizing enterocolitis, bacterial and fungal nosocomial infection, pulmonary air leak, 
patent ductus arteriosus, retinopathy of prematurity, number of days of positive 
pressure ventilation, number of days of oxygen supplementation, use of postnatal 
steroids, and use of non-study antibiotics. 
 

 
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Interventions and Duration  
After obtaining parental consent, 2 tracheal aspirates and one nasopharyngeal aspirate 
will be obtained at least 2 hours apart for Ureaplasma culture and PCR and the infant 
randomized to either placebo or azithromycin 20 mg/kg IV x 3 days. Follow-up cultures 
will be obtained 2 and 4-5 days post last dose and at 21d postnatal age (PNA). 
Additional tracheal aspirates for inflammatory cytokine measurements will be obtained 
on the same schedule if the infant remains intubated. We have completed collection of 
1-3 samples of residual blood specimens between 4-6 h, 8-24 h, and 69-120h post first-
dose from a sub-group of 30 subjects. Subjects will be followed to discharge for safety. 
Physiologic BPD will be assessed at 36±1 wk PMA by a timed-oxygen reduction test. At 
6, 12, and 22-26 months adjusted age, a pulmonary outcome questionnaire will be 
administered by phone or in person interview. At 22-26 months adjusted age, 
neurodevelopmental outcomes will be assessed by 1) Bayley Scale of Infant and 
Toddler Development, 3rd edition (BSID-III); 2) Amiel-Tison neurologic examination; 3) 
Gross Motor Function Classification System; and 4) medical record review for hearing 
impairment with or without amplification and vision impairment (vision <20/200). For 
infants who are unable to return for an inpatient assessment, the Ages and Stages 
Questionnaire-3 (ASQ-3) will be administered by phone. 
Sample Size and Population  
We will limit the recruitment after January 2016 to infants 240-266 wk gestation who are 
at highest risk for Ureaplasma respiratory tract colonization and BPD. All admissions to 
the NICUs of the 7 clinical sites <72 h age with GA 240-266 wk will be screened for 
study eligibility and parental consent of eligible subjects will be obtained. With a 54% 
colonization rate, a total of 140 subjects will be enrolled to achieve 30 Ureaplasma-
positive subjects in each arm. Subjects will be stratified by center. Infants will be 
randomized according to a permuted-block design to receive azithromycin or placebo. 
1. STUDY OBJECTIVES 
1.1 
Primary Objective 
To determine the safety and microbiological efficacy of a multiple dose course of IV 
azithromycin to eradicate respiratory tract Ureaplasma infection that might lead to 
physiologic BPD in preterm neonates in a Phase IIb multicenter, randomized, double-
blind, placebo-controlled clinical trial. We hypothesize that intravenous (IV) AZI therapy 
will prevent BPD in Ureaplasma-colonized preterm infants by accelerating pathogen 
clearance and/or down-regulating the pulmonary inflammatory response. 
 1.2 
Secondary Objectives 
To analyze the potential of early multiple dose azithromycin to prevent physiologic 
defined BPD  
o To evaluate the relationship of microbiologic eradication of Ureaplasma 
respiratory tract colonization in azithromycin treated infants and clinical outcome of 
physiologic BPD at 36 wks post-menstrual age 
o To analyze the effect of IV azithromycin on other secondary endpoints including 
overall mortality, number of days of mechanical ventilation and oxygen 
supplementation, exposure to non-study antibiotics, and incidence of co-morbidities 
of prematurity 

 
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To refine the population PK model of the multiple doses of azithromycin in preterm 
infants 24-28 weeks gestation. 
 
To analyze the potential of multiple dose AZI to improve the pulmonary outcomes at 6, 
12, and 22-26 months adjusted age assessed as number and duration of 
hospitalizations, number of physician and emergency visits for respiratory illness, 
frequency of pulmonary medication use, and Infant Lung Health Questionnaire parental 
report of recurrent wheezing or chronic cough. 
 
To analyze the impact of multiple dose AZI on risk for neurodevelopmental impairment 
at 22-26 months adjusted age. 
2. BACKGROUND AND RATIONALE 
2.1 BPD: Major Morbidity of Prematurity   
Bronchopulmonary dysplasia (BPD) was first described over 40 years ago as a 
progression of characteristic chest radiographic findings that correlated with pathologic 
changes of acute and chronic lung inflammation, fibrosis and bronchial smooth muscle 
hypertrophy in premature, ventilator-dependent infants 15,16. With recent improvements 
in perinatal care, BPD has become a disease limited to the most immature infants 17, 
occurring in 30% of infants ≤28 wks gestation, but in only 3% of infants >28 weeks 18. 
Compared to the lung histology observed in the ventilated preterm lung during the pre-
exogenous surfactant era, the ‘new’ BPD is characterized by more uniform inflation, 
fewer but larger alveoli, and less fulminant, but persistent inflammation 19. Studies of 
human infants and experimental animal models indicate that the central event in BPD 
pathogenesis is the interruption of normal developmental signaling during early stages 
of lung development by lung injury that is often initiated in utero by intrauterine infection 
and a subsequent dysregulated inflammatory response 17. 
Despite considerable effort focused on reducing BPD in infants ≤28 wks gestation who 
are at highest risk, recently studied therapies (post-natal steroids 20, vitamin A 21, 
caffeine 22,23, inhaled nitric oxide 24,25, and early CPAP 26,27) have had only modest 
impacts on the incidence and severity of BPD. Furthermore, the reduction in early 
pulmonary morbidity may come at the cost of higher neurodevelopmental handicaps 
later in life 28. The effects of these therapies on later pulmonary health are currently 
being evaluated 29,30. None of these therapies address the potential contribution of 
infections to BPD. 
Persistent Ureaplasma respiratory tract colonization: Risk factor for BPD: 
Ureaplasma parvum (serotypes 1, 3, 6, and 14) and U. urealyticum (serotypes 2, 4, 5, 
7-13) are closely related species of the Mollicutes class that are among the smallest 
free-living, self-replicating cells 2,31,32. All serovars lack cell walls, exhibit limited 
biosynthetic abilities, hydrolyze urea to generate ATP, and adhere to human mucosal 
surfaces 2. U. parvum is the most common species isolated from clinical specimens 2,33. 
Although Ureaplasma spp. are considered commensals in the adult GU tract, they have 
been implicated in complications of human pregnancy and neonatal outcomes. 
Ureaplasma urogenital tract colonization has been causally linked to infertility 34,35, early 
pregnancy loss 36, stillbirth 37,38, and preterm birth 39,40. Ureaplasma spp. are the most 
common organisms isolated from amniotic fluid obtained from women who present with 
preterm labor (POL) with intact membranes 41, preterm premature rupture of 

 
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membranes 42, and short cervix associated with microbial invasion of the amniotic cavity 
43, and from infected placentas 42. Recovery of Ureaplasma from the chorion increased 
with duration of rupture membranes, suggesting an ascending route of infection 39. 
However, Ureaplasma has also been detected in 22% of placentas with duration of 
membrane rupture < 1h, suggesting the possibility of a pre-existing infection. Indeed, 
Ureaplasma species have been detected in amniotic fluid as early as the time of genetic 
amniocentesis (16-20 weeks) in up to 13% asymptomatic women 44-47. Placentas with 
the lowest rate of Ureaplasma recovery were from women delivered for pre-eclampsia 
or intrauterine growth restriction 39. 
Vertical transmission is the only known route of infection in the newborn. The rate of 
respiratory tract colonization with Ureaplasma in infants <1500g birth weight ranges 
from 20-45%, depending on study entry criteria, frequency of sampling and detection 
methods 48,49. In a recent cohort of infants <33 wks gestation, Ureaplasma spp. were 
detected during the first week of life in tracheal aspirates or nasopharyngeal specimens 
in 35% of infants 50. Ureaplasma respiratory tract colonization is associated with a 
peripheral blood leukocytosis 51 and early radiographic emphysematous changes of 
bronchopulmonary dysplasia (BPD) 52,53. These findings may be explained, in part, by 
an in utero onset of the inflammatory response and lung injury. The contribution of 
Ureaplasma respiratory tract colonization to the development of BPD has been debated; 
however, a meta-analysis of 17 clinical studies published before 1995 supported a 
significant association between Ureaplasma respiratory tract colonization and 
development of BPD defined as oxygen dependence at 28 to 30 d postnatal age 48. In a 
meta-analysis of 36 published studies involving ~3000 preterm infants, Schelonka et 
al.49 observed a significant association between Ureaplasma respiratory colonization 
and development of BPD whether defined as oxygen-dependence at 28 d or at 36 wk 
post menstrual age (PMA). Studies published since the last meta-analysis support the 
association between Ureaplasma respiratory colonization and BPD 54,55, particularly for 
the subset of Ureaplasma-colonized infants exposed to chorioamnionitis and 
leukocytosis at birth 55. Some Ureaplasma serovars have been implicated in adverse 
pregnancy outcomes more commonly than others 56-59. Although U. parvum is more 
commonly isolated from clinical specimens 57,59, there is a higher rate of BPD in U. 
urealyticum colonized infants 57. Whether there are differences in the in vivo 
response to antibiotic therapy among the serovars or species of Ureaplasma is 
unknown. 
U. urealyticum modulates the pulmonary immune response: Evidence from studies 
of human preterm infants 60, and intrauterine infection models in mice 61, sheep 62,63, 
and non-human primates 64,65 as well as in vitro studies 66-69 support that Ureaplasma 
infection is pro-inflammatory and pro-fibrotic and results in a BPD phenotype. In a 
review of lung pathology of archived autopsy specimens from Ureaplasma-infected 
preterm infants, we observed moderate to severe fibrosis and increased elastin fiber 
accumulation in all Ureaplasma-infected infants compared to non-infected gestational 
controls and infants who died with pneumonia from other causes 70,71. There were 
increased numbers of myofibroblasts and TNF-alpha and transforming growth factor ß1 
(TGFß1)-immunostaining in the lungs of all Ureaplasma-infected infants 70,71. The 
increase in fibrosis and elastin fiber accumulation in the distal lung correlated spatially 
and temporally with the presence of macrophages positive for TGFß1, suggesting that 
these are closely linked 71. Preterm infants with Ureaplasma respiratory colonization 
have elevated tracheal aspirate IL-1ß, TNF-alpha, and monocyte chemoattractant 
protein-1 (MCP-1) concentrations and neutrophil chemotactic activity during the first 

 
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weeks of life compared to non-colonized infants 60,72,73. Experimental murine intrauterine 
U. parvum exposure stimulated fetal lung cytokine expression and augmented 
hyperoxia-induced lung injury 61. In the 125 d immature baboon model, we observed 
sustained recruitment of neutrophils and macrophages into the lung, increased 
expression of pro-inflammatory (TNF-alpha, interleukin (IL)-1ß ) and pro-fibrotic 
cytokines (TGFß1; oncostatin M), extensive fibrosis, and increased myofibroblast 
phenotype in lungs of antenatal Ureaplasma-infected animals compared to gestational 
controls or non-infected ventilated animals 64,74. In a study of rhesus macaques, 
histologic changes in the fetus’ lungs depended on the duration of intrauterine exposure 
to U. parvum 65. Infection exposure duration less than 136 h resulted in neutrophil 
infiltration without epithelial injury. With progressive duration of exposure there was an 
influx of neutrophils and macrophages, epithelial necrosis, and type II cell proliferation. 
For exposure duration >10 d, increased collagen and thickened alveolar walls were 
evident. These data confirm that Ureaplasma infection contributes to chronic 
inflammation and fibrosis in the preterm lung. Moreover, these data suggest that 
Ureaplasma acts as a co-inflammatory stimulus by causing an augmented, 
dysregulated inflammatory response to subsequent inflammatory insults such as 
hyperoxia and volutrauma. We propose that Ureaplasma may contribute to lung 
injury and fibrosis by modulating the local immune response to produce 
sustained chronic inflammation.  
Potential role of AZI to prevent Ureaplasma-mediated neonatal lung injury: Despite 
in vitro susceptibility of Ureaplasma to erythromycin 75 and favorable PK activity 76, trials 
of erythromycin therapy in Ureaplasma-colonized preterm infants have failed to 
demonstrated efficacy to prevent BPD 4,5 or to eradicate respiratory tract colonization 6. 
The failure to prevent BPD in these studies may have been due to the small sample 
size of each study, or to initiation of erythromycin therapy to late to prevent the lung 
inflammation and injury that contribute to the pathogenesis of BPD. Demonstration of 
efficacy of antibiotic therapy to prevent BPD in Ureaplasma-colonized preterm infants 
will require carefully designed, adequately powered studies.  
The 14-membered macrolides that are derivatives of erythromycin and the related 15-
member azalides have immunomodulatory effects, including effects on neutrophil 
function (e.g. chemotaxis, cell adhesion, oxidative burst, phagocytosis) and inhibition of 
release of cytokines involved in acute inflammation (e.g., IL-1ß, IL-8, TNF-alpha) 77-79 
and chronic inflammation (IL-12p40) 80 and NO2
- production in vitro 81. The macrolide 
antibiotics may exert these immunomodulatory effects in the setting of infection, and 
may occur independently of a direct bactericidal effect 82. AZI inhibits neutrophil influx 
and chemoattractant/cytokine release in murine lung noninfectious 7,9, as well as 
pneumonia 8,83 injury models. In addition, AZI exhibits higher potency than erythromycin 
against clinical Ureaplasma isolates in vitro 10,75,84. Pharmacokinetic studies in mice 85 
and humans have shown that AZI is preferentially concentrated in pulmonary epithelial 
lining fluid and alveolar macrophages 11,12. Since neutrophil recruitment and activation 
has been implicated in BPD pathogenesis 86-88, the experimental effects observed with 
AZI in vitro and in vivo indicate that this drug may be beneficial in the treatment of 
Ureaplasma infection and the prevention of BPD in preterm infants. Taken together, 
these properties suggest that AZI is an ideal candidate for therapy to prevent 
Ureaplasma-mediated lung injury in preterm infants at high risk for BPD.  
Since Ureaplasma-mediated lung injury may be initiated in utero and augmented 
postnatally by exposure to mechanical ventilation and hyperoxia, therapy to prevent 
BPD should be initiated as soon as possible after birth in infants at risk. This contention 

 
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was supported by the recent study by Walls et al. 13 demonstrating that AZI, but not 
erythromycin prophylaxis improved outcomes and reduced inflammation in a murine 
neonatal Ureaplasma infection model. However, in a single center RCT pilot study, AZI 
(10 mg/kg/d x 7d followed by 5 mg/kg/d up to 6 wks) did not reduce the incidence of 
BPD at 36 wk PMA 89. In a follow-up larger trial, Ureaplasma clearance and BPD rates 
were similar in the placebo and AZI-treated groups 90. These results underscore the 
importance of performing appropriate PK/pharmacodynamic studies preparatory 
to phase II/III trials to assess microbiological and clinical efficacy of AZI. 
Safety and Tolerance of AZI in the Pediatric Population: The safety and tolerability 
of oral AZI has been evaluated in 3995 patients aged 2 to 94 91. The overall rate of side 
effects is 12% with gastrointestinal symptoms the most common. Ototoxicity, although 
rare, has been reported with IV AZI in adults and included reversible tinnitus and 
hearing loss 92. A review of 43 open label, randomized pediatric trials performed outside 
the US that included 2655 patients treated with a 3 day course (10 mg/kg once daily) 
oral AZI for upper respiratory tract infections reported adverse effects in 8.7% subjects, 
primarily gastrointestinal symptoms 93. In a recent PK study of a single dose of IV AZI in 
32 children ages 6 months to 16 years, there were no serious adverse events. Nausea 
was the most common treatment related symptom, reported in 25% patients.  
Association of AZI with infantile hypertrophic pyloric stenosis: Although treatment 
with erythromycin in the first few months of life has been associated occasionally with 
the development of infantile hypertrophic pyloric stenosis (IHPS) 93,94, a recent report of 
an analysis of the association of prescriptions for oral azithromycin, erythromycin, or 
cephalexin during the first 90 days of life and diagnosis of pyloric stenosis (IHPS) in a 
retrospective cohort study of children born 2001-2012 utilizing the military health system 
database suggested an increased risk for IHPS with AZI exposure in the first 14 days of 
life95. Of 1,074,236 infants reviewed, 2,466 developed IHPS in the study period 
(2.29/1000). Of 4,875 infants prescribed oral azithromycin, 8 developed IHPS (1.64 per 
1000). All 8 affected infants were male. The median number of days IHPS developed 
after AZI prescription was 29.5 d (9-45 d). The relative risk for developing IHPS with AZI 
exposure during first 90 days of life: 0.71 (95%CI 0.36-1.43; P=0.34. The highest rate of 
IHPS occurred when AZI exposure was within the first 14 d of life (3/148 (2%); aOR 
8.26; 95%CI 2.62-26, P<0.01). In all cases, oral AZI was prescribed for treatment for 
Chlamydia conjunctivitis or pneumonia and treatment/prophylaxis against Bordetella 
pertussiss. Of note, in a separate report analyzing the association of prematurity and 
IHPS in the same cohort, there was an increase in IHPS for preterms 33-37 weeks 
gestation, but not in preterms ≤32 weeks gestation 96. This study did not include data on 
inpatient macrolide exposures and did not address IV azithromycin. Due to immaturity of 
intestinal motilin receptors, oral erythromycin has little effect on intestinal motility in 
preterm infants <32 weeks gestation and intravenous erythromycin does not affect 
intestinal motility in preterms97. So it is unlikely that either oral or IV azithromycin will 
affect intestinal function and risk for IHPS in the very preterm study population. 
Azithromycin and the risk of potentially fatal heart rhythms: Azithromycin is 
proarrhythmic with prior reports of occurrences of QT-interval prolongation, and 
torsades de pointes 98. Recently, a retrospective study of a large Tennessee Medicaid 
cohort detected a small absolute increased risk of cardiovascular death (hazard ratio, 
2.88; 95% CI, 1.79-4.63) in adults who took a 5 day course of azithromycin compared to 
individuals who took no antibiotics 98. There were 47 additional cardiovascular deaths 
per 1 million azithromycin courses. The increased risk for cardiovascular death was 
highest among patients with a high baseline risk for cardiovascular disease.  

 
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The FDA independently reviewed the study data and released a Drug Safety 
Communication on 3/12/2013 summarizing their findings and recommendations. The 
FDA noted important limitations of the study including 1) lack of randomization so 
patients who received different drugs might have differed in ways that biased the 
results; 2) study limited to patients treated as outpatients, so patients treated for severe 
or life-threatening infections were likely not included; 3) cardiovascular deaths were 
determined by death certificates rather than full medical records; and 4) some 
limitations to the statistical methods. However, the FDA found that the overall finding of 
excess risk of cardiovascular death in the azithromycin treated patients was valid and 
that the excess risk of cardiovascular death, especially of sudden death, is consistent 
with arrhythmias from drug-related QT prolongation. In addition the FDA evaluated the 
results of a clinical QT study conducted by the manufacturer assessing the effects of 
azithromycin on the QT interval in adults. In a randomized, placebo-controlled parallel 
trial, 116 healthy subjects received either chloroquine (1000 mg) alone or in 
combination with azithromycin (500 mg, 1000 mg, and 1500 mg once daily). In 
comparison to chloroquine alone, the maximum mean (95% upper confidence bound) 
increases in QTcF were 5 (10) ms, 7 (12) ms and 9 (14) ms with the co-administration 
of 500 mg, 1000 mg, and 1500 mg azithromycin, respectively (see attached Drug label, 
p. 7), indicating that co-administration of azithromycin increases the QTc in a dose- and 
concentration-dependent manner. The FDA identified the following groups at higher risk 
of torsades de pointes and fatal arrhythmia:  
o Patients with known prolongation of the QT interval, a history of torsades de 
pointes, congenital long QT syndrome, bradyarrhythmias, or uncompensated 
heart failure 
o Patients on drugs known to prolong the QT interval 
o Patients with ongoing proarrhythmic conditions such as uncorrected hypokalemia 
or hypomagnesemia, clinically significant bradycardia, and in patients receiving 
Class IA (quinidine, procainamide) or Class III (dofetilide, amiodarone, stalol) 
antiarrhythmic agents 
 
The implications of this study for azithromycin use in newborns and children are 
unclear. There is a single case report in the literature of cardiac arrest with suspected 
prolonged QT in a 9 month old who inadvertently received 50 mg/kg azithromycin 
intravenously over 20 minutes 99. Prolonged QT interval is rare in newborns with an 
incidence of the heritable long QT syndromes estimated between 1 per 3000 and 1 per 
5000 births 100. Based on a retrospective study of prolonged QT interval in neonates, 
Villain and co-workers 101 suggest that infants presenting with a QTc<0.5 second 
normalize their QTc over time while infants with a QTc>0.6 second are at risk for severe 
arrhythmias and sudden cardiovascular death. Prolonged QTc  (>0.45 s) was not 
detected in any of the 171 preterm infants screened for the 10 mg/kg and 20mg/kg 
single dose studies or any of the 192 infants screened for the 20 mg/kg x 3d multi-dose 
study.  
There are no known interactions of AZI with other drugs. The potential for these adverse 
outcomes will be carefully evaluated in our clinical trials of AZI in preterm infants. 
Pharmacokinetic/Pharmacodynamic of single and multiple doses of AZI in 
preterm infants: 
We have completed a Phase I open-label, pharmacokinetic (PK) study characterizing 
the single dose PK, safety, tolerability, and biologic effects of 10 and 20 mg/kg IV AZI in 
mechanically ventilated 24-28 wk gestation preterm neonates who are at high-risk for 

 
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Ureaplasma respiratory tract colonization and BPD. A two-compartment structural 
model with the clearance and volume of peripheral compartment (V2) allometrically 
scaled on body weight best described the PK of AZI in preterm neonates. The single 10 
mg/kg and 20 mg/kg dose regimens were safe, but did not suppress pulmonary 
inflammatory responses. The 10 mg/kg single dose was insufficient to eradicate 
Ureaplasma, but there were no treatment failures in the 20 mg/kg group. 
Pharmacokinetic simulations indicate that even multiple dose administration of 10 mg/kg 
AZI would be inadequate to maintain AZI plasma concentrations above the Ureaplasma 
MIC50, while multiple doses of 20 mg/kg might provide a favorable AUC24/MIC90 ratio. 
We subsequently completed the AZIP2 trial characterizing the multiple dose PK, safety, 
and biologic effects of 20 mg/kg x3 days regimen. Fifteen subjects 24-28 wk gestation 
(7 of whom were Ureaplasma-positive pre-dose) were enrolled and received all 3 doses 
of study drug and all were followed to the primary clinical endpoint of BPD at 36 weeks 
PMA. The population PK model was refined with the addition of the 15 subjects from the 
multi-dose study. The best model to describe the PK parameters of AZI in preterm 
infants based on 40 subjects (10 mg/kg single dose (N=12); 20 mg/kg single dose 
(N=13); and 20 mg/kg multiple dose (N=15)) is the 2 compartment model with all 
parameters scaled on body weight. Although the percent relative standard error was low 
for each PK parameter, the intersubject variability is high due to the small number of 
subjects in the multiple dose group. 
 
Table 1. Population Pharmacokinetic Parameter Estimates in Preterm Infants 
(N=40) 
The mean observed 
plasma concentration of 
AZI in the 15 subjects in 
the multiple dose study 
(Fig. 1) exceeded the 
MIC50 of 1 µg/ml for 
Ureaplasma isolates 14. 
This may explain in part 
the effective eradication of 
Ureaplasma from the 
respiratory tract in infants 
who received 20 mg/kg x 3 
day regimen (Fig. 2). 
There were no follow-up 
positive cultures and only one sample that was PCR positive 4-5 days post past dose in 
the Ureaplasma-infected group. There was no increase in frequency of events 
potentially related to azithromycin use such as feeding intolerance, sepsis, or hearing 
loss with the higher dose. The doses were well-tolerated and there were no adverse 
events attributed to the drug. 
Parameter 
Estimate (%RSE) 
%ISV (%RSE) 
CL (L/hr) 
0.15 x WT (kg)0.75 (10) 
58.1 (25) 
V1 (L) 
1.88 x WT (kg) (11) 
78.2 (41) 
Q (L/hr) 
1.79 x WT (kg)0.75 (10) 
64.3 (37) 
V2 (L) 
13.00 x WT (kg) (12) 
78.1 (30) 
Residual Error 
28 (24) 
--- 
CL: Clearance 
V1: Volume of distribution of first compartment 
V2: Volume of distribution of second compartment 
RSE: relative standard error 
ISV: intersubject variability calculated as the square root 
of the estimated variance f intersubject variability 
WT: body weight in kilograms 

 
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2.2 
Study Rationale 
Although not all epidemiologic studies linking Ureaplasma spp. respiratory tract 
colonization with BPD evaluated the duration of colonization 2,3, Catro-Alcaraz et al. 102 
observed that persistent, not transient respiratory colonization increased the risk for 
BPD at 36 wk PMA. In their cohort, transient colonization occurred in 35% while 
persistent colonization occurred in 45%. Late acquisition was found in 20%. In a recent 
prospectively recruited cohort of 112 infants <32 wk gestation with serial respiratory 
cultures, we observed transient colonization (defined as 1-2 positive cultures in the first 
wk followed by 1 or more negative cultures > 1 wk of age) rate of 25%. It is likely that 
persistently positive serial cultures represent true infection and/or ineffective immune 
response while transient detection represents colonization. To determine the 
microbiologic efficacy of IV azithromycin to eradicate Ureaplasma from the preterm 
lung, we will conduct a multicenter, randomized, double-blind, placebo-controlled clinical 
trial of azithromycin 20 mg/kg x 3 days. This endpoint is important to plan for a Phase III 
clinical trial to determine if ureaplasmal eradication improves short-term (BPD) and 
long-term pulmonary outcomes. 
From 11/2013 to 12/2016, 94 subjects have been enrolled in this Phase IIb trial. Initially, 
2 GA strata (240-266 and 270-286 wk) were included, but the Ureaplasma respiratory 
colonization rate was 16% in the higher GA stratum and 54% in the lower GA stratum. 
To limit enrollment to the GA stratum with the highest Ureaplasma respiratory 
colonization rate and to reduce the exposure of non-colonized infants to the potential 
risks associated with azithromycin, eligibility criteria will be changed in January 2016 to 
limit the study population to infants 240-266 wk gestation who are at highest risk for 
Ureaplasma respiratory tract colonization and BPD. We will utilize the UAB Diagnostic 
Mycoplasma Laboratory as a central reference laboratory for Ureaplasma culture, 
genotyping, and azithromycin susceptibility testing. Therefore, infants will receive study 
drug before Ureaplasma colonization status is known. This approach is justified since 
0
50
100
150
100
1000
10000
MIC90
MIC50
Time (hr)
Mean observed AZI CONC (ng/mL)
ure 1. Plasma AZI concentrations 
er time in infants treated with 20 
/kg AZI IV x 3d relative to MIC50  
mg/ml and MIC90 (4 µg/ml). 

 
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the antibiotic will be most beneficial if given early; non-colonized infants may benefit 
from the anti-inflammatory effects of azithromycin. 
3. STUDY DESIGN 
The study design will be a double-blind, placebo-controlled clinical trial to test the 
efficacy and safety of azithromycin 20 mg/kg IV x 3 days to eradicate Ureaplasma spp 
from the respiratory tract of preterm infants 240 to 266 weeks gestation (after January 
2016; 240-286 wk GA January 2016 or earlier) exposed to positive pressure ventilation. 
A total of 140 infants will be randomized to either azithromycin (N=70) or placebo 
(N=70) stratified by center. The primary outcome will be survival with microbiological 
eradication of Ureaplasma defined as survival to discharge or transfer with 3 negative 
cultures obtained post-therapy. Secondary outcomes will include physiologic BPD at 36 
weeks post-menstrual age (PMA), overall mortality, incidence of co-morbidities of 
prematurity such as intraventricular hemorrhage, periventricular leukomalacia, 
necrotizing enterocolitis, bacterial and fungal nosocomial infection, pulmonary air leak, 
patent ductus arteriosus, retinopathy of prematurity, number of days of positive pressure 
ventilation, number of days of oxygen supplementation, use of postnatal steroids, and 
use of non-study antibiotics. Subjects will be enrolled in 7 level III NICUs (3 sites in 
Baltimore, MD (University of Maryland Medical Center, Mercy Medical Center, Johns 
Hopkins Hospital); Christiana Care Health Services, Newark, DE; University of Virginia, 
Charlottesville, VA; University of Alabama at Birmingham, Birmingham, AL; and Monroe 
Carell Jr. Children¹s Hospital at Vanderbilt, Nashville, TN). Subjects will be followed to 
22-26 months adjusted age. The University of Maryland will provide data management 
and laboratory coordination. The Diagnostic Mycoplasma Laboratory under the direction 
of Dr. Ken Waites will serve as the central laboratory for all Ureaplasma culture, PCR, 
and antibiotic susceptibility testing. 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS  
The expected race/ethnicity distribution of subjects from the 7 sites is 52% white, 45% 
African-American, 3% Asian, and 12% Hispanic/ Latino. 
4.1 
Inclusion Criteria 
 Gestational age 240-266 weeks by best obstetrical estimate 
 <72 h age  
 Positive pressure ventilation for at least 1 hour duration during the first 72 
hours of life  
 Presence of indwelling intravenous line for drug administration 
 
All participants must meet all of the inclusion criteria to participate in this study. 
4.2 
Exclusion Criteria 
 Any patient judged to be non-viable or for whom withdrawal of life support 
is planned 
 Patients with major lethal congenital anomalies 
 Triplets or higher order multiples 
 Patients delivered for maternal indications (low risk of Ureaplasma 
colonization) 

 
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 Patients with electrocardiogram QT interval corrected for heart rate (Qtc) ≥ 
450 ms 
 Patients with significant hepatic impairment (direct bilirubin >1.5 mg/dL) 
 Patients exposed to other systemic macrolide 
 Patients with clinically suspected Ureaplasma CNS infection or other 
confirmed bacterial/viral infection  
 Patients participating in other clinical trials involving investigational 
products.  
All screened infants meeting any of the exclusion criteria at baseline will be excluded 
from study participation. 
4.3 
Study Enrollment Procedures  
All admissions to the NICUs of the 7 clinical sites <72 h age with gestational age 240 to 
266 wk (after January 2016; 240-286 wk GA January 2016 or earlier) will be screened 
daily by research staff for study eligibility and parental consent of eligible subjects will 
be obtained (Procedure Schedule, Appendix B). All admissions <72h age and 240 to 266 
weeks gestation by best obstetric estimate will be further screened for eligibility. For 
each screened potential subject, an eligibility checklist (CRF AZIP01) will be completed 
and entry added to a screening log with site-specific numeric patient identification 
number (PID) and letter code (LETCODE) (Sample Screening Log, Appendix A). The 
screening log with patient identifiers is maintained at each site in a secure location and 
is not submitted to the Data Coordinating Center. As part of the screening process, an 
electrocardiogram (EKG) QTc interval will be calculated from lead II from a potential 
subject’s monitor and a copy of rhythm strip and QTc calculation submitted on CRF 
AZIP02 for enrolled subject.  
If the answer to all inclusion criteria is yes and answer to all exclusion criteria is no, 
study personnel will approach parent(s) to discuss the research study and request 
informed consent. If a potential subject is eligible, but consent is not obtained, a reason 
(e.g. missed, maternal age <18, parent/LAR refused consent, non-English speaking 
parent/LAR) will be required on the submitted eligibility checklist CRF (AZIP01). If an 
infant qualifies, the PI or coordinator will obtain informed parental consent prior to 
initiation of study procedures. An EKG for QTc measurement will be obtained after 
consent if not done previously during the screening process. If the PI is also responsible 
for the clinical care of a potential subject, he/she will not participate in the consent 
discussion with the parents to avoid undue pressure on the parents to agree to the 
study. The information provided in the consent will cover the elements listed in the CFR 
Part 50.25 and be approved by the site Institutional Review Board (IRB). This includes 
the investigational nature and objectives of the trial; the procedures and treatments 
involved and their attendant risks, discomforts, and benefits; and the potential 
alternative therapies, alternative to not participate and right to withdraw without penalty 
will be explained. The study staff personnel will offer to answer any questions.  
In the consent process, banking (storage) of Ureaplasma samples isolated from positive 
subjects will be explained to parents. They can decline banking of their child’s 
specimens and still participate in this study. They can indicate on the consent form 
whether they would be willing or not to be contacted for future studies of their child’s 
banked specimens. For each question (banking vs no banking; future contact or not), 
the parent will initial their choice. 

 
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If a parent consents to their child’s participation, the parent will sign and date the 
consent form. One copy of the signed consent form will be provided to the parents, 
another copy will be placed in the child’s medical record and the original signed consent 
form will be kept in a secure file at each site. Written documentation of the consent 
process will be placed in the medical record. The subject will be assigned a study 
identification number. The study identification number will be recorded on all study 
forms. (See Section 16.1 for model consent form). 
4.3.1 Randomization procedure:  
There will be separate randomization schedules for each center. Infants will be 
randomized according to a permuted-block design to receive azithromycin or placebo. 
Twins will be randomized together to the same study arm. Randomization schedules 
prepared by the UMB biostatistician will be provided to the AZIP Randomization Center 
located at Axio Research (Seattle, WA) that will establish a web-based randomization 
site available 24 hours/day and seven days a week. Separate randomization schedules 
for each stratum within each clinical site will be prepared. The randomizations on the 
schedules will be in varying block sizes of two, four and six to force approximate 
balance of azithromycin and placebo treatment assignments at each clinical site. The 
University of Maryland Investigational Drug Service (UMB Coordinating Pharmacy) will 
receive the randomization number assignments for each site to prepare study drug 
shipments. The UMB Coordinating Pharmacy will ship to the study pharmacist at each 
site numbered study drug kits for active drug and sealed envelopes each labeled with a 
site-specific randomization number and containing a label for “azithromycin” or 
“placebo”. Each randomization is documented for time of issue on the computer system 
and under whose user ID authority the allocation is issued (i.e., who requested the 
randomization). Once a randomization number is assigned and study pharmacist is 
notified, he/she will open the corresponding envelope, prepare the appropriate drug or 
placebo, and attach the label from the opened envelope on the drug accountability log. 
For study drug, one vial per dose will be reconstituted with D5W to final concentration of 
2 mg/ml. For placebo, an equivalent volume of D5W will be prepared. Axio Research 
will cooperate with the UMB Coordinating Pharmacy in kit preparation, labeling and 
shipping to maintain an adequate inventory at each clinical site. In the event of 
enrollment of twins, the system will specify two numbers containing the same, blinded 
treatment. Axio Research randomization application will provide unblinded reports to 
assist UMB Coordinating Pharmacy staff in maintaining appropriate levels of drug kits at 
each site. A customized web page will allow the unblinded pharmacist to indicate to the 
application which kits have been sent to and received by the clinical sites (i.e., available 
for dispensing at randomization). 
5. STUDY INTERVENTIONS  
5.1 
Interventions, Administration, and Duration  
5.1.1 Drug Dose Selection Rationale 
We have selected a multiple dose for evaluation in this proposal based on the 
population PK model that includes the 10 mg/kg single dose (N=12) and 20 mg/kg 
single (N=13) and multiple dose (N=15) PK, and the clinical experience in older children 
with respiratory tract infections. The PK properties of AZI support short courses of 
therapy. The drug has a long elimination half-life (>50 h), is concentrated in phagocytic 
cells with good tissue penetration, especially in lung, and persists in tissues for much 

 
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longer than in plasma 103. Pharmacodynamic studies suggest that it is the 
AUC24(u)/MIC90 ratio that is most predictive of AZI efficacy 104. Increasing the dose is 
more effective than increasing duration of treatment for increasing the AUC24/MIC90 
ratio. Results of experimental infections in animal models suggest that increasing the 
dose early in the infectious process will result in clearance of even marginally 
susceptible organisms 105.  
Although optimal AUC24(u)/MIC90 ratios for AZI to eradicate other common organisms 
were reported to range from 0.5 to 14.8 h 106, the optimal AUC24(u)/MIC90 for AZI to 
effectively eradicate Ureaplasma is still unknown. Based on our recent clinical study in 
preterm neonates, a dosage regimen of 20 mg/kg/d x 3d IV AZI was deemed safe and 
effective and resulted in an AUC24(u)/MIC90  ratio of 5.69 as indicated in Table 2. 
Table 2. AUC24 and AUC24/MIC90 for potential dose regimens for Ureaplasma 
therapy. 
As such, we predict that an 
AUC24(u)/MIC90 ratio of > 5.69 h is needed 
in order for AZI dosage regimen to 
eradicate ureaplasma in preterm 
neonates. 
The data from the 20 mg/kg multiple dose 
Phase IIa study confirmed the simulations 
and Ureaplasma was eradicated from the 
respiratory tract of all subjects colonized 
pre-dose. The proposed short course will 
reduce development of drug resistance 
and limit the risk for increased mortality 
and necrotizing enterocolitis (NEC) recently reported for preterm infants exposed to a 
prolonged duration of initial empirical antibiotic treatment 107. The 3 day 20 mg/kg oral 
AZI regimen is well tolerated in older children 93, and is more effective in treating 
recurrent or persistent otitis media than high dose amoxicillin-clavulanate 108 and group 
A streptococcal tonsillopharynigitis than 3 d 10 mg/kg dose oral AZI or 10 d penicillin V 
109,110. 
5.1.2 Drug Dosage, Administration, and Schedule  
After baseline safety laboratory tests and 2 tracheal aspirate samples at least 2 h apart 
and one nasopharyngeal sample for Ureaplasma culture and PCR from intubated 
infants, or 2 NP samples at least 2h apart from non-intubated infants have been 
obtained, study infants will receive according to randomization assignment, either 
azithromycin administered at a concentration of 2 mg/ml in D5W or placebo of same 
volume D5W as a single intravenous infusion over 60 ± 5 min using a functioning 
intravenous line within 24 h of enrollment. The dose will be calculated as 20 mg/kg 
actual body weight. Each subject’s actual body weight at the time of study entry will be 
used for determining the injection volume of study drug. For study drug, one vial per 
dose will be reconstituted with D5W to final concentration of 2 mg/ml. For placebo, an 
equivalent volume of D5W will be prepared. A total of 3 doses will be administered at 24 
± 0.5 h intervals. A pharmacist from the Investigational Drug Service Pharmacy for each 
site will be responsible for dispensing and accounting for study drug. A NICU nurse will 
administer the study drug. 
5.1.3 Duration of Subject Participation 
Dose Regimen 
AUC24(U)
** 
(µg*h/ml) 
AUC24(U)/MIC90 
(h) 
10 mg/kg/d x 
1d (observed) 
12.00 
3.00 
20 mg/kg/d x1d 
(observed) 
21 
5.25 
20 mg/kg/d x 
3d (observed) 
22.8 
5.69 
**assuming 30% of AZI dose is bound to 
plasma proteins and MIC90 for AZI against 
Ureaplasma is 4 µg/ml 

 
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Enrolled subjects will receive 3 doses of azithromycin or placebo and will be followed for 
safety until discharge or transfer or 44 weeks post-menstrual age (PMA), whichever 
comes first. Respiratory cultures for Ureaplasma will be obtained pre-dose and 2d, 4-5 d 
post last dose and 21 d of age. A room air challenge test will be performed when the 
infant is 36 ± 1 weeks post-menstrual age or at discharge if earlier. A respiratory 
outcome questionnaire will be administered at 6, 12, and 22-26 months corrected age 
by phone or in-person interview. A comprehensive neurodevelopmental assessment will 
be performed at 22-26 months adjusted age. 
5.1.4 Stopping Rules for Individual Subjects 
Discontinuation Infusion of Study Drug: Study drug administration will be delayed or 
discontinued if any subject has evidence of an arrhythmia on the cardiac monitor (they 
will all have continuous cardiopulmonary monitoring) or an ongoing proarrhythmic 
condition such as hypokalemia (serum potassium < 3.5 mEq/L), hyperkalemia (serum 
potassium >6.5 mEq/L on non-hemolyzed specimen), hypomagnesemia (total serum 
Magnesium ≤ 1.6 mg/dL), hypocalcemia (ionized calcium < 3.0 mg/dL(<0.75 mmol/L)) 
or hypothermia (core temperature ≤ 35°C) until corrected by the clinical care team. 
Infusion of study drug will be discontinued for any of the following changes in vital signs 
unless it can be remedied by a simple maneuver (e. g., correcting the patency of the 
airway, reestablishment of artificial airway, simple stimulation for apnea of prematurity, 
etc.): Decrease in BP that requires fluid bolus or pharmacologic support, or sustained 
HR less than 80 bpm. If Study Drug is permanently discontinued due to an AE, including 
the above, this will be recorded on the AE CRF and the subject continued to be followed 
until discharge or transfer. 
5.1.5 Plans for Study Interruption Based on Safety Monitoring 
The DSMB members will review all deaths and all adverse reactions or suspected 
adverse reactions and make recommendations concerning continuation of the trial. The 
sites are required to submit reports of all adverse events that are serious and 
unexpected regardless of relationship to study drug within 5 business days to the 
sponsor. The sponsor will forward all reports to Dr. Agthe the clinical monitor for review 
of possible relationship to drug and to the DSMB chair. The DSMB chair may request 
treatment assignment after reviewing the initial report or at the time of the monthly 
summary report.  A summary of cumulative serious adverse events by study arm will be 
provided by the Data Coordinating Center to the DSMB chair monthly. The DSMB will 
meet to review safety data after 50% enrollment. The DSMB recommended that there 
be no pre-specified stopping rules for the study, but rather agreed to review SAEs as 
they occur. The DSMB chair may call a DSMB meeting to specifically review safety 
concerns. The DSMB recommendations may include study termination, study 
continuation with protocol modifications (e.g., change in eligibility criteria), or temporary 
suspension of enrollment until further evaluation is completed. Per DSMB 
recommendations, any death that occurs within 30 d of drug administration will be 
reviewed by the DSMB with each death occurrence.  
5.1.6 Dose Adjustments 
No dose adjustments will be made in this study. If the existing IV line stops functioning 
during the study drug infusion, the remainder of the drug infusion will be given via a 
second IV site. If all IV access is lost during the study drug infusion, the amount infused 
will be recorded and the remainder of study drug returned to the investigational study 
pharmacy. 

 
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5.2 
Handling of Study Interventions  
5.2.1 Packaging and Formulation 
Azithromycin will be manufactured and packaged by American Pharmaceuticals 
Partners (Schaumburg, IL) and shipped using appropriate clinical trial drug distribution 
procedures. Azithromycin for injection will be supplied in lyophilized form under a 
vacuum in a 10 ml vial equivalent to 500 mg of azithromycin for intravenous 
administration. For study drug, one vial per dose will be reconstituted with D5W to final 
concentration of 2 mg/ml. An initial solution of 100 mg/ml azithromycin for injection will 
be prepared by adding 4.8 ml of sterile water for injection to the 500 mg vial and 
shaking the vial until all of the drug is dissolved. To provide the azithromycin in the final 
concentration of 2 mg/ml, 1 ml (100 mg) of the reconstituted drug will be transferred to a 
syringe containing 49 ml dextrose 5% injection and the syringe will be inverted several 
times to mix completely. When diluted in this manner, azithromycin for injection is stable 
for 24 hrs at or below room temperature (30°C or 86°F) or for 7 days, if stored under 
refrigeration (5°C or 41°F). Placebo will be an equal volume of D5W.  
5.2.2 Labeling 
Information presented in the label for study medication will comply with hospital 
investigational pharmacy and local regulatory requirements. 
5.2.3 Storage 
The reconstituted azithromycin will be stored in a refrigerator. The reconstituted drug is 
stable for 7 days if stored under refrigeration (5°C or 41°F). For the study, one vial per 
dose will be prepared. Vials of azithromycin or placebo will not be used if any particulate 
matter or discoloration is observed. Records of the actual storage conditions during the 
period of the study will be maintained. 
5.2.4 Preparation 
The azithromycin will be supplied in lyophilized form under a vacuum in a 10 ml vial 
equivalent to 500 mg of azithromycin for intravenous administration. Each vial also 
contains sodium hydroxide and citric acid. To prepare each dose of azithromycin, using 
aseptic technique, 4.8 ml of sterile water for injection will be added to the 500 mg vial 
and mixed by shaking the vial until all drug is dissolved. Each mL of reconstituted sol-
ution will contain 100 mg azithromycin. This solution is stable for 24 hours when stored 
below 30°C or 86°F. To provide azithromycin in the final concentration of 2 mg/ml, 1 ml 
of the 100 mg/ml azithromycin solution will be added to 49 ml dextrose 5% injection ml 
of 5% dextrose in water in a syringe. The patient specific dose of azithromycin 2 mg/ml 
is drawn up and dispensed as a syringe to be infused intravenously over 60±5 min. If 
the subject’s weight is ≤ 0.94 kg, the dose may be ordered to the 1/10 of 1 ml using a 10 
ml syringe for administration with a syringe pump (example 0.94 kg x 10 ml/kg= 9.4 ml 
of 2 mg/ml azithromycin or equal volume of D5W). For subjects >0.94 kg, a 20 ml 
syringe with 1ml gradiations will be required, and the ordered drug volume should be 
rounded to nearest ml (example subject weight 1.23 kg x 10 ml/kg=12.3 ml, so 12 ml 
should be ordered). For placebo, an equivalent volume of D5W will be administered IV 
over 60±5 min. 
5.2.5 Supply and Return of Drug 
The University of Maryland Medical Center (UMMC) Investigational Drug Service will be 
Pharmacy Coordinating Center for the study. Azithromycin study drug kits will be 
packaged, labeled, and distributed to the study site pharmacies by the UMMC 

 
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Coordinating Pharmacy. Only 1 study drug kit will be used for each enrolled subject; 
each kit will have a unique number. At study initiation, and as needed thereafter, 
azithromycin kits will be shipped to a responsible person (e.g. pharmacist) at the 
Investigational Drug Service of each participating center, who will check the amount and 
condition of the study drug and enter this data into a Proof of Receipt form and 
Investigational Product Accountability Record (see next section). The Proof of Receipt 
form should then be faxed to the UMMC Coordinating Pharmacy. The Investigational 
Drug Pharmacy will record the final disposition of all study drug. 
5.2.6 Drug Accountability 
The study pharmacist at each site will submit monthly accountability logs to the UMB 
Coordinating Pharmacy. The PI Dr. Viscardi is responsible for the accountability of all 
used and unused trial supplies. The following information will be recorded: 
a. The batch number, dates and quantities of the drug received from supplier 
b. Subject’s identification (subject number, letter code, and study drug number) 
c. Date and quantity of drug dispensed 
d. The initials of the dispensing individual 
e. Date and quantity of drug returned to the investigator/pharmacy 
5.3 
Concomitant Interventions  
5.3.1 Allowed Interventions 
All medications except other macrolide antibiotics are allowed as clinically prescribed. 
5.3.2 Required Interventions None 
5.3.3 Prohibited Interventions: Any other macrolide antibiotic (e.g. 
erythromycin, clarithromycin, clinically prescribed azithromycin) 
5.4 
Adherence Assessment  
NICU medical staff will be responsible for administering all doses of study drug and 
recording the exact times of the initiation and completion of drug infusion in the CRF. If 
study drug administration is incomplete or discontinued, the reason will be provided on 
the drug administration CRF and a protocol deviation report will be submitted. 
6.0 Study Schedule 
 
6.1 Study Schedule

 
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6.2 
Description of Evaluations 
6.2.1 Screening Evaluation 
All infants admitted less than 72 h of age with gestational age 240 to 266 weeks gestation (after 
January 2016; 240-286 wk GA January 2016 or earlier) by best obstetric estimate will be screened 
for eligibility. A QTc interval will be calculated from the lead II of the heart rate monitor either before 
consent procedure or after consent is obtained, but before any other study procedures commence. 
If the QTc is >450 ms, the subject is ineligible for participation in the study. For each screened 
potential subject, an eligibility checklist (CRF AZIP01) will be completed and entry added to a 
screening log with site-specific numeric patient identification number (PID) and letter code 
(LETCODE). The screening log with patient identifiers is maintained at each site in a secure 
location and is not submitted to the Data Coordinating Center. 
6.2.2 Consenting Procedure 
If the answer to all inclusion criteria is yes and answer to all exclusion criteria is no, study 
personnel will approach parent(s) to discuss the research study and request informed consent. If a 
potential subject is eligible, but consent is not obtained, a reason (e.g. missed, maternal age <18, 
parent/LAR refused consent, non-English speaking parent/LAR) will be required on the submitted 
eligibility checklist CRF (AZIP01). If an infant qualifies, the PI or coordinator will obtain informed 
parental consent prior to initiation of study procedures. An EKG for QTc measurement will be 
obtained after consent if not done previously during the screening process. If the PI is also 
responsible for the clinical care of a potential subject, he/she will not participate in the consent 
discussion with the parents to avoid undue pressure on the parents to agree to the study. The 
information provided in the consent will cover the elements listed in the CFR Part 50.25 and be 
approved by the site Institutional Review Board (IRB). This includes the investigational nature and 
objectives of the trial; the procedures and treatments involved and their attendant risks, 
discomforts, and benefits; and the potential alternative therapies, alternative to not participate and 
right to withdraw without penalty will be explained. The study staff personnel will offer to answer 
any questions. 
In the consent process, banking (storage) of Ureaplasma samples isolated from positive subjects 
will be explained to parents. They can decline banking of their child’s specimens and still 
participate in this study. They can indicate on the consent form whether they would be willing or 
not to be contacted for future studies of their child’s banked specimens. For each question 
(banking vs no banking; future contact or not), the parent will initial their choice. 
 If a parent consents to their child’s participation, the parent will sign and date the consent form. 
One copy of the signed consent form will be provided to the parents, another copy will be placed in 
the child’s medical record and the original signed consent form will be kept in a secure file at each 
site. Written documentation of the consent process will be placed in the medical record. The 
subject will be assigned a study identification number. The study identification number will be 
recorded on all study forms. (See Section 16.1 for model consent form). 
6.2.3 Screening 
All screening evaluations, completion of eligibility checklist, and informed consent must be 
obtained within 72 h of birth. All screened infants must meet all of the inclusion criteria to 
participate in this study. All screened infants meeting any of the exclusion criteria at baseline will 
be excluded from study participation. 

 
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Specific inclusion criteria to be determined: 
 Gestational age 240-266 weeks (after January 2016; 240-286 wk GA January 2016 or earlier) 
by best obstetric estimate: Infant must be between 24 weeks 0 days and 26 weeks and 6 
days gestation to be eligible for the study. The gestational age estimate will be based on a 
hierarchy of the available information with the order from most to least accurate estimate 
(date of intrauterine insemination > fetal ultrasound before the 14th week; fetal ultrasound at 
14 or more weeks (if no earlier ultrasounds) > last menstrual period (LMP) without fetal 
ultrasound> gestational age by Ballard exam in absence of fetal ultrasounds or LMP. 
 Positive pressure ventilation for at least 1 h duration during the first 72 h of life. This is 
defined as receipt of positive pressure by intubation and mechanical ventilation or nCPAP 
for at least 1 continuous hour. The criterion will be met if the hour of positive pressure is by 
one of the modes of ventilation (mechanical ventilation or nCPAP) or combination (e.g. 
intubation for exogenous surfactant administration followed by extubation to nCPAP). 
 Presence of indwelling intravenous line for drug administration 
Specific exclusion criteria to be determined: 
 Any patient judged to be non-viable or for whom withdrawal of life support is planned. 
Viable, as it pertains to the neonate, means being able, after delivery, to survive (given the 
benefit of available medical therapy) to the point of independently maintaining heartbeat and 
respiration, in the opinion of the attending neonatologist. Neonates for whom death is 
considered imminent or are unresponsive to medical cardio-respiratory support defined as 
persistent hypoxemia (PaO2<40), hypercarbia (PaCO2>80) and acidosis (pH< 7.0) for >12 h 
will be considered non-viable for study criteria. 
 Major lethal congenital anomalies: Those anomalies that will likely result in death such as 
renal agenesis, pulmonary hypoplasia, cyanotic congenital heart disease. 
 Triplets or higher order multiples are excluded 
 Patients delivered for maternal indications. Maternal indications include, but are not limited 
to pre-eclampsia, eclampsia, pulmonary hypertension, trauma, or malignancy. Patients 
whose mothers have these conditions (e.g. pre-eclampsia, chronic hypertension), but are 
delivered for fetal indications such as preterm labor or premature preterm rupture of the 
membranes are eligible. 
 Patients with significant hepatic impairment (Direct Bilirubin >1.5 mg/dL): A fractionated 
bilirubin should be obtained and documented in medical record prior to enrollment. If a 
fractionated bilirubin is not available during screening prior to consent, then informed 
parental consent can be obtained first and a fractionated bilirubin obtained prior to 
enrollment and randomization. 
  Exposure to other systemic macrolide: Prior exposure of the infant to systemic 
erythromycin, clarithromycin, telithromycin, other macrolides, or clinically ordered 
azithromycin is an exclusion criterion. Exposure to ophthalmologic erythromycin ointment is 
not considered a systemic exposure. 

 
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 Participation in other clinical trials involving investigational products. Participation in other 
clinical trials of investigative drugs is an exclusion criterion. Participation in clinical research 
studies not involving investigational product is allowed. 
 EKC QTc ≥ 450 ms. Measure EKG QTc interval (See CRF AZIP 02): 
Calculation of EKG QTc (average 3 measurements): 
0. Print strip of lead II from potential subject monitor (paste copy to page) 
1. May calculate manually or use http://en.ecgpedia.org/wiki/QTc_Calculator 
2. Measure QT interval (duration from the beginning of the QRS complex to the end of 
the T wave): count the number of small squares, then multiply by 0.04 seconds(s) x 
1000: ______ms 
3. Measure RR interval (interval from the peak of one R wave to next R wave peak), 
count the number of small squares, then multiply by 0.04 seconds(s):_______ 
4. QTc= QT(ms)/√ RR (s) 
5. Is the Qtc ≥450 ms? ______Yes _______No 
The EKG can be completed during screening or after consent is obtained, but prior to 
any study procedures.  
6.3 
Enrollment, Baseline, and/or Randomization 
6.3.1 Enrollment 
The enrollment date is the date and time all screening criteria have been met and the parent 
signed the consent form. Baseline assessments, including tracheal and NP aspirates, 
randomization, and the first dose of study drug should be initiated within 24 h of obtaining informed 
consent. 
6.3.2 Baseline Assessments 
 Demographics, obstetric history, maternal macrolide exposure, neonatal characteristics 
including respiratory support mode and supplemental oxygen at study entry 
 Morbidities of prematurity: Morbidities associated with prematurity are protocol-specified 
potential serious adverse events. These are events that are anticipated to occur in the 
preterm population even in the absence of drug exposure. All morbidities present from the 
time of birth up to the start of Study day 1 (first dose study drug infusion) will be recorded. 
 Clinical laboratory tests: Values for electrolytes, total/direct bilirubin, CBC with differential 
cell count, and platelets obtained within 24 h pre-first dose will be recorded. 
 Physical examination: The site PI or other physician listed on local protocol and FDA1572 
form will complete the physical assessment and complete section on CRF AZIP 07. 
 Tracheal and nasopharyneal aspirates: For subjects who are intubated at time of 
enrollment, 2 tracheal aspirates (first for cytokine sample and second for Ureaplasma 
culture with separate suction catheters) will be obtained. The first NP sample will be 
obtained for culture at the time of first tracheal aspirate. At least 2 hours after the first 

 
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suction episode, a second culture sample will be obtained from the ET tube if subject is still 
intubated and from the nose if subject is extubated. For subjects who are extubated at the 
time of enrollment, 2 NP samples will be obtained at least 2 h apart for culture only. A TA 
sample may be obtained for the second sample if infant is intubated at time of second 
sampling. 
6.3.3 Randomization 
The enrolled subject will be randomized once all screening procedures are completed and parental 
informed consent obtained. Randomization should occur within 22 h of enrollment to allow 
sufficient time for investigational pharmacy to prepare the study drug. The first dose of study drug 
should be infused no later than 24 h after enrollment.  
6.3.4 Blinding 
The study pharmacists will be unblinded, but will not reveal treatment assignment to study staff 
administering the study drug. In the (unexpected) event of a clinically urgent reason for unblinding 
of a subject’s assignment, the clinical site investigator or any other individual will be directed in the 
kit instructions to contact (in order of preference) Dr. Viscardi, Dr. Terrin, the UMB Coordinating 
Pharmacy, or Axio Research to document the reasons for unblinding, assure that there is truly 
compelling cause to unblind, and obtain the treatment information which will be available from all 
four sources. Date and time of any unblinding as well as reasons for unblinding will be documented 
by the source providing the unblinding information. Because of the nature of azithromycin and the 
restriction of the patient population to experienced NICUs, we expect that there will be few or no 
unblinding events in this study. 
6.3.5 Follow-up Visits 
A total of 3 doses of study drug will be administered at 24 ± 0.5 h intervals. Follow-up tracheal/NP 
aspirates will be obtained 2 and 4-5 d post last dose and on postnatal day 21. Safety and tolerance 
will include vital signs (temperature, pulse, respiratory rate, blood pressure), frequency of apnea 
(cessation of breathing for > 20 seconds), frequency of bradycardia (heart rate < 80 beats per 
minute for > 10 sec), cardiac rhythm, pulse oximetry, clinical laboratory testing and Adverse Events 
(AE). Safety assessment for subject vital signs, frequency of apnea and bradycardia episodes, 
pulse oximetry, and cardiac rhythm will be collected 1 and 4 h after the initiation of azithromycin 
infusion. On study day 3 (±1 d), 7 (± 2 d), 14 (± 2 d) and every 2 wks (± 2 d) until discharge, 
transfer, or 44 weeks PMA, whichever occurs first, subjects will be assessed for morbidities 
associated with prematurity, and adverse events. Clinically relevant AEs that will be specifically 
evaluated include: local site reaction including phlebitis, gastrointestinal signs (abdominal 
distension, gastric aspirates, emesis, heme-positive stools), intestinal obstruction including 
radiographic-confirmed pyloric stenosis, culture-proven sepsis with other bacterial or nonbacterial 
pathogens, necrotizing enterocolitis (NEC), and cardiac arrhythmias. We will also specifically 
record adverse events that may signal potential proarrhythmic effects in our study population such 
as sudden death, ventricular tachycardia, ventricular fibrillation and flutter, and seizures. A hearing 
screening test as part of clinical care will be performed before discharge. Electrocardiograms will 
be performed pre-dose in all subjects and post-dose if a cardiac arrhythmia is noted on the patient 
continuous monitor. Clinical laboratory testing (complete blood count with manual differential, 
electrolytes, liver function, and renal function) will be performed during the 24 hours pre- first dose, 
and study day 3 (±1 d), and 7 (± 2 d), 14 (± 3 d), and 28 (± 3 d). Blood draws for study only labs 

 
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will be minimized by utilizing lab values from clinically indicated lab draws within the sampling 
periods. Relevant systemic medications received by subjects during the 7 days post-first dose 
administration will be recorded. 
 
Table 4.Timing of endotracheal and nasopharyngeal cultures 
 
Samples 
Pre-infusion 
Day 0 
2 Day 
Post-last 
dose 
4-5 Day 
Post-last 
dose 
postnatal d21 of age 
TA culture/PCR 
X (2) 
X 
X 
X 
NP culture/ PCR 
X 
X* 
X* 
X* 
TA cytokines 
X 
X 
X 
X 
*Collect only in the event that the infant is not intubated and a TA is not available  
 
 
Study D1 (first drug dose): 
§ Vital Signs: Vital signs (HR, RR, and oxygen saturation (SpO2) will be obtained within 1 
h pre-dose, within 20 minutes post-dose and 2h ± 20 min and 4h ± 20 min post-dose  
§ Treatment Administration: The investigational pharmacy at each site will prepare study 
drug vial as assigned by randomization of final concentration of 2 mg/ml and will 
dispense study drug for ordered volume to provide 20 mg/kg. The subject will receive 
azithromycin administered as a single intravenous infusion over 60± 5 min using a 
functioning intravenous line within 24 h of enrollment. The dose will be calculated as 20 
mg/kg actual body weight. Each subject’s actual body weight at the time of study entry 
will be used for determining the injection volume of study drug. A NICU nurse will 
administer the study drug. 
§ Concomitant Medications: Relevant systemic medications received by subjects during 
the 7 days post-first dose administration will be recorded. 
§ Adverse Events: Subjects will be assessed for morbidities associated with prematurity, 
and adverse events. Clinically relevant AEs that will be specifically evaluated include: 
local site reaction including phlebitis, gastrointestinal signs (abdominal distension, gastric 
aspirates, emesis, heme-positive stools), intestinal obstruction including radiographic-
confirmed pyloric stenosis, culture-proven sepsis with other bacterial or nonbacterial 
pathogens, necrotizing enterocolitis (NEC), and cardiac arrhythmias. 
 
Study D2 (second drug dose): 
§ Vital Signs: Vital signs (HR, RR, and oxygen saturation (SpO2) will be obtained within 1 
h pre-dose, within 20 minutes post-dose and 2h ± 20 min and 4h ± 20 min post-dose  
§ Treatment Administration: The investigational pharmacy at each site will prepare study 
drug vial as assigned by randomization of final concentration of 2 mg/ml and will 
dispense study drug for ordered volume to provide 20 mg/kg. If the subject’s weight is ≤ 
0.94 kg, the dose may be ordered to the 1/10 of 1 ml using a 10 ml syringe for 
administration with a syringe pump (example 0.94 kg x 10 ml/kg= 9.4 ml of 2 mg/ml 

 
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azithromycin or equal volume of D5W). For subjects >0.94 kg, a 20 ml syringe with 1ml 
gradiations will be required, and the ordered drug volume should be rounded to nearest 
ml (example subject weight 1.23 kg x 10 ml/kg=12.3 ml, so 12 ml should be ordered). 
The subject will receive azithromycin administered as a single intravenous infusion over 
60± 5 min using a functioning intravenous line within 24 ± 0.5 h of first dose. The dose 
will be calculated as 20 mg/kg actual body weight. Each subject’s actual body weight at 
the time of study entry will be used for determining the injection volume of study drug. A 
NICU nurse will administer the study drug. 
§ Concomitant Medications: Relevant systemic medications received by subjects during 
the 7 days post-first dose administration will be recorded. 
§ Adverse Events: Subjects will be assessed for morbidities associated with prematurity, 
and adverse events. Clinically relevant AEs that will be specifically evaluated include: 
local site reaction including phlebitis, gastrointestinal signs (abdominal distension, gastric 
aspirates, emesis, heme-positive stools), intestinal obstruction including radiographic-
confirmed pyloric stenosis, culture-proven sepsis with other bacterial or nonbacterial 
pathogens, necrotizing enterocolitis (NEC), and cardiac arrhythmias. 
 
Study D3 (Third drug dose): 
§ Vital Signs: Vital signs (HR, RR, and oxygen saturation (SpO2) will be obtained within 1 
h pre-dose, within 20 minutes post-dose and 2h ± 20 min and 4h ± 20 min post-dose  
§ Treatment Administration: The investigational pharmacy at each site will prepare study 
drug vial as assigned by randomization of final concentration of 2 mg/ml and will 
dispense study drug for ordered volume to provide 20 mg/kg. The subject will receive 
azithromycin administered as a single intravenous infusion over 60± 5 min using a 
functioning intravenous line within 24 ± 0.5 h of second dose. The dose will be calculated 
as 20 mg/kg actual body weight. Each subject’s actual body weight at the time of study 
entry will be used for determining the injection volume of study drug. A NICU nurse will 
administer the study drug. 
§ Concomitant Medications: Relevant systemic medications received by subjects during 
the 7 days post-first dose administration will be recorded. 
§ Clinical laboratory tests: Complete blood count with manual differential, electrolytes, liver 
function, and renal function will be performed, and study day 3 (±1 d). 
§ Adverse Events: Subjects will be assessed for morbidities associated with prematurity, 
and adverse events. Clinically relevant AEs that will be specifically evaluated include: 
local site reaction including phlebitis, gastrointestinal signs (abdominal distension, gastric 
aspirates, emesis, heme-positive stools), intestinal obstruction including radiographic-
confirmed pyloric stenosis, culture-proven sepsis with other bacterial or nonbacterial 
pathogens, necrotizing enterocolitis (NEC), and cardiac arrhythmias. 
 
Study D5 (2 d post last dose): 
 Respiratory samples: Tracheal aspirate (intubated subject) or NP aspirate (non-
intubated subject) for follow-up Ureaplasma culture, PCR, and antibiotic susceptibility 
testing. Separate tracheal aspirate will be collected for cytokine analysis if subject is 
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 Concomitant medications: Relevant systemic medications received by subjects 
during the 7 days post-first dose administration will be recorded. 
 Adverse Events: Subjects will be assessed for morbidities associated with 
prematurity, and adverse events. 
 
Study D6-7 (4-5 d post last dose):  
 Respiratory samples: Tracheal aspirate (intubated subject) or NP aspirate (non-
intubated subject) for follow-up Ureaplasma culture, PCR, and antibiotic susceptibility 
testing. Separate tracheal aspirate will be collected for cytokine analysis if subject is 
still intubated. 
 Concomitant medications: Relevant systemic medications received by subjects 
during the 7 days post-first dose administration will be recorded. 
 Clinical laboratory tests: Complete blood count with manual differential, electrolytes, 
liver function, and renal function will be performed on study day 7 (± 2 d). 
 Adverse Events: Subjects will be assessed for morbidities associated with 
prematurity, and adverse events. 
 
Postnatal Day 21: 
 Respiratory samples: Tracheal aspirate (intubated subject) or NP aspirate (non-
intubated subject) for follow-up Ureaplasma culture, PCR, and antibiotic susceptibility 
testing. Separate tracheal aspirate will be collected for cytokine analysis if subject is 
still intubated. 
 
36 weeks postmenstrual age/pre-transfer: 
 BPD assessment: For neonates at 36 ± 1 week or pre-discharge who are on positive 
pressure support or receiving > 30% supplemental oxygen with oxygen saturations 
between 90% and 96%, the diagnosis of BPD will be assigned with no further testing. 
For neonates at 36 ± 1 week or pre-discharge who are receiving oxygen < 30% with 
oxygen saturations between 90% and 96% or receiving oxygen ≥ 30% with oxygen 
saturations >96% will undergo a timed step-wise reduction by 2% increments every 5 
minutes to room air and a period of observation in room air for 30 minutes as 
described by Walsh et al 111. For infants receiving supplemental oxygen by nasal 
cannula, the delivered oxygen concentration or “effective FiO2” 111 will be calculated 
by the technique described by Benaron et al. 112 that was used in the STOP-ROP trial 
113 which is based on weight, oxygen liter flow, and oxygen concentration. Failure of 
the room air challenge will be defined as oxygen saturations 80-89% for 5 
consecutive min or <80% for 15 s. No BPD will be defined as treatment with room air 
with oxygen saturation ≥90% or passing the timed, oxygen-reduction test. Infants will 
be continuously monitored and observed throughout the reduction test. Study 
subjects who are transferred or discharged at ≤ 35 wk on supplemental oxygen will 
be considered to have BPD. 
 
Pre-discharge/transfer 
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performed before discharge. Test results will be recorded as passed, refer one ear, 
or refer both ears. If a brainstem auditory response test is requested for clinical care, 
the results will be recorded as passed or failed. 
 Cumulative days of respiratory support: The total number of days of mechanical 
ventilation, nCPAP, and supplemental oxygen will be recorded. 
 Adverse events: Subjects will be assessed for morbidities associated with 
prematurity, and adverse events. 
 Pulmonary Follow-up 
 Respiratory questionnaires: Data will be collected by structured parental interviews 
before discharge and follow-up phone interview at 6, 12 and 22-26 months adjusted 
age. With permission, we will utilize the validated Tucson Children’s Respiratory 
Study questionnaires that were designed to elicit a complete history of possible 
covariates such as family history of asthma or atopy, and important environmental 
(e.g., smoking) and infectious exposures and detailed interval respiratory health 
history. Recurrent wheezing and chronic cough will be defined as occurring more 
than twice per week. 
 The ideal times of visits and points of reference for data collection on long term 
pulmonary outcomes are six months, one year and two years adjusted age. To 
assure that patient contact information can be used regardless of dates of actual 
contact with the babies and their families, we have established contiguous windows 
for the three follow-up visits. The first of these follow-up visit windows extends from 
five months to nine months adjusted age with an ideal visit date at six months 
adjusted age; the second window from 10 months to 18 months with an ideal visit 
date at 12 months; and the third from 19 months to 30 months with the ideal date at 
24 months. Whenever within a data collection window the baby and family are 
contacted, the information collected about the baby's pulmonary health on that day 
may be used in the data collection window. If a baby and family is contacted more 
than once within a data collection window, the data closest to the ideal date are the 
data to be used. For example if a baby and family are contacted at 10 months and at 
13 months, the data on the baby's pulmonary health collected at the 13-month visit 
are to be used for the 12-month visit. If data were not collected within a specified 
window but the baby and family are contacted in a subsequent window, the data 
collected that day should be processed for the current window, and inquiry made as 
to the baby's pulmonary health at the ideal time of the window in which data were not 
previously collected. If the baby's family can supply answers in response to the 
inquiry, those data should be entered for baby's pulmonary questionnaire CRF at the 
time of the ideal visit date for which information had been missing previously. For 
example, if the first follow-up contact for a baby is completed at 11 months adjusted 
age, the pulmonary health information collected about that day may be used for the 
12-month visit, and answers to questions about the baby's pulmonary health at six 
months adjusted age may be used for late completion of the six-month visit 
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6.3.6 Completion/Final Evaluation 
 22-26 months adjusted age 
 Neurologic examination: A developmental specialist or neurologist blinded to 
treatment assignment will complete a comprehensive neurologic examination 114 that 
includes a standardized assessment of reflexes, muscle tone and strength. The 
neurologic assessment will be classified as normal or abnormal and whether the child 
has cerebral palsy (CP) and CP severity as defined in the MOP. 
 Gross Motor Function Classification Score (GMFCS): The degree of motor 
impairment will be assessed using the 5 level GMFCS 115. 
 Bayley Scales of Infant and Toddler Development, 3rd edition (BSIDIII): A certified 
psychologist blinded to treatment assignment will complete the BSIDIII 116. 
 Ages and Stages Questionnaire, Third Edition (ASQ3): Developmental milestones in 
five domains (communication, fine motor, gross motor, problem-solving ability, and 
personal-social functioning) will be assessed using a parent questionnaire 
administered by phone by study coordinators when a child is unable to return for in 
person assessments. This tool has high sensitivity and negative predictive value 
compared to the BSID-III at 24 months in preterm infants <32 weeks gestation 1. The 
window for this assessment is from 19 months to 30 months with the ideal date at 24 
months. 
 Pulmonary questionnaire: Data will be collected by structured parental interviews 
during the clinic visit. With permission, we will utilize the validated Tucson Children’s 
Respiratory Study questionnaires that were designed to elicit a complete history of 
possible covariates such as family history of asthma or atopy, and important 
environmental (e.g., smoking) and infectious exposures and detailed interval 
respiratory health history. Recurrent wheezing and chronic cough will be defined as 
occurring more than twice per week. 
 Review medical records: Medical records will be reviewed and parents interviewed to 
assess vision and hearing. Records from subspecialty ophthalmology and audiology 
services will be reviewed to determine whether the child is receiving services for 
vision or hearing impairment and results of vision and audiological examinations. 
 Neurodevelopmental impairment will be assigned for any of the following: moderate 
to severe CP, bilateral blindness, bilateral hearing impairment requiring amplification, 
GMFCS ≥ 2, BSIDIII cognitive or motor score <70, or ASQ ≤2SD on any domain. 
 
Early Termination: Subject will be classified as early termination if subject was terminated 
from study prior to 36 weeks PMA or discharge/transfer whichever comes first. A subject 
may be terminated for following reasons: patient enrolled, but not dosed; death; parent or 
LAR withdrawal of consent; investigator decision; or study chairman decision. Partial 
termination may occur when a parent requests no further study procedures, but can be 
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7. SAFETY ASSESSMENTS  
7.1 
Specification of Safety Parameters 
The subjects will be monitored for safety until discharge/transfer. The severity of adverse events  
and out of range laboratory values will be assigned as per the neonatal toxicity tables (Appendix D) 
and severity assessment of lab values  (Appendix E). 
7.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters 
The safety and tolerability of oral azithromycin has been evaluated in 3995 patients aged 2 to 94 
91. The overall rate of side effects is 12% with gastrointestinal symptoms the most common. 
Ototoxicity, although rare, has been reported with IV azithromycin in adults and included reversible 
tinnitus and hearing loss 92. A review of 43 open label, randomized pediatric trials performed 
outside the US that included 2655 patients treated with a 3 day course (10 mg/kg once daily) oral 
azithromycin for upper respiratory tract infections reported adverse effects in 8.7% subjects, 
primarily gastrointestinal symptoms 93. In a recent pharmacokinetics study of a single dose of IV 
azithromycin in 32 children ages 6 months to 16 years, there were no serious adverse events. 
Nausea was the most common treatment related symptom, reported in 25% patients. Although 
treatment with erythromycin in the first few months of life has been associated occasionally with 
the development of infantile hypertrophic pyloric stenosis (IHPS) 94, recent retrospective studies of 
a large cohort suggest that there is an increased risk of IHPS in infants >32 weeks gestation 
exposed to oral azithromycin or erythromycin 95,96. There was not an increases risk in preterm 
infants ≤32 weeks gestation. 
The macrolides are associated with prolongation of the Q-T interval that can lead to serious 
ventricular arrhythmias. Recently, a retrospective study of a large Tennessee Medicaid cohort 
detected a small absolute increased risk of cardiovascular death (hazard ratio, 2.88; 95% CI, 1.79-
4.63) in adults who took a five day course of azithromycin compared to individuals who took no 
antibiotics 98. There were 47 additional cardiovascular deaths per 1 million azithromycin courses. 
The increased risk for cardiovascular death was highest among patients with a high baseline risk 
for cardiovascular disease.  
The implications of that study for azithromycin use in newborns and children are unclear. There is 
a single case report in the literature of cardiac arrest with suspected prolonged QT in a 9 month old 
who inadvertently received 50 mg/kg azithromycin intravenously over 20 minutes 99. Prolonged QT 
interval is rare among newborns with an incidence of the heritable long QT syndromes estimated 
between 1 per 3000 and 1 per 5000 births 100. Based on a retrospective study of prolonged QT 
interval in neonates, Villain and co-workers 101 suggest that infants presenting with a QTc<0.5 
second normalize their QTc over time while infants with a QTc>0.6 second are at risk for severe 
arrhythmias and sudden cardiovascular death. Prolonged QTc was not detected in any of the 171 
preterm infants we screened for the 10 mg/kg and 20mg/kg single dose studies. However, we will 
continue careful screening of potential subjects for prolonged QT interval in the current protocol of 
multi-dose azithromycin. In consideration for safety, we will delay or discontinue study dosing if any 
subject has evidence of an arrhythmia on the cardiac monitor (they will all have continuous 
cardiopulmonary monitoring) or an ongoing proarrhythmic condition such as hypokalemia (serum 
potassium < 3.5 mEq/L), hyperkalemia (serum potassium >6.5 mEq/L on non-hemolyzed 
specimen), hypomagnesemia (total serum Magnesium ≤ 1.6 mg/dL), hypocalcemia (ionized 

 
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calcium < 3.0 mg/dL) or hypothermia (core temperature ≤ 35°C) until corrected by the clinical care 
team. We will also specifically record adverse events that may signal potential proarrhythmic 
effects in our study population such as sudden death, ventricular tachycardia, ventricular fibrillation 
and flutter, and seizures. 
There are no known interactions of azithromycin with other drugs. The potential for these adverse 
outcomes will be carefully evaluated in the Phase IIb trial of azithromycin in preterm infants. 
Adverse events will be recorded on case report forms with specific focus on potential events with 
reported (expected) relationship to the drug (e.g. gastrointestinal symptoms, local IV site reactions, 
cardiac arrhythmias). Hearing loss will be assessed by a pre-discharge hearing screen. Clinical 
laboratory tests will be monitored throughout the first 28 d of the study. On study day 3 (±1 d), 7 (± 
2 d), 14 (± 2 d) and every 2 wks (± 2 d) until discharge or transfer, subjects will be assessed for 
morbidities associated with prematurity, and adverse events. Clinical laboratory testing (complete 
blood count with manual differential, electrolytes, liver function, and renal function) will be 
performed during the 24 hours pre- first dose, and study day 3 (±1 d), and 7 (± 2 d), 14 (± 3 d), and 
28 (± 3 d).  
Cranial ultrasound and retinopathy of prematurity reports of all subjects will be reviewed for 
completeness and accuracy at the time of site visits or will be provided to the data coordinating 
center upon request. Independent review of CUS images will be requested if differences are noted 
in these outcomes by site or treatment assignment. 
7.3 
Adverse Events and Serious Adverse Events  
a. Adverse event: Any untoward medical occurrence in a patient or clinical 
investigation in which a subject is given a pharmaceutical product; does not 
necessarily have a causal relationship with such treatment, or  
b. Any unfavorable and unintended sign (including abnormal laboratory findings), 
symptom, or disease temporally associated with the use of a medicinal 
(investigational) product; not necessarily related to the product. 
c. Change-from-baseline AEs can take 2 forms:  
i. Appearance of a new symptom or sign, or  
ii. Increased severity or frequency of an existing symptom or sign 
d. Severity assessment: Severity refers to the intensity of the event and is generally 
indicated as mild (Grade I), moderate (Grade II), severe (Grade III), or life-threatening 
(Grade IV). See appendices E and F, Neonatal Toxicity Tables for severity grading of 
common events and laboratory abnormalities. For events not listed, grading 
definitions are: 
i. Mild (Grade I): mild transient symptoms, only requiring monitoring or 
symptomatic treatment, and clinically significant 
ii. Moderate (Grade II): Moderate illness or condition which requires new or 
significantly altered specific therapy 
iii. Severe (Grade III): Severe illness or condition: unresponsive to medical 
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iv. Life-threatening: Life-threatening illness or condition. Complicated by acute, 
life-threatening metabolic or cardiovascular complications (such as circulatory 
failure, hemorrhage, sepsis); life-threatening physiological consequences; or 
need for intensive care or emergent invasive procedure (e.g. requires major 
surgery). 
e. Causality refers to the likelihood and extent that the investigational agent being 
studied contributed to the development of an AE. In making determination, consider 
temporal relationship to drug exposure, other possible exposures as etiology, known 
effects of the investigational agent, anticipated (expected) side effects derived from 
preclinical studies, and what is known about similar drugs (See drug insert). 
 
Table 5. Relatedness of AEs to an Intervention (Agent)  
Definite (must have all 4)  
 
Has a reasonable temporal relationship to the intervention 
 
Could not have readily been produced by the subject's clinical state or have been due to 
environmental or other interventions 
 
Follows a known pattern of response to intervention 
 
Disappears or decreases with reduction in dose or cessation of intervention and recurs with 
re-exposure 
Probable (must have 3)  
 
Has a reasonable temporal relationship to the intervention 
 
Could not have readily been produced by the subject's clinical state or have been due to 
environmental or other interventions 
 
Follows a known pattern of response to intervention 
 
Disappears or decreases with reduction in dose or cessation of intervention 
Possible (must have 2)  
 
Has a reasonable temporal relationship to the intervention 
 
Could not have readily been produced by the subject's clinical state 
 
Could not readily have been due to environmental or other interventions 
 
Follows a known pattern of response to intervention 
Unlikely (must have 2)  
 
Does not have a temporal relationship to the intervention 
 
Could readily have been produced by the subject's clinical state 
 
Could have been due to environmental or other interventions 
 
Does not follow a known pattern of response to intervention 
 
Does not reappear or worsen with reintroduction of intervention 

 
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f. Duration of event: Onset of signs and symptoms as well as resolution of the event 
should be recorded, typically defined as the point when all signs and symptoms have 
subsided Some events continue or change in severity over time. Capturing this 
information is also important. Changes in severity such as when an event improves 
from severe to mild should be recorded as separate events, with separate intensities 
and durations collected. 
g. Adverse reaction: Defined as an adverse event caused by a drug. Therefore, 
adverse reactions are a subset of all suspected adverse reactions for which there are 
reasons to conclude that the drug caused the event. 
h. Suspected adverse reaction (21 CFR 312.32(a)): Suspected adverse reaction 
means any adverse event for which there is a reasonable possibility that the drug 
caused the event. For the purposes of IND safety reporting, ‘reasonable possibility’ 
means there is evidence to suggest a causal relationship between the drug and the 
adverse event. A suspected adverse reaction implies a lesser degree of certainty 
about causality than adverse reaction, which means any adverse event caused by a 
drug. 
i. Unexpected (21 CFR 312.32(a)): An adverse event or suspected adverse reaction is 
considered “unexpected” if it is not listed in the investigator brochure (or drug insert) 
or is not listed at the specificity or severity that has been observed; or, is not 
consistent with the risk information described in the general investigational plan or 
elsewhere in the protocol. “Unexpected” also refers to adverse events or suspected 
adverse reactions that are mentioned in the investigator brochure as occurring with a 
class of drugs or as anticipated from the pharmacologic properties of the drug, but 
are not specifically mentioned as occurring with the particular drug under 
investigation (e.g. adverse events associated with other macrolides, but not 
specifically azithromycin such as pyloric stenosis). In addition, adverse events that 
would be anticipated to occur as part of the disease process are considered 
‘unexpected’ for the purposes of reporting because they would not be listed in the 
investigator brochure. 
j. Serious (21 CFR 312.32(a)): An adverse event or suspected adverse reaction is 
considered ‘serious’ if, in the view of either the investigator or sponsor, it results in 
any of the following outcomes: death, a life-threatening adverse event, inpatient 
hospitalization, or prolongation of existing hospitalization, a persistent or significant 
incapacity, congenital anomaly/birth defect, or important medical event that may, 
based on appropriate medical judgment, jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the other outcomes listed in 
the definition.  
k. Life-threatening (21 CFR 312.32(a)): An AE or suspected adverse reaction is 
considered ‘life-threatening if, in the view of the investigator or sponsor, its 
occurrence places the patient or subject at immediate risk of death. 
l. Morbidities Associated with Prematurity 
The population to be studied in this protocol is at high risk for morbidities associated with 
prematurity. Events listed below will, for the purposes of this clinical trial, be considered events 

 
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commonly associated with prematurity. The following morbidities associated with prematurity will 
be entered on the morbidities CRF. 
 Air leak syndrome (e.g. pulmonary interstitial emphysema, pneumothorax, 
pneumomediastimum) 
 Anemia of prematurity 
 Apnea of prematurity 
 Bradycardia 
 Cystic periventricular leukomalacia (confirmed by cranial ultrasound) 
 Feeding intolerance (e.g. gastric residuals, abdominal distension) 
 Gastroesophageal reflux 
 Gastrointestinal perforation (confirmed by radiology and surgical pathology to not be 
associated with necrotizing enterocolitis) 
 Hyperbilirubinemia 
 Necrotizing enterocolitis (confirmed by radiology) 
 Patent ductus arteriosis (confirmed by echocardiography) 
 Periventricular or intraventricular hemorrhage (confirmed by cranial ultrasound) 
 Progressive hydrocephalus requiring shunting 
 Respiratory Distress Syndrome 
 Sepsis (culture-confirmed from sterile site e.g., blood, CSF, urine) 
 Retinopathy of prematurity 
7.4 
Reporting Procedures 
a. Reporting Procedures for all Adverse Events 
i. For adverse events that are level I or level II severity, a separate CRF AE form will be 
submitted for each occurrence. All AEs that occur after the initial study drug 
administration until discharge or transfer will be reported. All submitted AEs will be 
tabulated and submitted to DSMB, FDA annual report, and to each investigator for 
inclusion in annual reports to local IRBs. 
ii. A separate SAE form will be completed for each serious adverse event 
reported/observed until discharge or transfer. Each site investigator must report 
immediately (within 5 business days) to the sponsor (Viscardi) all serious adverse 
events, regardless of whether the investigator believes that they are drug related, 
including those events listed in the protocol as anticipated to occur in the preterm 
population independent of drug exposure (e.g. morbidities of prematurity), or in the drug 
insert as predicted to occur with the drug.  

 
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iii.  Dr. Alex Agthe, study medical monitor will review event information and determine if 
event qualifies as SAE, severity, and relationship to drug. He may contact site research 
team for additional information to make this determination. He will be responsible for 
preparing FDA safety reports and notifying Dr. Viscardi, the study chairman and IND 
investigator-sponsor. Dr. Viscardi will be responsible for submitting the safety reports to 
the FDA within the time requirements, the UMB IRB, and to site investigators for 
submission to local IRBs. Dr. Viscardi will forward all initial and follow-up SAE reports to 
the DSMB chair Dr. Jonathan Davis with each occurrence. The DSMB chair may request 
treatment assignment from the Data Coordinating Center at the time of the initial report 
or will review treatment assignment in the monthly safety reports of cumulative SAEs. 
Reports of deaths will be submitted to the DSMB chair with each occurrence. 
iv. FDA IND Safety Reports: 
a. Sponsor (Rose Viscardi, M.D.) must submit reports to FDA for those instances 
where there is evidence to suggest a causal relationship between the drug 
and the adverse event (i.e. adverse reaction of suspected adverse reaction). 
b. Reports may be submitted after  
1. a single occurrence because the event is uncommon, but is known to 
be strongly associated with drug exposure (e.g. anaphylaxis, Stevens-
Johnson Syndrome) 
2. one or more occurrences: a single occurrence or a small number of 
occurrences of a SAE that is uncommon in the study population, but not 
commonly associated with drug exposure and event(s) occur in 
association with other factors strongly suggesting causation (e.g. strong 
temporal association). 
3. Aggregate analysis of specific events. Morbidities of prematurity are 
examples of protocol-specified serious adverse events. If the frequency 
of a specific SAE occurs in statistically greater frequency in the 
treatment then placebo arm and the events occur in association with 
other factors strongly suggesting causation (e.g. strong temporal 
association), a safety report will be submitted. 
c. Serious adverse event reports for any cases of NEC meeting criteria for 
seriousness (e.g., deaths, infants requiring intestinal resection) will be reported 
to the FDA regardless of relationship to study drug as an IND safety report no 
later than 15 days after the sponsor is informed of a NEC case that qualifies 
as serious. Fatal or life-threatening cases of NEC will be reported no later than 
7 days after the sponsor is notified. With these NEC SAE reports, the 
investigational site where the event occurred and the number of trial 
participants enrolled in the trial will be identified. If available, the number of 
trial participants enrolled at the site where the SAE occurred will be provided.  
d. An expedited IND safety report will be submitted when any of the following 
criteria are met for serious and unexpected suspected adverse reaction: 
1. Suspected adverse reaction (reasonable possibility that the drug 
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2. Serious 
3. Unexpected 
e. Any unexpected and related fatal or life-threatening suspected adverse 
reactions must be reported to the FDA by telephone or FAX within seven (7) 
calendar days after the sponsor’s initial receipt of the information. The FDA 
Medwatch 3500A mandatory form will be completed and submitted 
(www.fda.gov/medwatch). 
f. A written report will be submitted to FDA within 15 calendar days for any 
serious unexpected adverse event considered related to study drug including 
those that have already been reported under the 7-day rule. 
7.5 
Follow-up for Adverse Events 
Any relevant additional information that is submitted as follow-up to a specific SAE, will be 
submitted to FDA as a “Follow-up IND Safety Report” as soon as the information is available. 
7.6 
Safety Monitoring  
An independent Data and Safety Monitoring Board (DSMB) has been appointed and reports to the 
PI. Members include a neonatologist, a biostatistician or a clinical trials epidemiologist, and 
infectious disease specialist. The DSMB will: 1) review and analyze the progress of the study, 2) 
approve amendments to the trial protocol, if warranted, 3) monitor the safety of the study 
treatments, 4) review data quality, 5) review interim analyses and recommend early stopping or 
continuation of the trial.  
Monitoring for serious or unexpected adverse events (SAEs) will be performed throughout the 
hospitalization period or until 44 weeks PMA whichever comes first. The site investigator will 
submit completed SAE CRFs to Data Coordinating Center, the PI, and Dr. Alex Agthe, who will be 
the study medical monitor. Dr. Agthe will review event information and determine if event qualifies 
as SAE, severity, and relationship to drug. He may contact site research team for additional 
information to make this determination. He will be responsible for preparing FDA safety reports and 
notifying Dr. Viscardi, the IND investigator-sponsor. Dr. Viscardi will be responsible for submitting 
the safety reports to the FDA within the time requirements. The Data Coordinating Center will 
prepare summaries of these cases that will be submitted to the DSMB. SAEs that are study-related 
or are cause for urgent concern will be reported to the DSMB, the site IRBs, and FDA immediately 
after recognition of their importance. The DSMB may review patient safety based on an individual 
report or accumulating evidence. Cumulative reports of all AEs and SAEs will be provided at each 
DSMB meeting. The DSMB will meet before subject recruitment begins and will subsequently meet 
every 6 months in person or by teleconference to review the study progress and AE and SAE 
reports. 
8. INTERVENTION DISCONTINUATION  
Subjects may be withdrawn from the study for the following reasons: 1) at the request of the 
subject’s parent(s) or guardian(s), or at the request of other legally authorized representative; 2) if, 
in the investigator’s opinion, continuation in the study would be detrimental to the subject’s well 
being; or 3) at the specific request of the Sponsor, the National Institute of Child Health and 
Human Development (NICHD). Although the decision to remove subjects from the study is the sole 

 
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responsibility of investigator, he/she is urged to discuss situations with Dr. Viscardi prior to 
removing participants from the study. If study drug is discontinued, subjects will continue to be 
followed for safety assessments. All randomized subjects will be included in the intent-to-treat 
analyses to the fullest extent possible. A 5% missing information rate (including early termination of 
study drug or consent withdrawal) is anticipated. In all cases, the reason for withdrawal will be 
recorded in the case report form (CRF) and in the subject’s medical records. If the reason for 
withdrawal was an adverse event (AE), the subject will be followed up and the AE must be 
reported in accordance with the procedures in Section 7.4.  
9. STATISTICAL CONSIDERATIONS  
9.1 
General Design Issues  
To determine the microbiologic efficacy of IV azithromycin 20 mg/kg x 3 days to eradicate 
Ureaplasma from the preterm lung, we will conduct a multicenter, randomized, double-blind, 
placebo-controlled clinical trial. This endpoint is important to plan for a Phase III clinical trial to 
determine if ureaplasmal eradication improves short-term (BPD) and long-term pulmonary 
outcomes. 
We hypothesize that compared to placebo azithromycin 20 mg/kg x 3 days will improve survival 
without persistence of Ureaplasma in preterm infants with Ureaplasma respiratory colonization. 
Primary Efficacy Variable: Survival without persistence of Ureaplasma respiratory tract 
colonization: To determine the efficacy of the IV azithromycin to eradicate Ureaplasma respiratory 
tract colonization, all randomized infants will have follow-up cultures of TAs if still intubated or NP 
aspirates if extubated at sampling times (2 and 5 days post third dose and 21 d of age). These 
samples will be frozen and shipped to UAB for analysis by culture and PCR. The MIC of 
azithromycin will be determined for each isolate. This will assess if Ureaplasma isolates develop 
azithromycin resistance over time or resistance varies among centers. Since Ureaplasma is only 
transmitted vertically, neonates who are positive by culture or PCR at any time point will be 
considered a colonized neonate. Although PCR may be more sensitive for detection of all 
Ureaplasma positive neonates, it does not distinguish between live and killed organisms. 
Therefore, we will define bacterial clearance as 3 negative cultures. The primary outcome will be 
survival with microbiological eradication of Ureaplasma defined as survival to discharge or transfer 
with 3 negative cultures obtained post-therapy.  
Secondary Efficacy Variables: 
Physiologic Test for BPD: Secondary outcomes will include the rate of physiologic defined BPD at 
36 wks PMA in placebo controls (Ureaplasma colonized, and non-colonized) and azithromycin 
responders (Ureaplasma eradicated) vs non-responders (persistent Ureaplasma). For the BPD 
endpoint, the recently reported physiologic definition of BPD based on oxygen-saturation 
monitoring will be used 111. 
Additional secondary endpoints will include overall mortality, incidence of co-morbidities of 
prematurity such as intraventricular hemorrhage, periventricular leukomalacia, necrotizing 
enterocolitis, bacterial and fungal nosocomial infection, pulmonary air leak, patent ductus 
arteriosus, retinopathy of prematurity, number of days of positive pressure ventilation, number of 
days of oxygen supplementation, use of postnatal steroids, and use of non-study antibiotics. The 

 
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treatment groups will be compared for the frequency of event outcomes and medians (treating 
deaths as bad outcomes) for durations. 
 
9.2 
Sample Size  
Table 6. Power to detect a difference between survival with Ureaplasma eradication in treated and control 
groups using a two-sided .05 level test under various assumptions and true difference 
Number of 
Ureaplasma-
colonized 
subjects/group 
Anticipated 
frequency of Survival 
without persistence 
of Ureaplasma 
among azithromycin 
group 
Anticipated frequency 
of Survival without 
persistence of 
Ureaplasma among 
placebo group 
Lower limit of power1 
Upper Limit of Power2 
30 
.70 
.20 
88% 
93% 
.65 
.20 
83% 
90% 
.60 
.20 
76% 
83% 
.55 
.20 
67% 
74% 
 
1 Power if there was perfect correlation between twins 
2 Power if there was no correlation between twins. 
 
Sample Size Considerations: We chose our sample size (30 Ureaplasma-colonized subjects per 
group) in order to have good power to detect an effect of treatment on Ureaplasma respiratory tract 
eradication. To calculate the power we will have, we simulated thousands of data sets under 
various assumptions about the rates of survival without persistence of Ureaplasma in the control 
and intervention groups. For each simulated data set, we calculated a Mantel-Haenszel Chi 
Square, and determined the proportion of data sets for which the p-value was less than .05. These 
simulations were based on the assumption that 20% of the infants were in twin pairs. Since twins 
will be randomized to the same treatment group, and assuming the outcomes in twins are 
positively correlated, the power of the study regarding the treatment effect is less than what it 
would be had all the infants in the study been singletons (independent). To assess the range of 
power, the power was calculated for the two extremes of perfect correlation between twins and no 
correlation between twins.  The actual correlation is likely to be between 0 and 1, so the actual 
power should be somewhere in between the limits as shown in Table 6. These power calculations 
also assume that 5% of the children will withdraw from the study before the endpoint is assessed. 
The actual power of our study will be somewhat higher than in these simulations because the 
actual analysis will glean information from both members of each twin pair using multiple 
outputation.  
Power calculations were performed assuming an 80% survival rate, a 54% Ureaplasma 
colonization rate and a 25% rate of clearance among the placebo group to determine a sample 
size as shown in Table 6. Assuming that survival and Ureaplasma clearance are independent 
outcomes and 25% of the infected children will spontaneously clear the infection, then the 
expected rate of survival without microbiological persistence of Ureaplasma in the absence of 
antibiotic therapy is 0.2 (0.8 x 0.25). In Table 6, we provide the power we will have to detect 
differences in survival without microbiological persistence of Ureaplasma between azithromycin 
and placebo groups with a sample of 60 Ureaplasma-colonized infants under various assumptions, 

 
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using a two-sided, .05-level test. Thus we will have good power to detect effects that are typical of 
appropriately chosen antibiotics.   
9.3  Definition of Populations 
The primary analyses will be performed to test the hypothesis of no difference in survival and 
Ureaplasma eradication between subjects randomly assigned to placebo versus IV azithromycin. 
All primary analyses will be carried out according to the principle of intention-to treat. If the null 
hypothesis is not rejected, 95% confidence intervals will be constructed about the differences 
observed to demonstrate how large the differences could be in either direction. 
9.4 
Interim Analyses and Stopping Rules 
Since the sample of Ureaplasma colonization is small (~30 in each arm), no interim analysis for 
efficacy is planned. 
There will be no pre-specified stopping rules, but rather the DSMB will review SAEs as they occur. 
The DSMB chair may call a DSMB meeting to specifically review safety concerns. The DSMB 
recommendations may include study termination, study continuation with protocol modifications 
(e.g., change in eligibility criteria), or temporary suspension of enrollment until further evaluation is 
completed. The DSMB will review each death occurrence. Cumulative SAEs for each study arm 
will be provided to the DSMB to determine whether there are statistical as well as clinical concerns. 
These findings may be reviewed by the DSMB in a closed session and the findings will be used to 
determine what steps will be taken.  
9.5 
Outcomes  
9.5.1 Primary Outcome  
The primary outcome will be survival to discharge and eradication of Ureaplasma respiratory tract 
colonization. 
9.5.2 Secondary Outcomes  
Secondary outcomes will include the rate of physiologic defined BPD at 36 wks PMA in placebo 
controls (Ureaplasma colonized, and non-colonized) and azithromycin responders (Ureaplasma 
eradicated) vs. non-responders (persistent Ureaplasma). For the BPD endpoint, the recently 
reported physiologic definition of BPD based on oxygen-saturation monitoring will be used 111. 
Additional secondary endpoints will include overall mortality, incidence of co-morbidities of 
prematurity such as intraventricular hemorrhage, periventricular leukomalacia, necrotizing 
enterocolitis, bacterial and fungal nosocomial infection, pulmonary air leak, patent ductus 
arteriosus, retinopathy of prematurity, number of days of positive pressure ventilation, number of 
days of oxygen supplementation, use of postnatal steroids, and use of non-study antibiotics. The 
treatment groups will be compared for the frequency of event outcomes and medians (treating 
deaths as bad outcomes) for durations. 
9.6 
Data Analyses  
Analysis of Primary Outcome: The primary outcome will be survival to discharge and eradication of 
Ureaplasma respiratory tract colonization. The null hypothesis will be tested using the Mantel-

 
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Haenszel chi-square test, taking into account different clinical sites. The test for differences in the 
primary outcome between the treatment groups will be conducted at an overall 2-tailed alpha-level 
of 0.05. Enrolled patients who have died will be counted as having adverse outcomes (i.e., as 
treatment failures) in the primary analysis. Death must be taken into account as an adverse 
outcome to avoid the possibility declaring an advantage for a treatment that is associated with 
improved Ureaplasma clearance but also with greater mortality. In addition to calculating a p-value, 
we will use a Mantel-Haenszel Odds Ratio to estimate the size of the effect of treatment and 
calculate its variance (and corresponding confidence interval) based on the method of Robins et al. 
117. 
Although an issue not addressed in many neonatal clinical trials 118, one complication to the 
analysis is the fact that some of the children will be in twin sets. This violates the assumption of 
independence required by the Mantel-Haenszel approach. To handle this complication, we will use 
the approach of “Multiple Outputation” 119. This approach was previously used to address this 
complication in the NO CLD study 24. This approach involves the random selection of one child 
from each twin pair. Then the analysis is performed only using the randomly selected single child 
from each set of twins. These steps (random selection and subsequent analysis) are repeated 
1000 times. The final estimate is the average of the estimates found in the multiple analyses. The 
variance of the multiple estimates can be used to appropriately adjust the variance of the final 
estimate to take into consideration the fact that each of the multiple analyses were only based on a 
subset of the data.  
Prior to performing the primary outcome analysis, the Breslow-Day test for homogeneity of odds 
ratios will be used to assess if there is significant (at alpha = 0.05) evidence of differences in the 
effect of treatment according to stratum or clinical site. The proposed approach to interactions will 
maintain the overall alpha level for the primary outcome analysis at 0.05. If a clinical site interaction 
that is not consistent with an assumption of homogeneity of treatment effect is detected, the clinical 
site's performance and results will be reviewed to determine the nature of the differences in 
treatment effects. Significant differences in stratum or clinical site treatment effects will be reported.  
Secondary Analyses Including Subgroup Analyses: A similar approach (Mantel-Haenszel tests and 
estimation using multiple outputation) will be used to estimate the effect of the treatment on BPD 
rates. The BPD analysis will be done using all children (with and without Ureaplasma), and then 
separately in strata defined by the presence of Ureaplasma. In the unlikely event that there are 
important imbalances between the randomly assigned treatments in clinical or laboratory variables 
recorded (e.g., selected co-morbid states, concomitant medications), secondary analyses will be 
conducted using logistic regression to estimate treatment effects controlling for confounding 
factors. Model assumptions will be examined, and models will be tested for goodness of fit. If the 
assumptions required for a logistic regression model are rejected, alternate models will be 
explored. Again, the approach of multiple outputation will be used to obtain the final estimates and 
their variance. 
Additional secondary analyses will assess the treatment effect on other outcome variables. 
Secondary outcomes to be considered include death which will be approached with survival 
analysis (time to event) methods; co-morbid outcomes which will be approached with logistic 
regression; number of days of treatment with positive pressure ventilation and number of days of 
oxygen supplementation which will be approached with rank or normal scores statistics to be able 
to assess outcomes with and without taking deaths into account. 
Pulmonary and Neurodevelopmental Outcomes Analysis: Given the duration of follow-up required 

 
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for these aims, drop-out needs to be considered.  So, initially, we will identify predictors of drop 
out, and compare the study groups with respect to drop-out rates.  If there is moderate difference 
between the groups with respect to drop-out rates, then variables associated with drop-out will be 
controlled for in subsequent analyses. 
The primary approach to analysis will be to compare the treatment groups with respect to the 
distribution of 6-month, 12 month, and 22-26 month pulmonary outcomes (number and duration of 
hospitalizations, number of physician and emergency visits for respiratory illness, frequency of 
pulmonary medication use, and parental report of recurrent wheezing or chronic cough).  Initially, 
these comparisons will be performed informally using side-by-side box plots and simple tables.  
More formal statistical analyses will be performed using the methods described for the primary 
outcome.  Specifically, for binary outcomes (e.g., “ever hospitalized”), we will use Mantel-Haenszel 
methods, stratifying by center.  If we need to control for confounding variables, we will analyze 
binary outcomes using logistic regression models including terms for center.  For quantitative 
outcomes we will use multiple regression models including terms for center and potential 
confounders.  To assess whether observed associations with treatment group are mediated by 
clearance of Ureaplasma infection, we will also fit models including a term for persistent infection. 
Final inferential statistics will be based on analyses using multiple outputation methods described 
above to account for correlation within twins. A similar approach for neurodevelopmental 
impairment at 22-26 months will be used to compare the difference for binary outcome survival 
free of neurodevelopmental impairment. 
Pharmacokinetic Evaluation: The plasma concentration versus time data from the a) single 10 
mg/kg (N=12), b) single 20 mg/kg (N=13) and c) multiple 20 mg/kg (N=30) will be compiled and 
analyzed using the nonlinear mixed-effects modeling software, NONMEM. The first-order 
conditional estimation procedure (FOCE) will be used for the analysis. Open one-, two-, and three- 
compartment structural disposition models with first order elimination, as implemented in the 
PREDPP library of models provided in the NONMEM software, will be evaluated to fit the data. 
Previously, a two-compartment structural model with the clearance and volume of peripheral 
compartment (V2) allometrically scaled on body weight (WT) best described the pharmacokinetics 
of i.v. azithromycin in preterm neonates. This model will be first used for the compiled PK data 
analysis. The selection of the final base model will be determined by the Akaike information criteria 
(AIC) and visual inspection of the goodness of fit scatter plots. The additive, proportional and 
exponential pharmaco-statistical models as well as a combination of the additive and proportional 
models will be evaluated to characterize the inter-individual variability and the residual random 
variability. During the covariate model-building process, the stepwise forward inclusion procedure 
will be applied to build the full model. The likelihood ratio test will be applied to discriminate the 
alternative nested models using the model selection criteria with a significance level of 0.05. 
Stepwise backward procedure will be applied to breakdown the full model to the final model with a 
stringent statistical criterion (p<0.01). The improvement in the diagnostic scatter plots (population 
predicted versus observed concentrations, individual predicted versus observed concentrations, 
weighted residuals versus time, weighted residuals versus population predicted concentrations and 
ETA plots) as well as the decrease in the inter-individual and residual variances will be used to 
evaluate the goodness of fit throughout the model building process. Potential covariates include 
birth weight, gestational age, gender, race, current weight, length, body surface area, postnatal 
age and post-menstrual age (gestational age plus postnatal age). Consolidating data from 55 
neonates [n=12 (single 10 mg/kg), n=13 (single 20 mg/kg) and n=30 (extending N for multiple 20 

 
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mg/kg)] will allow better examination of the influence of these covariates on the PK of azithromycin. 
Any potential drug/drug interactions caused by concomitant medications administered to mother in 
the 7 days prior to delivery (e.g., antenatal steroids, antibiotics, tocolytics) and to the subject from 
birth until 7 days (within the drug sampling period) post-azithromycin dosing (e.g., caffeine, 
diuretics, indomethacin, postnatal steroids) will also be examined. The final proposed PK model 
will be internally evaluated using posterior predictive check and simulating the data from which it 
was built. After the final model is developed, Bayesian estimates of the individual patients’ PK 
parameters will be calculated, including plasma clearance, volume of distribution, maximum 
plasma concentration (C max), time of Cmax (Tmax), area under the plasma concentration–time curve 
from zero to infinity (AUC 0-∞), the distribution rate constant, and elimination rate constant.  
Pharmacodynamics evaluation: Microbiological outcome will be classified as eradication or 
persistence of Ureaplasma respiratory tract colonization. Eradication will be defined as all negative 
follow-up cultures and failure defined as the presence of one or more follow-up positive respiratory 
culture for Ureaplasma. Pharmacodynamic parameters of AUC24/MIC90, Cmax/MIC, and %T>MIC 
will be determined for Ureapalsma culture positive subjects. Classification and regression tree 
(CART) analysis will be applied to determine breakpoints of PD indexes, e.g. AUC/MIC, and their 
statistical significances will be validated by Fisher’s exact test. Logistic regression analysis will be 
performed to determine the probability of clinical success or bacterial eradication based on the 
significant predictors, such as AUC/MIC, and T%>MIC, etc. 
10. DATA COLLECTION AND QUALITY ASSURANCE 
10.1 Data Collection Forms  
Data Capture: Data capture includes all procedures required to incorporate study data into a 
central database. Data capture and management of high quality data are key responsibilities of Dr. 
Tracy and the Data Management Core. The general principles that will be followed are: 1) 
continuous time windows to indicate ideal and permissible dates for follow-up visits; 2) permissible 
minimal and maximal time limits between specified data collection visits; 3) identical follow-up 
schedule for all treatment groups for measurement of variables used to assess outcome to avoid 
bias inherent in a schedule involving more frequent contact with one treatment group relative to the 
other; 4) tested data collection forms; 5) document contacts of participants, record reasons for 
missed visits via use of a missed visit form; 6) trained and certified data collectors; and 7) 
appropriate audit trails for all changes to data forms. The study coordinators at each site who are 
blinded to study treatment assignment, will be responsible for completing the case report forms. All 
data recorded by hand will be collected with paper case report forms designed and processed by a 
software application called Teleform (Cardiff, San Marcos, CA) that utilizes optical character 
recognition (OCR) and mark-sensitive technologies that allow data to be captured via electronic 
scanning rather than direct-keyed data entry. Teleform offers functions such as database 
validations, user-defined dictionary look ups, numeric range tests, data, currency and character-
specific formatting, as well as “Always review” and “Entry required” field designations. Once data 
are processed through the Teleform suite of programs, they are committed directly to the secure 
project database. 
Confidentiality 
Subject confidentiality will be maintained in accordance with the Health Insurance Portability and 
Accountability Act (HIPAA) of 1996. Only the subject number and letter code will be recorded in the 
CRF. Study findings stored on a computer will be stored in accordance with local data protection 

 
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laws. Patient data shall be stored with a unique numeric identifier to insure confidentiality. Names 
and addresses corresponding to each identification number will be kept in a secure file. Access to 
all computer files will be restricted to designated personnel through the use of passwords. 
Direct Access to Source Data/Documents 
The subjects’ parents/guardians will be told that representatives of the NICHD, IRB, or FDA may 
inspect their child’s medical records to verify the information collected, and that all personal 
information made available for inspection will be handled in strictest confidence and in accordance 
with local data protection laws. 
10.2 Data Management  
Clinical site data collection 
Clinical sites will be responsible for accuracy and completeness of CRFs and submission of 
completed forms to the Clinical and Translational Research Informatics Center within pre-specified 
windows. They will also be responsible for maintaining source documents and retention of original 
CRFs securely at their site. They will respond to edit checks and submit corrections on CRF 
modification/update/deletion form. Sample case report forms are provided in Appendix F. 
Case Report Forms as Source Documents 
Study personnel trained in the proper completion of case report forms (CRFs) will record study 
data on the CRFs and all forms will be signed and dated by the study personnel who completed 
the form. In some instances data for which there is no prior written or electronic record will be 
recorded on CRFs and these will be considered source documents. This will be limited to the 
following CRFs: 
1. AZIP06 (Tracheal/NP Aspirate Samples Pre-dose) 
2. AZIP07 (Physical Exam Study Day 1) 
3. AZIP08 (Drug Administration) (with supplementary attachments)  
4. AZIP09 (Vital Signs) 
5. AZIP13 (Pharmacokinetics Plasma Sample Collection) 
6. AZIP14 (Tracheal/NP Aspirate Samples Post Dose) 
7. AZIP15 (Adverse Events) (with supplementary attachments) 
8. AZIP16 (Serious Adverse Events) (with supplementary attachments) 
9. AZIP17 (Physiologic BPD Assessment) 
10. AZIP18 (Early Termination)  
11. AZIP19 (Final Disposition Form) 
12. AZIP 20 (Protocol Deviation Form) 
13. AZIP21 (Pre-Discharge Questionnaire) 
14. AZIP22 (Six Month Adjusted Age Questionnaire) 
15. AZIP23 (CRF Modification/Update/Deletion Request) 
 
Clinical and Translational Research Informatics Center 
The CTRIC under the direction of Dr. Kate Tracy will provide data management services for the 
study. They will develop the case report forms, data edit and audits, and prepare monthly 
performance reports. 
 

 
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Data confidentiality, security, and backup: All data collection instruments are designed by the 
CTRIC to exclude the transmittal of personal identifiers to the study database. Participant names 
and other directly identifiable information will not be captured in the research database. Each 
participant will be assigned a participant ID (PID) and a randomly generated alpha code at the 
initiation of screening. These two identifiers will uniquely identify participants on all forms and only 
these identifiers will be used when communicating with clinical sites regarding data edits, data 
audits, and concerns regarding an individual participant. Study data will be stored in a suitably 
designed relational database housed on a password protected departmental server. Access to the 
database will be restricted to authorized personnel and require the user to enter an appropriately 
complex password that is changed on a regular basis. Any modifications, updates or deletions will 
be implemented only by the database manager, and only when the proper documentation has 
been provided by the PI. A record of each change to the database will be maintained in a 
transaction log to allow for recovery of the original data if needed. Incremental backups of the 
database are performed daily and the database in its entirety is backed up weekly and maintained 
on tapes that are moved to a fire-proof safe off location to guard against catastrophic loss. These 
tapes are kept for a minimum of 12 months. This backup mechanism allows for restoration of the 
server to any desired state in the past. Thus, in the event of a catastrophic mishap, the data 
system can be rolled back to a state just before the error occurred. All backups are stored in 
secure locations and on password-protected computers. 
Quality assurance: As data are collected, validity and accuracy checks that will be pre-
programmed will be performed. Post-capture validity checks may also be programmed to identify 
other data quality issues. Reports of missing forms will be produced, and percent of important 
variables missing will be counted. Queries will be generated for resolution at the data collection 
point level. There will be a timely flow of reported information so that the PI or her designate can 
take corrective action when needed to address data quality issues. 
Data edits and audits: Edits are run on the database on a regular basis. Edits include checking 
for unusual or inconsistent values, as well as examining missing values. The PI or her designate is 
notified of the edits found and subsequently must make suitable updates to the forms, complete 
the necessary Modification/ Update/ Deletion (MUD) Log, and respond regarding resolution of the 
edits. Items previously queried in the data editing process that are legitimate (e.g., missing data 
from a previous visit that cannot be gathered at this point in time) are removed from future lists of 
data edits. Approximately 5% of the scanned forms are randomly selected and a list of requested 
forms is sent to the PI. Copies of the original forms are marked for re-scanning for comparison with 
the records in the existing database. The forms are also examined for other problems (e.g., 
improper documentation of changes made on the form). Discrepancies within the database must 
be resolved. 
Performance monitoring: The performance of the clinical sites is monitored via a variety of 
reports that are produced on a regular basis to assist the PI and project staff in monitoring study 
progression and recruitment, including information on what forms have been completed for each 
participant at each assessment time point, summaries of data edits and audits, the number of 
patients screened over time, the number of patients enrolled over time, and the timeliness of 
submitting forms for processing. Reports will be stratified by clinical site and/or important 
covariates as appropriate. 

 
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10.3 Quality Assurance 
10.3.1 Training 
The principal investigator, Dr. Viscardi will train the study coordinators in the procedures for 
tracheal aspirate and nasopharyngeal culture collection and the room air challenge test. All study 
procedures will be reviewed during the investigator meeting to be held prior to initiating the study. 
The PI will conduct site initiation visits to all additional sites to review the manual of procedures 
and sample collection techniques.  
For the neurodevelopmental assessments, a primary neurologic examiner will be identified who will 
certify annually and certify other examiners at their respective sites. Training DVDs of neurologic 
exams to include normal neurologic exam, CP, GMFCS ≥ level 2, and neurodevelopmental 
impairment will be prepared and each primary examiner will score each exam independently and 
their inter-rater agreement with identified Gold Standard examiners will be determined. 
10.3.2 Quality Control Committee  
Drs. Viscardi and Terrin will review monthly performance reports for site-specific enrollment, CRF 
completion, data edits (number of error messages), sample collection, and protocol deviations. 
They will contact site research staff if specific problems are identified in order to improve study 
performance. 
10.3.3 Metrics 
The UAB Mycoplasma Diagnostic Laboratory will be the central laboratory for all Ureaplasma 
culture, PCR, and antibiotic susceptibility testing and will conduct all testing according to strict 
standard operating procedures. They will provide 10B broth to all sites for collection of respiratory 
specimens. Appropriate controls will be run with each culture and PCR to identify false positive and 
false negative results.  
Assignment of physiologic BPD is an important secondary outcome. The investigators will be 
trained in the timed oxygen reduction test and all case report forms will be assessed for completion 
and accuracy. The PI will review whether the BPD assessment algorithm was applied accurately in 
each case of infant who survived until 36 weeks PMA. 
10.3.4 Protocol Deviations 
Protocol violations may jeopardize the study by breaching assurances made to the subjects or by 
diminishing the validity of the study. Major violations are those that endanger patients, such as 
failure to adhere to appropriate inclusion/exclusion criteria or undermine the conduct of the study 
such as failure to obtain blood and respiratory samples at the appropriate times. Minor violations 
are those that impede the progress of the study, such as not completing CRFs in a timely fashion. 
If blood draws or tracheal aspirate samples are outside the appropriate time range, an explanation 
will be documented. The principal investigator, site investigators and coordinators will problem-
solve to limit future protocol violations. Protocol violations will be reported to the IRB and the 
DSMB. 
10.3.5 Monitoring 
The site PI will be responsible for assuring protocol compliance and data quality at their sites. Dr. 
Viscardi will visit each site annually to review records, consent forms and adherence to procedures 
as outlined in the Manual of Procedures.  

 
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11. PARTICIPANT RIGHTS AND CONFIDENTIALITY  
11.1 Institutional Review Board (IRB) Review  
This protocol and the informed consent document (Section 16.1) and any subsequent 
modifications will be reviewed and approved by the IRB or ethics committee responsible 
for oversight of the study.  
11.2 Informed Consent Forms 
If an infant qualifies, the PI or coordinator will obtain informed parental consent prior to 
initiation of study procedures. An EKG for QTc measurement will be obtained after 
consent if not done previously during the screening process. If the PI is also responsible 
for the clinical care of a potential subject, he/she will not participate in the consent 
discussion with the parents to avoid undue pressure on the parents to agree to the 
study. The information provided in the consent will cover the elements listed in the CFR 
Part 50.25 and be approved by the UMB Institutional Review Board (IRB). This includes 
the investigational nature and objectives of the trial; the procedures and treatments 
involved and their attendant risks, discomforts, and benefits; and the potential alternative 
therapies, alternative to not participate and right to withdraw without penalty will be 
explained. The study staff personnel will offer to answer any questions. Consent will be 
documented by parental signature on the IRB approved consent form. A copy will be 
given to each legal guardian and this fact will be documented in the participant’s record.  
11.3 Participant Confidentiality  
Any data, specimens, forms, reports, and other records that leave the site will be 
identified only by a participant identification number (Participant ID, PID) to maintain 
confidentiality. All records will be kept in a locked file cabinet. All Teleform entry and will 
be done using PIDs only. Information will not be released without written permission of 
the participant, except as necessary for monitoring by IRB, the FDA, and the NICHD. 
11.4 Study Discontinuation  
The study may be discontinued at any time by the IRB, the NCCAM, the OHRP, the 
FDA, or other government agencies as part of their duties to ensure that research 
participants are protected.  
12. COMMITTEES 
Study Chair: The responsibilities of the Study Chair (Dr. Viscardi) are to: 1) Provide overall 
organization of the Clinical Consortium; 2) Serve as Chair, Steering Committee; 3) Oversee the 
subcontracted activities of the UAB Diagnostic Mycoplasma Laboratory, and the functional units 
within UMB (the UMB Coordinating Pharmacy, Data Management Core, UMB Pharmacokinetic 
Core, and UMB Cytokine Core Laboratory); 4) Work with the Clinical Sites, Laboratories and Cores 
to maximize collaboration and to arrange study meetings and to provide collaborators with 
information about the progress of the trial; 5) Hold the IND and submit study progress reports 
annually to the NICHD and FDA; and 6) Lead the presentation and publication of study results for 
the scientific and lay press. 

 
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Steering Committee: A Steering Committee will be responsible for overseeing the study. This 
committee will make major organizational and policy decisions. The committee will include the 
Study Chair, Deputy Chair (Dr. Terrin), Laboratory and Core Directors, and the Clinical Site 
Directors. This group will have quarterly conference calls and meet yearly prior to the annual 
Clinical Site Directors and Coordinators meeting. 
Publications and Ancillary Studies Committee: A Publications and Ancillary Studies Committee will 
be formed from the study leadership (PIs and Laboratory/Core Directors) and leading Clinical Site 
Directors. This committee will review all proposals for and final versions of research abstracts, 
presentations, and manuscripts to be submitted to journals and national meetings. The committee 
will also review proposals for ancillary studies. Since the success of the trial depends upon the 
collaboration of the Clinical Sites, credit will be assigned to them (as the “AZIP Research Group”) 
in the authorship of reports from the study. Each Clinical Site Director and Clinical Site Coordinator 
will be named in an appendix to the main report. 
13. PUBLICATION OF RESEARCH FINDINGS  
Publication of the results of this trial will be governed by the policies and procedures 
developed by the Steering Committee.  
14. REFERENCES 
1. 
Kerstjens JM, Nijhuis A, Hulzebos CV, et al. The Ages and Stages Questionnaire and 
Neurodevelopmental Impairment in Two-Year-Old Preterm-Born Children. PLoS One 2015;10:e0133087. 
2. 
Waites KB, Katz B, Schelonka RL. Mycoplasmas and ureaplasmas as neonatal pathogens. Clin 
Microbiol Rev 2005;18:757-89. 
3. 
Viscardi RM, Hasday JD. Role of Ureaplasma species in neonatal chronic lung disease: 
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15. SUPPLEMENTS/APPENDICES 
16. CONSENT FORM 
Protocol Title: Azithromycin to Prevent Bronchopulmonary Dysplasia in Ureaplasma-Infected 
Preterms: A Phase IIb Randomized, Placebo-Controlled Trial of Azithromycin to Eradicate 
Ureaplasma Respiratory Tract Infection in Preterm Infants 
 
Study No.: HP-00054998 
 
Principal Investigator: Rose M. Viscardi, M.D. 
Contact Information (410-328-6003) 
 
Sponsor: National Institutes of Health 
 
You are being asked to allow your baby to participate in this research study because your baby was born early 
(prematurely). Your baby’s participation in this study is voluntary and you may ask questions at any time. If you are 
consenting for someone else- a child or someone unable to provide consent themselves- then the word “you” means 
that person. 
 
PURPOSE OF STUDY 
Approximately 54% of babies born very early (before the 27th week of pregnancy) have a lung infection with the 
bacteria Ureaplasma urealyticum. This infection may lead to a chronic lung condition called Bronchopulmonary 
Dysplasia (BPD). There is no known effective treatment for this infection in preterm babies. Azithromycin is an 
antibiotic approved for use by the Food and Drug Administration (FDA) to treat infections caused by other bacteria in 
older children and adults, but has not been approved by the FDA for treating Ureaplasma infections in premature 
babies. This research study is being done as part of the FDA approval process for the use of azithromycin for this 
purpose. Azithromycin has been shown to be effective against Ureaplasma in test tube tests. By measuring blood 
levels of the drug in 40 preterm babies who received azithromycin as a single dose of either 10 mg or 20 mg or 3 doses 
of 20 mg for about every 2 pounds of body weight as part of a research study, we found that the 3 doses of 20 mg for 

 
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about every 2 pounds of body weight was most effective to eliminate the Ureaplasma bacteria from the lungs of babies 
who had the infection. There were no side effects attributed to the antibiotic. 
Babies with and without the Ureaplasma lung infection may participate in this study to evaluate azithromycin in 
preterm babies. 
The purpose of this study is to evaluate whether 3 doses of azithromycin 20 mg/kg daily based on body weight will 
effectively eliminate the Ureaplasma bacteria from the lungs of babies who have the infection and to make sure 
azithromycin is safe in very preterm babies. To determine the effectiveness of azithromycin, this study will compare 
the active drug to a placebo. A placebo is an inactive substance made to look like an active medicine. Your child will 
either get the study drug, azithromycin or a placebo. Researchers use a placebo to see if the study drug works better or 
is safer than not taking anything. In addition, the study will find out how 3 doses of azithromycin or placebo affect 
markers (indicators) of inflammation in the lung in all treated babies. Finally, the study will find out how 3 doses of 
azithromycin or placebo affect lung health status at 6 months adjusted age (6 months plus number of weeks born 
early), 12 months adjusted age (12 months plus number of weeks born early) by a phone interview and lung health and 
development status at 22-26 months adjusted age (22-26 months plus number of weeks born early) during a clinic visit. 
A total of 140 subjects at 7 medical centers will be asked to participate in this study. Your baby will be one of 
approximately 40 subjects to be asked to participate at this location.  
The research will be conducted at the following location(s): University of Maryland Baltimore, University of Maryland 
Medical System, and other sites. 
 
PROCEDURES 
This is a double-blinded study, which means that neither you or the study doctor or the study staff will know which 
treatment you are receiving.  However, in an emergency, study doctor can get this information. 
If you agree to allow your baby to participate in this study and your baby is eligible for participation you will be asked 
to read and sign this document. If you agree for your baby to participate, the following procedures will be performed. 
1) Nose or respirator tube sample: The fluid from the baby's nose or respirator tube will be tested for the Ureaplasma 
bacteria 5 times: twice before the baby gets the study drug, once two days after the baby gets the study drug, once from 
4 to 5 days after the drug, and once when the baby is 21 days old, or right before the baby is discharged, whichever 
comes first. These samples will be sent to the Diagnostic Mycoplasma Laboratory at the University of Alabama, 
Birmingham for bacterial testing. Fluid samples collected at the same times will be tested for indicators of 
inflammation. These samples will be obtained during routine suctioning (cleaning) of your baby's nose or the tube in 
your baby's windpipe that is connected to the breathing machine. You will not be contacted with the results of the 
bacteria tests or indicators of inflammation testing. 
2) Electrocardiogram (EKG): If the baby's heartbeat is not regular on his/her bedside monitor, an electrocardiogram 
test (test that measures the electrical activity of the heartbeat) will be done before study drug is given. If a special 
pattern called prolonged QT interval is seen on the EKG, the drug will not be given. If the baby’s blood potassium, 
magnesium, or calcium levels are low or the blood potassium level is high, the drug will not be given until the blood 
levels are corrected. 
 
3) The treatment your child receives will be chosen by chance, like flipping a coin. Neither you nor the study doctor 
will choose what treatment your child gets. Your child will have an equal chance of being given each treatment. 
Neither you nor the study doctor will know which treatment you are getting. Your baby will receive either the study 
drug (azithromycin) or placebo (an inactive substance made to look like an active medicine) once a day for 3 days by 
infusion, which means that it will be given through an intravenous line (line inserted in a vein). Your baby will receive 
the study drug dose of 20 mg/kg based on their body weight or the same volume of placebo. Each dose will be given 
over a 1 hour period of time.  
 
4) Test for BPD: If your baby is still in the hospital between 35 to 37 weeks gestation (1 month before the due date) 
and is still receiving extra oxygen through a tube to the nose, he/she will be tested to find out if he/she is ready to stop 

 
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the extra oxygen. The extra oxygen will be decreased every 5 minutes by 2% at a time until the extra oxygen is 
stopped and the baby is breathing room air for 30 minutes. The decrease in oxygen will be stopped if the baby's oxygen 
levels are 80-89% continuously for 5 minutes or less than 80% for 15 seconds at any time. Your baby will be 
monitored continuously throughout the test for BPD by study staff and returned to their starting oxygen treatment as 
soon as the test is done. 
 
5) Medical Records: The study doctors will review your baby's medical record. The study doctors will review the 
mother's medical record to make note of the treatments she received within one week of delivery. 
6) Complete questionnaires: Before your baby leaves the hospital, a study staff member will contact you to ask 
questions concerning family history of lung problems such as asthma, and exposures such as smoking in the home. 
You will be asked to provide the name and contact information for your child’s health care provider and permission to 
contact them after your child reaches 6 months adjusted age. Adjusted age is the age your baby would be if born on his 
or her due date. At 6 months adjusted age, 12 months adjusted age, and between 22 and 26 months adjusted age, a 
study member will contact you by phone to obtain a lung health history for your child by asking about breathing 
problems, illnesses, hospital stays, emergency room or doctor visits, and prescribed treatments. We will call your 
health care provider at this time to confirm your child’s medical history. 
7) Development assessment: When your baby is between 22 and 26 months adjusted age, the study staff will schedule 
a visit at the Mount Washington Pediatric Hospital Outpatient Clinic located at 1708 W. Rogers Ave., Baltimore, MD 
to evaluate your child’s development. He/she will have a complete physical exam including a neurologic exam of 
his/her muscle tone, strength, and reflexes. The developmental test will evaluate your child’s developmental 
milestones. You will receive a report about your child at the end of this visit.  
Your baby will receive the same medical care whether s/he joins the study or not. In addition to the regular care for 
prematurity, the study doctors and nurses will follow your baby closely to make sure the infusions are safe. 
 
If the Ureaplasma bacteria is obtained from the baby's nose or respirator tube culture, your child may be eligible for 
future studies of Ureaplasma bacteria as a result of his/her participation in this study. However, you may decline 
permission for future use of his/her samples and still agree to their participation in this study. 
 
I ______ agree ______ do not agree to have my child's bacteria samples stored for possible use in future studies 
(please initial). 
 
Please ______ do ______ do not contact me for future studies (please initial). 
WHAT ARE MY RESPONSIBILITIES IF I TAKE PART IN THIS RESEARCH? 
If you take part in this research, you will be responsible to: 
§ 
Ask questions about anything you do not understand 
§ 
For your child to complete all research procedures unless you formally request to withdraw from the study 
§ 
Bring your child to the follow-up clinic for scheduled visit between 22 and 26 months adjusted age 
§ 
Notify study staff of all changes to your contact information including your address, phone numbers, and e-mail address 
POTENTIAL RISKS/DISCOMFORTS: 
Fluid from the baby's nose or respirator tube will be collected at the time of routine suctioning by the baby's nurse. The 
discomfort from this procedure is minimal. 
 
As the extra oxygen is decreased for the BPD test, the baby may experience brief periods of lower oxygen levels or 
brief periods of lower heart rate. The baby’s oxygen level will be monitored for the entire BPD test and the extra 
oxygen will be adjusted if needed. 
 

 
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The most common side effects reported in older children and adults treated with azithromycin are gastrointestinal 
symptoms such as nausea, vomiting, and diarrhea (about 10%), pain at the injection site (6%), and less often vaginitis 
(vaginal discharge or infection), abdominal (belly) pain, loss of appetite, rash, and itching. Fewer than 1 in 100 babies 
had side effects of burping, passing gas, stomach upset, mouth yeast infection (thrush), headache, sleepiness, 
bronchospasm (temporary narrowing of airways into the lungs), and funny taste. In most cases these symptoms were 
mild. Rarely, heartbeat abnormalities, allergic reactions, pseudomembranous colitis (inflammatory condition of the 
large intestine due to imbalance of "good" and "bad" bacteria in the intestine) and reversible hearing loss have been 
reported. Blood tests for liver and kidney function were out of normal range for 6% or less in older children and adults 
treated with azithromycin. Low numbers of white blood cells (cells that fight infection) and platelets (cells that form 
blood clot) have occurred in less than 1% of treated individuals. Laboratory blood tests went back to normal in other 
clinical trials of azithromcyin. Side effects seen in infants with oral (by mouth) azithromycin and similar antibiotics 
include pyloric stenosis (narrowing of the stomach connection to the small intestine). Pyloric stenosis can lead to 
stomach blockage requiring surgery to relieve the blockage. Since azithromycin is an antibiotic, there is the risk of 
other bacteria that are resistant to the antibiotic starting a new illness. 
 
There is a risk to confidentiality. This risk will be minimized by storing your baby’s personal and medical information 
collected as part of this study in a secure location (locked file in the PI's office) and electronic data will be password 
protected. 
 
There are no other additional known risks and discomforts associated with this study other than those of the infusion 
over and above the risks of being extremely premature and in the NICU. There may be unforeseen risks of 
participation in the study. If you have any questions about the risks, please ask your baby's doctor, the study doctor, or 
the study staff. 
 
POTENTIAL BENEFITS 
Your child may or may not benefit by taking part in this study. There is no guarantee that your child will receive direct 
benefit from his/her participation in this study. The benefits of participating in this study may be a chance that 
azithromycin may decrease inflammation in the lungs or help get rid of the Ureaplasma bacteria from your baby's 
lungs if there is an infection. Your baby's participation may provide information that may benefit other babies with 
Ureaplasma lung infection in the future by helping to determine whether 3 doses of azithromycin is effective in 
eliminating the infection. Still, your baby may get no direct benefit from this study. You need to decide if your child’s 
participation in this research study is in your child’s best interest. 
 
ALTERNATIVES TO PARTICIPATION 
The following alternative procedures or treatments are available if you choose not to allow your baby to participate in 
this study: There is no approved drug to treat Ureaplasma lung infections in babies now. 
 
COSTS TO PARTICIPANTS 
It will not cost you anything to take part in this study. 
 
PAYMENT TO PARTICIPANTS 
You will receive a $50 gift card after the completion of your child’s neurodevelopmental assessment and lung health 
questionnaires at the end of the study when your child is 22-26 months adjusted age. 
 

 
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CONFIDENTIALITY AND ACCESS TO RECORDS 
All of the baby’s samples will be coded with a study number rather than their name or other identifier. We will keep 
your name and contact information on file to contact you at 6 months adjusted age, 12 months adjusted age and 
between 22 to 26 months adjusted age and in case we need to notify you of any important information about the study. 
Only the principal investigator, Dr Rose Viscardi and her staff will have access to your baby’s private information. The 
data from the study may be published. However, you will not be identified by name.  
Efforts will be made to limit your personal information, including research study and medical records, to people who 
have a need to review this information. People designated from the institutions where the study is being conducted and 
people from the sponsor will be allowed to inspect sections of your medical and research records related to the study. 
Everyone using study information will work to keep your personal information confidential. Your personal 
information will not be given out unless required by law. We cannot promise complete secrecy. Organizations that 
may inspect and copy your information include the IRB and other representatives of this organization, the Food and 
Drug Administration, Department of Health and Human Services, and National Institutes of Health. The monitors, 
auditors, the IRB, and the Food and Drug Administration will be granted direct access to your medical records for 
verification of the research procedures and date. By signing this document you are authorizing this access. 
A description of this clinical trial is available on http://www.ClinicalTrials.gov, as required by U.S. Law. This Web 
site will not include information that can identify you. At most, the Web site will include a summary of the results. 
You can search this Web site at any time. 
 
RIGHT TO WITHDRAW 
Your participation in this study is voluntary. You do not have to take part in this research. You are free to withdraw 
your consent at anytime. You will be told of any significant new findings which develop during the study which may 
affect your willingness to participate in the study. There are no adverse consequences (physical, social, economic, 
legal, or psychological) if you decide to withdraw from the research. Refusal to take part or to stop taking part in the 
study will involve no penalty or loss of benefits to which you are otherwise entitled. If you are an employee or student, 
your employment status or academic standing at UMB will not be affected by your participation or non-participation in 
this study. If you decide to stop taking part, or if you have questions, concerns, or complaints, or if you need to report a 
medical injury related to the research, please contact the investigator Dr. Rose Viscardi at 410-706-1913 during the 
day and at 410-328-6003 after hours. If you withdraw from this study, already collected data may not be removed from 
the study database. You will be asked whether the investigator can collect data from your routine medical care. If you 
agree, this data will be handled the same as research data.  
 
CAN I BE REMOVED FROM THE RESEARCH? 
The person in charge of the research study or the sponsor can remove you from the research study without 
your approval. Possible reasons for removal include a treatment becomes available for him/her that may be 
better for him/her than the care available in the study, or he/she has a serious reaction during the study. 
The Food and Drug Administration or the sponsor can also end the research study early. The study doctor 
will tell you about this and you will have the chance to ask questions if this were to happen. 
 
UNIVERSITY STATEMENT CONCERNING RESEARCH RISKS  
The University of Maryland, Baltimore (UMB) is committed to providing participants in its research the rights due 
them under State and federal law. You give up none of your legal rights by signing this consent form or by 

 
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participating in the research project. This research has been reviewed and approved by the Institutional Review Board 
(IRB). Please call the Institutional Review Board (IRB) if you have questions about your rights as a research subject. 
 
Participating in research may result in an injury, as explained above. If you suffer an injury directly related to your 
participation in this project, UMB and/or one of its affiliated institutions or health care groups will help you obtain 
medical treatment for the specific injury and provide referrals to other health care facilities, as appropriate. UMB 
and/or its affiliated institutions or health care groups will not provide you with financial compensation or 
reimbursement for the cost of care provided to treat a research-related injury or for other expenses arising from a 
research-related injury. The institution or group providing medical treatment will charge your insurance carrier, you, or 
any other party responsible for your treatment costs. If you incur uninsured medical costs, they are your responsibility. 
The HRPO will assist you in contacting the sponsor if you have an injury caused by the sponsor’s drug or device under 
study. Uninsured medical costs to treat research related injuries not caused by the drug or device under study are your 
responsibility. The study staff can give you more information about this if you have a study injury. 
 
By signing this Consent Form, you are not giving up any legal rights. If this research project is conducted in a 
negligent manner and you are injured as a direct result, you may be able to recover the costs of care and other damages 
from the individuals or organizations responsible for your injury. 
 
If you have questions, concerns, complaints, or believe you have been harmed through participation in this research 
study as a result of researcher negligence, you can contact members of the IRB or the Human Research Protections 
Office (HRPO) to ask questions, discuss problems or concerns, obtain information, or offer input about your rights as a 
research participant. The contact information for the IRB and the HRPO is: 
 
University of Maryland School of Medicine 
Human Research Protections Office  
620 W. Lexington Street, Second Floor 
Baltimore, MD 21201 
410-706-5037 
 
Signing this consent form indicates that you have read this consent form (or have had it read to you), that your 
questions have been answered to your satisfaction, and that you voluntarily agree to participate in this research study. 
You will receive a copy of this signed consent form. 
 
If you agree to participate in this study, please sign your name below. 
 
 
 
___________________________________ 
Participant’s Signature 
 
 
Date:______________________________ 
 
___________________________________ 
Signature of Parent/Guardian 
(When applicable) 
 
Relationship:________________________ 
 
Date:______________________________ 
 
 

 
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___________________________________ 
Investigator or Designee Obtaining Consent 
Signature 
 
Date:______________________________ 
 
Time: _____________________________ 
 
 
 
 
 
___________________________________ 
Witness* 
 
Date:______________________________ 
 
 
 
 
 

 
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16.1 RESEARCH CONSENT FORM 
 
17. 
OTHER APPENDICES 
 
 
A. Screening Log 
 
 
B. Study Schedule 
 
 
C. Effective FiO2 Table 
 
 
D. Neonatal Toxicity Table 
 
 
E. Severity Assessment of Clinical Laboratory Values 
F. Case Report Forms 
G. Ages and Stages Questionnaires 22 month-27 months