Azithromycin to Prevent Post-discharge Morbidity and Mortality in Kenyan Children
Phase: PHASE4
Conditions: Pneumonia, Diarrhea, Malaria, Co-infection, Death, Malnutrition
Sponsor: University of Washington

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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Azithromycin (AZM) to prevent post-discharge  
Morbidity and mortality in Kenyan children 
(Toto Bora Trial) 
 
NCT02414399 
 
 
 
 
 
 
Sponsored by 
National Institute of Health (1 R01 HD079695) 
 
 
 
 
 
 
 
 
Protocol Chairs 
Judd Walson, MD, MPH 
University of Washington, Department of Global Health 
Seattle, Washington, USA 
 
Benson Singa, MBChB, MPH 
Kenya Medical Research Institute 
Nairobi, Kenya 
 
 
 
December 12, 2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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Table of Contents 
STUDY INVESTIGATORS .......................................................................................... 3 
ABBREVIATIONS & ACRONYMS ............................................................................. 3 
SUMMARY .................................................................................................................. 4 
BACKGROUND .......................................................................................................... 5 
JUSTIFICATION ......................................................................................................... 7 
STUDY OBJECTIVES ................................................................................................. 7 
METHODS ................................................................................................................... 8 
Study Design ...................................................................................................................... 8 
Eligibility ............................................................................................................................. 8 
Sample Size ........................................................................................................................ 9 
PROCEDURES ......................................................................................................... 11 
Recruitment ...................................................................................................................... 11 
Enrollment ........................................................................................................................ 11 
Specimen collection ........................................................................................................ 12 
Randomization ................................................................................................................. 12 
Intervention ...................................................................................................................... 12 
Follow-up Procedures ..................................................................................................... 13 
Laboratory procedures and specimen collection and storage ................................... 13 
Data Storage ..................................................................................................................... 14 
Data verification and Quality assurance ........................................................................ 15 
Data Analysis ................................................................................................................... 15 
Data Safety and Monitoring Board Review .................................................................... 16 
Study timeline .................................................................................................................. 16 
Potential Challenges and Limitations ............................................................................ 17 
ETHICAL CONSIDERATIONS ................................................................................. 17 
Ethical approval ............................................................................................................... 17 
Risks to subjects ............................................................................................................. 17 
Adequacy of protection against risks ............................................................................ 18 
Confidentiality .................................................................................................................. 18 
Potential benefits of the proposed research to the subjects and others ................... 18 
Importance of the knowledge to be gained ................................................................... 19 
REFERENCES .......................................................................................................... 20 
 
 
 
 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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STUDY INVESTIGATORS 
 
Protocol Co-Chairs 
Judd Walson, MD, MPH 
University of Washington 
Seattle, Washington, USA 
 
Benson Singa, MBChB, MPH 
Kenya Medical Research Institute 
Nairobi, Kenya 
 
Co-investigators 
Liru Meshack Wekesa, MBChB MS 
Homa Bay District Hospital 
Homa Bay, Kenya 
George Bogonko, MBChB 
Kisii Teaching and Referral Hospital 
Kisii, Kenya 
 
Patricia Pavlinac, PhD, MS 
University of Washington 
Seattle, Washington, USA 
Barbra Richardson, PhD MS 
University of Washington 
Seattle, Washington, USA 
 
 
Christine J. McGrath, PhD, MPH 
University of Washington 
Seattle, Washington, USA 
Rebecca Brander, MPH 
University of Washington 
Seattle, Washington, USA 
 
Kirkby Tickell, MBBS, MPH 
University of Washington 
Seattle, Washington, USA 
Grace John-Stewart, MD, PhD 
University of Washington 
Seattle, Washington, USA 
 
Ruth Nduati, MbChB, MMed, MPH 
University of Nairobi 
Nairobi, Kenya  
 
Joseph Babigumira PhD MBChB MS 
University of Washington 
Seattle, Washington, USA 
 
 
ABBREVIATIONS & ACRONYMS 
 
AZM 
 
Azithromycin 
CBC 
 
Complete blood count 
CDC 
 
Centers for Disease Control and Prevention 
CRP 
 
C-reactive protein 
DBS 
 
Dried Blood Spot 
ERC 
 
Ethical Review Committee 
EQA 
 
External quality assurance 
GCLP  
Good Clinical and Laboratory Practice 
HAART 
Highly active antiretroviral therapy 
 
HIV  
 
Human Immunodeficiency Virus 
IRB 
 
Institutional Review Board 
IRIS 
 
Immune reconstitution inflammatory syndrome 
KEMRI  
Kenya Medical Research Institute 
MCH  
Maternal Child Health 
PBMC  
Peripheral blood mononuclear cell 
PITC  
Provider-initiated testing and counseling 
PMTCT 
Prevention of Mother-to-Child Treatment 
RNA   
Ribonucleic Acid 
SAE 
 
Serious Adverse Event 
TB 
 
Tuberculosis 
UW 
 
University of Washington 
VL  
 
Viral load (HIV-1 RNA copies/ml) 
WHO   
World Health Organization 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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SUMMARY 
An estimated 3.5 million deaths occur annually in children less than 5 years of age in sub-Saharan 
Africa, approximately 70% of which are due to infectious causes.[6] One-year mortality rates as high as 15% 
have been documented following hospital discharge in sub-Saharan Africa, a rate that is 8-fold higher than 
non-hospitalized children.[7-9] Children being discharged from hospital in Africa may represent an accessible 
high-risk population in which to target interventions to reduce mortality. 
A recent trial of mass drug administration of azithromycin reduced childhood mortality by half among 
children in Ethiopia in communities receiving the intervention.[10, 11] However, concerns about the potential 
for the emergence of antimicrobial resistance, possible toxicity, and the feasibility of delivery are all barriers 
to community-wide distribution of antibiotics.  
Targeted chemotherapeutic interventions, including the use of cotrimoxazole among HIV-infected 
children and the use of amoxicillin or cefdinir among malnourished children, have been shown to reduce 
mortality in these specific vulnerable populations.[12-15] Children who have been recently hospitalized are a 
high-risk population in which a similar targeted approach to azithromycin distribution may optimize benefit 
while reducing both individual and population level risks.  
The mechanisms by which azithromycin may impact morbidity and mortality have not been well 
described. Among high-risk pediatric populations with history of recent illness, azithromycin may act by 
treating residual disease not eliminated during inpatient therapy, by providing prophylaxis from future 
infectious exposures during a time of immune suppression and vulnerability following illness, by treating 
underlying enteric dysfunction and associated mucosal immune/gut barrier disruption and inflammation, 
and/or by clearing asymptomatic carriage of potentially pathogenic organisms. 
We propose a randomized, double-blind, placebo-controlled trial of a 5-day course of azithromycin in 
children age 1 to 59 months discharged from health facilities in Kisii and Homa Bay Counties in Western 
Kenya to reduce post-discharge re-hospitalizations and mortality, to explore possible mechanisms by which 
azithromycin has benefit and risk, and to identify correlates and intermediate markers of re-hospitalization 
and death in the post-discharge period.  
 
 
The proposed Toto Bora study will examine a pragmatic post-discharge intervention that could be 
used in low resource settings to lower mortality and re-hospitalization rates in high-risk children.  In addition 
the trial will also explore a number of scientific questions including the effect of antibiotics on enteric and 
nasopharyngeal infections, inflammation, and the causes of post-discharge mortality. Kisii and Homabay 
counties are the ideal areas in Western Kenya to test this intervention because the child mortality rate is high 
in Nyanza province yet the health facilities in which this Toto Bora study will be conducted have established 
pediatric research infrastructure.   
 
 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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BACKGROUND 
Interventions to reduce child mortality in sub-Saharan Africa (SSA) are not achieving necessary 
impact. Mortality in children under 5 remains extremely high in SSA, exceeding 100 deaths per 1,000 live 
births.[12] Three quarters of these deaths (73.2%) are due to preventable and treatable infectious causes, 
including diarrhea, pneumonia, and malaria.[12, 13] Although some progress has been made in decreasing 
child mortality over the last decade in parts of SSA, it is unlikely that the global community will meet child 
mortality targets stated in the Millennium Development Goals without additional effective interventions.[6, 13, 
14] Novel interventions targeting highest risk individuals are urgently needed to reach these goals. 
The health care encounter is an important point of intervention for reducing morbidity and mortality. 
The health care encounter represents a point of critical opportunity, where children have already accessed 
health care services and interventions targeting severe illness can save lives (Figure 1).[3-5] The majority of 
deaths in children under 5 in SSA are due to 
infectious diseases, including respiratory 
infections, diarrheal disease, and malaria.[13-
15] Interventions to improve survival from 
these diseases have largely focused on 
improving 
community 
diagnosis 
and 
management of these illnesses and on 
increasing 
access 
to 
effective 
medical 
care.[16, 17] While a substantial portion of 
children with these illnesses die prior to 
seeking care, significant numbers of children 
in SSA with severe illness do encounter the 
health care system. Studies of care seeking 
have shown that up to one-half of children with acute respiratory illness and diarrheal disease do seek health 
care in most SSA countries and that care seeking rates may be significantly higher (up to 80%) among 
children with severe disease.[18, 19] Interventions to improve health care seeking are important, but are 
limited by the availability and affordability of transportation, access to health care providers, cost of care, and 
social and cultural barriers.[20] Expanding access to clinical services has been shown to be the most effective 
method to reduce under-5 mortality; resulting in larger reductions in mortality than outreach, family or 
community-based service programs.[21, 22]  
 Many children in sub-Saharan Africa who seek medical care suffer recurrent illness and/or death in the 
immediate period after the health care encounter. Although accessing health care interventions can reduce 
immediate risk, recurrent morbidity and mortality among those children who survive hospitalization remains 
extremely high. The risk of death among children with history of recent hospitalization is 6 to 8-fold higher 
than similarly aged children in the community. While this increased risk is highest in the period immediately 
following discharge (up to 30 days), mortality risk remains elevated up to two years period post-discharge 
(Tables 1a and 1b).[7-9, 23-25] Re-hospitalizations are also associated with as much as a 23.6-fold increased 
risk of death in SSA settings.[7] In addition, the cost of a hospitalization for diarrhea or malaria often exceeds 
the average household monthly income ($60 USD) in Western Kenya and this impact on the economic 
stability of a household further contributes to malnutrition and poor health.[26-28] 
 
Table 1a. Post-discharge mortality in low-resource settings 
Study 
Location 
Population 
Post-
discharge 
mortality 
Risk factors 
Moisi 
2011[7] 
Kilifi, Kenya 
Children <15 years discharged from 
hospital  
3.3% within 
12 months 
Age <5 years, hospital stay >13 days, previous 
discharges, severe malnutrition, meningitis, pneumonia  
Snow 
2000[9] 
Kilifi, Kenya 
Children followed in surveillance 
cohort with a hospital admission 
1.4% within 3 
months  
Gastroenteritis  
Zucker 
1996[23] 
Western 
Kenya 
Children <5 years admitted to hospital  
15% within 8 
weeks 
Young age, severe anemia 
 
Veirum 
2007[8] 
Guinea-
Bissau 
Children admitted to hospital  
7.5% within 
12 months 
Young age, ethnicity, self- discharge, poor housing 
quality, low maternal education, anemia, diarrhea  
Phiri  
2008[29] 
Malawi 
Children 6-60 months hospitalized with 
severe anemia 
12.6% within 
18 months 
HIV, younger age 
Roy  
1983[24] 
Bangladesh 
Children 3 months-3 years discharged 
following treatment for diarrhea 
4.2% within 
12 months 
Age 24-35 months, moderate and severe malnutrition 
Villamor 
2005[30] 
Tanzania 
Children 6-60 months hospitalized with 
pneumonia 
10.4% within 
24 months 
Age <24 months, HIV, stunting, low MUAC, anemia, 
and lack of water supply in the household 
Islam 
1996[31] 
Bangladesh 
Children 1-23 months hospitalized with 
diarrhea 
7.5% within 3 
months  
Age < 6 months, malnutrition, not breastfeeding, lack of 
immunization 
 
 
15-30% 
20-60% 
10-30% 
Total mortality 
No care seeking 
In hospital/facility 
mortality 
Post Discharge 
Mortality 
Figure 1. Mortality due to malaria, pneumonia and diarrhea[3-5] 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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Table 1b. Hospital re-admission in low-resource settings 
Study 
Location 
Population 
Re-admission 
Moisi 
2011[7] 
Kilifi, Kenya 
Children <15 years discharged from hospital  
 17.7% over follow-up period (avg 2.8 years)  
Snow 
2000[9] 
Kilifi, Kenya 
Children followed in surveillance cohort with a hospital 
admission 
40.9% over total follow-up period (9% within 3 
months) 
Veirum 
2007[8] 
Guinea-
Bissau 
Children admitted to hospital  
20.1% re-admitted over 1 year follow-up 
Phiri 
2008[29] 
Malawi 
Children 6-60 months hospitalized with severe anemia 
9.4% within 18 months (17.2% among anemia 
cases) 
 
Illness following discharge from hospital is often the result of preventable and treatable infectious 
diseases. Infectious morbidity and subsequent mortality following hospitalization may be due to incompletely 
or inadequately treated infection, nosocomial infections that present following discharge, or new infections 
resulting from repeated exposure once the child returns to the community. In a large study of 1148 children 
admitted to hospital in Kenya, more than a quarter (27%) were readmitted following discharge, with over half 
of those admission occurring within 6 months. Of these admissions, almost half (47.8%) were due to malaria, 
26.4% due to acute respiratory disease, and 8.2% due to gastroenteritis.[9] In Western Kenya, over three 
quarters of all early deaths following discharge occurred among children who were originally hospitalized with 
malaria or bacteremia, while in Bangladesh, all children who died following hospitalization for diarrhea had 
persistent symptoms of gastroenteritis preceding death.[17, 23, 24] Nosocomial infections also contribute 
significantly to recurrent illness following hospitalization and are often unrecognized at discharge. In Kenya, 
nosocomial bacteremia was documented in almost 6/1000 admissions, with a mortality rate that was more 
than twice that seen for community-acquired bacteremia (53% vs. 24%).[32] Finally, children discharged from 
hospital usually return to the communities and exposures from which they originally came. As a result, 
repeated exposure to community-acquired infections (including malaria, acute respiratory infection, and 
diarrhea) may contribute to post-hospitalization morbidity and mortality. For example, in Kenya, diarrheal 
disease and malaria are the most significant diseases contributing to mortality within three months of 
discharge.[9, 23] Targeted antimicrobial interventions to cover residual infection, reduce carriage of 
pathogens, and provide prophylaxis against common exposures may significantly impact infectious morbidity 
and mortality in the post-discharge period. 
Underlying conditions associated with increased risk of recurrent infectious disease are common 
among hospitalized children in Africa. Many children hospitalized in Africa have underlying malnutrition, HIV-
infection or -exposure, and/or enteric dysfunction, placing them at high risk of recurrent illness and death 
from infectious causes.[33-38] In Kenya, two thirds or more of all pediatric hospital admissions, and up to half 
of all pediatric deaths among hospitalized children, are attributable to malnutrition.[39, 40] Malnourished 
children have a 14-fold increased risk of death following hospitalization for diarrhea, compared with well-
nourished children.[40] In addition, enteric dysfunction as a result of malnutrition, environmental enteropathy, 
HIV, or parasitic infection affects many children in SSA and may significantly impact risk of diarrheal disease 
and invasive infection.[41, 42] Despite increased efforts to improve the in-hospital diagnosis of these 
conditions, many children remain undiagnosed despite being hospitalized.[43-45] 
Targeted antimicrobial interventions have demonstrated benefit in these high-risk pediatric 
populations. Antimicrobial prophylaxis has been shown to decrease mortality in selected high-risk pediatric 
populations including children with HIV and those with malnutrition. Both amoxicillin and cefdinir have been 
shown to reduce risk of death among children with severe malnutrition who did not have evidence of systemic 
infection.[46] Similarly, prophylactic cotrimoxazole has dramatically reduced mortality among HIV infected 
children in SSA.[36] Many hospitalized children with underlying comorbidities are not being diagnosed with 
these comorbidities or experience delays in the initiation of care and treatment. The administration of 
antibiotics to all children at hospital discharge may be particularly effective among those children with 
underlying comorbid conditions.[47] 
Community mass drug administration of antimicrobials has been shown to reduce child mortality in 
SSA. The mass drug administration of azithromycin to communities in Ethiopia with endemic trachoma was 
recently shown to reduce mortality among children 1-9 years of age by almost half (49%).[10, 11] Child 
mortality in these communities was largely due to infectious causes, including malaria, fever, diarrhea or 
respiratory infection.[10]  In another study, a single oral dose of azithromycin provided to children in the 
context of trachoma control resulted in an 80% reduction in infectious mortality among children 1-5 years of 
age.[11] Single-dose community-administered azithromycin was associated with decreased mortality over 
one year of follow-up.  Although the mechanism for this observation is not well defined, the broad-spectrum 
antibiotic coverage and potential effect on pathogen carriage suggest potential benefit in children at risk of 
re-hospitalization or death. 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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Mass administration of azithromycin leads to the rapid emergence of widespread macrolide resistance 
within communities. In a randomized comparison of resistance in nasopharyngeal pneumococcus isolates, 
communities randomized to azithromycin had increased resistance at 12 months (46.9%) compared to those 
receiving no azithromycin (9.2%).  However, administration of azithromycin did not increase resistance to 
clindamycin, penicillin, or tetracycline.[48] Although resistance to azithromycin among pneumococcal isolates 
appears to subside when treatment programs are stopped, the potential impact of community-wide antibiotic 
administration on prevalence of resistance and treatment failure is of concern.[49, 50] The risk of developing 
resistance is lower with targeted administration of antibiotics than with community-wide administration. By 
targeting subgroups of high-risk individuals for empiric antibiotic administration, such as children recently 
discharged from hospital, the risk of community level resistance can be reduced considerably.  
JUSTIFICATION 
Many children in sub-Saharan Africa who seek medical care suffer recurrent illness and/or death in 
the immediate period after the health care encounter. Although accessing health care interventions can 
reduce immediate risk, recurrent morbidity and mortality among those children who survive hospitalization 
remains extremely high. Illness following discharge from hospital is often the result of preventable and 
treatable infectious diseases. Additionally, many hospitalized children with underlying comorbidities are not 
being diagnosed with these comorbidities or experience delays in the initiation of care and treatment. As 
such, the administration of antibiotics to all children at hospital discharge may be particularly effective among 
those children with underlying comorbid conditions. Therefore, targeted antimicrobial interventions to cover 
residual infection, reduce carriage of pathogens, and provide prophylaxis against common exposures may 
significantly impact infectious morbidity and mortality in the post-discharge period. 
Although some progress has been made in decreasing child mortality over the last decade in parts of 
SSA, it is unlikely that the global community will meet child mortality targets stated in the Millennium 
Development Goals without additional effective interventions.[6, 13, 14]. Therefore, novel interventions 
targeting highest risk individuals are urgently needed to reach these goals. Our study aims to reduce post-
discharge re-hospitalizations and mortality, to explore possible mechanisms by which azithromycin has 
benefit and risk, and to identify correlates and intermediate markers of re-hospitalization and death in the 
post-discharge period as a novel intervention towards decreasing child mortality. 
STUDY OBJECTIVES 
Primary Aims 
Aim 1. To compare rates of re-hospitalization and mortality in the 6 months following hospital discharge 
among Kenyan children receiving 5-day azithromycin vs. placebo. 
Hypothesis: The provision of a 5-day course of azithromycin provided at discharge will reduce hospital 
re-admission and death within the 6 months following discharge, as compared to placebo. 
Aim 2a. To evaluate possible mechanism(s) by which azithromycin may affect morbidity and mortality, by 
comparing reasons for re-hospitalization, prevalence of pathogen carriage, and markers of enteric 
dysfunction between the randomization arms.  
Hypothesis: Children treated with azithromycin will experience fewer hospitalizations due to diarrhea, 
acute respiratory infection, and malnutrition, will be less likely to have respiratory and gastrointestinal 
carriage of potentially pathogenic organisms, and will have less evidence of enteric dysfunction, as 
compared to children treated with placebo in the 6 months following hospital discharge.  
Aim 2b. To determine whether empiric administration of azithromycin at hospital discharge increases risk 
of antimicrobial resistance in commensal Escherichia coli (E. coli) and Streptococcus pneumoniae (S. 
pneumoniae) isolates from treated children and their household contacts. 
Hypothesis: Isolates of commensal E. coli and S. pneumoniae from children treated with azithromycin 
and their household contacts will have higher levels of macrolide and β-lactam resistance, compared to 
the placebo group, after 90 days of follow-up, but resistance in the 2 arms will be similar by 6 months.  
Aim 3. To identify correlates and intermediate markers of post-discharge mortality and hospital 
readmission among hospitalized Kenyan children. 
Hypothesis: Children younger in age, with enteric dysfunction, higher levels of bacterial pathogen 
carriage, immune dysfunction, and malnutrition will experience more frequent re-hospitalizations and 
deaths.  
Aim 4. To determine the cost-effectiveness of post-discharge administration of a 5-day course of 
azithromycin in settings of varying antibiotic use, re-hospitalization rates, and mortality rates. 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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Hypothesis: The provision of a 5-day course of azithromycin provided at discharge is cost-effective in 
settings with moderate to high re-hospitalization and mortality rates and this cost-benefit is sensitive to 
modest changes in individual and community levels of drug resistance. 
METHODS 
Study Design 
A randomized, double-blind, placebo-controlled clinical trial.  Table 4 outlines outcomes, eligibility and 
follow-up proposed for the randomized trial cohort and Figure 3 outlines the longitudinal design. 
 
Table 4. Study summary 
Study Design:  
Randomized, double-blind, placebo-controlled clinical trial 
Primary Outcome: 
Mortality or hospital re-admission 
Inclusion criteria: 
 
 
Children (age 1-59 months) admitted to hospital for non-trauma conditions and 
subsequently discharged who weigh 2 kg or more and plan to remain in study 
area for at least 6 months.  
Exclusion criteria: 
 
Contraindication to azithromycin use (the use of another macrolide antibiotic [i.e. 
erythromycin or clarithromycin]); known macrolide allergy; current use of 
Lopinavir; residence in an orphanage or children’s home; previous participation in 
the Toto Bora Trial; sibling enrolled in Toto Bora Trial on same day.  
Target enrollment: 
1400 (700/arm) 
Intervention 
5-day course of azithromycin or placebo 
Duration of follow-
up: 
6 months (enrollment, 90 days, and 6 months) (Figure 3) 
Sampling framework: All children hospitalized for non-traumatic cause, and subsequently discharged, 
from study sites during recruitment period 
 
Eligibility 
Children age 1 to 59 months old who weigh at least 2 kg and have been hospitalized and subsequently 
discharged from hospitals in Kisii or Homa Bay County for non-traumatic conditions will be eligible for the 
RCT (Aim 1). We will exclude children in whom azithromycin is contraindicated, such as those children 
currently taking another macrolide antibiotic, such as erythromycin or clarithromycin, those with a known 
macrolide allergy, and those who do not plan to remain in the study site catchment area for at least 6 months. 
A random subset of 300 children (150 from each arm) will be selected into the Enteric Function Cohort 
(Aims 2 and 3). We will also enroll a primary caregiver of each the 300 children enrolled in the Enteric 
Function Cohort in the Contact Cohort (Aim 2b). These caregivers must be at least 18 years of age or 
classified as an emancipated minor by Kenyan law, plan to remain in study site catchment area for at least 6 
!
RCT 
Enrolled 
1400 children 
Discharged from hospital following admission for non-
traumatic condition 
Treatment Arm 
700 Children 
Randomized to azithromycin 
10mg/kg/day X 1 day, then 5mg/kg/day 
X 4 days 
1st dose directly observed 
Placebo Arm 
700 Children 
Randomized to placebo X 5 days, 
1st dose directly observed 
 
Enteric Function Cohort 
150 Children 
Treatment arm 
Enteric Function Cohort 
150 Children 
Placebo arm 
Contact Cohort 
150 Primary Caregivers 
of children in treatment arm of the 
enteric function cohort 
 
AST testing of isolated 
S. pneumonia and E.coli 
Contact Cohort 
150 Primary Caregivers 
of children in treatment arm of the 
enteric function cohort 
 
AST testing of isolated 
S. pneumonia and E.coli 
90-Day Follow Up 
6-Month Follow Up 
200 Children 
AST testing of isolated 
S. pneumonia and E.coli 
200 Children 
AST testing of isolated 
S. pneumonia and E.coli 
Figure 3. Proposed study design 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
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months and be willing/able to provide a stool and nasopharyngeal sample at enrollment, 90 days and 6 
months of follow-up if randomized to the Contact Cohort.  
 
Sample Size 
Aim 1. To compare rates of re-hospitalization and mortality in the 6 months following hospital 
discharge among  children receiving 5-day azithromycin vs. placebo. The total sample size required was 
calculated for the primary endpoint of 
time to death or hospital re-admission 
within the 6 month post-discharge 
period, assuming an alpha level of 
0.05, power of 0.80, and a ratio of 
treatment 
to 
placebo 
random 
assignment of 1:1. In sub-Saharan 
Africa, it is estimated that 2-15% of 
children aged less than 5 years died 
within 6 months of hospital discharge 
and 
15.5% of children who survived 
discharge from the district hospital 
were 
re-admitted with the same diagnosis 
within 6-months[7, 9, 25] Assuming 
that 
an additional 5-10% of children are re-admitted for other conditions, we expect that re-hospitalizations will 
occur in 20.5 to 30.5% of children enrolled in the study. Combined with our expected fatality rate (2-15%), 
we expect the cumulative incidence of the combined endpoint to range from 22.5 to 45.5%.[25] Based on a 
previous trial of mass drug administration of a single dose of azithromycin in which a single dose of the 
antibiotic was associated with a 49% reduction in risk of death, we calculated sample sizes using estimates 
of reduction in risk ranging from 30-50% with the cumulative incidence range of 22.5 to 45.5%, and found the 
sample size required ranged from 90 to 550 children per treatment arm.[10] Using the most conservative 
estimates of a hazard ratio of 0.70 and 22.5% prevalence of re-admission/death, we need to enroll 1100 
children in the study (550 per arm) to achieve adequate power. We will recruit an additional 300 children 
(≈20%) to account for possible loss to follow-up, resulting in a total planned enrollment of 1400 children, or 
700 per treatment group. When considering mortality alone, and estimated mortality ranges of 2-15%, we will 
have >80% power to detect hazard ratios ≤0.5 for mortality rates of ≥ 8% and hazard ratios ≤0.6 for mortality 
rates ≥11% (Figure 6). Although the ability to determine differences in mortality between arms will require 
either a high mortality rate or a large measure of effect, in Kenya, re-hospitalization is associated with as 
much as a 23.6-fold increased risk of death, indicating that this outcome is itself a severe outcome and an 
intermediate marker of death.[7]  
Aim 2a. To evaluate possible mechanism(s) by which azithromycin may affect morbidity and mortality, 
by comparing reasons for re-hospitalization, prevalence of pathogen carriage, and markers of enteric 
dysfunction between the randomization arms. We calculated the minimum detectable association between 
treatment arm and cause-specific re-hospitalizations (hospitalization due to diarrhea, acute respiratory 
infection, malnutrition, or malaria vs. any other) among enrolled children, assuming an alpha level of 0.05, 
power of 0.80, and a ratio of treatment to placebo of 1:1.  Based on data from Kenya, re-hospitalization rates 
due to specific causes ranged from approximately 0.5% to 5.7% in the 6 month post-discharge period, with 
the lowest rate reported for gastroenteritis and the highest for malaria.[9] By not conditioning on the child 
having the same diagnosis at initial hospitalization, we expect the cumulative incidence of cause-specific re-
hospitalizations to range from 2.5% to 10%. With this range of outcome rates, we will be able to detect hazard 
ratios of 0.48 to 0.70 for the effect of azithromycin on specific severe morbidities.  
Among the 1400 children in whom nasopharyngeal swabs and stool samples are cultured, we expect 
56% of children in the placebo group to have S. pneumoniae identified based on data from Kenya, providing 
≥80% power to detect a prevalence ratio of 0.85 (or 1.15) between the two treatment arms at each time 
point.[51-53]  Because there is a strong link between carriage of S. Pneumoniae and development of 
associated disease, and because empiric azithromycin has been shown to prevent reduce clinical pneumonia 
by as much as 75% in an outbreak setting, we believe that our ability to detect a 15% reduction in risk is 
appropriately sensitive.[54, 55] Based on prevalence of Salmonella spp., Shigella spp., Campylobacter spp., 
or diarrheagenic E.coli (ETEC, EHEC, EPEC, and EAEC) among asymptomatic children in Western Kenya, 
we expect 20% of children in the placebo group to have a bacterial pathogen isolated at each time point, 
resulting in ≥80% power to detect differences in enteric pathogen prevalence of 0.68 (1.36) at each time 
point.[48]  
Figure 6. Power and detectable hazard ratios given a range of mortality rates 
0
.2
.4
.6
.8
1
Esimated Power
0
.2
.4
.6
.8
1
Hazard Ratio
2%
14%
5%
17%
8%
11%
Cumulative Incidence

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
10 
To determine the number of children needed to evaluate the effect of azithromycin on biomarkers of 
enteric inflammation children in the Enteric Function Cohort, we assumed an alpha level of 0.05, power of 
0.80, and a ratio of treatment to placebo of 1:1. For the outcome of enteric inflammation, and assuming a 
clinically relevant unit mean difference of 2.8mg/L, and a standard deviation of 7.8mg/L, 250 individuals would 
need to be enrolled to achieve adequate power to detect associations between the intervention groups.[56, 
57] We will enroll 300 children (150 per arm) in this cohort in order to account for 20% potential loss to follow-
up. 
Aim 2b. To determine whether empiric administration of azithromycin at hospital discharge increases 
risk of antimicrobial resistance in commensal Escherichia coli (E. coli) and pneumococcal isolates from 
treated children and their household contacts. We will select a random selection of 400 E. coli and 400 S. 
pneumoniae isolates (200 per arm) for β-lactam and macrolide resistance testing. We will also store all S. 
pneumoniae, E. coli isolates and other isolated bacteria from stool for potential future testing in the event that 
resistance prevalence is lower than expected. As shown in Table 7, we will have > 80% power to detect 
prevalence ratios > 1.1, with an ability to detect the smallest effect sizes when the prevalence of resistance 
in the placebo group is highest.  

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
11 
While we expect that all 300 adults in the Contact Cohort will have E. coli isolated and available for resistance 
testing, ranges for adult S. pneumoniae carriage in Sub-Saharan Africa range from 5-55%. Therefore, we 
expect 15 to 165 S. pneumoniae isolates to be available for resistance testing.[51, 58, 59] Assuming an alpha 
of .05, a 1:1 ratio of testable isolates, and a prevalence 
of resistance of 50% in the placebo arm, we will have 
80% power to detect a 1.4-fold higher prevalence to 
1.9-fold higher resistance prevalence in the contacts 
of azithromycin-treated children.  
Aim 3. To identify correlates and intermediate 
markers of post-discharge mortality and hospital-
readmission 
among 
hospitalized 
 
children. 
Conservatively estimating a 20% loss-to-follow-up 
rate in the RCT and a cumulative incidence of death 
or re-hospitalization of 22.5%, we will have >80% 
power to detect hazard ratios ≥1.3 between correlates 
and the outcome with exposure prevalence of ≥20% 
or more and hazard ratios ≥1.5 for exposure 
prevalence <20%.  
PROCEDURES 
Recruitment 
We will recruit children who have been hospitalized, and subsequently discharged, from health 
facilities in Kisii County, including Kisii Teaching & Referral Hospital, Christamarriane Hospital, Tabaka 
Mission Hospital and Homa Bay County, including Homa Bay District Hospital, Kendu Adventist Hospital, 
and St. Paul Mission Hospital. Study staff will work closely with hospital staff to determine when a potentially 
eligible child is going to be discharged from the hospital. After a child has been scheduled for discharge from 
hospital, the study nurse will approach the primary caregiver of the child and describe the study procedures. 
A script will be read to the caregiver to obtain consent for pre-screening of medical records and verbal 
screening. Study staff will explain that the child/caregiver might be randomly selected to provide additional 
samples (stool/rectal swab and nasopharyngeal swab) and therefore all caregivers who agree to participate 
will have agreed to take part in the Enteric Function and Contact Cohorts if randomized to these cohorts. 
If the caregiver is interested in participating and indicates consent for screening, the study staff will screen 
the child for eligibility, and if eligible, will obtain informed consent for study participation.  Informed consent 
will be conducted in the language of the respondent’s choosing (English, Kiswahili, Kisii, or Luo). The parent 
or guardian (primary caregiver) must sign written informed consent (or provide a witnessed thumbprint if not 
literate) prior to enrollment. We expect to enroll approximately 40 children in the RCT cohort per month from 
each of the two proposed study sites to reach the target enrollment of 1400 children within 36 months. Each 
of the proposed study sites records approximately 120 non-trauma pediatric admissions per month.  A sub-
set of 150 children from each randomization arm (Enteric Function Cohort) and their primary caregiver 
(Contact Cohort) will be randomly selected to participate in the nested cohorts. We will enroll approximately 
10 children and 10 primary caregivers per month into these cohorts to reach a target enrollment of 300 
children and 300 primary caregivers within 24-36 months. 
Enrollment 
At enrollment, primary caregivers will be interviewed to assess demographic information, medical 
history, and detailed contact information for the child. Medical records will also be used to abstract information 
from the hospitalization (including presenting diagnosis, medical management, length of stay, procedures 
performed, relevant medical history, physical examination, and laboratory data). All enrolled participants will 
undergo a physical examination performed by the study clinician, including measurement of height/length, 
weight, and mid-upper arm circumference (MUAC). The height, weight, and MUAC of the caregiver will also 
be collected. If laboratory results are not available from the hospitalization, HIV testing will be performed as 
indicated by Kenya NASCOP Guidelines (HIV testing should be performed in all children presenting to a 
healthcare facility).[60] Any child newly diagnosed with HIV will be referred to the HIV Care Clinics at the 
study sites for follow-up care and treatment. Because enrollment procedures may take up to an hour or more 
and on days of multiple enrollments, enrolled caregivers/children may have to wait to complete the enrollment 
procedures, caregivers will be provided with 200-500KSH ($2-5USD) to cover the cost of their transportation 
home.   
Table 7. Power (%) to detect prevalence ratios of macrolide 
and β-lactamase resistance in 200 E.coli and 200 S. 
pneumoniae isolates per treatment group  
 
Resistance prevalence (%) in placebo 
group 
Resistance 
Prevalence 
(%) 
10 
20 
30 
40 
50 
60 
70 
10 
20 
80 
30 
>99 
64 
40 
>99 
99 
55 
50 
>99 
>99 
98 
48 
60 
>99 
>99 
>99 
>99 
52 
70 
>99 
>99 
>99 
>99 
98 
55 
80 
>99 
>99 
>99 
>99 
>99 
99 
64 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
12 
 
Specimen collection 
Enrollment, 90-days, and 6 months 
All children will also be asked to provide a whole stool for enteric pathogen identification and storage. 
If a child cannot produce a bowel movement, flocked rectal swabs will be collected.  Stool samples/swabs 
will be divided as follows:  1) Sample for transport to microbiology lab for bacterial culture and AST testing 2) 
Used immediately for point-of-care parasite testing 3) placed in sample stabilizing reagent for shipment to 
UW/KEMRI freezers for -80°C storage for future determination of pathogen or commensal flora using 
standard culture and sensitivity or molecular identification. One nasopharyngeal swab will also be collected 
from all enrolled children at each time point for pathogen detection using bacterial culture or molecular-based 
methods. All children randomized to the Enteric Function Cohort (300 children) will have an additional 
portion of stool, or additional swabs collected, shipped to UW/KEMRI in Nairobi for -80°C storage for eventual 
enteric inflammation assessment. Primary caregivers in the Contact Cohort will also be asked to provide a 
stool sample (or 2 rectal swabs) and nasopharyngeal sample at each visit for testing as described above and 
for future determination of pathogen or commensal flora using standard culture and sensitivity or molecular 
identification. Venous blood (up to 1 teaspoon [5mL]) will be collected from enrolled children at each time 
point. This blood will be used for malaria and HIV testing, if not already performed during the hospitalization, 
and will be stored for future testing of inflammatory, immune, intestinal biomarkers, and sickle-cell disease.  
Randomization 
Block randomization (1:1) in random sized blocks of no more than 10, stratified by site, will be used. 
Primary randomization will also include allocation to the Enteric Function and Contact Cohorts. 
Randomization for selection into the nested cohort will occur within the treatment arms, at a ratio of 1:5 
(resulting in 150 per arm). The randomization code will be maintained at the UW/KEMRI central office (in 
Nairobi) by the Principal Investigator, an external entity who will also be responsible for labeling of study drug. 
Treatment allocation (once assigned) will remain blinded to the participant, the study staff and the hospital 
clinicians during all data collection phases of the study. Resistance testing will occur after all participants are 
enrolled. We will randomly select 200 E. coli and 200 S. pneumoniae isolates from children from each of the 
enrollment, 90-day and 6-month follow up visits for antimicrobial susceptibility testing and other isolates will 
be stored for possible future determination of pathogen or commensal flora using standard culture and 
sensitivity or molecular identification. All isolates from Contact Cohort participants will be tested for antibiotic 
susceptibility. 
Intervention 
Caregivers of enrolled children will be 
provided a 5-day course of oral suspension 
formulation azithromycin (Zithromax® from Pfizer) 
(10 mg/kg on day 1, followed by 5mg/kg/day on days 
2-5) or identically appearing and tasting placebo at 
discharge. 
Study 
staff 
will 
directly 
observe 
administration of the first dose and will provide 
information-motivation-behavior (IMB) theory based 
counseling on the importance of adhering to the 
intervention.[61] Daily text message reminders will be 
sent for the four days following discharge and 
caregivers asked to respond with whether or not the 
child took the daily dose. Five day dosing results in 
detectable levels of azithromycin that persist beyond 
72 hours following the final dose (Fig. 4).[62] The long 
half-life of azithromycin exceeds that of other antibiotics, such as amoxicillin (approximately 1 hour), 
contributing to the effectiveness of relatively short course dosing regimens.  
Figure 4. Azithromycin concentration post dosing[1, 2] 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
13 
Follow-up Procedures 
All enrolled children and primary caregivers will be scheduled to return to the health facility at 90 days and at 
6 months following enrollment to collect clinical information and stool and nasopharyngeal samples. 
Anthropometric measurements will be obtained from all children at both follow up visits (height/length, weight, 
MUAC) and caregivers will be asked about any hospitalizations occurring since the last time the child was 
seen by study staff. A flowchart of follow-up and sample collection is shown in Figure 5. Transportation cost 
will be reimbursed at each follow-up visit. If the participant does not return at their scheduled time, study staff 
will attempt to make contact with the primary caregiver via cell phone; if no telephone number is provided, or 
if the participant cannot be reached, study staff will trace the child to the household within 2 weeks of the 
scheduled follow-up time. Study staff will use a standardized questionnaire to ascertain history of recent 
illness/morbidity, post-discharge medication use including antibiotic treatment, and current condition of the 
child (any hospitalizations, admission and discharge date of any hospitalization, alive or dead, date of death 
if applicable). If caregivers report a hospitalization, study staff will work with hospital staff to pull the hospital 
record of the re-admitted child and will abstract relevant information from the hospitalization, including use of 
antibiotics and other medications. In addition, study staff will work with hospital staff to monitor hospital 
admissions on a weekly basis to determine whether a previously enrolled child is re-admitted at the health 
facility and will follow-up on reported admissions. Study staff will capture and transcribe admission and 
discharge dates, date of death (if occurring in facility), diagnosis or causes of admission, medication 
administration, and length of stay from patient medical records. Caregivers will be encouraged, at enrollment 
and at each subsequent contact, to bring the child to the health facility where enrollment occurred for 
assessment and possible treatment of any medical conditions. If a child is admitted at a facility other than the 
designated study site, study staff will contact facility personnel at the relevant site to obtain information. If a 
caregiver or staff member reports that a child has died, study staff will review hospital records (if available) 
or conduct a standardized verbal autopsy at the household to ascertain the cause of death. The height, 
weight, and MUAC of the caregiver will be collected at the follow up visit as well. 
Laboratory procedures and specimen collection and storage 
Stool (rectal swabs), nasopharyngeal swabs and DBS will be collected as described above and undergo 
laboratory testing as described in Table 5. All biological samples will be collected by staff trained in biosafety 
and GCLP. Samples will be processed in Kenya when technology is available at one of the following 
laboratories: Kenya Medical Research Institute (KEMRI) (Wellcome Trust or Centre for Microbiology 
Research [CMR]) or at the University of Nairobi (Microbiology Department).  Metagenomic analyses and/or 
analyses that require technology not available in Kenya will be performed at the University of Washington. If 
stool culture results report Shigella infection, the study staff will contact the child’s caregiver and encourage 
the caregiver to bring the child back for treatment if the child is symptomatic. 
 
 
 
 
 
 
 
 
Figure 5. Data collection throughout follow-up 
 
 
Stool/ rectal swab collection 
RCT Study Population 
1,400 children at hospital 
discharge 
Enrollment 
Questionnaire/ anthropometry 
Abstract Medical Records 
Blood 
Stool/ rectal swab collection 
Nasopharyngeal swab collection 
 
Questionnaire/anthropometry 
Abstract medical records (if re-
hospitalized)  
Verbal autopsy (or abstracted 
from medical records)  
Blood 
Stool / rectal swab collection 
Nasopharyngeal swab collection 
6-month follow-up 
90-day follow-up 
Questionnaire/anthropometry 
Abstract medical records (if re-
hospitalized)  
Verbal autopsy (or abstracted 
from medical records) 
Blood 
Stool/ rectal swab collection 
Nasopharyngeal swab collection 
Enteric Function 
Cohort 
Subset of 300 children 
Contact Cohort 
Primary caregivers of 300 
children in Enteric 
Function Cohort 
Stool collection 
Nasopharyngeal swab collection 
 
Stool collection 
 
Stool collection 
Stool collection 
Nasopharyngeal swab collection 
Stool collection 
Nasopharyngeal swab collection 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
14 
 
Table 5. Laboratory testing of stool, nasopharyngeal swab and blood specimens collected from participants at 
enrollment, 90-days, and 6 months 
Specimen 
Collected 
Participants 
Purpose 
Tests Performed 
Stool/ flocked 
rectal swabs 
All enrolled 
children and 
adults in the 
Close Contact 
Cohort  
Bacterial ID and 
storage for AST 
Fresh or frozen stool samples/rectal swabs will be evaluated 
for enteric bacteria and antimicrobial susceptibility testing 
(AST) will be performed using standard microbiologic or 
molecular diagnostic protocols.  Colonies of Escherichia coli 
(E.coli) and other enteric pathogens will be stored for future 
microbiologic or molecular testing.  
 
All enrolled 
children  
Parasite detection 
Fresh or frozen stool will be tested for parasites using 
microscopy or antigen test kits such as ImmunoCard STAT! 
Cryptosporidium/Giardia (Meridian Bioscience, Inc), Xpect 
Giardia/ Cryptosporidium (Remel, Inc), Quik CheK or similar 
tests on stored stool/rectal swab samples. If available, 
molecular testing will be performed.  
 
Children 
enrolled in 
Enteric 
Function 
cohort, and 
adults in the 
Close Contact 
Cohort 
Enteric 
inflammation 
Commercially 
available 
enzyme-linked 
immunosorbent 
assays such as neopterin (NEO), alpha-anti-trypsin (AAT), 
myeloperoxidase (MPO), or similar tests, will be used to 
measure enteric inflammation on fresh or frozen stool 
samples..   
 
All enrolled 
children and 
adults in the 
Close Contact 
Cohort 
Storage (eventual 
enteric infection 
detection and 
quantification) 
Stool/ flocked swabs will be immediately placed in sample 
stabilizing reagents and stored at -80°C in the University of 
Washington/KEMRI storage facilities in Nairobi. Stored stool 
will be used for one or more of the following: enteric infection 
detection and quantification using multiplex real-time PCR 
methods, DNA extracted for metagenomic analysis to 
determine the composition of the microbial communities.  
Nasopharyngeal 
Swabs 
All enrolled 
children and 
adults in the 
Close Contact 
Cohort 
Bacterial isolation, 
storage, and 
resistance testing 
Pneumococcal colonies will be isolated using standard 
microbiologic 
or 
molecular 
diagnostic 
protocols 
and 
susceptibility testing performed using standard microbiologic 
or molecular techniques.  
Blood 
All enrolled 
children 
Storage (eventual 
cytokine and 
inflammatory 
marker 
assessment), 
HIV, and Malaria 
testing 
Pro-inflammatory cytokines (such as C-reactive protein, IFNγ, 
TNFα, IL-2, IL-17A, IL-17F, IL-22, IL-23 and IL-1β),  anti-
inflammatory cytokines (such as IL-4, IL-5, L-9, IL-6, IL-10, IL-
13, IL-27, IL-37 and TGFβ), markers of intestinal damage 
(intestinal 
fatty 
acid 
binding 
protein) 
and 
microbial 
translocation (soluble CD14),  and activity of the growth 
hormone axis (insulin-like growth factor [IGF-1] )and insulin-
like growth factor binding protein measured using enzyme-
linked immunosorbent assays performed at the University of 
Washington. HIV testing will be performed per Kenyan 
National Guidelines and malaria microscopy performed using 
standard methods. Sickle-cell disease  will be tested using 
Sickle SCAN (BioMedomics, Inc), HEMECHIP (Hemex 
Health, Inc) or with hemoglobin electrophoresis. 
  
Data Storage 
Data will be directly entered into a comprehensive and secure web-based clinical trials data management 
platform, such as RedCap. The database will be password-protected and web-based, The database will be 
hosted by University of Washington Institute of Translational Health Services (ITHS) and is Compliant with 
U.S. Food and Drug Administration (FDA) 21 CFR Part 11 and Good Clinical Practices (GCP). Each study 
staff member will be assigned a unique password that allows secure access to the centralized data. Rights 
to enter, access, and modify data will be restricted and managed by the Seattle-based data manager. Within 
the RedCap platform, all demographic, clinical and laboratory data for each patient are uniquely linked using 
a unique patient identifier (PID) number. The data are encrypted and stored in a restricted access server. 
The code linking the PID to individual identifying information will be kept in a separate secure location. The 
clinical staff at the sites will be able to access the identifying information linked to the PID only to match the 
laboratory results to individual children, as results become available, and to follow-up children and contacts. 
No identifying information other than the PID will be captured on the data collection forms and within the 
computer databases. All study staff are trained in data protection and privacy and are required to have 
certified CITI training in human subjects research (including data protection). All data and study files will be 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
15 
stored in locked cabinets within locked study offices at each site. Study offices are accessible only to study 
staff. All databases are password protected. Only clinical files will contain patient identifiers and non-clinical 
study staff (including analysts) will receive only coded data. The link between identifiers and unique study ID 
numbers will also be kept in locked cabinets within the locked study offices and will be destroyed 3 years 
after study completion (in 2023). 
Data verification and Quality assurance 
A dedicated study data team will be responsible for all data entry, management and storage of study data. 
Each site will have a dedicated data clerk responsible for data entry and synchronization with the central 
server. The data team will be in close communication with the Seattle-based statistical team (under Dr. 
Richardson) for reporting, monitoring, and analysis. Entered data will be synchronized with the central 
database system on a daily basis and verified by the Seattle-based data management team. RedCap allows 
study staff at different locations to directly enter and review data online, with a complete auditing trail system 
tracks changes to data, and allows numerous real-time built-in data validation/quality checks with prompts 
during entry to ensure accuracy. 
Data Analysis 
The primary study endpoint is a combined outcome of mortality and hospital readmission, as re-
hospitalization is highly associated with risk of subsequent poor outcome. Additional analyses will be 
conducted individually for these two outcomes. The secondary endpoints of cause-specific morbidity 
(diarrhea, acute respiratory infection, malnutrition, or malaria), pathogen carriage in stool or nasopharyngeal 
swabs and markers of enteric dysfunction will be used to better elucidate the mechanism by which 
azithromycin may effect the primary endpoints of mortality and re-hospitalization.  
Aim 1. To compare rates of re-hospitalization and mortality in the 6 months following hospital 
discharge among Kenyan  receiving 5-day azithromycin vs. placebo. Primary analyses will be intent-to-treat 
(ITT) based on randomization allocation to the 5-day course of azithromycin versus placebo. Cox proportional 
hazards regression analysis will be used to compare the rate of the mortality or first re-hospitalization, 
between the randomization groups. If the baseline assessment of randomization reveals an imbalance in 
characteristics between the treatment groups, we will evaluate these variables as potential confounders in a 
sub-analysis secondary to the ITT. Potential baseline confounders will be added stepwise in a multivariable 
model and maintained in the model if adjustment changes the hazard ratio by more than 10%. Mediating 
variables, such as post-discharge antibiotic use, will be considered in secondary analyses as time-varying 
covariates to elucidate possible mechanisms of effect. The rate of death or first re-hospitalization will be 
compared at 90-days and at 6 months post-randomization using Kaplan-Meier (K-M) survival analysis. In 
addition, we will conduct Cox regression and K-M survival analyses for time to mortality and time to re-
hospitalization as separate endpoints to understand intervention effects on these outcomes individually. 
Aim 2a. To evaluate possible mechanism(s) by which azithromycin may affect morbidity and mortality, 
by comparing reasons for re-hospitalization, prevalence of pathogen carriage, and markers of enteric 
dysfunction between the randomization arms. To evaluate the association between azithromycin and rate of 
cause-specific re-hospitalizations (hospitalization due to diarrhea, acute respiratory infection, malnutrition, or 
malaria) we will use Anderson-Gill proportional hazards modeling with previous re-hospitalizations included 
as time-dependent covariates in the model to capture the dependent structure of recurrence times. Because 
we will not have granularity in the time points other than 90-days and 6-months for assessment of pathogen 
carriage, we will compare the prevalence of pathogen carriage at 90-days and 6 months by randomization 
arm using generalized estimating equations (GEE) with a Poisson link, exchangeable correlation structure, 
and will adjust for baseline presence of a bacterial pathogen. To determine whether an observed association 
between the intervention and pathogen carriage wanes over time, we will test the hypothesis that the 
prevalence ratios comparing carriage in intervention arms are the same at the two follow-up time points using 
a chi-squared test. For the random subgroup of children in the Enteric Function Cohort, we will create a 
composite disease activity score ranging from 0 to 10 as previously described.[63] We will model the mean 
composite intestinal inflammation score by treatment group using linear regression. If the baseline 
comparison of randomization arms reveals an imbalance in characteristics between the treatment groups, 
we will additionally adjust for these variables, including post-discharge antibiotic use, in the regression 
models. Additionally, we will compare baseline differences between children randomized into the Enteric 
Function Cohort to those who were not, in order to evaluate representativeness of the random sample. 
Aim 2b. To determine whether empiric administration of azithromycin at hospital discharge increases 
risk of antimicrobial resistance in commensal E. coli and pneumococcal isolates from treated children and 
their household contacts. Among children and adult household contacts in whom commensal E. coli and/or 
S. pneumoniae are isolated, we will compare the proportion of β-lactam or macrolide resistance, or both, 
between randomization arms and Contact Cohorts for each arm, at 90-days and 6 months using GEE with a 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
16 
Poisson link and exchangeable correlation structure. A chi-squared test will be used to determine whether 
the association between intervention arm and antimicrobial resistance wanes over time. Because the 
likelihood of having a bacterial pathogen isolated may depend on baseline factors, including intervention arm, 
we will conduct secondary analyses utilizing propensity scores to account for the potential differential 
likelihood of having antimicrobial susceptibility testing performed, which will allow us to make inference to the 
entire study population and their contacts. 
Aim 3. To identify correlates and intermediate markers of post-discharge mortality and hospital-
readmission among hospitalized Kenyan children. Enrollment hospital admission diagnosis, indicators of 
malnutrition, age, and randomization arm will be assessed in a multivariable Cox regression model to identify 
correlates of the primary endpoint of death and/or hospital-readmission independent of the treatment effect.  
In addition, Cox regression models will also be built for correlates of mortality and correlates of re-admission 
individually to understand distinct cofactors for each of these outcomes. 
Aim 4. To determine the cost-effectiveness of post-discharge administration of a 5-day course of 
azithromycin in settings of varying antibiotic use, re-hospitalization rates, and mortality rates. Costs analysis: 
We will assess the cost of all services and equipment necessary to implement the intervention (direct medical 
costs). The perspective will be the healthcare provider, i.e. Kenya’s Ministry of Health. Using WHO guidelines 
and its ingredients approach, we will quantify the resources and associated unit costs required to deliver a 
5-day course of azithromycin, organized in standard expenditure categories: personnel (salaries), supplies 
including drugs, equipment, services, space and overhead. We will also measure the costs incurred by severe 
child hospitalizations, the costs for the different types of personnel employed (e.g. nurses/doctors) and the 
time demanded from them for conducting the intervention.[64] When data are missing, they will be 
complemented by data extracted from the literature and other available sources. Full incremental costs will 
be derived, with estimation of the potential healthcare cost-savings realized in avoiding severe 
hospitalizations. Costs will be measured in local currency (Kenyan Shilling) and converted into US$. Our 
main metric will be cost per child treated. Cost-effectiveness analysis (CEA): we will develop a CEA 
mathematical model, and estimate incremental costs and cost-effectiveness for implementation of the 
intervention. The model will include two components: costs (described immediately above) and health 
benefits. The study will provide clinical outcomes (mortality/morbidity) over a 6-month follow-up period. 
Subsequently, deaths averted, life-years saved and disability-adjusted life years (DALYs) averted by the 
intervention will be estimated. We will estimate: a) incremental costs and b) incremental cost-effectiveness 
of the intervention vs. status quo. Incremental costs are the net sum of the costs to implement the intervention 
compared with status quo, and the costs averted due to the decrease in severe child hospitalizations. The 
main metric will be cost per child treated. Incremental cost-effectiveness ratios (ICERs) will be estimated in 
cost per death averted, cost per life-year saved, and cost per DALY averted. We will use recent estimates 
for the disability weights for DALYs.[65, 66] Short-term (over study follow-up i.e. 6 months) and longer-term 
time horizons (extrapolated to 1, 5, and 10 years) will be used. DALYs and costs will be discounted at 3% 
per year, consistently with CEA guidelines (undiscounted results will also be presented). Sources of 
uncertainty in the results will be explored in univariate and probabilistic sensitivity analysis. [67, 68] Finally, 
we will compare our findings to CEA estimates for other health interventions in sub-Saharan Africa.[69, 70] 
Inclusion of the potential impact of antibiotic resistance: based on the results of Aim 2b, we will revisit and 
adjust our CEA estimates to account for the potentially observed increases in antibiotic resistance at the 
individual and the community level. 
Data Safety and Monitoring Board Review 
A Data Safety and Monitoring Board (DSMB) will be established at study initiation to monitor severe 
adverse events (SAEs) and to evaluate the statistical analysis plan and associated stopping rules for benefit, 
futility, or harm determined using O’Brien-Fleming stopping boundaries. The DSMB membership will include 
expertise in clinical trials, statistics, child mortality assessment, ethics, and pediatric care in resource limited 
settings. Severe adverse events (SAEs) will be monitored in real-time and will be summarized and reported 
to study investigators and relevant institutional review boards (IRBs) within 48 hours of occurrence. On a 
monthly basis, frequencies and descriptions of SAEs will be pooled and circulated to investigators and IRBs.  
When half of the person-time is accrued in the study, the DSMB will review an interim data analysis by arm 
to determine whether stopping boundaries have been crossed. Dr. Barbra Richardson will oversee 
preparation of interim analyses and presentation to the DSMB. 
Study timeline 
We anticipate that this study will take approximately 5 years to complete. In the first six months, we 
will apply for and obtain ethical approval from relevant institutions, develop and refine study tools, and finalize 
standard operating procedures. We will also conduct community sensitization and outreach to ensure the 
community is aware of the purpose of the project. Following IRB approval, field site preparation will take 3 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
17 
months, including hiring and training of site staff. We will recruit and enroll participants and conduct follow-
up over a 36-month period. Approximately 6-9 additional months will be needed for data verification and 
cleaning, ascertainment and transcription of any outstanding hospital and laboratory records, data analysis 
and manuscript preparation.  
 
Table 8. Approximate study timeline 
 
Jan-May ‘ 15 
Jun-Dec ‘ 15 
Jan-May ‘ 16 
Jun-Dec ‘ 16 
Jan-May ‘ 17 
Jun-Dec ‘ 17 
Jan-May ‘ 18 
Jun-Dec ‘ 18 
Jan-May ‘ 19 
Jun-Dec ‘ 19 
Jan-May ‘ 20 
Jun-Dec ‘ 20 
Jan-May ‘ 21 
IRB approvals 
 
 
 
 
 
 
 
 
 
 
 
 
 
CRF development 
 
 
 
 
 
 
 
 
 
 
 
 
 
Database development 
 
 
 
 
 
 
 
 
 
 
 
 
 
SOPs  
 
 
 
 
 
 
 
 
 
 
 
 
 
Patient enrollment 
 
 
 
 
 
 
 
 
 
 
 
 
 
DSMB Interim Analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data cleaning 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
Manuscript prep and 
publications 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Potential Challenges and Limitations 
In order to ensure adequate power to detect a discernable clinically relevant difference between study 
groups in the primary outcome, we have combined hospital readmission with death. Preliminary studies 
suggest that sufficient numbers of children will reach this combined outcome. However, we have incorporated 
an interim analysis by the DSMB to review the accrued data and an adapted sample size could be considered 
if the combined event frequency is less than predicted. It is possible that since most children receive 
antibiotics during hospitalization, the benefit anticipated with the use of azithromycin based on previous trials 
of mass drug administration will not be observed. However, most hospitalized children are treated with 
penicillins, cephalosporins, gentamicin, or cotrimoxazole while in hospital and the broad spectrum of activity 
(including malaria prevention) and long half-life of azithromycin suggest that there may be additive treatment 
and/or prophylactic benefit. Interim analysis will allow us to determine whether children receiving specific 
agents during inpatient treatment are less likely to benefit and will allow us to adapt our study design, sample 
size, or approach if necessary. After discharge, it is difficult to ensure adherence with the full 5-day treatment 
course. We will attempt to improve adherence using the IMB model, as described, and will measure 
adherence using three different measures. In addition, the mortality benefit of azithromycin observed in 
Ethiopia was from a single dose and we will be able to directly observe the first dose.[10] While relying on 
caregiver report of mortality and morbidity may lead to bias due to outcome misclassification, this 
misclassification should not differ between randomization arms and therefore will be non-differential. Finally, 
resistance prevalence may be lower than predicted, limiting power to detect clinically relevant differences in 
resistance prevalence between the intervention arms. We will store all isolates in the event that a greater 
number of isolates are needed for antimicrobial resistance testing.  
ETHICAL CONSIDERATIONS 
Ethical approval 
Study approvals will be obtained from the KEMRI SERU  and the University of Washington IRB.  
Risks to subjects 
The study will involve care givers and children recruited from health facilities after discharge at hospitals in 
Kisii and Homabay Counties. This study carries a minimum risk to participants.  Laboratory testing done 
for the study involves blood test that may involve bruising and possible infection at the site of needle entry.  
There is some risk that confidential information may be disclosed; however, all clinical and laboratory 
providers and other staff involved in the evaluation will be provided special training on confidentiality 
procedures and the importance of keeping personal information private.  All study providers (nurses and 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
18 
laboratory technicians) will be trained in confidentiality and privacy of information, consenting of patients, 
administration of the questionnaire, specifics of nasopharyngeal swab specimen collection, and appropriate 
HIV and Malaria testing prior to the start of the study.  All study staff will be trained and receive certification 
in Good Clinical Practice standards.   
 
Azithromycin arm: Azithromycin is a well-tolerated drug that is used frequently in pediatric patients. It is 
recommended for prophylaxis against pertussis in children as young as 1 month of age by the Centers for 
Disease Control and Prevention and is generally thought to be safe in young children.[71] Over 200 million 
doses of azithromycin have been distributed for treatment of trachoma, with many of these doses being 
provided to children as young as 6 months of age[72, 73]. Common side effects of azithromycin are largely 
gastrointestinal symptoms, including diarrhea, nausea, vomiting and abdominal pain. Azithromycin may 
cause a prolonged QT interval, with a possible increased risk of cardiac arrhythmia. As noted above, millions 
of doses of azithromycin have been distributed in trachoma endemic areas with significant decreases in 
overall childhood mortality. The short term (5 day) dosing of azithromycin is not expected to increase risk of 
arrhythmia in this population. 
Placebo arm: Children randomized to the placebo arm may be at increased risk for persistent infection, 
recurrent infection, or new infection. The study is being conducted to determine if hospitalized children may 
benefit from azithromycin at discharge. 
 
Adequacy of protection against risks 
All study staff will be trained on proper infection prevention procedures and techniques for blood specimen 
collection and nasopharyngeal swabs to minimize any chances of bruising or infection. Participant files will 
be accessible only to investigators and will be stored in a locked office.  All clinical personnel involved in the 
study will be trained in identifying and reporting of adverse events.  Caregivers will be educated regarding 
symptoms that require urgent medical attention and counseled on the importance of contacting the study 
staff in the event of any change in a child’s health. Any adverse events directly attributable to participation in 
the study will be managed by the appropriate clinic site, and if necessary, hospitalization. The costs of this 
care will be borne by the study. The clinical sites involved in this study include referral Hospital settings, all 
of which can provide emergency care if necessary. A Data Safety Monitoring Board (DSMB) will be instituted 
to monitor study progress and review safety data regularly to ensure continued benefit of the study to 
participants 
Confidentiality 
Data forms will contain no individually identifiable information. Databases will not include patient identifiers 
and will be encrypted and password protected. Study staff will keep signed consent forms and completed 
study questionnaires in a locked study cabinet.  Each participant will be assigned a unique identification 
number at the time of enrollment.  Case report forms, and laboratory samples will be identified with this 
number.  The final study data will only have the participant’s unique identification number with no link to their 
name or other personally identifying information.  Once the study has been completed and data analyzed 
and disseminated, the IRB and bioethics committees will be informed of closure, and links and consent forms 
will be destroyed (within 3 years of study completion).  All study staff involved will receive training and 
certification on standards of Good Clinical Practice before being allowed to work in this study.  
Potential benefits of the proposed research to the subjects and others 
Direct benefits: Direct benefits of participation in the study include individualized care and follow-up during 
the course of the study. In addition, transportation costs for return visits due to illness or scheduled follow up 
will be borne by the study. Transportation reimbursement is calculated based on the village of residence but 
averages about 400 Kenyan Shillings ($5) per visit. During screening for eligibility, if it is determined that a 
child did not receive HIV or malaria testing during admission, such tests will be conducted at the expense of 
the study. In addition, participants will receive diagnostic testing (bacterial culture of stool and 
nasopharyngeal samples) and some children will receive additional diagnostic testing (enteric function 
including intestinal inflammation and gut absorptive capacity from stool and urine samples) that may result in 
the identification of otherwise undiagnosed conditions. If participants have potential bacterial pathogens 
identified in stool or nasopharyngeal swabs, or if participants are found to have evidence of intestinal 
inflammation or malabsorption on stool or urine testing, these participants will be referred to hospital staff for 
management as appropriate. All children will benefit from experiencing additional exposure to the healthcare 
system during enrollment and follow-up, which may result in increased/improved identification and diagnosis 
of serious illness (via laboratory tests associated with the study, or referral to health facility for assessment 
and management as the result of an encounter with a study staff). 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
19 
Indirect benefits: It is not known if the provision of antimicrobials to all children at hospital discharge can 
reduce morbidity and mortality and this study has the potential to dramatically impact national and 
international treatment guidelines for the management of hospitalized children in low-resource settings. Also 
by testing all children at baseline for resistance to macrolides and β-lactam resistance in this study we will 
have data on baseline rates of drug resistance. This information will be disseminated to hospital staff and we 
will conduct trainings on appropriate antibiotic use.  
Importance of the knowledge to be gained 
This study has the potential to benefit millions of children in low-resource settings who face high risk of 
morbidity and mortality after discharge from hospital. If beneficial, the provision of azithromycin to children at 
hospital discharge would be an inexpensive and practical intervention that could readily be delivered to 
reduce child morbidity and mortality in many settings. This study will also elucidate possible reasons why 
children recently discharged from hospitals are at a high risk of death in resource-limited settings. Finally, 
this study will provide important data on the risk of individual and household-level antimicrobial resistance 
from prophylactic antibiotic use which will be useful in guiding recommendations for such an intervention. 
The results of this trial will inform national and international treatment guidelines. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

AZM _IRB-Protocol_12Dec2017 
 
 
 
 
 
 
 
20 
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