Clinical trial NCT04315272
Conditions: Malaria
Sponsor: University of California, San Francisco
Start date: 2020-08-21; Completion date: 2022-06-01
Enrollment: 449 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Childhood mortality is decreasing worldwide. However, many sub-Saharan countries still have high children under 5 mortality rates. The MORDOR trial in Niger, Tanzania, and Malawi demonstrated a near 14% decrease in all-cause child mortality following biannual azithromycin in children 1-59 months. Current trials in Burkina aim to replicate these results from the MORDOR study with mass azithromycin treatment.

The investigators conducted an individually randomized placebo-controlled trial in Burkina Faso called the Gut and Azithromycin Mechanisms in Infants and Neonates Trial (GAMIN: NCT03676751) to evaluate the effect of a single dose of azithromycin (20 mg/kg) on potential mediators of the effect of azithromycin on all-cause mortality and to evaluate changes in the gut microbiome longitudinally (results pending). Here, the investigators propose to conduct an expansion of the original GAMIN trial. In GAMIN II, the investigators will evaluate 450 additional 1-59 month old children longitudinally for 6 months with a focus on stool collection and malaria status.

Objectives:

1\. To determine the effect of a single dose of azithromycin for children aged 8 days-59 months on malaria. The investigators hypothesize that a single dose of azithromycin will result in a reduced malaria status within the treatment group compared to the placebo group after a 14 day period within children ages 8 days-59 months.

The study will be conducted in Nouna Town in northwestern Burkina Faso.

Detailed description:
The investigators' previous MORDOR I research demonstrated a significant reduction in all-cause child mortality after biannual mass azithromycin distribution. In three sub-Saharan Africa countries, (including Niger, Tanzania, and Malawi) mass azithromycin treatment over 2 years resulted in a 14% reduction in child mortality. Moreover, 1 in 5-6 deaths were shown to be averted within Niger alone1. Similar findings were demonstrated in a previous study for trachoma control in Ethiopia with mass azithromycin distribution. This study in rural Ethiopia noted a nearly 50% decrease in all-cause childhood mortality5. However, neither of these studies evaluated the longitudinal impact azithromycin has on the gut microbiome. The MORDOR II trial in Burkina Faso will further evaluate the efficacy of biannual azithromycin treatment. The under-5 child mortality rate in Burkina Faso is approximately 110 per 1,000 live births. Major causes of child mortality in this area are infectious mostly due to malaria, diarrhea, and upper respiratory tract infections. In addition, malnutrition contributes to a high burden of child mortality and morbidity within this region as well. By treating underlying conditions, the use of routine antibiotic treatment could reduce diverse health outcomes leading to morbidity and mortality. The investigative team proposes to conduct this study alongside the MORDOR II trial in the town of Nouna where a majority of childhood deaths are attributable to infectious causes and malnutrition.

The World Health Organization is considering adopting the presumptive use of azithromycin and other antibiotics as a recommendation to reduce childhood mortality in areas with a high infectious disease burden. Many questions remain unanswered surrounding the use of mass antibiotic treatment in areas with high child morbidity and mortality. This study will add to the current knowledge of mass azithromycin distribution from our previous MORDOR I research. The investigators propose to evaluate how azithromycin will impact childhood growth and to assess the changes that occur in the intestinal microbiome following a single dose of azithromycin treatment. The goal is to contribute more scientific literature that could assist future guidelines regarding antibiotic use.

The role of antibiotics on the gut microflora is unclear. Longitudinal studies have been recommended to further investigate the role of antibiotics on the microbiome. The investigators propose a longitudinal study designed to improve our knowledge about the changes in the intestinal microbiome following the course of a single dose of antibiotic in a setting with high childhood mortality and morbidity. More specifically, the investigators propose to follow 450 children for a 6-month time period that are between the ages of 8 days old and 59 months old. Children in this age bracket are at the highest risk for mortality from infectious causes, and furthermore, they are at the highest risk for malnutrition. This group of children would receive the greatest benefit from this intervention. The causal changes in the microbiome are vastly understudied in regards to changes in the gut microbiome following a course of antibiotics. Additionally, this study will provide valuable data on the effect of azithromycin for malaria status within 2 weeks of treatment.

Arms: arm 1: A single dose of azithromycin will be administered to children between the ages of 8 days and 59 months old. arm 2: A single dose of placebo will be administered to children between the ages of 8 days and 59 months old.
Interventions: intervention 1: Azithromycin intervention 2: Placebo
Intervention descriptions: intervention 1: Zithromax® for oral suspension is supplied in bottles containing azithromycin dehydrate powder equivalent to 1200mg per bottle and the following inactive ingredients: sucrose; tribasic anhydrous sodium phosphate; hydroxypropyl cellulose; xanthan gum; FD\&C Red #40; and flavoring including spray dried artificial cherry, crème de vanilla, and banana. After constitution, a 5mL suspension contains 200mg of azithromycin. intervention 2: Placebo

Protocol documents:
Prot_000.pdf: 
Gut and Azithromycin Mechanisms in Infants and Neonates in Burkina 
Faso 
Manual of Operations and Procedures 
Version 2.7 July 1, 2021 
Centre de Recherche en Santé de Nouna 
University of Heidelberg 
Francis I. Proctor Foundation, University of California, San Francisco 
Catherine Oldenburg, ScD MPH 
Ali Sié, MD PhD 
Thomas Lietman, MD 
Mamadou Bountogo, MD 
Boubacar Coulibaly, PhD 
Cheikh Bagagnan, MS 
Alphonse Zakane, MS 
Mamadou Ouattara, MD 
Thierry Ouedraogo Andiyam 
Fla Koueta, MD 
Idrissa Kouanda 
Mariam Seynou 
Clarisse Dah 
Constantin Sow 
Till Bärnighausen, MD ScD 
Thuy D Doan, MD, PhD 
Jeremy Keenan, MD MPH 
Travis Porco, PhD MPH 
Benjamin Arnold, PhD MPH 
Kieran O’Brien, MPH
Elodie Lebas, RN 
Jessica Brogdon, MPH 
Catherine Cook, MPH 
Ariana Austin, MS 
Ying Lin, MSPH 
William Godwin, MPH 
Fanice Nyatigo, BS 
Huiyu Hu, MS  
NCT04315272 


2 
 
 
Table of Contents 
 
 
1 
Chapter 1: Overview ..............................................................................................................................6 
1.1 
Executive Summary .....................................................................................................................6 
1.2 
Objectives .....................................................................................................................................6 
1.3 
Study Partners...............................................................................................................................7 
1.4 
Study Site .....................................................................................................................................7 
2 
Chapter 2: Context .................................................................................................................................7 
2.1 
Background ..................................................................................................................................7 
3 
Chapter 3: Study Design ........................................................................................................................8 
3.1 
Recruitment and Eligibility ..........................................................................................................9 
3.2 
Sample Size ............................................................................................................................... 10 
3.3 
Baseline Measurement .............................................................................................................. 10 
3.4 
Randomization .......................................................................................................................... 11 
3.5 
Outcomes ................................................................................................................................... 11 
3.6 
Participant Timeline .................................................................................................................. 12 
4 
Chapter 4: Procedures ........................................................................................................... 14 
4.1 
Treatment ...................................................................................................................... 16 
4.2 
Field Worker Safety Assessment .................................................................................. 16 
4.3 
Rectal Swab Visits..................................................................................................................... 16 
4.4 
Anthropometry Assessment ...................................................................................................... 16 
4.5 
Whole Blood ............................................................................................................................. 16 
4.6 
Blood Smears ............................................................................................................................ 17 
4.7 
Swabs ........................................................................................................................................ 17 
4.8 
Vital Status Assessment ............................................................................................................ 17 
4.9 
Adverse Events .......................................................................................................................... 17 
4.10 
Summary of Sample Collections ............................................................................................... 17 
5 
Chapter 5: Study Medication .............................................................................................................. 18 
5.1 
Study Medication Description ................................................................................................... 18 
5.2 
Dosage Information ................................................................................................................... 18 


3 
 
5.3 
Medication Procurement ........................................................................................................... 19 
5.4 
Medication Quality Control ...................................................................................................... 19 
5.5 
Directly Observed Treatment .................................................................................................... 19 
5.6 
Adverse Events .......................................................................................................................... 19 
5.7 
Background on Infantile Hypertrophic Pyloric Stenosis in Neonates ....................................... 20 
5.8 
Screening for IHPS .................................................................................................................... 21 
6 
Chapter 6: Swab Specimen Collection ............................................................................................... 22 
6.1 
Nasopharyngeal Swabs ............................................................................................................. 22 
6.2 
Rectal Swab Collection for Culturing/Microbiological Testing ............................................... 24 
7 
Chapter 7: Fresh Frozen Stool Specimen Collection .......................................................................... 25 
8 
Chapter 8: Blood Specimen Collection .............................................................................................. 27 
8.1 
Thin and Thick Smears ............................................................................................................. 27 
8.2 
Measuring Body Temperature ................................................................................................... 29 
8.3 
Venipuncture Blood Draw ........................................................................................................ 29 
9 
Chapter 9: Anthropometry .................................................................................................................. 37 
9.1 
Measuring Length and Height ................................................................................................... 37 
9.2 
Measure Length ......................................................................................................................... 38 
9.3 
Measure Standing Height .......................................................................................................... 40 
9.4 
Measuring Weight ..................................................................................................................... 42 
9.5 
Measuring Mid-Upper Arm Circumference (MUAC) .............................................................. 43 
10 
Chapter 10: Vital Status ................................................................................................................. 44 
11 
Chapter 11: Data Collection, Management, and Security ............................................................. 44 
11.1 
Data Collection .............................................................................................................. 44 
11.2 
Data Management and Security .................................................................................... 44 
11.3 
Data Quality and Monitoring .......................................................................................... 44 
12 
Chapter 12: Protection of Human Subjects ................................................................................... 44 
12.1 
Institutional Review Board Approval ............................................................................. 44 
12.2 
Informed Consent .......................................................................................................... 45 
12.3 
Risks and Benefits of Study Procedures ....................................................................... 45 
13 
Chapter 13: Data Safety and Monitoring Committee .................................................................... 46 
13.1 
Data and Safety Monitoring Committee Charter ........................................................... 46 
13.2 
Primary Responsibilities of the DSMC .......................................................................... 46 


4 
 
13.3 
DSMC Membership ....................................................................................................... 46 
13.4 
Conflicts of Interest ........................................................................................................ 46 
13.5 
Timing and Purpose of the DSMC Meetings ................................................................. 47 
13.6 
Procedures to Ensure Confidentiality and Proper Communication................................ 47 
13.7 
Statistical Monitoring Guidelines ................................................................................... 48 
14 
Chapter 14: Study Team Roles and Responsibilities ..................................................................... 48 
15 
Revision History ................................................................................................................ 50 
16 
References ........................................................................................................................ 51 


5 
 
ABBREVIATIONS 
 
CRSN: Centre de Recherche en Santé de Nouna 
CSPS: Centre de Santé et de Promotion Sociale 
DSMC: Data Safety and Monitoring Committee 
FFS: Fresh Frozen Stool 
IRB: Institutional Review Board 
MUAC: mid-upper arm circumference 
NP swabs: nasopharyngeal swabs 
PCR: polymerase chain reaction 
UCSF: University of California San Francisco 
WHO: World Health Organization 


6 
 
1 Chapter 1: Overview 
 
1.1 
Executive Summary 
Globally, childhood mortality has shown a promising downward trend in recent years, however, 
many sub-Saharan countries still have relatively high child mortality rates. In previous studies 
within Niger, Tanzania, and Malawi, mass azithromycin treatment to children aged 1-59 months 
old effectively reduced all-cause childhood mortality1. A similar study will be conducted in 
Burkina Faso to replicate the results of mass azithromycin treatment. 
 
We propose an individually randomized placebo-controlled trial alongside the MORDOR II 
Burkina Faso trial to evaluate the effect of a single dose of azithromycin (20 mg/kg) on potential 
mediators of the effect of azithromycin on all-cause mortality. Many questions surround the 
mechanism behind azithromycin’s effect on reducing childhood mortality. Further questions 
exist regarding antibiotic resistance and how mass antibiotic administration can impact intestinal 
microflora. The goal of this study is to demonstrate the changes in the gut microbiome after 
antibiotic administration and to measure the growth of children after receiving a single dose of 
azithromycin. Additionally we will measure resistance markers, inflammatory markers, and 
IgA-bound bacteria throughout the study. We hypothesize that a single dose of azithromycin will 
lead to a significant increase in child growth and that the gut microbiome will be significantly 
different in children who received azithromycin compared to those who received placebo. 
 
 
1.2 
Objectives 
 
1: 
To determine the effect of a single dose of azithromycin for 
children aged 8 days-59 months on longitudinal changes in the 
intestinal microbiome over a 6-month period. We hypothesize that a 
single dose of azithromycin will result in a significant difference in the 
intestinal microbiome within the treatment group compared to the 
placebo group after a 6-month period within children ages 8 days-59 
months. 
 
2: 
To determine the effect of a single dose of azithromycin for 
children aged 8 days-59 months on child growth over a 6- month 
period. We hypothesize that a single dose of azithromycin will increase 
child growth over a 6-month period in children aged 8 days-59 months. 
 
3. 
To determine the effect of a single dose of azithromycin for children 
aged 8 days to 59 months on the presence of macrolide genetic 
resistance determinants within the first two weeks post- 


7 
 
treatment. The investigators hypothesize that a single dose of 
azithromycin will increase the presence of macrolide resistance 
determinants over a 2-week period in children aged 8 days to 59 
months. 
 
1.3 
Study Partners 
CRSN and UCSF are equal partners in the production of this protocol. This study was designed 
together and will be implemented by both parties. The Bill and Melinda Gates Foundation is 
providing funding for this study. 
 
1.4 
Study Site 
 
This study will be conducted in the northwestern region of Burkina Faso in the town of Nouna. 
Nouna is approximately 300 kilometers from the capital city of Ouagadougou. Nouna town has a 
population of approximately 28,000 which includes about 4,600 pre-school aged children. There 
is currently a hospital that serves all 7 sectors of Nouna town. We will enroll up to 1500 children 
presenting to the hospital. 
 
 
2 Chapter 2: Context 
 
2.1 
Background 
 
Our previous MORDOR I research demonstrated a significant reduction in all-cause child 
mortality after biannual mass azithromycin distribution. In three sub-Saharan Africa 
countries, (including Niger, Tanzania, and Malawi) mass azithromycin treatment over 2 years 
resulted in a 14% reduction in child mortality. Moreover, 1 in 5-6 deaths were shown to be 
averted within Niger alone1. Similar findings were demonstrated in a previous study for 
trachoma control in Ethiopia with mass azithromycin distribution. This study in rural Ethiopia 
noted a nearly 50% decrease in all-cause childhood mortality5. However, neither of these studies 
evaluated the longitudinal impact azithromycin has on the gut microbiome. The MORDOR II 
trial in Burkina Faso will further evaluate the efficacy of biannual azithromycin treatment. The 
under-5 child mortality rate in Burkina Faso is approximately 110 per 1,000 live births. Major 
causes of child mortality in this area are infectious mostly due to malaria, diarrhea, and upper 
respiratory tract infections3. In addition, malnutrition contributes to a high burden of child 
mortality and morbidity within this region as well4. By treating underlying conditions, the use of 
routine antibiotic treatment could reduce diverse health outcomes leading to morbidity and 
mortality. We propose to conduct this study alongside the MORDOR II trial in the town of 
Nouna where a majority of childhood deaths are attributable to infectious causes and 
malnutrition. 


8 
 
 
The World Health Organization is considering adopting the presumptive use of 
azithromycin and other antibiotics as a recommendation to reduce childhood mortality in 
areas with a high infectious disease burden2. Many questions remain unanswered surrounding 
the use of mass antibiotic treatment in areas with high child morbidity and mortality. This study 
will add to the current knowledge of mass azithromycin distribution from our previous 
MORDOR I research. We propose to evaluate how azithromycin will impact childhood growth 
and to assess the changes that occur in the intestinal microbiome following a single dose of 
azithromycin treatment. The goal is to contribute more scientific literature that could assist future 
guidelines regarding antibiotic use. 
 
The role of antibiotics on child growth is unclear. Recent studies indicate that antibiotic use 
could impact child growth, but a previous study in Niger failed to find a statistically significant 
correlation between antibiotic treatment with azithromycin and stunting, underweight, or MUAC 
of pre-school aged children. Longitudinal studies have been recommended to further investigate 
the role of antibiotics on child growth6. Meanwhile some studies suggest antibiotics may create 
modifications in the gut microbiota impacting nutrient absorption and weight gain7.We propose 
to measure child growth through anthropometric measurements longitudinally over a 6-month 
period to see if azithromycin treatment impacts child development. We hypothesize that children 
receiving a dose of azithromycin will have more growth and development in terms of height, 
weight, and mid-upper arm circumference compared to children who receive placebo. 
 
We propose a longitudinal study designed to improve our knowledge about the changes in 
the intestinal microbiome following the course of a single dose of antibiotic in a setting with 
high childhood mortality and morbidity. More specifically, we propose to follow 450 children 
for a 6-month time period that are between the ages of 8 days old and 59 months old. Children in 
this age bracket are at the highest risk for mortality from infectious causes, and furthermore, they 
are at the highest risk for malnutrition. This group of children would receive the greatest benefit 
from this intervention. The causal changes in the microbiome are vastly understudied in regards 
to changes in the gut microbiome following a course of antibiotics. We hypothesize that children 
receiving a dose of azithromycin will have a higher prevalence of anti-pneumococcal bacterial 
resistance in nasopharyngeal samples, decreased bacterial diversity, and a higher likelihood of 
identification of bacterial resistance genes in stool and nasopharyngeal samples. 
 
 
3 Chapter 3: Study Design 
 
We propose to prospectively follow 1500 children treated with azithromycin or placebo for 6 
months in the town of Nouna. Our goal is to evaluate intestinal microbiome changes and child 
growth longitudinally. We will individually randomize eligible children to a single dose of 


9 
 
azithromycin or placebo at the Nouna town hospital. At days 0, 14, 180, and at 2 years post 
enrollment we will collect a series of samples to measure both outcomes. Anthropometry and 
vital status will be collected at each follow up visit. In addition, we will collect a blood smear at 
each visit, record body core temperature, collect a nasopharyngeal swab, a rectal swab, fresh 
frozen stool, and perform whole blood venipuncture. We will also randomly select 25 children 
per arm (n=50) to receive a rectal swab at days 0, 2, 4, 6,8,10, 12, 14, and 180 days after 
treatment. As per the Bill and Melinda Gates Foundation guidelines, all biological samples 
collected will be stored and made available to other investigators at the finish of the study. 
 
GAMIN: 
 
 
Azithromycin 
Placebo 
n=725 
n=725 
 
 
 
Supplementary rectal swab group: 
 
 
Azithromycin 
Placebo 
n=25 
n=25 
 
 
3.1 
Recruitment and Eligibility 
 
3.1.1 
Recruitment 
Prior to enrollment in the study, mobilization will be conducted in up to four secteurs of Nouna 
Town (administrative districts). The mobilization will be done house-to-house within the 
designated secteurs. Caregivers with children under 59 months will be invited to participate. All 
study activities will take place at a local primary hospital. Community mobilizers will inform 
caregivers where to go for enrollment in the trial. Enrollment will occur at the Nouna hospital. 
 
At the hospital, a dedicated study team member will enroll the child. The study team member 
will be an existing member of the enrollment site staff who will be trained for the study or an 
external study team member who will be placed at the site for the duration of the study. Trained 
staff will collect the biological specimens. The team member will verify consent from the parent 


10 
 
or guardian and review the eligibility of the child. Following enrollment, each child will be 
provided with a study card and unique identification number to be entered in the tablet at each 
follow-up visit. 
 
3.1.2 Eligibility 
 
Eligible children for this study are those who are at least 8 days old but younger than 59 months 
old. Child must live within Nouna town and report to the local Nouna town hospital during the 
study period. 
 
Inclusion Criteria 
• Between 8 days and 59 months old 
• Primary residence within catchment area of study site 
• Available for full 6 month study 
• No allergy to macrolides/azalides 
• Appropriate written informed consent from at least one parent or guardian 
• Able to feed orally 
• No prior enrollment in the NAITRE study 
Exclusion Criteria 
• <8 days old or >59 months 
• Primary residence outside catchment area of study site 
• Not available for full 6 month study 
• Known allergy to macrolides/azalides 
• No written informed consent from at least one parent or guardian 
• Unable to feed orally 
• Prior enrollment in the NAITRE study 
 
3.2 
Sample Size 
We will enroll up to 1500 children into the two study arms (725/arm). In the sub-study, 25 
children per arm will be randomized. This sample size is based on the primary outcome, 
Simpson’s alpha diversity in the intestinal microbiome. We will base some statistical 
assumptions on preliminary data from the Nouna HDSS. For instance, we assume a 15% loss to 
follow-up, a baseline effective number of 9, and a standard deviation of 0.7. 
 
3.3 
Baseline Measurement 
After enrollment and informed consent but prior to randomization, baseline samples will be 
collected. Children will undergo anthropometry (height, weight, mid-upper arm circumference), 
whole blood venipuncture, a finger prick for malaria blood smears, 2 NP swabs, a rectal swab 
and they will provide a fresh frozen stool. In addition, study staff will administer a baseline 
questionnaire inquiring about the child’s age and sex as well as the mother’s maternal age, 
education, number of children, and breastfeeding. 


11 
 
 
3.4 
Randomization 
 
A list of potentially eligible children will be asked a series of questions to determine their 
eligibility. After completion of the baseline assessment, children will be randomized to receive a 
single dose of directly observed oral azithromycin or placebo. 
 
UCSF investigators will generate the randomization sequence. Children will be randomized in a 
1:1 fashion to a single dose of azithromycin or placebo. 
 
This study will be a double blind study to both the participants and the staff (both Nouna and 
UCSF). 
 
After consent is obtained, the hospital worker will enter the child's information into a computer 
tablet or smart phone. The application "Survey Solutions" will be installed on the computer. The 
application will ask the hospital worker if consent has been obtained and information to 
determine the child's eligibility status. Once the child is determined fully eligible for the study, 
the application will tell the health worker which treatment letter to provide the child with. For 
example, the screen on the tablet may say "treat with AA" or "treat with BB". The hospital will 
be equipped with bottles of azithromycin donated from Pfizer that will be pre-labeled with 
treatment letters such as AA or BB. The application will be pre-programmed with the 
randomization scheme, but study staff and participants will remain blinded. 
 
 
3.5 
Outcomes 
 
Primary Outcome 
• Changes in intestinal microbiome over a 6-month period (Objective 1). Microbiome 
diversity will be measured at all follow-up time points. 
o Whole Blood Venipuncture. Whole blood venipuncture will be collected at all 
follow-ups. We will measure cell-free DNA and inflammatory markers such as C- 
reactive protein. 
o Nasopharyngeal Swabs. NP swabs will be collected at all follow-up time points. 
We will collect data on phenotypic resistance of S. pneumoniae, evaluate the 
microbiome using next-generation sequencing, and monitor genotypic resistance 
among various organisms. 
o Fresh Frozen Stool. FFS will be collected at all follow-ups. We will be looking 
for IgA-bound bacteria from the small intestine and evaluating inflammatory 
markers such as C-reactive protein. 


12 
 
o Rectal Swabs. We will collect rectal swabs at all time points. We will monitor 
genotypic resistance of various organisms with targeted PCR and evaluate the 
microbiome using next-generation sequencing. 
• Presence of macrolide genetic resistant determinants (Objective 3). Macrolide 
resistance will be measured from a sample of participants.  
o Rectal Swab Cohort. We will collect rectal swabs from 50 children at days 0, 2, 
4, 6, 8, 10,12, 14 and 180 post-treatment. Macrolide resistance will be analyzed 
using DNA sequencing. 
o 2 Year Timepoint. We will collect rectal swabs from all kids 2 years post-
enrollment to evaluate macrolide genetic resistance determinants.  
 
Secondary Outcomes 
• Child growth over a 6-month period (Objective 2). Anthropometry measurements will 
be collected at all follow-up time points. Weight gain will be defined as grams per 
kilogram per month (g/kg/month). 
o MUAC. Mid-upper arm circumference will be measured at all follow-ups 
o WAZ. Weight will be measured at all follow-ups and weight-for-age z-scores will 
be calculated 
o WHZ. Weight and height will be used to calculate weight for height z-scores 
o HAZ. Height or length will be measured at all follow-ups and height-for-age z- 
scores will be calculated. 
• Mortality by 6-months. Vital status will be assessed at all follow-up visits. Mortality 
will be defined as death within the study period. Date of death will be collected. 
• Adverse events. Adverse events will be reported at all follow-ups. 
• Malaria. Thin and thick smears will be collected at all follow-up time points to 
determine malaria status. Additionally, we will measure each child’s core body 
temperature. 
• PERMANOVA L-1, L-2. Norm distance on bacterial reads (intestinal) for 50 children 
within the first 2 weeks post-treatment. 
• Campylobacter. Changes in normalized reads for Campylobacter species will be 
evaluated using DNA-seq from rectal swabs of 50 kids collected within the first 2 weeks 
after treatment. 
• Resistome. Chao1 total resistance gene determinant richness using DNA-sequencing 
from rectal swabs of 50 kids collected within 2 weeks after treatment. 
  
 
3.6 
Participant Timeline 


13 
 
Figure 1. Participant Timeline and Flow 
 
 
Children Enrolled in the Study 
 
Eligibility Criteria: 
• 
Between 8 days and 59 months old 
• 
Primary residence within catchment area of study site 
• 
Available for full 6 month study 
No allergy to macrolides/azalides 
• 
Appropriate written informed consent from at least one parent or guardian 
 
 
 
 
 
 
 
Baseline assessment: 
• 
Baseline questionnaire (feeding practices, household survey, maternal survey) 
• 
Anthropometry (height, weight, MUAC) 
• 
Malaria (thin and thick smears) and temperature 
• 
Whole Blood Venipuncture 
• 
NP Swab 
• 
Fresh Frozen Stool 
• 
Rectal Swab 
 
Randomized 
 
Randomized to single dose azithromycin 
Randomized to placebo 
 
50 randomly selected children (25 per arm) will have an additional rectal swab collected every other day post- 
treatment. 
 
Day 0, 2, 4, 6, 8 , 10, 12, 14, and 180 : Rectal swab 
 
 
 
 
Children <28 days old at time of treatment 
 
Day 7, 14, 21: IHPS and Adverse Event Follow-up 


14 
 
 
 
 
 
4 Chapter 4: Procedures 
 
Table 1 provides an overview of all study procedures. The eligibility assessment, enrollment, and 
randomization are explained in Chapter 3. See the appendix for all study forms described in the 
table. 
All Participants 
Day 14: 
• 
Anthropometry (height, weight, MUAC) 
• 
Adverse Events 
• 
Vital Status 
• 
Malaria (thin and thick smears) and temperature 
• 
Whole Blood Venipuncture 
• 
NP Swab 
• 
Fresh Frozen Stool 
• 
Rectal Swab 
Day 180 (Includes window of 120-210 days): 
• 
Anthropometry (height, weight, MUAC) 
• 
Vital Status 
• 
Malaria (thin and thick smears) and temperature 
• 
Whole Blood Venipuncture 
• 
NP Swab 
• 
Fresh Frozen Stool 
• 
Rectal Swab 
 
Day 730 (2 Year Follow-Up) 
• 
Rectal Swab  


15 
 
Table 1: Overview of Procedures 
 
 
Day (since treatment) 
Activity 
Study Form 
Day 0, 2,4,6,8,10,12, 14, 180 
 Treatment 
 Form 1: Eligibility 
 Rectal swab for 50 
 Form 2: Baseline 
randomly selected 
children 
 
 
Form 6: Treatment 
Form 8: Rectal Swab 
Cohort 
0 (Baseline) 
 Eligibility Assessment 
 Form 1: Eligibility 
 Enrollment 
 Form 2: Baseline 
 Randomization 
 Form 4: Exams 
 Anthropometry 
 Form 6: Treatment 
 Whole Blood 
 Blood Smear 
 Temperature 
 NP swab 
 Rectal Swab 
 Fresh Frozen Stool 
 Treatment 
Day 7 
 IHPS follow-up on 
neonates 
 Form 7: IHPS 
Day 14 
 Anthropometry 
 Form 3: Follow-up 
 
 
 
 
Whole Blood 
Blood Smear 
Temperature 
NP swab 
 
 
Registration 
Form 4: Exams 
Form 7: Vital 
Status,AE, IHPS 
 Rectal Swab 
 Fresh Frozen Stool 
 IHPS follow-up on 
neonates 
 Vital Status 
 Adverse Events 
survey 
Day 21 
IHPS follow-up on 
neonates 
 Form 7: IHPS 
Day 180 (primary outcome) 
 Anthropometry 
 
 
 
Form 3: Follow-up 
Registration 
Form 4: Exams 
Form 7: Vital 
Status,AE, IHPS 
*Includes window of 120-210 days 
 Whole Blood 
 Blood Smear 
 Temperature 
 NP swab 
 Rectal Swab 
 Fresh Frozen Stool 
 Vital Status 


16 
 
Day 730 (2 Year Follow-Up) 
 
 Rectal Swab 
Form 4: Exams 
 
4.1 
Treatment 
 
Details on the study medication are provided in Chapter 5. No child will be treated before 8 days 
of life or after 59 months. Once the child is treated, a team member will record if the treatment 
was given or if the child did not receive treatment. If the child was not treated, the team member 
will provide the reason why the child was not treated. 
4.2 
Field Worker Safety Assessment 
 
3 weekly home visits will be performed by a field worker for any participants younger than 28 
days old at the time of treatment. The visits will be performed 7,14, and 21 days after treatment. 
Specifically, the team will be asking a series of questions to the parent or guardian in an attempt 
to screen for infantile pyloric stenosis. See Chapter 6 for more information on IHPS. The field 
worker will ask if the child has vomited since treatment, if the vomit was projectile, and if the 
child is gaining or losing weight. Those who answer affirmatively will be referred to the study 
pediatrician for closer examination and follow-up. 
 
All caregivers will be given a pamphlet in the local language explaining IHPS in detail. Contact 
information and instructions on what to do if the child presents symptoms will also be provided. 
 
4.3 
GAMIN QUOD: Rectal Swab Home Visits 
 
50 children enrolled in the study will be randomly selected to provide additional rectal swabs at 
days 0, 2, 4, 6, 8, 10, 12, 14, and 180 days after treatment. We will select 25 children per study 
arm and conduct a home-visit for these given days.  
 
4.4 
Anthropometry Assessment 
 
We will record every participant’s weight, length/height, and mid-upper arm circumference at 
baseline, day 14, and day 180 after treatment. Weight will be measured using the SECA scale. 
Length/height will be measured using a Shorrboard to the nearest cm. See chapter 9 for the 
anthropometric measurements protocol. 
 
4.5 
Whole Blood 
Every participant will undergo a blood drawl procedure at baseline, day 14, and day 180 after 
treatment. Venipuncture will be done by a trained professional using a sterile butterfly-winged 
needle. See chapter 8 for the venipuncture protocol. 


17 
 
4.6 
Blood Smears 
Thick and thin blood smears will be collected for every enrolled child at baseline, day 14, and 
day 180 after treatment. The child’s finger will be pricked with a lancet by a trained professional. 
We will use the smears to determine the child’s malaria status. Additionally, we will record the 
child’s core body temperature. If the child has a fever, we will administer a rapid diagnostic test. 
If the child is positive for malaria, we will give the child a referral to the health center. See 
chapter 8 for more on the blood protocol. 
 
The RDT utilized depended on local availability. The brand used is described below:  
SD Bioline Malaria PF (PfHRPII) from November 19-28, 2019 for baseline (407 children) 
CareStart malaria PF (PfHRPII) from November 29-30, 2019 for baseline (43 children) 
Onsite (PfHRPII and pLDH) from December 3-16, 2019 for day 14 visits.  
 
4.7 
Swabs 
 
Nasopharyngeal and rectal swabs will be collected from each participant at baseline, day 14, day 
180, and 2 years after treatment. Flocked pediatric swabs will be used for the NP collection. See 
chapter 6 for more information on the swab protocol. 
 
4.8 
Stool 
 
We will collect fresh frozen stool from participants at each visit. Infants will be given a diaper to 
produce the sample while older children will be provided with miniature potties. The potties will 
have plastic bags placed around the opening to catch the sample. More details about stool collection 
are found in chapter 8. The potties will be sanitized after each use.  
 
At baseline, children that cannot produce the sample will be asked to stay at the hospital for up to 2 
hours. At the follow-up visits, the field team will schedule appointments for the child to come back 
and produce the sample. Alternatively, the field team will go to the child’s home to obtain the 
sample. The sample will then be placed in RTIC coolers and immediately transported back to the 
laboratory freezer.  
 
 
4.9 
Vital Status Assessment 
A vital status assessment will be conducted at each follow-up visit (days 14 and 180). Field 
workers will record on the tablet if the child is alive, died, moved, or unknown. Vital status will 
also be conducted during the neonatal follow-up visits at days 7, 14, and 21 after treatment. 
 
4.10 Adverse Events 
At the 14-day follow-up visit, field workers will conduct a survey of adverse events, including if 
the child had any of the following symptoms: 
• Fever 


18 
 
• Diarrhea 
• Vomiting 
• Abdominal pain 
• Skin rash 
• Constipation 
 
The field worker will also assess if the caregiver had sought care for the child since the last time 
they spoke to the study team, and if so, what the reason for the health care visit and if the child 
was hospitalized. 
 
4.11 Summary of Sample Collections 


19 
 
Summary of laboratory and other assessments in Nouna longitudinal study 
Organism/Outcome 
Test 
Sample 
Time Point 
(Days) 
Processing 
Site 
0 
14 
180 
Malaria 
Thick and Thin Blood 
Smears 
Finger stick 
✓ 
✓ 
✓ 
Nouna 
Phenotypic 
resistance: S. 
pneumoniae 
Kirby Bauer (or 
Etest) 
NP swabs 
✓ 
✓ 
✓ 
Nouna 
Genotypic resistance: 
E. coli, S. 
pneumoniae, S. 
aureus, 
Campylobacter 
sppKlebsiella spp 
Targeted PCR: erm, 
mef, mph 
Rectal and NP 
swabs 
✓ 
✓ 
✓ 
UCSF 
Microbiome 
Next-generation 
sequencing 
Rectal and NP 
swabs 
✓ 
✓ 
✓ 
UCSF 
Inflammatory markers 
C-reactive protein 
Whole blood 
and frozen stool 
✓ 
✓ 
✓ 
Nouna 
and/or 
Gates labs 
IgA-bound bacteria 
from small intestine 
BugFACS 
Whole blood 
and frozen stool 
✓ 
✓ 
✓ 
Gates labs 
Nutritional status 
Anthropometric 
measurements 
Weight, height, 
MUAC 
✓ 
✓ 
✓ 
Nouna 
Vital status 
Mortality, 
hospitalization 
Caregiver 
questionnaire 
✓ 
✓ 
Nouna 
 
 
 
5 Chapter 5: Study Medication 
 
Enrolled participants will be offered weight-based, directly observed, oral suspension 
azithromycin or placebo. Adverse events will be monitored. 
 
5.1 
Study Medication Description 
Zithromax® for oral suspension is supplied in bottles containing azithromycin dehydrate powder 
equivalent to 1200mg per bottle and the following inactive ingredients: sucrose; tribasic 
anhydrous sodium phosphate; hydroxypropyl cellulose; xanthan gum; FD&C Red #40; and 
flavoring including spray dried artificial cherry, crème de vanilla, and banana. After 
constitution, a 5mL suspension contains 200mg of azithromycin. 
 
5.2 
Dosage Information 
All children will receive a single dose of azithromycin or placebo. Dosage will be weight-based, 
based on the baseline weight assessment as part of the anthropometry measurements. Dosing will 
follow the WHO recommendations for treatment of active trachoma: 
• Single dose of 20mg/kg in children (up to the maximum adult dose of 1g) 
Children who are allergic to macrolides/azalides will be excluded from this study. 


20 
 
 
5.3 
Medication Procurement 
Study medication will be procured in the United States and shipped to the study site. All 
medications will be prepared as an oral suspension. Participants will not incur any costs 
associated with the study or the study medication. 
 
5.4 
Medication Quality Control 
The study medication will be stored at the Centre de Recherche en Sante de Nouna in a 
temperature monitored storage room. The Nouna team and site staff will regularly check the 
expiration dates and all expired medication will be discarded appropriately. The study 
coordinator will ensure the hospital has adequate stock of study medication. 
 
5.5 
Directly Observed Treatment 
Each child will take the oral suspension of azithromycin or placebo under direct observation by a 
hospital staff member. All doses will be electronically recorded on the study tablet. 
 
5.6 
Adverse Events 
Azithromycin is generally well tolerated. The most common side effects include abdominal pain, 
diarrhea, and vomiting. These symptoms occur in less than one in twenty people who receive it. 
Additional side effects are rare, but include allergic reactions and abnormal liver function. 
Diarrhea due to Clostridium difficile has been reported in uncommon cases. 
At each monitoring visit, staff members will screen all enrolled children for any adverse events. 
Parents or guardians of the participants will be advised to report any adverse events by phone or 
in person. Study staff will inquire about the child’s experience of adverse events during each 
follow-up visit and record if the child has had any of the following symptoms: 
• Fever 
• Diarrhea 
• Vomiting 
• Skin rash 
• Constipation 
• Abdominal Pain 
 
The study team will also inquire whether the parent or guardian sought care for the child since 
the last monitoring visit. If so, the team will find out what the reason for the health care visit was 
and if the child was hospitalized. 
 
The definition of serious adverse events will include death, hospitalization, or any other life- 
threatening situation. Within 24 hours, serious adverse events must be reported to the Medical 
Monitor. The study team member conducting the follow-up visit must email Dr. Ali Sie, the 
Director of CRSN, who will immediately email the Medical Monitor and UCSF team. The 
Medical Monitor will evaluate the adverse event and determine if the event could be reasonably 


21 
 
related to azithromycin and report the results of this determination to the investigators and the 
DSMC. All information on adverse events, both serious and non-serious, will be kept on data 
collection forms through the mobile application. 
 
5.7 
Background on Infantile Hypertrophic Pyloric Stenosis in Neonates 
 
IHPS is a condition in which the pylorus of the stomach becomes thickened, resulting in gastric 
outlet obstruction.8 In developed countries, the incidence of IHPS is approximately 2 per 1,000 
infants in the general population. While surgery is generally curative, the condition is lethal in 
the absence of surgery. The cause of IHPS is unknown and likely complex, and both genetic9,10 
and environmental11,12 factors are thought to contribute to its development. Male neonates are 
disproportionately affected by IHPS, with a 4-5:1 male to female ratio.8,10,11 Other risk factors for 
IHPS include prone sleeping position, bottle/formula feeding,12,14,15 preterm birth11,12 cesarean 
section delivery11,12 and birth order.8,11-13 IHPS incidence appears to be decreasing over time, 
which some have attributed to public health interventions promoting supine sleeping position and 
exclusive breastfeeding.16,17 
 
In addition to genetic and environmental factors, erythromycin is associated with increased risk 
of infantile hypertrophic pyloric stenosis (IHPS).18 Given that azithromycin is a related 
compound, there is some concern that azithromycin may lead to increased risk of IHPS in 
neonates. Limited evidence exists of the risk of IHPS among neonates treated with 
azithromycin.19 Two randomized controlled trials have assessed the use of intravenous 
azithromycin for prevention of bronchopulmonary dysplasia (BPD) in low birthweight infants 
(<1,250g) compared to placebo within 72 hours of birth.20,21 Of 263 neonates enrolled in the two 
studies (N=130 receiving IV azithromycin), no cases of IHPS were reported. A third non-placebo 
controlled randomized trial of azithromycin prophylaxis for BPD in premature neonates 
additionally reported no cases of IHPS among 53 neonates receiving azithromycin.22 An 
observational study of 58 infants receiving azithromycin following exposure to a healthcare 
worker with pertussis did not identify any cases of IHPS.23 The largest study of azithromycin 
exposure in neonates is a retrospective cohort of more than one million infants in the TRICARE 
Management Activity military health system, which reported an overall IHPS rate of 2.3 per 
1,000 (95% CI 2.2 to 2.4) among children in the first 90 days of life.24 Of 4,875 infants 
prescribed azithromycin, there were 8 cases IHPS, 3 of which occurred when azithromycin was 
prescribed during the first 14 days of life and 5 of which during the first 15-42 days of life. 
Overall, there was no significant difference in IHPS rate in infants treated with azithromycin 
versus IHPS rates among infants who had not received an antibiotic during the first 90 days of 
life were not presented by time since birth. Table 2 lists the rates of IHPS following 
azithromycin, erythromycin, or cephalexin prescription. 


22 
 
Table 3: Unadjusted rate of IHPS in infants with oral azithromycin, per 1,000 
Azithromycin 
Erythromycin 
Cephalexin 
0-14  
days1 
15-42  
days1 
43-90  
days1 
Overall 
20.3 (4.2- 
59.2) 
6.9 (2.2-16.1) 
0 (0-0.9) 
1.6 (0.7-3.2) 
30.9 (14.1- 
58.7) 
9.3 (3.0-21.8) 
2.8 (0.6-8.1) 
8.9 (5.2-14.3) 
2.2 (0.05- 
12.2) 
3.1 (1.0- 
7.2) 
0 (0-1.6) 
1.4 (0.5- 
3.0) 
 
 
There is relatively little evidence of the epidemiology of IHPS in sub-Saharan Africa. A study in 
Nigeria documented 57 cases of IHPS from 1978-2008 at a university teaching hospital, with 
only a single case from 2003-2008.17 A study of 102 cases of IHPS over a 5-year period in 
Tanzania documented a 4.9% mortality rate despite surgical intervention.26 The risk of mortality 
was higher in infants under 2 weeks of age and those with delayed presentation to care. A 
retrospective study at a tertiary hospital in Ethiopia found that 12.9 per 1,000 admissions were 
due to IHPS, with a 3.3% mortality rate.13In Ouagadougou, an unpublished case series of 32 
infants treated for IHPS showed a 6.3% mortality rate, which was attributed to late presentation 
due to the infant traveling from far outside the city. 
 
5.8 
Screening for IHPS 
 
Field workers will conduct home visits with caregivers every week for 3 weeks following 
treatment of any neonate < 28 days old. Any child suspected of having IHPS will be immediately 
referred to the study pediatrician for evaluation. Evaluation will include ultrasonography and 
physical exam. Physical exam will include assessment of the pyloric olive (a thickened and 
elongated pylorus). Any child with a positive physical exam for a pyloric olive will be 
immediately transferred to the pediatric surgical unit in Ouagadougou or Bobo. 
 
Images will be taken including the longitudinal pylorus with canal length measurement, 
transverse pylorus with muscle thickness measurement, and the relationship of the pylorus to the 
gallbladder. IHPS will be strongly suspected in infants with a permanently closed pylorus and 
exaggerated, retrograde gastric peristalsis. Diagnostic measurements include pyloric muscle 
thickness (diameter of a single muscular wall on a transverse image) >3 mm, length (longitudinal 
measurement) >15 mm, pyloric volume >1.5 cc, and pyloric transverse diameter >13 mm.25 The 
child should be placed with their right side down, and the pylorus watched to determine if it 
opens. Small infants not below the pathologic limits with a permanently closed pylorus will also 


23 
 
be considered for further workup.25 Any child with a pyloric muscle thickness >3 mm will be 
immediately transferred to the pediatric surgical unit in Ouagadougou. 
 
All children will be assessed for electrolyte disturbance and dehydration, and rehydration and 
correction of electrolyte disturbance will occur prior to surgery. 
 
Any child participated in the study that is suspected of IHPS will receive free transportation 
provided by the study to a tertiary hospital for clinical evaluation. If the participant is clinically 
diagnosed, all medical and transportation costs incurred by the child will be fully covered by the 
study. 
 
6 Chapter 6: Swab Specimen Collection 
 
6.1 
Nasopharyngeal Swabs 
 
Nasopharyngeal swabs will be collected from all study participants at 3 different time points (day 0, day 
14, day 180). 
 
The examiner will: 
 
1. 
Place a pediatric flocked swab with a nylon tip through the right nostril and down the 
nasopharynx of each participant. Note that if the swab is not perpendicular to the frontal plane of 
the face, it is likely not in the inferior turbinate. 
 
2. 
Once you reach the nasopharynx, rotate the swab 180° as you remove the swab from the nose. 
 
3. 
Place the swab in a tube containing 1.0 mL DNA/RNA shield media by Zymo or STGG (skim 
milk, tryptone, glucose, and glycerin) media, cut the handle off using sterile scissors, and close 
the cap of the tube with the swab immersed. 
 
4. 
The nasopharyngeal swab samples in STGG will initially be stored in the field at 4°C using an 
insulated storage bag with Fisher brand ice gel packs, and then transferred to -20°C. The 
nasopharyngeal swab samples in DNA/RNA shield media will be stored in ambient temperature 
in the field. Then transferred to a refrigerator or freezer. 
 
5. 
The scissors used to cut calcium alginate swabs will be sterilized with alcohol pads or cleaned 
with bleach wipes between participants. When collecting specimens in DNA/RNA shield, 
scissors will be cleaned between participants - first with bleach wipes, and then with alcohol 
pads. 
 
Do not attempt to collect the NP swab if you are not successful after three attempts. 


24 
 
Nasopharyngeal swabs will be stored in DNA/RNA shield media by Zymo or STGG media, and standard 
microbiologic techniques will be used to isolate S. pneumoniae and test for resistance to erythromycin, 
tetracycline, sulfa-trimethoprine, oxicillin, and clindamycin. Resistant isolates will be assessed for the 
most common genetic resistant determinants (ermB and mefA) using a PCR-based assay. Serotype will be 
assessed using a nested PCR reaction for the most common serotypes, followed by the Quellung reaction 
for any untyped isolates. 
 
5.1.1 Materials for Swab Collection for Resistance Testing 
 
Swabs 
NP specimens will be collected using sterile, individually-wrapped pediatric flocked swabs with a plastic 
swab shaft (manufactured by Copan). Nasal sites will be swabbed with a sterile, Dacron polyester-tipped 
swab with a plastic shaft (manufactured by Fisherbrand). 
Sample Tubes 
All field samples for DNA testing will be collected into sterile 2.0ml microcentrifuge tubes, manufactured 
by Sarstedt®. (DNA-free tubes will be used for collection in DNA/RNA shield.) 
Cooler Bags with Frozen Ice Packs 
Insulated cooler bags will be used to carry samples to and from the field. In addition, frozen gel ice packs 
designed to thaw slowly will be used to maintain the temperature in the cooler bags during transport. 
 
-80°C Freezer 
A standard -80°C freezer located at the CRSN microbiology laboratory will be used for the storage and 
freezing of ice packs and samples. This freezer is kept in a locked room on the grounds of the CRSN, 
which is under 24-hour security guard supervision. 
 
5.1.2 Protocol for Tubing and Handling of Samples 
 
The tubing and handling protocol must be carefully followed in order to prevent contamination and 
ensure the safe transport of the samples back to the CRSN microbiology laboratory and/or to the US for 
processing. The person in charge of labeling, tubing, arranging, and handling the samples needs to 
perform this task in the most orderly and attentive manner. 
 
1. 
Both hands of the tuber should be gloved at all times. The tuber's gloves only need to 
be changed when any potential contamination of the gloves occurs. The tuber opens 
the capped, hinged lid of a microcentrifuge tube, which has been labeled with the 
participant’s random identification number. 
2. 
The swab is inserted by the examiner into the microcentrifuge tube held by the tuber. 
The swab shaft should only be inserted until the swab head is fully in the tube. The 
tuber will cut the swab shaft with sterile scissors. 
3. 
The tuber should screw the cap of the microcentrifuge tube tightly, flick the tube to 
mix the sample with the media (for tubes with DNA/RNA shield media), and place it 
in the sample collection box, located in the cooler bag filled with frozen ice packs. The 
flap of the cooler bag should be closed between each patient. The cooler bag should be 
in as cool a place as possible in the field, in a shaded area out of the sun. 


25 
 
 
Upon returning from the field each day, the samples in STGG will be immediately taken to a local health 
center and stored in a commercial -80°C freezer, reserved solely for storage of specimens and ice packs. 
All samples will be in sample boxes, labeled with the village name for easy future identification. 
 
6.2 
Rectal Swab Collection for Culturing/Microbiological Testing 
 
Rectal swabs are collected at baseline, day 14, day 180, and 2 years after treatment. A small 
cohort of 50 randomly selected children will receive an additional rectal swab on days 0, 2, 4, 
6,8,10, 12, and 180 post treatment.. 
The test will require that the child’s parent and examiners work together to obtain a good 
sample. Is it important to describe the test to the parent so that they can best assist with 
keeping the child still during the procedure, if necessary. 
In place of stool specimens, rectal swabs can be collected in the following way: 
1. 
Put on a clean pair of gloves. 
2. 
Partially open the fecal swab package and remove the top section of the collection 
vial (this can be discarded). 
3. 
Position the child: 
• 
Lie the child on his/her back, hold legs in the air (it is useful to have assistance). 
• 
Or have the child lay on his/her stomach across the mother/guardian’s lap 
4. 
Remove the swab from the package. Take care that the cotton tip is not touched. If it is 
touched, throw the swab away and begin with a new one. 
5. 
Insert the tip of the swab into the child’s anus only as far as needed to contact fecal 
material (1-3cm) and rotate 180 degrees. The tip should be a brownish color when 
removed. 
6. 
Place swab into the preservative in the collection tube. Make sure the swab is fully 
submerged in the liquid preservative and then break the swab off using the pre-scored 
breaking point. 
7. 
Screw the cap back on the tube and make sure that it’s tightened. Wrap the area 
where the cap meets the tube with Parafilm to ensure that the sample will not leak, 
and then place the tube into the appropriate sample box. 
• 
If the swab cannot be broken off while the tip is fully submerged in the liquid, try 
twirling the swab in the liquid first (to release the contents of the sample into the 
preservative) before breaking it off. Avoid rubbing the sample on the tip of the 
swab off on the side of the tube where there is no liquid. 
8. 
Place a random number label on the collection tube. 
9. 
Place the tube the rectal swab container. 
10. Swab storage for Genetic analysis: Store samples at room temperature. According to 
the manufacturer, the preservative in the tube will preserve DNA for 5 months at room 


26 
 
temperature (7 days for RNA), and thereafter can be frozen (-20°C or -80°C) for long- 
term storage. 
 
 
5.2.1 Materials for Rectal Swab Collection 
Swab 
An individually-wrapped Copan flocked swab with a plastic shaft will be used to collect the 
rectal swab and then placed into a Stool Nucleic Acid Collection and Transport Tube containing 
Norgen Stool Preservative or Amies Transport Medium. 
 
 
Sample Tube with Media 
The specimen will be in a sterile Stool Nucleic Acid Collection and Transport Tube containing 
Norgen Stool Preservative or Amies Transport Medium with a cap that will be tightened firmly. 
 
 
5.2.2 Quality Control Measures for Specimen Collection 
 
Negative Field Controls 
Negative field control swabs for NP and stool will be taken in each community to assess for 
contamination: one control swab each (NP and stool/rectal) are taken before specimen collection 
begins in a community; and another (NP and stool/rectal) upon completion of specimen 
collection. 
1. For each negative field control, the examiner will open a new swab as described above. 
2. Wave the swab in the air, without making contact with anyone/anything. 
3. Tube the swab in media, as described above. 
 
 
 
7 Chapter 7: Fresh Frozen Stool Specimen Collection 
 
 
Fresh frozen stool will be collected to evaluate IgA-bound bacteria and inflammatory markers from the small 
intestine.  
 
6.1 Materials for FFS Collection 
• Stool specimen (10-20 grams or ml) 
• Pre-printed PID labels (4 plus 1 extra) 


27 
 
• Plastic disposable transfer pipette for liquid stools 
• Cotton tipped wooden stick 
• Wide-mouthed plastic container suitable for collecting stools 
• Wooden spatula 
• Frozen ice packs 
• Cold box 
• Tube rack 
• Disposable latex gloves 
• Disposable diaper 
• Sealable plastic bags 
• Plastic spoon 
• Pen 
• Stool Field Collection Form (SFC) 
 
6.2 Collection Procedure 
 
Stool sample will be collected three times. Once before the treatment administration, once 
14 days after the treatment administration and the last time 180 days after treatment 
administration. 
 
The guardian will be asked to come to the hospital for the child to be enrolled in the study, 
examinated, and treated. The guardian will be asked to come back for each visit to the 
hospital (Baseline, Day 14 and day 180). The guardian will be reminded the day before that 
she/he has to come to the hospital for examination. The stool sample will be collected on site, not 
at the child’s home. 
 
The mother / participant’s primary caretaker/ participant will be provided with the labeled stool 
container, diaper (for infants), gloves, plastic spoon, and 2 plastic bags when she arrives at the 
station.The caretaker will be instructedto use the plastic spoon to collect 3-4 spoons of stool 
within 20 minutes of defecation and place it in the stool container, close the lid tightly, 
and place the container in the plastic bag. 
 
6.3 Temporary Storage and Transport Procedures 
 
Instruct the caretaker to hand the plastic bag with the stool to the CRSN personnel immediately 
after collection (maximum time: 20 minutes). Collect the stool specimen as soon as possible and 
document if the sample was in a cold environment on the requisition CRF. Also document if 
specimen is acceptable (estimated quantity, lid closed, and no leakage). 
 
6.4 Processing 
 
Label the original stool container with SID labels. Write the date of collection 
(DD/MM/YY) and time of collection (hh:mm; 24 hour time scale) on the label. 
 
6.5 Collection and Transport 


28 
 
Initial collection of fecal samples should be made in a suitable sterile container after 
which smaller aliquots of fecal material should be transferred by the field worker (within 20 
minutes of defecation) into pre-labeled, sterile 2ml cryo-safe tubes. Tube labels should 
minimally include the Participant’s Sample ID (SID) and the date of collection, or as 
specified by the BEED manual of procedures. 
 
Wearing clean, disposable latex gloves, fill each 2 ml cryo-vial approximately one-half to two- 
thirds full using a sterile spatula and cap tightly. Do not add any buffers, preservatives or 
additives to the sample. [Note that use of screw cap vials and not overfilling them minimizes the 
potential for cross-contamination of samples during transport and storage].Immediately place 
capped vials into liquid nitrogen pre-charged ‘dry shippers’ (for transport back to the laboratory. 
Specimens should be transferred to dry shippers within 20 minutes of defecation. [Note: dry 
shippers can be reused between charges so long as they are checked each morning following the 
manufacturer’s instructions to ensure sufficient liquid nitrogen is present to complete the 
intended sampling needs for the day]. Upon return to the laboratory, empty the vials from the dry 
shipper into a bucket of dry ice to prevent thawing while sorting and transferring the vials to 9x9 
freezer boxes for longer term storage and transport. 
 
 
8 Chapter 8: Blood Specimen Collection 
 
 
8.1 
Thin and Thick Smears 
 
7.1.1 Materials for Fingerstick 
 
Thin and thick blood smears will be read using standard microbiological techniques at the CRSN 
laboratory. 
 
Fingerprick 
Gloves 
Disposable lancets 
Alcohol wipes 
Cotton balls 
Gauze 
10% household bleach or 4% chlorhexidine solution to clean spills 
Absorbent material for spills 
Sharps container 
 
7.1.2 Fingerstick Procedure 
 
Inform the mother that her child’s finger will be pricked to obtain blood to test for malaria and 
anemia. Describe the finger prick procedure, reassure her, and answer all questions. The blood 
specimen should be collected as described below to minimize the discomfort of the child and to 
ensure sufficient blood volume collection. 


29 
 
A finger stick of capillary blood will be collected for thin and thick blood smears to assess for 
malaria. Blood will be collected by a gloved health worker using aseptic technique. Gloves will 
be changed between each participant. The fingerprick or heelstick site will be disinfected using a 
70% isopropyl alcohol swab. 
 
1. Prepare the disposable lancet. Use a NEW disposable lancet for each child. Do not re-use 
lancets! 
2. The recorder will scan the child’s QR code, and place a random number sticker on the 
TropBio filter paper and the (right edge of the) slide. 
3. Position the child for the finger stick. Make sure that the child’s right hand is warm and 
relaxed. Hold the child’s thumb, middle, or ring finger on his/her right hand (from the top 
of the knuckle to the tip of the finger) between your left thumb and finger and disinfect in 
small outward circles with an individually packaged alcohol wipe. 
4. After the alcohol dries, use the thumb to lightly press the child’s thumb or finger from the 
top of the knuckle towards the fingertip to stimulate blood flow towards the sampling 
point (puncture site). For the best blood flow and least pain, prick the side of the 
thumb/fingertip, not the center. While applying light pressure towards the 
thumb/fingertip, hold the lancing device in your hand and prick the thumb/finger. If the 
finger prick is performed properly, a single prick should be sufficient to collect the 
required amount of blood. 
5. Allow the blood to ooze out. Wipe away the first 2 or 3 drops of blood with gauze. If 
necessary, re-apply light to moderate pressure towards the thumb/fingertip 
(approximately 1 cm behind the site of the finger prick) until another drop of blood 
appears. 
Note: Do not squeeze forcefully. Avoid “milking” as it may dilute the blood with tissue 
plasma. 
 
 
7.1.3 Smears Processing and Transport 
1. Label the slide with a random number sticker. 
2. For the thick blood smear: 
a. Place a drop of blood in the center (1 cm from the edge of the slide) of a clean, 
dust-free, and grease-free slide. 
b. Spread the drop of blood evenly with a disposable wooden applicator or with 
another clean slide into a circle with a diameter of 1 cm. 
c. The blood smear should be about 1cm away from the edge of the slide. The 
correct thickness of a thick blood smear is one through which newsprint is barely 
visible when the blood is still wet. 
3. For the thin blood smear: 
a. Place a smaller drop of blood on the slide. 
b. Using another slide angled at 45°, create a feathered edge before reaching the 
other end of the slide. 
4. Allow the blood smears to air dry flat. Do not heat the slides, as this will damage the 
parasites. Be sure to protect the slide from dust and insects. Do not refrigerate slides, as 
this may cause the smears to detach from the slide during the staining procedure. 
5. When dry, place the thick and thin blood smears into the slide box. 


30 
 
6. Smears will be transported at room temperature each day to a diagnostic facility near the 
study area. 
7. Within 24 hours of thick and thin blood smear collection, the smears will be stained with 
2% Giemsa stain for 30 minutes. (The thin smear will be fixed by submerging it in 100% 
methanol for 30 seconds and then let it air dry for 1-2 minutes prior to the Giemsa stain.) 
8. Parasite density will be measured by a masked reader using a microscope at the 
diagnostic facility. 
 
Smears will be stored at room temperature. 
 
7.1.4 Thick and Thin Blood Smears Materials 
 
Glass slides 
Slide box 
Rolls of random number stickers 
 
8.2 
Measuring Body Temperature 
Measuring temperature: 
Place a clean lens filter (cover) over the end of the device. Press the Start button and push the 
«I/O» button to clear the previous reading. During the internal self-check, the display shows all 
segments. Then the last temperature taken will be displayed together with «MEM». Wait for the 
ready signal to beep and the ready symbol in the display. Fit the probe snuggly into the canal of 
the child’s right ear, then push and release the Start button. If the probe has been securely 
inserted into the ear canal during the complete measuring process, a long beep will signal the end 
of the measuring process. The thermometer detects that an accurate temperature measurement 
has been taken. The result is shown on the display. 
 
 
If the probe has not been constantly placed in a stable position in the ear canal, a sequence of 
short beeps will sound, the «ExacTemp» light will go out and the display will show an error 
message («POS» = position error). 
For the next measurement, eject the used lens filter (push the ejector) and put on a new, clean 
lens filter. Clear the display by pushing the «I/O» button once. Wait for the ready signal. Fit the 
probe snuggly into the ear canal, then push and release the Start button 
The ear thermometer turns off automatically after 60 seconds of inactivity. The device can also 
be turned off by pressing the «i/O» button for at least three seconds. 
Collect used lens filters to be cleaned at the end of the day. 


31 
 
 
8.3 
Venipuncture Blood Draw 
 
 
 
Protection of Examiner and Study Participant 
Prior to examinations at the blood station and the swab station, the examiner and 
tuber must be gloved. The examiner will put latex gloves on both of his/her hands 
prior to touching the participant and a new pair of gloves will be used for each 
participant in order to avoid transmitting infection between participants. Purell® 
Instant Hand Sanitizer will be available for hand sanitization when needed. 
It is important to handle all blood specimens with care 
to minimize risk of infection 
Wear gloves. New gloves must be worn for each child. 
Clean spills. In the event of a blood spill or splash, clean immediately with approved 
disinfectant (10% bleach or chlorhexidine solution) and wipe with absorbent material. 
Disposal of sharps. All lancets must be disposed of properly in sharps containers. 
No food. Food and drink are not allowed at the blood collection station. 
 
 
Determination of the maximum allowable blood draw volume 
See the chart below in order to determine the maximum allowable blood draw volume from 
children and infants, based on body weight. 
 
Body wt in kg 
Max drawn in one blood 
draw 
2.5% of total blood volume 
1 kg 
2.5 ml 
2 kg 
4.5 ml 
3 kg 
6 ml 
4 kg 
8 ml 
5 kg 
10 ml 
6 kg 
12 ml 
7 kg 
14 ml 
8 kg 
16 ml 


32 
 
9 kg 
18 ml 
10 kg 
20 ml 
11 thru 15 kg 
22 - 30 ml 
16 thru 20 kg 
32 - 40 ml 
21 thru 25 kg 
42 - 50 ml 
26 thru 30 kg 
52 - 60 ml 
31 thru 35 kg 
62 - 70 ml 
36 thru 40 kg 
72 - 80 ml 
41 thru 45 kg 
82 - 90 ml 
46 thru 50 kg 
92 - 100 ml 
Greater than 50 kg 
100 ml 
 
 
Citation for above chart: Seattle Children’s Hospital Powered by Mayo Medical Laboratories. 
“Maximum Allowable Blood Draw from Infants.” 
https://seattlechildrenslab.testcatalog.org/show/1000721-1 
 
 
Cell-Free Plasma extraction 
In order to prepare whole blood for downstream molecular biology techniques, plasma with an 
absence of lysed blood cells must be prepared from the whole blood. Plasma is prepared as 
follows, according to the Streck tube protocols. 
1- Centrifuge whole blood at 1,600 x g for 10 minutes at room temperature (20-25ºC). 
2- Carefully pipette the upper plasma layer without disturbing the lower blood cell layer or 
pipetting up any red material. Transfer the plasma to a separate conical-bottom tube. 
*Discard the remaining blood cells and tube in Biohazardous Waste. 
3- Centrifuge the plasma at 16,000 x g for 10 minutes. 
4- Carefully pipette the upper plasma layer (translucent), avoiding the opaque lower layer, 
and aliquot it into labeled cryo-tubes, with 200uL in each tube. 
*Discard the remaining fluid and tube in Biohazardous Waste. 
5- Store extracted plasma at ≤ -80ºC for future use in molecular biology protocols. 
 
7.2.1 Blood Draw Protocol 
 
Identification 
 
Identify the child by scanning her/his study identity card. 
 
Verify the identity of the child with the mother/guardian of the child: check the name, study 
identification number, sex, age and the name of the mother and father of the child. 
Supplies 
2 chairs 
1 examination table 


33 
 
1 pillow 
1 Cotton ball 
1 Alcohol wipes 
1 Wing needle 23G with tubing and holder 
1 needle container 
1 trash bag 
1 small bandage 
1 pair of gloves 
1 tube to collect blood (streck brand Cell Free DNA tubes) 
1 tourniquet 
1 tablet 
1 unique identification number 
1 blue pad to put under the arm of the child 
 
Immobilization 
Ask if the mom/guardian of the child would like to help by holding the child. If the parent 
accepts to hold the child, provide full instructions: 
- 
Stand on opposite side of the table 
- 
Ask the person holding the child to: 
o Stretch an arm across the table and place the child on its back, with its head on top of 
the outstretch arm 
o Pull the child close, as if you were cradling with the child 
o Grasp the child’s elbow in the 
outstretched hand 
o Use your other arm to reach 
across the child and grasp its 
wrist in a palm up position 
(reaching across the child anchors 
the child’s shoulder, and thus 
prevents twisting and rocking 
movements) 
o If the child can sit on its own: 
▪ Sit the child on your lap, facing the examiner 
▪ Hold the legs of the child between your legs 
▪ Extend the child’s arm and grasp the child’s elbow 
▪ With your other hand grasp the child’s wrist in a palm up position 
 
Use the pillow to make the child more comfortable or to hold is elbow. Comfort is very 
important for the child to feel safe. 


34 
 
Explaining the person who is helping you how to hold the child is very important, make sure the 
person understands what you need from him/her 
Reassure the child, talk to the child with a calm voice, and touch the child gently 
 
Procedure 
 
1. Collect supplies and equipment 
2. Wash your hands 
3. Look at the arms of the child to try to find a good venipuncture site 
4. Wash your hands 
5. Immobilize the child 
6. Put the tourniquet on the child about 2 fingers widths above the venipuncture site chosen 
7. Put on your gloves 
8. Attach the end of the tubing to the end of the holder 
9. Remove the plastic sleeve from the needle 
10. Using the alcohol swab, disinfect the venipuncture site and allow it to dry 
11. Draw the skin using your tumb and stick the needle in the vein 
12. Push the tube onto the holder until the needle is in it 
13. Blood should begin to flow into the tube 
14. Fill the tube until it is full, remove the tube 
15. Release the tourniquet 
16. Place a dry coton ball over the venipuncture site and withdraw the needle 
17. Ask the parent/guardian to continue applying mild pressure 
18. Dispose of the needle in the sharps container 
19. Dispose of all contaminated supplies in the trash 
20. Label the tube with a label containing a unique number 
21. Put a bandage on the child’s arm and ask the mom to remove it 1h later 
22. Gently and completely invert the tube 10 times in order ro mix blood with the tube 
preservation agents 
23. Remove gloves, put them in the trash 
24. Wash your hands 
 
 
 
Illustrations 


35 
 
 


36 
 
 


37 
 
 


38 
 
Tips and Warnings 
 
Make sure the right child is in front of you, verify his/her identity with the mom/guardian 
 
Take your time to choose your venipuncture site. Choose the venipuncture site while the child is 
still in his/her mom’s arm, when s/he feels safe. 
Keep the child warm, it will help dilate the vein and increase the blood flow: use swaddling 
blanket, let the mother hold the child/infant, warm the area of puncture with warm cloths. 
Ask the person helping you holding the child if they can rhythmically tighten and release the 
child’s wrist to ensure that there is an adequate flow of blood 
Let the child suck on something (pacifier, breast) during the venipuncture if needed 
Distract the attention of the child away from the puncture 
Do not touch the cleaned site, if the cleaned site is contaminated, repeat the disinfection 
Do not let the tourniquet in place more than 2 minutes 
Do not attempt the venipuncture more than 3 times 
 
9 Chapter 9: Anthropometry 
 
Anthropometry will be collected for all children at baseline, 14 days, and 180 days after the completion of 
the treatment. We will measure length or height, weight, and MUAC. 
 
9.1 
Measuring Length and Height 
 
A lightweight measuring board will be used to measure the participant’s height to the nearest 0.1 cm. 
 
Length or Height will be assessed with a ShorrBoard. Depending on a child’s age and ability to stand, 
measure the child’s length or height. 
 
Length: If a child is less than 2 years old, or cannot stand alone, measure recumbent length. A child’s 
length is measured lying down (recumbent). 
 
Height: If a child is able to stand, we will measure standing height. 
 
8.1.1 Procedure 
 
If the child has braids or hair ornaments that will interfere with length/height measurements, remove them 
if possible. Check that any sandals, shoes, or socks have also been removed. 


39 
 
Whether measuring length or height, the mother/guardian is needed to help with measurements and to 
soothe and comfort the child. Explain to the mother the reasons for the measurements, and describe the 
steps in the procedure. Answer any questions she might have. Show her and tell her how she can help 
you. Explain that it is important to keep the child still and calm to obtain the best measurement. 
ShorrBoard Set-Up 
1) Remove ShorrBoard from bag. 
2) Stand the ShorrBoard upright. You can step on the base of the board to keep it stable. 
3) As you face the board, turn the non-removable bolt counterclockwise to release the extension 
piece. Note: the bolt remains attached to the back of the extension piece – do NOT remove it. 
4) Slide the extension piece into the top end of the main board and fasten the clasp on the back of 
the board. Make sure the clasp is fastened properly. 
5) The auto-lock sliding head/footpiece is stored in the base of the main board and can be moved up 
and down the length of the measuring board. It should stay in place on its own wherever you 
position it. 
6) The measuring board must be placed against a firm surface for standing height (e.g. wall, table, 
tree, against a vehicle, etc.). Make sure the board is stable. If necessary, place items such as small 
rocks underneath the height board to stabilize it during the measurement. 
7) Clean the equipment with alcohol swabs at the beginning of each day. 
 
Note: when you set up the board each day, examine each of the pieces to check for damage. 
 
9.2 
Measure Length 
 
Cover the length board with a chuck for hygiene and for the baby’s comfort. Explain to the mother that 
she will need to place the baby on the length board and then help to hold the baby’s head in place while 
the measurement is taken. Show her where to stand when placing the baby down (i.e. opposite you, on the 
side of the length board away from the tape). Also show her where to place the baby’s head (against the 
fixed headboard) so that she can move quickly and surely without distressing the baby. When the mother 
understands your instructions and is ready to assist: Ask her to lay the child on his back with his head 
against the fixed headboard, compressing the hair. 
 
Quickly position the head so that an imaginary vertical line from the ear canal to the lower border of the 
eye socket is perpendicular to the board. (The child’s eyes should be looking straight up.) Ask the mother 
to move behind the headboard and hold the head in this position. 
 
Speed is important. Standing on the side of the length board where you can see the measuring tape and 
move the footboard: Check that the child lies straight along the board and does not change position. 
Shoulders should touch the board, and the spine should not be arched. Ask the mother to inform you if the 
child arches the back or moves out of position. Hold down the child’s legs with the one hand and move 
the footboard with the other. Apply gentle pressure to the knees to straighten the legs as far as they can go 
without causing injury or distress. 
 
Note: it is not possible to straighten the knees of newborns to the same degree as older children. Their 
knees are fragile and could be easily injured, so apply only minimum pressure. 


40 
 
 
If a child is extremely agitated and both legs cannot be held in position, measure with one leg in position. 
While holding the knees, pull the footboard against the child’s feet. Upon reading the measurement, the 
examiner will clearly call out the number to the recorder. Record the child’s length in centimeters to the 
last completed 0.1 cm. (1.0 mm). Keeping the child in place, release the sliding footboard, and prepare to 
repeat the measurement. Re-position the child for a second and third measurement. 
 


41 
 
9.3 
Measure Standing Height 
 
Ensure that the height board is on level ground. Working with the mother, and kneeling in order to be at 
the level of the child. Help the child stand on the baseboard with the weight of the child evenly distributed 
on both feet. The heels of the feet are placed together with both heels touching the base of the vertical 
board.  Place the feet pointed slightly outward at a 60 degree angle. The back of the head, shoulder 
blades, buttocks, calves, and heels should all touch the vertical board. Arms should hang freely by the 
sides of the body with the palms facing the thighs. 
Note: Standing with all body parts touching the board may be difficult for some children, in which case, 
help the child to stand on the board with one or more contact points touching the board. 
Ask the mother to hold the child’s knees and ankles to help keep the legs straight and feet flat, with heels 
and calves touching the vertical board. Ask her to focus the child’s attention, soothe the child as needed, 
and inform you if the child moves out of position. Position the child’s head so that a horizontal line from 
the ear canal to the lower border of the eye socket runs parallel to the baseboard. Ask the child to inhale 
deeply and to stand fully erect without altering the position of the heels. If necessary, push gently on the 
belly to help the child stand to full height. 
Still keeping the head in position, use your other hand to pull down the headboard to rest firmly on top of 
the head and compress the hair. Upon reading the measurement, the examiner will clearly call out the 
number to the recorder. Record the child’s height in centimeters to the last completed 0.1 cm (1.0 mm). 
Keeping the child in place, release the sliding headboard, and prepare to repeat the measurement. Re- 
position the child for a second and third measurement. 


42 
 
 
 
 
 
 
8.3.1 Dismantling the ShorrBoard 
 
1) Stand the board upright: face the board and step on the base with one foot to keep it stable. 
2) Slide the head/footpiece into the base of the main board. 
3) Release the clasp on the back of the extension piece and remove it. Push the clasp FLAT against 
the extension piece. 
4) To attach the extension piece to the main board, turn the front of the extension piece inward and 
place it against the front of the main board. Make sure that all sides of the extension piece are 
straight and in line with the main board. 
5) Push on the bolt that is on the back of the extension piece and screw it into the main board. 


43 
 
6) Put the board back inside of the carrying case for storage until your next use. 
 
9.4 
Measuring Weight 
 
The SECA 874 scale will be used to weigh infants and children to the nearest 0.01 kg. Infants and young 
children can also be weighed simultaneously with their parent or guardian by the unique “mother-baby” 
function (parent or guardian is weighed and then the infant or child is weighed while held by the parent). 
Explain to the mother that we want to weigh her child to see how he or she is growing. If she has a baby 
or a child who is unable to stand, she will hold the child on the scale. If the child is 2 years or older/can 
stand alone, the child will be weighed alone. Children should be wearing only light clothing, no shoes or 
sandals, no hair ornaments, and no jewelry. Explain that the child needs to remove outer clothing and 
shoes/sandals in order to obtain an accurate weight. If the baby is wearing a diaper, the diaper should be 
removed. If any heavy clothes remain on the child, make a note in the Notes section. 
Seca 874 Scale 
1) Remove scale from bag. 
2) Be sure that the scale is placed on a flat, hard, even surface. All 4 legs of the scale should make 
contact with the ground surface, without wobbling. It may be helpful to place a piece of plywood 
on the ground underneath the scale. 
3) When batt appears in the display, you should change the batteries. Remove the old batteries and 
insert 6 new batteries. 
Turn the power on the scale when you are ready to begin weighing. If the child is unable to stand on the 
scale, you will use the 2 in 1 weighing function (called tared weighing). The 2 in 1 function enables the 
weight of babies and small children to be determined while an adult holds them. Identify a suitable area 
that is flat for horizontal placement of the scale. Press the start key with no load on the scale. The scale is 
ready for use when it sets to 0.00. If necessary, switch the weight display to KG: hold down the 2 in 1 key 
for about 3 seconds. Press the start key with no load on the scale. Wait until the display shows 0.00. 
Ask the adult to remove his/her shoes and stand in the middle of the scale without the child. S/he should 
remove any long garments, as these can cover the display and also lead to variable measurements. After 
the adult’s weight appears on the display, tell him/her to remain standing on the scale. Press the 2 in 1 key 
to activate the function. The scale stores the weight of the adult and the display returns to zero. When 0.00 
and NET appear in the display, hand the child to the adult. The scale will determine the weight of the 
child. Once the value is stable for about 3 seconds, the weight is measured. 
Note: If an adult is very heavy (e.g. more than 100 kg) and the baby’s weight is relatively low (e.g. less 
than 2.5 kg), the baby’s weight may not register on the scale. In such cases, have a lighter person hold the 
baby on the scale. 
 
The positioner will clearly call out the child’s weight to the recorder. Record the child’s weight to the 
nearest 0.01 kg. Repeat the measurement 2 more times. Note that only the baby needs to removed from 
the scale; the adult should remain on the scale the entire time. To turn off the 2 in 1 function, press the 2 
in 1 key. The 2 in 1 function remains on until you press the 2 in 1 key again, or until the scale switches 
off automatically. If several children are to be weighed consecutively with the same adult holding the 
babies, it is important that this person’s weight does not change (e.g. due to a piece of clothing being 
removed/added). If the child is able to stand on the scale, you will weigh the child alone. Talk with the 
child about the need to stand still. Communicate with the child in a sensitive, non-frightening way. Press 


44 
 
the start key with no load on the scale. The scale is ready for use when it sets to 0.00. Ask the child to 
stand in the middle of the scale. Once on the scale, the child must stand still. The HOLD function is 
automatically activated for weights over 1.5 kg/3.3 lbs. The display flashes until a stable weight has been 
measured. The display is then frozen until the next weighing operation. 
Note: If the child jumps on the scale or won’t stand still, you will need to use the tared weighing 
procedure instead. 
 
The positioner will clearly call out the child’s weight to the recorder. Record the child’s weight to the 
nearest 0.01 kg. Repeat the measurement 2 more times. Have the child move completely off the scale and 
then stand again on the scale. If no further weighing operations are performed, the scale switches off 
automatically after 2-3 minutes. 
 
8.4.1 Scale Calibration 
 
In order to monitor the calibration of the scales over time, each team will weigh a 5 kg test weight at the 
beginning and end of the day. This measurement will take place at the site of the scale storage so that the 
weights do not need to be carried to the field. In some cases, these weights will be kept in a project 
vehicle. 
 
9.5 
Measuring Mid-Upper Arm Circumference (MUAC) 
 
The child’s MUAC will be measured one time. MUAC measurements will be taken at the midpoint of the 
left arm between the tip of the shoulder and the tip of the elbow using non-stretch MUAC tapes. 
 
First, find the approximate midpoint of the upper arm. Have the child stand up 
straight with feet together, and the right arm bent 90 degrees at the elbow, palm 
facing up. The examiner is positioned behind the child. If it is helpful, mark the 
midpoint with a permanent marker. To measure mid-upper arm circumference, have 
the child stand up straight with the arms relaxed at the sides. The examiner will stand 
facing the child’s right side. The measuring tape is placed around the upper arm at the marked point. 
Wrap the tape around the arm, pulling it to lie flat against the surface of the skin. Be careful not to pull 
the tape too tightly (to compress the skin). 
 
The line for reading measurements is clearly labeled on the MUAC tape 
(READ (cm)). Read the number aligned with the measurement line on the 
tape Upon measuring, the examiner will clearly call out the number to the 
recorder. Record to the nearest 0.1 cm (0.1 cm = 1 mm). Keeping the child 
in place, release the MUAC strip. 
 
If the MUAC is <11.5cm refer the child to the nearest health center. 


45 
 
10 Chapter 10: Vital Status 
 
Vital status will be recorded at all follow-up visits, including whether the child is alive, has died, or has 
moved. Date of death and location of move will be recorded. 
 
 
 
11 Chapter 11: Data Collection, Management, and Security 
 
11.1 Data Collection 
Data will be collected on enrolled children at baseline (time of enrollment), and follow-up at days 14 and 
180. Written informed consent will be collected on paper and the study nurse will administer the 
treatment letter based on the tablet app. All data will be collected electronically on mobile devices using 
a mobile application. 
11.2 Data Management and Security  
Electronic data will be uploaded daily to a secure, password-protected, cloud-based server. All devices 
used for data collection will be password-protected, as will the mobile application itself. Paper forms will 
be stored in locked cabinets accessible only by specific study team members at each enrollment site. 
No identifiers will be collected electronically. Study data will only be accessible by study team members 
and investigators in order to protect confidentiality. 
11.3 Data Quality and Monitoring  
All study team members collecting data will undergo an initial training to learn how to use the mobile 
devices as well as best practices for data collection. Data collection will be monitored on a weekly basis 
by the study team. Concerns over data quality and completeness will be relayed to the local study team by 
email, and refresher trainings and/or additional supervision of data collection by local investigators will 
be planned as needed. The study team will send quarterly progress reports to the DSMC, including 
aggregate data on enrollment and follow-up status and adverse events. 
 
 
 
12 Chapter 12: Protection of Human Subjects 
 
Before the study begins, each site will obtain formal ethical approval from their respective national ethics 
committee. In addition, local staff will approach community leaders to describe the study and answer any 
questions. Study staff will proceed only if local leadership consents to participate. We will obtain written 
informed consent for each child’s participation from their parent or guardian. If, at any time, a parent or 
guardian elects to withdraw a family member from the study, they will be free to do so. Children with 
wasting or stunting will be referred for appropriate treatment by trained study personnel to the nearest 
health center. 


46 
 
12.1 Institutional Review Board Approval 
 
University of California, San Francisco (UCSF) Committee on Human Research 
UCSF’s Committee on Human Research will annually review the study protocol for ethical approval. 
 
Comité d’Ethique du Burkina Faso 
The study protocol will be reviewed and granted ethical approval by the Comité d’Ethique du Burkina 
Faso before any study activities begin. 
12.2 Informed Consent 
 
Study personnel fluent in relevant local languages will approach parents or guardians of eligible children. 
In a private setting, the study team member will explain the objectives, risks, and benefits of the study as 
well as detailed information about what study participation entails for the child and the parent or 
guardian. The study team member will clarify that participation in the study is voluntary, that 
participation may be stopped at any time, and that all collected data will be kept confidential and securely 
stored. The study team member will ensure comprehension by inviting the parent or guardian to ask 
questions and will provide time for the parent or guardian to consider participation. When providing 
consent, both the study team member and the parent or guardian will sign two copies the consent 
document. One copy will be given to the parent or guardian and the other will be kept for study reference. 
12.3 Risks and Benefits of Study Procedures 
 
11.3.1 Procedures 
 
Nasopharyngeal swabs: There are minimal risks to the participant who receives nasopharyngeal 
swabbing. Participants may experience some temporary discomfort, but the swabbing involves minimal 
risk. Any adverse effects, such as nose-bleeds, will be treated immediately by the examiners. Other health 
care will be provided at no cost to the study participant if necessary to address a study-related adverse 
health event. 
Stool samples: Fresh frozen stool collection is a simple procedure with very minimal risk. 
Stool sample collection is a non-invasive procedure with no associated risks. 
Anthropometric Measurements: There are minimal risks associated with the measuring board, scale, or 
MUAC tapes aside from anxiety during the measurements. Examiners will do their best to ensure that the 
parent/guardian of the child understands the process of assessing anthropometric measurements. The 
examiners will attempt to minimize discomfort for all study participants before, during, and after the 
measurements are taken. Children with wasting or stunting will be referred for appropriate treatment at 
the nearest health center. 
Venipuncture: The standard risks of drawing blood from a vein include discomfort at the site of the 
puncture, bruising, swelling around the site, and rarely infection. Trained staff will administer the 
venipuncture and attempt to mitigate any associated risks. 
Rectal Swabs: There are minimal risks associated with rectal swab collection. Some discomfort may 
occur. 
Fingerstick (Blood smears): A lancing device will be used to obtain a small sample of blood. There are 
minimal risks associated with this procedure. 


47 
 
Temperature: Discomfort may be associated with taking the child’s core body temperature. 
 
 
 
 
13 Chapter 13: Data Safety and Monitoring Committee 
 
13.1 Data and Safety Monitoring Committee Charter 
 
This Charter is for the Data Safety and Monitoring Committee (DSMC) for the longitudinal MORDOR II 
Burkina Faso component. 
 
The Charter will define the primary responsibilities of the DSMC, its relationship with other trial 
components, its membership, and the purpose and timing of its meetings. The Charter will also provide 
the procedures for ensuring confidentiality and communication, statistical monitoring guidelines to be 
implemented by the DSMC, and an outline of the content of the Open and Closed Reports that will be 
provided to the DSMC. 
13.2 Primary Responsibilities of the DSMC 
The DSMC will be responsible for safeguarding the interests of trial participants, assessing the safety and 
efficacy of the interventions during the trial, and monitoring the overall conduct of the trial. The DSMC 
will provide recommendations about stopping or continuing the trial. To contribute to the integrity of the 
trial, the DSMC may also formulate recommendations relating to the selection/recruitment/retention of 
participants, to protocol-specified regimens, and the procedures for data management and quality control. 
 
The DSMC will be advisory to the trial leadership group, hereafter referred to as the Steering Committee 
(SC). The SC will be responsible for promptly reviewing the DSMC recommendations and determining, 
whether to continue or terminate the trial, and to determine whether amendments to the protocol are 
required. If needed, the DSMC may seek the advice of a content expert outside of the committee. 
  
13.3 DSMC Membership 
The DSMC is an independent multidisciplinary group consisting of epidemiologists, biostatisticians, 
bioethicists, and clinicians that collectively has experience in pediatrics, the management of infectious 
diseases, and in the conduct and monitoring of randomized clinical trials including subsaharan Africa. 
13.4 Conflicts of Interest 
The DSMC membership has been restricted to individuals free of apparent conflicts of interest. The 
source of these conflicts may be financial, scientific, or regulatory. Thus, neither study investigators nor 
individuals employed by the sponsor, nor individuals who might have regulatory responsibilities for the 
trial products, are members of the DSMC. 
 
The DSMC members will disclose to fellow members any consulting agreements or financial interests 
they have with the sponsor of the trial, with the contract research organizations (CRO) , or with other 


48 
 
sponsors having products that are being evaluated or that are competitive with those in the trial. The 
DSMC will be responsible for deciding whether these consulting agreements or financial interests 
materially impact their objectivity. 
 
The DSMC members will be responsible for advising fellow members of any changes in any of the 
membership requirements that occur during the course of the trial. It may be appropriate for DSMC 
members who develop significant conflicts of interest resign from the DSMC. 
 
DSMC membership is to be for the full duration of the trial. If any members leave the DSMC, the SC, in 
consultation with the DSMC, will promptly appoint a replacement. 
13.5 Timing and Purpose of the DSMC Meetings  
 
Organizational Meeting 
The initial meeting of the DSMC will be an Organizational Meeting. This is during the final stages of 
protocol development and the purpose is to provide advisory review of scientific and ethical issues 
relating to study design to discuss the standard operating procedures and to discuss the format and content 
of the Open and Closed Reports that will be used to present trial results. 
 
The Organizational Meeting will be attended by all DSMC members, lead trial investigators, and the trial 
biostatistician. The DSMC will be given the drafts of the trial protocol, the Statistical Analysis Plan, the 
DSMC Charter, and the current version of the case report forms. At subsequent meetings, committee 
members will receive Open and Closed Data Reports. 
13.6 Procedures to Ensure Confidentiality and Proper Communication 
To enhance the integrity and credibility of the trial, procedures will be implemented to ensure the DSMC 
has access to all emerging information from the trial regarding comparative results of efficacy and safety, 
aggregated by treatment arm. 
 
Closed Sessions 
Sessions involving only DSMC members and, where appropriate, those unmasked trial investigators (on 
the Data Coordinating Committee) who generate the Closed Reports (called Closed Sessions) will be held 
to allow discussion of confidential data from the trial, including information about the relative efficacy 
and safety of interventions. 
 
At a final Closed Session, the DSMC will develop a consensus on its list of recommendations, including 
that relating to whether the trial should continue. 
 
Open Session 
In order for the DSMC to have access to information provided, by study investigators, or members of 
regulatory authorities, a joint session between these individuals and DSMC members will be held 
between the Closed Sessions. 


49 
 
Open and Closed Reports 
For each DSMC meeting, Open and Closed Reports will be provided. Open Reports, will include data on 
recruitment and baseline characteristics, pooled data on eligibility violations, and completeness of follow- 
up and compliance. The study statistician (TCP) will prepare these Open Reports. 
 
Closed reports, available only to those attending the Closed Sessions of the meeting, will include analyses 
of primary and secondary efficacy endpoints, including subgroup and adjusted analyses, AEs and 
symptom severity, , and Open Report analyses that are displayed by intervention group. These Closed 
Reports will be prepared by the study biostatistician. 
 
The Open and Closed Reports should provide information that is accurate, with follow-up that is 
complete to within two months of the date of the DSMC meeting. The Reports should be provided to 
DSMC members approximately three days prior to the date of the meeting. 
 
Minutes of the DSMC Meeting 
The research team will prepare minutes for the open portion of the meeting, including the DSMC’s 
recommendations. 
 
Recommendations to the Steering Committee (SC) 
At each meeting of the DSMC during the trial, the committee will make a recommendation to the Steering 
Committee to continue or terminate. This recommendation will be based primarily on safety and efficacy 
considerations and will be guided by statistical monitoring guidelines defined in this Charter. 
 
Recommendations to amend the protocol or conduct of the study made by the DSMC will be considered 
and accepted or rejected by the SC. The SC will be responsible for deciding whether to continue or to stop 
the trial based on the DSMC recommendations. 
 
The DSMC will be notified of all changes to the protocol or to study conduct. The DSMC concurrence 
will be sought on all substantive recommendations or changes to the protocol or study conduct prior to 
implementation. 
 
The SC may communicate information in the Open Report to the sponsor and may inform them of the 
DSMC recommended alterations to study conduct or early trial termination in instances in which the SC 
has reached a final decision agreeing with the recommendation. The SC will maintain confidentiality of 
all information it receives other than that contained in the Open Reports until after the trial is completed 
or until a decision for early termination has been made. 
13.7 Statistical Monitoring Guidelines 
The SC will propose statistical rules for a futility stopping rule (requested by the sponsor) and an efficacy 
stopping rule at the first DSMC meeting. A decision will be made whether the efficacy stopping rule is 
appropriate for the study. 


50 
 
14 Chapter 14: Study Team Roles and Responsibilities 
• 
Investigators (UCSF): UCSF will be responsible for study design and implementation, data 
management and monitoring, data analysis, and dissemination of results. The UCSF investigators 
will design and implement trainings for study procedures and maintain frequent communication 
with local Nouna study staff 
• 
Investigators (Nouna): The Nouna investigators will contribute to the study design, 
implementation, data management and monitoring, data analysis, and dissemination of the results. 
The Nouna team will oversee all local study activities, including training and regular supervision 
and monitoring of all local study staff. 
• 
Medical Monitor: The Medical Monitor will provide clinical oversight for the enrolled children. 
Study team members will report serious adverse events to the Medical Monitor within 24 hours of 
occurrence. The Medical Monitor will determine if the event is likely to be related to 
azithromycin and provide clinical guidance for the adverse event as needed. 
• 
Data and Safety Monitoring Committee: The Data and Safety Monitoring Committee will provide 
independent oversight of data quality and patient safety during the course of the study. 
• 
Study Team Members (Nouna): Study team members in Nouna will be responsible for 
implementing all study procedures including recruitment, consent, enrollment, randomization, 
drug administration, and collection of all study data. 
o Hospital Staff: Hospital staff will be trained to determine the randomization allocation of 
each enrolled child by utilizing the study tablets and administer azithromycin if indicated 
o Outcome Assessor: One outcome assessor will be trained to manage consent, enrollment, 
and collection of all study data, including outcome assessments


51 
 
 
 
 
15 Revision History  
 
1/30/19: Addition of Will Godwin 
1/31/19: Addition of new inclusion/exclusion criteria (enrollment in NAITRE) 
2/1/19: Addition of new Fresh Frozen Stool Protocol, re-formatting, addition of rectal swab risk and 
fingerstick risk to 11.3.1. 
3/11/19: Addition of secondary outcome measure to section 3.5 of malaria. We will measure core body 
temperature as well as blood smears. Addition of temp to 3.6 participant timeline, chapter 3 baseline 
information, 7.2 temperature protocol, 11.3 discomfort as risk added 
3/14/19: Addition of rectal swab collection in 20 randomly selected children at day 2,4,6,8,10, 12 after 
treatment. Addition of Chapter 4 to better describe procedures 
4/15/19: Change study site information. Will only enroll at Nouna hospital now. Addition of Eric Nebie, 
MD. Section 4.6-Addition of rdt and referral if child has a fever 
5/30/19: Rectal swab group no longer has other procedures done. Changes made to section 3.5, 3.6, 
Chapter 4 Table 1, 4.3, and 6.2. 
7/3/19: Removal of the following exclusion criteria: 
• Antibiotic use in the past 7 days 
• Clinical complications requiring antibiotic treatment 
• Clinical complications requiring inpatient treatment 
• Congenital abnormality or chronic debilitating illness that would lead to 
predictable growth faltering or reduce likelihood of treatment benefit (such as 
cerebral palsy, Down syndrome, congenital heart disease, cleft lip/palate, etc.) 
7/19/2019: Rewording for 3.1.1. Removed the word “census” and replaced with “mobilize” 
7/29/2019: Changed the sample size for the supplementary rectal swab group from 25 to 50. 
Edited Figure 1, Table 1, Sections 3.0, 4.3, and 6.2. Also deleted in section 3.4 the sentence “we 
will ask about baseline antibiotic consumption.” 
8/30/2019: Addition of Ben Arnold, Zijun Liu, Ariana Austin as authors. Addition of RECTAL 
primary and secondary outcomes (Section3.5). Addition of RECTAL objective (section 1.2). 
9/10/2019: Removal of “home” rectal swabs. Rectal swabs will be collected at home OR at the 
hospital for the rectal cohort of 50 kids. 
10/3/2019: Addition of Emily Colby  
11/26/2019: Addition of Victoria Le, Zhaoxia Zhou, Kevin Ruder, Cindi Chen 
3/18/20: Increase sample size (section 1.4, Chapter 3 diagram, 3.2) 
3/20/2020: Addition of 6 month window (120-210 days 
2/10/21: Addition of RDT used to section 4.6, addition of 4.8 “Stool” because this section was 
mistakenly omitted , addition of language to Chapter 7 regarding children unable to produce 
stool.  
7/1/21: Addition of 2 year follow-up timepoint with a rectal swab collection.  
 
 


52 
 
 
 
16 References 
 
1 
Keenan JD, Bailey RL, West SK, et al. Mass azithromycin distribution for reducing childhood 
mortality in sub-Saharan Africa. N Engl J Med 2018; In press. 
2 
World Health Organization. (2018, May 29). Presumptive use of azithromycin. Retrieved from 
http://www.who.int/maternal_child_adolescent/guidelines/development/provision-of- 
azithromycin-to-infants/en/ 
3 
Wang H, Bhutta ZA, Coates MM, et al. Global, regional, national, and selected subnational levels 
of stillbirths, neonatal, infant, and under-5 mortality, 1980–2015: a systematic analysis for the 
Global Burden of Disease Study 2015. The Lancet 2016; 388: 1725–74. 
4 
Beiersmann C, Bountogo M, Tiendrébeogo J, et al. Malnutrition in young children of rural 
Burkina Faso: comparison of survey data from 1999 with 2009. Tropical Medicine & 
International Health 2012; 17: 715–21. 
5 
Porco TC, Gebre T, Ayele B, et al. Effect of Mass Distribution of Azithromycin for Trachoma 
Control on Overall Mortality in Ethiopian Children: A Randomized Trial. JAMA 2009; 302: 
962–8. 
6 
Amza A, Yu SN, Kadri B, Nassirou B, Stoller NE, Zhou Z, et al. (2014) Does Mass 
Azithromycin Distribution Impact Child Growth and Nutrition in Niger? A Cluster-Randomized 
Trial. PLoS Negl Trop Dis 8(9): e3128. https://doi.org/10.1371/journal.pntd.0003128 
7 
Angelakis E, Merhej V, Raoult D, 2013. Related actions of probiotics and antibiotics on gut 
microbiota and weight modification. Lancet Infect Dis 13: 889–899.</ 
8 
Ranells JD, Carver JD, Kirby RS. Infantile Hypertrophic Pyloric Stenosis: Epidemiology, 
Genetics, and Clinical Update. Advances in Pediatrics 2011; 58: 195–206. 
9 
Everett KV, Ataliotis P, Chioza BA, Shaw-Smith C, Chung EMK. A novel missense mutation in 
the transcription factor FOXF1 cosegregating with infantile hypertrophic pyloric stenosis in the 
extended pedigree linked to IHPS5 on chromosome 16q24. Pediatr Res 2016; 81: 632–8. 
10 Peeters B, Benninga MA, Hennekam RCM. Infantile hypertrophic pyloric stenosis —genetics and 
syndromes. Nature Publishing Group 2012; 9: 646–60. 
11 Svenningsson A, Svensson T, Akre O, Nordenskjöld A. Maternal and pregnancy characteristics 
and risk of infantile hypertrophic pyloric stenosis. Journal of Pediatric Surgery 2014; 49: 1226– 
31. 
12 Zhu J, Zhu T, Lin Z, Qu Y, Mu D. Perinatal risk factors for infantile hypertrophic pyloric 
stenosis: A meta-analysis. Journal of Pediatric Surgery 2017; 52: 1389–97. 


53 
 
13 Tadesse A, Gadisa A. Infantile hypertrophic pyloric stenosis: A retrospective study from a 
tertiary hospital in Ethiopia. East Cent Afr J Surg 2014; 19: 120–4. 
14 Wayne C, Hung J-HC, Chan E, Sedgwick I, Bass J, Nasr A. Formula-feeding and hypertrophic 
pyloric stenosis: is there an association? A case–control study. Journal of Pediatric Surgery 2016; 
51: 779–82. 
15 Krogh C, Biggar RJ, Fischer TK, Lindholm M, Wohlfahrt J, Melbye M. Bottle-feeding and the 
Risk of Pyloric Stenosis. Pediatrics 2012; 130: e943–9. 
16 Sommerfield T, Chalmers J, Youngson G, Heeley C, Fleming M, Thomson G. The changing 
epidemiology of infantile hypertrophic pyloric stenosis in Scotland. Archives of Disease in 
Childhood 2008; 93: 1007–11. 
17 Osifo DO, Evbuomwan I. Does Exclusive Breastfeeding Confer Protection Against Infantile 
Hypertrophic Pyloric Stenosis? A 30-year Experience in Benin City, Nigeria. Journal of Tropical 
Pediatrics 2008; 55: 132–4. 
18 Murchison L, Coppi P, Eaton S. Post-natal erythromycin exposure and risk of infantile 
hypertrophic pyloric stenosis: a systematic review and meta-analysis. Pediatric Surgery 
International 2016; 32: 1147–52. 
19 Smith C, Egunsola O, Choonara I, Kotecha S, Jacqz-Aigrain E, Sammons H. Use and safety of 
azithromycin in neonates: a systematic review. BMJ Open 2015; 5: e008194–8. 
20 Ballard HO, Anstead MI, Shook LA. Azithromycin in the extremely low birth weight infant for 
the prevention of Bronchopulmonary Dysplasia: a pilot study. Respir Res 2007; 8: 1793–9. 
21 Ballard HO, Shook LA, Bernard P, et al. Use of azithromycin for the prevention of 
bronchopulmonary dysplasia in preterm infants: a randomized, double-blind, placebo controlled 
trial. Pediatr Pulmonol 2010; 46: 111–8. 
22 Gharehbaghi MM, Peirovifar A, Ghojazadeh M, Mahallei M. Efficacy of azithromycin for 
prevention of bronchopulmonary dysplasia (BPD). Turk J Med Sci 2012; 42: 1070–5. 
23 Friedman DS, Robinette Curtis C, Schauer SL, et al. Surveillance for Transmission and antibiotic 
Adverse Events Among Neonates and Adults Exposed to a Healthcare Worker With Pertussis. 
Infect Control Hosp Epidemiol 2015; 25: 967–73. 
24 Eberly MD, Eide MB, Thompson JL, Nylund CM. Azithromycin in Early Infancy and Pyloric 
Stenosis. Pediatrics 2015; 135: 483–8. 
25 Said M, Shaul DB, Fujimoto M, Radner G, Sydorak RM, Applebaum H. Ultrasound 
Measurements in Hypertrophic Pyloric Stenosis: Don’t Let the Numbers Fool You. The 
Permanente Journal 2012; 16: 25–7. 

SAP_001.pdf: 
GAMIN II
MORDOR Project
April 9, 2021
Version 1.2
Statistical Analysis Plan
UCSF Francis I. Proctor Foundation
Centre de Recherche en Sant´e de Nouna
University of Heidelberg
NCT04315272 


Introduction
This document (Statistical Analysis Plan, SAP) describes the planned analysis and re-
porting for the clinical trial, GAMIN II: Gut and Azithromycin Mechanisms in
Infants and Neonates in Burkina Faso. It includes speciﬁcations for the statistical
analyses and tables to be prepared for the interim and ﬁnal Clinical Study Report. This
study is a Phase IV clinical trial to compare methods to reduce childhood mortality using
mass administration of azithromycin (Pﬁzer, CAS 83905-01-5) compared to placebo. The
content of this Statistical Analysis Plan meets the requirements stated by the US Food
and Drug Administration and conforms to the American Statistical Association’s Ethical
Guidelines.
The following documents were reviewed in preparation of this Statistical Analysis Plan:
• Azithromycin for the reduction of child and infant mortality: Longitudinal Component,
Manual of Operations and Procedures
• GAMIN: Gut and Azithromycin Mechanisms in Infants and Neonates in Burkina Faso,
Statistical Analysis Plan
• Statistical Analysis Plan, MORDOR Trial
• ICH Guidance on Statistical Principles for Clinical Trials
The planned analyses described in this SAP will be included in future manuscripts.
Exploratory analyses not necessarily identiﬁed in this Statistical Analysis Plan may be per-
formed to support the analysis. Unplanned analyses not delineated in this Statistical Anal-
ysis Plan will be documented as such in the ﬁnal Clinical Study Report and manuscripts.
Note that unplanned analyses will be broadly grouped into two categories:
1. Sensitivity analyses for pre-speciﬁed outcomes
2. Other unspeciﬁed analyses
2


Sensitivity analyses are deﬁned as alternate ways of modeling the primary outcome to ensure
the ﬁnding was not dependent on the analytic plan, and these will not be subject to a multiple
comparisons correction. Other unspeciﬁed analyses will be declared hypothesis generating
or subject to an alpha level of 0.001.
This document will be reviewed prior to the enrollment of patients.
All subsequent
changes will be indicated by detailed change log in the Appendix.
3


Contents
1
Summary
5
1.1
Longitudinal Trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
2
Statistical Analysis
5
2.1
Planned Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
2.1.1
Longitudinal Trial
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
3
Sample Size Considerations
6
4
Randomization
7
5
Abbreviations
8
6
Revision History
9
4


1
Summary
1.1
Longitudinal Trial
The trial proﬁle is given in the Manual of Operations. In the longitudinal trial, children are
randomized to two arms: Azithro and Control. The trial is a placebo-controlled, double-
masked (i.e., double-blind), individual-randomized clinical trial.
Objectives.
Determine the eﬀect of a single dose of azithromycin for children aged 8 days-59 months
on rapid diagnostic tests (RDT) for malaria positivity over a 14-day period. Children under
5 years of age will be randomized to a single dose of azithromycin or placebo. We hypothesize
that children randomized to a single dose of azithromycin will have signiﬁcantly lower RDT
positivity after a 14-day period, compared to those randomized to placebo.
Determine the eﬀect of a single dose of azithromycin for children aged 8 days-59 months
on longitudinal changes in the intestinal microbiome over a 6-month period. We hypothesize
that children randomized to a single dose of azithromycin will exhibit a signiﬁcant diﬀerence
in the intestinal microbiome after a 6-month period, compared to those randomized to placebo.
2
Statistical Analysis
2.1
Planned Analyses
2.1.1
Longitudinal Trial
Four hundred ﬁfty children between the ages of 8 days and 59 months may be enrolled
in the trial and be oﬀered placebo or azithromycin in a masked fashion. Relevant indicators
will be collected using ﬁnger prick and fresh frozen stool at days 0, 14, and 180. Children
followed up within day 120 and day 240 will be included in primary day 180 analysis.
5


Primary Analyses. We will compare betweens arms RDT positivity using logistic regres-
sion. We will use permutation tests to estimate coeﬃcient p-values.
We will compare proﬁle of Campylobacter, C. upsaliensis and C. hominis based on L1,
L2 distance, and PERMANOVA. We will compare diversity between arms at days 14 and
180 separately, using Shannon’s Index and Simpson’s Index.
Subgroup analysis. We will restrict the data set to participants RDT negative at baseline
and compare between arms RDT positivity at 14-day visit using logistic regression. We will
use permutation tests to estimate coeﬃcient p-values.
Statistical considerations.
• Model adequacy will be checked by examination of residuals or other goodness of ﬁt
tests as needed. Inadequate model ﬁt will prompt us to report alternative models.
• Multiple imputation will be used in case of missing baseline covariates (if applicable).
Missing outcome variables will be handled by sensitivity analysis and reporting of
conditional results.
• False Discovery Rate will be calculated for each estimated signiﬁcant eﬀect.
3
Sample Size Considerations
We assume 80% power to detect a signiﬁcant eﬀect. We also assume a sample size of 225
per group, no loss to follow-up, and RDT positivity prevalence in the control group of 10%.
Given these assumptions, we estimate a detectable eﬀect diﬀerence of -0.0659 in proportion
of RDT positive participants.
6


4
Randomization
The randomization will be conducted using R. The function sample with option replace=FALSE
will be used to conduct the random shuﬄing. Note that the choice of the random number
seed completely determines the randomization.
7


5
Abbreviations
DSMC Data and Safety Monitoring Committee
RDT Rapid Diagnostic Tests for Malaria
SAP Statistical Analysis Plan
8


6
Revision History
02 Jul 2020 Example revision.
9